pa ge 1 pa ge 37 american journal of life science and innovation (ajlsi) frequency of non-specific cervical pain experienced by the adult population in peshawar a review article muheebur rehman1*, babar ali2, shahzad waqar sethi3, abdul malik1, muhammad ismail khan4, faheem ullah5, mohammad shoaib khan1 volume 2 issue 2, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i2.1950 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: august 01, 2023 accepted: august 31, 2023 published: september 11, 2023 non-specific cervical pain is a prevalent health concern affecting individuals across various age groups and populations. this study aims to investigate the frequency of non-specific cervical pain experienced by the adult population in peshawar. a cross-sectional survey was conducted, gathering data from a diverse sample of adults residing in peshawar. the results revealed valuable insights into the prevalence and characteristics of non-specific cervical pain in this region. the findings emphasize the need for public health interventions and awareness campaigns to address this issue effectively. keywords non-specific cervical pain, cervical pain prevalence, adult population, ergonomics physical activity, posture introduction non-specific cervical pain, characterized by discomfort or pain in the neck area without a specific underlying cause, is a common musculoskeletal problem affecting a substantial portion of the adult population globally. its impact on daily life activities, work productivity, and overall well-being cannot be overlooked. while research on cervical pain has been conducted in various parts of the world, there is a gap in knowledge regarding its frequency and prevalence in peshawar, pakistan. this study aims to fill this gap by exploring the frequency of non-specific cervical pain in the adult population of peshawar. methodology a cross-sectional survey was conducted over a period of six months. a well-structured questionnaire was developed, consisting of demographic information, lifestyle factors, and questions related to the presence of non-specific cervical pain. the survey was administered to a random sample of adults aged 18 and above residing in different areas of peshawar. data was collected through face-to-face interviews and analyzed using appropriate statistical methods. results the study out of the 1000 participants surveyed, 42.5% reported experiencing non-specific cervical pain at some point in their lives. among those who reported cervical pain, 65% were females and 35% were males. the most commonly affected age group was 30-50 years. the majority of participants described the pain as intermittent and mild to moderate in intensity. factors such as poor posture, prolonged sitting, and lack of physical activity were identified as potential contributors to cervical pain. discussion the findings of this study indicate a significant prevalence of non-specific cervical pain in the adult population of peshawar. the higher prevalence among females and individuals with sedentary lifestyles is consistent with existing literature. the study highlights the need for public health campaigns aimed at promoting awareness about cervical pain prevention strategies, including maintaining proper posture and engaging in regular physical activity. access to ergonomic workstations and neck exercises could also be beneficial in reducing the frequency and impact of cervical pain. conclusion non-specific cervical pain is a noteworthy health issue affecting a substantial proportion of the adult population in peshawar. this study provides valuable insights into its prevalence, characteristics, and associated factors. the findings underscore the importance of adopting preventive measures and promoting healthy lifestyle habits to mitigate the impact of cervical pain on the wellbeing of individuals in the region. 1 abasyn university peshawar, pakistan 2 khyber teaching hospital, pakistan 3 rehman college of rehabilitation sciences, pakistan 4 cecos university peshawar, pakistan 5 riphah international universit islamabad, pakistan * corresponding author’s e-mail: muheebrehman18@gmail.com pa ge 38 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 37-38, 2023 references allander, e. (1974). prevalence, incidence, and remission rates of some common rheumatic diseases or syndromes. scandinavian journal of rheumatology, 3(3), 145153. https://doi.org/10.3109/03009747409097141 balague, f., troussier, b., & salminen, j. j. (1999). nonspecific low back pain in children and adolescents: risk factors. european spine journal, 8, 429-438. https:// doi.org/10.1007/s005860050201 bouhassira, d., attal, n., fermanian, j., alchaar, h., gautron, m., masquelier, e., ... & boureau, f. (2004). development and validation of the neuropathic pain symptom inventory. pain, 108(3), 248-257. https:// doi.org/10.1016/j.pain.2003.12.024 chard, m., hazleman, r., hazleman, b. l., king, r. h., & reiss, b. b. (1991). shoulder disorders in the elderly: a community survey. arthritis & rheumatism: official journal of the american college of rheumatology, 34(6), 766-769. https://doi.org/10.1002/art.1780340619 driscoll, t., jacklyn, g., orchard, j., passmore, e., vos, t., freedman, g., ... & punnett, l. (2014). the global burden of occupationally related low back pain: estimates from the global burden of disease 2010 study. annals of the rheumatic diseases, 73(6), 975-981. https://doi.org/10.1136/annrheumdis-2013-204631. picavet, h. s. j., & schouten, j. s. a. g. (2003). musculoskeletal pain in the netherlands: prevalences, consequences and risk groups, the dmc3-study. pain, 102(1-2), 167-178. https://doi.org10.1016/s03043959(02)00372-x pope, d. p., croft, p. r., pritchard, c. m., & silman, a. j. (1997). prevalence of shoulder pain in the community: the influence of case definition. annals of the rheumatic diseases, 56(5), 308-312. https://doi.org/10.1136/ ard.56.5.308 rareshide, e. h., & amedee, r. g. (1990). referred otalgia. the journal of the louisiana state medical society: official organ of the louisiana state medical society, 142(6), 7-10. thelin, a., holmberg, s., & thelin, n. (2008). functioning in neck and low back pain from a 12year perspective: a prospective population-based study. journal of rehabilitation medicine, 40(7), 555-561. https://doi. org/10.2340/16501977-0205 webb, r., brammah, t., lunt, m., urwin, m., allison, t., & symmons, d. (2004). opportunities for prevention of ‘clinically significant’knee pain: results from a population‐based cross sectional survey. journal of public health, 26(3), 277-284. https://doi. org/10.1093/pubmed/fdh162 pa ge 1 pa ge 17 american journal of life science and innovation (ajlsi) comparative morphological analysis of pellonula leonensis fished from isaba creek and new calabar river, niger delta region, nigeria nwafili s.a1*, eloho sorhor1 volume 2 issue 2, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i2.1549 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: april 12, 2023 accepted: june 09, 2023 published: june 16, 2023 the samall-toothed pellonula, pellonula leonensis is ecologically and economically important. it is good source of protein to rural coastal and riverine communities because of its taste. despite the economic significance, information on its biology are scarce. we evaluated the morphometric and meristic traits of the species from two water bodies in the niger delta region. the population obtained from isaba creek, delta state were significantly (p˂0.05) smaller in all the morphometric traits analysed relative to the population from new calabar river with the exception of pectoral fin length (pfl) and pelvic fin length (pvfl). the range of total length, tl and weight respectively of 3.70-8.52cm (x ̅=5.14±0.034cm) and 0.20-3.40g (x ̅=0.75±0.022g) for isaba creek population and 6.5-9.80cm (x ̅=8.35±0.06cm) and 1.7-7.30 g (x ̅=3.84±0.083g) for new calabar river population was observed. the range of meristic characters significantly differed between the two waters bodies (p<0.05). the observed intraspecies differences could be attributed to environmental variables, fishing pressure and genetic factors. this study reveals the need for developing conservation strategies and domestication programs for species distributed in different water system. keywords pellonula leonensis, isaba creek, new calabar river, morphological traits, geographic isolation, intraspecies differences introduction the fresh and brackish waters of tropical africa boast of about 20 species of clupeids and one of them is pellonula leonensis with wide spread distribution in both remote sahelian and coastal basins (reid and sydenham, 1979, teugels, et al., 1992; gourene & teugels, 2003). pellonula is essentially a riverine fish that can inhabit both fast-flowing and calm, muddy habitats, lakes and creeks (lowe-mcconnell, 1987), near the shore (turner, 1994). information on the biology of pellonula leonensis are scarce in the rivers and coastal areas of the niger delta region. however, ikomi (1995) and kingdom and allison, (2007a; b) have reported on aspects of the species’ biology in the warri and nun rivers, respectively. the small-toothed pellonula is of commercial importance in communities around new calabar river (ncr), rivers state and isaba creek (isc), delta state, fetching good income on basis of account of its tasty characteristics. this study evaluates and compares the morphometric and meristic characteristics of p. leonensis from the two water bodies (ncr and isc) in the niger delta region. there is no record of any research on the species from the two water bodies. materials and methods samples collection the samples of pellonula species were procured from fishermen operating in isaba creek, delta state and new calabar river, rivers state in the niger delta region (table 1). 1 department of fisheries, faculty of agriculture, university of port harcourt pmb, 5323, nigeria * corresponding author’s e-mail: sylvanus.nwafili@uniport.edu.ng table 1: sampling location and size sampling location latitude longitude n isaba creek 5.42749° or 5° 25' 39" north 5.69516° or 5° 41' 43" east 614 new calabar river 4.41939° or 4° 25' 10" north 7.02001° or 7° 1' 12" east 168 total 782 the species was identified according to gourène and teugels (1991, 2003) and specimens collected were stored in ethanol (97% alcohol). the morphometric characteristics were measured on each individual with a metre rule (cm) and the body weight (g) was measured with a mini-digital weighing scale for the following parameters: anal fin length (afl), body depth (bd), caudal fin length (cfl), caudal peduncle (cp), distance between the pectoral and anal fin (dbpaf), dorsal fin length (dfl), eye diameter (ed), head depth (hd), head length (hl), pectoral fin length (pfl), pelvic fin length (pvfl), pre-dorsal length (prdl), prepectoral length (prpfl), pre-pelvic length (prpvfl), pre-orbital length (prol), standard length (sl), fork pa ge 18 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 17-22, 2023 length (fl), total length (tl) (fig. 1) and body weight (bw). the meristic characteristics were the anal fin ray count (afr) and dorsal fin ray count (dfr). statistical analysis past 326b (paleontological statistics software) was used to analyze the descriptive statistics such as the minimum (min.), maximum (max), mean (x ̅), standard errors (se), standard deviation (sd), variance (v) and coefficient of variation (cv) of each characters, and students’ t-test was run to compare between means of each characters by recording the significant difference of each character parameter recorded from both locations. figure 1: morphometric measurements of pellonula leonensis used for study results and discussion morphological characteristics of p. leonensis from isaba creek and new calabar river. a total of 782 individuals were sampled, 614 individuals from isaba river and 168 from new calabar river (table 1). the range of mean (x ̅), standard error (se), standard deviation (sd), variance (v), and coefficient of variation (cv) of the morphological characters of p. leonensis are shown in table 2. the size range of tl and weight respectively of 3.70-8.52cm (x ̅=5.14±0.034cm) and 0.20-3.40g (x ̅=0.75±0.022g) for isaba creek population and 6.5-9.80 cm (x ̅=8.35±0.06cm) and 1.7-7.30 g (x ̅=3.84±0.083g) for new calabar river population were observed. it can be seen that individuals from the new calabar river possessed higher comparative range and mean values for each character relative to isaba creek. for all 19 morphometric characters evaluated, 17 were significantly different between the isaba creek and the new calabar river (p<0.05), except for the pfl and pvfl (p>0.05). meristic characteristics of p. leonensis from isaba river and new calabar river table 3 shows the descriptive statistics for the meristic characteristics of p. leonensis from isaba creek and new calabar river. the range of mean (x ̅) standard error (se), standard deviation (sd), variance (v) and coefficient of variation of meristic characters observed in p. leonensis. the afr and dfr showed significant differences between the two water bodies (p<0.05). for isaba river population, the anal fin ray (afr) has a range count of 10-22, and a mean value of 17.33 ± 0.073, and the dorsal fin ray (dfr) has a range count of 9-22, and a mean value of 15.97 ± 0.085. while for the population of new calabar river, the anal fin ray has a range count of 10-25 and a mean value of 19.18 ± 0.172, and the dorsal fin ray has a range count of 12-22 and a mean value of 16.89 ± 0.168. table 2: summary descriptive statistics for the morphometric characteristics of p. leonensis from isaba creek and new calabar river c ha ra ct er isaba river (n=614) new calabar river (n=168) sig min max ±se sd v cv min max ±se sd v cv t uneq. var. t afl 0.20 1.40 0.70 ± 0.009 0. 23 0. 05 32.83 0.6 1.9 1.11 ± 0.02 0.23 0.05 20.32 ** ** pa ge 19 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 17-22, 2023 bd 0.50 2.85 1.10 ± 0.011 0. 30 0. 09 26.99 1.2 2.6 1.83 ± 0.020 0.26 0.07 14.32 ** ** cfl 0.30 1.80 0.77 ± 0.01 0. 33 0. 11 42.57 0.6 2.2 1.23 ± 0.026 0.33 0.11 27.08 ** ** cp 0.20 0.90 0.44 ± 0.004 0. 11 0. 01 24.13 0.4 5.00 0.67 ± 0.027 0.36 0.13 52.85 ** ** dbpaf 1.2 5.00 2.21 ± 0.031 0. 76 0. 58 34.46 2.4 4.1 3.23 ± 0.028 0.36 0.13 11.06 ** ** dfl 0.60 2.20 0.67 ± 0.01 0. 27 0. 07 40.57 0.5 1.4 0.98 ± 0.015 0.19 0.04 19.25 ** ** ed 0.2 1.5 0.40 ± 0.005 0. 11 0. 01 27.89 0.35 0.65 0.49 ± 0.003 0.05 0.002 9.15 ** ** hd 0.3 1.6 0.77 ± 0.009 0. 22 0. 05 28.77 0.9 2.1 1.44± 0.017 0.22 0.05 14.90 ** ** hl 0.4 2.00 1.03 ± 0.011 0. 2 0. 08 26.56 1.00 2.2 1.61± 0.019 0.24 0.06 14.91 ** ** pfl 0.05 1.15 0.11 ± 0.002 0. 05 0. 00 2 43.56 0.09 0.15 0.10± 0.0006 0.008 0.00006 7.61 n . sig # pvfl 0.08 1.15 0.11 ± 0.002 0. 00 5 0. 00 2 43.41 0.1 0.15 0.10± 0.0006 0.008 0.0006 7.56 n . sig n. sig prdfl 1.1 3.6 2.16 ± 0.017 0. 43 0. 18 19.82 2.7 4.20 3.51± 0.024 0.32 0.10 8.98 ** ** prpfl 0.3 2.00 1.12 ± 0.012 0. 30 0. 09 26.81 1.10 2.50 1.73± 0.263 0.26 0.07 15.24 ** ** prpvfl 0.7 5.00 2.09 ± 0.021 0. 53 0. 28 25.27 2.3 4.1 3.35± 0.025 0.33 0.11 9.74 ** ** prol 0.2 2.6 0.43 ± 0.011 0. 28 0. 08 66.58 0.4 0.9 0.54± 0.006 0.08 0.006 14.50 ** ** sl 3.00 7.30 4.43 ± 0.03 0. 78 0. 69 17.48 5.2 8.5 6.90± 0.047 0.60 0.36 8.74 ** ** fl 3.2 7.7 4.72 ± 0.033 0. 82 o .6 7 17.29 5.6 8.7 7.28± 0.05 0.65 0.42 8.92 ** ** tl 3.70 8.52 5.41 ± 0.034 0. 86 0. 74 15.92 6.5 9.80 8.35± 0.06 0.71 0.50 8.50 ** ** bw(g) 0.20 3.40 0.75 ± 0.022 0. 56 0. 31 74.27 1.7 7.3 3.84 ± 0.083 1.08 1.16 28.04 ** ** where, min=minimum, max=maximum, = mean, se= standard error, sd= standard deviaton, v= variance, cv= coefficient of variation, t=t-test, **significant at p<0.05, #=p=0.05, n sig= no significant difference p>0.5 pa ge 20 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 17-22, 2023 table 3: ssummary descriptive statistics for the meristic characteristics of p. leonensis from isaba creek and new calabar river chrs isaba river (n=614) new calabar river (n=168) sig min max ±se sd v cv min max ±se sd v cv t uneq. var. t afr 10 22 17.33 ± 0.073 2.11 4.45 13.21 12 22 16.89 ±0.166 2.23 4.97 11.63 ** ** dfr 9 22 15.97 ± 0.085 2.11 4.45 13.21 12 22 16.89± 0.166 2.16 4.65 12.77 ** ** where, min=minimum, max=maximum, = mean, se= standard error, sd= standard deviaton, v= variance, cv= coefficient of variation, t=t-test **significant at p<0.05, n sig= no significant difference the morphometric and meristic characters of p. leonensis from isaba creek and new calabar river were observed and compared in this study. a total of 782 individuals were sampled in this study. the size range of tl and total weight recorded for isaba creek population and new calabar river population fall within the range reported by ezenwaji and offish (2003) and ezenwaji (2004) for the anambra river. yao et al. (2015) also reported individual weight ranging from 0.26 g to 3.22 g, with an average of 0.81 ± 0.44 g and length varying from 31.67 to 65.35 mm with an average of 43.38 ± 6.31 mm in taabo lake. the result from this study clearly demonstrates the variations between the two populations. for all the characters evaluated, individuals from the population from new calabar river were bigger than the population those from isaba creek. kurniawan et al. (2020) detected highly significant morphometric difference in osteochilus spilurus from two separate water bodies. solomon et al., (2015) reported abundant significant variations in morphometric characters of clarias gariepinus from different populations. factors responsible for the differences between populations observed in this study are many and varied, including environmentally induced morphological and meristic changes due to stressors or pollution (kurniawan et al. (2020). the two water bodies by their location in the niger delta have been under the influence of illegal exploration and exploitation of crude oil resulting in oil pollution of the water bodies, which is one of the causes of serious damages to water quality. disparity in levels of exposure to these environmental stressors may underpin changes in phenotype and genotype that culminate in fish adaptation. swain and foote (1999) observed that the phenotypic variation in morphological characters and meristic count may not only be genetic but may be environmentally induced. krabbenhoft et al. (2009) described the environmental factors underlying the morphological changes as water clarity, water depth and flow, food availability and physical complexity. environmentally induced differences can also include diet or feeding habit, resulting in within species morphological variations in fish (ehlinger, 1988; fermon and cibert 1998; parker et al., 2009). morphological divergence and intraspecific difference induced by habitat are reported to be common in fishes. for example, alteration in shape and fins can be influenced considering whether the water body is lentic or lotic. physical characteristics can determine evolutionary changes and ecological conditions can drive changes in the morphological characteristics of resident fish populations (haas et al., 2010). secondly, fishing pressure and utilization differential may mount spatial pressure on populations in natural habitats resulting in pronounced morphological differences (kurniawan et al. (2020 citing yang et al., 2003). thus, we assert that fishing intensity may be higher in isaba creek relative to new calabar river. geographic isolation is another reason for intraspecific morphometric differences. over time isolation by distance and barriers to gene flow play major role in these differences. the ncr and isc are connected to the ocean at different points along the coastal waters of nigeria, which has created an effective barrier to gene flow between the two populations. with dna, more light could be shed from these species when these rivers and creeks came to be separated. sexual dimorphism observed in some species can manifest in wide range of morphometric differences between populations (parker, 1992; breder and rosen. 1998; kim et al., 2008; im et al., 2016; pramono et al., 2019). therefore, we suggest that in the studies employing the use of morphometric traits, individuals should identified by sex. this was not taken into cognizance in executing this research, which may account for the differences if sexual dimorphism exist in the species. the meristic counts showed a very clear difference between the samples from the two population. although it has been established that the meristic characters are independent of the fish size; hence, they should not change during growth (strauss 1985). this study has shown that the population from isaba creek is far small in size than that from new calabar river, this significant difference in size might be due to high level of fishing of mostly immature stock from the isaba creek. however, the morphometric and meristic variations examined in this reports are preliminary and provide an insight into pa ge 21 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 17-22, 2023 the population of the same species from two different water bodies. conclusion studies on the basis of morphometric traits with respect to aquaculture and potential aquaculture species and species of interest to capture fisheries should be initiated with the aim of determining sexual dimorphism. such studies would be expedient and useful for developing conservation strategies and domestication programs. conflict of interest. the authors confirm that this article content has no conflicts of interest. acknowledgements we thank professor s.n. dekae of department of fisheries, rivers state university of science and technology, rivers state for assisting with identification. reference breder, c. m. & rosen, d. e. 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(2009). interplay of morphology, habitat, resource use, and genetic relationships in young yellow perch. transactions of american fisheries society, 138, 899-914. parker, g. a. (1992). the evolution of sexual size dimorphism in fish. journal of fish biology, 41, 1-20. pramono, t. b., arfiati, d., widodo, m. s. & yanuhar, u. (2019). sexual dimorphism in morphometric characters of mystus singaringan from klawing river in central java, indonesia: strategic instruction for conservation development. biodiversta, 20(4), 1133-1139. reid, g. m. g & sydenham, d. h. j. (1979). a checklist of lower benue river (west africa). journals of natural history 13, 41-67. solomon, s. g, okomoda, v. t. & ogbenyikwu, a. i. (2015). intraspecific morphological variation between cultured and wild clarias gariepinus (burchell) (clariidae, siluriformes) – arch polish fisheries 23, 53-61. strauss, r. e. (1985). evolutionary allometry and variation in body form in the south american catfish genus corydorus (callichthydae). systematic biology, 34, 381-396. swain, d. & foote, c. j. (1999). stocks and chameleons: the use of phenotypic variation in stock identification. pa ge 22 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 17-22, 2023 fishery research, 43, 113-128. teugels, g. g., reid, g. m. g. & king, r. p. (1992). fishes of the cross river basin (cameroon nigeria): taxonomy, zoogeography, ecology and conservation. annules sciences zoologues, 266, 1-732. turner, g. f. (1994). lake kainji fisheries statistic and stock assessment. nigerian/german (gtz) kainji fisheries promotion project., 0. yao, a. a., da costa, k. s. & dietoa, y. m. (2015). small pelagic fish pellonula leonensis boulenger, 1916 (pisces; clupeidae) fishery in taabo lake: typology, yield and socioeconomic characteristics. international journal of fisheries and aquatic studies, 3(1), 392-399. pa ge 1 pa ge 15 american journal of life science and innovation (ajlsi) extraction and evaluation: the viability of the three varieties of banana (musa paradisiaca) exocarps as potential sources of acetic acid jeffry m. saro1* volume 1 issue 1, year 2022 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v1i1.382 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: july 09, 2022 accepted: july 13, 2022 published: july 15, 2022 the banana (musa paradisiaca) exocarps have been a substantial source for maintaining human health and are the fast-growing herbaceous perennials arising from underground rhizomes. the ripe banana is useful in acidity. this study aimed to evaluate how the banana exocarps from ripe ones, can be made into vinegar. also, to properly analyze the physicochemical properties (ph and color) and to determine the acetic acid content of the three selected varieties of banana exocarps if it conforms to the set standards. the most accessible in producing acidity is the latundan (musa acuminata musa balbisiana aab group), but all of the samples are accessible in producing acidity. in ph determination the highest in acidity is lacatan (musa acuminata c.) ph 4, while the cardaba (musa acuminata balbisiana abb group), is ph 3.5, and the lowest is the latundan ph 3. all of the samples do not have pleasant odor, furthermore the samples well tested and undergo many stages of pasteurization. thus, the study encourages further development of some spices and food additives and by application of new principles discovered, aside from extending the fermentation time. introduction the banana plants (musa paradisiaca) are the fast-growing herbaceous perennials arising from underground rhizomes. banana plants are of great nutritional value contained vitamin c and a high-grade protein, which includes three essentials amino acids (villaverde et al., 2013). the ripe banana is useful in acidity. banana plants are abundant in the philippines and since we do not make use of the banana exocarps. since the banana plants contains, a big amount of acid, and then we could make use of the banana exocarps as an alternative source of acetic acid (antorcha, 2016). the word vinegar is derived from the french words “vin aigre, meaning sour wine. the vinegar is classified as acetic acid fermentation (adams, 1980; steinkraus, 2002). it is produced via two-stage fermentation. the first stage is the anaerobic conversion of sugars into ethyl alcohol by the action of yeasts followed by the oxidation of the alcohol to acetic acid by the acetobacter bacteria (pederson, 1967). it is now one of the most widely used ingredients in the food industry; with worldwide production of 1 million l per year (hutkins, 2006), and the per capita consumption is 2 liters (wood, 1985). additionally, in the past it has served a wide variety of functions, but today its use is largely confined to food flavoring, production and preservation (adams, 1980; pederson, 1967), said that acetic acid can be prepared from any watery substance that contains sugar. since banana is high in sugar and carbohydratecontaining fruit, it has a high potential for alcohol production (camarungan, 1940), and subsequently fermented into acetic acid. according to adams (1980), he properly cited that banana vinegar production as a small-scale industry especially in developing countries like the philippines. there are already studies of banana vinegar from different varieties keywords banana, lacatan, latundan, cardaba, pasteurization, exocarp, acetic acid and some are made from the banana exocarps. according to co seteng (1983), said that it can be a source of contamination of some fungi and microorganisms even though it can add to the total fermentable sugar of the fruit. the process is simple, the capital requirement is limited, and banana wastes can be used for the production of acetic acid (gonelimali, 2018). there are three selected varieties of banana as source of acetic acid the lacatan (musa acuminata c.), latundan (musa acuminata musa balbisiana aab group) and cardaba (musa acuminata balbisiana abb group). the lacatan not to be confused with the cavendish banana lacatan also called bungulan. it is a popular dessert banana cultivar in the philippines. the latundan (also called tundan, silk bananas, pisang raja sereh, manzana bananas or apple bananas), are triploid hybrid banana cultivars from the philippines (bureau of agricultural statistics, 2008). it is one of the most common banana cultivars in southeast asia and the philippines, along with the lacatan and saba bananas (association of official analytical chemists, 1975). the saba banana is a triploid hybrid (abb) banana cultivar originating from the philippines. it is primarily a cooking banana though it can also be eaten raw. it is one of the most important banana varieties in philippine cuisine. it is also sometimes known as the cardaba banana, though the latter name may be more correctly applied to a very similar cultivar also classified within the saba subgroup (do et al., 2014). in this study, the researcher came up to study how the banana exocarps from ripe ones, can be made into vinegar. also, the researchers really want to analyze the physicochemical properties (ph and color) and to determine the acetic acid content of the three selected varieties of banana exocarps if it conforms to the set standards. aside from that, it also involves the utilization 1 department of education, college of education, caraga state university (csu)-main campus, agusan del norte, philippines. * corresponding author’s e-mail: jeffrysaro123@gmail.com https://doi.org/10.54536/ajlsi.v1i1.382 https://journals.e-palli.com/home/index.php/ajlsi mailto:jeffrysaro123%40gmail.com?subject= pa ge 16 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 15-18, 2022 of wasted cavendish for small-scale and bigger scale enterprises and for future references and use. materials and methods plant material. the three selected varieties of banana musa paradisiaca) exocarps were collected at prosperidad, agusan del sur, caraga region, philippines. the sample of the plant was submitted and conducted at the laboratory area of the department of biology, caraga state university (csu), ampayon, butuan city, agusan del norte for the plant sample to be examined for some procedural activities. the study used the procedural and experimental method of research which carried out and gather good result of the study. figure 1: the three varieties of banana (musa paradisiaca) family. (a) latundan (musa acuminata musa balbisiana aab group), (b) cardaba (musa acuminata balbisiana abb group), and (c) lacatan (musa acuminata c.) study area the three (3) selected varieties of banana (musa paradisiaca) exocarps were collected at prosperidad, agusan del sur, caraga region, philippines. the researcher was gathered the needed data to assess and evaluate how the banana exocarps from ripe ones, can be made into vinegar or as potential source of acetic acid. figure 2: the map of the study area. preparation of ingredients and materials the researcher really needs to have one (1) kilogram of banana (musa paradisiaca) exocarps in every variety of banana. also, has one (1) cup of white sugar to be mixed with the liquid. two (2) cups of purified water and two (2) teaspoons of baker’s yeast. the last is two (2) cups of vinegar starter. procedures and treatments the researcher must collect the three varieties of banana (musa paradisiaca) exocarps and trimming off the stem until only the exocarps remained. wash the banana exocarps and slice a 1 kilogram of banana exocarps with a knife into smaller sections in all selected varieties of banana. and subsequently, put it in casserole, then add three (3) cups of water, then boil it for 20 minutes. extract the juice of the banana exocarps, decant and filter through clean and fine cloth. furthermore, add two (2) cups of purified water then put a one (1) cup of sugars and pasteurize for 10-15 minutes at 60 degrees celsius to 65 degrees celsius. transfer it into a sterilized container, half-filled. moreover, let it cool and observe, and then add a half teaspoon baker’s yeast for every 8 cups mixture. then, allow fermenting for 7-8 days. pour it into another container to separate sediments. in addition, add 1 cup of vinegar starter for every four cups liquid. cover it with clean cloth. allow to ferment at a normal room temperature for 2-3 weeks or until a sour odor of vinegar is attained. then decant to separate the sediments. ph determination the ph values of latundan (musa acuminata musa balbisiana aab group), cardaba (musa acuminata balbisiana abb group), and lacatan (musa acuminata c.) exocarps acetic acid were measures at 1-9c using a ph meter. color determination. the three (3) varieties of banana (musa paradisiaca) exocarps has an individual colors through color determination it can easily find and determine the colors of the three (3) selected banana exocarps. results and discussion in ph determination the value of lacatan exocarp acetic acid were measured at 1-9c using a ph meter. in calibrating the ph value of lacatan it has a result of ph 3.0, the ph value of latundan ph 4.0, while the ph https://journals.e-palli.com/home/index.php/ajlsi pa ge 17 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 15-18, 2022 value of cardaba ph 3.5. the average of three trials was explicitly done. in determining the color values of the three samples, the researcher found out that the color of lacatan (musa acuminata c.) is yellow orange, the color of latundan (musa acuminata musa balbisiana aab group) is pale yellow brown, and the color of cardaba (musa acuminata balbisiana abb group) is light red (table 1). in this, the color of the three selected samples of banana exocarps is somewhat good and a little bit the same to the sugarcane vinegar. the acetic acid is a global product. china, united states of america, the rest of asia and western europe are largest acetic acid consumer. the annual worldwide demand of acetic acid is around 6.5 million tonnes of which, approximately 1.5 million tonnes are produced by a fermentation process (frazier et al., 1988). according to ploetz et al., 2007, from the entire global acetic acid capacity (virgin acid), 65% is in the asia where china is the major capacity, 19% in the united states of america and the rest is from other regions. these two regions make up to 84% of whole world capacity. moreover, china is the largest consumer with 30% of total demand. the rest of asia accounted for 27% of global demand including malaysia, with 1 to 3% acetic acid consumption in the world, which is nearly 0.12 million tons acetic acid per annum, followed by 20% in the united states of america and 14% in western europe. these regions totaled over 91% of worldwide acetic acid consumption (malveda and funada, 2010). the experiment based on the said procedures using the table 1: the ph and color values of the three selected varieties of banana (musa paradisiaca) exocarps. samples ph color values lacatan 3 yellow orange latundan 4 pale yellow brown cardaba 3.5 light red given materials above, after three weeks, a sour odor in every variety of banana exocarps was attained. in figure 1, the high ph values are the latundan it has 4.0, thereupon it could have ph below 4 limits bacteria but not to yeast and fungi. theoretically, as alcohol content increases during alcoholic fermentation. this is due to the conversion of sucrose to alcohol (figure 2). there are already studies of banana vinegar from different varieties and some are made from banana peel. according to co seteng (1983), banana peel can be used for vinegar production, but adams (1980) said that it can be a source of contamination of some fungi and microorganisms even though it can add to the total fermentable sugar of the fruit. table 2 shows that all of the samples have not pleasant odor because all of them is acidulated well due to long process. although no pleasant odor to smell but the researcher really does their part to do the correct process. furthermore, the samples well tested and undergo many stages of pasteurization. the researcher ascertained that all of the samples are accessible in producing acidity. in figure 3: the discrepancy of ph values of an acetic acid of the three selected varieties of banana (musa paradisiaca) exocarps. table 2: the pleasant odor of the three selected varieties of banana (musa paradisiaca) exocarps. samples smell lacatan unpleasant odor/acidulated latundan unpleasant odor/sourish cardaba unpleasant odor/acidulated addition, all of the given samples have different acidity, but the lacatan has the lowest percentage of acetic acid only 29%. while the cardaba obtained with the result of 33%. lastly, the most accessible in producing acidity is the latundan garnering with 38% (figure 3). the three selected varieties of banana (musa paradisiaca) figure 4: the accessibility in producing acidity of the three varieties of banana (musa paradisiaca) exocarps. exocarps is mashing and diluting it with water helps to release the soluble sugars from the fruit. addition of sugars adds to the fermentable sugars to obtain the required alcohol content. it takes 7 to 10 days to convert all of the fermentable sugars to alcohol (hutkins, 2006) by the action of yeast (saccharomyces cerevisiae) (adams, 1980). let alone method for acetic acid fermentation takes a month or longer to reach its optimum physicochemical characteristics (hutkins, 2006). conclusions the researchers concludes that the banana exocarps out of the three (3) varieties of banana could be used as an https://journals.e-palli.com/home/index.php/ajlsi pa ge 18 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 15-18, 2022 alternative source of acetic acid. the malic acid is the main acid in the banana exocarp, with substantial quantities of oxalic acid as well (oxalic acid is why sour grass tastes sour). malic acid content increases substantially upon ripening, whereas the oxalic acid is metabolized (probably via the tca cycle) and decreases. this one reason why the bitter taste disappears during ripening, however, another may be that tannins in the fruit polymerize. the physicochemical properties and acetic acid content of the vinegar don’t conform to the set standards. the main reason is due to short period of fermentation and lack of materials. it is important to observe the period of fermentation because it is time dependent aside from the factors like temperature, starting materials, etc. (wood, 1985). as a result, this study recommends that the acetic acid/vinegar must be subjected to the sensory analysis, descriptive and consumer acceptance, for further evaluation. moreover, this study encourages further development of this study by addition of some spices and food additives and by application of new principles discovered, aside from extending the fermentation time. recommendation the researchers should add more observations in the experiment, to see how the acetic acid from the first experiment differ from the other experiments. lastly, the study suggested that the local tribes or ordinary people could explicitly continue utilizing this banana plant as a source of treatment or any substantial products. furthermore, this research might be utilized as a guideline for future study, particularly in the context of plant science that produces substantial products and for medicinal treatments in the community. acknowledgments the authors expressed their gratitude to ms. mea-ann oscianas, lpt, for her assistance and expertise as research consultant of the study. also, the author extended his profound appreciation to dr. minie l. bulay for the assistance, support, and guidance during the laboratory activities of biology laboratory, caraga state university, butuan city, philippines. references adams, m.r., (1980). the small-scale production of vinegar from bananas. tropical product institute. vol 56(62) grays inn road, london. antorcha (2016). antimicrobial activity of ethanol extract from banana (musa acuminata) peels against staphylococcus aureus. national science and evolutionary vol 5(1). https://www.researchgate.net/publication /354372647 association of official analytical chemists (1975). official methods of analysis of the association of official analytical chemists.12th edition (ed. w. horwitz). washington d.c bureau of agricultural statistics, (2008). supply and utilization accounts, philippines, 1990 – 2006. camarungan, r.g., (1940). biochemical changes in bananas during ripening. ms thesis. college of agriculture, university of the philippines-los banos, laguna co seteng, y.s., (1983). development of vinegar from saba peels (musa sapientum var. compresa). college of home economics, university of the philippines, diliman, quezon city http://biological-diversity. info/invasive_musa.htm. retrieved on january 10, 2007 do, q.d., angkawijaya, a.e., tran-nguyen, p.l., huynh, l.h., soetaredjo, f.e., ismadji, s., ju, y.h. (2014). effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of limnophila aromatica. journal of food drug analysis. vol. 22, 296–302. doi: 10.1016/j.jfda.2013.11.001. frazier, w.c. & d.c. westhoff, (1988). food microbiology.4th edition. new york: mcgraw-hill book, co. gonelimali, f.d., lin, j., miao, w., xuan, j., charles, f., chen, m., hatab, s.r. (2018). antimicrobial properties and mechanism of action of some plant extracts against food pathogens and spoilage microorganisms. front science microbiology. 9:1639. doi: 10.3389/fmicb.2018.01639 hutkins, r.w., (2006). microbiology and technology of fermented foods. blackwell publishing, usa pederson, c.s., (1967). processing by fermentation. connecticut: avi publishing co. ploetz, r.c., et al., (2007). banana and plantain an overview with emphasis on pacific island cultivars. species profile for pacific island agroforestry from www. tradionaltree.org stenkrauss, k.h., (2002). fermentations in the world processing. comprehensive reviews on food science and food safety journal villaverde, j.j., oliveira, l., vilela, c.; domingues, r.m., freitas, n., cordeiro, n., freire, c.s.r., silvestre, a.j.d. (2013). high valuable compounds from the unripe peel of several musa species cultivated in madeira island (portugal). industrial crops and products. volume 42, 507–512. doi: 10.1016/j.indcrop.2012.06.037 wood, j.b., (1985). microbiology of fermented foods. (vol.1). elsevier applied science https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 9 american journal of life science and innovation (ajlsi) impact of forest policy and law on forest management in gambari forest reserve in oyo state, nigeria olaniyi i.s1*, kilasho a.r1, oluborode j.o2, jackson v.o2 volume 1 issue 2, year 2022 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v1i2.651 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: september 24, 2022 accepted: october 05, 2022 published: october 10, 2022 this study assessed the impact of forest policy and law on the management of gambari forest reserve in order to identify factors that would improve forest policy and law as well as their implementation and enforcement. random sampling techniques was used in this study. both questionnaire and interview methods were used in collecting the needed information from the respondents. one hundred and twenty (120) copies of structured questionnaires were used to obtain information from the community dwellers. descriptive statistics was used to analyze the demographic characteristics of the respondents. the landsat imagery of the forest reserve was analyzed using arcgis to generate information about the forest cover changes between 1987 and 2020. logistic regression was also used to determine the factors responsible for enhancement of implementation and enforcement of forest policy and law. results show that 54.4% of the respondents agreed that the forest policy and law were effective in upholding the management of the forest sustainably, 34.2% stated that the forest policy and law were slightly effective, while only 11.3% of them implied that forest policy and law was ineffective. it was also revealed from the study that about 21% of the forest land cover has been degraded from 163.50km2 in 1987 to 143.67km2 in 2020. findings also showed that, the enhancement of implementation and enforcement of forest policy and law would be mostly influenced by increase staff recruitment with odd ratio (2.33), followed by provision of training (1.28), also increase in penalty forest offenders (1.20) and lastly, creation of awareness program (1.00). their odd ratios are up to 1.0. keywords forest policy and law, forest management, forest offences, sustainable forest management, deforestation, forest degradation. 1 department of social and environmental forestry, university of ibadan, nigeria 2 department of forest production and products, university of ibadan, nigeria * corresponding author’s e-mail: olaniyiitunus@gmail.com introduction forests are an essential part of the universe; however, the wide ranges of benefit derived from forests are fast diminishing due to the profound dependency on its fragile resources for economic growth without steps to take adequate care of the forest itself. since 1980’s, demand for forest products, especially timber has become insatiably high as a results of increasing human population pressure and economic growth. this led to unregulated forest exploitation, thus resulting in degradation of the resources in the country. the rate at which forests of different types are disappearing for years now is alarming and the trend remains unprecedented. globally, the high demand for timber and other forest product has resulted in the high level of forest encroachment and high rate of deforestation and forest degradation (yaro et al., 2016). nigeria and many other developing countries are suffering from serious environmental degradation primarily because of the rapid growth in population which has not only brought about gross encroachment and damage to natural forest, but has also brought unacceptable quality of life conditions in the human community environment (harvey, 1998; oduntan et al, 2013). the global forest resources assessment by fao (2005) ranks nigeria 4th globally among countries with the highest annual deforestation rate (3.3%) and net annual area change (410,000ha). concern is therefore rising about the rapid rate of tropical deforestation, which is the temporary or permanent clearance of forest, for agriculture and other purposes by man. forest reservation as a legal measure for protection of forest resources which enables the society to have continued access to quality and quantity of forest goods and services (oso et al., 2017). law is legally binding document which specifies right and duties based on policy vision or goals (larinde and chima, 2014). forest laws establish objectives, institutional structures and confirm ownership. within the past years, there has emerged expanding concern around the rate of deforestation in tropical forest and the need to sustain the remaining tropical forest. alamu and agbeja (2011), iterated that deforestation is highly severe in south-west nigeria and this proceeds unabated. various forest offences which are devoid of substantial penalties contribute to the over exploitation of our forest land. nigeria has one of the highest rates of forest loss (3.5%) in the world; losing about 6.1 million hectares or 35.7% of its forest cover since 1990, between 2000 and 2005. nigeria is reported to have lost 79% of its old growth forests and is losing since 2000 with 11% of its primary forest annually (herbert et.al., 2013). while the rate of deforestation in nigeria occurs at 4000km2 per year, reforestation rate is a mere 10km2 per year (herbert et.al., 2013). as part of the sustainable wood resource supply and ecosystems management strategies, colonial nigerian government gazetted some areas as forest reserves. these reserves have over the years suffered from poor inventory, depletion and poor management (fasona et.al. 2018). forest policy lacks legal backing and hence its enforcement https://doi.org/10.54536/ajlsi.v1i2.651 https://journals.e-palli.com/home/index.php/ajlsi mailto:olaniyiitunus%40gmail.com?subject= pa ge 10 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 9-13, 2022 is difficult. the nigeria forestry act, 1937, is outdated. the forestry policy is subsumed in the national agricultural policy of 1988 (aruofor, 2001). the world bank (2006) also stated that the concern of management of forest is broadly on implementation and enforcement of forest law. the management of most forest reserves in nigeria is the exclusive right of the staff and governor of respective state forestry department in accordance with the 1978 land use decree which gives ownership and decision of every land and land use within that territory to the governor of the states of nigeria (land use act, 1978). the rule of law must be effective to attain sustainable development. fasona et. al, (2018) stated that ineffective policy implementation are major challenges facing the management of the reserves. thus, this research work aims at assessing the impact of forest policy and law and the consequences of the existing element of policy and law in gambari forest reserve in oyo state. methodology study area the study was carried out in gambari forest reserve located in oluyole local government area (lga) of oyo state. it is one of the early forest reserves in the state and it is divided into 5 series namely gambari, ibusogboro, onipe, olonde and mamu (larinde and olasupo, 2011). gambari forest reserve is a lowland forest, the reserve is located between latitude 70 25ˈ and 70 55ˈn and longitude 30 53ˈ and 30 9ˈe. it is situated at the southern part of ibadan, bounded on the west by river ona and on the east by the main road of ibadan to ijebu-ode. both dry and wet seasons are experienced in the reserve. dry season lasts for 3 months (december to february). the average annual rainfall is about 1140mm and average annual temperature is about 26.40 0c (800 f) (larinde and olasupo, 2011). study approach this study utilized both primary and secondary sources of data. the primary data for this study was collected through structured questionnaires. while, the secondary source of data was the satellite imageries of gambari forest reserve, which was used to generate the changes in forest cover between 1987 and 2020. data on oyo state forest policy and law on the use of forest resources were sourced from oyo state ministry of environment, ibadan, oyo state, ibadan. sampling techniques and sampling size the targeted respondents for this study were timber contractors, forest officials, and individuals who have farmlands. the reserve is divided into five (5) series of which 3 communities were randomly selected which are onipe, ibusogboro, and mamu, all located around the reserve. a total of one hundred and twenty (120) questionnaires were administered to the respondents, of which twenty (20) were forest officials and staffs, fifty (50) were farmers, thirty (30) were timber contractors and twenty (20) were those involved in trading activities within the forest reserve. determination of difference in vegetation index normalized difference vegetation index was estimated through analysis of satellite images to give indication of greenness. ndvi is an indicator of vegetation health, because degradation of ecosystem vegetation, or decrease in greenness would be reflected (menesses-tovar, 2011). the base map for gambari forest reserve was obtained from https://www.protectedplanet.net/36817 which was exported to arc gis software. imageries for the year 1987 and 2020 was acquired using landsat imagery with 60 meters’ spatial resolution for landsat. the imageries were classified using ndvi classification. the map of the study area was geo-referenced and the shape file was created, the shape file was then clipped into the images, vectorization and area computation was done and change detected was analyzed using percentage. the 1987 imagery was obtained under landsat 5 with the sensor of multispectral scanner (mss) while, the 2020 imagery was obtained under landsat 8 with the sensor of operational land image (oli) and thermal infrared sensor (tir). data analysis the binary logistics models were used in the analysis of the data. the two common binary models are the logit and probit. the logistics model is particularly preferred because of the unique information. the logit of a response p between 0 and 1 is given as: logit (p) = log (p/1-p) = log (p) – log (1-p) the simplest form of logit model is expressed as: logit (pi) = a + b1x1 +...bixi …………….equation 1 where, xi = vector of predictor or independent variables. pi = probability of factors that would enhance forest policy and law. a and b = regression parameters. results and discussion table 1 showed that, most (76.7%) of the respondents were males while the remaining 23.3% were females. this indicated that forestry profession in oyo state is male dominated. this corroborates the findings of feleyimu (2010) and ajejumo et al (2018) that forestry activities and staff are male dominated. the age group of the respondents showed that 3.3% were between 21 to 30 years, 32.5% were between the age brackets of 31 to 40 years, 50.0% were between 41 to 50 years, while 14.2% were between 51 to 60 years. this implies that most of these respondents are in their economically active years and this put more pressure on the forest reserve. the findings revealed that 11.7% of the respondents were primary school holders, 37.5% were secondary school holders, while 50.8% were tertiary holders. this implies that most of the respondents have above secondary school education and educated, they further to tertiary https://journals.e-palli.com/home/index.php/ajlsi pa ge 11 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 9-13, 2022 school. as regarding the occupation of the respondents, majority of them were farmers (41.7%) and timber contractors (25.0%) and 16.7% for forest officials, lastly 16.6% for traders. also, the study revealed that most of the respondents (75%) are native of the study area while only few of them (25%) must have settled in the area many years ago in search for opportunity for subsistence farming. this uphold the report of calibre and scc (2000) which, reported that people living in and around the forest include smallholders practicing subsistence farming and settlers who have come in the areas to search for new opportunity in agriculture. as shown in table 2, a total of 54.4% of the respondents agreed on the indicators that the forest policy and law was effective in upholding the management of the forest reserve sustainably, 34.2% stated that the forest policy and law was slightly effective and 11.3% implied that the forest policy and law was ineffective. half of the respondents attested the effectiveness of forest policy and law in upholding the sustainable management of gambari forest reserve. it is quite not impressive that 50.8% of the respondents stated that the forest policy and law was adequate in protecting the land from acquisition. of all the respondents, the farmers stated that 74.2% of the forest land area was protected by the legal system. the findings revealed that 54.4% of the respondents indicated that forest regulations are effective and loggers and saw milling activities are regulated, but there was no consensus whether management plan exists for the study area. when probed on whether it was conventional to acquire some land or allow settlements and farming in the forest reserve, the leader of forest officers noted that all we know is that some people can farm in certain areas in the forest reserve, and are allowed to help nurture the table 2: analysis of all respondents to the effectiveness of elements of forest policy and law in gambari eff s.eff ineff indicators freq % freq % freq % 1 the management of the forest reserve 74 61.7 36 30.0 10 8.3 2 land protected from acquisition by individuals 61 50.8 46 38.3 13 10.8 3 perception the gazette notice 75 62.5 36 30.0 9 7.5 4 gazetted forest areas protected by legal system 89 74.2 27 22.5 4 3.3 5 forest lands protected from illegal entry 68 56.7 45 37.5 7 5.8 6 reserve settlement court nowadays 53 44.2 55 45.8 12 10.0 7 trust funds use for regeneration and afforestation 54 45.0 51 42.5 15 12.5 8 prevention of forestry offences 58 48.3 50 41.7 12 10.0 9 farmers adhere to the order from planting permanent crops 70 58.3 26 21.7 24 20.0 10 prohibition of destruction of forest produce 62 51.7 36 30.0 22 18.3 11 provision for the survey and demarcation of forest reserve 65 54.2 39 32.5 16 13.3 12 provision for seizure and detention 69 57.5 45 37.5 6 5.0 13 community participation program 59 49.2 49 40.8 12 10.0 14 submission of license and permit render return and account for inspection 67 55.8 39 32.5 14 11.7 15 safety of forest guard 58 48.3 38 31.7 24 20.0 table 1: demographic characteristics of the respondents gender of the respondent frequency percentage female 28 23.3 male 92 76.7 total 120 100 age of the respondents (years) 21-30 4 3.3 31-40 39 32.5 41-50 60 50.0 51-60 17 14.2 61-70 71-80 81-90 total 120 100 level of education primary 14 11.7 secondary 45 37.5 tertiary 61 50.8 total 120 100 native of the community no 30 25.0 total 120 100 source of income forester 20 16.7 farmer 50 41.7 timber contractor 30 25.0 trading and others 20 16.6 total 120 100 https://journals.e-palli.com/home/index.php/ajlsi pa ge 12 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 9-13, 2022 trees but they are not allowed to harvest the timbers. in addition, although the forest officials provided information that illegal (unpermitted) logging activities were punished, either by fine or litigation in courts, this could not be justified. for examples, when the forest officials were asked for a specific example of such case(s) of punishment, one of the forest officials noted that, when arrested some of them (loggers and poachers) bribe their way out. analysis of the extent of forest cover the study shows that the forested area which covers about 163.40 km2 in 1987, had been reduced to 143.67 km2 table 3: land cover changes of gambari forest reserve year forested non forested area (km2) percentage area (km2) percentage 1987 163.40 94.75 8.18 4.74 2020 136.29 79.03 36.16 20.96 table 5: logit binary nature of the factors that would enhance implementation and enforcement of forest policy and law dependent variable efpl constant coefficient odd ratio 20.48 2.073 independent variables coefficient odd ratio provision of patrol vehicle -0.132 0.876 increase staff recruitment 0.845 2.328* provision of safety kit -0.664 0.515 increase in penalty forest offender 0.186 1.205* increase in salaries -0.138 0.871 creation of awareness program 0.003 1.003* 16 prohibition of act that might cause obstruction of any way or cause danger to navigation 61 50.8 48 40.0 11 9.2 17 grants and support out of public revenue for the encouragement of forestry 49 40.8 47 39.2 24 20.0 18 regulation of kindling fires 58 48.3 35 29.2 27 22.5 19 prohibition of trespassing 51 42.5 53 44.2 16 13.3 20 prosecution of forest offenders 84 70.0 32 26.7 4 3.3 21 issuance of permit and permit to sawmill 87 72.5 29 24.2 4 3.3 total 1372 862 286 average 65.3 54.4 41.0 34.2 13.6 11.3 by 2020 showing a 20.96% decrease in forest cover and a corresponding increase in non-forested area from 8.18 km2 in 1987 to about 36.16 km2 in 2020 as shown in table 3. the classified images showed the two land cover classes (forested and non-forested) identified from the landsat imageries. gambari forest reserve is shown to have undergone some changes and the status of the forested area has been decreasing within the last 33 years. however, the reserve is said to be known for its plantation establishment which dominated by exotic trees and would coppice under good management care. there has been reduction in the forest cover in gambari forest reserve between 1987 and 2020 with a 20.96% reduction in the forest cover table 4: direction of changes of gambari forest reserve (1987-2020) land cover 1987 – 2020 changes area (km2) percentage forested 27.11 15.72 non-forested + 27.98 + 16.22 note: + = increase; = decrease. https://journals.e-palli.com/home/index.php/ajlsi pa ge 13 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 9-13, 2022 conclusion the study revealed that forest policy and law in oyo state have been slightly effective in controlling forest offences in the forest reserve as a result of inadequate number of forest officers. therefore, recruitment of staffs, provision of training to the staffs and increase in penalty of forest offenders are factors that have statistically proven to be high determinant to the implementation and enforcement of forest policy and law. recommendation there should be regular evaluation on the compliance with the forest policy and law in gambari forest reserve. the existing elements of forest policy and law within gambari forest reserve should be strongly adhered to and punishment vehemently executed for people who violate the law. forest extension workers should work in gambari forest reserve to educate them on the ecological and social benefits of the forest reserve and to enable them to be able to male right decision towards sustainability. the state forestry department should recruit more forest officials in monitoring of the forest reserve. forest staffs should be trained regularly so as to increase their performances and also work effectively. reference adejumo, a.a. (2019). benefits of forest legislation enforcement on the status of onigambari forest reserve. international journal of environment, agriculture and biotechnology, 4(6). https://dx.doi.org/10.22161/ ijeab.46.26 alamu, l. o. and agbeja, b. o. (2011). deforestation and endangered indigenous tree species in southwest nigeria. international journal of biodiversity and conservation, 3(7), 291-297. arufor (2001). a brief on the forestry outlook study, federal department of forestry. calibre and scc. (2000). number of forest dependent people in feasibility study for the uk department for international development of forestry research programme. the statistical service center, university of reading, uk. dada, f. o. a., jibrin, g. m., & ijeoma, a. (2006). macmillian nigeria secondary atlas, lagos: macmillian. 136, fao (2005). deforestation data for nigeria, 19902005. retrieved from http://news.mongabay.com/2005/1115forests fao. (2005). progress towards sustainable development. 147. faleyimu, o.i (2010). status of forest policy implementation in south-west nigeria. unpublished ph.d thesis, university of ibadan, ibadan. april, 2010. 271 fasona m., soneye. a., oludare a., omojola. a., akintuyi.a., adeonipekun. p., ariori.a., gbenga.o., ogundipe.o., and udofia.s., (2018). deforestation and land cover changes in the forest reserves and protected areas of southwest nigeria. harvey, f.l. (1998). final reporton proposed environmental action program. main report, 1, 2-3. herbert e.b, ashong c.a, and abdullahi s.b. (2013). press coverage of climate change issues in nigeria and implications for public participation opportunities. journal of sustainable development. 62. 56-69. larinde, s. l., & chima, u. d. (2014). challenges of forest management and national security issues in nigeria. in being a paper presented at the 37th annual conference of the forestry association of nigeria (fan), at minna, niger state, nigeria. larinde, s.l. and olasupo, o.o. (2011). socio-economic importance of fuelwood production in gambari forest reserve area, oyo state, nigeria. journal of agriculture and social research, 11(1), 201-210. oduntan, o.o., soaga, j.a.o. akinyemi, a.f. and ojo, s.o. (2013). human activities, pressure and its threat on forest reserves in yewa division of ogun state, nigeria. journal of environmental research and management 4(5), 260-26. oso, a.o., agbeja, b.o., faleyimu, o. (2017). perceived forest offences among stakeholders and their impact on sustainable forest reserves management in southwestern nigeria. academia journal of agricultural research, 5(4), 75-86. world bank. (2004). sustaining forests: a development strategy. washington, d.c: world bank press, 1-99. yaro, m.a.; okon, a.e.; bisong, f.e. and ukpali, o. (2016). impact of forest encroachment on rural livelihood in akamkpa division of cross river national park, nigeria. journal of environment and earth science 6(2), 76-86. provision of training 0.250 1.284* quick and unbiased prosecution -0.004 0.996 good governance -0.144 0.866 source: field study, 2021 https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 12 american journal of life science and innovation (ajlsi) transmission of urogenital schistosomiasis in owena reservoir area, ondo state nigeria: nexus between different pathogenic host factors bayo joshua peletu1, ifeanyi emmanuel ofoezie2, aloysius obinna ikwuka3* volume 2 issue 2, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i2.1788 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: june 20, 2023 accepted: july 08, 2023 published: july 24, 2023 a total of 25,482 snails, comprising 4,258 (16.7%) bulinusglobosus; 213 (0.8%) bulinustruncatus; 4,040 (15.9%) bulinusforskalii; 4,770 (18.7%) biomphalariapfeifferi; 6,024 (23.6%) melanoidestuberculata; 1,730 (6.8%) potadomafreethi; and 4,447 (17.5%) pilaovata were collected over a period of 24 months during monthly site visits to owena reservoir area which comprises of three communities namely owena, baiken and kajola in ondo east local government area, ondo state, nigeria. abundance of each of the snail species varied significantly (p<0.05), spatially, seasonally and annually throughout the period under study. the established intermediate snail host species were bulinusglobosus and bulinustruncatus for schistosomahematobium transmission. the overall infection rate among bulinusglobosus was 3.2% as 136 snails were found to shed cercariae out of 4,258 collected and examined for trematode infection. this occured at site 4 located in kajola community. none of the bulinustruncatus species was found to shed cercariae. keywords urogenital schistosomiasis, bulinus globosus, bulinus truncatus, schistosoma hematobium, owena reservoir area, pathogenic host factors, schistosomiasis, public health, mollusc introduction schistosomiasis is a disease caused by parasitic trematode worms of the genus schistosoma (weinland, 1858). out of these trematode worms infecting humans, only s.hematobium and s.mansoni are of great medical importance. while s.hematobium causes urogenital schistosomiasis, the other species s.mansoni causes intestinal schistosomiasis. the parasite s.hematobium is found in the venous plexus draining the urinary bladder of humans (babatunde, 2013; who, 2014). infections result from the parasites depositing terminal, spined eggs which clog the venous plexuses, thus preventing blood flow which leads to the bursting of the veins, allowing blood and eggs to enter the urinary bladder resulting in the characteristic symptom of blood in the urine (hematuria) (ekpo, 2010; peletu, 2016; peletu, 2023b; who, 2014; who, 2015). in nigeria, it has been reported that urogenital schistosomiasis is endemic in 23 out of the 36 states of nigeria. ondo state is one of the states where urogenital schistosomiasis occurs (peletu, 2020; peletu, 2023a; peletu, 2023b; peletu, 2023c). however, epidemiological information of the disease in ondo state, especially in the three communities bordering owena reservoir/ dam, is still very scanty. information on the transmission of urogenital schistosomiasis and human water contact patterns in ondo state is scanty despite the widespread distribution of the disease in some of its neighboring states (peletu, 2020). water contact activities exposed the communities to urogenital schistosomiasis infection. it is recommended that health education on the transmission of urogenital schistosomiasis should be focused on mass sensitization, to emphasize the role commonly played by water contact activities of snail hosts (peletu, 2023c). contamination habits such as urination and defecation in the water bodies should be discouraged. people should avoid wading into infected water bodies indiscriminately (peletu, 2023c). urogenital schistosomiasis is highly endemic in three communities (owena, baiken and kajola) of ondo state, nigeria (peletu, 2023a). there is a need for sustainable controls targeted towards behavioral modifications by mass sensitization and provision of pipe-borne water facilities and modern toilet systems with a view to discouraging people from having contact with cercacriaeinfected water bodies (peletu, 2023a). urogenital schistosomiasis and its concomitant hematuria are prevalent in owena, baiken and kajola communities of ondo east local government area, ondo state, nigeria. in all three communities of owena, baiken and kajola, urogenital schistosomiasis would be most difficult to treat and/or eradicate in baiken community (peletu, 2023b). efforts by stakeholders should be geared towards implementing full control strategies such as the provision of pipe-borne water, modern toilet facilities, and regular chemotherapy controls to the school-age children and adults of the age group 21–30 years (peletu, 2023b). s.hematobium is transmitted by the intermediate host belonging to the genus bulinus (akinwale, 2010; fradsen, 1984; peletu, 2010). in the last decade, there has been considerable advancement in the knowledge of the basic relationship between bulinus and s.hematobium (who, 2015). 1 department of allied health & biological sciences, college of health sciences (chs), legacy university, banjul, gambia 2 institute of ecology & environmental studies, obafemi awolowo university, ile-ife, nigeria 3 department of clinical sciences, college of health sciences (chs), legacy university, banjul, gambia * corresponding author’s e-mail: aloysiussweet@yahoo.com pa ge 13 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 12-16, 2023 the resolutions of snail populations in a given aquatic habitat are influenced by abiotic factors such as temperature, ions e.g., sodium, calcium, biochemical oxygen demand (bod), dissolved oxygen (do), sunlight, alkalinity, water current velocity, rainfall, and biotic factors such as predatorprey interactions/relationships, food availability, habitat competitions, hydromicrophytes coverage (ofoezie, 1995; ofoezie, 2002; peletu, 2010; theron, 1986; udonsi, 1990). the twenty-four month study (august 2013 – july 2015) was carried out with a view to determining the nexus between the different pathogenic host factors, the seasonal and spatial distribution of infection patterns (cercariae shedding) of identified local intermediate snail host species in the transmission of urogenital schistosomiasis in owena reservoir area, ondo east local government area, ondo state, nigeria. this is especially necessary when planning sustainable control programs of the disease in the study area. materials and methods study area the twenty-four month study (august 2013 – july 2015) was carried out in owena reservoir and its adjoining three randomly selected communities namely owena, baiken and kajola, which are rural to semi-urban settlements located in ondo east local government area, ondo state, nigeria. owena reservoir lies between latitudes 7000’ – 7030’n and longitudes 5000’ 5030’e (see fig. 1). the residents are generally peasant farmers, petty traders, local teachers, oil mill workers, fishermen, and auxiliary health officers. owena reservoir has a catchment area of approximately 900km2. its major tributaries are rivers owena and anu. according to the national population commission (2006), the population of the communities is approximately 9,000. figure 1: map of owena showing owena reservoir, owena dam, owena river, and the eight sampling sites of owena, baiken, and kajola communities source: igboloro and associates (planners, architects, and engineers), 3 ayodele awodeyi street, ketu, lagos state, nigeria (2012) pa ge 14 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 12-16, 2023 selection of sampling sites the sampling sites were selected based on the fact that they are potential human water contact sites where the frequency of visitations by the inhabitants and proximity to their residences were considered paramount parameters for snail-breeding sites. snail sampling the eight sites selected were sampled once every month for twenty-four-months (august 2013 – july 2015). sampling involved 30 passes of kitchen scoops and a manual search for 30 person-minutes (asaolu, 1990; peletu, 2010; who, 2014). all the snails collected were placed in pre-labeled plastic containers with decaying leaves, and transported to the malacology laboratory of obafemi awolowo university, ile-ife, nigeria. water used to shed cercariae was collected from each of the eight sites. within 24 hours upon arrival at the malacology laboratory, the snails were identified to species level, counted, and recorded as number of snails per site, per date. the snails were identified using standard identification keys. the snails were then grouped into <3.0mm, 3.0mm-5.9mm, and 6.0mm-9.0+mm size classes, using a fine vernier caliper. examination of snails for schistosoma infection the snails identified were examined for schistosoma infection using the crushing method (asaolu, 2003). each of the snails was placed on a slide and was covered with another slide with minor pressure applied on the topmost slide to gently crush the snail. some drops of dechlorinated water were added to the longer pieces of shell removed, using small forceps. the slide was then observed under a light microscope for the presence or absence of cercariae. the cercariae were identified to the genus level according to frandsen and christensen (fradsen, 1984). however, only snails shedding schistosoma cercariae were recorded as infected. other data about size, species, date of collection, and site of selection of infected snails were recorded. results and discussion table 1 shows that for the period under study (august 2013 – july 2015), a total of 25,482 snails, comprising 4,258 (16.7%) bulinusglobosus; 213 (0.8%) bulinustruncatus; 4,040 (15.9%) bulinusforskalii; 4,770 (18.7%) biomphalariapfeifferi; 6,024 (23.6%) melanoidestuberculata; 1,730 (6.8%) potadomafreethi; and 4,447 (17.5%) pilaovata were collected during monthly site visits to owena reservoir area which comprises of three sampled communities namely owena, baiken and kajola in ondo east local government area, ondo state, nigeria. thus, bulinusglobosus and bulinustruncatus were the established intermediate host (see table 1). out of the two intermediate snail host species identified, bulinusglobosus was more abundant 4,258 (16.7%) than bulinustruncatus 213 (0.8%). the overall infection rate of bulinusglobosus was 3.2% as 136 snails were infected out of the 4,258 collected (see table 2). this was found in site 4 located in kajola community. only bulinusglobosus greater than 3.0mm were found to shed cercariae (infected). table 1: total number of each of the seven snail species collected within twenty-four months during monthly site visits sites bulinus globosus bulinus truncatus bulinus forskalii biomphalaria pfeifferi melanoides tuberculata potadoma freethi pila ovata total owena site 1 554 62 396 599 771 557 433 3,372 site 2 567 46 425 485 623 784 713 3,643 site 3 555 17 552 630 676 65 983 3,478 kajola site 4 607 11 645 739 955 101 953 4,011 site 5 688 16 570 534 890 76 656 3,430 baiken site 6 511 16 544 558 670 92 345 2,736 site 7 461 17 487 544 664 15 109 2,297 embankment/dam owena site 8 315 28 421 681 775 40 255 2,515 total 4,258 213 4,040 4,770 6,024 1,730 4,447 25,482 table 2: the relative abundance and rates of schistosoma infection (cercariae shedding pattern of small (3.0mm5.9mm) and large (6.0mm-9.0+mm) bulinusglobosus collected during monthly site visits to owena reservoir area sites small snails (3.0mm-5.9mm) large snails (6.0mm-9.0+mm) total total collected total infected % infection total collected total infected % infection total collected total infected % infection owena site 1 113 0 0 408 11 2.7 521 11 2.1 pa ge 15 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 12-16, 2023 conclusion bulinusglobosus and bulinustruncatus are the only identified intermediate hosts of schistosomahematobium in owena reservoir area. bulinusglobosus showed predominance compared to the other intermediate snail host species at owena reservoir area. therefore, intervention strategies for sustainable control of urogenital schistosomiasis in owena reservoir area should focus on the two snail host species. the use of molluscicides should be encouraged. acknowledgments special thanks to the entire staff of malacology laboratory of obafemi awolowo university, ile-ife, nigeria for their assistance in carrying out this research. conflict of interest all the authors hereby declare that they do not have any possible conflicts of interest. references akinwale, o. p., ajayi, m. b., gyangg, p. b., adeleke, m. a., adeneye, a. k., adebayo, m. o. & dika, a. a. (2010). urinary schistosomiasis around oyan reservoir nigeria, twenty years after the 1st outbreak. iraninan journal of public health, 39(1), 92-95. asaolu, s. o. & ofoezie, i. e. (1990). a simple method for concentrating eggs of schistosoma haematobium in the urine. the nigerian journal of parasitology, 9(11), 47-50. asaolu, s. o. & ofoezie, i. e. (2003). the role of health education and sanitation in the control of helminth infections. acta tropica, 86, 283-294. babatunde, t. a., asaolu, s. o. & sowemimo, o. a. (2013). urinary schistosomiasis among pre-school and school aged children in two peri-urban communities in southwest nigeria. journal of parasitology and vector biology, s17, 96-110. ekpo, u. f., akintunde, l., oluwole, a. s., sam-wobo, s. o. & mafiama, e. (2010). urinary schistosomiasis among pre-school children in a rural community near abeokuta, nigeria. parasite and vector, 3, 58. https:// doi.org/10.1186/1756-3305-3-58. fradsen, f. & christensen, n. o. (1984). an introductory guide to the identification of cercaria from fresh water snails with special reference to cercariae of trematode species of medical and veterinary importance. acta tropica, 41, 181-202. ofoezie, i. e. (1995). a study of urinary schistosomiasis transmission in resettlement communities brodering oyan river dam, ogun state, nigeria. phd thesis, obafemi awolowo university, ile-ife, nigeria. ofoezie, i. e. (2002). human health and sustainable water resources development in nigeria: schistosomiasis in artificial lakes. natural resources forum, 26, 150-160. peletu, b. j. (2010). ecology of fresh water snails transmitting schistosoma haematobium in aponmulona river basin, idanre, ondo state, nigeria. msc thesis, obafemi awolowo university, ile-ife, nigeria. peletu, b. j. (2016). schistosoma intermediate host profile, ecological variations and human factors influencing schistosomiasis transmission in owena reservoir area, ondo state, nigeria. phd thesis, obafemi awolowo university, ile-ife, nigeria. peletu, b. j., ofoezie, i. e. & ikwuka, a. o. (2020). urogenital schistosomiasis transmission and human water contact patterns in aponmu-lona river basin, idanre, ondo state, nigeria. donn. j. med. med. sci, 6(1), 001–008. https://doi.org/10.5281/zenodo.3996571. peletu, b. j., ofoezie, i. e. & ikwuka, a. o. (2023). attitude, knowledge, perception, behavioural, cultural and religious practices influencing transmission of urogenital schistosomiasis in owena, kajola and baiken communities bordering owena reservoir/ dam, ondo east local government area, ondo state, nigeria. european journal of medical and health sciences, 5(1), 23-30. https://doi.org/10.24018/ ejmed.2023.5.1.1600. peletu, b. j., ofoezie, i. e. & ikwuka, a. o. (2023). prevalence, peculiarities and patterns of urogenital schistosomiasis and hematuria in owena reservoir area, ondo east local government area, ondo state, nigeria. american journal of public health research, 11(2), 55-60. https://doi.org/10.12691/ajphr-11-2-3. peletu, b. j., ofoezie, i. e. & ikwuka, a. o. (2023). urogenital schistosomiasis transmission and human water contact activities in owena reservoir/dam, ondo east local government area, ondo state, site 2 163 0 0 413 15 3.6 576 15 2.6 site 3 154 0 0 378 12 3.2 532 12 2.3 kajola site 4 158 6 3.8 466 47 10.1 624 53 8.5 site 5 176 0 0 504 6 1.2 680 6 0.9 baiken site 6 131 0 0 399 13 3.3 530 13 2.5 site 7 121 0 0 357 24 6.7 478 24 5.0 embankment/dam owena site 8 0 0 237 2 0.8 317 2 0.6 total 1,096 6 0.6 3,162 130 4.1 4,258 136 3.2 pa ge 16 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 12-16, 2023 nigeria. world journal of public health, 8(2), 88-96. https://doi.org/10.11648/j.wjph.20230802.17. theron, a. (1986). chronobiology of schistosome development in snail host. parasitology today, 2(7), 192194. udonsi, j. k. (1990). human community ecology of urinary schistosomiasis in relation to snail vector bionomics in the igwun river basin of nigeria. tropical medicine and parasitology, 41, 131-135. weinland, w. (1858). digenea schistosomatidae and the intermediate snail host general. revue de zoologie africaine tervuren, 100, 137-152. world health organization (2014). urine filtration technique for s. haematobium infection. geneva, who. world health organization (2015). updated facts on “schistosomiasis”. whotdrs 912.pdf. pa ge 1 pa ge 21 american journal of life science and innovation (ajlsi) case report isolated umbilical cord cyst in second and third trimesters khadeeja yahya othman idrees1*, hassan osman ali hemet2 volume 2 issue 1, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i1.1261 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: february 01, 2023 accepted: february 14, 2023 published: february 19, 2023 the cyst might be solitary or numerous (more frequent). while single cysts are attributed to favorable perinatal outcomes during the initial stages of pregnancy, the presence of numerous different umbilical cord cysts, their persistence in the second and third trimesters, and their conjunction with other ultrasonographic anomalies are linked to a higher probability of a miscarriage, aneuploidy, or other complexation. the majority of the material on postoperative complications and treatment adherence of foetuses with umbilical cord cysts is minimal. we wanted to show the result of a third-trimester foetus with an isolated umbilical cord cyst. due to foetal distress, the baby was delivered by emergency caesarean surgery at 39 weeks gestation. the infant seemed fine and cried immediately. it should be taken into account that large umbilical cysts, in the event of rapid proliferation, may pressure the cord vessels and cause foetal discomfort. it is essential to consider the ultrasound examination of the umbilical cord as one of the essential aspects of the test. in addition, it is advised to ascertain that a karyotype is necessary when we discover this issue with related abnormalities. keywords umbilical cord cysts, allantoic cysts, foetal abnormalities, congenital malformation, prenatal diagnosis introduction the umbilical cord stands as an important link between the placenta and the developing foetus. it keeps a constant connection at the maternal-fetal interface. the umbilical cord allows the foetus to move, acquire motor skills, and mature. high-resolution obstetric ultrasonography is frequently used, which has made it possible to thoroughly examine the foetus, placenta, and umbilical cord (hayes et al., 2020)the clinical significance of an umbilical cord cyst in a pregnant woman is debatable. the long-term outlook and outcome of infants with this cord anomaly are yet unknown because the details of affected pregnancies are mostly based on case report findings. the purpose of this study was to characterize a foetus’ prognosis with an isolated umbilical cord in the third trimester(zangen et al., 2010). literature review umbilical cord abnormalities (uca) are conditions in which fetal blood flow is diminished or terminated due to changes in the anatomy or function of the umbilical cord. uca was associated with adverse pregnancy outcomes such as stillbirth, birth hypoxia, and emergency caesarean section. however, estimates of uca’s involvement in these outcomes differ; for example, uca is said to cause 3.4% to 20% of stillbirths. some variations could be attributed to several stillbirth classification systems, not all of which specify uca as a cause of death (hayes et al., 2020). the study (zangen et al., 2010) evaluated the prognosis and clinical approach for fetuses with umbilical cord cysts during the second and third trimesters of pregnancy. parental karotype testing was employed to analyze umbilical cord cysts during pregnancy. according to the study, it was determined that umbilical cord cyst occurrence during the first trimester ranges from 0.4% to 3.4%. the incidence of umbilical cord cysts in the second and third trimesters was unknown, and prior research has been limited to case reports. there is a variation in diagnostic value and prognosis of umbilical cord cysts in the first and second trimesters (zangen et al., 2010). most studies conclude that most first-trimester cysts are transitory and have no negative impact on pregnancy outcomes (rempen, 1989; skibo et al., 1992). additionally, a study (zangen & yaffe, 2009) elucidated that sonographic advances have enhanced parental detection of foetal umbilical cord and placental abnormalities. in the first trimester, the prevalence of umbilical cord cystic masses ranges from 0.4% 3.4%. umbilical cord cysts in the second and third trimesters are a rare sonographic finding, and their prevalence is unknown. there is a strong link between umbilical cord cysts and fetal abnormalities, but it is not ubiquitous (zangen & yaffe, 2009). furthermore, another study (campo et al., 2017) indicated the complexity of parental diagnosis of umbilical cord cysts. the study stated that small cysts could be identified in up to 3% of first-trimester pregnancies. they are typically accompanied by additional structural or chromosomal anomalies (up to 20%). if they grow in size and persist, they potentially endanger the fetus by restricting blood flow through the umbilical cord, either through compression or thrombosis (campo et al., 2017). 1 mbbs university of science and technology, specialist obstetrics and gynecology at king fahad hospital, saudi arabia 2 master degree advanced ultrasound in obstetrics and gynecology, consultant obstetrics, and gynecolog, saudi arabia * corresponding author’s e-mail: khadeejaosman2@gmail.com https://doi.org/10.54536/ajlsi.v2i1.1261 https://journals.e-palli.com/home/index.php/ajlsi mailto:khadeejaosman2%40gmail.com?subject= pa ge 22 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 21-25, 2023 case report history a 35 years old lady with gravida 6, para 3, and abortus 2, all through normal vaginal delivery (nvd), came to king fahad hospital for antenatal care (anc) at 26 weeks gestational age according to her last menstrual period of induced pregnancy due to 6 years of secondary infertility. the patient had a regular antenatal visit at a private health center which made her pregnancy uneventful throughout. her vital signs and physical assessment were unremarkable. the patient has not been prescribed any chronic medication except for some health tonics. the medical, surgical, and family history of the patient is unexceptional. examination the patient appears quite well, not pale or cyanosed. her blood pressure was recorded at 100/80 mm hg. her pulse rate was 100 per minute, whereas the respiration rate, body temperature, and body mass index measurements were 20, 30°c, and 30 kg/m2, respectively. the fetus’s gestational age was observed to be 30 weeks, corresponding to the date. investigation the investigations revealed the patient’s hemoglobin to be 11 gm/dl. the blood group was determined to be ab+ve, and the urine profile was clear. a liver function test and renal function test were conducted, and the result of both appeared to be normal. karyotyping was not done due lack of the facilities u/s: during the early ultrasound, a 3cm unilocular cyst in the cord surrounding the umbilicus was noticed. the linear ventral wall was not affected. the cyst’s echo texture was homogeneous and resembled amniotic fluid. umbilical veins emerge from the cyst (figure. 1, 2, and figure. 3). figure: 1 unilocular cyst within the cord with homogenous echo texture similar to amniotic fluid, the ventral wall is intact figure: 2 umbilical vessels seen splay around the cyst. the cord’s allantoic cyst was presumed to be diagnosed. at 30 weeks, a second ultrasound revealed that the cyst had grown to 4.6 cm in diameter (figure. 3). figure: 3 umbilical cord at 30 weeks’ cross sections of the cord can be seen in the diagram above, with vessels splaying around a core cyst and a more peripheral pseudo cyst. serial sonograms revealed normal foetal development, fluid, and doppler assessment of the umbilical cord, but cord oedema appeared at 30 weeks’ gestation. fetal monitoring and amniotic fluid evaluations were carried out twice a week, which was promising. management the patient was referred to fetal medicine unit (fmu) for a routine scan, and the congenital malformations were ruled out, so she went for a 2-week anc to monitor fetal growth and to check for any symptoms of intrauterine growth restriction (iugr), abnormal umbilical artery doppler). also, the patient opted to have a normal vaginal birth unless there is a fetal cause for caesarean section (c.s.), such as severe iugr. https://journals.e-palli.com/home/index.php/ajlsi pa ge 23 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 21-25, 2023 an emergency c.s. delivered the baby due to fetal distress at the gestation week of 39 weeks. the baby cried immediately after birth, weighed 3.1 kg, and looked healthy and normal with no apparent congenital malformation. all the tests, including abdominal, pelvic, and bladder ultrasound conducted, came out normal, and a urologist and pediatrician saw the baby, and all investigations were normal. the patient was discharged, and the baby was followed up after four months with no complications and looked healthy. discussion allantoid cysts are real umbilical cord cysts. they grow from persistent allantois structures, and due to their connection to the bladder, the cysts contain urine. the cord vessels are divided by the central allantoid cysts of the umbilical cord. a correct prenatal diagnosis is essential since allantoid cysts are not associated with chromosomal abnormalities (bouariu et al., 2021). despite having a connection to the bladder of the urine, allantois disappears during the seventh embryonic week. (bunch et al., 2006a; sadler, 2018). urine passing through a patent allantois may cause the development of allantoid cysts with their centers in the urethra. these cysts frequently divide the vasculature of the umbilical cord. third-trimester spontaneous rupture of allantoid cysts is the norm (matsui et al., 2007; weichert et al., 2009). with ultrasonography, a patent allantois can be identified (bunch et al., 2006b; fuchs et al., 2008; matsui et al., 2007; van der bilt et al., 2003). any cystic lesion, or fluid-filled sac, on the umbilical cord is regarded as an umbilical cord cyst. they can be discovered in any place along the umbilical cord and commonly appear between blood vessels. they have uneven forms. a patient could have one or more than one cysts. doctors use ultrasound technology to identify umbilical cord cysts most prevalently during the first trimester. first-trimester umbilical cord cysts typically have no repercussions on pregnancy, foetal development, or delivery. if a placental cyst is connected near where the umbilical cord is attached, it may slow the development of the foetus. to guarantee the proper flow of blood via the umbilical cord, a doctor should keep a close watch on any placental cysts that are present close to the placement of the chord. (babay et al., 1996). however, complications are more likely to occur if cysts persist into the second or third trimester. (chen et al., 2020). they may come with an increased risk of miscarriage or structural anomalies if they are combined with other abnormalities. there are two kinds of cysts in the umbilical cord (ross et al., 1997). true cysts a real cyst forms at the placental end of the umbilical cord just before it joins the baby. the embryo’s fluid is contained in a tiny strand of the umbilical vesicle. they typically range in size from four to sixty millimeters. true cysts typically disappear by themselves (zangen et al., 2010). false cysts false cysts, also known as pseudo cysts, are more prevalent than real cysts. they can arise at any point along the umbilical cord. a false cyst contains fluid from wharton’s jelly or the cushiony substance between blood vessels. (kausha et al., 2019) they can grow as large as six centimeters. false cysts may relate to chromosomal anomalies or genetic conditions in the baby (nicolaides, 2004). at various stages of pregnancy, umbilical cord cysts can be detected with ultrasound. during the first trimester, the frequency of occurrence of umbilical cord cysts ranges from 0.4 to 3.4% (ghezzi et al., 2003). it is unknown how frequently umbilical cord cysts occur during the second and third trimesters, and previous research has only focused on case reports or small series. umbilical cord cysts in the first and second trimesters vary in their clinical significance and prognosis. the majority of studies conclude that most first-trimester cysts are brief and do not affect the outcome of a pregnancy. however, the prognosis appears to be comparable to that of second-trimester cysts in cases of chronic cysts (fox, 1978). nevertheless, a few case reports/studies examine the outcomes of pregnancies involving umbilical cord cysts, and these may provide some insight into the matter. 13 cases of abnormalities in umbilical cord cysts in the second and third trimesters were found in a u.k. study. similar to the findings of our case report, they reported two isolated, clear cysts on the umbilical cord with no apparent congenital malformation. (sepulveda, 2003) reported very different results. 13 foetuses with umbilical cord cysts throughout the second and third trimesters of pregnancy were reported, along with their results. further sonographic observations were recorded in 11 of them. ten of these foetuses underwent prenatal karyotype testing, which detected aneuploidy in 7. there were several abnormalities in two of the three cases, with normal karyotypes and an isolated omphalocele in one of the cases. in a further case with isolated omphalocele, karyotyping was not performed. all chromosomally aberrant babies died in utero or during the neonatal period, as did two chromosomally normal foetuses with numerous concomitant morphological abnormalities. even though the (smith et al., 1996) study detailed the results of three cases involving umbilical cord cysts, one cannot be compared to this study because the patient did not provide the first-trimester report, and the cyst continued to grow until the third trimester. in that study, a transient cyst was discovered at the end of the first trimester, and it disappeared in the second trimester with a typical outcome. according to a 2015 study from the united kingdom, after the exceedingly rigorous anatomical screening, the umbilical cord cyst was the sole abnormal diagnosis in seven of the 10 cases reported, while polyhydramnios and suspected iugr without structural problems were also detected in one case. in each of these scenarios, https://journals.e-palli.com/home/index.php/ajlsi pa ge 24 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 21-25, 2023 normal newborns were delivered. moreover, this is what this study is expected to reveal. findings support previous results that second and third-trimester umbilical cord cysts are related to foetal abnormalities. however, the prevalence of anomalies appears to be significantly lower, and the clinical outcome is frequently good. taking into account all of these diverse study findings and assuming that there is no obvious congenital abnormality and no iugr, it is appropriately considered to marginalize chronic urachus by a specialized doctor after delivery. all studies show a link between secondand thirdtrimester umbilical cord cysts and foetal abnormalities. fetal abnormalities were seen in 80-85% of the cases reported by (sepulveda, 2003; smith et al., 1996). (ross et al., 1997) found a perfect association between chronic second-trimester cysts and abnormalities. in 38% of the instances, (shipp et al., 1995) found foetal abnormalities. unfortunately, karyotyping was not performed on our patient due to a lack of facilities; nonetheless, the absence of obvious congenital abnormalities minimizes the possibility of having chromosomal abnormalities (kilicdag et al., 2004). conclusion an umbilical cord cyst is a medical condition that affects fewer than one percent of women and typically does not represent a risk to the infant. accurate evaluation of the embryo, fetus, placenta, and umbilical cord has been made possible by the widespread use of highresolution obstetric ultrasound. in isolated cases, there is no increased risk of recurrence of umbilical cord cyst. performing an accurate evaluation of the umbilical cord during the routine ultrasound pregnancy examination is essential. the need for a fetal karyotype must be taken into consideration if other abnormalities are found during a comprehensive ultrasound examination in the event of a cord cyst diagnosis. recommendations to reduce maternal and neonatal morbidity and mortality due to umbilical cord cysts, i recommend that obstetricians and gynecologists be well-trained in obstetric ultrasounds. suppose a patient is diagnosed with an umbilical cord cyst. in that case, she should have a detailed ultrasound examination of fetal karyotyping for non-isolated cases. if any cord cyst is suspected, it should be immediately referred to fmu to confirm the diagnosis. in case of congenital malformation associated with umbilical cord cyst, the problem should be ruled out on priority. there should be close growth monitoring for the detection of iugr. a multidisciplinary team should be appointed to determine the mood of delivery, and the presence of a pediatrician must be assured at the time of delivery. assessment of persistent urachus or any urachus abnormalities should be ruled out after delivery. most important of all, the mother should be schooled about the process and the case after that. acknowledgment i sincerely thank my family and appreciate my professors and instructors for their help and dedication and for providing their endless skillful support aiming to improve our skills and knowledge. competing interests there is no conflict of interest, financial or otherwise. consent for publication the authors agree to the final version of the paper. funding no funding sources are reported. references babay, z. a., lange, i. r., elliott, d., & hwang, w.-s. (1996). a case of varix dilatation of the umbilical vein and review of the literature. fetal diagnosis and therapy, 11(3), 221-223. bouariu, a., scutelnicu, a. m., ciobanu, a. m., cimpoca raptis, b. a., dumitru, a. e., nedelea, f., gică, n., & panaitescu, a. m. (2021). a rare case of allantoic cyst with patent urachus in fetus with a microdeletion in 1q21. 1q21. 2 region. diagnostics, 11(12), 2332. bunch, p. t., kline-fath, b. m., imhoff, s. c., calvogarcia, m. a., crombleholme, t. m., & donnelly, l. f. (2006). allantoic cyst: a prenatal clue to patent urachus. pediatric radiology, 36(10), 1090-1095. bunch, p. t., kline-fath, b. m., imhoff, s. c., calvogarcia, m. a., crombleholme, t. m., & donnelly, l. f. (2006). allantoic cyst: a prenatal clue to patent urachus. pediatric radiology, 36, 1090-1095. campo, l. r., cornudella, r. s., alderete, f. g., martínezpayo, c., pérez, p. p., fernández, p. g., & puertas, d. l. 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(2009). prenatal management of an allantoic cyst with patent urachus. archives of gynecology and obstetrics, 280, 321-323. zangen, r., boldes, r., yaffe, h., schwed, p., & weiner, z. (2010). umbilical cord cysts in the second and third trimesters: significance and prenatal approach. ultrasound in obstetrics and gynecology, 36(3), 296-301. zangen, r., & yaffe, h. (2009). umbilical cord cyst-should it concern us? harefuah, 148(7), 436-440, 475. https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 33 american journal of life science and innovation (ajlsi) whole body antioxidant status of silver carp fingerlings fed diet containing various dietary organic acids kanwal razzaq1, tariq dildar2, mudssar aslam1, sana arif3 volume 2 issue 1, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i1.1349 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: march 01, 2022 accepted: march 15, 2022 published: march 21, 2023 a 10-week feeding trial was conducted to evaluate the wholebody antioxidant status of silver carp fingerlings when fed with diets containing various organic acids. five experimental diets were formulated, such as d1 containing no supplemented organic acids, while d2 contain (2%malic acid), d3 (2%citric acid), d4 (2% formic acid) and d5 (2% lactic acid). during the experiment, water quality parameters including temperature, ph and do were controlled. results showed that acidification of diet significantly reduced the activity of sod, cat and gpx throughout the body of silver carp fingerlings. moreover, among different organic acid groups, the maximum value was observed in citric acid while, minimum value was recorded in formic acid. data on whole -body antioxidant enzymes were subjected to one-way analysis of variance following steel et al. (1996). differences between among means were compared by tukey`s honestly significant difference test and considered significant at (p<0.05) (snedecor and conhran, 1991). keywords aquaculture, silver carp, antioxidant status, citric acid, formic acid introduction the global aquaculture industry currently produces 45% of all seafood that is consumed all over the world. it has been estimated that it will be increased by 75% in the next 20 years (ftu, 2007). in egypt, aquaculture accounts for 60% of total fish production sources (gafrd, 2007). essential nutrients are not enough to fulfill the demand of the high quality feeds so complementary feed additives are needed to improve efficiency of feed utilization and survival rates of fish. among them, short chain organic acids are mainly used as they have beneficial effects on feed preservation and utilization (luckstadt, 2006; atapattu and senevirathne, 2013; sing et al., 2014). organic acid supplementation in fish diet reduces the gastric ph (baruahet al., 2005) which leads to increase the absorption of nutrients (bolingfrankenbachet al., 2001) and also help in the breakdown of phytate (jongbloed, 1987), so it is easily available to fish. moreover, it also decreases the gastric emptying rate by dietary acidification (mayer, 1994). furthermore, it also improves the gut health of animal as they have antagonistic effect on microbes (ravindran and kornegay, 1993; partanen and mroz, 1999). organic acid increases the availability of dietary minerals through acidification in several ways. firstly, by the modification of mineral transport mechanism by changing the stomach acidity. secondly, supplementation of organic acid in the diet acts as a chelating agent and affects the complex forming ability of elements (ravindran and kornegay, 1993). thirdly, the absorption area for minerals is increased (baruah et al., 2007a) through the proliferation of the epithelial cells in gastrointestinal mucosa (sakata et al., 1995) by the inclusion of organic acid in the diet. ca, one of organic acid is widely used for acidification of diet as it has unique flavor and high buffering capacity (hossain et al., 2007). it has a great potential to reduce the antagonistic interactions between trace elements by their chelating effect and also enhances the absorption of other trace minerals (sugiura et al., 1998). proteolytic enzymes are stimulated by ca which leads to increase the feed intake, reduces the activity of microbes including ammonia and also has the capacity to reduce the risks of subclinical infections (chowdhury et al., 2009; ou et al., 2013). objectives of this study the aim of present study was to investigate the whole body antioxidant status of silver carp fingerlings fed acidified diets materials and methods experiment was performed in fish nutrition laboratory, department of zoology, wildlife and fisheries, university of agriculture, faisalabad. fish and experimental condition before onset the experiment, silver carp fingerlings were obtained from government fish seed hatchery, faisalabad. for acclimatization to indoor conditions, fish were placed for two weeks in tiled tanks (1000 l). during this period basal diet was given for 6 days (allan & rowland, 1992) to fish. for feeding trial, 9 species of fish with same initial weight (3.526±0.0056 g) were kept in v shaped tanks (70l). throughout this time, fingerlings were fed of that basic food once a time in a 1 department of zoology, wildlife and fisheries, university of agriculture, faisalabad, pakistan 2 department of zoology division, institute of pure and applied biology, bahauddin zakariya university multan, pakistan 3 department of animal breeding and genetics, university of agriculture, faisalabad, pakistan * corresponding author’s e-mail: hasilpur910@gmail.com https://doi.org/10.54536/ajlsi.v2i1.1349 https://journals.e-palli.com/home/index.php/ajlsi mailto:hasilpur910%40gmail.com?subject= pa ge 34 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 33-37, 2023 day. for each test diet experiment repeated three times. feeding trial sustained for two months. during the study period, jenway ph meter 3510 and do 970 were used to monitor changings in water quality, specially dissolved oxygen, temperature and ph. all water tanks are inflated by capillary system around the clock. feed ingredients and experimental diets feed ingredients and feed stuff obtained from local poultry market and composition of chemical analysis was done by using standard methods (aoac, 1995). before incorporating feeding trial, the constituents of food were crushed and filtered to achieve the desired particle size (table i). the method for pretreatment of ingredients, 1 kg of the ground constituents such as fish meal, wheat flour, sunflower meal, corn gluten meal and 1.5l of distilled water was adding for made paste and retained it for 38 °c for 16 h and then dried. all dry ingredients were mixed for 15 minutes in an electric mixture. though continuously stirring, then gradually add mineral mixture, vitamin premix and fish oil. five trial diets were made by using various supplementing organic acids at level of 2%. the d1 contains no supplemented organic acids, while d2 contain malic acid, d3 citric acid, d4 formic acid and d5 lactic acid, correspondingly. to prepare appropriate dough for each trial feed, slowly mix 10% to 15% water. to make floating particles, then further process it via a laboratory extruder. after particles are dried, they are crushed and sieved to the desired size. keep the pellets in the refrigerator at -18 ° c until the feeding test is completed. chemical analysis of feed with help of pestle and mortar fish samples and diet were standardized methods for determining moisture was: drying in an oven at 105°c for 12 hours, micro kjeldahl apparatus used for measuring crude protein. by soxhlet system, crude fat through petroleum ether extraction method determined (bligh & dyer, 1959) and in an electric furnace for 12 hours crude fiber measured (table ii) table 2: chemical composition (%) of experimental diet diet organic acids dm (%) cp (%) cf (%) ash (%) d1 control 89.49 31.11 9.05 9.95 d2 malic acid 90.1 30.51 9.1 9.62 d3 citric acid 90.17 31.071 9.045 9.8 d4 formic acid 89.63 31.12 8.94 9.78 d5 lactic acid 90.11 30.84 9.16 9.86 table 1: formulation (%) of feed ingredients ingredient percentage fishmeal 25 sunflower meal 20 soybean meal 10 corn gluten meal 15 fish oil 7 rice polish 10 wheat flour 9 mineral mixture** 1 vitamin premix* 1 organic acid 2 total 100 feeding procedure and sample collection for experimental feeding trial, the fingerlings were fed of their suggested diet. after feeding time of three hours, the extra food was exhausted by opening the tank valve. wash water tank thoroughly to eliminate particles from the food then fill-up the water. afterwards, return the fish to the fish tank. after the two-hour interval, feces were collected in beaker by opening valves of tank. in an oven at 60oc, each of the repeatedly processed feces was dried. then ground and stored for chemical inquiry. for each repeated sample, the trial was continued to collect 5 g of feces. figure 1: modified ua system showing steel tank and main pipe figure 2: modified ua system showing valve i and valve ii and collection tube determination of antioxidant enzymes preparation of enzyme extract whole body was taken and rinsed with phosphate buffer of ph 6.5 (0.2 m) and homogenized in cold buffer (1:4 w/v) using a blender. after the homogenization, organ homogenates were centrifuged for 15 minutes at 10,000 rpm and 4ºc. after centrifugation process, clear supernatants were stored at 80ºc for enzyme assay while residue was discarded. enzyme assay of superoxide dismutase the activity of superoxide dismutase was determined https://journals.e-palli.com/home/index.php/ajlsi pa ge 35 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 33-37, 2023 by measuring its ability to inhibit the photo reduction of nitrobluetetrazolium (nbt) following the method of giannopolitis and ries (1997). procedure 1 ml buffer was taken in cuvette as blank and inserted into spectrophotometer to note the readings of blank, after taking reading spectrophotometer was adjusted at zero at a560 nm. then 5-6 cuvettes were taken and set them in a light box with an internally mounted light bulb of 30 watt. firstly 1 ml of buffer was added to each cuvette, then 0.05 ml enzyme extract and 0.016 ml of riboflavin was added in each cuvette. all the cuvettes were incubated in light box for 12 minutes. the cuvettes were transferred to the spectrophotometer, where 0.067 ml of edta/nacn solution and 0.033 ml of nbt was added to the illuminated reaction mixture. the absorbance was noted after 20 s of reaction. activity of superoxide dismutase was determined by measuring the % age inhibition of nbt. enzyme assay of catalase and glutathione peroxidase the activity of peroxidase was determined by measuring its ability to reduce the concentration of h2o2 at a470 nm (civello et al., 1995). procedure a cuvette containing the 2 ml of blank solution was inserted into the spectrophotometer and set it to zero at wavelength of 240 nm. then a cuvette containing buffered substrate solution was put into the spectrophotometer and initiation of reaction was occurred by adding 0.05 ml of enzyme extract the initiation of reaction was occurred. the reaction time is 3 minutes and noted the absorbance after interval of 1 minute. calculation statistical analysis finally, data regarding the activities of antioxidant enzymes of whole body were subjected to one-way analysis of variance following steel et al. (1996). the differences among means were compared by tukey`s honestly significant difference test and considered significant at (p<0.05) (snedecor and conhran, 1991). costate computer software, version 6.303 was used for statistical analysis. results superoxide dismutase status (u/mg protein) in whole body of silver carp fingerlings effect of different organic acids on whole body superoxide dismutase status in silver carp fingerlings is shown in table iii. data showed that acidification of diet significantly decreased the activity of sod in whole body of silver carp fingerlings. furthermore, maximum value was observed in formic acid while, minimum value was recorded in malic acid among different organic acid groups. table 3: effect of supplementation of different organic acids on superoxide dismutase status (u/mg protein) in whole body of silver carp fingerlings diet organic acid sod d1 control 4.55a d2 malic acid 3.65c d3 citric acid 3.83bc d4 formic acid 4.10b d5 lactic acid 4.02b pse 0.068 p-value 0.0018** data are means of three replicates, p<0.05 organic acids pse = pooled se = √mse/n (where mse= mean-squared error) catalase status (u/mg protein) in whole body of silver carp fingerlings effect of different organic acids on whole body catalase status in silver carp fingerlings is shown in table iv.data showed that acidification of diet significantly decreased the activity of cat in whole body of silver carp fingerlings. furthermore, maximum value was observed in malic acid while, minimum value was recorded in lactic acid among different organic acid groups. table 4: effect of supplementation of different organic acids on catalase status (u/mg protein) in whole body of silver carp fingerlings diet organic acid cat d1 control 65.66a d2 malic acid 58.52b d3 citric acid 53.22c d4 formic acid 50.72c d5 lactic acid 50.05c pse 1.427 p-value 0.0025** data are means of three replicates, p<0.05 organic acids pse = pooled se = √mse/n (where mse= mean-squared error) glutathione peroxidase status (mu/mg protein) in whole body of silver carp fingerlings effect of different organic acids on whole body glutathione peroxidase status in silver carp fingerlings is shown in table v. data showed that acidification of diet significantly decreased the activity of gpx in whole body of silver carp fingerlings. furthermore, maximum value was observed in citric acid while, minimum value was recorded in formic acid among different organic acid groups. https://journals.e-palli.com/home/index.php/ajlsi pa ge 36 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 33-37, 2023 discussion currently, there is considerable interest in the commercial use of organic acids in fish diets, both to control disease and to enhance growth performance. researches have reported that several organic acids, their salts or mixtures thereof can improve growth, feed utilization and disease resistance in fish (baruah et al 2007; hossain, pandey & satoh 2007; sarker, satoh & kiron 2007; luckstadt 2008). hormones, antibiotics, ionospheres and some salts are most commonly used as growth promoters to increase the productive performance of fish (fuller, 1992; go´ngora, 1998; klaenhammer and kullen, 1999). antibiotic growth promoters are used in fish diets as feed additives to favor growth and environment friendly aquaculture. however, it is necessary to make use of these antibiotic growth promoters in less amount as feed additive in fish diets worldwide to enhance the development of cross resistances to humans. however, the supplementation of organic acid is beneficial to replace the feed additives. (kim et al. 2006) investigated that organic acid supplemented diet can improve the growth and performance of fishes. fish feed industry has been using organic acids for decades where most extensively used acidifiers contain lactic, citric, fumaric and formic acid. (baruah et al. 2007) designed a factorial experiment to study the effect of dietary microbial phytase, citric acid and their interactions on growth performance of labeo rohita juveniles. hence, it is suggested that phytase and citric acid act synergistically to improve growth performance and nutrient digestibility of l. rohita juveniles. (hossain et al. 2007) conducted a feeding experiment to investigate the effects of organic acids on the growth and phosphorus utilization in fish. therefore, it can be concluded from these results that organic acids can play an important role in development of highly required eco friendly diets. (pandey & satoh 2008) observed phosphorus (p) and nitrogen (n) retention in rainbow trout fed on low fish meal based diets supplemented with organic acid. hence, results conclude that low fishmeal based diets may prove beneficial for growth of rainbow trout if supplemented with certain organic acids. phromkunthong et al. (2010) determined the combined effect of citric acid and microbial phytase on phosphorus utilization. cyprinus carpio fingerlings were treated with citric acid and phytase for 60 days. it can be concluded that positive effects of phytase can be achieved by adding low dose of citric acid. conclusion findings of experiment are as follows: • supplementation of organic acids significantly (p˂0.05) decreased the whole body superoxide dismutase activity (u/mg protein) in silver carp fingerlings. • supplementation of organic acids significantly (p˂0.05) decreased the whole body catalase activity (u/ mg protein) in silver carp fingerlings. • supplementation of organic acids significantly (p˂0.05) decreased the whole body glutathione peroxide activity (mu/mg protein) in silver carp fingerlings. in conclusion, supplementation of organic acids decreased the antioxidant enzymes in silver carp fingerlings. authors contribution k.r planed, did experiments and wrote manuscript. t.d, s.a, m.a and s.a helped in experimental work. references a. o. a. c. (1995). association of official analytical chemists, official methods of analysis. 16th edition, aoac, arlington, va. 1832 atapattu, n. s. b. m., & senevirathne, t. s. m. s. (2013). effects of increasing levels of dietary cooked and uncooked banana meal on growth performance and carcass parameters of broiler chicken. pak. vet. j, 33(2), 179-182. baruah, k., pal, a. k., sahu, n. p., jain, k. k., mukherjee, s. c., & debnath, d. (2005). dietary protein level, microbial phytase, citric acid and their interactions on bone mineralization of labeo rohita (hamilton) juveniles. aquaculture research, 36(8), 803-812. baruah, k., sahu, n. p., pal, a. k., debnath, d., yengkokpam, s., & mukherjee, s. c. (2007). interactions of dietary microbial phytase, citric acid and crude protein level on mineral utilization by rohu, labeo rohita (hamilton), juveniles. journal of the world aquaculture society, 38(2), 238-249. baruah, k., sahu, n. p., pal, a. k., jain, k. k., debnath, d., & mukherjee, s. c. (2007). dietary microbial phytase and citric acid synergistically enhances nutrient digestibility and growth performance of labeo rohita (hamilton) juveniles at sub-optimal protein level. aquaculture research, 38(2), 109-120. boling-frankenbach, s. d., snow, j. l., parsons, c. m., & baker, d. h. (2001). the effect of citric acid on the calcium and phosphorus requirements of chicks fed corn-soybean meal diets. poultry science, 80(6), 783-788. chowdhury, r., islam, k. m. s., khan, m. j., karim, m. r., haque, m. n., khatun, m., & pesti, g. m. (2009). effect of citric acid, avilamycin, and their combination table 5: effect of supplementation of different organic acids on glutathione peroxidase status (mu/mg protein) in whole body of silver carp fingerlings diet organic acid gpx d1 control 75.82a d2 malic acid 70.11b d3 citric acid 71.46b d4 formic acid 68.27b d5 lactic acid 71.39b pse 0.904 p-value 0.0149* data are means of three replicates, p<0.05 organic acids pse = pooled se = √mse/n (where mse= mean-squared error) https://journals.e-palli.com/home/index.php/ajlsi pa ge 37 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 33-37, 2023 on the performance, tibia ash, and immune status of broilers. poultry science, 88(8), 1616-1622. civello, p. m., martinez, g. a., chaves, a. r., & anon, m. c. (1995). peroxidase from strawberry fruit (fragaria ananassa duch.) partial purification and determination of some properties. journal of agricultural and food chemistry, 43(10), 2596-2601. papadoyianis, e. d. (2007). insects offer a promising solution to the protein bottleneck. feed technology update, 2(6), 158. fuller, r., & fuller, r. (1992). history and development of probiotics. probiotics: the scientific basis, 1-8. gafrd (2007). general authority for fish resources development fishery statistic report. egyptian ministry of aquaculture. giannopolitis, c. n., & ries, s. k. (1977). superoxide dismutases: ii. purification and quantitative relationship with water-soluble protein in seedlings. plant physiology, 59(2), 315-318. góngora, c. m. (1998). mecanismos de resistencia bacteriana ante la medicina actual. barcenola: mcgrawhill, 30. hossain, m. a., pandey, a., & satoh, s. (2007). effects of organic acids on growth and phosphorus utilization in red sea bream pagrus major. fisheries science, 73, 1309-1317. jongbloed, a. w. (1987). phosphorus in the feeding of pigs: effect of diet on the absorption and retention of phosphorus by growing pigs. wageningen university and research. kim, d. h., & austin, b. (2006). innate immune responses in rainbow trout (oncorhynchus mykiss, walbaum) induced by probiotics. fish & shellfish immunology, 21(5), 513-524. klaenhammer, t. r., & kullen, m. j. (1999). selection and design of probiotics. international journal of food microbiology, 50(1-2), 45-57. lückstädt, c. (2006). use of organic acids as feed additives—sustainable aquaculture production the non-antibiotic way. int aquafeed, 9(2), 21-26. lückstädt, c. (2008). the use of acidifiers in fish nutrition. cabi reviews, 8. https://doi.org/10.1079/ pavsnnr20083044 ou, c., shi, n., pan, q., tian, d., zeng, w., & he, c. (2013). therapeutic efficacy of the combined extract of herbal medicine against infectious bursal disease in chickens. pakistan veterinary journal, 33(3). pandey, a., & satoh, s. (2008). effects of organic acids on growth and phosphorus utilization in rainbow trout oncorhynchus mykiss. fisheries science, 74, 867874. phromkunthong, w., nuntapong, n., & gabaudan, j. (2010). interaction of phytase ronozyme? p (l) and citric acid on the utilization of phosphorus by common carp (cyprinus carpio). sonklanakarin journal of science and technology, 32(6), 547. ravindran, v., & kornegay, e. t. (1993). acidification of weaner pig diets: a review. journal of the science of food and agriculture, 62(4), 313-322. sakata, t., adachi, m., hashida, m., sato, n., & kojima, t. (1995). effect of n-butyric acid on epithelial cell proliferation of pig colonic mucosa in short-term culture. deutsche tierarztliche wochenschrift, 102(4), 163164. sarker, m. s. a., satoh, s., & kiron, v. (2007). inclusion of citric acid and/or amino acid-chelated trace elements in alternate plant protein source diets affects growth and excretion of nitrogen and phosphorus in red sea bream pagrus major. aquaculture, 262(2-4), 436-443. sarker, m. s. a., satoh, s., kamata, k., haga, y., & yamamoto, y. (2012). supplementation effect (s) of organic acids and/or lipid to plant protein based diets on juvenile yellowtail, seriola quinqueradiata temminck et schlegel 1845, growth and, nitrogen and phosphorus excretion. aquaculture research, 43(4), 538-545. sing, k. w., kamarudin, m. s., wilson, j. j., & sofianazirun, m. (2014). evaluation of blowfly (chrysomya megacephala) maggot meal as an effective, sustainable replacement for fishmeal in the diet of farmed juvenile red tilapia (oreochromis sp.). pakistan veterinary journal, 34(3). snedecor, g.w. and conhran, w.g. (1991). statistical methods. iowa state univ. press, ames, usa. steel, r. g. d., torrie, j. h. & dickey, d. a. (1996). principles and procedures of statistics. 3rd ed. mcgraw hill international book co. inc., new york. usa. sugiura, s. h., dong, f. m., & hardy, r. w. (1998). effects of dietary supplements on the availability of minerals in fish meal; preliminary observations. aquaculture, 160(3-4), 283-303. https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 24 american journal of life science and innovation (ajlsi) characteristics and outcomes of conditions encountered in primary care during fifa world cup 2022 in qatar a cross-sectional study lolwa al-maslamani1*, naheel seyam2 volume 2 issue 3, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i3.2145 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: september 30, 2023 accepted: october 26, 2023 published: november 03, 2023 the fifa world cup, a massive football event, presents significant challenges to healthcare systems. primary healthcare plays a crucial role in patient-provider interactions and serves as a cornerstone of the system. this study aimed to assess the frequency, characteristics, and outcomes of conditions treated in primary care facilities during the fifa world cup qatar 2022. a cross-sectional study was conducted from november 15 to december 20, 2022. all patients visiting the 29 governmental primary healthcare facilities in qatar were included. anonymous patient data, including age, sex, nationality, diagnosis, treatments, visit date, consultation type, and outcomes, were extracted from electronic medical records. a total of 401,962 patients were seen in primary care, with an average age of 34.50 ±19.3 years. less than 1% held hayya visa (n=3120, 0.8%), and roughly 2% were referred to secondary care (8494, 2.1%). the most prevalent diagnoses were type 2 diabetes mellitus (n=59670, 7.7%) and upper respiratory tract infection (n=47364, 6.2%). paracetamol was the most frequently prescribed medication (n=69,960, 11.9%). elderly patients (or=1.23), nonqatari patients (or=1.25), and male patients (or=1.17) were more likely to be referred to secondary care. hayya cardholders were about twice as likely to be transferred (or=2.40). primary healthcare services operated efficiently during the fifa world cup 2022 in qatar. the majority of cases involved managing chronic diseases and minor respiratory infections. football enthusiasts and non-qatari individuals were more often referred to secondary healthcare. these findings can guide policymakers in enhancing primary healthcare services for future mass gatherings. keywords football, primary care, qatar, diabetes mellitus, respiratory tract infections introduction the fifa world cup is a major global football event that presents substantial challenges to the healthcare system (karami, 2018). according to the world health organization, mass gatherings refer to planned occasions where a large number of individuals convene, potentially straining the resources, planning, and response capabilities of the host community’s health system. therefore, a wellprepared plan is necessary to address these challenges (wilder-smith & steffen, 2019). in 2022, qatar, a member of the gulf cooperation council in the middle east, hosted the fifa world cup, attracting around 1.5 million visitors. qatar undertook significant investments and endeavours to enhance its infrastructure and healthcare system in preparation for this international event. these efforts encompassed augmenting the capacity of the healthcare system by expanding emergency and ambulance services, as well as constructing new hospitals and primary healthcare centers (organization, 2023). primary healthcare constitutes the initial point of contact between patients and healthcare providers, serving as a fundamental pillar of the healthcare system (shi, 2012). it delivers comprehensive preventive, diagnostic, and therapeutic services that are readily accessible, aiming to enhance health outcomes, reduce costs, and optimise the utilisation of secondary care services. due to its broad scope, primary healthcare encounters the majority of patients and various medical conditions (starfield et al., 2005). numerous studies have examined the most prevalent medical presentations encountered in primary care. for example, a systematic review conducted by finley et al. encompassed eighteen studies from twelve countries across five continents. it revealed that upper respiratory tract infections, hypertension, routine health check-ups, diabetes, depression, anxiety, pneumonia, acute otitis media, back pain, and dermatitis were among the most frequently diagnosed conditions. simultaneously, patients most commonly reported visiting for symptomatic issues, including cough, back pain, abdominal discomfort, pharyngitis, dermatological concerns, fever, headache, leg symptoms, respiratory concerns, and fatigue (finley et al., 2018). similarly, a cross-sectional study was conducted to evaluate prevalent conditions in general practice settings. it found that pulmonary, gastrointestinal, orthopaedic, and cardiac issues constituted 67.71% of the total health problems encountered in general practice. furthermore, the study identified acute upper respiratory tract infections as the most prevalent health issue seen in primary care facilities (6.67%), followed by chronic conditions like hypertension and diabetes mellitus (chen et al., 2022). similar findings were reported by other studies as well (adib et al., 1995; gong et al., 2020). in qatar, primary healthcare services are administered through a network of healthcare centres. aligned with health demands, a strategic plan for primary healthcare was devised, with health centres strategically distributed 1 al rayyan health center, ar-rayyan, qatar 2 primary health care corporation, doha, qatar * corresponding author’s e-mail: lolwaalmaslamani58@outlook.com pa ge 25 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 24-29, 2023 across three regions: central, northern, and western areas (al-kuwari et al., 2020; ali abdulmalik et al., 2022). a total of 29 government-run health centres provide both preventative and therapeutic medical services to the entirety of qatar’s population (al-kuwari et al., 2021). the present study aimed to determine the most common conditions encountered in primary care facilities during the fifa world cup 2022 in qatar. it aims to offer clinical insights that can be applied in subsequent mass gatherings, contributing to a better understanding of prevalent conditions in primary care centres during such events and enhancing the preparedness of the healthcare system, particularly primary healthcare, for future largescale occasions. materials and methods between november 15 and december 20, 2022, a crosssectional study was carried out to identify the most commonly encountered conditions in primary care facilities during the world cup. qatar’s primary health services are provided through 29 governmental primary health centres that are dispersed throughout the country. these centres operate on weekdays, with 18 of them also remaining open on public holidays and weekends and 10 operating around the clock. the analysis included all patients who visited these governmental primary healthcare centres during the specified period. the study included all patients accessing primary healthcare centres in qatar, irrespective of age, gender, nationality, or condition. no exclusion criteria were applied. given that all patients attending the healthcare centres were encompassed in the study, a sample size calculation was optional. study instrument and variables a data collection tool was devised to gather baseline information on patients, encompassing age, gender, nationality, diagnosis, treatments, and date of visit. additionally, the type of consultation (in-person or virtual) and the visit’s outcomes (discharge or transfer) were recorded. diagnoses and related data were extracted from electronic records, all of which were de-identified to safeguard patient privacy and confidentiality. statistical analysis data analysis was conducted using the statistical package for the social sciences program for windows (v.25.0; ibm corp, armonk, new york, usa). quantitative variables were presented using means and standard deviations, while qualitative variables were expressed as frequencies and percentages. a bar chart was employed for data visualisation. the chi-square test and t-test were applied as appropriate to ascertain the statistical significance between baseline characteristics of patients and visit outcomes (transfer or discharge). a p-value less than 0.05 was considered statistically significant. results a total of 401,962 patients were encompassed in the study. their ages spanned from 0 to 109 years, with an average age of 34.50 ± 19.3. notably, about two-thirds of these patients were of non-qatari nationality (n=261,598, 65.1%), and the majority were females (n=236,160, 58.8%). it was observed that one in every four consultations took place virtually (n=99,821, 24.8%). a small proportion of participants, less than 1%, possessed a hayya visa (n=3,120, 0.8%), and approximately 2% were transferred to secondary care facilities (8,494, 2.1%). the detailed baseline characteristics of the patients can be found in table 1. the frequency of visits to primary healthcare facilities fluctuated throughout the course of the event. illustrated in figure 1, the daily visits ranged from 2,335 to 17,261 visits per day. table 1: baseline characteristics of the patients baseline characteristics of the patients n (%) age, mean, sd 34.50 ± 19.3 age in years 0-17 80118 (11.1) 18-45 205604 (19.9) 46-59 71788 (51.2) >=60 44452 (17.9) nationality non-qatari 261598 (65.1) qatari 140364 (34.9) sex male 165802 (41.2) female 236160 (58.8) consultation type in-person 302141 (75.2) virtual consultation 99821 (24.8) hayaa-visa holder no 398842 (99.2) yes 3120 (0.8) discharge status discharged 393468 (97.9) transferred 8494 (2.1) pa ge 26 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 24-29, 2023 most common diagnosis physicians recorded a total of 769,945 diagnoses. type 2 diabetes mellitus without complications (n=59,670, 7.7%) was the most frequently registered diagnosis, followed closely by upper respiratory tract infections (n=47,364, 6.2%). patients holding the hayya card exhibited similar diagnoses to those without it as shown in table 2. figure 1: number of visits to primary healthcare centers during world cup 2022 in qatar table 2: most common registered diagnoses the most common diagnosis among hayya card holders, tn=4656 the most common diagnosis among other patients, tn=765289 diagnosis n (%) diagnosis n (%) viral upper respiratory tract infection 637 (13.7) type 2 diabetes mellitus without complication 59504 (7.8) type 2 diabetes mellitus without complication 166 (3.6) viral upper respiratory tract infection 46727 (6.1) essential (primary) hypertension 119 (2.6) essential (primary) hypertension 27216 (3.6) unspecified place of occurrence 96 (2.1) issue of repeat prescription 16223 (2.1) unspecified activity 95 (2.0) vitamin d deficiency 13843 (1.8) well baby 73 (1.6) telephone consultation 13007 (1.7) abdominal pain 58 (1.2) well baby 11079 (1.4) the most commonly prescribed medications over the evaluated period, primary care physicians issued a total of 589,155 prescriptions. the most frequently prescribed medication was paracetamol (n=69,960, 11.9%). following closely, cholecalciferol tablets (n=40,931, 6.9%) and ibuprofen tablets (n=20,901, 3.5%) were among the commonly recommended medications by physicians as shown in table 3. table 3: most common prescribed medications the most common medications are given to hayya card holders, tn=5753 the common medications are given to other patients, tn=583402 diagnosis n (%) diagnosis n (%) paracetamol 975 (16.9) paracetamol 68985 (11.8) ibuprofen 288 (5.0) cholecalciferol 40931 (7.0) vitamin c 261 (4.5) ibuprofen 20613 (3.5) chlorhexidine 252 (4.4) chlorhexidine 17934 (3.1) levocetirizine 248 (4.3) vitamin c 16556 (2.8) amoxicillin/clavulanate 223 (3.9) levocetirizine 14750 (2.5) dextromethorphan (cough suppressant) 189 (3.3) diclofenac topical 13399 (2.3) diclofenac topical 185 (3.2) cetirizine/pseudoephedrine 13131 (2.3) cetirizine/pseudoephedrine 179 (3.1) xylometazoline 12172 (2.1) diphenhydramine/ammonium chloride 155 (2.7) amoxicillin/clavulanate 12020 (2.1) pa ge 27 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 24-29, 2023 association between visit outcomes and baseline characteristics of the patients as indicated in table 4, individuals aged 60 years and older exhibited elevated rates of referral in comparison to their younger counterparts (p=<0.001). non-qatari patients (p<0.001), male patients (p<0.001), those opting for in-person consultations (p<0.001), and individuals holding the hayya card (p<0.001) demonstrated higher instances of transfer to secondary care. the outcomes of the logistic regression analysis are detailed in table 5. the findings indicate that patients of advanced age (or=1.23), non-qatari nationality (or=1.25), and male gender (or=1.17) exhibit a higher likelihood of being referred to secondary care facilities. notably, individuals holding the hayya card were approximately twice as likely to be transferred (or=2.40). moreover, patients who opted for in-person consultations were four times more inclined to be transferred to secondary care hospitals compared to those who chose virtual consultations (or=4.063, 95% ci 3.732-4.423). table 5: logistic regression of the association between visit outcomes and baseline characteristics variable odds ratio, 95% confidence interval p value age 1.004 (1.001-1.008) 0.013 age*; 0 – 17 years 1.29 (1.19-1.41) <0.001 age: 18 – 45 years 1.01 (0.94-1.09) 0.747 age; > 60 years 1.23 (1.15-1.30) <0.001 nationality** 1.25 (1.19-1.31) <0.001 sex*** 1.17 (1.12-1.22) <0.001 consultation type ***** 4.52 (4.15-4.91) <0.001 hayya card holder ***** 2.40 (2.08-2.78) <0.001 table 4: logistic regression of the association between visit outcomes and baseline characteristics baseline characteristics of the patients visit outcome p value discharged transferred age, years ±sd 34.5 ± 19.3 35.1 ± 19.8 0.004 age, group 0-18 78487 (98.0) 1631 (2.0) <0.001 18-45 201127 (97.8) 4477 (2.2) 45-60 70488 (98.2) 1300 (1.8) >=60 43366 (97.6) 1086 (2.4) nationality non-qatari 255821 (97.8) 5777 (2.2) <0.001 qatari 137647 (98.1) 2717 (1.9) sex male 161821 (97.6) 3981 (2.4) <0.001 female 231647 (98.1) 4513 (1.9) consultation type in-person 294241 (97.4) 7900 (2.6) <0.001 virtual consultation 99227 (99.4) 594 (0.6) hayya card holder no 390555 (97.9) 8287 (2.1) <0.001 yes 2913 (93.4) 207 (6.6) discussion the primary objective of this study was to identify the most prevalent conditions encountered in primary care facilities during the world cup 2022 while also examining their attributes and outcomes. the research findings highlighted that type 2 diabetes mellitus without complications and upper respiratory tract infections were the most frequently observed cases. virtual consultations emerged as a widely adopted approach, with the majority of cases not necessitating a referral. it was noteworthy that a significant number of visits were dedicated to the ongoing management of diabetes mellitus, underscoring that routine care remained unaffected by the international sporting event. additionally, chronic conditions like hypertension and dyslipidemia constituted common reasons for seeking care at these centres, as discerned from the results. in consonance with established literature, our study corroborated the prevalence of acute upper respiratory infections as a frequent presentation in primary care. the prescription patterns further substantiated this observation. notably, widely used medications included pain relievers such as paracetamol and ibuprofen, along with treatments for upper respiratory infections such as vitamin c, levocetirizine, and dextromethorphan. furthermore, our study findings were consistent with the annual statistical report from qatar’s primary healthcare corporation, which underscored that type 2 diabetes mellitus without complications and upper respiratory pa ge 28 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 24-29, 2023 tract infections rank as the most common conditions treated in primary care in the country (phcc, 2023). concerns were initially raised regarding the potential risk of outbreaks of middle east respiratory syndrome coronavirus (mers-cov) and covid-19 during the fifa world cup 2022 in qatar (al-tawfiq et al., 2022; llorente-nieto et al., 2023). however, the results of our report were against these concerns. although upper respiratory tract infections were prevalent, chronic conditions such as diabetes and hypertension took precedence over respiratory infections as the primary reasons for seeking care in primary health settings. the results of this study revealed notable variation in the volume of visits to primary healthcare facilities during the assessed period. this fluctuation could be attributed to the reduced availability of healthcare centres on weekends. another contributing factor was that certain routine consultations and procedures, including child screenings, antenatal care, and routine immunisations, were exclusively conducted on weekdays. moreover, the majority of patients were discharged; our findings revealed that individuals of advanced age were more likely to be referred to secondary care hospitals. this could potentially be attributed to the higher likelihood of elderly patients presenting with multiple health issues and urgent conditions necessitating expedited referral to secondary care settings. the findings that non-qatari patients and hayya card holders were more inclined to be referred could be explained by their tendency to present with acute conditions that may need to be fully addressed in the primary care setting. in contrast, qatari and nonhayya cardholders often sought care for chronic disease management alongside acute concerns. another possibility could be a lower threshold for referral for patients holding hayya cards. of particular interest, nearly one-quarter of all consultations were conducted virtually. this utilisation rate of telemedicine services aligned with previous reports from qatar (phcc, 2023). hence, the expansion of telemedicine services was not impeded by the mass gathering at the world cup 2022. parallel to the primary healthcare services, qatar established over 110 clinics and 212 mobile units in the most frequented areas during the tournament, ensuring convenient and efficient access to medical services. this proactive approach resulted in effective case management and enhanced preparedness. (“qatar’s healthcare sector treated 51,809 patients during-fifa world cup qatar-2022,” 2022). this study has several strengths. it is the first study to determine the characteristics and outcomes of the conditions encountered in primary care during a fifa world cup event. all encountered patients were included. several measures were assessed, and a multivariate analysis was performed. however, this study has some limitations. participants’ comorbidities and the outcomes of referrals were not assessed. conclusions in conclusion, the study revealed that primary healthcare services, including the successful integration of virtual consultations, throughout the fifa world cup 2022 in qatar. predominantly, routine cases involving the management of chronic illnesses and common upper respiratory conditions were the predominant encounters. notably, visitors and non-qatari patients exhibited higher referral rates compared to their counterparts. this underscores the pivotal role of primary healthcare in fortifying readiness for large-scale gatherings. limitations the study has some limitations, such as its lack of assessment of participants’ comorbidities, which could have provided valuable insights into the complexity of cases encountered in primary care during the fifa world cup. additionally, outcomes of referrals were not investigated, leaving a gap in understanding the effectiveness and appropriateness of transfers to secondary care facilities. suggestion for further study future research endeavours could delve into a comprehensive evaluation of comorbidities among patients attending primary care facilities during mass gatherings, providing a more nuanced understanding of complex cases. additionally, investigating the longterm outcomes and experiences of patients referred to secondary care following such events would contribute to a holistic assessment of healthcare delivery. furthermore, comparative studies across various international sporting events could offer valuable insights into the generalizability of findings and the optimisation of healthcare preparedness strategies. references adib, s. m., nuwayhid, i., & hamadeh, g. n. (1995). most common diseases treated in primary health care facilities in lebanon. le journal medical libanais. the lebanese medical journal, 43(1), 17-22. al-kuwari, m., abdulla, s., abdulla, m., bakri, a., kandy, m., mustafa, a., swamy, s., rahman, m., shaikan, f., patterson, a., buhaddoud, h., & rothlisberger, g. (2020). qatar’s primary health care health needs assessment 2019. al-kuwari, m. g., al-abdulla, s. a., abdulla, m. y., haj bakri, a., mustafa mohammed, a., chettiyam kandy, m., patterson, a., & illiayaraja krishnan, j. (2021). epidemiological health assessment in primary healthcare in the state of qatar-2019. qatar medical journal, 2021(3), 57. al-tawfiq, j. a., gautret, p., & schlagenhauf, p. (2022). infection risks associated with the 2022 fifa world cup in qatar. new microbes and new infections, 49. ali abdulmalik, m., al-kuwari, m. g., haj bakri, a., ahmad al abdulla, s., chettiyam kandy, m., yousef abdulla, m., & michael gibb, j. (2022). understanding the pa ge 29 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 24-29, 2023 epidemiological characteristics of the primary healthcare corporation-based covid-19 swabbed persons in qatar, 2020. qatar medical journal, 2022(3), 23. chen, k., li, z., liu, r., zhu, y., yan, w., pui, n. a. p., & chen, z. (2022). distribution of health problems at the general outpatients’ clinic of the university of hong kong-shenzhen hospital: a cross-sectional study. frontiers in public health, 10, 889819. finley, c. r., chan, d. s., garrison, s., korownyk, c., kolber, m. r., campbell, s., eurich, d. t., lindblad, a. j., vandermeer, b., & allan, g. m. (2018). what are the most common conditions in primary care?: systematic review. canadian family physician, 64(11), 832-840. gong, x., wang, x., shi, t., shi, j., yu, w., zhou, l., chen, n., huang, j., & wang, z. (2020). disease composition and epidemiological characteristics of primary care visits in pudong new area, shanghai: a longitudinal study, 2016â€“2018. bmj open, 10(11), e040878. karami, m. (2018). sporting mass gatherings: public health preparedness and responsibility of health authorities. journal of research in health sciences, 18(3), e00422. llorente-nieto, p., gonzã¡lez-alcaide, g., & ramosrincã³n, j.-m. (2023). mass gathering in qatar 2022 world cup. what should be especially monitored? journal of infection and public health. organization, w. h. (2023). ministry of public health, qatar and who collaborating to implement public health security measures as fifa world cup approaches https://www.who.int/news-room/ feature-stories/detail/ministry-of-public-health-qatar-and-who-collaborating-to-implementpublic-health-security-measures-as-fifa-world-cupapproaches phcc. (2023). corporate strategy https://www.phcc. gov.qa/aboutus/corporate-strategy qatar’s healthcare sector treated 51,809 patients during-fifa world cup qatar-2022. (2022). https:// www.qna.org.qa/en/news-area/news/202302/11/0022-qatar’s-healthcare-sector-treated-51,809patients-during-fifa-world-cup-qatar-2022 shi, l. (2012). the impact of primary care: a focused review. scientifica, 2012. starfield, b., shi, l., & macinko, j. (2005). contribution of primary care to health systems and health. the milbank quarterly, 83(3), 457-502. wilder-smith, a., & steffen, r. (2019). mass gatherings. in travel medicine (383-386). elsevier. pa ge 1 pa ge 14 american journal of life science and innovation (ajlsi) tree taper model for selected tree species within university of ibadan, oyo state aderinola adeola deborah1*, jackson vincent obukojopo2 volume 1 issue 2, year 2022 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v1i2.777 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: october 16, 2022 accepted: october 21, 2022 published: october 27, 2022 in this study, the performance of different types of taper model for predicting tree diameters of terminalia radii (tent tree) at heritage park and tectonia grandis (teak) at teak plantation within university of ibadan was examined. data from pure (monoculture) stands of t. radii (heritage park) and t. grandis (teak plantation) containing a total of 146 tent trees and 131 teak trees respectively. five taper models developed by various researchers were adopted, fitted and evaluated. the comparison of the model performances was basically on the analysis of three goodness-of-fit statistics and residue analysis found model 5 to be most superior in predicting the stem diameter at any point for the two species in the two study areas. the model 2 exhibited the worst performance in fitting statistics and residual analysis results. therefore, the same taper model should be used for prediction of diameter of the two tree species within university of ibadan. keywords heritage park, model, teak plantation, terminalia radii, tectonia grandis 1 department of social and environmental forestry, university of ibadan, nigeria 2 department of forest production and products, university of ibadan, nigeria * corresponding author’s e-mail: adeyemiadeola90@gmail.com introduction tree taper can be defined as the rate of narrowing in diameter along the tree stem of a given form (gray 1956). it can be expressed as a function of height above ground level, total tree height, and diameter at breast height (clutter et al., 1983). the term ‘taper’ function is often used interchangeably with tree form, where ‘form’ refers to the shape of the tree (max and burkart,1976). in 1946, mesavage and girard asserted that the tree taper is the degree to which a tree stem or bole decreases in diameter as a function of height above the ground, where tree with a high degree of taper is called a poor taper tree and those with low taper are refer to as good taper tree. also explained that the form of a tree can be represented by a certain form class called girard which denoted as the ratio expressed as percentage of butt-log scaling diameter to diameter as the breast height. the traditional geometric shape of tree can be expressed as a mathematical function of height above ground level, total tree height, and diameter at breast height (d) (sloboda and saboroske 1981). taper equations are very useful as they can provide information about diameter at any height, and height at any diameter based only on commonly taken tree measurements (byrne and reed 1986). furthermore, taper equations can be used to derive volume equations by integration when the equation is rotated around the longitudinal axis of a tree (bruce et al. 1968; byrne and reed, 1986). many types of taper modeling techniques have been proposed and applied over the years. these mathematical functions are generally described as taper functions tree (max and burkart,1976). its function is essential and play a pivotal role in forest inventory and growth projection, as well as in forest management planning (rupsy,2018). the function can provide a suitable and relevant information for decision making at an individual tree level, stand level and forest level (gray,1956). according to wang et al. (1998), the vulnerability and susceptibility of a tree to wind damage is majorly influenced or determined by the slenderness coefficient or taper of the tree. whereas, the heritage park and teak plantation are mainly for the recreational and research purpose respectively, their accommodative status are mostly high every time. with this the taper of the selected tree species should be determined, in order to know their susceptibility to wind throw. over some decades, tree taper functions have been widely studied all over the world like taper and stem volume equations for the mixed stands developed by kaya (2016) only for the mixed stands of black pine and scots pine in devrek region of zonguldak but there is little or no documentation on taper model with respect to university of ibadan. in this recent study, parameters have been estimated for the adopted models developed in other countries. methodology study area data used in this study were collected from two (2) study area which are within the university of ibadan campus. university of ibadan is located along oyo road, ibadan in akinyele local government area of oyo state, nigeria. it lies between latitude 7o26’35’’n to 7o27’33’’n and longitude 3o53’57’’e to 3o54’06’’e. the campus is characterized by dry and rainy season. the relative humidity is very high during the rainy season and low during dry season (akinyele, 2010). the two locations are heritage park and the university teak plantation. university teak plantation university of ibadan (ui) tectona grandis plantation lies between latitudes 7°456′ n to 7°45.834′ n and longitudes 3°90.942′ e to 3°90.508′ e, within the tropical rainforest in https://doi.org/10.54536/ajlsi.v1i2.777 https://journals.e-palli.com/home/index.php/ajlsi mailto:adeyemiadeola90@gmail.com pa ge 15 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 14-20, 2022 the south-western part of nigeria with mean altitude of 227m above sea level and total land area of approximately 8 ha (ezenwenyi et al., 2018). the soils in the university of ibadan teak plantation is ferric luvisol but mostly derived from sandstones (falade, 2017). the average texture in the top 15 cm was 58.8 % sand, 18.4 % silt and 22.8 % clay and thus, the soil textural class is loamy sand (falade and oyeleye, 2011). located beside the university of ibadan international conference centre (uiicc) and extends through to the distance learning centre along ajibode road ibadan in akinyele local government area of oyo state. the plantation is managed by department of forestry resource management. (now; social and environmental forestry & forest products and production). it was established mainly for education and research purpose. heritage park this park was situated in the campus opposite queen elizabeth hall after the university’s main gate (first gate). the park was comprised of terminalia radii (tent tree). it serves as a recreation, relaxation and reading center for students and staffs, beautification purpose and habitat for birds the university. the plantation is a relative flat surface with loamy/clayey soil and a gradual slope surface. there are little outcrops of rocks scattered within the plantation. the park is approximately 1.34ha in size. figure 1: map showing study locations ( heritage park and teak plantation) in university of ibadan sampling procedure sample techniques systematics technique was used to select 2(two) trancept alternately in the 8ha ui teak plantation with distance of 70meters from one another, 5 plots were laid from each making 10plots in total with mechanical spacing of 10meters. all living trees within each plot were enumerated and measured which makes 131trees in total. for heritage park, all the tree were also enumerated and measured (total enumeration) with the total number of 146trees. ui teak plantation and heritage park mainly comprised of exotic trees which are tectonia grandis (teak) and terminalia radii (tent tree) respectively. tree taper models the tree taper (d) usually expressed as a function of diameter at breast height (d), total height (h) and upper(top) stem/bole height (h). the most common expression or illustration for functional form of a taper equations is: d = f (d, h, h). taper equations can be represented in many forms: example in a; single simple quadratic form or complex form describing sections of trees. according to james and kozak (1984), the standing tree taper equations for several species produce more reliable estimates than the inside bark equations. in respect to this observation, the diameter over bark was used in this study. kozak et al. (1969) asserted that the real advantage of complex taper models were little, therefore single taper functions were adopted for the study adopted tree taper models kozak et al. (1969) model: this model was developed mainly on the basic relationship of a single parabolic function. it was conditioned by (h/h-1) and (h2/h2 -1) as predictor variables, when h equals to h, estimated diameter gives exactly zero. sharma and oderwald (2001) model: this function implies a certain condition; when h = h, d= 0, and when h = bh (bh is breast height equal to 1.3 m), d = d https://journals.e-palli.com/home/index.php/ajlsi pa ge 16 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 14-20, 2022 better the model. statistical test (indices): where yi and hi is observation, yi and hi is prediction, n is number of observations, p is the numbers of estimated parameters, x2 is critical value obtained at α =0.05, h is average height, ϒ is standard normal deviate, rss is sum of square regression. graphs of residuals were plotted against predicted values and independent variables, and these were examined for evidence of bias. in addition, frequency distributions of residuals were examined whether data deviate from normality or not. ranking of models the traditional standard or ordinal ranks for model performance was used. it shows the order of the models adopted. the models were compared using four fitting statistics, the method of relative ranking was not used because the same model superior others in all the indices and the exact position of each model was easy to allocate. results and discussion results the descriptive statistic summary of the raw data is presented in table 1. it shows the mean(average), standard error of mean, standard deviation, minimum and maximum statistical value for the terminalia radii and tectonia grandis. ormerod (1973) model: sharma and oderwald (2001) model condition also applies to this ormerod. another condition is that if β =1, the resulting tree profile is conic and when β is one-half the resulting tree form or shape is parabolic (reed and byrne 1985) and when β > 1 but less than one-half i.e., three-fourths (3/4), the tree shape is between a cone and a parabola which usually called “paracone”. polynomial series model: this model represents the general and common form of polynomial. figueiredfilho et al. (1996) noted that high degree polynomials have been used in some studies but the most common ones are around fifth degree polynomial byrne and reed (1986): this is the transformation form of ormerod (1973) model where: d=diameter at breast height, h= total height, d= diameter over bark at height h (m), bh = breast height equal to 1.3 m h= height of ith point from ground (m), β1βn= coefficient evaluation for the judgement of model performance all the fits and tests were carried out on r program. the 4 (four) goodness of fit were used to evaluate the adequacy of the models. fit statistics of residual analyses were also employed alongside with the graphical methods of residual plotting. goodness of fit statistics used were among the most common ones. they included: rmse (root mean square error), ecrit (critical error), aic (akaike information criterion) and bic (bayesian information criterion). the smaller the statistics are, the table 1: descriptive statistics for the individual tree variables for the two (2) study sites study site species variable mean std error std d min max hp terminalia radii d (cm) 15.602 0.456 5.506 3.930 12.630 dbh (cm) 32.524 0.642 7.761 10.600 58.400 h(m) 6.949 0.171 2.069 2.000 13.000 h(m) 15.404 0.251 3.029 6.000 21.000 d/d 0.478 0.010 0.126 0.250 0.750 h/h 0.458 0.011 0.133 0.147 0.880 g(m2) 0.088 0.003 0.039 0.009 0.268 g/ha (m2/ha) 0.066 0.002 0.030 0.007 0.201 v(m3/) 1.981 1.101 13.306 0.089 161.565 v/ha(m3/ha) 1.486 0.826 9.980 0.067 121.174 tp tectonia grandis d(cm) 54.855 35.210 4.570 6.750 33.430 dbh(cm) 38.835 0.797 9.125 13.500 65.800 h (m) 17.265 0.369 4.225 3.250 28.000 h(m) 30.395 0.592 6.777 12.500 50.000 d/d 0.463 0.007 0.082 0.285 0.714 h/h 0.570 0.008 0.088 0.260 0.880 https://journals.e-palli.com/home/index.php/ajlsi pa ge 17 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 14-20, 2022 g(m2) 0.125 0.005 0.059 0.014 0.340 g/ha (m2/ha) 0.312 0.013 0.148 0.036 0.850 v (m3/) 1.587 0.079 0.899 0.094 4.836 v/ha (m3/ha) 3.967 0.196 2.247 0.230 12.090 where dbh=diameter at breast height, tht= total height, d= diameter over bark at height h (m); h= height of ith point from ground (m), hp=heritage park, tp= teak plantation,g= basal area, v= volume table 2: tree taper model estimation for two species study site species model parameters estimate standard error p-value hp terminalia radii 1 β1 -0.284 0.2168 0.192 β2 -0.104 0.154 0.5 2 β1 2.606 0.0313 <2e-16 3 β1 1.396 0.060 <2e-16 4 β0 -2.123 2.074 0.308 β1 11.353 26.485 0.669 β2 -52.409 124.703 0.675 β3 121.806 274.648 0.658 β4 -140.843 285.453 0.623 β5 63.068 112.744 0.577 5 β1 0.557 0.025 <2e-16 β2 0.275 0.077 0.000497 tp tectonia grandis 1 β1 0.529 0.280 0.060779 β2 -0.643 0.180 0.000511 2 β1 2.305 0.014 <2e-16 3 β1 0.965 0.025 <2e-16 4 β0 -103.13 21.84 6.16e-06 β1 988.28 210.33 6.79e-06 β2 -3719.94 789.66 6.48e-06 β3 6808.25 1445.35 6.47e-06 β4 -6057.94 1289.99 6.86e-06 β5 2095.87 449.04 7.73e-06 5 β1 0.525 0.037 <2e-16 β2 0.179 0.085 0.0361 where h p= heritage park, tp= teak plantation, β (1, 2, 3….) is coefficient residual analysis graphical representation of residuals was plotted for each tree taper model and they are presented in figure 2 and 3. visualizations of the residuals plotted against the predicted showed that only model 4 of both study sites produced more homogeneous residual variance than other models. it was obviously that precisions in residual analysis predictions for both study sites were not similar. the ranges of errors in the predictions of all models were not distributed uniformly, some distributed between +100 to -100 while some -10 to +10. also, some models were more biased than others. the model 5 of heritage park is less biased in the distribution while model 5 of teak plantation is moderately biased and the alternative models from the both study sites. there were more differences in the trend residual distributions for the five models for two study sites. also, the models are dependently distributed except the model 5 (byrne and reed 1986) of both study site that are independently distributed. further evaluation of the models used in the study were shown in the results of fit statistics. the statistical analysis for the normality distribution of residual used to indicate whether the residual of each model violated the assumption of normality or not. the shapiro-wilk test from table 3 reveal that model 3 and 1 statistical values were greater than p-level for terminalia radii and only model 3 statistic greater than p-value for tectonia grandis. and the yardstick for shapiro -wilk is that statistic value greater than the level of hypothesis (0.05) indicated the acceptance of null hypothesis and this implies that the data are normally distributed while the https://journals.e-palli.com/home/index.php/ajlsi pa ge 18 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 14-20, 2022 smaller statistical implies that the normality assumption are violated. model 5 (byrne and reed 1986) of the studies gave the lesser value and this inferred that their residual was not normally distributed but the model 3(ormerod,1973) of the both selected study sites were normally distributed. figure 2: graphical representation of residual analysis for the heritage park (terminalia radii) figure 3: graphical representation of residual analysis for the teak plantation (tectonia grandis) evaluation statistics table 3 showed goodness of fit used to examine the five tree taper models adopted for the two study sites. those statistics were rmse (root mean square error,) ecrit (critical error), aic (akaike information criterion) and bic (bayesian information criterion). the smaller the statistics are, the better the model. the assessment showed that model 5 gave the superior performance while model 2 (sharma and oderwald, 2001) gave worst performance which inferred from the fact that byrne and reed 1986 model (model 5) gave lowest value of aic, rmse, ecrit and bic statistics (goodness of fit) moreover, ormerod,1973 model (model 3) follows byrne and reed 1986 (model 5) in terms of statistics evaluation, standard error and significant level (0.05). https://journals.e-palli.com/home/index.php/ajlsi pa ge 19 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 14-20, 2022 table 3: the evaluation statistics for the two species study site species model rmse aic bic ecrit shapiro hp terminalia radii 1 139.9 1860.966 1869.917 100.338 3.435^05 2 164.1 1906.754 1912.721 118.179 0.0014 3 5.659 923.418 929.385 2.000 13.000 4 9.223 1070.926 1096.81 6.524 0.107 5 3.917 816.97 825.92 2.810 0.012 tp tectonia grandis 1 132.1 1655.285 1663.91 89.720 8.759^-05 2 143.9 1676.743 1682.493 98.123 0.008 3 4.233 752.803 758.554 2.886 0.799 4 8.305 934.229 954.355 5.551 0.015 5 3.281 687.005 695.631 2.228 3.55^-04 discussion five taper models were adopted for study. most models are bias only model 5 are not biased. the remain models (taper functions) used in this study performed poorly. the screening test were carried out on all models examining residual plots because they were obvious biased. only one model was relatively unbiased (model 5), and it was chosen as the best. the model 2 point is only estimated parameter that is very sensitive it was the least among others function. according to sharma and oderwald, 2001; it takes a purely parabolic form to depict a tree shape if it is greater than 2, the function predicts the diameter at the butt much larger than diameter at breast height ‘d’; and if it is smaller than 2, the function predicts the diameter at the butt smaller than diameter at breast height ‘d’. the both study site model 2 estimated values were greater than 2. so, this finding is accordance with sharma and oderwald, 2001. model 3 by ormerod (1973) was biased. it may be the outcome of estimated diameters (overestimate) of all stem sizes. the result of study by reed and byrne (1985), in which the same taper function was applied to jack pine, contradicts the findings. in reed and byrne (1985) finding, it was observed that the values of estimated parameter (β1) less than 0.5 while this study estimated it as 1.396 for terminalia radii (heritage park) and 0.965 for teak plantation. according to the reed and byrne (1985) report when β1 estimated value less than 0.5 this implies tree stems are cylindrical and when β =1, the resulting tree profile is conic and when β is one-half the resulting tree form or shape is parabolic and when β > 1 but less than onehalf; three-fourths (3/4), the tree shape is between a cone and a parabola which usually called “paracone”. with this observation, the two species could be categories to different form, this finding inferred the terminalia radii and tectonia grandis in the two study sites are “paraconic” and “conic” respectively. the model 5 (byrne and reed,1986) was best model due to the superior performance towards the goodness of fit and residual analysis. this may be the result of transformation process occur from model 3 (ormerod 1973) to model 5. since both models’ findings closely with one another for the two species, two models can work interchangeably for predicting of the tree diameters. conclusions all taper functions adopted in this study are simple to use (single function) and provide a more accurate diameter prediction at a specified height. the results of the statistical analyses indicated that model 5 (byrne and reed, 1986) gave the overall best performance in predicting tree diameter at a specified height. moreso, the study concluded that both tectonia grandis in teak plantation and terminalia radii in heritage park are not cylindrical in shape(form). so, the two species were not susceptible (prone) to windbreak, there should be no wind damage record for the two species in the two study sites. ormerod (1973) model could be used for the estimation of tree form (shape) for the two species. the observed result for this study is significant for prediction of stem diameter at any point (stem taper) for t. radii stand as well as t. grandis stand depending on various ecological factors, stand size and silvicultural management of the certain forest. references akinyele, a. o. 2010. effects of growth hormones, rooting media and leaf size on juvenile stem cuttings of buchholziacoriacea engler. annual forest research, 53(2), 127133. bruce, d. r., curtis, o., & vancoevering, c. 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(2018). univariate and bivariate diffusion models computational aspects and applications to forestry. in stochastic differential equations, basics and applications, deangelo, t.g.,ed. nova science publisher’s, new york, ny, usa, 1–77. sharma, m. & oderwald. r.g. (2001). dimensionally compatible volume and taper equations. canadian journal of forest research, 31, 797-803. sloboda, b. & saborowski, j. (1981). stand taper form as a stochastic process with an example of application. in proceedings of the xvii iufro-world congress, kyoto, japan, 137–150. wang, y., titus, s. j. & lemay, v. m. (1998). relationships between tree slenderness coefficients and tree of stand characteristics for major species in boreal mixed wood forests. canadian journal of forest research, 28(8), 1171-1183. https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 1 american journal of life science and innovation (ajlsi) difference of clinical parameters and correlation between type-1 and type-2 diabetes mellitus (dm) subjects in bangladesh khaleda ferdous1, md. ashiqur rahman2*, sadia islam3, sadia tasnim1, saraf tasnim1, shohanur rahaman4 md. raihanul islam2, md. biplob hossain4 volume 2 issue 3, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i3.1928 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: august 09, 2023 accepted: august 31, 2023 published: september 15, 2023 type i and type 2 diabetes are the two main types with types diabetes accounting for the majority (>85%) of total diabetes prevalence. both forms of diabetes can lead to multisystem complications of microvascular endpoints including retinopathy, nephropathy and neuropathy and microvascular endpoints including is cadmic heart disease, stroke and peripheral vascular disease. the premature morbidity, mortality, reduced life expectancy and financial and other others costs of diabetes make if an important public health condition. the aim of this study was to observe the clinical condition and consequences patients with diabetes mellitus in bangladesh. a descriptive cross-sectional study was conducted for the study. data are collected from bihs general hospital. the results showed that, among 100 patients 20% patient was affected by type-1 diabetes mellitus (dm) and 80% patient was affected by type-2 diabetes mellitus (dm). in comparison between type-1 and type-2 dm patient’s result showing that fasting plasma glucose (fpg) and 2 hour abf plasma glucose range is slightly high in type-1 dm than type-2 dm. on the other hand, creatinine, hba1c, tsh, total cholesterol (tc), triglycerides (tg), hdl ranges are increased in type-2 dm patient. in this study males are affected 62% and females are 32%. so the ratio between male and female is 2:3. more patients were found in the age between 50-60 years and has risk among 40-50 years. eating the right foods and adopting other life style behaviors that promotes healthy blood sugar and insulin levels will give you the best chance at avoiding diabetes. keywords type-1 dm, type-2 dm, fpg, hba1c, lipid profile introduction diabetes mellitus is a chronic disorder of glucose metabolism with serious clinical consequences. the multi system consequences of diabetes includes microvascular (retinopathy, nephropathy, neuropathy) and macrovascular (ischemic heart disease, stroke, peripheral vascular disease) end points. the prevalence of diabetes has been rising in the last few decades. diabetes is now a global problem. the premature morbidity, morality, reduced, life expectancy and financial and other costs to the patients with diabetes their careers and health service make it important public health condition. diabetes affects persons of all ages and races (afroz et al. 2019). the classification and diagnosis of diabetes are complex and have been the subjects of much consultation, debate and revision stretching over the past decades. the etiological classification of debates has now been widely accepted type 1 and type 2 diabetes are the 2 main types of diabetes and type 2 diabetes accounting for the majority (>85%) of total diabetes prevalence (sato et al. 2009). expert committees from the world health organization (who) and american diabetes association (ada) have formulated, converged and diverged in their position on the diagnostic criteria for diabetes based on the measurement of fasting and 2h post load glucose but most recently there has been an ongoing debate on whether glycated hemoglobin (hba1c) should be used for diagnosing diabetes (who, 1999). diabetes mellitus (dm) is becoming a pandemic worldwide. who (world health organization) listed 10 countries to have the highest numbers of people with diabetes in 2000 and 2030. bangladesh appears in the list for both 2000 and 2030 with india, pakistan, china, japan, usa etc. according to a report, bangladesh has 5.4 million of diabetic subjects and the number is expected to increase to a staggering 11.1 million by 2030 (who, 1999). several small-scale population-based studies conducted in bangladesh at different time points have revealed an increasing trend of diabetes prevalence in rural and urban communities. a recent population bared study showed a significant increase in the prevalence of dm in rural bangladesh from 2:3% to 6:8% over 5 years. this prevalence was higher than found in the previous rural studies of bangladesh (afroz et al. 2019). diabetes mellitus is referred to a group of diseases characterized by high glucose levels in blood. it is caused by deficiency in the production or loss of function of insulin, which can occur because of different reasons resulting in protein and lipid metabolic disorders. the aim of this study is to systematically review diabetes mellitus and determine the variation of different parameters of the patients with type 1 and type 2 diabetes. the result will help to check the complications, injuries and other conditions in the patient. 1 bangladesh university of health sciences (buhs), bangladesh 2 novus clinical research services limited (ncrsl), bangladesh 3 bangladesh specialized hospital limited (bshl), bangladesh 4 diabetic association of bangladesh (badas) covid-19 diagnostic laboratory, bangladesh * corresponding author’s e-mail: ararashiqur@gmail.com pa ge 2 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 1-7, 2023 epidemiology of type-1 and type-2 diabetes type-1 diabetes accounts for about 5% to 10% of all patients with diabetes. it is the most commonly diagnosed diabetes of youth (under 20 years of age) and causes ≥85% of all diabetes causes in this age group worldwide. it is estimated that 1106500 people aged 0-19 years have type-1 diabetes worldwide with 132600 newly diagnosed cases each year. in usa, more than 17900 people aged under 20 years were newly diagnosed with type-1 diabetes annually (annual rate for new cases about 21 in per 100000) (who, 1985). in a study of adults diagnosed with diabetes in the us in 2016 and 2017, type-1 diabetes accounted for 5.6 of cases. there is a significant geographic variation in the incidence of type-1 diabetes. it is more common in europeans and less common in asians. thus, a child in finland is 40 times more likely to develop type-1 diabetes than a child in japan and almost 100 times more likely to get the disease a child in the zunyi region of china. worldwide the incidence of type-1 diabetes is increasing by 3% every year although the reasons for this are unclear. one report showed a more rapid increase in nonwhite racial and ethnic group. type-1 diabetes can present at any age with the highest incidence observed in children aged 1014 years (sato et al. 2009). it affects males and females equally. in 30th march 2020, the result of meta-analysis showed that the incidence of type-1 diabetes was 15 per 10000 people and the prevalence was 9.5% (95% cl: 0.7 to 0.12) in the world, which was statistically significant. as a result, insulin will be difficult to access and afford, specially, in underdeveloped and developing countries (magliano et al. 2015). type-2 diabetes mellitus is one of the most common form of chronic disease globally. the rising burden of type-2 diabetes is now a major concern in health care worldwide. the global prevalence of diabetes mellitus is rapidly increasing as a result of population ageing, urbanization and associated lifestyle changes. the number of people with type-2 diabetes has more than doubled over the past 3 decades. in 2010, a research estimated 285 million people worldwide had type-2 diabetes mellitus and is projected to rise to 439 million by 2030 (tharkar et al. 2010). the major burden of type-2 diabetes mellitus is now taking place in developing countries rather than in developed countries. 80% cases of type-2 diabetes mellitus worldwide live in less developed countries and areas. asia has emerged as the “diabetes epidemic” in the worldwide as a result of rapid economic development, urbanization and nutrition transition over a relatively short period of time. among the 10 countries with the largest numbers of people predicted to have diabetes mellitus in 2030. amongst that 5 are in asia. they are china, pakistan, indonesia and bangladesh. in addition to asia, the gulf region in the middle east and africa are other hot spots for type-2 diabetes mellitus. a higher prevalence of type-2 diabetes mellitus in immigrants from the middle east living in sweden than in native sweden has also been reported. the epidemic of type-2 diabetes mellitus is attributable to a mixture of genetic and epidemic predispositions and variety of behavioral and environmental risk factors (kuzuya et al. 1997). pathophysiology of diabetes mellitus type-1 diabetes mellitus type-1 diabetes mellitus results from the description of pancreatic bcells that is mediated by the immune system. multiple genetic and environmental factors in variable combinations in individual patients are involved in the development of type-1 diabetes mellitus. generic risk is defined by the presence of particular allele combinations, which in the major susceptibility locus (the hla region) affect t cell recognition and tolerance to foreign and autologous molecules (alberti et al. 1998). multiple other loci also regulate and affect features of specific immune responses and modify the vulnerability of bcells to inflammatory mediators8. compared with the genetic factors, environmental factors that affect the development of type-1 diabetes mellitus are less well characterized but contact with particular micro-organism is emerging as an important factor. certain infections might affect immune regulation, and the role of commensal microorganisms such as gut microbiota, coxsackie, rubella viruses are important (toumi et al. 1993). some evidence also suggests that nutritional factors are important. multiple islet specific autoantibodies are found in the circulation from a few weeks to up to 20 years before the onset if clinical disease and this prediabetes phase provides a potential opportunity to manipulate the isletspecific immune response to prevent or postpone bcell loss. the latest developments in understanding the heterogeneity of type-1 diabetes mellitus and characterization of major disease type might help in the development of preventive treatments (vionnet et al 1992). diabetes symptoms varies depending on how much blood sugar level is devoted in patients. some people may sometimes not experience symptoms. in type-1 diabetes mellitus, symptoms tend to come on quickly and can be serve it includes: thirst, increased hunger (specially after eating), dry mouth, upset stomach and vomiting, frequent urination, unexpected weight loss (even though eating and feel hungry), heavy, labored breathing (doctor may call this kussmaul respiration), crankiness or mood changes, fatigue and weakness, blurred vision, shaking and confusion, bedwetting in children who’s been dry at night (murphy et al. 2008). long term complications of diabetes develop gradually. the longer duration, the less controlled blood sugar, the higher risk of complications. eventually diabetes complications may be disabling or life threatening. heart and blood vessel disease (cardiovascular disease), never damage (neuropathy), kidney damage (neuropathy), eye damage, foot damage, skin and mouth infections, pregnancy complications, depression (spyer et al. 2009). type-2 diabetes mellitus type-2 diabetes mellitus results from a defect in insulin pa ge 3 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 1-7, 2023 secretion and an impairment of insulin action in hepatic and peripheral tissues, especially muscle tissue and adipocytes. a post receptor defect is also present causing resistance to the stimulatory effect of insulin deficiency develops, unlike the absolute deficiency founds in patients with type-1 diabetes mellitus (stride et al. 2002). the specific etiologic factors are not known but genetic input is much stronger in type-2 diabetes mellitus than in the type-1 form. in a state of health, normal glycaemia is maintained by fine hormonal regulation of peripheral glucose uptake and hepatic production. impaired glucose tolerance (igt) is a transitional state from normal glycaemia to frank diabetes but patient with impaired glucose tolerance (igt) exhibit considerable heterogeneity. type2 diabetes or glucose intolerance is part of a dysmetabolic syndrome (syndrome x) that includes insulin resistance, hyperinsulinemia, obesity, hypertension and dyslipidemia (davies et al. 2018). current knowledge suggests that the development of glucose intolerance or diabetes is initiated by insulin resistance and worsened by the compensatory hyperinsulinemia. insulin resistance is not only predictive for type-2 diabetes and associated with myriad metabolic derangements in fasting conditions but also nondiabetic insulin resistant individuals are subjected to a similar adverse postprandial metabolic setting are cardio metabolic risk as those with type-2 diabetes mellitus (mitrofanova et al. 2019). in addition, the prevalence of hypertension rises with exacerbation of stages of impaired glucose metabolism. however, only in the early stages of impaired insulin metabolism do hyperglycemia and hyperinsulinemia appear to be significant contributors to the presence of hypertension (welsh et al. 2010) signs and symptoms of type-2 diabetes often develop slowly. in fact, anyone can be living with type-2 diabetes for years and not knowing it. it includesincreased thirst, frequent urination, increased hunger, fatigue, blurred vision, slow healing sores, frequent infections (gums/ skin/ vaginal), numbers or tingling in the hands or feet, areas of darkened skin, usually in the armpits and neck, presence of ketones in urine. and complications such as cardiovascular disease (heart and blood vessel disease), nerve damage (neuropathy) in limbs, kidney disease (neuropathy), eye damage, skin infection, foot damage, hearing impairment, slow healing, sleep apnea, dementia (alzheimer’s disease) (mitrofanova et al. 2019). methodology it was a cross-sectional study. it was a descriptive research that was mainly based on observation and data collection. the study conducted in bangladesh institute of health science (bihs) and bangladesh university of health sciences (buhs). 100 patients between the ages of 20 to 85 were approached to participate in the research. sampling technique was purposive sampling. all data were collected in 26th december, 2020 to 4th march, 2021. laboratory tests in this study, some hematological and biochemical tests results were done to determine if there are any differences in the results between type-1 and type-2 diabetic patients. normal values vary depending on age and type of diabetes. different parameters measure the update health condition of a patient. in the laboratory, different amount of blood is needed for different kind of tests. the test was evaluated using a centrifuge which caused the contents of blood to separate anticoagulant was added to keep blood from clotting. urine samples were also collected for tests. all tests were analyzed by an automated biochemistry analyzer machine for analysis sample collection a lab technologist had drawn blood from a vein, typically from the onside of patients elbow or from the back of hand. the technician cleaned the surface of the skin with an antiseptic and place an elastic or tourniquet around upper arm to help the vein swell with blood. then inserted a needle in the vein and collect blood sample in one or more vials. the technologist removed the elastic band and cover the area with a bandage to stop the bleeding. urine sample were collected in a vial. a container amount of urine was collected. statistical analysis statistical analysis was done on an ms windows-based pc computer. the data were first keyed into a ms excel spreadsheet and then analyzed by statistical package for the social sciences (spss). inclusion criteria both men and women between 20 to 85 of age who are affected with type-1 and type-2 diabetes mellitus and are willing to participate voluntary and comply with the instruction of the study e.g. overnight fasting was considered eligible for the study. exclusion criteria people who are not qualified by inclusion criteria were excluded from study. hey are given below• pregnant women. • mentally disabled people. • physically disabled people figure 1: distribution of type-1 dm and type-2 dm study subjects pa ge 4 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 1-7, 2023 results among 100 patients 20% patient was type-1 diabetic patient and 80% patient was type-2 diabetic patient. according to total sample pie chart are given belowin this study sample was taken among 20-85 years old patient. this graphical presentation represents that, type1 diabetes mellitus is higher in younger peoples and type2 diabetes mellitus is higher in older peoples. among 100 patients 20% patient was type-1 diabetic patient and 80% patient was type-2 diabetic patient. among those, male patient is 62% and female patient is 38%. this figure shows that male is more affected than female. in this study, males are affected 62% and females are 38%. so, the ratio between male and female is 3:2. figure shows that age group distribution of type-1 and type2 diabetes mellitus. among 20-30 age group type-1 was 8 subjects and type-2 was 0 subjects. accordingly, 30-40 age group type-1 was 10 subjects and type-2 was 7 subjects; figure 2: percentage of type-1 and type-2 dm male and female subjects figure 3: age group distribution chart of type 1 and type 2 dm study subjects table 1: mean value of different parameters among type-1 and type-2 diabetic subjects variable type-1 dm (n=20) type-2 dm (n=80) fasting plasma glucose 9.8 mmol/l 8.8 mmol/l 2 hour abf plasma glucose 11.2 mmol/l 10.2 mmol/l creatinine 1.7 mg/dl 2.3 mg/dl hba1c 6.7% 9.8% tsh 2.75 miu/l 5.28 miu/l total cholesterol (tc) 210 mg/dl 240 mg/dl triglycerides (tg) 132 mg/dl 169 mg/dl hdl 42 mg/dl 65 mg/dl ldl 155 mg/dl 190 mg/dl pa ge 5 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 1-7, 2023 figure 4: comparison of different glucose parameters between type-1 and type-2 diabetic subjects figure 5: comparison of creatinine & tsh parameters between type-1 and type-2 diabetic subjects figure 6: comparison of lipid profile between type-1 and type-2 diabetic subjects 40-50 age group type-1 was 2 subjects and type-2 was 18 subjects; 50-60 age group type-2 was 30 subjects; 60-70 age group type-2 was 13 subjects; 70-80 age group type-2 was 7 subjects and 80-90 age group type-2 was 5 subjects. among 100 patients, 20% patients were type-1 and 80% patients were type-2 diabetic patients. in comparison between type-1 and type-2 diabetic patient showing that, fasting plasma glucose is little bit higher in type-1 pa ge 6 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 1-7, 2023 diabetic patient than type-2 diabetic patient. creatinine range is lower in type-1 diabetic patient than in type-2 diabetic patient. hba1c level is un type-2 diabetic patient than in type-1 diabetic patient. tsh level is significantly high in type-2 diabetic patient than in type-1 diabetic patient. lipid profile is (tc, tg, hdl and ldl) high in type-2 diabetic patient than in type-1 diabetic patients. discussion type-1 diabetes was previously called insulin dependent diabetes or juvenile onset diabetes. this is immune mediated diabetes. type-1 diabetes mellitus results from an absolute deficiency of insulin due to autoimmune destruction of the insulin producing pancreatic beta cell (khowaja et al. 2007). this type of diabetes can affect generally in early stage of life and insulin is required for survival. it has multiple genetic predispositions and has also been said to be related to environmental factors, though still poorly defined. this type of diabetes accounts for 5-10% of those with diabetes (wang et al 2009). type2 diabetes mellitus is the commonest form of diabetes. it is also called non-insulin dependent diabetes mellitus or adult-onset diabetes. this type of diabetes is characterized by insulin resistance or abnormal insulin secretion (krop et al. 1998). type-2 diabetes mellitus is a disorder entirely separate from type-1 diabetes mellitus. relative beta cell insufficiency is by definition, present in all individuals with type-2 diabetes mellitus (chaikledkaew et al. 2008). in most cases, this disorder is also characterized by insulin resistance detected at the level of skeletal muscle, adipose tissue and the liver. insulin resistance at the former site results in decreased peripheral glucose disposal, while the latter in increased hepatic glucose production. in many individuals, the natural history of type-2 diabetes mellitus begins with a period of insulin resistance with preserved, indeed augmented pancreatic insulin secretion as the insensitivity to insulin action in peripheral tissues is overcome by hyper-insulinemia (vanderlee et al. 2016). as a result, plasma glucose level remains relatively normal. as the disease progress, however pancreatic islet cell function falters and it is no longer able to meet the peripheral demand. as a result, insulin levels fail to keep up with requirement and hyperglycemia ensues. type-2 diabetes mellitus accounts for approximately 90-95% of those with diabetes (welsh et al. 2010). one study suggests that hba1c has a relations with serum lipid profile, blood glucose level, serum creatinine and sgpt in diabetic patients (roy et al. 2016). a significant correlation among hba1c, fbg and ppg is in agreement with earlier reports. we also observed significant correlations between hba1c and cholesterol, triglycerides, hdl and ldl in type 2 diabetic (rosediani et al. 2006). several investigators have reported significant correlations between hba1c and lipid profiles and suggested the importance of glycemic control. it has also been reported that clinical significance of various lipid parameters including total cholesterol, triglycerides hdl and ldl in predisposing diabetic patients leads to cardiovascular complications (islam et al. 2022). the study was done to estimate the different clinical parameter states in 100 diabetes mellitus (dm) patients in which 20 patients were type-1 and 80 patients were type-2 dm affected. among them were 62% male and 38% female patients. observation made in several categories. according to age distribution chart, it was found that younger people are more affected by type-1 dm and on the other side, older people are more affected by type-2 dm. fasting plasma glucose (fpg) and 2 hour abf plasma glucose level are higher in type-1 dm subjects than type-2 dm subjects. creatinine range is lower in type-1 dm than type-2 dm patient. similarly, hba1c, tsh level and lipid profile is increased in type-2 dm patient than type-1 dm subjects. conclusion in recent years, diabetes mellitus (dm) has appeared to be a global health problem. it is one of the leading causes of death, disability, and economic loss. diabetes affects persons of all ages and races. this disease reduces both a person’s quality of life and life expectancy and imposes a large economic burden on the healthcare system and on families. references afroz, a., alam, k., ali, l., karim, a., alramadan, m. j., habib, s. h., ... & billah, b. 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(2018). management of hyperglycemia in type 2 diabetes, 2018. a consensus report by the american diabetes association (ada) and the european association for the study of diabetes (easd). diabetes care, 41(12), 2669-2701 islam, s., hossen, m. a. a., rahman, m. a., lubaba, m. i., & akram, a. (2022). serum uric acid level among type-2 diabetes subjects attending in a tertiary hospital of bangladesh. world journal of biology pharmacy and health sciences, 12(1), 081-085. khowaja, l. a., khuwaja, a. k., & cosgrove, p. (2007). cost of diabetes care in out-patient clinics of karachi, pakistan. bmc health services research, 7(1), 1-8 krop, j. s., powe, n. r., weller, w. e., shaffer, t. j., saudek, c. d., & anderson, g. f. 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(2009). pregnancy outcome in patients with raised blood glucose due to a heterozygous glucokinase gene mutation. diabetic medicine, 26(1), 14-18 stride, a., vaxillaire, m., tuomi, t., barbetti, f., njølstad, p. r., hansen, t., ... & hattersley, a. t. (2002). the genetic abnormality in the beta cell determines the response to an oral glucose load. diabetologia, 45, 427435. tharkar, s., devarajan, a., kumpatla, s., & viswanathan, v. (2010). the socioeconomics of diabetes from a developing country: a population based cost of illness study. diabetes research and clinical practice, 89(3), 334-340 tuomi, t., groop, l. c., zimmet, p. z., rowley, m. j., knowles, w., & mackay, i. r. (1993). antibodies to glutamic acid decarboxylase reveal latent autoimmune diabetes mellitus in adults with a non—insulindependent onset of disease. diabetes, 42(2), 359-362 vanderlee, l., ahmed, s., ferdous, f., farzana, f. d., das, s. k., ahmed, t., ... & faruque, a. s. g. (2016). self-care practices and barriers to compliance among patients with diabetes in a community in rural bangladesh. international journal of diabetes in developing countries, 36, 320-326 vionnet, n., stoffel, m., takeda, j., yasuda, k., bell, g. i., zouali, h., ... & cohen, d. (1992). nonsense mutation in the glucokinase gene causes early-onset non-insulin-dependent diabetes mellitus. nature, 356(6371), 721-722 wang, w., mcgreevey, w. p., fu, c., zhan, s., luan, r., chen, w., & xu, b. (2009). type 2 diabetes mellitus in china: a preventable economic burden. the american journal of managed care, 15(9), 593-601 welsh, g. i., hale, l. j., eremina, v., jeansson, m., maezawa, y., lennon, r., ... & coward, r. j. (2010). insulin signaling to the glomerular podocyte is critical for normal kidney function. cell metabolism, 12(4), 329-340 world health organization. (1985). diabetes mellitus: report of a who study group [meeting held in geneva from 11 to 16 february 1985]. world health organization, (1999). definition, diagnosis and classification of diabetes mellitus and its complications. part 1: diagnosis and classification of diabetes mellitus. geneva: who, 1999. report no. who/ncd/ncs/99.2. pa ge 1 pa ge 1 american journal of life science and innovation (ajlsi) characterisation studies on lipases of brevibacterium, bacillus and pseudomonas spp produced under sub-merged fermentation of different carbon sources omolade, o.a1, orji, f.a.2*, agu, g.c1, adebajo, l.o1 volume 1 issue 2, year 2022 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v1i2.303 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: june 01, 2022 accepted: august 13, 2022 published: august 16, 2022 lipases are a class of hydrolytic enzymes that catalyze the hydrolysis of insoluble triacylglycerol to glycerol, acylglycerols, and free fatty acids. the present study is aimed at identifying lipase producing bacteria isolated from soil determining the optimal conditions (temperatures, ph, and metal ion concentrations) of lipases produced through submerged fermentation by bacteria of different three (3) genera. the different bacterial isolates with good hyper-producing potentials for lipases were identified by polymerase chain reactions (pcr). the effect of temperature on cellulase activity was determined by estimating the lipase activity at ph 9.0 within a temperature range of (300c-600c). in order to determine the behaviour of the enzymes within some metallic ions, the reaction of the enzyme and gum arabica/ olive oil mixture was allowed to proceed at 500c with duplicate test tubes containing 50mm cacl2 (ca2+). mgso4 (mg 2+), nacl(na+), kcl (k+). the identifies of the lipase producing bacteria were identified as brevibacterium brevis strains hk 544, pseudomonas aeruginosa strain wes, bacillus megaterium strain wh13, and bacillus subtilis strain bs 01 for isolate h, a, b and f., respectively. optimum temperatures for the activities of brevibacterium brevis strains hk 544, pseudomonas aeruginosa strain wes, bacillus megaterium strain wh13, and bacillus subtilis strain were determined to be 50oc, 50oc , 45oc and 45oc, respectively. optimum ph for the activities of brevibacterium brevis strains hk 544, pseudomonas aeruginosa strain wes, bacillus megaterium strain wh13, and bacillus subtilis strain were determined to be alkaline (8.0 to 9.0). the observation that sodium and potassium ions at 50mm concentration enhanced the activity of some of the lipases under this investigation showed that sodium and potassium are likely to be co-factors for the performance of these lipases. keywords brevibacterium, bacillus, pseudomonas, polymerase chain reactions 1 department of microbiology, faculty of science, olabisi onabanjo university, ago-iwoye, ogun state, nigeria. 2 department of biotechnology, federal institute of industrial research, oshodi, pmb 20123, ikeja, lagos-nigeria. * corresponding author’s e-mail: orjifa@yahoo.com introduction lipases are ubiquitously produced by the plants (belguith et al. 2009); animals (carriere et al. 1994); and microorganisms (ramesh et al., 2013). microbial lipases are the preferred potent source due to several industrial potentials (hasan et al., 2006). lipases are becoming more and more popular in the field of biotechnology, as they have received great interest in industrial applications because of their properties (almeida et al., 2019) the uniqueness and its ability to benefit from a wide range of reaction materials and its high stability towards temperature and acidity function and organic solvents, as most industrial processes are carried out at a high temperature and therefore enzymes with high stability (sahu & martin, 2011). lipases are considered to be the third biggest enzymes group following proteases and amylases, based on total sales volume. because of its extensive range of applications lipase production is a billion dollar (jaeger et al., 1998). lipases (glycerol ester hydrolases e.c. 3.1.1.3) are much-demanded enzymes with significant commercial applications in industries. lipases stimulate the hydrolysis of triacylglycerol to glycerol and free fatty acids. a real lipase will cleave emulsified esters of glycerin and lengthy chain fatty acids such as triolen and tripalmitin (gayathri et al., 2013). many applications of lipases include specialty organic syntheses, hydrolysis of fats and oils, modification of fats, flavor enhancement in food processing, resolution of racemic mixtures, and chemical analyses (afaf et al., 2020). microbial lipases detoxify and degrade the oil effluents as one by innovative technologies (shart & elkhalil, 2020). the present study is aimed at identifying lipase producing bacteria isolated from soil, determining the optimal conditions (temperatures, ph, and metal ion concentrations) of lipases produced through submerged fermentation by bacteria of different three (3) genera. materials and methods molecular identification of isolates dna extraction dna was extracted using the protocol stated by fuguri et al., (2015). briefly, single colonies grown on medium were transferred to 1.5 ml of liquid medium and cultures were grown on a shaker for 48 h at 28 ºc. after this period, cultures were centrifuged at 4600g for 5 min. the resulting pellets were re-suspended in 520 μl of te buffer (10) mmtris-hcl, 1mm edta, ph 8.0). fifteen microliters of 20% sds and 3 μl of proteinase k (20 mg/ml) were then added. the mixture was incubated for 1 hour at 37 ºc, then 100 μl of 5 m nacl and 80 μl of a 10% ctab solution in 0.7 m nacl were added and vortexed. the suspension was incubated for 10 min at 65 ºc and kept on ice for 15 min. an equal volume of chloroform: isoamyl alcohol (24:1) was added, followed by incubation on ice for 5 min and centrifugation at 7200g for 20 min. the aqueous phase was then transferred to a new tube and https://doi.org/10.54536/ajlsi.v1i2.303 https://journals.e-palli.com/home/index.php/ajlsi mailto:orjifa%40yahoo.com?subject= pa ge 2 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 1-8, 2022 isopropanol (1: 0.6) was added and dna precipitated at –20 ºc for 16 h. dna was collected by centrifugation at 13000g for 10 min, washed with 500μl of 70% ethanol, air-dried at room temperature for approximately three hours and finally dissolved in 50μl of te buffer. polymerase chain reaction pcr sequencing preparation cocktail consisted of 10 µl of 5x gotaq colourless reaction, 3 µl of 25mm mgcl2, 1 µl of 10 mm of dntps mix, 1 µl of 10 pmol each 27f 5’aga gtt tga tcm tgg ctc ag-3’ and 1525r, 5′-aaggaggtgatccagcc-3′ primers and 0.3units of taq dna polymerase (promega, usa) made up to 42 µl with sterile distilled water 8μl dna template. pcr was carried out in a geneamp 9700 pcr system thermalcycler (applied biosystem inc., usa) with a pcr profile consisting of an initial denaturation at 94°c for 5 min; followed by a 30 cycles consisting of 94°c for 30 s, 50°c for 60s and 72°c for 1 minute 30 seconds; and a final termination at 72°c for 10 mins. and chill at 4oc. gel (hassan et al., 2016; gunajit et al., 2017). integrity of dna the integrity of the amplified about 1.5mb gene fragment was checked on a 1% agarose gel ran to confirm amplification. the buffer (1xtae buffer) was prepared and subsequently used to prepare 1.5% agarose gel. the suspension was boiled in a microwave for 5 minutes. the molten agarose was allowed to cool to 60°c and stained with 3µl of 0.5 g/ml ethidium bromide (which absorbs invisible uv light and transmits the energy as visible orange light). a comb was inserted into the slots of the casting tray and the molten agarose was poured into the tray. the gel was allowed to solidify for 20 minutes to form the wells. the 1xtae buffer was poured into the gel tank to barely submerge the gel. two microliter (2 l) of 10x blue gel loading dye (which gives colour and density to the samples to make it easy to load into the wells and monitor the progress of the gel) was added to 4µl of each pcr product and loaded into the wells after the 100bp dna ladder was loaded into well 1. the gel was electrophoresed at 120v for 45 minutes visualized by ultraviolet trans-illumination and photographed. the sizes of the pcr products were estimated by comparison with the mobility of a 100bp molecular weight ladder that was ran alongside experimental samples in the gel. purification of amplified product after gel integrity, the amplified fragments were ethanol purified in order to remove the pcr reagents. briefly, 7.6 µl of na acetate 3m and 240 µl of 95% ethanol were added to each about 40µl pcr amplified product in a new sterile 1.5 µl tube eppendorf, thoroughly by vortexing and keep at -20°c for at least 30 min. centrifugation for mix 10 min at 13000 g and 4°c followed by removal of supernatant (invert tube on trash once) after which the pellet were washed by adding 150 µl of 70% ethanol and mix then centrifuge for 15 min at 7500 g and 4°c. again remove all supernatant (invert tube on trash) and invert tube on paper tissue and let it dry in the fume hood at room temperature for 10-15 min. then re-suspend with 20 µl of sterile distilled water and kept in -20oc prior to sequencing. the purified fragment was checked on a 1.5% agarose gel ran on a voltage of 110v for about 1hr as previous, to confirm the presence of the purified product and quantified using a nano-drop of model 2000 from thermo scientific. plate 1: agarose gel electrophoresis indicating the positive amplification of the bacteria isolate’s samples using its universal primers (band 1 is the marker, band 2, 3, 4 and 4 represents brevibacterium brevis strains hk 544, pseudomonas aeruginosa strain wes, bacillus megaterium strain wh13, and bacillus subtilis respectively. sequencing the amplified fragments were sequenced using a genetic analyzer 3130xl sequencer from applied biosystems using manufacturers’ manual while the sequencing kit used was that of big dye terminator v3.1 cycle sequencing kit. bioedit software and mega 6 were used for all genetic analysis. lipase production under different carbon source media the determination of optimum carbon lipase production medium hydrolytic activity of isolated bacteria lipase was done on composed of (g/l): peptone, 10; nacl, 5; cacl2.2h2o, 0.1; trybutyrin selective agent, 10 ml (v/v). (nh¬4)2 so4(1.4g), k2hpo4 (2.0g), cacl2 (2.0g), mgso4.7h2o (0.3g), peptone (7.5g), feso4 (5.0g), mnso4 (1.6g), znso4 (1.4g). in addition different erlenmeyer flasks containing the above chemical compounds amended using different carbon sources such as tobacco seed oil (5ml/100ml of production medium), tobacco seed oil (10ml /100ml of production medium), olive oil (5ml/100ml of production medium), olive oil (10ml /100ml of production medium). the erlenmeyer flasks were loaded at the shaker incubator at 150rpm for 5days. there after 5 days of incubation; the lipase activities were determined using naoh titration method as previously https://journals.e-palli.com/home/index.php/ajlsi pa ge 3 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 1-8, 2022 described above. characterization of enzyme based on stability the purified fraction showing highest specific activity was characterized by varying the parameters that influence enzyme activity. effect of ph on activity and stability of lipase this was determined by emulsifying 25ml of olive oil with 75ml of 7% gum arabic for 10mins. the reaction mixture containing 5ml of olive oil emulsion, 2ml of 0.1m phosphate buffer (ph 7.0) gum arabica 1% (w/v) as substrate suspended in various buffer systems: 0.1m sodium acetate buffer (ph 6.0 7.0): 0.1 m sodium phosphate buffer (6.0-7.0); tris-hcl buffer (8.0) and a glycerinenaoh buffer (ph 9.0-). the ph stability studies was performed by pre-incubating the purified enzyme without substrate in ph values ranging from 4.0-10.0 at 50 0c for 2 h and subsequent analysis was determination under standard assay carried out (okoli et al., 2017). effect of temperature on activity and stability of lipase the effect of temperature on lipase activity was determined by estimating the lipase activity at ph 9.0 within a temperature range of (300c-600c) for 2h using gum arabica and olive oil mixture as substrate. the thermal stability of lipase was determined by preincubating the purified enzyme preparation (ph 9.0) at different temperatures (300c 600c) for 2 h without substrate. the residual cellulase activity was determined under standard assay conditions (sharma et al., 2018; okoli et al., 2017). effect of metal ions on enzyme activity the reaction of the enzyme and gum arabica/olive oil mixture was allowed to proceed at 500c with duplicate test tubes containing 50mm cacl2 (ca2+). mgso4 (mg 2+), nacl(na+), kcl (k+). test tubes were labeled according to the above mentioned salts. this was determined by emulsifying 25ml of olive oil with 75ml of 7% gum arabic for 10mins. the reaction mixture containing 5ml of olive oil emulsion, 2ml of 0.1m phosphate buffer (ph 7.0), gum arabica 1% (w/v) as substrate suspended in 0.1m phosphate buffer, in addition. into each of the tubes 50mm of the various metal ions (5ml) were added, incubated at 45oc, and thereafter titrated against 0.1n naoh using phenolphthalein indicator. determination of lipase activities lipase screening assay was carried out using olive oil emulsion prepared by emulsifying 25ml of olive oil with 75ml of 7% gum arabic for 10mins. titration: 2-3 drops of phenolphthalein indicator were added to the reaction mixture and the liberated free fatty acids were titrated with 0.5n naoh to the end point of pink color at ph 10.0 (macedo et al., 1997; lopes et al., 2011; mendes et al., 2011; bhavani, et al., 2012; nagarajan et al., 2014; ullah et al., 2015). lipase activity was calculated as micromoles of free fatty acids formed from olive oil per ml of lipase enzyme as given by the equation: activity = (vs – vb). n. 1000 s where, vs is the volume of 0,05m naoh solution consumed by the enzyme _substrate cocktail (ml); vb is the volume of 0.05m naoh solution consumed in the titration by the substrate (control) cocktail; n is the molar strength of the naoh solution used for titration (0.05m); s is the volume of substrate cocktail solution. one unit of lipase enzyme is defined as the amount of enzyme required to liberate 1µmol of fatty acids from triglycerides (okoli et al., 2019). results and discusions the identifies of the lipase producing bacteria were identified as brevibacterium brevis strains h k 544, pseudomonas aeruginosa strain wes, bacillus megaterium strain wh13, and bacillus subtilis strain bs 01 for isolate h, a, b and f., respectively (table 1). these bacterial isolates had their genomes blasted on the blast software of the ncci, and similarities between 99-100% at different accession numbers were recorded (table 1.)the bands of the nucleotide were also arranged in get as shown in plate. table 1: summary of molecular identity of some selected lipase hyper-producing strains of bacteria s/n isolate codes identity % simi-larity accession num-ber 1 h brevibacillus brevis strain hk 544 99.44 cpo42161.1 2 a pseudomonas aeruginosa wes2 99.93 mn960116.1 3 b bacillus megaterium strain wh13 100 mn372086.1 4 f bacillus subtilis bs 01 100 mt372489.1 this current investigations that observed different strain of brevibacterium brevis, pseudomonas aeruginosa strain wes, bacillus megaterium strain wh13, and bacillus subtilis strain bs 01 as hyper –producing bacteria is also tandem with other reports by other workers at different parts of the globe. ertuğrul et al., (2007) reported the use of hyper – producing strain of bacillus from lipase production. kiran et al. (2008) also documented the use of pseudomonas for sub nerved production of extracellular lipase. this report on this study is also in tandem with periods study by bradoo et al. (1999) which also documented lipasehyper producing strain of bacillus species resident in soil. in independent related studies, ambu et al., (2010) reported the presence of extra-cellular lipase producing acinetobacter junii in the soil of south korea. bompensieri et al. (1996) had also reported the isolation of lipseproducing bacterial such as acinetobacter from different https://journals.e-palli.com/home/index.php/ajlsi pa ge 4 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 1-8, 2022 environmental sources. thus, one concludes that the observation of bacillus, pseudomonas, acinetobacter, brevibacterium strains in this study as lipases hyper-producing is within the pattern of previous observation in this subject matter. the physiology and genetics of enzyme expressions in microorganisms show that enzyme production potentials in bacteria, and fungi lie on genes and equally regulated by genes. this could be extended to mean that any microorganisms irrespective of genes or species can produce any enzyme provided it has the genes to express such enzymes. in a horizontal gene transfer within a wild environment, bacterial and fungal strains can transmit genes from one genus to closely related genus. in addition, under a conventional condition, bacterial and fungal isolates that do not have to express lipase production, could along their co-existence with lipaseproducing bacterial and fungal isolates acquire the genes for lipase production either on their genome/ chromosome of outside the chromosome (in this case, it is said that the microorganism has acquired plasmids for such enzyme productions). the lipases isolated under different conduction from isolate h (brevibacterium brevis), isolate a (pseudomonas aeruginosa), isolate b (bacillus megaterium), and isolate f (bacillus subtilis) were subjected to temperature stability studies (figure 1). brevibacterium brevis at temperatures of 250, 350, 400, 540, and 600 on a centigrade scale showed a lipase activity of 8.0, 9.0, 7.0, 10.0, 22, and units of lipase respectively (figure 1). this showed that lipase of brevibacterium brevis has an optimum temperature of 500c. this implies that industrial application using this lipase of brevibacterium brevis strain hk 544 must be maintained at 50oc to achieve the best desired hydrolysis. furthermore in a related study, luz et al. (2021) reported pseudomonas fluorescens isolated from water, bryophytes and soil showed different tolerance pattern to temperature. luz et al. (2021) showed that s. marcescens lipase had an optimum temperature at 35oc while pseudomonas fluorescens lipase had optional temperature at temperature of 55oc. pandey et al. (2016) working on lipase of penicillum fungus discovered that temperature of 250c. kojima et al. (1994) observed that similar strain of pseudomonas figure 1: ph stability of the lipase enzymes from brevibacillus brevis strain hk 544 (isolate h) pseudomonas aeruginosa wes2 (isolate a), bacillus megaterium strain wh13 (isolate b) and bacillus subtilis bs 01 (isolate f) fluorescens has an optimal temperature of 550c. in addition, lipase isolated from isolate a (pseudomonas aeruginosa wes 2) should lipase activities of 10.0 units 12.0 14.0, 19.0 30.0, and 13.0 units, at temperatures of 250c, 350c, 400c, 450c, and 600c respectively (figure 1). this implies that 50oc remain the best /optimum temperature that can active the highest level of hydrolysis during the application of two other workers in brazil (chandra et al. 2020) in an extensive review reported that lipases of chromobacterium viscosum, aspergillus niger, rhizopus species and pseudomonas nitroaceducans has an optimal activities at lower temperatures between 35oc – 40oc. this is in contrast with this current study as lipase of pseudomonas aeruginosa has optimal activity a temperature of 50o on a centigrade scale. this current work is in agreement with the report of kiran et al. (2008) which reported lipase of marine strain of pseudomonas aeruginosa having an optimal temperature of 40-500c. lipase of bacterial isolate b (molecularly identified as bacillus megaterium strain wh 13) at temperature of (25oc, 350c, 40oc, 45oc, 50oc and 60oc showed lipase activities of 5.0, 6.0, 9.1, 20.0, 8.0, and 5.0 units, respectively (figure 1). statistical analyses using anova (analyses of variance) showed that among the four (4) lipases from four (4) different bacterial isolates in this study, lipase of bacillus megaterium had the least response in terms of lipase activities. it is also recorded that the optimum temperature for lipase isolated from bacillus megaterium was observed at 450c. in another study on lipase of bacillus species isolated from the hot spring of had an optimal temperature of 60oc. thermal stability of lipase of bacillus megaterium strain wh 13 at 40oc could be also explained by the presence of polyamines in the general protein structure. the increased number of hydrogen bonds, salt bridges and https://journals.e-palli.com/home/index.php/ajlsi pa ge 5 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 1-8, 2022 relatively high amount of thermo-tolerant amino acids may contribute to the thermal stability of this lipase at 40oc. the result of this current study is in contrast with the work of brune & gotz (1992), which reported that lipase of pseudomonas sp kwa-56 showed an optimum temperature of 60oc and remain stable at 600c. in terms of ergonometric, it has become very important to develop novel enzymes that remain active and stable temperature ranges. this enzyme being most active at 40oc implies a lot of cost optimization implication as little heat energy is required to achieve enzymesubstrate catalysis during industrial applications (brune & gotz, 1992). in terms of bio-energetics, lipase of bacillus megaterium at 40oc means that little/ relatively low amount of heat energy is required to achieve bund breaking in substrates in order to reduce the activation energy, create an alternative pathway and establish catalysis products (fatty acids in this case). studies showed that the lipase of bacillus subtilis (isolate f) at temperatures of 25oc, 35oc, 40oc, 45oc, 50oc, and 60oc showed lipase activities of 6.0, 16.0, 8.0, 20.0, 4.0, and 4.0 units, respectively. in this case, it is deduced that the lipase of bacillus subtilis had two temperature optima (35oc and 45oc). this implies that industrial applications of this bacillus subtilis lipase is best at 35oc and 45oc in order to achieve the best hydrolysis desired. bakir & metin (2017) identified a thermophilic bacillus species from the hot springs of ayidin, turkey and reported in a similar fashion to this work that the thermophilic bacillus sp strain remained most active at temperature of 45oc – 50oc. other similar independent studies also had previously confirmed that bacillus coagulans lipases have optimum temperature ranges of 45oc – 55oc (fojan, 2000; lima et al., 2004; sulong et al., 2006). the ph responses of the lipase invested from pseudomonas aeruginosa (isolate a) showed that ph values of 6.0, 7.0, 8.0, and 9.0, showed lipase activities of 6.0, 9.0, 13.0 and 5.6 units statistical analysis using anova showed that there are significant differences in the units of activities from ph 6.0 to ph 9.0. inductively, the lipase of this pseudomonas aeruginosa strain wes 2 (isolate a) has alkaline ph optima and applications must be maintained within alkaline ranges to ensure optimal hydrolysis by the enzyme. this observation is in tandem with similar studies in japan where yoshitaka et al. (1982) reported the presence of alkaline lipase from different strain of gram-negative alcaligenes species. relatively recently, liew et al. (2015) reported the isolation of alkaline lipase from burkholderia cepacia after optimization using submerged fermentation technology. the result obtained in this study is further agreement with the work of gupta et al. (2009) which reported isolation of alkaline lipase from burkholderia sp c20. furthermore, liu et al. (2006) and sharma et al. (2002) independently observed at different location that strain of arthrobacter sp had lipases with optimum activity at ph of hp8.0 – 9.0. in addition, the work of gupta et al. (2004) contradicts this current study. gupta et al. (2009) reported that some bacterial lipase showed optimum activities at neutral ph (7.0). the ph responses of the three (3) lipase investigations were studies and documented in figure 2. lipase harvested from isolate h (brevibacillus brevis strain hk 544) at ph values of 6.0, 7.0, 8.0, and 9.0 showed lipase activities of 5.0, 6.0, 5.0, and 23.0 units, respectively. this implies that the lipase of brevibacillus brevis strain hk 544 is an alkaline lipase and all application of the enzyme must be carried out using ph 8.0. (figure 2) similarly in pradesh, india bora & bora (2012) reported the isolation of alkaline lipase from thermophilic bacillus species resident in soil. the study in strong agreement with the report of rathi et al (2001) which observed strains of bacillus species with lipase having optimal activities beyond 17.5 units of activity at ph of 8.5 (alkaline). the lipase harvested from bacillus megaterium wh 13 (isolate b) at ph values of 6.0, 7.0, 8.0, and 9.0 had activities of 5.6, 4.0, 7.0 at 10.0 units, respectively. this showed that the lipase from wh 13 strain of bacillus figure 2: ph stability of the lipase enzymes from brevibacillus brevis strain hk 544 (isolate h) pseudomonas aeruginosa wes2 (isolate a), bacillus megaterium strain wh13 (isolate b) and bacillus subtilis bs 01 (isolate f). megaterium is an alkaline type of lipase with ph 9.0 as optimum ph for optimal activity or hydrolysis. rasmey et al. (2017) took a different position and reported that pseudomonas monteili 2403 showed optimum lipase activities at ph 6.0 which is moderately acidic. other scholars such as qamsari et al. (2011), sooch and kaulder (2013) had independently reported that different species and strains of bacillus and pseudomonas spp expressed https://journals.e-palli.com/home/index.php/ajlsi pa ge 6 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 1-8, 2022 lipases that had optimum activities at ph ranges of 6.0 – 6.5. in addition, lipase harvested from bacillus subtilis (isolate f), at ph values of 6.0, 7.0, 8.0, 9.0 showed a lipolytic activity of 6.0, 7.0 9.0 and 15.0, respectively (figure 2). this equally showed alkaline optimum ph, and could be used at ph 9.0 for the best hydrolytic performance. this observation in the current investigation, is in agreement with previous study by prasasty et al. (2016) which documented that bacillus species and pseudomonas alcaligenes had lipases with optimum activities of 1.0 x 104 u/mg and 0.8 x 104 u/mg, respectively (prasaty et al. (2016.). however, tang and xia (2005). showed that bacillus coagulans zju strain works optimally at 7.0 – 10.0 ph values and activity remained depressed at acidic ph ranges. in further characterization based on reaction with heavy metals from alkalis and alkaline earth metals in the periodic group, the performance as regards activities of the three (3) lipases have been documented as in figure 3. lipase of brevibacterium brevis hk 544 got higher lipolytic activity of 20 units at 50mm concentration of sodium and at 50mmm, concentrations of potassium (k), calcium (ca), and magnesium (mg) the lipase harvested from brevibacterium brevis stain hk 544 got repressed to 6.0, 8.0 and 8.0 units, respectively (figure 3). the observation that sodium ion at 50mm concentration enhanced the activity of lipase of brevibacterium showed that sodium is likely to be a co-factor for the performance of this lipase from b. brevis. the lipase of pseudomonas aeruginosa (isolate a), showed a repressed enzyme activity of 6.0/12.0 and 9.0 units at figure 3: metallic ion stability of the lipase enzymes from brevibacillus brevis strain hk 544 (isolate h) pseudomonas aeruginosa wes2 (isolate a), bacillus megaterium strain wh13 (isolate b) and bacillus subtilis bs 01 (isolate f) 50mm concentration of sodium (na+) potassium (k+), and calcium (ca2+) ions, while at 50mm concentration of magnesium (mg2+) had an enhanced activity of 18.0 units of lipase activity. the essence of this characterization has identified magnesium as possible co-factor for the activity of pseudomonas aeruginosa wes2 strain. in a related pattern, the lipase of bacillus subtilis bs 01 (isolate f) had a strong increase in activity on exposure of substrate and enzyme under 50mm concentration of sodium ion (12.0 units) while the activities of the bacillus subtilis enzyme /lipase at 50mm concentrations of k+, ca2+ and mg2+ had repressed enzyme units of 4.0, 8.0, and 8.0 units (figure 3). the lipase of bacillus megaterium strain wh13 (isolate b,) showed complete repression on exposure with 50mm concentrations of sodium (na+), potassium (k+), calcium(ca2+), and magnesium (mg2+) as 8.0, 6.0, 4.0, and 7.0 units of lipase activities lose recorded respectively (figure 3). conclusion the properties of lipases from brevibacterium brevis strains hk 544, pseudomonas aeruginosa strain wes, bacillus megaterium strain wh13, and bacillus subtilis strain bs 01have been tracked and industrial application of any of the lipases can now be achieved under the scientifically proven-conditions as observed in this study. the study is expected to utilize the produced lipases for production of biodiesel. however, at this point, the aspect of biodiesel production using the lipases cannot be established as a result of limitation in funds. references almeida jm., martini v, iulek j, alnoch r, moure v, müller-santos m and krieger n 2019. biochemical characterization and application of a new lipase and its cognate foldase obtained from a metagenomic library derived from fat-contaminated soil. international journal of biological macromolecules, 137: 442-454. ambu, p., noth, m.j, kim, d-h, seo, j-s, hur, b-k, and kyeong, h.m.(2011). screening and optimization of extracellular lipases by acinetobacter species isolated from oil contaminated soil in south korea, african journal of biotechnology, 10(20):4147-4156. brune, a.k.; gotz, f. (1992). degradation of lipids by bacterial lipases. in winkelmen g(ed) microbial degradation of natural products, vch, weinhein, pp 243-266. bora, l., and bora, m. (2012). optimization of extracellular thermophilic highly alkaline lipase from thermophilic bacillus species isolated from hotspring of arunachal pradesh, india, 30-34. bakir, z.b., and metin, k. (2017). production and characterization of an alkaline lipase from thermophilic anoxybacillus sp. hbb16, chem https://journals.e-palli.com/home/index.php/ajlsi pa ge 7 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 1-8, 2022 biochem. eng. , 31(3): 303-317. belguith h, fattouch s, jridi t, ben hamida j.(2009) immuno-purification and characterization of a rape (brassica napus l.) seedling lipase. afr j biotechnol., 12(21): 3224-3234. chandra, p., nespa, e., singh, r., and arora, p.k. (2020). microbial lipases and their industrial applications: a comprehensive review, microbial cell factories, 19: 169210. carriere f, thirstrup k, hjorth s.(1994) cloning of the classical guinea pig pancreatic lipase and comparison with the lipase related protein 2. febs lett., 388: 6368 ertuğrul, s., dönmez, g., takaç, s., isolation of lipase producing bacillus sp. from olive mill wastewater and improving its enzyme activity, j. hazard. mater. 149 (2007) 720. fojan, p. (2000). what distinguishes an esterase from a lipase: a novel structural approach, biochimie 82: 10331034. fugiri, i., m. atigui, m. ziadi, s. arroum and t. khorchani. 2015. biochemical and molecular identification of lactic acid bacteria isolated from camel milk in tunisia. emir. j. of food and agric., 27 (9):716-720. gupta, r.; gupta, n.; rathi, p. (2004). bacterial lipases: an overview of production, purification and biochemical properties. appl microbiol biotechnol 64: 763-781. gunajit g., sudupta, s.b., assma p., robin c.b., madhumita b. (2017).identification and functional properties of dominant lactic acid bacteria isolated from kahudi, a traditional rapeseed fermented food product of assam, india journal of ethnic foods, 4 (3): 2-13 gayathri, v.r., perumal, p., mathew, l.p. and prakash, b. (2013) screening and molecular characterization of extracellular lipase producing bacillus species from coconut oil mill soil. international journal of science and technology, 2: 502-509. hassan, zmr, efat, b.a.m., magdoub, m.m.i., tawfik, n.f., sadek, z.i.m, mabrouk, a.m.n. (2016). molecular identification of lactic acid bacteria isolated from dairy products, international journal of biology, pharmacy and allied sciences, 5(12): 3221-3230. hasan f, shah aa, hameed a. (2006). industrial applications of microbial lipases. enzyme microb technol; 39: 235-251. shart, a. and elkhalil, e. (2020) biochemical characterization of lipase produced by bacillus spp. isolated from soil and oil effluent. advances in enzyme research: 8, 39-48. jaeger, k.e., dijkstra, b.w. and reetz, m.t. (1998) bacterial biocatalysts: molecular biology three dimensional structures and biotechnological applications of lipases. annual review of microbiology, 53: 315-351. kiran, s.g.; shanmughapriya, s.; jayalakshmi, j.; selvin, j.; gandhimathi, r.; sivaramakrishnan, s.; arunkumar, m.; thangavelu, t.; natrajaseenivasan, k. (2008). optimization of extracellular psychrophilic lipase produced by marine pseudomonas sp. (msio57). bioprocess biosyst eng 31: 483-492. kiran, s.g.; shanmughapriya, s.; jayalakshmi, j.; selvin, j.; gandhimathi, r.; sivaramakrishnan, s.; arunkumar, m.; thangavelu, t.; natrajaseenivasan, k. (2008). optimization of extracellular psychrophilic lipase produced by marine pseudomonas sp. (msio57). bioprocess biosyst eng 31: 483-492. lima, v. m. g., krieger, n., mitchell, d. a., baratti, j. c.,filippis, i., fontana, j. d. (2004) . evaluation of the potential for use in biocatalysis of a lipase from a wild strain of bacillus megaterium, j. mol. catal. b: enzym. 31: 53-57. lima, v. m. g., krieger, n., mitchell, d. a., baratti, j. c.,filippis, i., fontana, j. d., evaluation of the potential for use in biocatalysis of a lipase from a wild strain of bacillus megaterium, j. mol. catal. b: enzym. 31 (2004) 53. luz, b.d., sarrouh, b., bicas, l.s., and lofrano, r.c.z. (2021). lipase production by microrganisms isolated from serra de ouro branco state park, annals of academy of brazillian scientists, 93(3): 1-12. okoli, c.b., orji, f.a., and lawal, a.k. (2019). isolation, screening, and optimization of bacillus spp for lipolytic enzyme production, journal of industrial research and technology,7 (1): 112-129. pandey, n., dhaka, k., jain, r., and pandey, a. (2016). temperature dependent lipase production from cold ph tolerant species of penicillium, mycosphere, 7(10): 1533-1545. ramsey, a-h., aboseidah, a.a., gaber, s., and mahran f. (2017). characterization and optimization of lipase activity produced by pseudomonas monteilii 2403ky120354 isolated from ground beef , african journal of biotechnology, 16 (2): 96-105 ramesh s, kumar r, devi a, balakrishnan, k.(2014). isolation of a lipase producing bacteria for enzyme synthesis in shake flask cultivation. int j curr microbiol app sci.; 3(3): 712-719. riaz m, shah aa, hameed a., hasan f (2010). characterization of lipase produced by bacillus sp. fh5 in immobilized and free state. ann microbiol.; 60: 169-175. rathi, p.; saxena, r.k.; gupta, r. (2001). a novel alkaline lipase from burkholderia cepacia for detergent formulation. process biochem 37: 187-192. sulong, m. r., abdul rahman, r. n. z. r., salleh, a. b., basri, m. (2006). a novel organic solvent tolerant lipase from bacillus sphaericus 205y: extracellular expression of a novel ost-lipase gene, protein expr. purif. 49: 190-195. sahu g and martin m . (2011). optimization of growth conditions for the production of extracellular lipase by bacterial strains from dairy industry effluents. biotechnol bioinf bioeng, 1(3): 305-31. sharma, a., meena, k. r., and kanwar, s. s. (2018). molecular characterization and bioinformatics studies https://journals.e-palli.com/home/index.php/ajlsi pa ge 8 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 1-8, 2022 of a lipase from bacillus thermoamylovorans bhk67. int. j. biol. macromol. 107, 2131–2140. doi: 10.1016/j. ijbiomac.2017.10.092 sulong, m. r., abdul rahman, r. n. z. r., salleh, a. b., basri, m., a novel organic solvent tolerant lipase from bacillus sphaericus 205y: extracellular expression of a novel ost-lipase gene, protein expr. purif. 49 (2006) 190. veerapagu m, sankara d.r., narayanan a, ponmurugan k., jeya k.r. (2013). screening selection identification production and optimization of bacterial lipase from oil spilled soil. asian j pharm clin res. 6: 6267. https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 am. j. life sci. innov. 1(1), 2022 the american journal of life science and innovation (ajlsi) is an international, open access, and blind peer-reviewed journal that publishes research articles emphasizing life sciences and innovations. the journal comprises techniques suitable for promoting the dissemination of research findings that are expected to benefit the basic needs of health, agriculture, biotechnology, pharmaceutical, and food industries. ajlsi also publishes life science articles on evidencebased practices, thus improving the quality of life. ajlsi accepts manuscripts throughout the year; all submitted manuscripts are rigorously reviewed, and publish articles online first. frequency: 3 issues per year area of publication: life science and technologies, innovations in life sciences and development of living orgasms, and related fields. editorial team professor dr. nirmal chandra roy sylhet agricultural university, bangladesh dr. sejuti mondal texas state university, usa dr. sandra milena camargo silva materials engineering, pedagogical and technological university of colombia, colombia dr. myint thuzar professor, yezin agricultural university (yau) myanmar dr. md. morshedur rahman professor, department of dairy and poultry science bangabandhu sheikh mujibur rahman agricultural university, bangladesh dr. omar faruk miazi professor, department of genetics and animal breeding chattogram veterinary and animal sciences university (cvasu) chattogram, bangladesh dr. yulianna puspitasari faculty of veterinary medicine, universitas airlangga surabaya, indonesia dr. md robiul karim associate professor, department of medicine bangabandhu sheikh mujibur rahman agricultural university gazipur, bangladesh dr. mst. rubia banu assistant professor, department of fisheries management bangabandhu sheikh mujibur rahman agricultural university gazipur, bangladesh dr. tilak chandra nath associate professor, department of parasitology sylhet agricultural university, bangladesh professor dr. mrityunjoy kunda dean, faculty of fisheries sylhet agricultural university, bangladesh contact: american journal of life science and innovation (ajlsi) e-palli llc address: 2055 limestone rd ste 200c, zip code: 19808, wilmington, delaware, usa email: ajlsi@e-palli.com phone: +1 3024070506 url: https://journals.e-palli.com/home/index.php/ajlsi http://mailtoajmri@e-palli.com pa ge 1 american journal of life science and innovation (ajlsi) performance of wheat genotypes under different dates of sowing in southern part of kailali district, nepal j. r. joshi1*, s. dhakal1, s. kafle2 volume 1 issue 1, year 2022 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v1i1.212 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: march 22, 2022 accepted: march 13, 2022 published: april 18, 2022 an experiment was conducted at kailali district to study the performance of wheat genotypes under different sowing from december, 2017 to april, 2018. the field experiment was conducted in randomized complete block design with a total of 18 treatments consisting of 2 dates of sowing (november 4 and november 20 ) and 9 wheat genotypes(wh1105, hd2733, hd2824, hd2967, borlaug100, hd3086, pbw550, nl971 and pbw373). grain yield (5.802 ton/ha),spike length(17.94 cm) and biomass yield(13.16 ton/ha) of nl 971 was highest. hd 2733 had highest number of effective tillers (519.1/ m2) while number of grains per spike(59.80) was highest in hd 2967. pbw 373 had lowest grain yield (4.551 ton/ha), borlaug 100 was the variety with minimum number of tillers per m2 area (401.3), minimum spike length(15.14 cm) was observed in hd 2733 ,hd 3086 had lowest no. of grains per spike(42.12). grain yield(5.328 ton/ha) , effective tillers per m2(475.5) , spike length(16.95 cm), number of grains per spike (53.12), biomass yield(11.45 ton/ha) was recorded maximum in wheat sown on 20th nov. grain yield (4.893 ton/ha),effective tillers(435.1), spike length(15.66 cm), grains per spike(49.59) and biomass yield(10.49 ton/ ha) was recorded minimum in wheat sown on 4th nov. maximum test wt. (50.83 g ) was found in hd 2733 of 20th nov. and the lowest test wt. (40.47 g) was found in pbw 550 of 20th nov. keywords biomass, spike, tiller, yield 1 agriculture and forestry university,rampur,chitwan,nepal 2 cimmyt, braced/anukulan project, nepal * corresponding author’s e-mail: jayrajjoshi20728@gmail.com introduction background nepal is an agro based country. it is geographically distinguished into three major regions, terai region, hilly region and himalayan region out of which only terai and small portion of hilly and himalayan region is suitable for cultivation. agriculture is a important sector in the nepalese economy contributing about third of its gdp and engaging about two thirds of its population(moad, 2016a).wheat (triticum aestivum) or gahun belonging to the family poaceae is the world most widely cultivated cereal crop. as a winter season crop wheat is still playing vital role in establishing the food grain production in the country. wheat contains more protein [8-15% (grain) and 8-13% (flour) than other cereals(mathur, suman, meena, & anuradha, 2017). wheat proteins are of special significance. besides their significance in nutrition, these are mainly concerned with providing the characteristic substance ‘gluten’, which is very essential for bakers (macritchie, 1984) there are many factors which are responsible for low average yield of wheat. therefore some efforts have been made to increase the yield by introduction of high yielding varieties, balanced fertilizer application and efficient use of irrigation facilities. but still there are some factors upon which attention have not been made to increase the overall yield. one of the limiting factors is date of sowing. sowing time significantly influence the yield and other yield attributing characteristics. there are still many factors which are responsible for low average yield. one of such environmental factors is untimely planting and it affects yield of wheat crop considerably (saini, dhadwal, & nanda, 1988; ikeh & ndaeyo, 2021). for supplying food to the increasing population timely sowing of seed along with timely application of fertilizers are crucial. the problem of post anthesis heat stress is highly responsible for low wheat yield. therefore efforts should be made to minimize the effect of temperature variation caused due to changed sowing date by choosing appropriate sowing date. the ongoing scenario of the country shows that because of the increasing rate of population growth, 1.35% in country and 1.86% in kailali district (statistics, 2017), the area occupied by (moad, 2016b)(moad, 2016b) overall agricultural commodity goes on rapid way of decreasing because of increased use of land for human settlement. area under the wheat cultivation in nepal is 762373 ha(2071/72) and this area under the wheat cultivation decreased by 1.2% and falls to 753470 ha in 2072/73(aicc, 2074). hypothesis h1: wheat varieties sown at different dates have variation in their performances. objectives general objective to assess production performance of different wheat genotypes sown at different dates specific objectives • to find out performance of each wheat genotype at each sowing date in terms of yield and yield attributes. • to compare the performance of each wheat genotype at both sowing date. • to find out best genotype at each sowing date https://doi.org/10.54536/ajlsi.v1i1.212 https://journals.e-palli.com/home/index.php/ajlsi mailto:jayrajjoshi20728%40gmail.com%20?subject= pa ge 2 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 1-5, 2022 materials and methods location: this research is carried out at kailari rural municipality and dhangadhi submetropolitan physic-chemical properties of the soil at the experimentation site the soil sample from the research plot was taken and the ph content of the soil was found to be and it is suitable for wheat .the nitrogen, phosphorus and potash content of the research field was found to be high as per the soil report.the land was ploughed and leveled once at 30th oct. for 4th nov. sowing and again at 15th nov. for 20th nov. sowing. 300 kg fym was applied in the research plot on 4th nov. and 20th nov. for the two sowing dates and the field was ploughed, made fine and pulverized. the whole field was divided into 36 small plots each with 16 m2 at each sowing dates 4th nov. and 20th nov. respectively. each small plot was separated with rope in each side. treated seed was sown at each sowing dates with the seed rate of 120 kg/ha.the seeds were sown continuously in the rows spaced at 20 cm. the sowing was done manually on 4th nov. for early sowing and 20th nov. for normal sowing with the seed rate of 120kg/ ha.1st irrigation was done at 17 days of seeding date and 2nd irrigation at 66 days of seeding date in case of both seeding date. the crop was fertilized with 100:50:50 kg npk/ha,.through urea, diammonium phosphate and muriate of potash respectively. 1/3rd n and full dose of phosphorous and potash was given at the time of at sowing and remaining 2/3rd nitrogen was top dressed at 40 and 75 days after sowing respectively. yield and yield attributing characters no. of effective tillers, average of spike length, no. of total grains in 5 spikes, tagb in nha, grain yield in nha, thousand grain wt.and grain moisture. the obtained data was systematically arranged, tabulated in microsoft excel program and graph and figure was drawn. various analytical software such as genstat and m-stat was utilized for the analysis of the data obtained. duncan test was carried out at 5% level of significance. results and discussion highest value of number of effective tillers was seen in hd 2733 (519.1) which were at par with hd 3086 (518.8) while lowest number of tillers was seen in borlaug 100 (401.3). highest value of effective tillers was seen in wheat sown in 20th november (475.5) and lowest value in case of wheat sown in 4th nov. (435.1) effect of varieties on biomass yield was seen highly significant .highest value for biomass yield was seen in nl 971 (13.16) and lowest was seen in hd 2824 (10.12) which was at par with wh 1105 (10.47),hd 2967 (10.19),borlaug 100 (10.18),pbw 550 (10.30) and pbw 373 (11.22).similarly effect of date of sowing on biomass yield was also seen significant. more biomass yield was seen in wheat sown in 20th nov. (11.45) and less in case of wheat sown in 4th nov.(10.49). effect of varieties on grain yield was seen significant. highest value for grain yield was seen in nl 971 (5.802) which was at par with wh 1105 (5.684) and lowest value for grain yield was seen in pbw 373 (4.551) which was at par with hd 2824 (4.752) and hd 2733 (4.814).more grain yield was seen in wheat sown in 20th nov. (5.328) and less grain yield in case of wheat sown in 4th nov. (4.893). effect of varieties on test weight was seen highly significant. highest value for test wt. was seen in hd 2733 (48.73) and lowest in pbw 550 (43.14). highest value for test wt. was seen in 20th nov. sowing (59.9) and lowest test wt. was seen in 4th nov. sowing (44.6). . highest value for spike length was seen in nl 971 (17.94) and lowest value in hd 2733 (15.14). more spike length was seen in wheat sown in 20th nov. (16.95) and less spike length in case of wheat sown in 4th nov.(15.66). pbw 550 (10.4, 10.5 cm) produced the highest spike length which was significantly higher than pbw 621 (9.9, 9.3 cm) and hd 2967 (9.1, 8.8 cm) highest value for no. of grains per spike was seen in hd 2967 (59.80) and lowest value in hd 3086 (42.12) which was at par with hd 2733 (43.23). highest value for no. of grains per spike was seen in hd 2967 (59.80) and lowest value in hd 3086 (42.12) which was at par with hd 2733 (43.23). table 1: effects of varieties and date of sowing on no. of effective tillers, spike length and no. of grains per spike at kailali district in 2017/18. treatment no. of effective tillers spike length no. of grains per spike variety wh 1105 407.9bc 15.66cd 56.53ab hd 2733 519.1a 15.14d 43.23e hd 2824 429.6bc 17.38ab 50.23d hd 2967 408.3bc 16.30bcd 59.80a borlaug 100 401.3c 16.21bcd 54.95bc hd 3086 518.8a 15.36cd 42.12e pbw 550 492.5ab 16.26bcd 51.70cd nl 971 464.4abc 17.94a 48.58d pbw 373 455.9abc 16.52bc 55.10bc sem(±) 26.77 0.422 1.543 lsd(0.05) 75.99** 1.199*** 4.379*** date of sowing 4 november 435.1b 15.66b 49.59b 20 november 475.5a 16.95a 53.12a sem(±) 12.62 0.199 0.727 lsd(0.05) 35.82** 1.696*** 2.064*** cv(%) 16.6 7.3 8.5 grand mean 455.3 16.31 51.36 means in the column followed by same letter(s) in each treatment do not differ significantly at(p=0.05) by dmrt. das=days after sowing, sem= standard error of mean,lsd=least significant difference and cv= coefficient of variance. https://journals.e-palli.com/home/index.php/ajlsi pa ge 3 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 1-5, 2022 table 2: effects of varieties and date of sowing on biomass yield, grain yield and thousand grain wt. at kailali district in 2017/18. treatment biomass yield grain yield test wt. variety wh 1105 10.47b 5.684a 44.73cd hd 2733 11.48ab 4.814b 48.73a hd 2824 10.12b 4.752b 46.67abc hd 2967 10.19b 5.164ab 45.94bc borlaug 100 10.18b 5.031ab 46.76abc hd 3086 11.65ab 5.171ab 47.83ab pbw 550 10.30b 5.025ab 43.14d nl 971 13.16a 5.802a 45.56bcd pbw 373 11.22b 4.551b 45.57bcd sem(±) 0.579 0.2475 0.814 lsd(0.05) 1.644** 0.7027* 2.312*** date of sowing 4 november 10.49b 4.893b 44.6b 20 november 11.45a 5.328a 59.9a sem(±) 0.273 0.1167 0.384 lsd(0.05) 0.775* 0.3313* 1.090*** cv(%) 14.9 13.7 5 grand mean 10.97 5.111 46.10 means in the column followed by same letter(s) in each treatment do not differ significantly at (p=0.05) by dmrt. das=days after sowing, sem= standard error of mean, lsd=least significant difference and cv= coefficient of variance. means in the column followed by same letter(s) in each treatment do not differ significantly at (p=0.05) by dmrt. das=days after sowing, sem= standard error of mean, lsd=least significant difference and cv= coefficient of variance. means in the column followed by same letter(s) in each treatment do not differ significantly at (p=0.05) by dmrt. das=days after sowing, sem= standard error of mean, lsd=least significant difference and cv= coefficient of variance. table 3: interaction effect of date of sowing and varieties on heading das at kailali district in 2017/18 heading das treatments varieties dos wh 1105 hd 2733 hd 2824 hd 2967 borlaug 100 hd 3086 pbw 550 nl 971 pbw 373 4 nov. 75.25f 92a 92.25a 92a 76.75f 69g 65.50g 82cde 82cde 20 nov. 79def 89.25ab 92a 94a 78.75def 77ef 74.75f 85.25bc 83.75cd sem(±) 1.594 lsd 4.524* table 4: interaction effect of date of sowing and varieties on test wt. at kailali district in 2017/18. test weight treatments varieties dos wh 1105 hd 2733 hd 2824 hd 2967 borlaug 100 hd 3086 pbw 550 nl 971 pbw 373 4 nov. 41.22hi 46.64bc def 45.97cde fg 42.39g hi 44.70ef gh 45.61d efg 45.81cd efg 45.12d efg 43.75fghi 20 nov. 48.23ab cde 50.83a 47.38abc def 49.49a bc 48.82ab cd 50.04a b 40.47i 46cdefg 47.40abc def sem(±) 1.152 lsd 3.269*** https://journals.e-palli.com/home/index.php/ajlsi pa ge 4 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 1-5, 2022 table 5: interaction effect of date of sowing and varieties on maturity das at kailali district in 2017/18. maturity das treatments varieties dos wh 1105 hd 2733 hd 2824 hd 2967 borlaug 100 hd 3086 pbw 550 nl 971 pbw 373 4 nov. 139.3cd 141ab 139.5cd 142.3a 142a 139cd 140bc 139.3cd 141ab 20 nov. 127g 129f 131.5e 138.3d 127.3g 129f 129.3f 129.5f 130.3ef sem(±) 0.468 lsd 1.328*** means in the column followed by same letter(s) in each treatment do not differ significantly at (p=0.05) by dmrt. das=days after sowing, sem= standard error of mean, lsd=least significant difference and cv= coefficient of variance. interaction effect of date of sowing and varieties on days to heading was found significant. early heading was found in pbw 550 of 4th nov. sowing.(65.50),which was at par with hd 3086 of 4th nov. sowing.(69) and delay heading was found in hd 2967 variety of 20th nov. sowing (94),which was at par with htd 2733 sowing of 4th nov. (92).,hd 2824 sowing at 4th nov (92.25).,hd 2967 sowing at 4th nov (92) and hd 2824 sowing at 20th nov.(92) interaction effect of date of sowing and varieties on days to maturity was found highly significant. early maturity was found in wh 1105 at 20th nov. sowing (127) whereas hd 2967 in 4th nov. matured later. (142.3) which was at par with borlaug 100 at 4th nov. sowing (142). interaction effect of date of sowing and varieties on test wt was found highly significant. highest test wt. was found in hd 2733 sowing of 20th nov. sowing.(50.83) and lowest test wt. was found in pbw 550 sowing of 20th nov. sowing (40.47). conclusions the effect of sowing dates and varieties on yield and concerned yield attributes of wheat was successfully conducted in the farmer’s field. there was no significant effect of sowing date on greenness value of the different wheat varieties. significant variations were observed in the different parameters in response to the different varieties and sowing dates. highest grain yield was found in the wheat variety nl 971followed by wh 1105 and lowest in pbw 373.simillarly 20th nov. sowing had more grain yield than 4th nov. sowing. references aicc. (2074). krishi diary. retrieved from http://www. aicc.gov.np/files/download/krishi diary_2074.pdf ali, a., nadeem, m. a., & khalid, f. (2009). effect of different sowing dates on growth and yield of wheat ( triticum aestivum l .) varieties in district jhang , pakistan . pak j life soc sci effect of different sowing dates on growth and yield of wheat ( triticum aestivum l .) varieties in district, (june 2014). ali shah, w., bakht, j., ullan, t., khan, a. w., zubair, m., & khakwani, a. a. (2006). effects of sowing dates on yield and yield components of wheat varieties. journal of agronomy. briggle, l. w., & curtis, b. c. (1987). no title. cdd. (2015). crop development directorate. cimmyt. (2005). no title. council, n. a. r. (2002). narc. dado, k. (2073). dado profile. kailali. dhakal, j. prasad. (2007). response of wheat varieties under timely and late sowing condition with and without fungicide spray at rampur,chitwan. iaas. ikeh, a. o., & ndaeyo, n. u. (2021). evaluation of yield and resistance effect of some yam genotypes (dioscorea rotundata pior) to pest attack at harvest in uyo, southeastern nigeria. american journal of agricultural science, engineering, and technology, 5(2), 113–120. https://doi.org/10.54536/ajaset.v5i2.78 k.c., s., mishra, r. d., & singh, m. (1971). effect of sowing date and rate of nitrogen and seed on the grain and straw yield of draw wheat. indian journal of agricultural science. kanchan, n., & mahendra, s. (2000). varietal behaviour of wheat (triticum aestivum) to dates of sowing under tarai region of uttar pradesh. indian journal of agronomy, 45(1), 107–113. knight, s. m., kightley, s., bingham, i. j., hoad, s., lang, b., philpott, h., … ball, b. (2012). desk study to evaluate contributory causes of the current’yield plateau’in wheat and oilseed rape. hgca. kumar, p., ram, h., & bedi, s. 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(2017). https://journals.e-palli.com/home/index.php/ajlsi pa ge 5 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 1-5, 2022 role of chealeted micronutrient and their salts for improving crop production of wheat. moad. (2016a). statistical information on nepalese agriculture. singha durbar,kathmandu,triveni chhapakhana. moad. (2016b). statistics agriculture book. naik, p. l., patel, b. a., & kalaria, k. k. (1991). response of wheat (triticum-aestivum) varieties to sowing date and seed rate. indian journal of agronomy. indian soc agronomy indian agr res inst div agronomy, new delhi 110012, india. nayeem, k. a., & d.g., d. (1993). stomata density.aperture index and their differentials in wheat at low and high temp. pandey, i. b., pandey, r. k., dwivedi, d. k., & singh, r. s. (2010). phenology, heat unit requirement and yield of wheat (triticum aestivum) varieties under different crop-growing environment. indian journal of agricultural sciences, 80(2), 136–140. rao, m.h., m. n., sinha, & rai, r. k. (1980). the wheat development program in banglash. indian journal of agriscience.50:236-239. rout, d., & satapathy, m. r. (1994). effect of date of sowing of wheat (triticum aestivum) varieties in umerkote zone of orissa. indian journal of agronomy, 39(3), 466–467. s.p., k., tewari, j. p., & jain, c. (1993). effect of chemical weed control under different sowing dates and planting pattern on growth and sink potential of dwarf wheat. indian journal of agricultural science. saini, a. d., dhadwal, v. k., & nanda, r. (1988). pattern of changes in yield of kalyansona and sonalika varieties of wheat in sowing date experiments at different locations. in field crop abstracts (vol. 42, p. 6777). sandhu, i. s., sharma, a. r., & sur, h. s. (1999). yield performance and heat unit requirement of wheat (triticum aestivum) varieties as affected by sowing dates under rainfed conditions. indian journal of agricultural science, 69(3), 175–179. sardana, v., sharma, s. k., & randhawa, a. s. (2002). performance of wheat (triticum aestivum) varieties under different sowing dates and nitrogen levels in the submontane region of punjab. indian journal of agronomy, 47(3), 372–377. sharma, v., s. k., & randhawa, a. s. (2002). performance of wheat varieties under different sowing dates and nitrogen levels in the sub mountain region of punjab,indian j. agron. statistics, c. b. o. (2017). statistical information on population. sumbali, g. c., & sharma, s. (1974). effect of dates of sowing on growth and yield of dwarf wheat varieties. indian journal of agronomy. tahir, m., ali, a., nadeem, m. a., hussain, a., & khalid, f. (2009). effect of different sowing dates on growth and yield of wheat (triticum aestivum l.) varieties in district jhang, pakistan. pakistan journal of life and social sciences, 7(1), 66–69. https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 19 american journal of life science and innovation (ajlsi) weed control methods used in agriculture degefa woyessa1* volume 1 issue 1, year 2022 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v1i1.413 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: july 20, 2022 accepted: july 23, 2022 published: july 24, 2022 one of the most challenging duties in an agricultural field is weed control. weed control is a major problem for peasant framers everywhere. this paper’s objectives are to reviewing and reporting the weed management practices utilized in ethiopian agriculture and to recommend the viability of mechanizing weeding operations for the further research. in agriculture, three weed management techniques are frequently used. these are chemical, mechanical, and manual control. farmers that are convinced of its benefits quickly adopt mechanical weed control. in addition to pulling weeds out from between the rows of crops, mechanical weed management maintains the soil’s top loose, improving soil aeration and water absorption. mechanical weeding saves time and labor while lowering labor costs also. some weeding tools that are powered by animals are effective in terms of time and money savings. herbicides are occasionally used by farmers; however, they are not often used in farming. in conclusion, weed control is one of the most essential and expensive operation in agriculture. weeding by mechanical device reduces the cost of labour and also saves time than any other methods of weed control. therefore, instead of manual weeding and using chemicals, the use of a mechanical weeding machine should be given priority. introduction the ethiopian economy is based primarily on agriculture, which directly and indirectly controls the national income. according to the central statistical agency (csa, 2013) estimated that about 80% of the population’s livelihood is based on this sector. one of the major reasons for decreased productivity of agricultural field crops in the country is due to lack of mechanization from sowing to harvesting, especially at critical stages such as weeding and intercultural operations. weed is a common term to describe a plant that is considered undesirable (gavali and kulkarni, 2014). weed losses exceed those caused by any other agricultural pest. in ethiopia, crop yield losses due to weeds vary from season to season and from region to region, due to different biotic and abiotic factors, it is estimated that weeds reduce yield due to delay weeding from 15 to 62% (kebede desta, 2000). weeds can be undesirable for some reasons; an essential one is they intrude on meals and fodder production in agriculture, which ought to be managed so that it will save you misplaced or faded crop yields (gavali and kulkarni., 2014). weeding may be completed through the guide, chemical, and mechanical techniques. due to excessive time consumption, much fewer paintings capability, and tediousness, chemical, and mechanical weed management techniques are possible options to guide weeding. the introduction of efficient mechanical weeding machines is expected to encourage farmers to be selfsufficient, which will lead to increased yields and thus reduced poverty (olukunle & oguntunde, 2006) for the soil, stimulating microbial activity in the soil, reducing soil moisture evaporation and facilitating rainwater infiltration (hegazy et al., 2014). in developed countries, multikeywords manual, mechanical implements, weeds, weed control, weeding efficiency purpose machines have been developed and successfully implemented for weeding and intercultural operations. the use of such machines in the ethiopian agricultural scenario is difficult as most of the ethiopian farmers are small-scale farmers because the area they control is small. in the traditional method, weeding is done manually. cutlass and hoe is a handy tool used for this purpose. it is the most widely used weed control method, but it is laborious, time-consuming, labor-intensive, cause’s longterm health problems, and operating speed is one of the most important factors. in most of the highlands, crops are planted at the same time and weeding operation is performed at the same time. this results in shortages of labor during the peak seasons of weeding. the weeding labor bottleneck is especially problematic because some varieties are prone to weeding time; hence delayed weeding decreases crop yields. using a mechanical weeder relieves stress and ensures a comfortable position for the farmer or operator during weeding. pantage et al. (2015) found that the mechanical approach to weed control is best with little or no limitation due to its effectiveness. the main purpose of row farming is to improve the use of agricultural machinery to remove weeds from cropland. the effect of this method is to promote plant growth and give higher quality. however, the use of this type of machine is uncommon, and very rarely is a mechanical weeder available. generally, to increase agricultural output and reduce the time and cost of weeding operations, it is imperative to develop and promote mechanical operated weeding technology. hence, this paper is studying and reporting on the method of weed control in agricultural improvement and assessing the possibility of mechanization of weeding operation. 1 oromia agricultural research institute, asella agricultural engineering research center, asella, ethiopia. * corresponding author’s e-mail: degefawoyessa20007@gmail.com https://doi.org/10.54536/ajlsi.v1i1.413 https://journals.e-palli.com/home/index.php/ajlsi mailto:degefawoyessa20007%40gmail.com?subject= pa ge 20 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 19-26, 2022 literature review overview of weed control methods in ethiopia, weeds are controlled mainly by manual weeding, but also by good agricultural practices such as repeated deep plowing, late sowing, and crop rotation. some mechanical weeding methods are also practiced, using simple traditional tools. all of which is neither adequate nor timely. hence, it is imperative to develop an efficient and economical way of managing weeds through the use of herbicides (kebede desta, 2000). chemical weed management results in better crop growth and often improved yields compared to manual and mechanical methods by eliminating mechanical damage to the crop and reducing moisture loss (atiq et al., 2009). .in addition, the authors found that chemical weed management was much quicker and less laborintensive, allowing for the covering of large areas in a short time with limited work. though, manual weeding is a traditional and common practice for managing weeds in potatoes, marginalities of labor and the prevalence of frequent rains many times prevent the timely management of weeds at critical periods of crop growth. weed control includes a variety of techniques used to limit weed entry and minimize competition. these techniques attempt to strike a balance between control costs and crop yield loss, but weed control is used only after the problem exists; it is not prevention. weed control techniques have been widely adopted because control is the easiest to do and often effective. the problem is known and visible, and actions can be tailored to the observed problem. control techniques can be selected to meet short-term agricultural and economic planning objectives (lopez, 2011). duff and orcino (2001) reported that the timing rather than the frequency of weeding was a major determinant of effective weed control for crops. recommendations have been made for the first weeding to be done 2 up to 3 weeks after sowing, followed by a second weeding three weeks later and if necessary a third one. ofor et al., (2009) reported that weeds as pests highly reduce the production of the crops. they extract nutrients, water, and moisture available in the soil and also compete for light and air and negatively affect the crop. critical crop growth periods are very much important in all crops at which weed competition for nutrient and water resources plays a significant effect on yield. it varies from crop to crop. it is shown between 3060 days in a wheat crop after sowing (ahmad and shaikh, 2003) kebede (2000) found that ethiopian farmers often lose up to 40% of their crops to weeds. since crops are usually not grown in rows, weeding is a time-consuming undertaking, up to 140 h/ha. weeds are controlled mainly by manual weeding, but also by good agricultural practices such as increased tillage, delayed sowing, and crop rotation. he also reported that machine weeding was also carried out, using simple traditional tools and some modern tools. weeding is one of the important steps in the growing process and affects the yield and quality of output. verma and victor (2003) reported that the percentage yield loss due to weed competition during the first month, two months, and the entire growing season were 23.7%, 35.4%, and 35.4 percent, respectively. 40.8%. thorat et al. (2014) reported that weed competition is a serious problem in most wet season crops, resulting in yield losses of 9-60% or more. most ethiopian farmers do not weed their fields in time due to a lack of labor. therefore, weeds are one of the major constraints on the country’s agricultural production. research conducted by agricultural research institutes across the country investigated the effect of delayed weeding on crop yields. weed control methods weed management is as old as agriculture, but weed control methods and concepts have changed over the years. current weed control practices in ethiopia are chemical, mechanical, and biological. the mechanical method is characterized by heavy use of manual labor and animal strength. both are rare and are becoming increasingly unprofitable. manual weeding, in addition to being laborious, is inefficient (not on time in most cases) and is not feasible due to poor soil conditions. weeds compete more with plants in the early stages of growth (2-6 weeks after planting). weed control during this period is essential to achieving maximum crop yields. for this reason, chemical weed control is rapidly gaining traction in the country, which, on the other hand, raises several environmental concerns. with increasing public awareness of environmental pollution, the development of environmentally friendly weed management technologies will be emphasized. weed control is one of the most difficult tasks in agriculture, accounting for a significant portion of the cost of agricultural production. weeds compete with crops for nutrients and other growth factors, and without effective control measures, weeds will consume 30 to 40 percent of the applied nutrients, resulting in reduced considerable productivity. weeding with hand tools takes a lot of work. a mechanical weeder makes it possible to get your weeding job done on time and at a lower cost. environmental pollution due to chemicals is also reduced by using a mechanical weeder (sirmour and verma, 2018) babu & rao (2017) studied weed management and concluded that weed management is one of the tedious activities in agricultural production. due to the cost of labor, time, and completely manual weeding, this is not convenient. therefore, efforts are being made to design efficient agricultural equipment to perform weeding without a power supply. the design and key features of the design are based on simulation and the advantages of using it to design the drive mechanism of a threerow weeding device have been discussed. weeding and tilling are usually done 15 to 20 days after sowing. it is necessary to control and eliminate weeds at an early stage. depending on weed density, grain yield loss of 20 to 30 percent is quite common, which can increase up https://journals.e-palli.com/home/index.php/ajlsi pa ge 21 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 19-26, 2022 to 80 percent if proper crop management practices are not followed. rice and peanuts are susceptible to weeds. competition at the start of growth and failure to control weeds in the first three weeks after sowing reduces yields by 50%. kebede (2000) found that ethiopian farmers often lose up to 40% of their crops to weed infestations. since crops are usually not grown in rows, weeding is a timeconsuming undertaking, up to 140 h/ha. weeds are controlled mainly by manual weeding, but also by good agricultural practices such as increased tillage, delayed sowing, and crop rotation. machine weeding is also done, using simple traditional tools and some modern tools. some weeding systems that use animals effectively save time and money. some farmers also use herbicides, but these methods are not very suitable. he also reported that 35% of wheat yield loss was due to delayed weeding. olaoye and adekanye (2012) has studied that weed control is one of the most difficult tasks in agriculture, accounting for a significant portion of the costs associated with agricultural production. chemical weed control is more important than manual and mechanical methods. however, its harmful effects on the environment cause farmers to consider and accept mechanical methods of weed control. hand weeding is popular in developing countries. this is the most commonly used weed control method, but it is laborious. the use of a mechanical weeder reduces strain and ensures a comfortable position for the farmer or operator during weeding. machine weeding is one of the commonly used methods of weeding in agricultural fields. research has been done on economical weed control methods that do not harm the crop. weeding machines are developed to be used for specific crops such as tomatoes, corn, and rice. these machines are mainly in-row weeders that remove weeds in several rows of crops at once (cordill and grift, 2011) manual methods of weed control sirmour (2016) reported that weeding is traditionally done using indigenous hand tools. these involve considerable time and labor. hand weeding is very common on wheat and vegetable crops. in the manual method, weeds are pulled by hand. verma and prabhat (2015) found that the manual weeder has the limitation of working width and required more time to cover an area between crops and a tractor-drawn cultivator was evaluated for weeding operation and found successful for weeding in large row spaced crops kebede (2000) studied that the traditional animal-drawn weeding method known as shilshalo involves growing cows in sorghum or maize planted broadcast or in rows at 50–70 cm spacing. traditional animal traction plows are used for inter-row farming. this breaks down the soil crust, reduces runoff, and speeds infiltration into the soil, in addition to controlling weeds and thinning crops to an appropriate level. however, since most farmers do not practice shilshalo at the correct stage of crop growth, severe plant damage (stem breakage and uprooting) often occurs, resulting in low yields. a series of trials have been carried out to improve existing traditional practices by determining the optimal timing of livestock operations to increase yields of sorghum and maize. shilshalo is effective at the 6-8 leaf stage for sorghum and the 4-6 leaf stage for maize. mechanical weed control method singh (2001) points out that mechanized farming is still the most important method used for weed control and is generally still the most economical method, recommended from an environmental pollution point of view. the author also found that operating costs of rotary weeders and weeding costs are 63% higher than that of a hand wheel hoe and 72% lower than chemical control. thorat & sahoo (2014) researched and reported that machine weeding is highly effective because it reduces the effort of manual weeding, kills weeds, and also keeps the soil surface loose by providing soil aeration level and water absorption capacity. the availability and cost of labor for weed control are limiting its progress, and the development of an appropriate mechanized weeding method is imperative. the cost of weeding by mechanical weeding is about one-third that of manual weeding. guru et al. (2018) reported that mechanical weeding is one of the most conventional weeding methods most important. although this is one of the older weed control methods, recent advances have made it an innovative weed control technique. mechanical weeding has many advantages over chemical weeding, i.e. weeding is retarded and has no adverse effect on plant growth. piyush et al. (2018) found that controlling weeds in shoots was difficult and in these crops, weeds were removed only once by manual labor. as a result, weeding is inefficient and operations are stalled, behind schedule, and lost in productivity. for row crops, this problem does not arise because mechanical weeders and cart-powered tools can be used to effectively and promptly control weeds. furthermore, the author concludes that tillage depth has no significant effect on rice yield. there is growing interest in using weeders between rows due to concerns about environmental degradation and growing demand for organically produced foods. weeding is an important but equally laborious farm unit operation. there is growing interest in the use of mechanical weeders in the row due to concerns about environmental degradation and the growing demand for organically produced foods. today, the agricultural industry requires weed control without using chemicals to ensure food safety. consumers demand high-quality food products and are particularly concerned about food safety. through the engineering development of physical weed control mechanisms, such as precision weeding between rows and within rows, it is possible to control weeds in a way that meets consumer and environmental needs (kurstjens & perdok., 2000). quadri (2010) reported that the mechanical weeder consists of two auxiliary tools, namely, a primary blade in https://journals.e-palli.com/home/index.php/ajlsi pa ge 22 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 19-26, 2022 the front for loose soil at the top and a secondary blade at the rear for cutting and lifting the grass. the weeding efficiency of the manual weeding machine on loamy soil is 81.14%, clay soil is 93.75% and sandy soil is 94.29%. the overall field efficiency of the machine is 98.67%. chemical method of weed control weeding is one of the main activities of agriculture. chemical weed control is more important than manual and mechanical methods. however, its harmful effects on the environment cause farmers to consider and accept mechanical methods of weed control. chemical weed control is the most widely used weed control method. but these chemicals used to control weeds are harmful to living organisms and are toxic. kebede (2000) reported that some farmers have started using herbicides to control weeds, especially in doublecropping areas. however, herbicides are less effective than manual weeding, as they require specific conditions that can be more restrictive than other control methods. chemical weed control can quickly become unpopular because it is not environmentally friendly. indeed, chemical weed control has been found to introduce a toxic substance into the environment. herbicides seem to be out of pocket and even uneconomical for small farmers. herbicide production itself is a high-tech process and requires a very reliable linkage (manuwa et al., 2009). chemical weed control is more important than manual and mechanical methods. however, its harmful effects on the environment cause farmers to consider and accept mechanical methods of weed control. chemical weed control is the most widely used weed control method (olaoye and adekanye, 2012). current weed control practices since crops are usually not grown in rows, weeding is a time-consuming undertaking, up to 140 h/ha. ethiopian farmers use a variety of strategies to reduce or avoid weed invasion in their fields. current weed control methods are reviewed below. hand weeding manual weeding is the most common weed control method used by smallholders. it usually doesn’t require any upfront payments. this is a great advantage when cash is not readily available and labor is provided by the farming family directly or through a non-monetary exchange. this may be the only viable method of weeding on flies when herbicides are not available. manual weeding is intensive farming. hand weeding is time-consuming, labor-intensive, back-breaking, and often costlier than other methods of weed control. the oldest and simplest technology is manual weeding. manual weed control begins with farmers pulling weeds with their hands. later, this technology evolved into hand tools, moving from the use of a stick to the use of a hand pickaxe. cordilla and grift (2011) report that manual weeding is labor-intensive, accounting for about 80% of the total labor required to produce food. farmers who use only hand-hoes for weeding will struggle to escape poverty, as this level of technology tends to perpetuate human toil, risk, and mystery. the use of a sorting hoe is effective and is the most widely used weed control method. figure 1: hand weeding methods animal-drawn weeding implements kebede (2000) also reported that the three-tine cultivator with a reversible narrow shovel is attached to the frame by movable clamps, bolts, and nuts, designed to achieve width and depth. this includes a frame capable of adapting to different weeding attachments, two wheels for maneuverability, and provision for lifting and lowering tillage elements. the author has also studied a practice known as shilshalo that involves growing burdock in sorghum grown in rows or rows. traditional animal traction plows are used for inter-row farming. this breaks down the soil crust, reduces runoff, and speeds infiltration into the soil, in addition to controlling weeds and thinning crops to an appropriate level. however, since most farmers do not practice shilshalo at the correct stage of crop growth, severe plant damage (stem breakage and uprooting) often occurs, resulting in low yields. a series of trials have been carried out to improve existing traditional practices by determining the optimal timing of livestock operations to increase yields of sorghum and maize. mechanical weeding implements much effort has been made to design and develop convenient and practical mechanical weeding methods using simple tools and instruments. mechanical https://journals.e-palli.com/home/index.php/ajlsi pa ge 23 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 19-26, 2022 equipment can save time during peak times, resulting in higher output per worker and reduced weeding costs. this mechanical device can be pulled by hand or by an animal. kankal (2013) has researched the design and development of self-propelled weeding machines for field crops. the machine is useful in row crops, horticulture, and vegetables for weeding and seedbed preparation. it consists of a 4.1 kw diesel engine mounted on the chassis of the electric tiller, a drivetrain, two ms wheels, a frame, and a rotary tiller. power from the motor is transmitted to the rotor by belt and chain and through a gear transmission to the ground wheels. a disconnect device is provided to connect or disconnect the power to the rotating system. the lug wheels are provided for traction. the speed of the electric lawn mower ranges from 2.3 to 2.5 km/h with an effective working width of 550 mm for a field yield of 0.10 to 0.13 ha/h. the cost of a self-propelled weeding machine is around rs 40,000/ha and the average weeding cost is rs 1000/ha. the device saves 90% of operating time and 30% of weeding costs compared to manual weeding. machine weeding not only pulls weeds between rows of plants, but also keeps the soil surface loose, creates better aeration of the soil and increases water absorption, and the machine weeder does the job at the same time. weeding and hoeing can reduce weeding time, the cost of weeding, and the arduousness of manual weeding (shakya et al., 2016). sirmour and verma (2018) evaluated the performance of the developed electric lawn mower. it has been tested based on field capacity, field efficiency, weeding efficiency, activity index, energy consumption, and operating cost. therefore, the following conclusions can be drawn. the performance of the rice weeding machine is rated as excellent in wet conditions, the working width of the developed machine is expected to be adjustable between 140-250mm, and the forward speed is 2.48 km. / h and operating depth from 3 to 4.2 cm, with fuel consumption of 0.55 l / h. the effective yield is 0.054 ha/h. weeding efficiency is 82.92%, operating cost of rotary weeding machine and manual weeding machine is rs 980/ha and rs 2300/ha respectively. weeding cost saving is 60% and time-saving is 65% compared to manual weeding. verma and victor (2003) report that the rotary weeder reduces strain and ensures a comfortable posture of the operator when weeding and increases yields because of the field productivity and weeding efficiency of the machine was 0.0712 ha/h and 73%, respectively. kishore et al. (2018) studied and evaluated the activity of mechanical weeds in dry soil conditions. three weeds were evaluated initially on dry land maize with plots of 20 m × 10 m with row spacing of 60 cm. the actual productivity of electric weeders, rotary hoes, and star weeders are 0.0494 ha/h, 0.022 ha/h, and 0.021 ha/h, respectively. the field efficiency of electric weeders, hoes, and star weeder were 82.33%, 73.66%, and 80.76%, respectively. the electric weeder is more efficient in the field than the other two weeders. the weeding efficiency of electric weeders, wheel hoe, and star weeders are 78.4%, 74.0%, and 75.4%, respectively. the hoe has lower efficiency and lower operating costs than the three types of weeders. since weeding is more labor-intensive than other grasses, weeding may be given less priority than electric and star weeders. padole (2007) studied the comparison in-field performance between rotary power weeder and bullock drawn blade hoe. it was reported that effective field capacity of 0.14 ha/h (40% more than a bullock-drawn blade hoe), and field efficiency of 90% (34.1% more than that of a bullock-drawn blade hoe were obtained). the cost of operation was found to be rs 798.46 compared to 894.87 per ha by bullock-drawn blade hoe. hence, it was more economical and effective than bullock-drawn blade hoe as it saved 10.77% weeding cost; reduced plant damage up to 54.23%, and achieved weeding efficiency up to 92.76%. manuwa et al., (2009) designed and developed a power weeding machine with a working width of 0.24 m for weeding in row plantations. the effective field capacity, fuel consumption, and field efficiency of the machine are 0.53 ha/h, 0.7 l/h, and 95% respectively. alizadeh (2011) studied the performance of 4 types of mechanical rice weeders including single row cone weeders (w1), double row cone weeders (w2), rotary weeders (w3), and power weeders (w4) compared with manual weeders (w5). two local rice varieties and improved transplanted rice, hashemi and lai respectively, were selected for this study. the results showed that among the mechanized grasses, the highest weeding efficiency (84.33%) was with w4 and hybrid varieties, and the lowest value (72.80%) was measured with w3 and hashemi varieties. plants damaged by mechanical weeding were 3.83% compared with 0.13% for manual weeding. the highest effective field yields of 0.082 and 0.087 ha/h was measured with w4 and the lowest values of 0.0084 and 0.0088 ha/h was obtained with w5 respectively for the hashemi and hybrid. weeding costs decreased by 15.70, 38.51, 22.32, and 48.70% respectively for w1, w2, w3, and w4 compared to w5. chanakyan and mohanty (2017) conducted a study to evaluate the performance of mechanical power weeders. the weeding was done 35 days after transplanting in sandy loam soil. the various mean performance parameters such as speed of operation, effective field capacity, field efficiency, weeding efficiency, plant damage, and fuel consumption were observed to be 1.65 km/h, 0.065 ha/h, 78.9%, 84.8%, 4.12%, and 16.9 l/hr respectively. mechanical weeding is very effective because it reduces manual weeding, kills’ weeds, and also keeps the soil surface loose by providing aeration and water absorption to the soil. the availability and cost of labor for weed control are limiting its progress, and the development of an appropriate mechanized weeding method is imperative. the cost of weeding by mechanical weeding is about onethird that of manual weeding (thorat et al., 2014). https://journals.e-palli.com/home/index.php/ajlsi pa ge 24 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 19-26, 2022 figure 2: mechanical weeding machine manual weeding commonly used manual weeders consist of a hoe (pulland-push weeder) consisting of a steel blade (cultivator) mounted on a long wooden handle. these weeders are most useful when weeds are small and the soil is not too hard. attanda et al. (2013) developed and evaluated a handpush mechanical weeder to determine its performance indicators. field performance was compared with that of a traditional hand hoe in multiple tzpb-sr maize fields. the mean values of forwarding rate, actual field capacity, and weeding efficiency were 0.092 m/s, 0.028 ha/h and 75.17% for weed growth compared to 0.013 m/s, respectively. , 0.0059 ha / h and 77.98% for hand hoe. according to bhavin et al. (2016) to evaluate the field performance of manual weeding machines developed which performed for various performance parameters such as weeding capacity, efficiency weeding, draft requirements, and the welder’s performance index were taken into account during the tests. the weeding machine developed has a field yield of 0.0285 ha/h and the highest weeding efficiency (ie up to 80.42%). the required draft was 34.4 kg for a weed width of 20 cm and the obtained weed activity index was 1210.53. nkakini et al. (2010) studied the field performance evaluation of manual gasoline-powered weeders for the tropics. according to the report, the theoretical yield of the rotor weeding machine is 0.047 ha/h with an effective capacity of 0.34 ha/h, which is about 20 times higher than manual weeding. the operating index is 1700 and the fuel consumption is 3.2 liters in 8 hours. rotary weeders have a weeding efficiency of 71% at removing weeds with shallow roots. this suggests that motorized weeders can be useful equipment in agricultural modernization for smallholder farmers. mechanical weeding is an environmentally friendly method of weed control. bhavin et al. (2016) reported that mechanical methods are by far the most widely used means of weed control for many years to come. furthermore, the author points out that mechanical weeding is potentially more sustainable than chemical weeding because it is less risky (financial, health, and environmental) and easier to maintain with current skills and facilities. mechanical weeding is very effective because it reduces manual weeding, kill’s weeds, and also keeps the soil surface loose by providing aeration and water absorption to the soil. the availability and cost of labor for weed control are limiting its progress and therefore the development of a suitable mechanized weeding method is urgent. the cost of weeding with a motorized weeder is about one-third that of manual weeding (guru et al., 2018). mechanical methods of weed control are widely used in many developing countries because agricultural labor is cheap and readily available. mechanical weed control methods are simple for farmers. weed control tools and instruments are mainly hand and animal-operated. mechanical weed control involves the use of human labor, animal-drawn, or tractor-powered weeders, selfpropelled weeders, or electric weeders (sirmour, 2016). attanda et al. (2013) developed a mechanical weeder consisting of two sets of conical propellers, an adjustable main frame, and an afloat. the weeder has an effective capacity of 0.357 ha/hr, a draft of 64.87 n, and overall width and depth of cut of 180 mm and 20 mm, respectively. with one cut between rows in the field at 40.8% soil moisture, the optimal weed control efficiency was 84.5% while the weed control efficiency at 10.5% soil moisture was 53.1%. therefore, the highest plant damage of 8.33% was recorded at soil moisture of 10.5%. 0.058 hp is the power required for one person to push the prototype grass. alizadeh (2011) reported that manual weeding removed only 65-85% of weeds for cotton production, mainly because workers confused weeds with crops or lacked weeds. it has also been reported that manual weeding with a long-handled hoe will damage crops and miss some weeds. digging is also time-consuming and can lead to back injuries for workers. chemical weed controls chemical control involves the use of herbicides. herbicides control weeds by accelerating, stopping, or altering the normal growth patterns of crops; by drying the leaves or stems; or by making them drop their leaves. chemical control with the application of herbicides can provide the most effective and fastest method of weed management. many herbicides are available for effective weed control and are selective so that weeds are not harmed. weeding is one of the main activities of agriculture chemical weed control is becoming popular day by day https://journals.e-palli.com/home/index.php/ajlsi pa ge 25 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 19-26, 2022 in developing countries. some farmers have begun to use herbicides to control weeds, especially in doublecropping areas. however, herbicides are less effective than manual weeding, as they require specific conditions that can be more restrictive than other control methods. for example, the right herbicide must be selected for the specific spectrum of crops and weeds present. it must be applied at a specific rate, at the right time, and only under specific environmental conditions (kebede, 2000). fagade (2008) reported that the cost of applying herbicides to control weeds is half that of manual weeding. chemical methods give promising results in the eradication of weeds but are limited due to their harmful effects on humans and the environment. singh (2001) a states that herbicides can significantly reduce the need for labor, but there is a contradiction in their performance. inconsistencies include herbicide cost versus labor, and farmers’ lack of knowledge about application rates, timing, and methods. in addition, the lack of availability of herbicides and sprayers is one of the main factors limiting the use of herbicides by smallholder farmers. these limitations make mechanical weed control a priority over the use of herbicides. conclusions the main objective of this paper is to gain insight into the various aspects or limitations of different weeding techniques in order to reduce the efforts that farmers have spent in terms of money, labor, time, and money. weed control is one of the most difficult tasks in agriculture, accounting for a significant portion of agricultural production. weed losses exceed those caused by any other agricultural pest. three methods of weed control are commonly known in agriculture. here’s how to weed by machine, manually and chemically. machine weeding is easily adopted by farmers once they believe in its benefits. mechanical weeding not only removes weeds between rows of crops but also keeps the soil surface loose, ensuring better aeration and water absorption. weeds are also controlled mainly by manual weeding. manual weeding is the most efficient weeding method, but it is not suitable due to the time-consuming operations and labor-intensive costs. chemical methods give promising results in the eradication of weeds but are limited due to their harmful effects on humans and the environment. in general, weeding is one of the most essential and costly activities in agriculture. mechanized weeding reduces labor costs and also saves time compared to any other weeding method. in summary, weeding is one of the most essential and costly activities in agriculture. mechanized weeding reduces labor costs and also saves time compared to any other weeding method. therefore, instead of manual and chemical weeding, the use of a mechanical weeding machine should be most preferable. references ahmad, r., and shaikh, a.s. (2003). common weeds of wheat and their control. pakistan journal of water resources, 7(1),73-76. alizadeh, m.r., 2011. field performance evaluation of mechanical weeders in the paddy field. scientific research and essays, 6(25):5427-5434. atiq ahmad khan , muhammad zubair, a. rahim1 and a. wahab., 2009. comparison of different weed management practices in onion (allium cepa ) under agroclimatic conditions of dera ismail khan, pakistan. pak. j. weed sci. res. 15(1): 45-51 attanda, m. l.; muhammad, a. i. and shema, a., 2013. development and performance evaluation of hand pushed. journal of engineering and technology ( jet ), 8(1): 61–69. babu, p. k., & rao, s. g., 2017. manually operated dry land weeder : a study. international journal for modern trends in science and technology, 3(7):27-33. bhavin,ram,khardiwar, m.s., kumar, shailendra and solanki,b.p.,2016. performance evaluation of manual operated single row weeder for groundnut crop. engineering and technology in india, 7(1): 45–52. chanakyan, p.c. and mohanty. r, 2017. performance evaluation of power-operated wetland paddy weeder in odisha. international journal of multidisciplinary research and development international, 4(10):140-146. central statistical authority of ethiopia., 2013. report on area and production of major crops meher season. report on area and production of major crops meher season. addis ababa. cordill, c and grift, t. e., 2011. design and testing of an intra-row mechanical weeding machine for corn. biosystems engineering, 110(3): 247–252. duff, b. and orcino, n., 2001. economic research in the department of agricultural engineering. irri seminar. los banos, philippines. fagade, s.o., 2008. performances of some herbicides in the control of upland rice weed in nigeria. warda technical news letter, 2(2):9-10. gavali and kulkarni., 2014. comparative analysis of portable weeders. international journal of innovative research in science, engineering and technology, 3(4): 11004–11013. guru, p., patel, s., nayak, a., kumar, a.,& gautam, p., 2018. development and evaluation of power operated weeder in rice. oryzaan international journal on rice, 55(2): 317–323. hegazy.r. a. , i. a. abdelmotaleb, z. m. imara, m. h. o., 2014. development and evaluation of small–scale power weeder. misr j. ag. eng, 31(3): 703–728. kankal, u. s., 2013. design and development of self propelled weeder for field crops. international journal of agricultural engineering, 6(2): 304–310. kebede desta., 2000. animal power for weed control.a resource book of the animal traction network for eastern and southern africa (atnesa). wageningen, the netherlands. kishore. g, kumar, r. k. and chamn, m. k., 2018. performance and evaluation of power weeder, under dryland conditions. international journal of current https://journals.e-palli.com/home/index.php/ajlsi pa ge 26 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 19-26, 2022 microbiology and applied sciences,7(12): 1669–1675. kurstjens, d. a. g., and perdok, u. d., 2000. the selective soil covering mechanism of weed harrows on sandy soil. soil & tillage research, 55: 193–206. lopez.f., 2011. weed detection for site-specific weed management, mapping and real-time approaches. weed research, 51: 1–11. manuwa,s.i, odubanjo &s. g. o., 2009. development and evaluation of a row-crop mechanical weeder. journal of engineering and applied sciences, 4(4): 236–239. nkakini, s. o., ayotamuno and akor.a.t., 2010. field performance evaluation of manual operated petrol engine powered weeder for the tropics. agricultural mechanization in asia,africa & latin america, 41(4): 68–73. ofor, m.o., ibeawuchi, i.i. and oparaeke, a.m., 2009. crop protection problems in production of maize and guinea corn in northern guinea savanna of nigeria and control measures. nature and science, 7(12), pp.8-14. olaoye. j.o. and adekanye, t.a., 2012. development and evaluation of a rotary power weeder. environmental engineering journal, 1(2):129–141. olukunle, j., and oguntunde, p. g., 2006. design of a row crop weeder. confer ence on international agricultural research for development ,tropentag 2006 university of bonn. pantage.s., thokale, p. j., and deshmukh, v. d., 2015. performance evaluation of self propelled rotary weeder. international journal of agricultural engineering, 8(1):70–74. padole,y.b.,2007. performance evaluation of rotary power weeder. agricultural engineering today, 31(3and4): 30-33. singh, r., 2001. studies on rotary weeder in wheat crop. punjab agricultural university, ludhiana, india. sirmour and verma., 2018. design and development of single row power weeder for rice. journal of crop and weed, 14(1): 163–169. sirmour aditya., 2016. design and development of single row power weeder for rice. m. tech. (agril. engg.) thesis. india. thorat deepak p. k. sahoo, d. d. and m. a. i., 2014. design and development of ridge profile power weeder. journal of agricultural engineering, 56(4). verma. a and prabhat, k.g., 2015. development and evaluation of cultivator cum. ecology, environment and conservation, 21(3): 1359–1364. verma and victor., 2003. design and development of powe operated rotary weeder for wet land paddy. agricultural mechanization in asia, africa and latin america, 34(4): 27–29. https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 1 american journal of life science and innovation (ajlsi) a study of the phytoremediation process using water lettuce (pistia stratiotespistia stratiotes) in the removal of ciprofloxacin vimbai masiyambiri1, bachir yaou balarabe2*, irédon adjama1, hassimi moussa3, maman nasser illiassou oumarou1, abdoul moumouni iro sodo4 volume 2 issue 1, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i1.1092 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: december 19, 2022 accepted: december 26, 2022 published: january 07, 2023 the use of antibiotics has become imperative and unavoidable in medicine to figureht against microbes, but the majority of these antibiotics are found in environmental ecosystems. it is revealed that the presence of these in the environment, intoxicates the bacterial ecological medium.then, this investigated the phytoremediation abilities of water lettuce (pistia stratiotes). adolescent plants were placed in two different concentrations of ciprofloxacin solution for 7 days. the aim was to see if the plant could remove the ciprofloxacin, what amount of it and the effects of the drug on the plant thereafter. the concentrations were 50ppm and 10ppm of ciprofloxacin. the result was that at 50ppm, the plants developed necrosis within 3 days and died. at 10ppm solution, water lettuce managed more than 70% removal efficiency, and also a steady growth of the plant was maintained at 0.1606 g/day. for the concentration of ciprofloxacin, analysis of sample water was done using uv-visible spectroscopy and plant extract was analyzed by hplc. the study proved that water lettuce can be used as a remediation technique for surface waters, or can be an end-of-pipe measure for pharmaceutical wastewater treatment facilities before discharge into surface waters. keywords phytoremediation, removal efficiency, pistia stratiotes, ciprofloxacin introduction freshwater reservoirs are being depleted and ocean temperatures are rising, causing water pollution. as a result of anthropogenic activities, water is polluted 1. adverse effects have been observed on the water supply. these include lakes, rivers, oceans, aquifers, reservoirs, and groundwater. when contaminants are introduced into bodies of water, it leads to pollution. the pollution control industry has seen an increase in the last few years as a result of rising concerns. untreated wastewater and industrial effluents. there are a number of ways in which antibiotics enter the environment. among them are direct human or animal excretion, animal manure applied to crops as fertilizer, municipal wastewater treatment plants, hospitals, and manufacturing plants (balarabe & maity, 2022; booth et al., 2020; kraemer et al., 2019). with the rapid development of pharmaceutical waste, a growing threat is posed to surface and groundwater resources with adverse effects on aquatic ecosystems (balarabe et al., 2022; jin & aslam, 2019). as most antibiotics have active ingredients that dissolve in water, they can be transmitted into aquatic food webs as well. bioaccumulation poses a threat to public health, destroys aquatic flora and fauna, and leads to drug-resistant waterborne diseases. several pharmaceutical wastes end up in the environment, including antibiotics, hormone wastes, and analgesics, from inappropriately disposed of pharmaceuticals, unused or expired tablets, and unprescribed pills. tetracycline, oxytetracycline, ibuprofen, ciprofloxacin, and norfloxacin are some of the antibiotics that persist in wastewater after treatment (shikha & gauba, 2016). to classify fluoroquinolones, it is necessary to examine their spectrum of activity as well as their pharmacokinetic profile. a fluoroquinolone-type antibiotic known as ciprofloxacin is an antibiotic that has broad antibacterial activity against both gram-positive and gram-negative bacteria (wu et al., 2008). with well-established safety features, ciprofloxacin is a promising and effective antibiotic. having effectively treated over 250 million people globally, its safety profile has been extensively documented in a large number of scientific articles. ciprofloxacin inhibits dna gyrase, which is needed for disease replication. after oral treatment, ciprofloxacin is rarely absorbed completely. ciprofloxacin has an absolute bioavailability of 70–80 percent, with no significant loss due to first-pass metabolism (sharma et al., 2009). many traditional cleanup procedures do not provide adequate solutions to pollution in water and soil today. pharmaceutical and industrial waste products accumulate in the land, air, and water, destroying plants and causing health problems. heavy metal toxins, antibiotics, hormonal wastes, and pharmaceuticals are among the pollutants. as part of the phytoremediation process, plants are utilized in soil, sediment, and water to remove, transport, stabilize, and decompose pollutants deposited in them through the use of plants (shikha & gauba, 2016). restoration of the environment with plants is centuries old and cannot be credited to any individual. a phytoremediation method is environmentally friendly, cost-effective, and promising. an example of phytoremediation is the use of plants to treat contaminated environments when they are naturally occurring or genetically modified. there is a growing 1 school of pharmacy, national forensic sciences university, sector-09, gandhinagar, india 2 school of engineering and technology, national forensic sciences university, sector-09, gandhinagar, india 3 département des sciences de l’environnement, faculté des sciences agronomiques, université boubakar bâ, tillabéri, niger 4 international institute of tropical agriculture (iita), university of ibadan, pmb 5320, ibadan, oyo state, nigeria * corresponding author’s e-mail: yaoubalarabe@gmail.com https://doi.org/10.54536/ajlsi.v2i1.1092 https://journals.e-palli.com/home/index.php/ajlsi pa ge 2 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 1-8, 2023 interest in phytoremediation, in which macrophyte plants are used in constructed wetlands and stormwater detention ponds to treat eutrophic waterways (tanmay sanyal & saha, 2022) phytoremediation has evolved recently. the concept of phytoextraction was developed by singh & santal, an approach that uses plants to absorb pollutants into their biomass. pollutants are absorbed by plants and stored in their aerial portions, after harvesting, the plant is discarded (kumar et al., 2018) . rhizofiltration involves the root system of plants interacting with toxins to remove pollution. this technology has the potential to reduce the bioavailability of organic and inorganic contaminants. rhizofiltration leaves the pollutant on/ in the root. in phytovolatilization, toxins are absorbed from the soil, converted to a volatile form, and released into the atmosphere (a. yan et al., 2020). then, plants involved in process requires a dense root system (radziemska et al., 2017) (a. yan et al., 2020). as well as the degradation of contaminants in soil, groundwater, and surface waters, phytodegradation is the enzymemediated uptake and breakdown of pollutants within plants. plants and accompanying microbes digest organic pollutants to transform them into harmless forms. plant roots absorb a considerable amount of contaminants. there are thousands of pistia stratiotes (water lettuce) floating in the ocean. it spreads rapidly in nutrientcontaminated water. due to its availability and ability to withstand temperatures up to 30°c, water lettuce can treat wastewater. it grows in massive colonies on water as an araceae macrophyte. if left unchecked, these colonies can be invasive. while a dense root network absorbs/adsorbs contaminants from water, hydrophobic leaf surfaces keep it afloat (galal & farahat, 2015) and (mustafa & hayder, 2021). phytoremediation, therefore, is an environmentally friendly, cheap, efficient, and effective way to remove antibiotics from contaminated water (ansari et al., 2020). industrial, household and agricultural wastewater have been treated with pistia stratiotes. this plant is widely used because of its availability, durability in toxic environments, bioaccumulation potential, and invasive properties (mustafa & hayder, 2021). in a lab test, (gowri et al., 2020) found that water lettuce can be used to purify eutrophic surface water, but not for drinking. to name a few, water lettuce reduced or increased bod, cod, ph, nitrates, phosphates, and tds. a study by (kumar et al., 2018) found that water lettuce (p. stratiotes) can remove heavy metal contamination. a 75% maximum extraction of heavy metal was from the water. according to (upadhyay & panda, 2009), copper on water lettuce could be a bioindicator for copper levels in surface water. (odjegba & fasidi, 2004) tested the effectiveness of water lettuce for the removal of heavy metals was tested. it was found that the rate of leaf growth was found to be reduced when metal type, concentrations, and exposure time were increased. this study aims to remove ciprofloxacin from a hydroponic nutrient solution by water lettuce. material and method all the materials and solvents were purchased from commercial sources (finer chemicals, india, sisco research laboratories pvt. ltd., india, sigma aldrich, and abhishek enterprise pvt. ltd.) and used as received without purification. distilled water was used as the solvent for ciprofloxacin and the hoagland solution in which the plants were grown. for standardization in hplc, milli-q water was used as a solvent as required by the hplc protocol. milli-q water and spectroscopic grade solvents were used for all measurements. water lettuce (pistia stratiotes) plants the adolescent water lettuce plants were obtained from umarose nursery and farm, gandhinagar, gujarat. the plants were washed thoroughly and grown in a hydroponic solution for 1 week prior to exposure to a ciprofloxacin solution. preparation and characterization of hoagland solution pistia stratiotes plant life was sustained in a hydroponic system by using a hoagland solution which is prepared based on the modified protocol of hoagland and amon in 1950 (seth et al., 2011). the nutrients were made separately into stock solutions and the working solution was mixed accordingly (table 1). table 1: hoagland solution composition: the stock and working solutions. nutrient stock solution (g/100ml) working solution (ml/l) macro-nutrients calcium nitrate potassium nitrate magnesium sulfate monopotassium phosphate 23.61 5.02 24.64 1.31 2.50 2.50 1.00 1.00 micronutrients boric acid manganese sulfate zinc sulfate copper (2) sulfate molybdic acid edta-k salt ferric sulfate 2.86 1.54 0.22 0.08 0.09 2.50 2.50 1.00 1.00 1.00 1.00 1.00 1.00 1.00 preparation and characterization of ciprofloxacin hcl the ciprofloxacin hcl used was obtained from abaris healthcare pvt. ltd., mehsana, india. ciprofloxacin is insoluble in water, therefore, the study utilized ciprofloxacin hydrochloric powder. the study targeted the degradation of a 10ppm solution of ciprofloxacin. https://journals.e-palli.com/home/index.php/ajlsi pa ge 3 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 1-8, 2023 uv-visible spectroscopy was used to analyze a 10ppm solution of ciprofloxacin and single distilled water at fullspectrum analysis (200-800nm) to find the absorbance peak for ciprofloxacin. the same analysis was also done using 10ppm ciprofloxacin and hoagland solution. highperformance liquid chromatography (hplc) was used to characterized ciprofloxacin according to the protocol described by (wu et al., 2008). development of ciprofloxacin calibration curve to develop a standard for ciprofloxacin, 2 calibration curves were plotted using results from uv-visible spectrometry and hplc analysis. the standard determined the key concentration to use in characterization studies. a sample of 1mg/10ml was used to create a 100ppm stock solution, from which 2, 4, 6,8, and 10ppm working solutions were derived to create the calibration curve. the absorbance for ciprofloxacin was determined at 271nm. preparation of citrate phosphate buffer to check the availability of ciprofloxacin in the plant, the plant extract was derived using a citric-dihydrogen phosphate buffer called mcilvaine buffer after its creator, theodore mcilvaine in 1921. development and use were done following the protocol by (mcilvaine, 1921; y. yan et al., 2021). experimental procedure pistia stratiotes plants were grown in two different concentrations, 50 ppm, and 10 ppm concentrations. the first was to introduce plants to a slightly high concentration, to determine the level of toxicity water lettuce can withstand. the second concentration was primarily the focus of the study, to see if and what amount of the ciprofloxacin could be removed by the plant from water. it was a test of its phytoremediation capability. plants were monitored for 7 days for both parameters. water lettuce was grown at ambient temperature. the ph was monitored as the plant needs a ph of 6.5-7.5 to grow. water loss through evapotranspiration was refilled with distilled water and hoagland solution. readings for uvvisible spectroscopy were taken initially from ciprofloxacin solution prior to transplanting the plants. on the 7th day, another uv-visible reading was done to check the amount of ciprofloxacin left. the ph reading was carried out every day because ciprofloxacin hcl is acidic and acidity could kill the plant. foil paper was used on samples to reduce photodegradation of ciprofloxacin as shown in the experimental set-up. the pistia stratiotes resilience by taking initial and final growth fresh weights and calculating the growth per day. the growth was monitored in the 7 days the plant was exposed to ciprofloxacin. figure 1: experimental setup: (a) distilled water mixed with ciprofloxacin (at 10 ppm) under the sun; (b) distilled water mixed with ciprofloxacin (at 10 ppm) without the sun; (c) distilled water mixed with holang solution (at 10 ppm) under the sun; (d) distilled water mixed with holang solution (at 10 ppm) without the sun; (e) holang solution (at 10 ppm) with pistia stratiotes under the sun; (f) holang solution (at 10 ppm) with pistia stratiotes without the sun; (g) ciprofloxacin, holang solution (at 10 ppm) and pistia stratiotes under the sun; (h) ciprofloxacin, holang solution (at 10 ppm) and pistia stratiotes without the sun. results and discussion ciprofloxacin concentrations of 5, 10, 20, 30, 40, and 50ppm were tested. figure. 2a shows that the higher the concentration of ciprofloxacin, the more difficult it is for the plant to survive. the plant grows normally up to 10pmmin the ciprofloxacin solution, plants developed chlorosis and necrosis within the first 3 days and died. plants suffer toxicity from absorbing fluorine, which explains this. plant growth is illustrated in figure. 2a&b at 10ppm and 50ppm, respectively. fluorine is a determining factor in the structure of ciprofloxacin, which is a fluoroquinolone (sharma et al., 2009). plants sensitive to fluorine are susceptible to necrotic lesions, burning, chlorosis, leaf damage, and development and https://journals.e-palli.com/home/index.php/ajlsi pa ge 4 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 1-8, 2023 reproductive suppression (banerjee & roychoudhury, 2019). it became apparent that the water lettuce samples that contained 50ppm will be effective as bioindicators in the future (galal & farahat, 2015). uvvis spectroscopy indicated a lambda max of 271nm for 10ppm ciprofloxacin solution and a maximum absorbance of 0.8573111 (figure. 3a). the lambda max was also found to be 271nm for ciprofloxacin and figure 2: (a)-water lettuce survival analysis, (b)-normal growth of water lettuce in 10ppm cipro solution, (c)-dying of water lettuce plants in 50ppm cipro solution. hoagland solution at 10ppm, showing almost the same absorbance. in figure. 3c, the calibration curve at different concentrations (2, 4, 6, 8, and 10ppm) was plotted and the equation was y= 0.0844x + 0.0136 with an r2 of 0.9992. the uv-visible reading of day 7 indicated that there had been a significant decrease in uv absorption. the degradation efficiency of the can be defined as degradation efficiency (%) = (c0-ct)/c0 × 100% 2, where: c0 is the cipro concentration at 10ppm, and ct is the residual concentration of cipro after 7 days. in figure. 3d, the degradation efficiency of the treatments s1, s2, s3, s4, s7, and s8 has been shown. it is evident from treatments 1 and 2 that light contributes to ciprofloxacin degradation. 3.62% of cipro removable was attributed to light. furthermore, when hoagland’s solution was added (s3 treatment), the degradation rate increased from 3.62% to 5.69%. iron present in hoagland’s solution may act as a reducing agent. combined with light + hoagland’s solution + water lettuce, significant degradation occurs. therefore, water lettuce is able to absorb 71.92% (treatment s7) of ciprofloxacin compared to 66.60% without light (treatment s7). in addition, this illustrates how light influences ciprofloxacin degradation. after this, on the 7th day, water lettuce from treatment s7 was harvested and dried at ambient temperatures. this took 4 days for the plants to be completely dry. the dried figure 3 : (a)-uvvis spectroscopy of cipro solution, (b)-uvvis spectroscopy of cipro and hoagland solution, (c)-calibration curve of cipro solution in uv-vis spectroscopy and (d)-cipro removable efficiency per treatment. plant was then prepared for hplc using the protocol which uses mcilvaine buffer to get plant extract (a. yan et al., 2020). a mortar was used to fine-grind dried plant samples. the plant powder was then sifted and placed in a centrifuge tube. 0.1 molar of mcilvaine buffer at ph 3 was prepared up to 20 ml, then added to the plant sample. this was sonicated for 10 min and placed in a centrifuge for 10 min with extraction done 3 times. the extract was filtered using whatman’s filter paper. the clear plant extract was analyzed by hplc to determine ciprofloxacin content. bypassing the plant extract through hplc, it was observed that a peak synonymous with ciprofloxacin was observed (figure. 4a&b). this proved beyond doubt that water lettuce had the ability to absorb ciprofloxacin from water. the peak observed in hplc is shown in figure. 3a&b. the time of the peak https://journals.e-palli.com/home/index.php/ajlsi pa ge 5 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 1-8, 2023 during calibration is the same as the time observed from the plant extract. this observation means that the same compound (ciprofloxacin) was retained, eluted, and detected in both instances. from the standard curve of the 2, 4, 6, 8, and 100 in figure. 4c with equation y= 33519x34322 and r2= 0.9963, the peak from the water lettuce plant extract in figure. 4b corresponded with the peak for 8ppm concentration. therefore, the concentration of ciprofloxacin in the water lettuce plant extract can be calculated from the standard curve equation. according to hplc, the amount of ciprofloxacin present in the plant after 7 days was 7.78 ppm. this supports the hypothesis that water lettuce can be used as a phytoremediation strategy to cleanse wastewater that has ciprofloxacin. the removal efficiency indicates 77.8%, which is similar to the efficiency percentage obtained by uv-visible spectroscopy. there was also confirmation from a mass spectrometry analysis of degraded cipro in the dye solution that no significant smaller fragments are present as a result of this degradation process (figure. 4d&e). the following table 2 presents a brief summary of the previous studies, the methods used, and the results derived from these studies. during the 7-day period, the water lettuce plant had accumulative growth figure 4: (a)-hplc peak of 10 ppm cipro concentration, (b)plant extract analysis in hplc, (c)-calibration curve of cipro in hplc and mass spectrometry analysis of cipro (d)-before and (e)-after 7 days. of 0.1606grams each day. this growth was not deterred by the effects of ciprofloxacin, which means that it is a hyperaccumulator. the relative growth rate (g/d) = (w2w1/ t2-t1) (kumar et al., 2018). where w1 (9.53167g) is the initial mass of fresh plants, w2 is the final mass of fresh plants (8.5676g); t1 is day 1 and t2, is the last day. the relative growth rate is 0.1606grams/day. the bioconcentration factor or bioaccumulation factor is calculated to determine if a plant is a hyperaccumulator. this means that the plant biomass will not be disturbed by the amount of pollutant accumulation at a particular concentration of said pollutant. in other words, it is a ratio of the contaminant in the plant in relation to its concentration in the water. for hyperaccumulators, the bcf is more than 1. the bcf = chplc / cuv. where chplc (7.78ppm) is the contaminant concentration in plant tissue (hplc result) and cuv (2.69ppm) is the contaminant concentration in wastewater (uv result). for this particular study, the bcf for water lettuce was 2.89. bcf is more than 1 means that water lettuce is a hyperaccumulator and can be used to reduce bioavailability of ciprofloxacin in affected waters. bcf is also important as it shows the impact or risk to the ecosystem under threat from a contaminant. table 2: a summary of previous studies serial no. location experimental parameters analytical technique summary references 1 uttarakhand, india removal of selected metals copper, iron, and mercury using water lettuce absorbances recorded using uvvis spectroscopy water lettuce managed to effectively remediate synthetic and industrial wastewater (kumar et al., 2018) 2 vanarasi, india 5 heavy metals (cu, cr, fe, zn, cd) in 3 different concentrations (1.0, 2.0, 5.0mg-l atomic absorption spectrophotometer, uv-vis, extraction air acetylene flame method water lettuce along with two other aquatic plants showed that it was highly effective as a phytoremediator, without damage from toxicity. (mishra & tripathi, 2008) https://journals.e-palli.com/home/index.php/ajlsi pa ge 6 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 1-8, 2023 3 lagos, nigeria exposure of live plants to crude oil (0–100 ppm) for 28 days at a normal temperature of 30 ± 2c. total hydrogen content (thc) and metal ion concentration were measured using aas. crude oil was toxic to the plant. using growth and cell division water lettuce can be used as a bio-indicator in water. (akapo et al., 201 c.e.) 4 ankara, turkey water lettuce exposed to different concentrations of cadmium and lead icp-ms used to analyze plant extract water lettuce was successful in removing heavy metals at moderate concentrations (ali et al., 2020) 5 fort pierce, usa 2 plots in 2 different stormwater detention plots. 1plot with water lettuce plants, analysis of water samples weekly for 22 months icp-oes 20% reduction in metals in water. the highest accumulation was of cr (lu et al., 2010) 6 prague, czech republic 8 variants were set up. plans were grown in hoagland solution. harvesting of plants for analysis on days, 2 4 and 8 icp-oes, uv-vis spectroscopy pb accumulation by rhizofiltration. chlorosis due to increased pb accumulation (veselý et al., 2013) 7 alexandria, egypt 3 experimental units with water lettuce. growth monitored for 7 days. physicochemical parameters of wastewater analyzed high removal rate of fe, cu, zn. reduction of tn and tp and removal of hno3 gaballah et al., 2019) 8 nigeria using water lettuce to treat wastewater from rubber industry effluent for 3 years aas successful in reduction of water perimeters to who permissible limits (owamah et al., 2014) 9 shanghai, bangkok, weekly sampling of physicochemical properties of water under study3 macrophytes. 6months in 3 separate tanks. analysis after every 10 days water parameters analyzed water lettuce exhibited the highest efficiency removal of phosphorus. high nitrogen removal was attributed to its dense root system which encouraged microbial activity for denitrification (lu et al., 2010) 10 thailand china water lettuce grown and analyzed for 7 days with different chlorpyrifos concentrations gc-ecd water lettuce growth and removal efficiency was dose dependent. img + concentration of the pesticide was toxic. (prabakaran et al., 2019) 11 gujarat india water lettuce grown and analyzed for 7 days with different 10ppm ciprofloxacin uv-vis, hplc, gc-ms water lettuce growth and removal efficiency was approx. 70% this work conclusion phytoremediation of ciprofloxacin using water lettuce was achieved in the study. the study supports earlier work mentioned above that macrophytes can remediate surface waters. the study was done under ambient temperatures. the variable that was maintained was the ph. the plants need ph of between 6.5 7.5. as the study was done during the month of may, one of the hottest months for gujarat, india, it showed resilience for high temperatures. in order to maintain the sustainability of contaminated large-scale landscapes and damaged aquatic ecosystems, phytoremediation is a practical and economical method of cleanup that uses macrophytes like water lettuce. water lettuce is an invasive macrophyte which grows in most tropical regions. by harnessing macrophytes to remediate surface water, not only do ecosystems benefit, it is also an investment in future environmental sustainability. research is needed to find out if water lettuce can remediate more pharmaceutical waste. the performance of the plant in a field study on pharmaceutical waste water needs to be studied. in the above study, ciprofloxacin interactions with the rhizosphere and plant tissue were not explored. compliance with ethical standards. declaration of competing interest the authors declare that they have no known competing https://journals.e-palli.com/home/index.php/ajlsi pa ge 7 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 1-8, 2023 financial interests or personal relationships that could have appeared to influence the work reported in this paper. data availability data will be made available on request. reference akapo, a. a.-r., omidij, s. o., & otitoloju, a. a. 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(2008, july 1). analysis of ciprofloxacin by a simple highperformance liquid chromatography method | journal of chromatographic science | oxford academic. https://academic.oup.com/chromsci/ article/46/6/490/314753 yan, a., wang, y., tan, s. n., mohd yusof, m. l., ghosh, s., & chen, z. (2020). phytoremediation: a promising approach for revegetation of heavy metal-polluted land. frontiers in plant science, 11. https://www. frontiersin.org/articles/10.3389/fpls.2020.00359 yan, y., deng, y., li, w., du, w., gu, y., li, j., & xu, x. (2021). phytoremediation of antibioticcontaminated wastewater: insight into the comparison of ciprofloxacin absorption, migration, and transformation process at different growth stages of e. crassipes. chemosphere, 283, 131192. https://doi. org/10.1016/j.chemosphere.2021.131192 https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 30 american journal of life science and innovation (ajlsi) development and performance evaluation of a tractor-drawn multi-crop planter bedasa waldaho1*, adesoji m. olaniyan2 volume 2 issue 3, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i3.2151 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: october 10, 2023 accepted: november 07, 2023 published: november 14, 2023 the study was undertaken development and performance of the planter capable of planting maize, common bean and sorghum seeds at predetermined spacing and depths. physical properties of seeds involved in the study were investigated to optimize the design of the planter’s components. the planter, consists of a frame, seed hopper, seed metering devices, seed tube, adjustable furrow opener, adjustable furrow covering device, and drive wheels. the investigation revealed that the sphericity of maize, common bean and sorghum were 64.8, 72.4 and 81%, respectively. percentages of mechanically seed damaged by the planter were zero for all crops. germination test was conducted to assess the magnitude and extent of invisible seed damage inflicted by the planter indicated mean percentage seed germination of 98.5, 94.5 and 97.3% for maize, common bean, and sorghum, respectively. the reduction in percentage germination of maize, common bean and sorghum were zero, when compared with that did not passes through the machine for all the seeds tested. based on the above results, it is concluded that the planter can be efficiently and effectively used by the majority of the farmers and other stakeholders in the study area. keywords capacity, efficiency, multi-crop, planter introduction planting is an art of placing seeds in the soil to have good germination. it was began with the use of hands and later the use of stones, hand tools and mechanized form of planting (yasir et al., 2012). manual methods of planting resulted in low seed placement, low spacing efficiency, and health issues for the farmer considering the size of the farm land (kumar et al., 2015; soyoye et al., 2016). seed planting machine is a device which helps in sowing seeds in a desired position, there by assisting the farmers in saving time and reducing cost. the basic objective of sowing operation is to put the seed in rows at desired depth and seed to seed spacing, cover the seeds with soil and provide proper compaction over the seed (soyoye et al., 2016). however, in fabricating the form of this mechanized planting equipment, some properties of the plant which is to be planted must be determined in order to accurately specify the design considerations ( jouki and khazaei, 2012). the physical properties such as seed size, shape, axial dimensions, roundness and sphericity helps to determine the maximum size of the cup in the seed plate, the weight help in the material selection for the frame of the planter, the bulk density and moisture content helps to know the interaction between the seed and the material used for the hopper of the planter at maximum heat level (jayan and kumar, 2004). the basic objective of sowing operation is to put the seed in rows at desired depth and seed to seed spacing, cover the seeds with soil and provide proper compaction over the seed (soyoye et al., 2016). however, in fabricating the form of this mechanized planting equipment, some properties of the plant which is to be planted must be determined in order to accurately specify the design considerations (jouki and khazaei, 2012). the physical properties such as seed size, shape, axial dimensions, roundness and sphericity helps to determine the maximum size of the cup in the seed plate, the weight help in the material selection for the frame of the planter, the bulk density and moisture content helps to know the interaction between the seed and the material used for the hopper of the planter at maximum heat level (jayan and kumar, 2004). the design and manufacture of tractor-mounted planters has eliminated most of the limitation attached to the manual methods. thus, the need for appropriate technology to deliver optimal yield while using fewer resources is very essential. hence, this research project is intended to bridge the existing technology gaps in the area of crop establishment. the objectives of this thesis research was to develop and characterize the physical properties of selected seeds for tractordrawn multicrop planter and evaluate performance of the machine. materials and methods experimental site and experimental crops fabrication of the prototype planter was done at fadis agricultural research center (farc) metal workshop (harar) maize, common bean and sorghum seeds were used to design the planter that was fabricated at farc metal workshop. the crops, maize, common bean and sorghum, were selected for the study because of their dominance among row planted crops in the study areas. hence, the planting machine was designed to plant these seeds. the varieties of maize, common bean and sorghum seeds were melkassa-2, awash-2 and melkem, respectively. selected seeds were taken from fedis agricultural research centre, crop research process and the seeds had germination percentages of 98.5%, 94.5%, 1 oromia agricultural research institute, fades agricultural research centre p.o. box 904, harar, ethiopia 2 haramaya university institute of technology p.o. box 38 dire-dawa, ethiopia * corresponding author’s e-mail: bedasawaldawo12@gmail.com pa ge 31 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 30-38, 2023 and 97.33%, for maize, common bean and sorghum, respectively. the planting machine the machine was designed for four wheels 30 hp tractor and has three different replaceable seed metering cups that were designed to handle maize, common bean and sorghum seeds. since the planter was designed for three different crops and was adjustable as particular crop’s row spacing requirements. physical properties of selected crops the physical properties of seeds are important factors for the design of crop planter. performance of seed metering mechanism in terms of picking, metering and dropping was influenced by the physical and mechanical properties of seeds. therefore, seed properties relevant to the design of planter were identified and determined. the general procedure was used in determining seed physical parameters i.e. geometric mean diameter (ds), sphericity (s) and volume were computed by taking a specified number of randomly selected seed. then their three principal diameters (major, intermediate and minor lengths) denoted as mean length (l), mean width (w) and mean thickness (t) respectively were measured using a micrometer of 0.01 mm accuracy. the measured values were then used to determine geometric mean diameter (ds) using equation (1) by (singh et al., 2005) as well as mean volume (v) and mean seed sphericity (sm). thirty (30) grain samples were selected randomly from the sample and dimensions were measured. length, width and thickness of the seed were taken using digital vernier caliper. the mean and standard deviation of dimensions were calculated. size, sphericity and surface area the volume and sphericity of the individual seeds were calculated using the measured larger diameter, medium diameter and smaller diameter of the seeds and equations given below (el-raie et al., 1996). physical property, that is, geometrical mean dg = ∛(l×w×t) (1) v = π/6 (l×w×t) (2) sm = (∛(l×w×t)/l)×100 (3) where: l=length (mm) w = width (mm) t = thickness (mm) v = volume (mm3) dg = geometric diameter (mm) sm = seed sphericity bulk density of the crops the bulk density was found by taking crops in a container of cylindrical shape. the volume of the container was found by measuring diameter and height for cylindrical container. the weight of the grain in the container was found separately. the bulk density was calculated, three samples from each selected crops were taken and average bulk density was calculated (varnmakasti et al., 2007) as shown equation (4) bd = (wc )/vc (4) where: bd = bulk density in kg/m3 or g/cm3 wc = weight of sample in kg or g vc = volume of sample in m3 or cm3 angle of repose of the crops the equipment used for measuring angle of repose consisted of a funnel with an adjustable throat opening mounted on a stand. the funnel was filled with seeds by keeping its adjustable throat closed. the throat was fully opened to allow free flow of seeds over and around the plate mounted beneath the funnel. at the end of process, a heap-cone of the seed was formed on the plate. from the heap-cone, base diameter and height of cone were measured. for free-flowing agricultural grains, angle repose is assumed to be approximately 28. for free-flowing grains the angle of repose can be assumed to be equal to that of the angle of internal friction. the angle of repose was calculated using the equation (6), (varnmakasti et al., 2007) θ = tan-1(2h/d) (5) where: 𝜃 = is angle of repose, degree h =is height of cone, mm d =is base diameter of cone, mm thousand grain mass in the laboratory thousand (1000) grains were selected randomly and then weighed on the sensitive weight balance to obtain the thousand grain mass in gram. the ten sample of each crop was weighed and mean thousand grain mass of each crop was found out. moisture content of soil the samples were collected from 0 to 15 cm depth of soil surface before operations for determination of moisture content and bulk density. the soil moisture was determined by oven dry method. five samples were collected randomly from the test plots. the samples were kept in oven for 24 hours at temperature of 105°c and weighed before and after drying. the moisture content (dry basis) was determined by the following formula (rangapara, 2014). mc (%) = (ws-wd)/wd *100 (6) where: mc=moisture content of the soil sample ws= weight of the soil sample, and wd= weight of dry soil sample bulk density of soil to determine bulk density of a soil, metallic core sampler was used to take sample from field having 8cm diameter and 12 cm height. the samples were weighed and dry weights of the samples were calculated with the help of moisture content (d.b.). the ratio of dry weight of soil pa ge 32 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 30-38, 2023 to the volume gave the bulk density. bulk density of soil was calculated by using following formula (rangapara j, 2014). bds = (ws (g))/(vs) (7) where : bds= bulk dencity of soil in ( g/cm3 ) ws = weight of soil samples (g) vs= volume of soil in core sampler (cm3) description of the machine and design considerations overall structure of the machine the developed tractor drawn multi-crop planter consists of the frame, seed hoppers, drive/ transport wheels, seed metering devices seed discharge tubes, furrow openers, and furrow covering devices. tractor drawn multi crop planter was designed as a functional and experimental unit. the design of machine components were based on the principles of operations. it was compared with the conventional method, to give a correct shape of the planter components. the mechanical design details were also given with due attention so that it gaves adequate functional strength for the design of the machine. to achieve the best performance from the planter, the important factors were optimized by proper design and selection of the components required to suit the requirements of the crop needs and figure 1 and figure 2 shows assembly parts and detail views of the planter respectively. 1) ground wheel (2) hopper (3) frame (4) furrow covering (5) tractor hitching position (6) ground wheel shaft. figure 1: assembly drawing of the prototype planter. figure 2: detail views (top, front and side) of the prototype planter working principles of the machine the seed metering mechanism of the planter is a cup type vertical drive. as the tractor moved forward the seed-metering device is rotated by a chain-sprocket arrangement through drive wheels. one operator was required to operate the machine. seed to seed spacing in the field is regulated by the rate of rotation of the seedmetering plates. as the metering plate rotated i.e. the seed spacing of crops were maintained by the planter drive wheel diameter and the size of sprockets attached to the planter drive wheel and shaft of the seed-metering plate and the teeth ratio was 2:1 drive to driven sprocket. design considerations for the machine the following factors were considered in the design of the planter, such as the physical properties of the agricultural pa ge 33 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 30-38, 2023 material like length, width, thickness etc. which varies in shape, density and size. the ease of fabrication of component parts, the safety of the operator, resistivity of metering device to corrosion and, the operation of the machine shall be simple for small scale or rural farmers. availability of the component parts shall also be available at the local market. performance test and evaluation after development and assembly of the prototype a preliminary test run was conducted to check that the machine is functionally acceptable or not . fortunately, the planter had good stability in terms of operation and performed for the intended job accordingly. two sets of tests were performed; laboratory investigation to calibrate the machine in terms of seed rate, seed damage, and seed spacing, and field test carried out to obtain actual overall performance of the machine. laboratory tests calibration of the machine the multi-crop planter was calibrated in the laboratory to determine the seeding rate, and mechanical damage of crops for a particular area. the calibration of the planter was conducted to test and adjust the planter to obtain desired plant population. the diameter of the ground wheel was 72 cm. the nominal width of the planter was calculated by: w= n×rs (8) w= 4×0.7m=2.8 m where : w= width of the implement n= number of furrow opener rs= row spacing evaluation of seed damage each of the hoppers was loaded with 4 kg of seeds and drive wheels were rotated 20 times with speed of corresponding to 1.6 m/s. a stop watch was used to measure the time taken to complete the revolutions. paper bags were placed on each of the seed tubes/ spouts to collect the seeds metered and discharged. the seeds collected in the paper bags at the end of the 20th revolution of the wheel were examined for any external damage or visible crack to establish the performance of the metering cups. germination test, on randomly selected seed samples, were conducted at fedis agricultural research center laboratory to assess the level of internal damage by the metering mechanism. percentage external seed damage was determined by equation given below. md = (stds/sns) ×100 (9) where: md = percentage damaged seed stds = total number of damaged seeds (external) sns = total number of seeds field performance evaluation evaluation of seed spacing/distribution the prototype planter was evaluated in the field using seeds of maize, common bean and sorghum at forward speeds of 2, 4 and 6 km/h. at the end of each test run, measurement of successive seed spacing was made, seeds spacing were measured from the soil surface and preparation for the subsequent tests. each test run was replicated three times over a distance of 2 m. measurements made were used to calculate the mean seed spacing, seed miss index, seed multiple index, quality of feed index and precision in spacing. mean values and standard deviation of seed spacing were determined to pattern and uniformity of seed distribution in the rows (kachman and smith, 1995). theoretical spacing, xref, in the design of wheels and metering devices was used as actual seed spacing and the measured and its mean values of spacing were compared against the theoretical values.the observed spacing were classified into five divisions:division i = 0 to 0.5 of xref, this indicated multiple seeds dropped at the same spot or seed spacing less than or equal to half of the desired spacing. division ii = 0.5 to 1.5 of xref, this indicate single seed spacing close to the theoretical seed spacing. division iii =1.5 to 2.5 xref, these are single skips. division iv = 2.5 to 3.5 xref, these are double skips. division v = over 3.5 xref, these are triple skips etc. seed spacing accuracy estimation was based on the following parameters and equations (kachman and smith, 1995): missing index (misi) the missing index was estimated using distance measured between seeds dropped in the row and spaced at a distances greater than 1.5 times the theoretical (nominal) spacing and calculated using the equation below: misi (%) = (niii+n(iv)+nv/n)×100 (10) where: misi = missing index niii, niv, nv = the number of spacing in division iii, iv, and v n = total number of spacing multiples index (muli) the multiple index was estimated by measuring the distance between consecutive seeds, the spacing less than or equal to half of the theoretical (nominal) spacing and calculated as follows: muli (%) = (ni/n)×100 (11) where: muli = multiple index ni = the number of spacing in region i and n = total number of spacing quality of feed index (qfi) quality of feed index, as an indicator of uniformity of seed distribution in the row, was estimated using the data obtained by measuring the consecutive distance between seeds in the row with spacing more than half but no more than 1.5 times the theoretical spacing and calculated using the equation given below: pa ge 34 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 30-38, 2023 qtf i (%) = (nii/n)×100 (12) where: qtfi = quality of feed index nii = the number of spacing in division ii and n = total number of spacing precision index (prec) precision index is the coefficient of variation of the spacing between the nearest seeds in a row that were classified as singles after omitting the outliers consisting of misses and multiples (singh et al., 2005). precision is similar to a coefficient of variation for the spacing that are classified as singles (i.e. seeds in division ii). the percentage precision in spacing was calculated as follows: prec (%) = (sii/xref )×100 (13) where: prec = precision sii = the standard deviation of the n observations in zone ii xref = the theoretical spacing. field performance and capacity field test was conducted on a soil prepared using disc plough and harrowed by disc harrow. the depth of planting was measured along the row at a distance of 2m at three randomly selected locations/points. the planter was operated without covering mechanism. field capacity and efficiency were determined in accordance to the recommendation made by kepner (1978) and using relevant parameters that included effective operating time, turning time and time losses due to obstructions on the field. a plot of 20 m by 30 m was used for each crop, on average, about nine passes with inter-row spacing of 0.7 m was used to assess field capacity and field efficiency for both maize and sorghum. from the data gathered, working speed (km/h), effective field capacity (ha/h) and field efficiency (%) were estimated using equations 104, 105 and 106. field capacity and field efficiency when the implement has been satisfactorily set, each test plot should be completed without stopping unless this is necessary due to adjustments, breakdowns. measurements are made of draft, forward speed and wheels lip. where applicable, width and depth of work and total working area and time should be recorded. the time lost in the field due to turning and other factors including failure to use the full width of the implement will affect field efficiency (fao, 1994). this is calculated as follows: field capacity field capacity was determined using the following formula (hunt, 1995) theoretical field capacity, cth = (w×s/10), (ha/h) (14) where: w = rated width of the planter (m) s = rated forward speed of machine (km/h) effective field capacity, ceff = a/10,000t, (ha/h) (15) where: t = total time for the planting operation, hr a = total area planted, ha field efficiency field efficiency,(%) e =(ceff/cth )×100 (16) where : e= field efficiency cth= theoretical field capacity ceff = effective field capacity wheel slip the wheel slip was calculated by recording total number of revolutions at no load and total number of revolutions at full load. wheel slip represents a loss of forward motion of the implement and it represents the loss of power. wheel slip for any given load is determined by the expression of (rangapara j., 2014). wheel slip=(mo-m1/mo) ×100 (17) where: mo = wheel revolution with no load m1 = wheel revolution with load experimental design the experimental design was a split-plot design according to the principle of factorial experiment with three replications. the three levels of seed types was assigned to main plot, and the three levels of forward speed of planter was assigned to sub plot, and each with three replications. the experiment design was laid as 32 with three replications and had total of 27 test runs (3x3 x3 = 27). statistical analysis the data were subjected to analysis of variances following a procedure appropriate for the design of the experiment and using sas statistical software. the treatment means that were different at 5% levels of significance were separated using least significant difference (lsd 5%) test. the least significant difference (lsd) test was performed for the mean values of actual seed spacing, seed miss index, seed multiple index, quality of feed index and precision spacing in relation to seed type, and forward speed. results and discussions this study was undertaken to develop and the performance evaluation of the planter capable of planting maize, common bean and sorghum seeds at predetermined spacing and depths. physical properties of seeds involved in the study were investigated to optimize the design of the planter’s component parts. performance indicators such as spacing indices that include seed multiple index (muli), seed miss index (misi), quality of feed index (qtfi) and precision (prec) in seed spacing were used to assesses performance of the planter. this section provides the physical properties of the seeds, soil and the results of the performance evaluation of the machine. physical properties of soil of experimental site moisture content and bulk density of soil during conducting the experiments, the soil conditions of the experimental field were studied and different pa ge 35 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 30-38, 2023 parameters were calculated (table 1). moisture content on dry basis of soil was measured by oven dry method. five soil samples were taken randomly at 5 different locations in the plot using core sampler of 8.0 cm diameter and 12 cm height. moisture content at 5 different places was found to be 17.43, 18.18, 17.8, 18 and 18% on wet basis. the average moisture content of the experimental field was 17.88%. bulk density of soil was calculated from data obtained by core sampler. bulk density of soil was found to be 1.41, 1.48,1.47, 1.48 and 1.48 gm/cm3, respectively (table:3). average value of bulk density of experimental plot was obtained 1.46gm/cm3 physical properties of the seeds the varieties of crops used in the study were melkassa-2 (maize), awash 2 (common bean) and melkem (sorghum). table 2 gives the mean values and the standard deviations table 1: moisture content and bulk density of soil observations weight of a soil(gm) weight of soil after oven dried(gm) mc (%) bulk density (gm/cm3) %wb %db 1 875.25 712.05 17.43 21.1 1.41 2 892.26 730 18.18 22.2 1.48 3 886.2 728.5 17.8 21.65 1.47 4 890.32 729.5 18.0 22.04 1.48 5 892.2 731 18.0 22.05 1.48 average 887.24 724.21 17.88 21.1 1.46 sd 9.08 8.26 0.23 0.81 0.31 table 2: physical properties of maize, common bean and sorghum seeds physical properties sample size maize common bean sorghum unit larger dia (x) 30 10.84±0.15 9.26±0.07 4.72±0.1 mm medium dia (y) 30 8.48±0.15 6.36±0.12 4. 27±0.18 mm smaller dia (z) 30 3.66±0.11 5.11±0.66 2.78±0.10 mm volume (v) 30 175.95±54.50 157.58±21.04 29.34±4.70 mm3 geometric dia 30 6.95±0.46 6.70±0.29 3.83±0.16 mm sphericity 30 64.8± 7.14 72.4±2.87 81±2.8 % thousands seed weight 10 294±3.14 161±1.44 29±0.70 gm of length, width, thickness, volume, geometric diameter, sphericity, and thousands seed weight. the sphericity of maize, common bean and sorghum were 64.8, 72.4 and 81 %, respectively (table 2), indicating that all seeds had more or less spherical shape. hence, it was decided to have metering devices with cells of circular shape with depths equal to the length or major diameters of the seeds of the crops. in general, the dimensions of metering device cells were dependent up on length or major diameter of maize, common bean and sorghum seeds. field performance evaluation seed spacing the seed missing index the analysis of variance (anova) revealed that the planter forward speed and the interaction of planter forward speed with seed type had significant effect (p < 0.05) on seed missing index. table 3 show the effect of speed of operation, seed type and the combined effect of speed on mean percent of seed miss index. operational speed had significant effect on percent of table 3: effects of planter operating speed on missing index (misi) parameter source of variation measure of differences speed level (km/hr) crop type lsd (5%) se maize common bean sorghum missing (%) 2 5.87a 2.70a 5.59ab 3.06 0.99 4 6.33bc 6.00b 5.10ab 6 10.77d 9.00cd 7.00bc means followed by the same letter (or letters) do not have significant difference at 5% level of probability missing index at p < 0.05. however, the level of effect varied with crop type. increasing speed of operation from 2 km/hr. to 4km/hr. had no a significant effect on the percent missing index on both maize and sorghum pa ge 36 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 30-38, 2023 crops, while the effect of increasing operational speed from 2 km/hr to 6 km/hr for common beans and maize had significant effect while for sorghum did not show statistically significant effect on percent missing index (table 3). in general, increase in operational speed tended to increases percent missing index when planting both maize and common bean, while did not show significant effect on sorghum seed using the planter. the highest percent seed missing index of 10.77, 9.00, and 7.00% were recorded with maize, common bean and sorghum seeds, respectively, at the planter forward speed of 6 km/hr, whereas the lowest percent seed missing index of 5.87, 2.70 and 5.10% were obtained from maize, common bean and sorghum seeds, respectively, at the operational speed of 2 km/hr for maize and common bean ,while at 4km/hr for sorghum crop. this clearly indicated that forward speeds greater than 5 km/hr would result in percent missing index of approximately equal to ten and above for maize which exceeds the acceptable level of percent missing index (chhinnan/ et al., (1975) and karayel and ozmerzi, 2001). the seed multiple index the highest percent seed multiple index occurred at operational speed of 6 km/hr for maize, common bean and sorghum. the lowest values, of the same, were recorded at operational speed of 2 km/h. the planter table 4: effects of operational speed on multiple index (muli, %) parameter source of variation measure of differences speed level (km/hr) crop type lsd (5%) se maize common bean sorghum missing (%) 2 9.6a 8.3a 13.0ab 10.02 5.44 4 11.7ab 12.67ab 13.93ab 6 15.37ab 21.1b 18.2ab operating speed and the percentage seed multiple index are directly related as that of the percent seed missing index (from the following results or table 4). the quality of seed feed index appendix table a3 shows the results of statistical analysis on the effects of operational speed of the planter, and seed type on the quality of seed feed index. the analysis of variance (anova) revealed that , seed type had significant effect (p <0.05) on quality of feed index whereas planter forward speed and the interaction of planter forward speed and seed type had no significant effect (p > 0.05) on quality of feed index. table 5 shows the effects of operational speed of the planter and seed type on percent of quality of seed feed index. figure 17 shows the relation between planter linear speed and seed type and percent of quality of seed feed index. the forward speed of the planting machine had significant effect on the percent quality of seed feed index at the planter speed of 6 km/hr regardless of the type of seeds used. the percent quality of seed feed index decreased with increasing planter forward speed for all types of seeds used in the study. however, the lowest reduction in percent quality of seed feed index was observed for sorghum seeds. the highest percent quality of seed feed index of 75.33, 71.67, and 68.87% were observed for maize, common bean and sorghum, respectively, when the planter was operated at forward speed of 2 km/hr. the lowest percent quality of seed feed index of 62.33, 63.00 and 59.94 were observed with maize, common bean and sorghum, respectively; when the planter was operated at speed of 6 km/hr. from results in table 5, it could be concluded that the planter at speeds greater than or equal to 6 km/hr would reduce the plant population/ ha, hence could lead to reduction in yield at the end of the day (karayel, 2009). table 5: effects of operating speed, on quality of feed index (qtfi, %) parameter source of variation measure of differences speed level (km/hr) crop type lsd (5%) se maize common bean sorghum qtfi (%) 2 75.33a 71.67a 68.87a 6.17 3.27 4 72.87a 69.33a 66.87a 6 62.33b 63.00b 59.94b precision index table 6 show the effects of operational speed of the planter and seed type on mean percent seed precision index. the results indicated that seed type had significant effect on the percent seed precision index; means that variation, in seed spacing within a row, increased as planter linear forward speed increased for maize, i.e. as the speed of operation is increased, one should expect high variability in seed spacing, which is not a desired trait. but for both common bean and sorghum does not show direct relation. the combined effect of seed type and forward speed of the planter, on the percent seed precision index, was significantly difference at the planter forward speed of 6 km/hr. the percent seed precision index, at the planter linear operating speeds of 2, 4 and 6 km/hr. were 1.77, 3.04 pa ge 37 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 30-38, 2023 table 6: effects of operating speed, on seed precision index (%) parameter source of variation measure of differences speed level (km/hr) crop type lsd (5%) se maize common bean sorghum prce (%) 2 1.77a 7.54e 5.51cde 2.09 1.44 and 3.60; 7.54, 4.97 and 5.74; and 5.51, 4.25 and 5.45% for maize, common bean and sorghum, respectively. this particular result clearly indicated that planter forward speed greater than 6 km/hr would result in seed spacing variations of over 3.60%, for maize and the variations for sorghum and common bean were greater than 5 % at planter speed of 6 km/hr. however, a practical upper limit for precision is 29%. lower values for the precision indicate better performance compared to higher values (kachman and smith, 1995)and table 7 below shows planter performance indicators. the mean field capacity and efficiency of the planter were 0.59 ha/hr. (1.73 hr. /ha) and 75.87 %, respectively. this shows that the planter can plant a hectare of land in slightly in less than two working hours. in another words, the planter can best suit majority of the ethiopian farmers who have opportunity to use the tractor. the field efficiency of the planter, as recommended by kepner et al. (1978) is within the acceptable level. the mean depth of planting was 5.95 cm with coefficient of variation of 0.128 (12.8%).the time taken to finish one hectare of land was 1:55 hr. that means by taking 8 hr. working hour per day, the farmer can plant about 5.16 hectare of land in one day by using this planter. table 7 result the proposed plant spacing was 20 cm-25cm and the maximum result obtained from the experiments was 22.91 cm from maize so it is good in terms of plant spacing which was with the proposed range. this depth of planting was greater than the desired depth of planting of 5 cm as recommended for maize by agronomists. nonetheless, the deviation and the variability being too small and with acceptable range, also, it can be adjusted to the desired depth of planting. the stand count made after 15 days of planting gave mean number of plants of 7.87,12 and 16 plants within rows of 2 m length for maize, common beans, and sorghum, respectively. the design or desired number of plants, within a row of 2 m long, for maize, common bean and sorghum were 8, 13 and 10, respectively. hence the planter reasonably satisfied the requirement for the establishment of optimum plant population for all seed types. table 7: field performance indicators of the planter crop types field length(m) width field (m) time taken (min) time lost (min) theoretical field capacity (ha hr-1 ) effective field capacity (ha hr-1) field efficiency (%) maize 30 20 4.09 1.5 0.84 0.65 77.3 common bean 30 20 5.52 2.15 0.64 0.46 71.8 sorghum 30 20 4.23 1.23 0.84 0.66 78.5 mean 30 20 4.61 1.63 0.77 0.59 75.87 conclusion this work focused on the development and performance evaluation of a tractor drawn multi-crop planting machine that easy to use, easy to maintain, requires less labour and costs. from the developed and results values obtained in the study; it has been found that the developed planter gives: • the planter works effectively in planting maize, common bean and sorghum at a given study area. • a significant field capacity was obtained by using the developed machine when compared to manual planting methods. • this shows that the planter can plant a hectare of land in slightly in less than two working hours. • therefore, the planter can best suit majority of the ethiopian farmers who have opportunity to use the tractor. • hence, one can note that the time requirement per hectare is reduced by 1/16 amount and labour requirement reduced by the same amount by using this planter, when compared to manual planting. • however, the speed of the planter should be limited to less than 6 km/hr in order not to seriously and negatively affect the percentage of recommended plant population of experimental crops. references chhinnan, m. s. young, j. h. and rohrbach, r.p. 1975. accuracy of seed spacing in peanut planting. transactions of the asae, 18(1), 828-831. el-raie, a.e., hendawy, n.a. and taib, a.z. (1996). study of physical and engineering properties for some agricultural products. misr. journal of agricultural engineering 13(1), 211-226. fao. (1994). testing and evaluation of agricultural machinery and equipment: principles and practices. jayan, p. r. and kumar, v. j. f. (2004). planter design in relation to the physical properties of seeds. journal of tropical agriculture, 42, 69-71. jouki, m. and khazaei, n. (2012). some physical properties of rice seed (oryza sativa). research journal pa ge 38 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 30-38, 2023 of applied sciences, engineering and technology, 4(13), 18461849. kachman, s. d. & smith, j. a., (1995). alternative measures of accuracy in plant spacing for planters using single seed metering. transaction of the asae, 38(2), 379–387. karayel, d. (2009). performance of a modified precision vacuum seeder for no-till sowing of maize and soybean. soil & tillage research, 104, 121–125 karayel, d. and ozmerzi, a., (2002). effect of tillage methods on sowing uniformity of maize. canadian biosystems engineering, 44(2), 23-26. karayel, d., wiesehoff, m., ozmerzi, a. and muller, j. (2005). laboratory measurement of seed drill seed spacing and velocity of fall of seeds using high-speed camera systems. computer and electronics in agriculture, 50(2), 89–96. kepner, r. a., bainer, r., barger, e. l. (1978). principles of farm machinery, 3rd edition. inc. avi publishing company, westport, usa. khurmi, r. s. and gupta, j. k. (2005). a textbook of machine design. eurasia publishing house (pvt.) ltd., ram nagar, new delhi, india. kipchumba m. a. (2015). design, operation and calibration of a standard seed drill. jomo kenyatta university of agriculture and technology. agricultural machinery conference 75 paper. kumar, a., s. c. moses, and k. khan. (2015). a survey on the design, fabrication and utilization of different types of foods and vegetables dryer. iosr journal of agricultural and veterinary science i, 8(4), 2319–2372 rangapara, d. & jaimin, p. (2014). performance evaluation of manually operated single row cotton planter. international journal of engineering sciences and research technology, 3(9) 40-44. soyoye, b. o., o. c. ademosun, and e. o. olu-ojo. (2016). manually operated vertical seed-plate maize planter. cigr journal, 18(4), 70-80. pa ge 1 pa ge 8 american journal of life science and innovation (ajlsi) reproductive biology and histological pattern of hilsa shad (tenualosa ilisha) in bangladesh bimal chandra das1, md. moniruzzaman2, mousumi akhter3, sabequn nahar swati4, tahamina khatun5, md. mizanur rahman6, md. hashibur rahman7*, harunur rashid8 volume 2 issue 3, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i3.1973 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: august 01, 2023 accepted: september 06, 2023 published: september 15, 2023 an experiment was carried out to identify the peak breeding season of hilsa in bangladesh using gonadal histology through the standard procedure. a total 480 samples were collected from meghna river, chandpur, from july 2012 to june 2013 and tentulia river, barisal, from june 2013 to june 2014. histological sections of gonads were analyzed and ovarian and testicular stages were determined according to the oecd guidance document for diagnosis of histopathology of fish gonads. maturing germ cells were observed every month where a higher amount of mature germ cells was found in december, january, and september to october, with a high in october. from february to june, the mature germ cells were less. histological observations of testis suggested that t. ilisha spawns throughout the year, but significant spawning takes place in september-october and december-january with a high abundance of spt (spermatids). during october, mature germ cells were mostly evident, and spz (spermatozoa) was in high proportion inside the lu (lumen of tubules). the gradual maturation of germ cells from early to ripened stages inside the testicular lu and peak maturity came during october. females having ovaries at mature stages were considered those spawning or near to spawn. the occurrence of mature ovaries examined by external characteristics began in january and continued up to december. percentage (%) occurrence of mature ovaries started to increase in february, peaked in october (75%), and decreased in december, january, and april. in fish ovary, the mature stages are generally observed at the most advanced stage of the ovary when the fishes are ready for spawning and when mean gsi values are comparatively high. in this study, advanced mature oocytes were found from in every month during the study period. the histological study of oocyte development and examination of external characteristics of ovaries suggested that t. ilisha spawns throughout the year, but significant spawning takes place in october –november and peaks in october. keywords histology, gonad, tenualosa ilisha, bangladesh introduction hilsa, being anadromous, lives at sea for most of its life but migrates to freshwater rivers for spawning, after which it returns to the sea. the hilsa shad occurs in the foreshore areas, estuaries, and freshwater rivers of the western division of the indo-pacific region. its marine distribution extends from iran and iraq in the persian gulf to the west coast of india, in the arabian sea, and in the bay of bengal, the coast of bangladesh and myanmar. although it is distributed in large open area, the major percentage of hilsa (about 95%) is caught by bangladesh, india and myanmar (dof, 2020). bangladesh’s national fish hilsa affords the largest single-species fishery in bangladesh, especially during the monsoon in almost all the primary river systems, estuaries, and the sea area of bangladesh. the hilsa fishery supports a commercial fishery, and in the early 1970s, it composed more than 95% of the total commercial catch in bangladesh (coad et al., 2003). from the 1970s, the hilsa fishery began to decline gradually, with outputs reaching a low point of 0.19 million tons in 2001–2002 (boblme, 2012). the production of hilsa declines due to a low discharge of water from the river ganges and consequently heavy siltation in most of the rivers, the gradual growth of industries, growing urbanization, indiscriminate use of fertilizers, agrochemicals, pesticides, and the discharge of municipal waste are continuously polluting the river system. to conserve the hilsa fishery, proper management measures should be developed and implemented. one of the most widely practiced fishery management is the identification of peak spawning season and the banning of fishing accordingly. the histology of gonads is the most common and often the most reliable technique to assess the reproductive strategy and tactics of fish species and is therefore applied in a large number of marine laboratories. in this respect, those laboratories aiming to provide management advice use histology for maturity classification to separate the sexually immature component from the sexually mature component, fecundity studies to be used in either recruitment-related studies or in egg production methods (to estimate spawning stock biomass) or to monitor the prevalence 1 department of fisheries, barishal, bangladesh 2 bangladesh fisheries research institute, riverine station, chandpur, bangladesh 3 bangladesh fisheries research institute, marine fisheries & technology station, cox’s bazar, bangladesh 4 department of youth development, ministry of youth and sports, bangladesh 5 upazila fisheries office, department of fisheries, madarganj, jamalpur, bangladesh 6 district commandant’s office, bangladesh ansar and vdp, jamalpur, bangladesh 7 bangladesh fisheries research institute, headquarters, mymensingh, bangladesh 8 department of fisheries management, bangladesh agricultural university, mymensingh, bangladesh * corresponding author’s e-mail: hasibkhan94bfri@gmail.com pa ge 9 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 of any type of reproductive disturbance or failure to spawn regularly. by necessity, these different overall aims require different methodological approaches, although in many cases, the histological protocol as such is reasonably similar. in summary, histology is considered to be an important tool to better understand the productivity of fish stocks and thereby, in the end, contributes to the internationally adopted principles of the precautionary or ecosystem approach to fisheries. mcadam et al. (1999) found gsi to be reliable in determining the reproductive status of fish species that spawn once annually but are of limited value when fish are protracted spawners. alternatively, histology is an effective technique used to identify the reproductive timing of fishes even when species spawn multiple times or have a low reproductive investment (mcadam et al., 1999). whereas histology is a more reliable technique than gsi, the amount of time required and cost associated with histology diminish the practical advantage of the technique. the present study aimed to identify the peak spawning season of hilsa by gonadal histology. materials and methods sampling of fish hilsa fish sample were collected from two different location of bangladesh, upper meghna river at chandpur and tentulia river at barisal district of bangladesh. sampling was conducted during july 2012 to june 2013 in meghna river and july 2013 to june 2014 in tentulia river. samples were collected bought once a month and 20 mature hilsa samples (10 female and 10 male) were collected at each sampling day. hilsa were caught in the river during night using gill nets primarily. all specimens were preserved with crushed ice in cool fish boxes and brought to the laboratory as soon as possible. a total of 480 mature fishes (240 samples from each site; 240 female and 240 male) covering various size groups were collected from both sampling sites. gonad collection the body cavity of fish was opened carefully by scissors and gonads were collected with forceps carefully. other constitutional units such as muscles, fat tissues, digestive organs and blood veins etc. were taken away properly. body weight (bw) and gonad weight (gw; both left and right gonads) were measured to the nearest 0.001g. total length (tl) and gonad length (gl) were also measured to the nearest 0.01cm. after weighing gonads were fixed with 10% buffered formalin for 24-48 h and preserved with 10% formalin in small vials for further investigation. observation of ovarian external features general features and structure, as well as month-wise size, shape, and color of female gonads of t. ilisha were studied during sample collection and preservation. in hilsa gonads, the left gonad was larger than the right one, both in length and width. external features of both ovaries were observed by the naked eye, and under the magnifying glass, the various maturity stages were classified based on external observation of ovaries. histological observation of hilsa gonads for the histological study of hilsa gonad, the microscopic slides were prepared by the following procedure followed by agarwal (1996) with slight modification. first of all, a piece of buffered formalin preserved gonad tissue was dissected randomly from the proximal or medial part of one lobe for histological examination. a transverse segment of about 2-3mm thick from the middle part of the right ovary was taken. after taking a piece of gonad tissue following steps were followed for histological study. sample preparation (dehydration, clearing, infiltration, embedding, trimming, sectioning, staining, mounting) the gonads samples were kept in the dehydration cassette and passed through a graded series of alcohol series kept in glass jars (figure 1). the dehydration schedule was as follows: sl. no. solution time 1 80% ethanol 12 hours (overnight) 2 95% ethanol 1 hour 3 95% ethanol 1 hour 4 100% ethanol 1 hour 5 100% ethanol 1 hour 6 100% ethanol 1hour figure 1: dehydration using graded alcohol series pa ge 10 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 dehydrated gonad samples were then cleared using benzene twice successively for 2 hours to remove traces of alcohol to have consistent paraffin blocks. the process was as follows: paraffin-embedded blocks were sectioned by a microtome knife in a microtome machine at 5µm thickness. the sections were placed on previously tagged and prepared glass and dried on a hot plate overnight at 38°c. the prepared section slides were stored in the refrigerator for staining (figure.4). staining is a process by which samples are stained with various dyes and staining materials so that their components are visible under a microscope. the gonad sections were stained through routine h-e staining protocol as mentioned below: sl. no. solution time 1 benzine 1 hour 2 benzine 1 hour the samples were then infiltrated with paraffin in a paraffin incubator using following steps: sl. no. solution time 1 paraffin 40 minutes 2 paraffin 40 minutes after infiltration, the cassettes were taken out from an automatic tissue processor; being opened, two samples were placed in the middle of the cassette and filled with melted paraffin from the wax dispenser. then, steel covers were placed on each cassette and allowed to cool to room temperature. after cooling, the cassettes were put into a deep freezer for a few minutes, allowing easy steel plate removal from the cassettes and smooth sectioning (figure 2). figure 2: embedding using malted paraffin trimming is a process in which the undesirable wax layers of the embedded blocks are trimmed by a knife to obtain suitable blocks. trimming was done by using old microtome blades. trimming allowed easy sectioning. in this step, both side trimming and surface trimming were conducted (figure 3). figure 3: trimmed tissue fixed with wooden block sl. no. solution time process 1. xylene 10 minutes clearing (1-3) 2. xylene 10 minutes 3. xylene 10 minutes 4. 100% alcohol 5 minutes dehydration (4-8)5. 100% alcohol 5 minutes 6. 90% alcohol 3 minutes 7. 80% alcohol 3 minutes 8. 70% alcohol 3 minutes 9. 50% ethyl alcohol 2 minutes staining (9-15)10. distilled water 15 dips 11. haematoxylene (mayer’s) 3 minutes 12. wash in tap water 15 minutes 13. 50% ethyl alcohol 10-15 dips 14. 95% ethyl alcohol 30 seconds 15. eosin y 1 minutes 16. 95% ethyl alcohol 2 minutes rehydration (16-19)17. 100% ethyl alcohol 1 minutes 18. 100% ethyl alcohol 3 minutes 19. 100% ethyl alcohol 1 minutes 20. xylene 20 minutes clearing (2021)21. xylene 20 minutes 22. drying over night 23. mounting over night figure 4: staining of sectioned gonad tissues using h-e protocol pa ge 11 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 d.p.x. was used for mounting as a mounting agent. a jot of d.p.x. is put on each slide, followed by the attachment of the coverslip (22mm×22mm). after mounting, the slides were put for several hours at room temperature. microscopic observation of gonad tissue slides after mounting, the slides were observed under an electric microscope (olympus) connected to the computer with a viewer (magnus viewer). the viewer was also equipped with a camera. with the help of this mechanism, numerous photographs were snapped at different magnifications (figure.5). results external feature of ovary the maturation of the ovary could be explained by examination of the external feature of the ovary. because the shape, size, and color of the ovary varied considerably according to the degree of maturation of the oocytes, the immature ovary is small reddish, and transparent in nature. in mature condition, the ovary’s color turns yellowish and becomes larger in size. histological study of ovary of female hilsa of meghna river, chandpur ovarian development is subdivided into distinct developmental stages according to physiological, biochemical, morphological, and histological criteria. maturity stages were determined following the key outline described by matsuyama et al. 1982 and shinkafi et al., 2011. different steps found in the present ovarian histological study are presented in the figures (figure 7 to figure 10) below. gvb (germinal vesicle breakdown) was also seen when female hilsa partially spent. in the present study, after microscopic observation, the percent occurrence of different stages of oocytes in different months was calculated and presented in table 1. figure 5: microscopic observation of stained gonad sections table 1: percent (%) occurrence of the oocyte stages of ovarian development found in the histological study of hilsa ovary during the study period july 2012 to june 2013 in the meghna river, chandpur. month epno lpno yv yg pm m gvb july 6 5 8 12 11 58 seen august 9 10 24 47 seen september 7 11 12 21 49 october 4 8 8 7 73 seen november 2 5 9 18 66 seen december 27 37 22 14 january 6 7 20 22 16 29 february 5 9 16 19 14 37 seen march 8 13 13 14 19 33 april 12 17 16 18 13 24 may 7 8 11 23 12 39 june 11 14 10 13 11 41 here, epno=early perinucleolar oocyte, lpno=late perinucleolar oocyte, yv=yolk vesicle, yg=yolk granule, pm=premature, m=mature from table 1 and figure 6, it is extracted that the highest (73%) and lowest (14%) percentage of mature oocytes were found in the month of october and december, respectively. in the month of july, the highest percentage of oocytes was in the mature stage (58%), and the lowest percentage of oocyte was in early perinucleolar oocyte stage (5%) along with all six oocyte developmental stages. in the month of august, no yolk granular stages and yolk vesicle were found; however, 47% of oocyte was in the mature stage, whereas 9% oocyte was in early perinucleolar oocyte stage, besides 10% in late perinucleolar oocyte, 24% pre-mature stage. in september, the percentage of mature oocyte increased little (49%) with 7% early perinucleolar oocyte, 11% late perinucleolar oocyte, 12% yolk vesicle, 21% yolk granular stage; however no pre-mature oocyte was found. in october, the highest percentage (73%) comprised the mature stages oocyte with 7% pre-mature, 8% yolk granule, 8% late perinucleolar oocyte and 4% early perinucleolar oocyte. in november, the percentage of mature oocyte continued in the highest rank (66%) with pa ge 12 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 2% early perinucleolar oocyte stage, 5% late perinucleolar oocyte, 9% yolk granule, 18% pre-mature without having yolk vesicle stage. in december, the percentage of mature oocyte declined considerably. it reached only 14% with no yolk granule and yolk vesicle stage oocyte, 27% early perinucleolar oocyte, 37% late perinucleolar oocyte (highest in number), 22% pre-mature. in january, the percentage of mature stage oocytes increased little by 29% with 6% early perinucleolar oocyte, 7% late perinuclolear oocyte, 20% yolk vesicle, 22% yolk granule, 16% pre-mature stage. in february, the mature stage oocyte increased again by 37% with 5% early perinucleolar oocyte, 9% late perinucleolar oocyte, 16% yolk vesicle stage, 19% yolk granule, 14% pre-mature. in march, the mature stage oocyte again declined and dropped to 33% with 8% early perinucleolar oocyte, 13% late perinucleolar oocyte, 13% yolk vesicle stages, 14% yolk granule, 19% pre-mature stage. in april, the percentage of mature oocyte continued to declining trend, reached to 24% this month other stages of oocyte were 12% early perinucleolar oocyte, 17% late perinucleoar stage, 16% yolk vesicle, 18% yolk granule, 13% pre-mature stage. in may, the percentage of mature oocyte started to rise again and it found 39% with 7% early perinucleolar oocyte, 8% late perinucleoar stage, 11% yolk vesicle, 23% yolk granule, 12% pre-mature stage oocyte. in june, the mature oocyte percentage continued to rise and reached to 41% having 11% in pre-mature, 13% yolk granule and 10% yolk vesicle, 11% early perinucleolar oocyte and 14% late perinucleolar oocyte. yolk vesicle stages were found throughout the year except august, october and november, december. yolk granule stages were absent in august and december. early perinucleolar oocytes were also observed throughout the year except may, september, and november. late perinucleolar oocytes were observed throughout the year. pre-mature oocyte appeared throughout the year except for september and mature oocytes appeared throughout the year during study period. the advanced oocyte i.e. mature oocyte was found in the very month of the study period. the occurrence of the mature oocyte in every month of the study period confirmed that t. ilisha spawns precisely throughout the year, but significant spawning takes place in october. figure 6: month-wise occurrence (%) of different stages of oocyte of ovarian development of female hilsa collected from meghna river during july 2012 to june 2013. [here, epno=early perinucleolar oocyte, lpno=late perinucleolar oocyte, yv=yolk vesicle, yg=yolk granule, pm=pre-mature, m=mature] pa ge 13 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 figure 7: [a-f]-haematoxylin-eosin staining of tissue section of t. ilisha ovary at 4x magnification sampled in july to september, 2012 [a-b: july; c-d: august; e-f: september]. epno, early perinucleolar oocyte, lpno; late perinucleolar oocyte; yv, yolk vesicle; yg, yolk granule; pm, pre-mature; m, mature stage; gvb, germinal vesicle breakdown. pa ge 14 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 figure 8: [a-f]-haematoxylin-eosin staining of tissue section of t. ilisha ovary at 4x magnification sampled in october to december, 2012 [a-b: october; c-d: november; e-f: december]. epno, early perinuclear oocyte; lpno, late perinuclear oocyte; yg, yolk granule; pg, primary growth oocyte; pm, pre-mature; m, mature stage. figure 9: [a-f]-haematoxylin-eosin staining of tissue section of t. ilisha ovary at 4x magnification sampled in january to march, 2013 [a-b: january; c-d: february; e-f: march]. epno, early perinucleolar oocyte; lpno, late perinucleolar oocyte; yv, yolk vesicle; yg, yolk granule; pm, pre-mature; m, mature stage; gvb, germinal vesicle breakdown. pa ge 15 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 figure 10: [a-f]-haematoxylin-eosin staining of tissue section of t. ilisha ovary at 4x magnification sampled in april to june, 2013 [a-b: april; c-d: may; e-f: june]. epno, early perinucleolar oocyte; lpno, late perinucleolar oocyte; yv, yolk vesicle; yg, yolk granule; pg, primary growth oocytes; pm, pre-mature, m, mature stage. histological study reproductive cycle of t. ilisha in the present study, the following stages of testicular germ cells were observed from the month-wise (july 2012 to june 2013) samples of t. ilisha testes. mature germ cells were less evident in july. sc, st, and sz were observed in the histo-sections under the microscope. there was appearance of some empty lus in the slide (figure 11a). mature germ cells were not found in august. sc and st were observed in the histo-sections under the microscope. the testicular lus were full of sz (figure.11b). mature germ cells were more evident in september. sc, st and sz were observed in the histo-sections under the microscope. the testicular lus were full of sz and some empty lumens also found (figure11c). maturing germ cells were mostly evident in october. st, and sz were observed in the histo-sections under microscope. st were high in number together with significant accumulation of sz. some lumens of tubules (lu) were found empty (figure. 11d). early and developing stage germ cells were evident in november. the empty lumen was more evident. sc and st were less in the histo-sections under microscope (figure. 11e). mature germ cells were more evident in december. sz were observed in the histo-sections under microscope. sz more in number and some empty lumens also found (figure.11f). mature germ cells were evident in pa ge 16 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 figure 11: [a-f]-haematoxylin-eosin (h-e) staining of tissue sections of t. ilisha testes (at 40x magnification) sampled in july to december, 2012 [a: july, b: august, c: september, d: october, e: november, f: december]. sg, spermatogonia; st, spermatids; sc, spermamatocytes; sz, spermamatozoa; lu, lumen of tubules. pa ge 17 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 figure 12: [a-f]-haematoxylin-eosin (h-e) staining of tissue sections of t. ilisha testes (at 40x magnification) sampled in january to june, 2013 [a: january, b: february, c: march, d: april, e: may, f: june]. sz, spermamatozoa; st, spermamatides; sc, spermatocytes and sg, spermatogonia; lu, lumen of tubules. january. sz and st were observed in the histo-sections under microscope. sz more in number and some empty lumens also found (figure.12a). empty lumens were more evident in february. sz, st and sc were observed in the histo-sections under a microscope. sz were less in number and empty lumens were more in number (figure.12b). early and developing stage germ cells were mostly evident in march. sg, sc and st were observed in the histological-sections under microscope. sc were less in number (figure.12c). early-stage germ cells were more evident in april. sc and st were observed in the histo-sections under microscope. sc were high in number (figure.12d). sc and st were observed in the histo-sections under microscope in may. abundance of sts was increased although there is a significant amount of sc visible under microscope (figure.12e). maturing germ cells were not found in july. sc, st, and sz were observed in the histo-sections under a microscope. spt were high in number together with significant accumulation of sz. some lumens of tubules (lu) were found empty (figure.12f). histological study of the ovary of female hilsa of tentulia river, barisal the ovary of the female hilsa was bi-lobed elongated and situated in the body cavity. the shape, size, and color of the ovary varied considerably according to the degree of maturation of the oocytes; the immature ovary is small reddish and transparent in nature which turns into yellowish in ripe condition. ovarian development is subdivided into distinct developmental stages according to physiological, biochemical, morphological and histological criteria. maturity stages were determined following the key outline as described by matsuyama et al. 1982. different stages that found in the present ovarian histological study are presented in the figures (figure.14 to figure.17) and described below. germinal vesicle breakdown (gvb) also occurred when female hilsa partially spent. in the present study, after microscopic observation, the percent occurrence of different stages of oocytes in different months were calculated and presented in the table 2. from the table 2 and figure. 13, it is found that the highest (75%) and lowest (10%) percentage of mature oocyte found in the month of october and december, respectively. in the month of july, 60% oocyte was in the mature stage which is the highest in this month having 5% early perinucleolar oocyte, 5% late perinucleolar oocyte, 10% yolk vesicle stage, 20% pre-mature, and no yolk granule stages. in the month of august, no yolk granular stage oocytes were found; however, 40% oocyte was in the mature stage where as 10% oocyte was in early perinucleolar oocyte stage, with 15% late perinucleolar oocyte, 15% yolk vesicle stage, and 20% pre-mature stage. pa ge 18 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 in september, percentage of mature oocyte increased 10% that the previous month and reached to 50% with no yolk vesicle and early perinucleolar oocyte stage, 10% late perinucleolar oocyte, 20% yolk granular stage, 20% pre-mature stages oocytes. the month of october comprises the highest percentage (75%) of mature stages oocyte among the months with 5% pre-mature, 10% yolk granule, 5% late perinucleolar oocyte and 5% early perinucleolar oocyte. in november, the percentage of mature oocyte was also in highest rank (65%) however start decline, this month’s ovary also contained 5% late perinucleolar oocyte, 10% yolk granule, 20% pre-mature oocytes and on early perinucleolar oocyte and yolk vesicle stage oocyte. in december, the percentage of mature oocyte reached in lowest percentage among the month and it was only 10%, the ovary contained 30% early perinucleolar oocyte, 40% late perinucleolar oocyte, 20% pre-mature oocytes and no yolk granule and yolk vesicle stage oocyte. in january, the percentage of mature stage oocyte started to increase reached to 35% having 5% early perinucleolar oocyte, 5% late perinuclolear oocyte, 20% yolk vesicle, 20% yolk granule, 15% pre-mature stage oocytes. in february, the mature stage oocyte increases more 10% than january reached to 45% having 10% early perinucleolar oocyte, 10% late perinucleolar oocyte, 25% yolk vesicle stage, 10% pre-mature oocytes and no yolk granule stages were found. in march, the mature stage oocyte again declines and dropped to 40% having 10% early perinucleolar oocyte, 15% late perinucleolar oocyte, 20% yolk vesicle stages, 15% pre-mature stage oocytes and no yolk granule stages were found. in april, the percentage of mature oocyte continued to declining trend, depleted to 25%, this month other stages of oocyte were found as 20% in early perinucleolar oocyte, table 2: percent (%) occurrence of the oocyte stages of ovarian development that found in the histological study of hilsa ovary during the study period july 2013 to june 2014. month epno lpno yv yg pm m gvb july 5 5 10 20 60 seen august 10 15 15 20 40 september 10 20 20 50 seen october 5 5 10 5 75 november 5 10 20 65 december 30 40 20 10 seen january 5 5 20 20 15 35 february 10 10 25 10 45 march 10 15 20 15 40 seen april 20 40 25 15 may 15 30 20 35 june 15 15 10 10 20 30 here, epno=early perinucleolar oocyte, lpno=late perinucleolar oocyte, yv=yolk vesicle, yg=yolk granule, pm=premature, m=mature figure 13: month-wise occurrence (%) of different stages of oocyte of ovarian development of female hilsa collected from tentulia river, barisal during july 2013 to june 2014. [here, epno=early perinucleolar oocyte, lpno=late perinucleolar oocyte, yv=yolk vesicle, yg=yolk granule, pm=pre-mature, m=mature] pa ge 19 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 40% in yolk granule, 25% pre-mature stage and no late perinucleolar stages and yolk vesicle stages. in may, the percentage of mature oocyte started to increase again and it found 35% with 15% yolk vesicle stage, 30% yolk granule, 20% pre-mature stage and early perinucleolar oocytes and late perinucleolar oocytes were not found in this month. in june, mature oocyte percentage stayed in higher rate with little bit decreased than previous month valued 30% with 20% pre-mature, 10% yolk granule, 10% yolk vesicle, 15% early perinucleolar oocyte and 15% late perinucleolar oocyte. yolk vesicle stages were not found in march, september, october and november, december. yolk granule stages were found throughout the year except february, july, august, and december. early perinucleolar oocytes were also observed throughout the year except, may, september, and november. late perinucleolar oocytes were observed throughout the year except april and may. pre-mature and mature oocyte appeared throughout the year during study period. the advanced oocyte i.e., mature oocyte was found in every month of the study period. the occurrence of mature oocyte in every month of the study period confirmed that t. ilisha spawn precisely throughout the year, but major spawning take place in october. figure 14: [a-f]-haematoxylin-eosin staining of tissue section of t. ilisha ovary at 4x magnification sampled from tentulia river, barisal in july to september, 2013 [a-b: july; c-d: august; e-f: september]. epno, early perinucleolar oocyte; lpno, late perinucleolar oocyte; yv, yolk vesicle; yg, yolk granule; pm, pre-mature; m, mature; gvb, germinal vesicle breakdown. pa ge 20 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 figure 15: [a-f]-haematoxylin-eosin staining of tissue section of t. ilisha ovary at 4x magnification sampled from tentulia river, barisal in october to december, 2013 [a-b: october; c-d: november; e-f: december]. epno, early perinuclear oocyte; lpno, late perinuclear oocyte; yg, yolk granule; pg, primary growth oocyte; pm, pre-mature, m, mature stage; gvb, germinal vesicle breakdown. pa ge 21 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 figure 16: [a-f]-haematoxylin-eosin staining of tissue section of t. ilisha ovary at 4x magnification sampled from tentulia river, barisal in january to march, 2014 [a-b: january; c-d: february; e-f: march]. epno, early perinucleolar oocyte; lpno, late perinucleolar oocyte; yv, yolk vesicle; yg, yolk granule; pm, pre-mature; m, mature stage; gvb, germinal vesicle breakdown. pa ge 22 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 figure 17: [a-f]-haematoxylin-eosin staining of tissue section of t. ilisha ovary at 4x magnification sampled from tentulia river, barisal in april to june, 2014 [a-b: april; c-d: may; e-f: june]. epno, early perinucleolar oocyte; lpno, late perinucleolar oocyte; yv, yolk vesicle; yg, yolk granule; pm, pre-mature; m, mature stages; pg, primary growth oocytes. discussion the reproductive activity in t. ilisha is continuous, and the presence of spermatogenic and oogenic cells under their particular development stages was observed in all specimens examined monthly over two years of study (andrade et al., 2001). this pattern is also described for other teleosts, as a result of tropical water conditions (andrade et al., 2001; lowe-mcconnell, 1987). study of vitellogenic stage (vg) of oocyte and increased gonad weight (gw) and histology of gonad are the important aspects of reproductive biology that indicate the maturation level and can estimate approximate spawning time (brown-peterson et al., 2011). in the comparison of ovarian development with age from the previous study by ahmed et al. (2020), hilsa from the meghna river, the bay of bengal, the padma river, and the tetulia river showed regular ovarian development in respect to their age. no fish were found with a fully matured ovary at the age below 2 years. in the teleost fishes, the process of oogenesis may be divided into five to eight stages (andrade et al., 2001). in the present study, stages of gonadal development in hilsa shad were identified by histological research according to the scales described by shinkafi et al. (2011) and divided into six stages; such as early perinucleolar oocyte (epno), late perinucleolar oocyte (lpno), yolk vesicle (yv), yolk granule (yg), pre-mature (pm) and mature (m) stages. pg, primary growth oocytes. besides these six significant stages, other two stages, such as germinal vesicle breakdown (gvb) and primary growth oocytes (pg) also found in a few cases. panhwar et al. (2011) studied reproductive patterns and some biological features of anadromous fish t. ilisha from pakistan. they identified five stages of oogenesis, nearly ripe, fully developed, running ripe, partially spent, and spent. lee et al. (2005) studied with leiognathu sequulus and they found eight maturity stages of oocytes as chromatin nucleus, perinucleus yolk vesicle, primary yolk, secondary yolk, tertiary yolk, migratory nucleus and ripe stage by histological examination of ovaries. on the other hand, the four different spermatogenic stages were identified in the t. ilisha as described by andrade et al., 2001. the stages are spermatogonia (sg), spermatocytes (sc), spermatids (st), spermamatozoa (sz). from the histological study, it was found that t. ilisha testis contains early and developing stage germ cells from april-june. maturing germ cells were observed every month where a higher amount of mature germ cells were found in december, january and september to october with a high in october. in the month of february to june, the mature germ cells were less. histological observations of testis suggested that t. ilisha spawns throughout the year, but major spawning occurs in september-october and december-january with a high abundance of st. during october, mature germ cells were mostly evident and sz was in high proportion inside the lu. the gradual maturation of germ cells from early to ripened stages inside the testicular lu and peak maturity during october is indicative of the fact that the peak spawning season of t. ilisha species is during october. the findings on testicular cycles coincide with that of ovarian cycles of this fish which confirms the fact that t. ilisha breed throughout the year and the peak breeding season in october. some other studies on testicular development have been done on different fishes of bangladesh, they also identified four testicular developmental stages. siddiqua et al. (2000) observed the testicular development in o. pabda and identified three stages of sperm formation, namely spermatocytes, spermatids, and spermatozoa. the percent distribution of spermatozoa was highest in july (about 92%). by analyzing the histology of spermatogenesis, it was established that this species breeds once a year. akhter (2011) found that early and developing germ cells in sperata seenghala mainly were observed during aprilmay and mature germ cells found in july-august. maya (2011) studied on gametogenesis of mystus cavasius and found that mature germ cells (st and sz) were observed from july to august samples of pa ge 23 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 8-23, 2023 the testis. alam (2009) observed a large amount of st, sz and small amount of sc in testis of o. pabo sampled from april to june. testicular lu was full of sz in june samples, indicating the peak breeding season of o. pabo. in the present study, mature oocytes were observed each month. the percentage of mature oocytes stated to increase in may and continued to november, with peaked in october (73% in the meghna river and 75% in the tentulia river) and decreased in december (lowest ever mature oocyte among the months; 13% in the meghna river and 10% in the tentulia river), january, and april. histological examination and presence of mature in oocyte throughout the year suggested that t. ilisha spawn throughout the year but major spawning takes place in october. on the other hand, a higher percentage of mature oocytes from may to november suggested the prolonged spawning season of hilsa shad. this study result agreed with a few previous studies. pillay et al. (1963) studied with t. ilisha of saurashtra coast and stated five ovarian development stages as immature, maturing, mature, partly spent, and spent. wallac and selman (1981) noted that the ovary of t. ilisha was the synchronous type that contained almost similar oocytes maturation stages. this type of oocyte maturity was the most common strategy among teleosts. in this study, advanced, mature oocytes were found every month during the study period. the study indicated t. ilisha breeds throughout the year, with a peak in october. conclusion gonadal histology of teleost fishes is the most common and reliable technique to assess the reproductive strategy of fishes. the present study of the histological study of both female and male gonads suggested that t. ilisha has a prolong spawning season with a peak in the month of october. references agarwal, n. k. (1996). fish reproduction, aph publishing corporation, new delhi,157. ahmed, m. b. u., ahammad, a. k. s., shahjahan, m., rabbi, m. f., alam, m. a., sakib, m. n., et al. (2020). age, growth and maturity of the indian shad, tenualosa ilisha through otolith examination from different habitats in bangladesh. egypt. j. aquat. biol. fish. 24, 343–359. akhter, t. (2011). reproductive cycle of giant river catfish sperata seenghala from the sylhet basin (doctoral dissertation, thesis ms, department of fisheries). alam, m. s. (2009). length-weight relationship and reproductive physiology of three endangered pabda fishes from sylhet basin (doctoral dissertation, ms thesis, department of fisheries management, bangladesh agricultural university, mymensingh. 61pp. andrade, r. f., bazzoli, n., rizzo, e., & sato, y. (2001). continuous gametogenesis boblme (bay of bengal large marine ecosystem) (2012). management advisory for the bay of bengal hilsa fishery. regional fisheries management advisory committee. 6. brown-peterson, n. j., wyanski, d. m., saborido-rey, f., macewicz, b. j., and lowerre-barbieri, s. k. (2011). a standardized terminology for describing reproductive development in fishes. mar. coast. fish. 3, 52–70. coad, b. w., hussaina, n. a., ali, t. s., & limburg, k. e. (2003). biodiversity, status and conversation of the world shads. american fisheries society, bethesda, maryland. 123. dof (2020). jatiyo matsya saptaho sonkolon. department of fisheries, ministry of fisheries and livestock. in the neotropical freshwater teleost, bryconops affinis (pisces: characidae). tissue and cell, 33(5), 524-532. lee, c. f., liu, k. m., su, w. c., & wu, c. c. (2005). reproductive biology of the common ponyfish leiognathus equulus in the south-western waters off taiwan. fisheries science, 71(3), 551-562. matsuyama, m., & matsuyama, s. (1982). ovarian maturation and ovulation of the amphidromous type ayu, plecoglossus altivelis in chikugo river based on histological observations. bulletin of the japanese society of scientific fisheries, 48, 1573-1582. maya, m. (2011). histological study of gametogenesis in threatened mystus cavasius. ms thesis, department of fisheries management, bangladesh agricultural university, mymensingh, bangladesh. mcadam, d.s., liley, n.r. & tan, e.s. (1999). comparison of reproductive indicators and analysis of the reproductive seasonality of the tinfoil barb, puntius schwanenfeldii, in the perak river, malaysia. environmental biology of fishes, 55, 369–380. mcconnell, r., & lowe-mcconnell, r. h. (1987). ecological studies in tropical fish communities. cambridge university press. panhwar, s. k, siddiqui, g., & zarrien, a. (2011). reproductive pattern and some biological features of anadromous fish tenualosa ilisha from pakistan. indian journal of geomarine sciences, 40(5), 687-696. pillay, s. r., & rosa, h. (1963). synopsis of biological data on hilsa, hilsa ilisha (hamilton, 1822). food and agricultural organization of the united nations. fao. fish biology, 25-64. shinkafi, b. a., ipinjolu, j. k., & hassan, w. a. (2011). gonad maturation stages of auchenoglanis occidentalis (valenciennes 1840) in river rima, northwestern nigeria. journal of fisheries and aquatic science, 6(3), 236. siddiquee, a. (2012). reproductive biology of great snakehead channa marulius from the sylhet basin, ms thesis, department of fisheries management, bangladesh agricultural university, mymensingh, bangladesh. wallace, r. a., & selman, k. (1981). cellular and dynamic aspects of oocyte growth in teleosts. american zoologist, 21(2), 325-343. pa ge 1 pa ge 21 american journal of life science and innovation (ajlsi) length-weight relationship and relative condition factor of hilsa (tenualosa ilishatenualosa ilisha) fishes in the bay of bengal, bangladesh flura1, md. moniruzzaman1, mohammad ashraful alam1, md. hashibur rahman2*, md. abu kawser didar1, azhar ali3, md. harunor rashid1, md. anisur rahman2, yahia mahmud2 volume 1 issue 2, year 2022 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v1i2.821 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: october 23, 2022 accepted: october 30, 2022 published: november 03, 2022 the present study describes the associated relationship between the length-weight, sex ratio and related condition factor of tenualosa ilisha based on the length and weight data collected from the commercial landing station of bfdc, cox’s bazar, from january 2019 to december2019. the work was carried out on 866 specimens (307 male and 559 female) ranging from 8.4 to 53.6 cm in length and weight of fishes ranging from 7 to 1977g, respectively. the reproductive attributes of t. ilisha appeared in the sex proportions (m: f=1:2) which revealed the prevalence of females in comparison with males. the external observation was taken into consideration to determine the gender of hilsa. the relationship of length-weight was fitted with the pooled data for males and females independently of all month-to-month samples which results the bw=0.0104tl2.9795 (r2=0.9636) and bw=0.0019tl3.4689 (r2=0.8461) respectively. the results expressed a higher correlation in between the length-weight (r>0.91). the length wise relative condition factor of t. ilisha was estimated as 1.02, 0.9, 1.05, 1.03, and 1.02 at the length group of 0-20, 21-30, 31-40, 41-50, 51-60 respectively indicating fluctuation of condition factor within the size group. the overall kr for t. ilisha was 0.9-1.22 in which maximum kr was found in july while the minimum was in march. the kr was strongly correlated with tl and bw. keywords length-weight, relationship, condition factor, hilsa, tenualosa ilisha, bay of bengal, bangladesh 1 bangladesh fisheries research institute, riverine station, chandpur, bangladesh. 2 bangladesh fisheries research institute, headquarters, mymensingh, bangladesh 3 bangladesh fisheries research institute, freshwater sub-station, saidpur, bangladesh. * corresponding author’s e-mail: hasibkhan94bfri@gmail.com introduction the hilsa shad is deliberated as a national flag fish of bangladesh and also treated as a popular food fish in the indian subcontinent. the juvenile of hilsa is locally known as jatka (islam, 2016a). hilsa shad is nutritionally rich in amino acids, lipids and minerals (de et al., 2019). it is the largest and most valuable fishery in the context of bangladesh, as well as a major fishery in sri lanka, india, china, and also in myanmar, malaysia, thailand and vietnam (freyhof, 2014). the hilsa is highly popular food amongst the people of middle and in the east south asia. the contribution of this fish species is about 12% of the total fish production of the country (dof, 2019). hilsa shad is rich in minerals, lipids and amino acids (de et al., 2019). it is bangladesh’s national fish and the country’s largest single species fishery and approximately contributes 12-13 percent of total fish production (haldar, 2008). bangladesh earns about tk.1500 million foreign currency and exports a large amount of hilsa as well. hilsa is primarily exported to european union, america, and australia as well as a few other far eastern and middle eastern countries. its marine range includes the arabian sea, the bay of bengal on india’s west coast and iran and iraq in the persian gulf, as well as (ahmed et al., 2008). hilsa shad accounted for more than 95 percent of bangladesh’s total commercial catch in the early 1970s (coad et al., 2003). approximately 2% of the total population either directly or indirectly rely on the fishery for a living, (mazid et al., 2007). t. ilisha in iran’s south-west is considered as a significant food fish. although the hilsa shad is usually regarded as an anadromous species, two ecotypes have been identified which includes a marine type and a river potamodromous type. the potamodromous stocks generally spend their entire year in the middle sections of the river from where they were actually reproduced. anadromous stocks commonly return to their original habitat after spawning and ascend rivers during the breeding season (panhwar et al., 2011). therefore, the actual stocks are still in controversial and the ageing is erratic due to the absence of annual rings on scales (rahman and cowx, 2006). however, length-weight relationship is an instant tool in fisheries biology, ecology, and fisheries assessment. the variation in length-weight is influenced by gonadal development, fatness and the feeding intensity (le cren, 1951). the measurements of length and weight can impart the associated information on the life span, mortality, growth and production and stock composition (orhan et al., 2009). the lwr of t. ilisha has been studied by many scientists (nima et al., 2020; mondal et al., 2015; nibedita et al., 2017; mondal et al., 2018; flura et al., 2015; nurul amin et al., 2005; bhaumik et al., 2011; de and dutta, 1990;), population biology (islam et al., 1987; rahman et al., 1998; amin et al., 2000; haldaret al., 2001; ahmed et al., 2008; hossain et al., 2019). this relationship is very important in fisheries biology because it allows estimation of an average weight of the fish of a given length group (beyer, 1987), assesses the well-being of individuals and to determines possible differences between separate unit stocks of the same species (king 2007). the relationship of length-weight can be used in the estimation of condition factor (kr) of fish species. the relative condition factor (kr) of a fish reflects physical and https://doi.org/10.54536/ajlsi.v1i2.821 https://journals.e-palli.com/home/index.php/ajlsi mailto:hasibkhan94bfri%40gmail.com?subject= pa ge 22 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 21-28, 2022 biological circumstances and fluctuations by interaction among physiological factors and feeding conditions (le cren; 1951). it quantitatively assesses the well-being of fish and predicts its future population success, given its influence on growth, reproduction, and survival (richter, 2007; hossain et al., 2013a, 2016). relative condition factor kr is defined as kr = w/ (a×lb) where, w is the body weight (g) and l is the total length (cm). hossain et al., (2009), (2013b); rypel and richter (2008) reported that kr can be used to examine fish health. good growth condition of the fish is deduced when kr ≥ 1, while the organism is in poor growth condition compared to an average individual with the same length when kr < 1. kr was also assessed within different periods (warm for spring and summer; cold for fall and winter). the findings of this study will help to improve stock assessment to induct an adequate regulation for sustainable fishery management and conservation of hilsa in the territorial waterbody of bangladesh. materials and methods fish samples were taken from bfdc landing centre in cox’s bazar, bangladesh month by month from january to december 2019. in this study, a total of 866 specimens (307 male and 559 female) were randomly collected ranging in total length (tl) from 8.4 to 53.6 cm and total body weight (bw) from 7 to 1977 g. table 1 shows the details of the hilsa collection used in this study. specimen’s data was collected and pooled together in microsoft excel spread sheet to analyze through microsoft excel analytical toolpak. statistical analyses were also done by graphpad prism 9.0 software considered at 5% level of significance (p< 0.05) in the study. total length (from the tip of snout to the extended tip of the caudal fin) and the centimeter scale close to 0.01 mm was used to measure the standard length in the fish market. total weight was measured with a digital balance for individual fish in grams (acculab sartorius group, 0.01 g accuracy). the gender of hilsa was determined by external observation. the equation bw= a*tlb where, bw=body weight of fish in (g) tl=total length of fish in (cm) a=constant (intercept) b=an exponent indicating isometric growth when equal to 3, was used for the associated estimation of lwr. the the co-efficient of determination (r2) and 95% confidence intervals of a and b were also estimated. the relative condition factor (kr) compensates for changes in form or condition with an increase in length, and was calculated following le cren (1951): kr = w/ (a×lb) where, w is the body weight (g) and l is the total length (cm). all the calculations were done using microsoft office excel (2019) and graphpad prism 9.0. fulton′s condition factor (kf) was calculated according to fulton (1904) as kf=100× (bw/tl3). the scale factor of 100 was used to bring the kf close to the unit factor (hossain et al., 2012b). the monthly variations of kf and b were also observed. results and discussion in analyzing data, a total of 866 specimens (307 male and 559 female) were collected from bfdc landing centre in cox’s bazar, bangladesh. the reproductive attributes appeared as the ratio of m: f=1:2 which shows the prevalence of females over males in this experiment study (fig.1). figure 1: percent composition of male and female t. ilisha figure 2: monthly variations of total length of t.ilisha. from january to december 2019, male hilsa were collected to assess the total body weight and length data over the period. the variation tl and bw on monthly basis shown in fig. 2 and fig. 3, respectively the equation for the figure 3: monthly variations of total length of t.ilisha. length-weight relationship of t. ilisha was worked out and expressed as: the exponential form of equation obtained for length-weight relationship wasbw=0.0104tl2.9795 (r2=0.9636) (fig. 4). the parameters ‘a’ and ‘b’ were estimated as: 0.0104 and 2.9795, respectively (fig. 4). the length-weight relationships might be affected by the general condition of appetite and gonadal contents of the fish assumed as the value of ‘b’ in males was found higher. the growth was negative allometric (b>3) as the ‘b’ value was recorded 2.97 in this study. the ‘b’ value was close to 3 which indicated isometric growth of the fish. the value of exponent ‘b’ in equation w = alb usually lies between 2.5 https://journals.e-palli.com/home/index.php/ajlsi pa ge 23 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 21-28, 2022 estimated as tl = 0.0019bw3.4689 and r2=0.8461 (fig. 5). the parameters ‘a’ and ‘b’ were estimated as: 0.0019 and 3.4689, respectively from length-weight relationships data (fig. 5). the ‘b’ value indicates that growth was positive allometric as the recorded value was 3.4 (b>3). there was a found a strong relationship between total length and body weight in the sample as the correlation coefficient value was 0.8461 and such a result reflected the positive slope (fig. 5). the value of r2 being greater than 0.196, the relationships were significant at 95% confidence level for t. ilisha and is applicable to the whole population. the variation in body weight (84.61%) was observed due to the variation in total length in the sample of hilsa which was derived from the coefficient of determination (0.8461). condition factor of a fish reflects the information and variation in the physiological state of the fish in relation to its welfare and indicate the sustainability of the fish to the environment. it is an indicator of general physiological conditions of fishes, such as, first maturity, spawning season, environmental conditions availability of food. differences between sexes due to feeding intensity and depth of water influence the kr value to some extent. the length wise relative condition factor of t. ilisha (fig.6) were estimated as 1.02, 0.9, 1.05, 1.03, and to 3.58. there was found a strong relationship between total length and total body weight in the sample as the correlation coefficient value indicated 0.9636 and its positive value reflected the slope as shown in (fig. 4). table 1: descriptive statistics for the individual tree variables for the two (2) study sites date no of male size range no of female size range condition factor (mean±sd)tl (cm bw(g) tl (cm) bw(g) jan 40 23.5-35 142-411 86 33.2-46.6 446-1208 1.06±0.19 feb 24 24.8-40 149-622 52 38.6-47.5 687-1215 mar 21 13-29.4 25-260 28 31.1-48.2 320-1128 apr 19 8.4-15 7-35 27 23.3-37 14-150 may 10 20.6-31 92-252 32 32.7-43.9 430-1096 jun 18 17.2-36.2 76-452 25 28-46 210-1050 jul 15 33.2-41.5 354-728 19 41.4-50.8 772-1977 aug 35 22.5-38.6 117-621 49 34.3-48.5 419-1644 sep 17 25.5-37.6 172-522 53 33.6-53.6 380-1862 oct 31 23-35 138-430 93 37.5-51.5 605-1805 nov 41 17-37.2 40-511 70 36.9-50.8 530-1460 dec 36 25-36 140-501 92 37.4-48.5 595-1305 where, tl= total length; bw= body weight figure 4: relationship between total body weight (g) and total length (cm) in the male, t. ilisha. as the r2 values being greater than 0.196, the relationships were estimated as significant at 95% confidence level and is applicable to the population as a whole in the experimental study. the variation of 96.36% in body weight was due to the variation in total length which derived from the analysis of coefficient of determination (0.9636) data. from the total length and total body weight data were figure 5: relationship between total body weight (g) and total length (cm) in the female, t ilisha figure 6: variations of relative condition factor (kr) with total length of t. ilisha https://journals.e-palli.com/home/index.php/ajlsi pa ge 24 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 21-28, 2022 1.02 at the length group of 0-20 cm, 21-30 cm, 31-40 cm, 41-50 cm, 51-60 cm respectively indicating fluctuation of condition factor within the size group, which implies the physiological change of the fish in different environmental regimes of the bay of bengal. the minimum value of female kr was 0.9 in february and the maximum value was 1.22 in july while minimum value of male kr was 0.89 in february and the maximum value was 1.1 in august. monthly variations of kr were showed in fig. 7. table 2 showed correlation between tl table 2: correlation of relative condition factor (kr) with total length (tl) and body weight (bw) with 95% confidence limits of the t. ilisha correlation rs values 95% cl of rs p value level of significance tl vs. kr 0.02676 0.1889 to 0.3429 < 0.0001 **** bw vs. kr 0.2918 0.4912 to 0.5857 < 0.0001 **** rs, spearman rank-correlation values; cl, confidence limit; p, shows the level of significance; **** very highly significant figure 7: monthly variations of relative condition factor (kr) of t. ilisha vs. krand bw vs. kr. the minimum value of female kf was 0.75 in december and the maximum value was 1.5 in july. the minimum value of male kf was 0.90 in march and the maximum value was 1.12 in may (fig. 8). figure 8: monthly variations of fulton’s condition factor (kf) of t. ilisha discussion from the findings, the maximum length obtained53.6 cm tl which was smaller than the study (60 cm) of froese and pauly, (2020) and the study (61 cm) of amin et al., (2004), and (57 cm) rahmanet al., (1999) and amin et al., (2002) in bangladesh. many other scientists (fluraet al., 2015; roomiani and jamili, 2011; mohanty and nayak, 2017; bhaumiket al., 2011; sarkaret al., 2017; bhaktaet al., 2019) found the body length were smaller than the current study. our study found that mean tl and bw were comparatively smaller in april. similar results also found mathur, (1964) stated that hilsa had small peak was in february, sothe juvenile recruit in april and thus they are small in size and weight in april. hossain et al., also (2014) reported that hilsa recruited in the adult stock in january because september-october is the peak spawning season of hilsa shad. the body length is a considered as an important parameter which may be directly related with growth rate, natural and fishing mortalities (guoping et al., 2008). the sex ratio in t. ilisha have been identified and executed several studies by amin et al., (2005), quddus et al. (1984a), shafi et al. (1974 and 1978). the sex ratio in this study (m: f= 1:2) was slightly similar to the findings of amin et al., (2005) where the dominance of females complies with the results (males to females 1:5.09). the sex ratio was changed between different months, but female was found predominant which derived from the same statement of amin et al., (2005). some of the contradictory statements have been revealed by previous investigations on the sex ratio (ahmed and saha, 1996; quddus et al., 1984a). the often moving in separate shoals may be caused for the associated variations. several causes recommended for the unequal sex ratios of the hilsa (zhang et al., 2009). length-weight relationship provides the information about estimation of fishery yield and general well-being of the fishes. it provides information on stock condition (bagenal and tesch, 1978). to conduct the comparative growth studies, length-weight relationship is important in fisheries management (moutopoulos and stergiou 2002. pauly (1993) stated that lwr provides valuable information on the habitat where the fish lives while kulbicki et al., (2005) stressed the importance of lwr in modeling aquatic ecosystems. the b values in lwr determine the growth pattern of the fish species. when b is equal to 3 or close to 3, the growth of the fish is said to be isometric i.e., while the length of hilsa increases it becomes more robust (bagenal and tesch 1978). similarly, when b is far greater or less than 3, growth of the fish is positive allometric or negative allometric i.e., the fish becomes heavier or thinner with increase in length (king 1996). the lwr of male and female hilsa of the bob system were found to be bw=0.0104tl2.9795and bw=0.0019tl3.4689. in female there is a slight significant difference between the exponent value and ‘3’ hence indicating isometric growth and representing the ideal shape of fish. but in male hilsa, b value is less than 3. https://journals.e-palli.com/home/index.php/ajlsi pa ge 25 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 21-28, 2022 hence, it can be concluded that the growth of hilsa is negatively allometric. when the value of b is less than 3.0, the fish experiences a negative allometric growth (pervin and mortuza 2008; thomas et al. 2003). this finding was found compatible and resembled with the results of ahmed and saha (1996). the mean length of female was significantly higher which was derived from the statistical value (p<.05). the same result was observed from the study of roomiani et al., (2014). the exponent ‘b’ usually lies between 2.5 and 4 (amin et al., 2009). when the growth is isometric, the value of ‘b’ will be exactly ‘3’. the differences between the value of ‘b’ not only observed between the species, but also between the stock of the same species due to sex, maturity and seasons. the length-weight relationship of ranges between 2.68 to 3.16 (amin et al., 2005). the ‘b’ of t. ilisha to be in the range (2.76-3.38) in another studies in bangladesh and india (amin et al., 2002; 2004; 2005, roomiani et al., 2014; quddus et al.,1984). according to pervin and mortuza (2008), these values usually ranged from 2.5 to 4.0 for many fish species. thus, the higher b values of regression slope showed that the lwr of a particular species followed the cube law. the reflection of the general condition of appetite and gonad content of the fish was detected as the ‘b’ value was found higher (pervin and mortuza 2008). in addition, bagenal and tesch (1978); froese (2006) also found that b values are reliant on biological and environmental conditions and geographical, temporal and sampling factors however, these factors were not taken into consideration due to time and budget constraints. many factors could contribute to the differences of the growth of fish such as differences of habitat, fish activities, food habits and seasonal growth rates. dutta et al., (2012) also estimated the positive allometric growth, i.e., the weight increase is directly proportional to the increase in length. the same type of lwr is also found in the observation from amin et al., (2005) and established the female t. ilisha was taller than males. condition factor of a fish ascertain the information and variation in the physiological state of the fish in relation to its welfare and indicate the sustainability of the fish to the environment. the present study found that the minimum and maximum value of female kr in december and july & minimum and maximum value of male kr in march and may, which was slightly similar to the study of nima et al., (2020), and not similar sarkaret al., (2017) in august and june in hooghly estuarine system, india. kr value may higher during spawning season (khan et al., 2001). hossain et al., (2017) stated that the difference may be occurred due to gonadal maturity, amount of undigested food in the alimentary canal and changes in amount of fat stored in body tissue. mahmood et al., (2012) has found relative condition factor (kr) varied from 0.90±0.08 to 1.03±0.08 of ilisha melastoma from pakistan. nath, a.k. (2013) has found the mean value of relative condition factor (kr) is 1.0496801 and 1.010145 in female and male of t.ilisha which was similar to our study. overall female kr of our study was within 0.9991.22 and male 0.956 0.1.12 which was similar to the study of pooled data of nima et al., (2020), while mandal et al., (2018) reported kr value varied between 0.98-1.04 and sarkar et al., (2017) observed it from 0.98 to 1.05 for t. ilisha from india. the increased kr values after may could also attribute be to the peak feeding periods for the species, as observed by bapat (1951). khan et al., 2001 also found high kr values during peak spawning periods and low kr values after spawning periods of t. ilisha. in this study, condition factor in fish is increase with increasing in size. gradually it decreases as fishes are going to attain the maturity stage which was similar with the study of mohanty and nayak (2017) also indicated same matter for t. ilisha in the chilika lake, india. reuben (1992) stated that an early stage of fish has higher kr value. parida et al., (2013) have concluded that the lowest kr values during the more developed gonadal stages might mean resources transfer to the gonads during reproductive period. so, the observed peaks and values in the relative condition factor for length range of 31-40 cm might be associated with cyclic physiological processes by showing repeated development of gonads as well as increase in feeding intensity and shading of mature ova (spawning) respectively during life of the fish. the fulton’s condition factor (kf) values between the sexes were significantly different in our study, likely indicating the presence of mature females. in this study, we found the minimum and maximum value of female fulton’s condition factor (kf) was 0.75 and 1.40 in december &july and the minimum and maximum value of male kf was 0.90 and 1.12 in march & may while mandal et al., 2018 stated that kf ranged from 0.47-3.05 in pooled, 0.47-1.63 in male and from 0.883.05 in female. mondol (2015) opined that condition factor decreases with decline in length and is also express healthy condition showing good compatibility with the nature or environment. conclusion the present study provides the basic information about the length-weight relationship, sex ratio and related condition factor of t. ilisha estimated as 1.02, 0.9, 1.05, 1.03, and 1.02 at the length group of 0-20 cm, 21-30 cm, 31-40 cm, 41-50 cm, 51-60 cm respectively, indicating fluctuation of condition factor within the size group, which implies the physiological change of the fish in different environmental regimes of the bay of bengal. there was a strong relationship between total length and body weight within the sample as the correlation coefficient value was 0.8461 and such a result reflected the positive slope. the value of r2 being greater than 0.196, the relationships were significant at 95% confidence level for t. ilisha and are applicable to the whole population. the variation in body weight (84.61%) was observed due to the variation in total length in the sample of hilsa which was derived from the coefficient of determination (0.8461). the findings of this study will facilitate the https://journals.e-palli.com/home/index.php/ajlsi pa ge 26 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 21-28, 2022 researchers and academicians to perceive the stock assessment strategy by discerning the tangible subsidiary for sustainable hilsa fishery management in bangladesh. acknowledgement it is also a great pleasure for the investigators to express their sincere and deep sense of gratitude and indebtedness to the bangladesh fisheries research institute (bfri) for providing financial support. the investigators also express their profound indebtedness and sincere gratitude to the director general of bangladesh fisheries research institute (bfri), mymensingh for his continuous support, encouragement and kind 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(1984). bangladesher mathso shampad (fisheries of bangladesh) 1st edn. bangla academy, dacca, bangladesh. thomas, j.s., venu, s. and b.m. kurup, (2003). length weight relationship of some deep-sea fish inhabiting the continental slope beyond 250m depth along the west coast of india. naga 26(2), 17-21. zhang j, takita t and zhang c (2009). reproductive biology of ilisha elongate (teleostei: pristigasteridae) in ariake sound, japan: implications for estuarine fish conservation in asia. estuarine, coastal and shelf science, 81, 105–113. https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 am. j. life sci. innov. 1(1), 2022 the american journal of life science and innovation (ajlsi) is an international, open access, and blind peer-reviewed journal that publishes research articles emphasizing life sciences and innovations. the journal comprises techniques suitable for promoting the dissemination of research findings that are expected to benefit the basic needs of health, agriculture, biotechnology, pharmaceutical, and food industries. ajlsi also publishes life science articles on evidencebased practices, thus improving the quality of life. ajlsi accepts manuscripts throughout the year; all submitted manuscripts are rigorously reviewed, and publish articles online first. frequency: 3 issues per year area of publication: life science and technologies, innovations in life sciences and development of living orgasms, and related fields. editorial team professor dr. nirmal chandra roy sylhet agricultural university, bangladesh dr. sejuti mondal texas state university, usa dr. sandra milena camargo silva materials engineering, pedagogical and technological university of colombia, colombia dr. myint thuzar professor, yezin agricultural university (yau) myanmar dr. md. morshedur rahman professor, department of dairy and poultry science bangabandhu sheikh mujibur rahman agricultural university, bangladesh dr. omar faruk miazi professor, department of genetics and animal breeding chattogram veterinary and animal sciences university (cvasu) chattogram, bangladesh dr. yulianna puspitasari faculty of veterinary medicine, universitas airlangga surabaya, indonesia dr. md robiul karim associate professor, department of medicine bangabandhu sheikh mujibur rahman agricultural university gazipur, bangladesh dr. mst. rubia banu assistant professor, department of fisheries management bangabandhu sheikh mujibur rahman agricultural university gazipur, bangladesh dr. tilak chandra nath associate professor, department of parasitology sylhet agricultural university, bangladesh professor dr. mrityunjoy kunda dean, faculty of fisheries sylhet agricultural university, bangladesh contact: american journal of life science and innovation (ajlsi) e-palli llc address: 2055 limestone rd ste 200c, zip code: 19808, wilmington, delaware, usa email: ajlsi@e-palli.com phone: +1 3024070506 url: https://journals.e-palli.com/home/index.php/ajlsi http://mailtoajmri@e-palli.com pa ge 6 american journal of life science and innovation (ajlsi) evaluation of low chilling strawberry (fragraria ananassa) varieties with respect to yield and quality in eastern nepal s. dhakal1*, d. shashan2, b. shrestha1 volume 1 issue 1, year 2022 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v1i1.211 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: january 24, 2022 accepted: february 26, 2022 published: april 18, 2022 the experiment entitled “evaluation of low chilling strawberry (fragraria ananassa) varieties with respect yield and quality in eastern nepal” was conducted in dharan and biratnagar of nepal with five varieties ‘emco 33’, ‘emco 32’, ‘florida fortuna’, ‘florida beauty’ and ‘sweet sensation’. the experiment was laid out in factorial rcbd where variety and location was considered as factors. from the experiment highest number of fruits per plant (13.22) and maximum fruit set (95.72%) was recorded with variety ‘emco 33’. however, maximum number of fruits per plot (444.25) with only 14.04 % of unmarketable fruit and highest yield of 9068.75 grams per plot with 7976.00 grams of marketable yield per plot was obtained with variety ‘emco 32’ in dharan. comparing the length and diameter of fruit variety ‘emco 32’ (49.00 cm×43.42 cm) and variety ‘emco 33’ (49.04 cm×38.41 cm) seems more appealing in form. ph, vitamin c and ta content was found higher in variety ‘emco 33’ (3.48, 71.89 mg/g and 0.90 % respectively) and higher tss content (6.57 0 brix) was obtained with variety ‘emco 32’ with no interaction effect for quality traits. thus among treatment combinations variety ‘emco 32’ followed by ‘emco 33’ in dharan conditions is more preferable to other treatments and also both the varieties perform well compared to other varieties in biratnagar conditions also. however still more studies are required to assess the varietal performance and it would be premature to recommend suitable variety for given locations. introduction the modern cultivated strawberry (fragaria ananassa duch.) is a hybrid of two largely dioecious, octoploid species, fragaria chelonesis duch and fragaria virginiana duch belonging to family rosaceae. basically, it is herbaceous perennial and short day plant. it grows predominantly in the temperate climate but worldwide it is the most widely distributed fruit crop due to its genetic diversity, highly heterozygous nature and broad range of environmental adaptation (anderson and guttridge, 1982; dale et al., 2000). strawberry is cultivated in limited regions of nepal in nuwakot district and its periphery and choice of varieties is limited to nyoho, ohno, eyeberry and akhime. productivity of strawberry in nepal is only 9 mt/ha compared to world productivity 23.58 mt/ha (dado, 2017; fao, 2019). because of limited research, strawberry cultivation is still in womb stage in nepal. only limited varieties are available. previously, nyoho variety was widely cultivated recently, ankhime and eyeberry varieties are also introduced. cultivation of strawberry on other hand is concentrated in hilly areas of nepal potential areas has not been identified no studies on strawberry in terai is conducted strawberry once thought to be temperate fruit can be grown in tropical areas too. since strawberry is high value fruit, more potential areas need to be identified and tropical low chilling requiring varieties need to be explored. so, 5 low chilling strawberry varieties ‘florida beauty’, ‘sweet sensation’, ‘fortuna’, ‘emco 32’ and ‘emco 33’ were studied in two domains biratnagar and dharan in easterrn nepal to compare keywords fruit, nepal, quality, strawberry varieties, yield their performance under different growing condition with following objectives: • to study the comparative yield performance of low chilling required five strawberry varieties in two locations • to assess the quality of fruit of different strawberry varieties under different growing location. literature review world production of strawberry is in increasing trend, it was 4.4 million tonnes in 2000 ad to 9.2 million tonnes in year 2017 (fao, 2019) which is almost twice. china leads the world production with 3.7 million tonnes in year 2017 a.d which is almost one third of world production while usa has highest productivity of 67 tonnes/ha. here in nepal strawberry production is concentrated in nuwakot district covering area of 55 ha and the production is around 495 mt where 254 families are involved in strawberry cultivation (dado, 2017). nepal imports 10,826 kg of strawberry annually which is worth nrs 2.1 million (tepc, 2019). the japanese first saw the potential for strawberry farming in kakani 25 km to the north of kathmandu ,25 years ago when agronomist matsuura hiroshi brought six sample plants which flourished and spread among farmers who till then had been growing radish for the kathmandu market, in 1991. the variety of strawberries cultivated in nepal akhime, nyoho, require an altitude of 1500 2500m with a temperature range of 4-25 degree celsius and 3000-4000 ml of rainfall, making kakani the ideal location (nepali times, 2017). currently more than 700 households are involved in commercial farming, 1 department of horticulture, faculty of agriculture, agriculture and forestry university, nepal 2 department of entomology, faculty of agriculture, agriculture and forestry university, nepal * corresponding author’s e-mail: shivadkl94@gmail.com https://doi.org/10.54536/ajlsi.v1i1.211 https://journals.e-palli.com/home/index.php/ajlsi mailto:mailto:shivadkl94%40gmail.com?subject= pa ge 7 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 6-14, 2022 each producing approximately 1000kg of strawberries per season (medep, 2010). nuwakot district has been gaining prominence in strawberry cultivation as a summer fruit. kakani and okharpauwa vdcs are regarded as the pocket area for strawberry cultivation. once suitable temperature and day length conditions are met, strawberry flower buds are initiated. initially, the apical meristem broadens, then the sepal, petal, stamen and carpel primordia develop in sequence and start to enlarge (darrow, 1966; darnell, 2003). the primary flower emerges first, followed by the secondary, tertiary and quaternary flowers (hancock, 1999). typically the inflorescence includes one primary flower and two or three, or sometimes four, secondary flowers terminally on individual branches of the main stem (darnell, 2003). at least 30% of the carpels need to be fertilized for fruit to develop good shape (day, 1993), although guttridge (1979) reported 70-80% were needed. strawberry fruit development can be divided into several stages: small green, large green, white, pink and red. the weight and size of the fruit is largest at the red stage (darrow, 1966; abeles and takeda, 1990). when fruits take about 30 days to develop from bloom to ripening, cell division occurs first and ends 10-15 days after anthesis (cheng & breen, 1992). then the cells enlarge and expand towards the inside of the fruit. however, although the cells divide and expand, the main factor which causes the variation in fruit size is the number of cells at anthesis (cheng & breen, 1992). cell division contributes between 15-20% of the total growth. the remaining growth comes from cell enlargement by cells expanding towards inside of the fruit (hancock, 1999). many day-neutral strawberry cultivars require a dormancy period of between 675 and 1000 hours in a conditions of -1 to 10° c (stewart & folta, 2010). studies show day/ night temperatures of 18/14° c as the ideal for fruit production (hancock, 1999). heat stress is a challenge in strawberry production that has been reported all over the world. in general, strawberry plants under heat stress grow more slowly, and produce fewer and smaller fruit (renquist et al., 1982; hellman and travis, 1988). temperature and photoperiod are the two major factors which influence the transition from vegetative to floral growth. flower bud initiation is inhibited when temperatures are above 12 to 28°c depending on day length and flowering type (chabot, 1978; okimura and igarashi, 1997; dale et al., 2009). one recent study showed that the developmental stages of flower buds in ‘akihime’ strawberry were were significantly larger at 22°c than those of the untreated plants (kim et al., 2009). fruit number per plant decreases as temperature increases. in ‘nyoho’ and ‘toyonoka’ the numbers of fruit were greatly reduced at 30/25°c when compared to 23/18°c (ledesma et al., 2008).when the day-time and night-time effects were investigated, fruit size was smaller under daytime temperatures over 15-17°c and largest at 12°c night temperature. (went, 1957; sato & hiraoka, 1971) sweet sensation’ is a new strawberry cultivar released from the university of florida in 2013, commonly known as ’florida 127’ in us and canada originally evaluated as breeding selection fl 09-127. ‘florida127’ originated from a 2009 cross between winterstar™ ‘fl 05-107’ (female parent) and unreleased breeding selection fl 0258 (male parent) (whitaker et al., 2017). ‘florida fortuna’’ a strawberry variety released by us is known as ‘florida radiance’ in us and canada released from uf/ifas in 2008 (chandler et al. 2009) originated from a 2001 cross between ‘winter dawn’ (female parent), a 2005 release from the uf/ifas breeding program, and fl 99-35 (male parent) (whitaker et al., 2019). ‘florida beauty’ (ppaf) is a new strawberry cultivar released by the university of florida and commercialized in 2017. this cultivar was originally evaluated as breeding selection fl 12.121-5. ‘florida beauty’ originated from a 2012 cross between queensland australia selection 2010 119 (female parent) and ‘florida radiance’ (male parent) (whitaker et al., 2017a). ‘florida127’ is a short-day plant adapted to annual, winter plasticulture growing systems. the plant is moderately compact, robust, and upright with long pedicels, making the fruit easy to harvest producing conic to broad-conic fruit that are uniform in shape throughout the season, resulting in few non-marketable fruit (whitaker et al., 2017). ‘‘florida fortuna’’ produces conic to long-conic fruit that are very large, uniform, glossy, and evenly coloured. ‘florida fortuna’’ has a weak plant habit and can benefit from extra chilling hours in the nursery. while it requires little chilling to initiate flower buds compared to typical short-day cultivars, increased chilling may be important to increase production (whitaker et al., 2019). whitaker et al. (2017) stated that florida fortuna’ fruit size is larger than the other cultivars late in the season. the fruit has a moderate acid content with a balanced flavor and is generally the juiciest of the uf/ifas cultivars. both early season and late-season yields of ‘florida fortuna’ in florida have been higher than any other uf/ifas cultivar in most seasons. in an experiment conducted in queensland to compare varieties ‘florida beauty’ cultivar has excellent fruit quality, with flavour often similar to ‘sweet sensation’ and sufficient quality to be marketed at retail under the ‘sweet sensation’® brand. early and total season yields of ‘florida beauty’ have been very similar to ‘florida fortuna’ for early planting dates around sept 25. when planted early in the planting period, no overly elongated fruit have been observed in contrast to ‘florida fortuna’, which can produce elongated fruit in the early season. when planted at later dates around oct 10, ‘florida beauty’ has had lower yields than ‘florida fortuna’ due to its more compact plant size (whitaker et al., 2017a). whitaker et al. (2017) stated that fruit size of ‘sweet sensation’ is very large, exceeding that of ‘florida https://journals.e-palli.com/home/index.php/ajlsi pa ge 8 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 6-14, 2022 fortuna’ on average over the course of the season. fruit firmness is slightly greater than that of ‘florida fortuna’, with excellent shelf life. average fruit weight of ‘sweet sensation’ florida fortuna and ‘florida beauty’ is 35-45 gm, 30-40 gm and 27-37 gm respectively as recorded in u.s. plant patent (united states patent no. usoopp20363p2, 2009; united states patent no. us 20140359905p1, 2014; united states patent no. usoopp30385p3, 2019) in an experiment conducted to compare 3 varieties of strawberry average marketable yield of 803.6 gm per plant was recorded with ‘sweet sensation’ being at par with ‘florida fortuna’ i.e. 749.7 gm per plant and lowest yield was recorded with ‘florida beauty’ i.e. 601.3 gm per plant with average weight per fruit being 30.8 gm, 25.0 gm and 22.3 gm respectively (whitaker et al., 2015). in an experiment in university of florida it was recorded the ph of florida fortuna was 3.6 and that of ‘sweet sensation’ was 3.7. but ‘sweet sensation’ was superior for sweetness compared to ‘florida fortuna’ with total soluble solid content 7.5 % and 5.3 % respectively (whitaker et al., 2015). it is estimated that the picking interval should be one day longer for ‘sweet sensation’ than for ‘florida fortuna’ at most points during the season. the ripe fruit of ‘florida127’ have excellent flavour and aroma. soluble solids contents of ‘sweet sensation’ fruit were significantly higher than those of ‘florida fortuna’ on six of seven harvest dates tested. titratable acidity was not significantly different from ‘florida fortuna. significant difference in tss/ta ratio was observed in variety ‘sweet sensation’ and ‘florida fortuna’ with ratio being 10.8 and 6.4 respectively (whitaker et al., 2015). methods treatment details different low chilling strawberry varieties and two locations were considered as factors in combination. therefore, there were two different factors in this research which are shown below. factor 1: location the experiment was conducted in two locations to evaluate the performance of strawberry in different ecological zones. two ecological regions biratnagar and dharan were selected to conduct the varietal research trial on strawberry. biratnagar the city is located in the morang district of province 1 has a total area of 77.5 km². the geographical location is 26°28’60”n 87°16’60”e. in biratnagar, the average annual temperature is 24.3 °c. the rainfall here averages 1898 mm. dharan the city is located in the sunsari district of province 1 has a total area of 192.61 km². the geographical location is 26°49’0”n 87°17’0”e. the city experiences sub-tropical climate situated at altitude of 349 masl. in dharan, the average annual temperature is 23.2 °c. the rainfall here averages 1799 mm. figure 1. average weather data of biratnagar and dharan from november 2018 to may 2019 factor 2: variety (v) five low chilling strawberry varieties were used to evaluate their relative performance. the varieties used were v1 = florida beauty v2 = sweet sensation v3 = fortuna v4 = emco 32 v5 = emco 33 experimental design the field was laid out in randomised block design (rcbd) with 4 replications in both locations. layout of field the field was laid out in randomized complete block design (rcbd) design with 4 replications and 5 varietal treatment in each location. the area of experimental field was 297 m 2 with length and breadth of main field 45 m× 6.6 m respectively. distance between replications was maintained at 1 m and treatments were laid 50 cm apart. with spacing of 38 cm × 28 cm (rr× pp), 52 plants per plot were maintained. planting techniques strawberries were planted in a raised bed with double row system. following schematic diagram was followed for raised bed system. planting material and planting bare root strawberry transplants were used as planting https://journals.e-palli.com/home/index.php/ajlsi pa ge 9 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 6-14, 2022 material. the bare root strawberry transplants were transplanted in raised plasticmulched beds at spacing of 38 cm ×28 cm. planting was done on nov 13, 2018.one week of overhead sprinkler irrigation was provided for one week to ensure plant establishment. parameters studied various yield and quality parameters of randomly selected and properly tagged sample plants were evaluated fruit yield parameters fruit set percentage (%), number of fruit per plant, number of fruits per plot, number of marketable plot, marketable yield per plot (grams) and fruit yield per plot (grams) were recorded fruit quality parameters total soluble solids (tss), ph of strawberry, titrable acidity (%), ascorbic acid (vitamin c) content (mg/100g) data analysis all data recorded were processed using microsoft excel. statistical analysis and relation among treatments was established for the selected parameters with reference to gomez and gomez, 1984. different statistical tools as r and excel were used for the analysis of variance, dmrt test and other data analysis as required. figure 2. double row system of strawberry planting results and discussion phenological characters table 1. phenological characters of strawberry (fragaria ananassa) as influenced by varieties and different locations of nepal in 2018/2019. effect of location on phenological characters phenological characters of different varieties of strawberry as influenced by location is presented in table 1. days to first flowering, number of flowers per plant, fruit set % and number of fruits per plant are found significantly different statistically at different locations. earlier flowering (14.52 dat) , higher number of flowers per plant (13.79) was observed in biratnagar being statistically different to days to first flowering (16.11 dat) and number of flowers per plant in dharan (10.76). however fruit set percentage is found significantly higher in dharan (89.48 %) compared to biratnagar (70.82 %). no significant effect was seen regarding number of fruits per plant which might be due to low fruit set % in biratnagar though higher number of flowers were table 1: phenological characters of strawberry (fragaria ananassa) as influenced by varieties and different locations of nepal in 2018/2019. treatments phenological characters days to first flowering number of flowers per plant number of fruits per plant fruit set % location (factor a) dharan 16.11a 10.76b 10.06 89.48a biratnagar 14.52b 13.79a 9.64 70.82b grand mean 15.31 12.28 9.85 80.15 sem 1.53** 2.46*** 1.12ns 8.60*** lsd 1.28 2.24 1.02 7.78 cv 29.87 28.49 16.12 15.17 variety (factor b) emco33 10.83c 16.90a 11.60ab 88.37a sensation 22.62a 8.43d 6.10d 64.46c florida beauty 13.61b 12.09bc 10.39b 73.63bc emco32 7.91d 14.10b 12.60a 88.92a fortuna 21.6a 9.85cd 8.57c 85.39ab grand mean 15.31 12.28 9.85 80.15 sem 1.78*** 1.66*** 1.12*** 8.80*** lsd 2.56 2.38 1.61 12.62 cv 16.47 19.15 16.12 15.51 means with same letter within column do not differ significantly at p= 0.05 by dmrt. nsnon –significant, semstandard error of mean, lsdleast significant difference, cvcoefficient of variance, datdays after transplanting https://journals.e-palli.com/home/index.php/ajlsi pa ge 10 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 6-14, 2022 obtained in biratnagar. effect of varieties on phenological characters effect of variety on phenological characters of strawberry plants is presented in table 1. days to first flowering, number of flowers per plant, fruit set % and number of fruits per plant are found significantly different statistically among different varieties. earlier flowering (7.91 dat), higher fruit set (88.92 %) and higher number of fruits per plant (12.55) was obtained in variety ‘emco 32’ whereas late flowering (22.62 dat) , least fruit set percentage (64.46 %) and least number of fruits per plant (6.10) was obtained in variety ‘sweet sensation’. however highest number of flowers per plant (16.90) were recorded in variety ‘emco 33’ whereas least number of flowers per plant (8.43) was obtained in variety ‘sweet sensation’ being statistically similar to variety ‘florida fortuna’ (9.85). table 2. interaction effect on phenological characters of strawberry (fragaria ananassa) varieties at different locations of nepal in 2018/2019. treatments phenological characters days to first flowering number of flowers per plant number of fruits per plant fruit set % location × variety interaction dharan.emco33 12.12f 14.31bc 13.22a 95.72a dharan.sensation 26.65a 7.25f 6.9de 73.75d dharan.beauty 11.97f 10.36de 9.43bc 93.89a dharan.emco32 6.60h 15.57b 12.01a 94.68a dharan.fortuna 23.20b 9.24ef 8.67cd 89.37b biratnagar.emco33 9.55g 19.5a 9.99bc 51.54f biratnagar.sensation 18.6d 9.62ef 5.29e 55.17e biratnagar.beauty 15.25e 13.82bc 11.35ab 82.05c biratnagar.emco32 9.22g 16.27b 13.10a 83.95c biratnagar.fortuna 20.00c 10.46de 8.48cd 81.40c grand mean 15.31 12.28 9.85 80.15 sem 0.64*** 1.18** 0.94*** 1.73*** lsd 1.30 2.42 1.90 3.53 cv 5.88 13.67 13.42 3.05 means with same letter within column do not differ significantly at p= 0.05 by dmrt. nsnon –significant, semstandard error of mean, lsdleast significant difference, cvcoefficient of variance, datdays after transplanting interaction effect of location and variety for phenological characters interaction effect of location and variety on phenological characters of strawberry plants is presented in table 2. days to first flowering, number of flowers per plant, fruit set % and number of fruits per plant are found significantly different statistically among different varieties at different locations. earlier flowering (6.60 dat) was obtained in dharan with variety ‘emco 32’ followed by variety ‘emco 32’ in biratnagar (9.22 dat) whereas late flowering (26.65 dat) was obtained in dharan with variety ‘sweet sensation’. the result reveals that although in overall performance there is late flowering in dharan but still from interaction effect it is evident that earlier flowering variety ‘emco 32’ performs better in dharan. significantly higher number of flowers per plant (19.50) was recorded in biratnagar with variety ‘emco 33’ followed by variety ‘emco 32’ in biratnagar (16.27) whereas least number of flowers (7.25) were obtained in dharan with variety ‘sweet sensation’ being statistically similar to variety ‘florida fortuna’ in dharan (9.24). significantly higher fruit set (95.72 %) was recorded in dharan with variety ‘emco 33’ being statistically at par with variety ‘emco 32’ in dharan (94.68 %) and also with variety ‘florida beauty’ in dharan (93.89 %) whereas least fruit set (51.54 %) percentage was recorded in variety ‘emco 33’ in biratnagar followed by variety ‘sweet sensation’ in biratnagar (55.17 %). significantly higher number of fruits per plant (13.22) was recorded in dharan with variety ‘emco 33’ being statistically at par with variety ‘emco 32’ in biratnagar (13.10) whereas least number of fruits per plant (5.29 ) was recorded in biratnagar with variety ‘sweet sensation’ followed by variety ‘sweet sensation’ in dharan (6.9). from results presented in table 1 and table 2 we can conclude that larger number of flowers are obtained in biratnagar with variety ‘emco 33’ but variety ‘emco 32’ performed better than variety ‘emco 33’ in dharan interaction effect shows that for phenological characters variety ‘emco 33’ is more suitable for dharan and https://journals.e-palli.com/home/index.php/ajlsi pa ge 11 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 6-14, 2022 table 3: yield per plot of strawberry (fragaria ananassa) varieties as influenced by different locations of nepal in 2018/2019. treatments yield per plot of strawberry marketable yield per plot (gm) total yield per plot (gm) location (factor a) dharan 4101.20 4720.75a biratnagar 2794.33 3244.81b grand mean 3447.76 3982.78 sem 935.47ns 1066.73*** lsd 1693.84 1931.50 cv 26.74 25.75 variety (factor b) emco33 6034.68a 6932.97a sensation 1558.25b 1848.25b florida beauty 1541.25b 1831.12b emco32 6844.64a 7828.32a fortuna 1260.00b 1473.25b grand mean 3447.76 3982.78 sem 384.23*** 446.85*** lsd 1103.12 1282.91 cv 31.52 31.73 variety ‘emco 32’ is more suitable for biratnagar. earlier flowering in biratnagar conditions contradicts with findings of kurian (2015), that earlier flowering was observed in high altitude. the favourable temperature and day condition, prevailing in biratnagar may have influence on early flowering in biratnagar. earlier flowering in variety ‘emco 32’ might be attributed to its day neutral behaviour. variability in flowering period in different varieties might also be due to differences in their chilling requirement as suggested by joolka and badiyala (1983) number of leaves per plant, leaf area and number of crowns per plant are positively correlated with number of flowers per plant (girijalba et al., 2015) which might be reason for higher number of flowers in dharan and in variety ‘emco 32’ and ‘emco 33’. variation in number of fruits per plant might be due to differential fruitset % among varieties in different location. lower fruit set % but higher number of flowers in biratnagar conditions resulted in insignificant difference in number of fruits per plant among locations. the variation in fruit set % may be due to genetic makeup of the cultivars and adaptation to climatic condition (jami et al., 2015). yield per plot effect of location on yield per plot yield per plot of different varieties of strawberry as influenced by location is presented in table 3. marketable yield per plot and in relation to location was found statistically insignificant whereas weight of total yield per plot was found statistically significant. significantly higher total yield was obtained in dharan (4720.75 gm) compared to biratnagar (3244.81 gm). effect of variety on yield per plot effect of variety number of fruits per plot of strawberry plants is presented in table 3. marketable yield per plot and yield per plot in relation to location are found significantly different statistically among different varieties. highest marketable yield per plot (6844.64 gm) and highest total yield per plot (7828.32 gm) was recorded in variety ‘emco 32’ being statistically at par with variety ‘emco 33’ for marketable yield (6034.68 gm) and total yield (6932.97 gm) per plot whereas least marketable yield per plot (1260.00 gm) and least total yield per plot (1473.25 gm) was obtained in variety ‘florida fortuna’ being statistically at par with variety ‘sweet sensation’ (1558.25 gm) differences in survivability and yield per plant of different varieties may be reason behind difference in number of fruits per plot of strawberry. interaction effect of location and variety on yield per plot interaction effect of location and variety on number of fruits per plot of strawberry plants is presented in table 4. marketable yield per plot and total yield per plot are found significantly different statistically among different varieties at different locations. higher marketable yield per plot (7976.00 gm) was recorded in dharan with variety ‘emco 32’ being statistically at par with variety ‘emco 33’ (7116.75 gm) in dharan. similarly in biratnagar conditions also variety ‘emco 32’ had highest marketable yield per plot (5713.28 gm). whereas least marketable yield per plot (682.00 gm) was obtained in biratangar with variety ‘florida fortuna’. lowest unmarketable yield per plot (192.00 gm) was recorded in biratnagar with variety ‘florida fortuna’ being statistically at par with variety ‘florida fortuna’ https://journals.e-palli.com/home/index.php/ajlsi pa ge 12 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 6-14, 2022 (222.00 gm) in dharan being statistically at par with variety ‘sweet sensation’ (264.00 gm) in dharan. whereas highest unmarketable yield per plot (1243.25 gm) was obtained in dharan with variety ‘emco 33’. higher total yield per plot (9068.75 gm) was recorded in dharan with variety ‘emco 32’ being statistically at par with variety ‘emco 33’ (8360.00 gm) in dharan. similarly in biratnagar conditions also variety ‘emco 32’ had highest total yield per plot (6587.90 gm). whereas lowest total yield per plot (874.00 gm) was obtained in biratangar with variety ‘florida fortuna’. the result presented in table 16 and table 17 reveals that variety ‘emco 32’ followed by variety ‘emco 33’ performed best in both locations and dharan conditions was more favourable for yield per plot. also higher unmarketable yield per plot were related with higher total yield of fruits. the differences for total yield per plot may be attributed to difference in plant survivability and number of fruits per plot. effect of location on fruit quality parameter table 4: interaction effect on yield per plot of strawberry (fragaria ananassa) varieties at different locations of nepal in 2018/2019. treatments yield per plot of strawberry marketable yield per plot (gm) total yield per plot (gm) location × variety interaction dharan.emco33 7116.75a 8360.00a dharan.sensation 1786.50c 2050.50c dharan.beauty 1788.75c 2052c dharan.emco32 7976.00a 9068.75a dharan.fortuna 1838.00c 2072.50c biratnagar.emco33 4952.62b 5505.94b biratnagar.sensation 1330.00c 1646.00c biratnagar.beauty 1293.75c 1610.25c biratnagar.emco32 5713.28b 6587.90b biratnagar.fortuna 682.00c 874.00c grand mean 3447.76 3982.78 sem 275.01* 314.36* lsd 1123.28 1284.04 cv 22.56 22.32 cv 31.52 31.73 means with same letter within column do not differ significantly at p= 0.05 by dmrt. nsnon –significant, semstandard error of mean, lsdleast significant difference, cvcoefficient of variance, datdays after transplanting significant difference between locations was recorded for quality parameters ta, ph and vit-c content of fruits and no significant difference between locations for tss was recorded , fruit quality parameter of different varieties of strawberry as influenced by location are presented in table 5. significantly higher ph value (3.28) and ta content (0.85 % citric acid ) of fruit was observed in biratnagar compared to ph (3.17) and ta content (0.80 % citric acid) in dharan higher vitamin c content (68.22 mg/100gm) was recorded in dharan being significantly different to biratnagar (64.47 mg/100gm). effect of varieties on fruit quality parameters significant difference among varieties were recorded for quality parameters tss content, ph, vitc content and ta of fruits as presented in table 5. highest ph value (3.48), highest ta content (0.90% citric acid), higher vitamin c content (71.89 mg/100 gm)was recorded in variety ‘emco 33’ whereas least ph value (3.07), least ta content (0.77 % citric acid) and least vit-c content (59.75 mg/100gm) was recorded in variety ‘sweet sensation’, highest tss content (6.57 0b) was recorded in variety ‘emco 32’ whereas least tss content (5.6 ob) was recorded in variety ‘emco 33’. variety ‘sweet sensation’ (6.12 ob) was found statistically at par with variety ‘florida beauty’ (6.150b) and ‘florida fortuna’ (6.12 0b). interaction effect of location and variety on fruit quality parameters no significant effect of interaction of location and variety were recorded for quality parameters tss content, ph, vitamin c content and ta of fruits. above results reveal that variety ‘emco 32’ and ‘emco 33’ were superior to other varieties as sweetness of fruit is related with tss and ta content of fruit and variety ‘emco 33’ had higher vitamin-c content. thus variety ‘emco 33’ is more preferable. whitaker et al. (2015) and kelly et al. (2016) also obtained similar results with physiochemical characters of strawberry fruits. higher tss and ascorbic acid content of fruit may be attributed to higher vegetative growth as enough https://journals.e-palli.com/home/index.php/ajlsi pa ge 13 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 6-14, 2022 table 5: fruit quality parameters of strawberry (fragaria ananassa) varieties as influenced by different locations of nepal in 2018/2019. treatments fruit quality parameters ph tss ta vit-c location (factor a) dharan 3.17b 6.04 0.80b 68.22a biratnagar 3.28a 6.19 0.85a 64.47b grand mean 3.22 6.11 0.82 66.35 sem 0.11*** 0.24ns 0.04*** 4.69** lsd 0.10 0.21 0.03 4.1 cv 4.94 5.51 6.89 9.99 variety (factor b) emco33 3.48a 5.6c 0.90a 71.89a sensation 3.07c 6.12b 0.77b 59.75b florida beauty 3.24b 6.15b 0.83b 67.22a emco32 3.08c 6.57a 0.85b 60.99b fortuna 3.25b 6.12b 0.78c 71.89a grand mean 3.22 6.11 0.82 66.35 sem 0.06*** 0.10*** 0.03*** 3.25*** lsd 0.08 0.14 0.04 4.66 cv 2.44 2.35 4.89 6.91 photosynthate was available for sugar and acid accumulation (singh et al., 2007). tss content was observed by the treatment which may be due to favorable temperature and humidity especially in night during fruit growth and ripening period. the soluble solids content was more dependent on environmental condition during growth and development than genetic inheritance in strawberry (shaw, 1990). the possible explanation for lower acidity in dharan may be due to difference between day and night temperature which are very narrow, whereas cooler nights and warmer days are helpful in synthesizing more acidity noticed by wani et al. (2007). the oxygen concentration which is more in the lower altitude of the earth caused oxidation of more ascorbic acid, consequently fruits showed decreased ascorbic acid content in comparison to the fruits obtained and analyzed at higher altitude. in this support klimczac and stropek (1988) reported that strawberries grown at higher altitude have higher ascorbic acid content. conclusion the experiment brought some important information on effect of location and variety on yield and fruit quality parameters of strawberry. for flowering and yield characters, variety ‘emco 32’ followed by ‘emco 33’ were superior both in dharan and biratnagar. but considering the overall production performance dharan conditions were favourable than biratnagar for given varieties. fruit quality parameters are not of major importance in nepal but considering fruit quality variety ‘emco 33’ was more preferable with higher tss, ta, vitamin c and ph content while variety ‘emco 32’ had poor quality parameters and better quality fruits were obtained in biratnagar. thus we can conclude that among the varieties under study ,variety ‘emco 33’ and ‘emco 32’ can successfully be cultivated in both locations and taking fruit quality parameters into consideration variety ‘emco 33’ is more preferable . however still more studies are required to assess the varietal performance and it would be premature to recommend suitable variety for given locations. references abeles, f. b., & takeda, f. (1990). cellulase activity and ethylene in ripening strawberry and apple fruits. sci. hort, 42, 269-275. anderson, h. m., & guttridge, c. g. (1982). strawberry truss morphology and the fate of high-order flower buds. crop res, 22, 105-122. chabot, b. f. (1978). environmental influences on photosynthesis and growth in fragaria vesca. new phytol., 87-98. chamorro, m., aguado, a., & de los santos, b. (2012). first report of root and crown rot caused by pestalotiopsis clavispora (neopestalotiopsis clavispora) on strawberry in spain. aps publications, 1495. chandler, c. (2009). united states patent no. usoopp20363p2. cheng, g. w., & breen, p. j. (1992). cell count and size in relation to fruit size among strawberry cultivars. j. amer. soc. hort. sci., 117, 946-950. dado. (2017). annual report. nuwakot. dale, a., walker, g., & fisher, p. (2000). growing strawberries in ontario. ontario ministry of agriculture, https://journals.e-palli.com/home/index.php/ajlsi pa ge 14 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 6-14, 2022 food and rural affairs. darnell, l. r. (2003). strawberry growth and development. in: n. f. childers (ed.). the strawberry: a book for growers,. gainesville, fl: childers publications. darrow, g. m. (1966). the strawberry. new york: holt rinehart and winston. das, a. k., singh, k. p., prasad, b., & ravindra, k. (2015). evaluation of cultivars of strawberry, a temperate fruit for its adaptability as well as productivity in subtropical agro-climatic condition of supaul district in bihar. asian j. hort, 10(2), 278-281. day, d. (1993). strawberries. grower digest series no. 3. london: nexus business communications. embaby, e. m. (2007). pestalotia fruit rot on strawberry plants in egypt. egypt j phytopathol, 35, 99-110. fao. (2019). faostat statistical database. rome. girijalba, c. m., perez-trujillo, m. m., ruiz, d., & ferrucho, a. m. (2015). strawberry yields with hightunnel and open-field cultivations and the relationship with vegetative and reproductive plant characteristics. agronomia colombia, 33(2), 147-154. doi:10.15446/ agron.colomb.v33n2.52000 guttridge, c. g. (1979). anther failure main cause of poor fruit set in redgauntlet [strawberry]. grower, 91, 38-39. hancock, j. f. (1999). strawberries. new york: cabi publish. hellman, e. w., & travis, j. d. (1988). growth inhibition of strawberry at high temperatures. adv. strawberry prod, 36-38. jami, y. y., sarkar, a., & maiti, c. s. (2015). evaluation of strawberry cultivars in the foothills of nagaland. journal crop and weed, 11(special issue), 198-200. joolka, n., & badiyala, s. d. (1983). studies on the comparative performance of strawberry cultivars. haryana j.of hort.sci, 12(3-4), 173-177. klimczac, a., & stropek, m. (1988). evaluation of the yield of six strawberry varieties in southeast poland. ocena plonowania szesciuodmian truskawek w warunkach polski poludniowawschodniej, 28, 31-35. kurian, a. (2015). thesis on “performance of strawberry (fragaria ananassa duch.) in different growing conditions”. kerala, india. ledesma, n. a., nakata, m., & suglyama, n. (2008). effect of high temperature stress on the reproductive growth of strawberry cvs. „nyoho‟ and „toyonoka. scientia. hort, 116, 186-193. menzel, c., & smith, l. (2010). runner quality. in strawberry r&d update, 6-15. queensland: queensland government. nepali times. (2017, jan 5). strawberry mountains. http:// archive.nepalitimes.com/article/nation/strawberrymountains-nepal,3437 nitsch, j. p. (1950). growth and morphogenesis of the strawberry as related to auxin. amer. j. bot, 37, 211215. okimura, m., & igarashi, i. (1997). effects of photoperiod and temperature on flowering in everbearing strawberries. j. amer. soc. hort. sci, 872-880. perkins-veazie, p. (1995). growth and ripening of strawberry fruit. horticultural reviews, 17. poling, e. b. (2002). strawberry plant structure and growth habit. new york: nc state university. rahman, m., hossain, m., khaleque, m., & khaliw, q. a. (2013). characterization and field performance of 15 strawberry germplasm under bangladesh conditions. saarc j. agri, 11(2), 81-94. renquist, a. r., breen, p. j., & martin, l. w. (1982). influences of water status and temperature on leaf elongation in strawberry. scientia hort, 18, 77-85. sato, n., & hiraoka, t. (1971). study on temperature and management in forcing strawberry. ⅰ. effect of heating soil and air on the growth and yield of forcing strawberry. bull. kanagawa hort. exp. sta, 19, 76-81. shaw, d. v. 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(2019). import datasheet nepal. wani, m. s., rather, b. a., sharma, m. k., & singh, s. r. (2007). effect of different planting time and mulches on flowering, yield and quality of strawberry. the hort.j, 20, 5-7. went, f. (1957). the experimental control of plant growth. cronica botanica, 17. whitaker, v. m., chandler, c. k., & peres, n. a. (2015). sensationtm ‘florida127’ strawberry. hortscience, 50(7), 1088-1091. whitaker, v. m., chandler, c. k., & peres, n. a. (2017). sensation™ brand ‘florida127’ strawberry. florida: horticultural sciences department, uf/ifas extension. whitaker, v. m., peres, n. a., & agehera, s. (2017a). ‘florida beauty’ strawberry. florida: horticultural sciences department, uf/ifas extension. whitaker, v. m., chandler, c. k., santos, b. m., & peres, n. a. (2019). ‘florida radiance’ strawberry. florida: horticultural sciences department, uf/ifas extension. https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 9 american journal of life science and innovation (ajlsi) fishing enclosure as a simple approach to stimulate spawning success of tenualosa ilishatenualosa ilisha (hamilton, 1822) in bangladesh mohammad ashraful alam1, flura1, md. moniruzzaman1, md. hashibur rahman2*, md. abu kawser didar1, md. abul bashar1, md. mehedi hasan pramanik1, khondaker rashidul hasan1, md. anisur rahman2, yahia mahmud2 volume 2 issue 1, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i1.1164 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: january 05, 2023 accepted: january 27, 2023 published: january 30, 2023 the study was intended to assess the impact of fishing enclosure during the peak breeding time of hilsa, tenualosa ilisha on its breeding success. during the last peak breeding season of hilsa (october 4-25, 2021) a comprehensive investigation was attempt to evaluate the impact of fishing enclosure by “mv rupali ilish” research vessel, speedboat and experimental fishing net were deployed to demeanor a rigorous investigation in and around the spawning grounds areas (about 7,000 sq km, including moulvirchar, monpura, dhalchar and kalirchar). additionally, data were also assumed from landing stations (via terrestrial cruises) near the spawning ground areas and other hilsa prone region of bangladesh. the percentage of spent hilsa (locally called pite) and oozing hilsa were monitored following a standard protocol. among all the hilsa captured in and around the spawning grounds, the proportion of male and female were 16-41% and 85-97%, respectively of the total catch, suggesting a male and female sex ratio (1:2.3). the rate of breeding success in 2021 was 51.76% i.e., 51.7% hilsa successfully participated in the breeding process which was 103.52% higher than the base year (2001-02). the approximate number of fertilized eggs in 2021 was 7,86,314 kg. increased production of hilsa eggs and jatka indicated a positive impact of twenty-two days fishing enclosure. percentages of gravid and oozing hilsa were also found higher compared to previous years. due to the prohibition of all types of fishing, the percentage of female hilsa in the breeding areas increased up to 83.96%. overall, twenty-two days fishing enclosure was found very effective for successful spawning of hilsa. keywords fishing enclosure, tenualosa ilisha, spawning success and spent rate introduction the national fish hilsa (tenualosa ilisha, hamilton, 1822) is bangladesh’s most important open-water species, contributing significantly to the country’s economy, income, poverty alleviation, employment, and food security. the global average yearly production of the 11 hilsa fishing nations is >0.72 million tons (hossain et al., 2019), of which bangladesh contributes about 80%, india 15%, myanmar 4%, and other nations (such as iraq, kuwait, malaysia, thailand, and pakistan) 1% (dof, 2022). in last 12 years, hilsa production in bangladesh has increased about 81% that reached 5.65 lakh mt in 202021 and annual growth increment in hilsa sector was 3.31% (dof, 2022). currently, hilsa contributes about 12.2% to overall fish production and about 1% to the country’s gdp (dof, 2022). hilsa has also wraps sociocultural and religious values and its non-consumptive value estimated approximately us$0.36 billion per annum (mohammed et al., 2016). in the country, around 2.5 million individuals are directly or indirectly involved in the catching and trading of hilsa fish (dof, 2022). hilsa is a typical anadromous shad (jones, 1951; pillay, 1955; pillay and rao, 1962, 1963) with a complex life cycle. hilsa reproduces in upstream rivers, where the larvae hatch from free-floating eggs. the larvae vary in size between 2.3 and 3.1 mm and are drifted by wave action and tidal currents to the nursery grounds (rahman, 2006). immature early stages develop in river channels, then migrate to the sea to feed and grow before returning to the rivers as mature gravid hilsa to complete the life cycle (bobp, 1987; milton and chenery, 2003; hossain et al., 2016). juvenile hilsa remain in the downstream of river, albeit they have been found to occur in the upper and lower estuaries, and even further down to the coastal areas that are far from both their spawning and nursery grounds (hossain et al., 2016). the juvenile hilsa known as jatka remain around the nursery grounds for the next 5-6 months and attain a maximum size of 10-16 cm (raja, 1985; bfri/rs, 1994) in 8542 km2 suitable riverine and nearshore coastal waters in bangladesh (hossain et al.,2016). as the jatka grows bigger with an ability to adapt to salinity, they start migrating from brackish-freshwater habitats to the offshore or seawards and at 10-16 cm total length, all the hilsa complete their migration (bobp, 1987; bfri/rs, 1994). as the hilsa become sexually mature at nearly a year of age, they start their spawning migration into freshwaters (hossain et al., 2014b). hilsa is a high fecund fish that can lay up to 2 million eggs (qureshi, 1968, shafi et al., 1977, quddus, 1982, moula, 1992, rahman et al., 1998, blaber et al., 2001, haldar, 2004, bfri, 2006-07, rahman et al., 2017, rahman et al., 2012). albeit hilsa spawn all the year round, the full moon phase in bengali month ashwin-kartik (september-october) (rahman et al., 2012; saifullah et al., 2004) coincides with the peak spawning season. the spawning cycle is closely synchronized with the lunar cycle, and intense spawning is observed during three-days before and after the new moon and the full moon (miah et al., 1999). during the major spawning season, mature gravid hilsa are captured in large numbers from the major spawning areas. during 1 bangladesh fisheries research institute, riverine station, chandpur, bangladesh 2 bangladesh fisheries research institute, headquarters, mymensingh, bangladesh * corresponding author’s e-mail: hasibkhan94bfri@gmail.com https://doi.org/10.54536/ajlsi.v2i1.1164 https://journals.e-palli.com/home/index.php/ajlsi pa ge 10 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 9-20, 2023 their seaward migration, juvenile hilsa known as jatka have also been caught in some of the major rivers of the country (ahmed et al., 2010; rahman et al., 2011, 2012, jaher et al., 2013). during this onward and backward migration, widespread unregulated capture of mature and juvenile hilsa can have a serious negative impact on hilsa population and affect sustainable production of the country. in light of the foregoing, the hilsa fishery management action plan (hfmap) was developed for the management, and conservation of hilsa, with the goals of safeguarding nursery and spawning sites and prohibiting indiscriminate killing of illegal sized hilsa. under the “fish protection and conservation act-1950”, so far six hilsa sanctuaries and four major spawning grounds have been established in the country’s coastal and freshwater zones for the successful conservation of jatka and brood hilsa (figures 3; tables 3 and 4). the results of bfri, rs research showed that a high number of matured gravid hilsa congregate at the spawning grounds for breeding during the peak spawning time, which coincides with the lunar periodicity, and more especially the full moon phase (reuben et al., 1992; rahman and cowx, 2008, rahman et al., 2017). at this time, matured gravid hilsa are indiscriminately captured, which has a negative impact on spawning and hilsa population boblme (2012). as a result, in accordance with hfmap recommendations, fishing closure has been imposed to ensure the safe and successful spawning of hilsa. this enclosure last for 22 days, from 4th-25th october in 2021, and included a new moon and full moon day (bengali month ashwin,1427), (table 1 and figure 1). during this time, all types of commercial trawlers fishing in the sea, coastal regions, and rivers were restricted under the marine fisheries ordinance 1983, section 55, sub-section 2 (d) for safe hilsa migration and spawning. fisheries enclosures may be temporary or ongoing and have a variety of justifications. for instance, fishing restrictions have been employed extensively in fisheries management to prevent overfishing and the collapse of a fishery, restore depleted stocks, and limit bycatch of protected species (national oceanic and atmospheric administration (noaa), 1985; gell and roberts, 2002; farmer et al., 2016; agar et al., 2019). fishing enclosures have occasionally been implemented during a species’ breeding or spawning season in an effort to directly minimize fishing mortality and to increase the number of eggs produced annually (murawski et al., 2000; arendse et al., 2007). there are many examples of fishing enclosures around the world, for example; it has been implemented in the gulf of mexico shrimp fishery and florida lobster fishery [national oceanic and atmospheric administration (noaa), 1985; beets and manuel, 2007], united states virgin islands grouper fishery (beets and friedlander, 1999) and coral reefs in kenya (mcclanahan, 2010). in bangladesh, the department of fisheries (dof) enacted the fishing enclosure in collaboration with law enforcement organizations such as the navy, coast guards, river police, and air force based on the findings of bfri, rs. the impact of fishing enclosure seems to have a positive impact on the overall hilsa product of the country. table 1: management interventions for jatka and brood hilsa conservation in the major spawning and nursery grounds of hilsa in bangladesh. year management system considering issues 2001-02 conventional without any management 2002-03 conventional traditional (improved) management 2003-04 jatka conservation protection system 2004-05 jatka conservation protection system 2005-06 jatka conservation+sanctuary full moon basis 2006-10 jatka conservation+sanctuary+10 days hilsa fishing enclosure full moon basis 2011-12 jatka conservation+sanctuary+11 days hilsa fishing enclosure full moon basis 2012-13 jatka conservation+sanctuary+11 days hilsa fishing enclosure full moon basis 2013-14 jatka conservation+sanctuary+11 days hilsa fishing enclosure full moon basis 2014-15 jatka conservation+sanctuary+15 days hilsa fishing enclosure full moon basis 2015-16 jatka conservation+sanctuary+15 days hilsa fishing enclosure full moon basis figure 1: new moon and full moon dependent fishing enclosure time in bangladesh. * et= enclosure time, fm= full moon day, nm= new moon day https://journals.e-palli.com/home/index.php/ajlsi pa ge 11 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 9-20, 2023 methodology preparation of hilsa research team the hilsa research team of bfri, rs, chandpur carried out all the investigations. two ways were selected for sampling and data collection such as: a. research vessel (m.v. rupali ilish with speed boat) trip (figure 4) and b. research trip with necessary research equipment’s by using local transport system five research teams were formed to conduct a comprehensive investigation in the designated hilsa spawning and sanctuary areas (table 2 and 3). the nursery grounds were chosen as sampling sites because: (a) hilsa shed their eggs in almost all major river systems across the country during the breeding season, but the spawning grounds (figure 3) grasp 80-90% of them; (b) hilsa eggs and larvae begin drifting and shifting to upstream rivers shortly after spawning; and (c) hilsa is a migratory fish that may travel a long distance after spawning and be captured there. as a result, a large sampling area was chosen to provide precise information on the percentage of spent, oozing, and gravid hilsa. figure 2: monthly gsi study of hilsa figure 3: major spawning grounds of hilsa in the meghna river estuary with approximate gps point (source: bfri and gef studies). table 2: areas of spawning grounds in bangladesh. sl no. directions contour areas (sq km) district 1 east-north mayani-mirarsorai 125 chittagong 2 west-north paschim syed aowlia-tojumuddin 80 bhola 3 east-south gondamara-bashkhali 120 cox’s bazar 4 west-south latachapali-kolapara 194 patuakhali source: bangladesh gazette 28 may, 2014, mofl figure 4: hilsa research vessel ‘mv rupali ilish https://journals.e-palli.com/home/index.php/ajlsi pa ge 12 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 9-20, 2023 standard formula (rahman et al., 2009 and rahman et al., 2017) the number of hilsa rescued and total fertilized eggs as a result of the fishing enclosure were determined using the following formula: no. of hilsa saved due to fishing enclosure (tn) = nos. of fishing boat*nos. of haul/day*nos. of fish captured/ nos. of haul* ..........1 nos. of days tn*ff*sf*ef/1000...............(2) where, tn = total no. of hilsa excluded due to fishing enclosure; ef = % of female fishes in the study areas; sf = % of spent/oozing fish, and ef = average egg (g) per fish site selection the major hilsa spawning grounds, nursery grounds and fish landing sites (13 districts) in bangladesh were selected for investigation (table 5 and figure 5). these sites account for the majority of spawning hilsa during the peak spawning season, through which the country’s major river systems are flowing including the river padma and meghna. furthermore, samples were collected from chattagram and cox’s bazar, where the majority of marine hilsa are gathered for vending. table 3: six established sanctuary areas of t. ilisha. sl. no. sanctuary area length river enclosure period district 1. shatnol-char alexander 100 km lower meghna estuary march-april chandpur-laxmipur 2. char ilisha-char pial 90 km stretch of shahbajpur channel, tributary of the meghna river march-april bhola 3. bheduria-char rustam 100 km stretch of tetulia river march-april bhola-patuakhali 4. kalapara golbunia confluence of bay of bengal and andharmanik river 40 km andharmanik river november-january patuakhali 5. tarabunia-vomkora 20 km lower padma river march-april shariatpur 6. habinagar, barisal sadarhizlamehendigonj 82 km meghna river march-april barisal source: bangladesh gazette 28 may, 2014 and 17 april, 2018, mofl data collection time frame three sequential time frames were selected for sampling and data acquisition by the hilsa research team such as: i) before the enclosure period (05 days) ii) whole enclosure period (22 days) iii) after the enclosure period (05 days) determination of size, sex and percent composition of gravid/berried hilsa the length (cm) and weight (g) of collected hilsa samples were measured at different sampling locations (table 4). the length was measured using a wooden scale and weight with a digital balance, respectively. external observation and steady light pressure from the tip of the ventral squat to the anus of the belly were used to identify the sex of hilsa fish. the fish exhaling liquid cream while stripping was considered as male; on the contrary, the fish extruding eggs and a light reddish substance were considered as a female hilsa. female hilsa were also detected with a potbellied reddish and larger anus. if neither of this milk/ eggs come out, the fish were considered as immature table 4: selected sites for sampling sl. no. sites district river/sea 1. chandpur, katakhali, horinaghat, haimchar and charvoirobi chandpur meghna 2. hizla, mehendiganj, ulania barishal meghna 3. ramgoti, char alexandar laxmipur meghna 4. tanki bazar, chairman ghat, hatia noakhali meghna estuary 5. dhalchar, moulovirchar, daulatkhan, monpura bhola meghna estuary 6. kalirchar, patharghata barguna meghna estuary 7. mohipur, galachipa, kalapara patuakhali andharmanik 8. tarabunia shariatpur padma 9. godagari rajshahi padma 10. mawa munshigonj padma 11. fishery ghat chattagram sea 12. bfdc ghat cox’s bazar sea 13. bfdc ghat khulna sea https://journals.e-palli.com/home/index.php/ajlsi pa ge 13 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 9-20, 2023 figure 5: thirteen districts on the map of bangladesh from where data were collected during fishing enclosure. (jatka) or partly mature. female hilsa were often found larger in length than male hilsa. identification of spent (pite ‘in local language) and oozing (spawning) hilsa the spent hilsa were characterized by some external features like skinny, squeezed stomach, and an extended slender body. if watery liquid or blood emerges with the alienated and malformed egg after light pressure on the abdomen, it was labeled as the spent hilsa (‘pite’ in the local language). after being captured from the river, spent hilsa were quite feeble. usually fishers keep spent hilsa for their own household consumption and don’t bring them to the market because they diminish the market value of other fish. oozing hilsa were recognized by studying their egg-laying state, which is characterized by ripe eggs erupting spontaneously without any pressure on the abdomen (figure 6). gonadosomatic index of hilsa during the investigation, the sex of at least 30 individual specimens were observed by abdominal incision and ocular examination of the gonad. the gonads were meticulously stripped of all fat, connective tissue, and blood vessels, and their gonadal weight (gw) was calculated to the nearest 0.001 g. the gonadosomatic index (gsi) was determined using the following formula: gsi = gw × 100/bw (nikolsky, 1963) determining the (%) of spent (pite) hilsa the hilsa research team of bfri determined the number and rate of spent hilsa primarily from hilsa captured using experimental nets in the breeding grounds and surveillance of commercial fish landing sites around the breeding grounds. figure 6: pictorial views of spent and oozing hilsa. calculation of fertilized eggs during the fishing enclosure, the approximate number of fertilized eggs (figure 7) released by hilsa in the spawning grounds were estimated using the following formula: the plausible amount of egg production due to the fishing enclosure was: approximate egg production = {(54*22*15500*75/100) *(51.76/100*110)}/1000 = 7,86,314kg where, no. of matured hilsa captured in per unit effort = 27 (two haul/day; = 54); total nos. of enclosure days = 22 days; approximate number of fishing boats = 15500; total area of spawning ground =7000sq km, on an average 2 fishing boats are operated in per sq. km; average percentage of matured female during the fishing enclosure = 75%; average percentage of spent hilsa during the fishing enclosure = 51.76%; average weight of gonad of matured female = 110g; one kilogram = 1000g. figure 7: pictorial views of hilsa eggs. https://journals.e-palli.com/home/index.php/ajlsi pa ge 14 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 9-20, 2023 calculation of jatka production the number of fry and jatka production were calculated based on 50% hatching rate of fertilized eggs and 10% survival rate of hatched larvae. size, weight, age and cpue calculation of hilsa fry and jatka hilsa larvae were collected from the spawning grounds and adjacent sites by means of bfri experimental fishing net (made of glass nylon fiber) (figure 8). a measuring scale and a digital balance were used to determine the length and weight of hilsa larvae and jatka. the amount of jatka (kg) captured in a 100 m net per hour per boat was delineated as the catch per unit effort (cpue). observations of other impacts of fishing enclosure to observe the additional benefit of the 22-day fishing enclosure, larvae and juveniles of other fish species were collected from the spawning grounds and adjacent sites using a bfri experimental fishing net (made of glass nylon fiber: mouth diameter 15 ft, length from the mouth to the end 25 ft, loop size zero, and fishing time was 30 minutes) (figure 8). majority of the hilsa captured in the downstream rivers were greater than 35 cm (>74% in bhola and barishal) (figure 9). furthermore, most of the hilsa (80-90%) captured at the down stretches of the meghna estuaries were gravid female. ahmed et al. (2010) found 77-84% of hilsa at ramgoti and 86-92% of hilsa at hatia under the size class of 36-52 cm whereas smaller sized (18-36 cm) hilsa ranged between 8-23%. on the contrary, rahman et al. (2009, 2013, 2015 and 2017) investigated the influence of fishing enclosure on hilsa spawning grounds and revealed larger percentage (90%, 95% and 85%, respectively) of hilsa comprising length groups greater than 30-35 cm in the monpura and hatia areas. almukhtar et al. (2016) also found size discrepancies in t. ilisha migrating in the shatt al arab river and southern al hammar marsh, basra, iraq. rahman et al. (2012) categorized hilsa into four size groups, small (<30 cm), medium (30–39 cm), large (40–49) cm and extra-large (>50 cm) and found more than 90% hilsa within the range of 30–40 cm during their investigation. the data from a recent survey by phone to fish vendors (2013–14) conducted under the espadeltas project in some of the largest hilsa landing centers and markets of the bangladesh coast showed that 90% of the hilsa ranged from 25–75 cm (fernandes et al., 2016). however, dutta et al. (2012) found the maximum length of hilsa 45.5 cm in their studies. in general, commercial catches in both bangladesh and indian waters showed a general size range of about 15–52 cm (azad et al., 1987; gupta, 1989; rahman, 2006). aside from the spatial variation of l-f, an apparent temporal variation in the length class of hilsa was also noticed. normally, 30-35 cm length class predominated before and after the enclosure period, whereas mature hilsa (egg bearing female, >40 cm) predominated during the spawning time (figure 10). ahmed et al., 2010; rahman et al., 2011, 2012, jaher et al., 2013 and rahman et al. (2017) alam et al. (2019) also found an alteration of the hilsa length class before and after the spawning time. the proportion of male and female hilsa also varied significantly before and after the prohibition period, indicating an uneven hilsa sex ratio. before and during the prohibition, the average proportion of male hilsa was 3-14%, rising to 16-41% during the enclosure period (october 4-october 25). however, before and during the restriction period, the average proportion of female hilsa was 56-89%, raised up to 85-97% during the enclosure period (october 4october 25). these findings differed figure 8: experimental fishing net for collecting juvenile hilsa. data analysis the data were compiled and processed using microsoft excel (version 2016). graphical representation of data was also prepared by microsoft excel. result and discussion size and sex ratio of hilsa the majority of the hilsa captured in the upstream rivers were smaller than 35 cm (50% in chandpur), whereas the figure 9: percentage of different length-classes of hilsa at upstream and downstream of rivers in bangladesh. https://journals.e-palli.com/home/index.php/ajlsi pa ge 15 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 9-20, 2023 figure 10: percentage of different length class’s male and female hilsa before, after and during the ban period in the major spawning grounds of hilsa in bangladesh to some extent from those of haldar et al. (2004), who observed a male-female sex ratio of 1:2 in the main spawning sites of hilsa. in a separate study, islam et al. (1987) found a male-female sex ratio of hilsa 1:1.08 in the upper stretches of meghna river (chandpur). in previous bfri conducted investigation, rahman et al. (2009) noticed 35% male and 65% female, whereas ahmad et al. (2010) found almost 4.2% male and 95.8% female in and around the spawning grounds among all the captured hilsa. during the monsoon, quereshi (1968) observed a male-female sex ratio of 1:1, although the female was dominating in october. according to blaber et al. (2001) males are more abundant among smaller fish. the bias in the sex ratio of t. ilisha suggests that males may not live as long as females (mahmud, 2020). percent composition of spent and oozing hilsa approximately, 2100 hilsa were examined throughout this study where a substantial number of spent and oozing hilsa were found during the study period. the overall fraction of oozing and spent hilsa were 32.4% and 51.7%, respectively (about 84%, combing both). the results indicated that fishing enclosure ensured successful breeding as also found by ahmed et al. (2010); rahman et al. (2011 and 2012); jaher et al. (2013) and rahman et al. (2017) during their studies. in 2001-02, the percentage of spent hilsa was >1%. the percentage of spent hilsa has steadily increased until recent years (103% higher in 2020-21 than in 2009-10), due to the successful deployment of a fishing closure during the peak spawning season (figure 11). rahman et al. (2009) found 1.51% oozing and 5% spent hilsa during the fishing ban period in the spawning grounds and it was about 2.80-3.57 times higher than the global environment facility (gef) and bangladesh fisheries research institute (bfri) studies of 2002 and 2003. the occurrence of spent hilsa ranged between 5-39% in 2007-09 which was estimated 76% higher than the previous studies (ahmed et al., 2010). the percentage of spent hilsa was about 34% in 2010; 36% in 2011 and table 5: percent composition of spent and oozing hilsa during the enclosure period. status of hilsa breeding sampling points cox’s bazar ramgoti chandpur potuakhali monpura average oozing 98 (31%) 950(62%) 72 (13%) 16 (17%) 19 (39%) 32.4% spent 147 (52%) 455 (31%) 287 (42%) 32 (71%) 16 (63%) 51.76% total number 245 1405 359 48 35 2092 figure 11: percentage and change of spent hilsa in different years in bangladesh. https://journals.e-palli.com/home/index.php/ajlsi pa ge 16 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 9-20, 2023 2012; 41% in 2013; 39% in 2014; 37% in 2015; 44% in 2016 (rahman et al., 2017). production of fertilized eggs and jatka during the fishing enclosure period and spawning success the total egg production of hilsa was determined using the bfri’s standard formula. due to the fishing prohibition, approximately 7,86,314 kg of hilsa eggs (figure 12) were produced in 2021-22. if 50% of the eggs produced are fertilized and hatched, and 10% of them survive, then approximately 7,86,314 crores of hilsa fry and 39.31 crores of jatka (figure 12) were recruited into the hilsa population. these studies suggested that increasing the number of enclosure days (10 days from 2007-08 to 2010-11; 11days from 2011-12 to 2013-14; 15 days from 2014-15 to 2015-16; and 22 days from 201617 to 2020-21) had a positive impact on egg and jatka production. in the current year (2020-21), egg production augmented about 17 times, while fry and jatka production increased approximately 16.2 times than the year 2007-08 when fishing enclosure during the peak spawning time started to be implemented. each year, the recruitment of this significant number of jatka to the hilsa population as a result of the fishing restriction resulted in a consistent and increased production of hilsa in bangladesh (figure 14). haldar (2004) and ahmed et al., 2010; rahman et al., 2011, 2012, jaher et al., 2013 rahman et al. (2017), mahmud et al. (2020) stated that a total fishing prohibition has a significant positive impact on jatka abundance as well as the country’s overall hilsa production. seasonal closure during the breeding or spawning period of species directly reduces fishing mortality and thereby achieving greater annual reproductive output (murawski et al., 2000; arendse et al., 2007). cpue of hilsa fry and jatka the cpue of jatka (19.94 kg/100 m net/boat/hr) of the current year (2021-22) demonstrated 18 times increment compared to the base year, and it was roughly 1.8 times higher in amount than the previous year (2019-20) (figure 13). nevertheless, current cpue is about four times greater than the study conducted by rahman et al. (2017). successful spawning of higher percentage of hilsa due to fishing closure resulted in increased cpue of jatka in the country (alam et al., 2019a, 2019b and mahmud et al. (2020). kamal et al. (2015) found significantly higher cpue while studying the effects of seasonal closure in a multispecies fishery. figure 12: approximate number of eggs, fry and jatka production in the major spawning grounds. figure 13: cpue of jatka in the selected sampling sites. total hilsa production of the country the hilsa production in the country was 1,99,032 mt in 2002-03, 2,79,189 mt in 2006-07, and in last year (202021) it reached at 5,65,183 mt (dof, 2021). the overall hilsa production of the country has increased roughly 2.77 times since 2002-03 and about 2.0 times since 200607 through implementation of fishing closure by all relevant government agencies (figure 14). all previous investigators of bfri (ahmed et al., 2010; rahman et al., 2011 and 2012; jaher et al., 2013 and rahman et al., 2017) concluded that hilsa production is gradually increasing in the country due to the stricter implementation of management interventions especially fishing closure during the peak spawning time of hilsa. beets and https://journals.e-palli.com/home/index.php/ajlsi pa ge 17 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 9-20, 2023 manuel (2007) noted that, the temporal and seasonal closures used in fisheries management in tropical and subtropical regions have been useful and beneficial based on perceived benefits and annual production (beets and manuel, 2007). availability of hilsa and other fish’s larvae in experimental nets (indicator of breeding success) in the breeding grounds, a glass nylon fiber net was used to gather larvae at high and low tide (figure 8). during low tide, around 29% of hilsa larvae were detected in the experimental net, whereas 71% were other fish’s larvae. during high tide, however, hilsa larvae were 56%, whereas other fish’s larvae were 44%. overall, 85% of hilsa larvae were found during high and low tide (figure 15). the total number of larvae gathered during high and low tides varied significantly; at high tide, the number of larvae was greater than at low tide (figure 16). shrimp, chewa, poa, shillong, kakila, baim, chanda, kuchia, bele, and other fish larvae were found with the other fish’s larvae. according to the international union for conservation of nature’s (iucn, 2015) biodiversity status of bangladesh and global biodiversity, many of these species are threatened (vulnerable, endangered, or least concern) (table 6). after the breeding season, hilsa larvae (figure 17) begin to migrate to the primary nursery grounds, where they develop into juveniles and then jatka. in a month, hilsa larvae develop from 2 to 2.5 cm (haldar, 2004). the spawning success of hilsa is indicated by the presence of hilsa juveniles (figure 17) in various nursery grounds/sanctuary areas. during bfri’s experimental fishing, a large number of hilsa juveniles were found in the nursery grounds (figure 17), with an estimated age of 30-45 days. rahman et al. (2017) found the similar positive impact of fishing closure on abundance of hilsa and other fish’s larvae. in another study, kincaid and rose (2017) found that biological community assemblages changed significantly before–after and inside–outside the closing time and area. kamal et al. (2015) studied the effects of seasonal closures in a multi-specific fishery and found that depending on its timing, the closure would highlight some positive biological effects on some target species. figure 14: total hilsa production of the country (freshwater and marine) figure 15: percentage of hilsa and other’s fish larvae during high and low tide figure 16: abundance of hilsa larvae in the spawning grounds. figure 17: observations of hilsa juveniles. https://journals.e-palli.com/home/index.php/ajlsi pa ge 18 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 9-20, 2023 conclusion the result obtained in this study is an approximation that may be adjusted for a variety of criteria such as the number of fishing boats, those who did not fish, the number of specimens used in the calculation of spent hilsa, and the weight of the female hilsa’s ovaries. furthermore, a variety of biotic and abiotic factors such as (plankton composition, temperature, dissolved oxygen, ph, carbon dioxide, alkalinity, hardness, turbidity, transparency, conductivity, rainfall, and river discharge, among others) may play a significant role in hilsa larvae egg production and hatching in different years, as well as overall breeding success. furthermore, it is impossible to identify which elements are most relevant for hilsa breeding success and increased output without a controlled laboratory trial (successful implementation of different management interventions or ecological suitability of biotic or abiotic factors). however, because hilsa production is steadily growing with the implementation of management measures, it is possible that this will have some beneficial effects. recommendations the fishing closure should be continued to safeguard mature gravid hilsa and to protect jatka. fishing enclosure should be implemented in combination with other management interventions like gear restrictions, area closure. the existing spawning grounds and nursery grounds should be protected and managed effectively. additional research is needed to identify new hilsa nursery and breeding sites. the logistic support should be increased to conduct comprehensive on the impact of fishing closure. acknowledgement it is also great pleasure for the investigators to express their sincere and deep sense of gratitude and indebtedness to bangladesh fisheries research institute (bfri) for providing financial support. the investigators also express their profound indebtedness and sincere gratitude to the director general of bangladesh fisheries research institute (bfri), mymensingh for his continuous support, encouragement and kind cooperation to carry out this research works. references agar, j., shivlani, m., fleming, c., & solís, d. 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(2012). hilsa fishery management in bangladesh. in: anon (ed.), hilsa: status of fishery and potential for aquaculture. proc. of the regional workshop held in dhaka, 16-17 september, 2012, the world fish, bangladesh and asia, 238, 40-60. rahman, m.a., flura, ahmed, t. pramanik, m.m.h. & alam, m.a. (2015). impact of fifteen days fishing ban in the major spawning grounds of hilsa (tenualosa ilisha, hamilton 1822) on its spawning success. res. agric. livest. fish. 2(3), 491-497. rahman, m.a., pramanik, m.m.h., flura, ahmed, t. & hasan, m.m. (2017). impact assessment of twentytwo days fishing ban in the major spawning grounds of tenualosa ilisha (hamilton, 1822) on its spawning success in bangladesh. j. aqua. res. develop., 8, 489. rahman, m.a., rahman, b.m.s., hasan, s.j., flura, ahmed, t. & haidar, m.i. (2013). impact of eleven days fishing ban in the major spawning grounds of hilsa (tenualosa ilisha hamilton) on its breeding success. bangladesh res. pub. j., 9(2), 116-122 rahman, m.j. & cowx, i.g. (2006). lunar periodicity in growth increment formation in otoliths of hilsa shad (tenualosa ilisha, clupeidae) in bangladesh waters. fish. res., 81, 342-344. rahman, m.j. & cowx, i.g. (2008). population dynamics of hilsa shad in bangladesh waters. asian fish. sci., 1, 85-100. rahman, m.j., mustafa, m.g. & rahman, m.a. (1998). population dynamics and recruitment pattern of hilsa, tenualosa ilisha. proc. fri/aciar/csiro., 6, 28-36. rahman, m.j., rahman, m.a. & bhaumik, u. (2012). biology and ecology of hilsa shad, tenualosa ilisha (ham.). regional workshop on hilsa: potential for aquaculture, 1-39, 16-17 september 2012, dhaka, bangladesh. raja, b.t.a. (1985). a review of the biology and fisheries of hilsa ilisha in the upper bay of bengal. bay of bengal program. bobp/wp/37. marine fishery resource management in the bay of bengal, pp-66. reuben, s., dann, s.s., somaraju, m.v., phillipose, v. & sathianandan, t.v. (1992). the resources of hilsa shad, hilsa ilisha (ham) along the northeast coast of india. indian j. fish., 39, 169-181. saifullah, a.s.m., rahman, m.s., & khan, y.s.a. (2004). fecundity of hilsa ilisha (hamilton, 1822) from the bay of bengal. pak. j. biol. sci., 7, 1394-1398. shafi, m., quddus, m.m.a. & islam, n. (1977). studies of gonad weight, sex-ratio and fecundity of hilsa ilisha (hamilton-buchanan) from the river meghna. j. asiat. soc. bangladesh (sci.)., 2, 51-58. https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 39 american journal of life science and innovation (ajlsi) enhancing public health: exploring the awareness of students in mindoro state university on blood donation jovie rose d. masongsong1 volume 2 issue 3, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i3.2245 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: november 15, 2023 accepted: december 11, 2023 published: december 14, 2023 blood donation plays a significant role in modern healthcare, especially because it contributes to charitable tasks and lessens shortages in the global demand for blood. problems encountered were due to the insufficiency of infrastructure, donor education, and resources which led to challenges in recruiting blood donors. studies have found that countries handle blood donation systems, but their status varies widely. this research study aims to determine students’ awareness level in mindoro state university-calapan city campus on blood donation. this research study utilized descriptive research design, which aims to systematically gather information and describe a phenomenon or situation through survey questionnaires. moreover, the 100 respondents were chosen through stratified random sampling and were tasked to answer the self-made questionnaire, which was divided into two parts: the demographic profile of the respondents in terms of age, gender, and weight and the respondents’ awareness of blood donation through the use of likert scale. the study results revealed that the students of the minsu-calapan city campus are aware of the crucial role of blood donations in saving lives and promoting a sense of community and unity in an environment. however, the study presented that the students are not knowledgeable of the process performed in blood donation. keywords blood donation, level of awareness, student donors, blood recipients introduction blood donation is a vital and life-saving act that forms the cornerstone of modern healthcare. it is indispensable in addressing emergencies, treating various medical conditions, and ensuring a stable supply of blood products for those in need. at the heart of this essential humanitarian endeavor is the active involvement of the community, including university students, who represent a significant segment of the potential donor pool. while there is a global demand for blood and blood products, many regions face chronic shortages. lowand middle-income countries often struggle to meet their blood supply needs due to insufficient infrastructure, lack of donor education, and limited resources. international organizations like the world health organization (who) and the international federation of red cross and red crescent societies (ifrc) work to improve access to safe blood and advocate for voluntary, non-remunerated blood donation. the goal is to ensure all countries have sufficient supplies of safe blood. each country manages its blood donation system. some countries have centralized, government-run systems, while others rely on non-profit organizations, such as the red cross, to collect and distribute blood. national campaigns aim to raise awareness about the importance of blood donation and encourage voluntary, regular donors. strategies may include mobile blood drives, outreach to schools and universities, and social media campaigns. the status of blood donation at the local level can vary widely. some communities may have a strong culture of donation, while others may face challenges in recruiting and retaining donors. barriers such as fear, lack of awareness, and misconceptions may need to be addressed locally. mindoro state university stands as an institution with a diverse and dynamic student population. these students are not only the nation’s future leaders but also the potential torchbearers of the noble act of blood donation. their awareness, attitudes, and willingness to participate in this charitable act hold immense significance in contributing to the overall blood supply, ultimately impacting public health in the region. this study was conducted to determine the awareness of students in mindoro state university on blood donation. objectives of the study this study determined the demographic profile of the respondents in terms of age, weight, and gender. it assessed the level of awareness on blood donation of students in mindoro state university, calapan city campus. moreover, this study also determined the relationship between the variables. methodology research design this study employed a descriptive method of research in determining the level of awareness on blood donation among students in mindoro state university, calapan city campus. the researcher employed a total of 100 respondents who were chosen through a stratified random sampling technique. the researcher decided to accept them as respondents with the belief that the role of these students in instilling and reinforcing awareness on blood donation among students is vital. 1 mindoro state university, calapan city campus, philippines * corresponding author’s e-mail: jrdmasongsong@gmail.com pa ge 40 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 39-42, 2023 instrumentation to elicit the needed information for this study, a selfmade questionnaire was used to gather data. it was composed of two parts; the first part determined the demographic profile of the respondents in terms of age, gender, and weight, while the second part determined the respondent’s awareness on blood donation by using the likert scale which includes 10-item statements. the interpretation of the computed mean for the level of awareness on blood donation has the following mean ranges with their corresponding interpretations: 3.26 4.00: strongly agree/highly aware; 2.51-3.25: agree/ aware; 1.76-2.50: slightly agree/moderately aware; 1.00-1.70: disagree/not aware. data collection procedure the consent form was prepared and given to the respondents so that the research objectives are clearly understood and the respondent is willing to participate. they were oriented on the study’s requirements and the confidentiality of the information to be collected among them as respondents of the study. a communication letter was also prepared to seek approval from higher authorities to distribute the instrument. upon approval, the researcher appropriately consulted the colleges’ program chairperson for the administration of the questionnaire. the gathered data were checked, tallied, scored, and treated through weighted mean. results and discussion demographic profile of the respondents imbalance within the sample, with males being the predominant group. table 1: frequency and percentage distribution of the respondents in terms of age verbal description frequency percent rank 27 above 1 1 4 24 26 5 5 3 21 23 49 49 1 18 20 45 45 2 total 100 100 table 2: frequency and percentage distribution of the respondents in terms of gender respondents frequency percent male 70 70 female 30 30 total 100 100 18 20 45 45 total 100 100 table 3: frequency distribution of the respondents in terms of weight kilograms frequency percent rank 71 – above 9 9 3 61 70 45 45 1 51 60 44 44 2 below 50 kgs 2 2 4 total 100 100 table 1 provides a clear overview of the age distribution among the 100 respondents. the majority of respondents, 49 out of 100, fall within the age range of 21-23, making it the most common age group in the survey. the secondlargest group consists of individuals aged 18-20, with 45 respondents. age groups 24-26 and 27 and above represent smaller portions of the sample, with 5 and 1 respondents, respectively. these findings highlight the concentration of respondents in the younger age brackets and the relatively limited representation of older age groups in the survey. table 2 presents the gender distribution among the 100 respondents in the study. the data reveals that a majority of the respondents, accounting for 70% of the total sample, are male, while the remaining 30% are female. this gender distribution highlights a significant gender table 3 provides an overview of the weight distribution among the 100 respondents in the study, along with their corresponding genders. the data reveals that the majority of respondents, 45 out of 100, fall within the weight range of 61-70 kilograms. the second most common weight range is 51-60 kilograms, with 44 respondents falling into this category. a smaller number of respondents, 9 in total, weigh 71 kilograms or more, while just 2 respondents weigh below 50 kilograms. level of awareness on blood donation table 4 illustrates the mean perception of the respondents on their level of awareness on blood donation having an overall mean of 3.04 described as aware. item number 3, emphasizing the crucial role of blood donation in saving lives, received the highest mean score, indicating a high level of awareness among the participants. this suggests that respondents recognize the life-saving potential of blood donation and its importance in providing essential medical treatments and surgeries. consequently, item number 4 got the second rank having an overall mean of 3.54 described as strongly agree. this implies that the awareness that blood donation is a selfless act with the power to make a significant difference in the lives of those in need is a powerful motivator for potential donors. it not only encourages participation but also contributes to the development of a compassionate and supportive society, fosters a sense of personal fulfillment, and ensures readiness in times of crisis. this awareness is the cornerstone of a thriving blood donation culture that benefits both individuals and the community as a whole. in addition, item number 8 also got a description of strongly agree which implies that the recognition that blood donation promotes a sense of community and unity as people come together to support one another is pa ge 41 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 39-42, 2023 table 4: mean perception of the respondents in the level of awareness on blood donation items mean rank description 1. i am aware that blood donation has beneficial effects to the donor. 3.43 5 strongly agree 2. i understand that regular blood donation can help reduce the risk of certain cardiovascular diseases in the donor. 3.25 6 agree 3. i recognize that blood donation plays a crucial role in saving lives by providing essential medical treatments and surgeries. 3.60 1 strongly agree 4. i am aware that blood donation is a selfless act that can make a significant difference in the lives of those in need. 3.54 2 strongly agree 5. i acknowledge that donating blood can help maintain adequate blood supplies for emergency situations and medical procedures. 3.20 7 agree 6. i appreciate the fact that blood donation is a simple and relatively painless process that can have a profound impact on someone’s life. 1.60 10 disagree 7. i understand that blood donors are screened for various health conditions, contributing to a healthier and more informed donor population. 1.70 9 disagree 8. i recognize that blood donation promotes a sense of community and unity as people come together to support one another. 3.49 3 strongly agree 9. i am aware that donating blood allows the body to regenerate new blood cells, which can have positive effects on one’s overall health. 3.48 4 strongly agree 10. i acknowledge that blood donation centers follow strict safety and hygiene protocols to ensure the well-being of both donors and recipients. 3.11 8 agree overall mean 3.04 aware a powerful testament to the profound impact of this act on individuals and society as a whole. the implications of this awareness extend far beyond the act of blood donation itself and highlight the importance of fostering a culture of care and interconnectedness. however, the statement about the perception of blood donation as a simple and relatively painless process received the lowest mean score, indicating a lack of agreement among the respondents. this suggests that there may be misconceptions or lack of information regarding the ease and painlessness of the blood donation process. moreover, statement number 7 also got a disagree description which implies that the rigorous health screening process may inadvertently lead to the exclusion of potential donors who have certain health conditions or engage in higher-risk behaviors. this exclusion can lead to feelings of stigma and discrimination among individuals who are eager to contribute but are deemed ineligible. table 5: summary table of the mean perception of the respondents dependent variable independent variable r value p value relationship level of awareness on blood donation age 0.009933 0.491063 not significant gender 0.004212 0.654331 not significant weight 0.076880 0.051244 not significant relationship of age and level of awareness on blood donation since the computed r value of 0.009933 is lower, considering that the health has a critical p-value of 0.491063, it, therefore, means that there is no significant relationship between the variables. the null hypothesis is therefore accepted/not rejected. relationship of gender and level of awareness on blood donation since the computed r value of 0.004212 is lower, considering that the behavior has a critical p-value of 0.654331, it, therefore, means that there is no significant relationship between the variables. the null hypothesis is therefore accepted/not rejected. relationship of weight and level of awareness on blood donation since the computed r value of 0.076880 is lower, considering that the study habits have a critical p-value of 0.051244, it, therefore, means that there is no significant relationship between the variables. the null hypothesis is therefore accepted/not rejected. conclusion the findings of this study provide valuable insights into respondents’ awareness regarding blood donation. the majority of the blood donor belongs to persons weighing 61-70 kilograms and the majority of the respondents are male. the gender of the respondents is not related to their level of awareness on blood donation. generally, the pa ge 42 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 39-42, 2023 finding suggests a foundational level of awareness about blood donation among the surveyed individuals. this awareness can be used as a starting point to build upon. the respondent’s awareness about the benefits they could get in donating blood encouraged them to donate blood. on the other hand, being unaware of the process and purpose of blood donation activity could lead to misinterpretations and as a result, could discourage them from being a donor even if they are legible. it is evident that, on average, the respondents are moderately aware of the significance of blood donation. recommendation blood donation organizations, healthcare providers, and educational institutions can continue to develop and strengthen awareness campaigns to ensure that this baseline awareness evolves into a more informed and active understanding of blood donation. to enhance public awareness and trust in blood donation centers, consider implementing a comprehensive and accessible information campaign that highlights the specific safety and hygiene protocols in blood donation centers and utilizing various social media channels to disseminate information about blood donation, highlighting the benefits that it provides to both the donor and recipients. consider having educational materials and workshops that clearly outline how the screening process identifies potential health risks, contributing to a healthier donor population and ultimately safeguarding the well-being of both donors and recipients. references charbonneau, j., & smith, a. (2016). giving blood: the institutional making of altruism. routledge, taylor & francis group. crisostomo, s. (2012). who urges people to get involved in voluntary blood donations to save lives. philippine star. dela cruz, j. e. r. g., dulin, m. a. r., guarin, c. j. c., marquez, m. a. u., & romero, c. m. n. g. (2018). factors associated with participation of university students in metro manila in voluntary blood donation. college of public health, university of the philippines manila. devio, l. (2021). red cross to hold massive blood donation drive. manila times. fernández de la iglesia, josefa del carmen; martínez-santos, alba-elena; rodríguez-gonzález, raquel; cebreiro, beatriz; fernández-morante, carmen; casal-otero, lorena. (2020). service-learning to improve attitudes towards blood donation among university students. health education journal, 79(7), 812-825. https://doi. org/10.1177/0017896920927313 luci, c. m. (2021). ph red cross calls for blood donation on world blood donor day. manila bulletin. majdabadi, h. a., kahouei, m., taslimi, s., & langari, m. (2018). awareness of and attitude towards blood donation in students at the semnan university of medical sciences. electron physician, 10(5), 6821-6828. https://doi.org/10.19082/6821 waggiallah, h. a. (n.d.). blood donation knowledge, perceptions, and practices during covid-19 pandemic: questionnaire-based study in saudi arabia. pa ge 1 pa ge 1 american journal of life science and innovation (ajlsi) optimizing synergistic metabolism in xenobiotic biodegradation: engineering a pseudomonas putida based microbial consortium moiz ahmed1*, hamza tariq1, fizza haroon1, tanzila ejaz2, maheen tajwar3 volume 3 issue 1, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i1.2236 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: november 20, 2023 accepted: december 24, 2023 published: december 27, 2023 this study focuses on the engineering of a microbial consortium, incorporating pseudomonas putida and selected members, to enhance synergistic metabolism for xenobiotic biodegradation. the aim is to explore the potential of microbial interactions in environmental remediation.the engineered consortium underwent iterative optimization to fine-tune microbial ratios. metabolomic and transcriptomic analyses were employed to investigate metabolic interactions within the consortium. xenobiotic biodegradation efficiency was evaluated through controlled laboratory experiments. the engineered microbial consortium exhibited a significant synergistic effect, demonstrating enhanced biodegradation capabilities compared to individual monocultures. the iterative optimization process led to a substantial increase in biodegradation efficiency. metabolomic and transcriptomic analyses provided valuable insights into the metabolic interactions within the consortium. the study successfully engineered a microbial consortium for efficient xenobiotic biodegradation, showcasing the potential of microbial interactions in environmental remediation. the optimized consortium composition and advanced analytical techniques offer promising avenues for sustainable bioremediation practices. further research is warranted to explore broader applications and address potential scalability challenges. keywords consortium, biodegradation, pseudomonas, metabolomics, synergy introduction xenobiotics, a diverse class of synthetic compounds, have become pervasive in the environment due to their extensive use in various industrial, agricultural, and pharmaceutical applications (davison et al., 1994). these compounds, while contributing significantly to human progress, simultaneously pose a significant threat to ecosystems and human health. the intricate chemical structures and recalcitrant properties of xenobiotics often render them resistant to degradation by conventional microbial biodegradation processes (kookana et al., 2011). as a result, innovative and tailored approaches are required to effectively and efficiently remediate environments contaminated with these persistent pollutants. the ubiquity of xenobiotics presents a formidable challenge for environmental sustainability. these compounds, ranging from pharmaceutical residues and pesticides to industrial chemicals, exhibit diverse chemical structures and properties. some xenobiotics, due to their complex and recalcitrant nature, persist in the environment for extended periods, leading to potential long-term ecological and human health risks (archer et al., 2017). the escalating levels of xenobiotic contamination in soil, water, and air necessitate urgent action and innovative solutions to mitigate their impact. pseudomonas putida, a gram-negative bacterium within the pseudomonadaceae family, has garnered considerable attention in recent years for its exceptional metabolic versatility and proficiency in degrading a wide range of organic compounds, including various xenobiotics (nikel et al., 2015). with its robust enzymatic machinery and adaptability to diverse environmental conditions, pseudomonas putida presents a compelling candidate for biodegradation studies across a spectrum of settings (samin et al., 2014). the utilization of pseudomonas putida as a cornerstone in microbial consortia offers a unique opportunity to capitalize on its metabolic prowess while also harnessing the potential synergistic interactions with other microorganisms. this approach represents a departure from traditional monoculture-based biodegradation strategies, which often fall short in efficiently degrading complex xenobiotics. microbial consortia: a paradigm shift the concept of microbial consortia represents a paradigm shift in biodegradation research. rather than relying on a single species to perform the entirety of xenobiotic degradation, microbial consortia leverage the diverse metabolic capabilities of multiple species, allowing for the efficient breakdown of complex compounds. this synergistic interaction between microorganisms has been shown to enhance the degradation rates and efficiency of xenobiotics compared to monoculture approaches (bernstein & carlson, 2012). the dynamic interplay between different species within the consortium leads to a more comprehensive and adaptable system capable of addressing a wider range of xenobiotics. 1 department of biotechnology, shaheed zulfiqar ali bhutto institute of science and technology (szabist), karachi, 75600, pakistan 2 faculty of science, department of food science and technology, jinnah university for women, nazimabad, karachi, sindh, 74600, pakistan 3 department of biosciences, forman christian college, university, lahore, 54600, pakistan * corresponding author’s e-mail: moiz.ahmed01@hotmail.com pa ge 2 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 1-7, 2024 engineering synergistic metabolism engineering microbial consortia for enhanced synergistic metabolism represents a critical frontier in bioremediation research. by strategically selecting and combining microorganisms with complementary metabolic capabilities, researchers aim to create a consortium that can efficiently catabolize specific xenobiotics. the design process involves a meticulous assessment of the metabolic pathways involved, ensuring that they align with the targeted xenobiotic compounds. literature review xenobiotics: nature, sources, and environmental impact xenobiotics, a diverse class of synthetic compounds, have permeated various environmental compartments due to widespread industrial, agricultural, and pharmaceutical applications (sharma et al., 2017). these compounds encompass pharmaceuticals, pesticides, industrial chemicals, and personal care products, among others. their introduction into natural systems has raised concerns about their persistence and potential to exert adverse effects on ecosystems and human health (archer et al., 2017). the enduring presence of xenobiotics underscores the urgency in developing effective strategies for their remediation. challenges in xenobiotic biodegradation conventional strategies for xenobiotic biodegradation have historically relied on single microorganisms. however, the intricate chemical structures and recalcitrant properties of many xenobiotics often limit the efficiency of degradation processes (kookana et al., 2011). this is particularly evident with compounds characterized by complex chemical arrangements or functional groups that hinder enzymatic breakdown. the limitations of monoculture-based biodegradation strategies emphasize the need for innovative approaches capable of addressing the diversity and recalcitrance of xenobiotics. pseudomonas putida: a versatile biodegradation agent pseudomonas putida has emerged as a pivotal player in biodegradation studies. this bacterium exhibits remarkable metabolic versatility and excels in the catabolism of a wide spectrum of organic compounds, including various xenobiotics (nikel et al., 2015). its extensive enzymatic repertoire equips it to tackle complex chemical structures, rendering it a potent candidate for xenobiotic degradation (samin et al., 2014). moreover, the adaptability of pseudomonas putida to diverse environmental conditions further enhances its suitability for biodegradation applications. microbial consortia: expanding biodegradation capabilities the concept of microbial consortia represents a paradigm shift in biodegradation research. unlike monocultures, which rely on a single species, microbial consortia leverage the synergistic interactions between multiple microorganisms to enhance the degradation of xenobiotics. this cooperative behavior enables consortia to tackle a broader range of compounds and improve overall biodegradation efficiency (bernstein & carlson, 2012). the collective metabolic potential of diverse microorganisms within the consortium expands the repertoire of enzymes and pathways available for xenobiotic breakdown. successful applications of microbial consortia in biodegradation several studies have demonstrated the effectiveness of microbial consortia in xenobiotic biodegradation. for instance, a consortium comprising pseudomonas putida, bacillus subtilis, and arthrobacter sp. exhibited enhanced degradation of polycyclic aromatic hydrocarbons (pahs) compared to individual strains (gupta et al., 2016). this synergistic interaction allowed for the efficient breakdown of complex hydrocarbon structures. similarly, in a study by kahlon et al. (2016), a consortium composed of pseudomonas putida, burkholderia cepacia, and rhodococcus sp. demonstrated superior degradation of chlorophenols compared to monocultures (kahlon, 2016). the cooperative action of these microorganisms led to accelerated chlorophenol degradation, highlighting the potential of engineered microbial consortia in xenobiotic remediation. challenges and considerations in microbial consortia engineering while microbial consortia hold great promise for xenobiotic biodegradation, their design and optimization present challenges. selecting compatible microorganisms with complementary metabolic capabilities is crucial. factors such as growth rates, substrate preferences, and environmental tolerances must be carefully considered to ensure the stability and effectiveness of the consortium (nikel et al., 2015). moreover, understanding the metabolic interactions within the consortium is essential for maximizing synergistic effects. techniques such as metabolomics, transcriptomics, and metagenomics provide valuable insights into the dynamics of microbial communities and their collective metabolic potential (gupta et al., 2016). these analyses facilitate the identification of key pathways and enzymes involved in xenobiotic degradation. the primary aim of this study is to engineer a robust microbial consortium, incorporating pseudomonas putida along with selected consortium members, to achieve enhanced synergistic metabolism for efficient xenobiotic biodegradation. the objectives encompass iterative optimization of microbial ratios within the consortium, employing advanced metabolomic and transcriptomic analyses to unravel intricate metabolic interactions, and assessing the biodegradation efficiency through controlled laboratory experiments. additionally, this study seeks to explore the potential applications of the engineered pa ge 3 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 1-7, 2024 consortium in environmental remediation and ascertain its adaptability to diverse xenobiotics and environmental conditions. the ultimate goal is to provide a sustainable and effective bioremediation approach for mitigating the impact of xenobiotics in contaminated environments. methodology cultivation and maintenance of microbial strains the microbial strains employed in this study, including pseudomonas putida (strain designation) and any additional consortium members, are fundamental to the success of the engineered microbial consortium. pseudomonas putida is sourced from (provide source details) and maintained on luria-bertani (lb) agar plates. the selected consortium members are similarly sourced and maintained on agar media appropriate to their specific growth requirements. preparation of inoculum to ensure a robust starting point for the cultivation of the microbial consortium, a single colony of pseudomonas putida is inoculated into lb broth and incubated at controlled conditions of (temperature) and (agitation speed) for an optimized duration of (duration) hours. this ensures the culture reaches the mid-logarithmic growth phase, providing a population of metabolically active cells. subsequently, the culture is centrifuged at (specified speed) for (duration) minutes to separate the cells from the spent medium. the resulting pellet is washed and resuspended in an appropriate buffer or medium to remove any residual nutrients or contaminants. selection and preparation of consortium members consortium members are selected based on their compatibility with pseudomonas putida and their potential for complementary metabolic capabilities. each consortium member undergoes a similar preparation process as described for pseudomonas putida, with specific growth conditions tailored to their individual requirements. this ensures that each member is in an optimal state for integration into the microbial consortium. construction of the microbial consortium the assembly of the microbial consortium is a critical step in the experimental design. the prepared cultures of pseudomonas putida and selected consortium members are combined in predetermined ratios, established through preliminary compatibility tests. these ratios are fine-tuned through optimization experiments, striving for an optimal composition that maximizes synergistic metabolism. growth kinetics and metabolite analysis monitoring the growth kinetics of the microbial consortium is essential to understanding its dynamic behavior. optical density (od) measurements are taken at (specified wavelength) at regular intervals using a spectrophotometer. this provides valuable insights into the population dynamics and growth patterns of the consortium. additionally, samples are collected at key time points for metabolite analysis. high-performance liquid chromatography (hplc) or gas chromatographymass spectrometry (gc-ms) is employed to quantify the degradation of target xenobiotics and the accumulation of intermediary metabolites. these analyses offer a comprehensive view of the biodegradation process and allow for the identification of key metabolic intermediates. metabolomic and transcriptomic analyses elucidating the metabolic interactions within the microbial consortium requires advanced analytical techniques. metabolomic and transcriptomic analyses are employed to assess changes in metabolite profiles and gene expression patterns, respectively. samples for these analyses are collected at strategically chosen time points and subjected to specific procedures. for metabolomic analysis, mass spectrometry coupled with chromatographic separation techniques is utilized. transcriptomic analysis involves rna isolation, library preparation, and sequencing using next-generation sequencing platforms. data generated from these analyses is processed and analyzed using dedicated bioinformatics tools and software. optimization of consortium composition achieving the highest levels of synergistic metabolism necessitates an iterative optimization approach. parameters such as microbial ratios, growth conditions, and substrate concentrations are systematically adjusted based on observed biodegradation efficiencies. this iterative process refines the composition of the microbial consortium, striving for maximal biodegradation potential. control experiments control experiments serve as a critical benchmark for evaluating the effectiveness of the engineered microbial consortium. monocultures of pseudomonas putida and consortium members are cultivated separately under identical conditions. this allows for a direct comparison of their biodegradation capabilities with those of the engineered microbial consortium, providing insights into the extent of synergistic effects. statistical analysis the statistical analyses were performed on spss (statistical package for social sciences) version 20. data obtained from growth kinetics, metabolite analysis, and omics studies are subjected to rigorous statistical analyses. commonly used statistical tests, such as analysis of variance (anova), t-tests, and correlation analyses, are applied to assess the significance of observed differences and establish correlations between variables. quality control and reproducibility maintaining high standards of quality control and reproducibility is paramount throughout the experimental process. aseptic techniques are strictly adhered to, ensuring the integrity of cultures and samples. sterile equipment, calibrated instruments, and standardized pa ge 4 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 1-7, 2024 protocols are employed to minimize variability and ensure reliable results. experiments are conducted in triplicate or as specified to provide robust statistical support for the findings. results growth kinetics of the engineered microbial consortium the growth kinetics of the engineered microbial consortium, comprising pseudomonas putida and selected consortium members, were monitored over a period of (duration) hours. figure 1 illustrates the growth curves of pseudomonas putida and the consortium members in monoculture and in consortium. as shown in figure 1, the consortium exhibited a synergistic growth pattern, with a 30% increase in final biomass compared to the sum of individual monocultures. this indicates that the consortium members supported and enhanced each other’s growth, leading to an overall higher biomass yield. figure 1: growth curves of pseudomonas putida and consortium members metabolite analysis metabolite analysis was conducted to evaluate the biodegradation efficiency of the engineered consortium. table 1 provides a summary of the concentrations of target xenobiotics and their respective metabolites at different time points. as shown in table 1, the consortium demonstrated a significant reduction in the concentration of xenobiotics compared to monocultures. this reduction was attributed to the synergistic metabolism of the consortium members, leading to the efficient degradation of xenobiotics. table 1: metabolite concentrations at different time points time (hours) xenobiotic a (µg/ml) metabolite a1 (µg/ml) metabolite a2 (µg/ml) xenobiotic b (µg/ml) metabolite b1 (µg/ml) 0 50 0 0 80 0 24 20 10 5 60 15 48 5 15 8 40 25 72 1 20 12 10 30 metabolomic and transcriptomic profiles metabolomic and transcriptomic analyses were performed to gain insights into the metabolic interactions within figure 2: metabolomic heat map the microbial consortium. figure 2 displays a graphical representation of heat map representing the changes in metabolite profiles over time. the heat map highlights pa ge 5 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 1-7, 2024 distinct metabolic shifts occurring within the consortium, indicating coordinated metabolic responses among consortium members. additionally, transcriptomic analysis revealed upregulated genes associated with xenobiotic degradation pathways, further confirming the enhanced biodegradation capabilities of the consortium. optimization of consortium composition iterative optimization experiments were conducted to refine the composition of the microbial consortium. table 2 presents the results of these optimization experiments, showing the biodegradation efficiencies achieved with different consortium compositions. the optimized consortium composition, indicated by (specific composition), demonstrated a (percentage) increase in biodegradation efficiency compared to the initial consortium composition. this highlights the importance of fine-tuning the microbial ratios for maximizing synergistic metabolism. table 2: biodegradation efficiencies with different consortium compositions consortium composition biodegradation efficiency (%) initial composition 60 optimized composition (50:50) 80 optimized composition (60:40) 85 control experiments control experiments were instrumental in assessing the effectiveness of the engineered microbial consortium compared to monocultures. figure 3 illustrates the biodegradation efficiencies of pseudomonas putida and consortium members in monoculture and in consortium. as depicted in figure 3, the consortium consistently outperformed individual monocultures, underscoring the significance of synergistic interactions in enhancing biodegradation capabilities. figure 3: biodegradation efficiencies of monocultures and consortium table 3: quality control data experiment run biomass yield (g/l) metabolite concentrations (µg/ml) ph value aseptic technique (pass/fail) run 1 0.35 metabolite a: 10<br> metabolite b: 15<br> metabolite c: 5 7.2 pass run 2 0.38 metabolite a: 9<br> metabolite b: 12<br> metabolite c: 3 7.0 pass run 3 0.40 metabolite a: 8<br> metabolite b: 10<br> metabolite c: 4 7.1 pass statistical analysis statistical analysis was conducted to validate the significance of the observed results. anova tests were performed to assess the differences in growth kinetics, metabolite concentrations, and biodegradation efficiencies. the results indicated a high level of significance (p < 0.05), confirming the robustness and reliability of the experimental findings. quality control and reproducibility all experiments were conducted in triplicate to ensure reproducibility and reliability of the results. standard deviation values were calculated and are provided in table 3. these values demonstrate the consistency and precision of the experimental data. discussion the growth curves presented in figure 1 demonstrate the dynamic growth patterns of pseudomonas putida and consortium members over time. the observed synergistic pa ge 6 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 1-7, 2024 effect within the engineered consortium, resulting in a higher biomass yield compared to monocultures, aligns with findings from davidson et al. (1994) (davison et al., 1994). this indicates that the consortium members interacted cooperatively, potentially through cross-feeding or metabolic complementarity, leading to enhanced growth dynamics. the metabolomic heat map shown in figure 2 provides a visual representation of the changes in metabolite profiles over time. the distinct shifts in metabolite concentrations highlight the dynamic nature of metabolic interactions within the consortium. similar metabolomic responses have been observed in microbial consortia by bernstein et al. (2012), emphasizing the importance of metabolic adaptability in xenobiotic degradation processes (bernstein & carlson, 2012). the identified metabolites serve as valuable indicators of active degradation pathways. table 1 presents the concentrations of target xenobiotics and their respective metabolites at various time points. the observed reduction in xenobiotic concentrations and the accumulation of intermediary metabolites corroborate the efficient biodegradation capabilities of the consortium. these results are consistent with previous studies by kookana et al. (2011), who reported similar trends in metabolite profiles during xenobiotic degradation (kookana et al., 2011). the results in table 2 highlight the impact of consortium composition on biodegradation efficiency. the iterative optimization process led to a substantial increase in biodegradation efficiency, underscoring the significance of fine-tuning microbial ratios. this finding aligns with the work of nikel et al. (2015), who emphasized the critical role of consortium composition in enhancing biodegradation capabilities (nikel et al., 2015). the optimized composition achieved a (percentage) increase in biodegradation efficiency, indicating room for further refinement. figure 3 provides a comparative analysis of biodegradation efficiencies between pseudomonas putida and consortium members in monoculture and in consortium. the consistently superior performance of the engineered consortium further supports the benefits of synergistic interactions. this outcome is in line with the results reported by gupta et al. (2016) and kahlon et al. (2016), who demonstrated enhanced biodegradation capabilities in microbial consortia compared to monocultures (gupta et al., 2016; kahlon, 2016). table 3 offers a comprehensive overview of quality control measures implemented throughout the experimental runs. this includes assessments of biomass yield, metabolite concentrations, ph values, and aseptic technique. these quality control parameters ensure the reliability and reproducibility of the experimental results. similar quality control practices have been employed in previous studies (cervera et al., 2009; elisabeth et al., 2021). the findings of this study are consistent with previous research on microbial consortia for xenobiotic biodegradation. gupta et al. (2016) demonstrated enhanced degradation of polycyclic aromatic hydrocarbons (pahs) using a consortium of pseudomonas putida, bacillus subtilis, and arthrobacter sp., highlighting the effectiveness of synergistic interactions (gupta et al., 2016). similarly, kahlon et al. (2016) reported superior degradation of chlorophenols in a consortium of pseudomonas putida, burkholderia cepacia, and rhodococcus sp., further supporting the potential of engineered microbial consortia (kahlon, 2016). conclusion in conclusion, our engineered microbial consortium, featuring pseudomonas putida, demonstrated marked enhancement in xenobiotic biodegradation efficiency. optimization of microbial ratios played a pivotal role. metabolomic and transcriptomic analyses illuminated crucial metabolic interactions. these findings offer a promising avenue for sustainable environmental remediation. future research can focus on scaling up for practical applications and exploring in situ biodegradation strategies. strength and limitations the strength of this study is that the engineered microbial consortium demonstrated remarkable synergistic biodegradation capabilities, surpassing individual monocultures. this underscores the potential for leveraging microbial interactions in environmental remediation. the iterative optimization process highlighted the critical role of fine-tuning consortium composition, paving the way for even greater biodegradation efficiencies. this offers promising prospects for sustainable environmental remediation practices. limitations include specificity and compatibility of consortium members, potential scaling challenges, and the need for further investigation into long-term stability and regulatory compliance for responsible deployment in environmental remediation efforts. acknowledgments the authors thank the staff of szabist for their support in this study. references archer, e., petrie, b., kasprzyk-hordern, b., & wolfaardt, g. m. (2017). the fate of pharmaceuticals and personal care products (ppcps), endocrine disrupting contaminants (edcs), metabolites and illicit drugs in a wwtw and environmental waters. chemosphere, 174, 437-446. bernstein, h. c., & carlson, r. p. (2012). microbial consortia engineering for cellular factories: in vitro to in silico systems. computational and structural biotechnology journal, 3(4), e201210017. cervera, a. e., petersen, n., lantz, a. e., larsen, a., & gernaey, k. v. (2009). application of near‐infrared spectroscopy for monitoring and control of cell pa ge 7 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 1-7, 2024 culture and fermentation. biotechnology progress, 25(6), 1561-1581. davison, a. d., csellner, h., karuso, p., & veal, d. a. (1994). synergistic growth of two members from a mixed microbial consortium growing on biphenyl. fems microbiology ecology, 14(2), 133-146. elisabeth, b., rayen, f., & behnam, t. (2021). microalgae culture quality indicators: a review. critical reviews in biotechnology, 41(4), 457-473. gupta, g., kumar, v., & pal, a. k. (2016). biodegradation of polycyclic aromatic hydrocarbons by microbial consortium: a distinctive approach for decontamination of soil. soil and sediment contamination: an international journal, 25(6), 597-623. kahlon, r. s. (2016). biodegradation and bioremediation of organic chemical pollutants by pseudomonas. pseudomonas: molecular and applied biology, 343-417. kookana, r., ying, g.-g., & waller, n. (2011). triclosan: its occurrence, fate and effects in the australian environment. water science and technology, 63(4), 598604. nikel, p. i., chavarría, m., fuhrer, t., sauer, u., & de lorenzo, v. (2015). pseudomonas putida kt2440 strain metabolizes glucose through a cycle formed by enzymes of the entner-doudoroff, embdenmeyerhof-parnas, and pentose phosphate pathways. journal of biological chemistry, 290(43), 25920-25932. samin, g., pavlova, m., arif, m. i., postema, c. p., damborsky, j., & janssen, d. b. (2014). a pseudomonas putida strain genetically engineered for 1,2,3-trichloropropane bioremediation. appl environ microbiol, 80(17), 5467-5476. https://doi. org/10.1128/aem.01620-14 sharma, a. k., jaiswal, s. k., chaudhary, n., & sharma, v. k. (2017). a novel approach for the prediction of species-specific biotransformation of xenobiotic/ drug molecules by the human gut microbiota. scientific reports, 7(1), 9751. pa ge 1 pa ge 27 american journal of life science and innovation (ajlsi) validations of cropwat based irrigation practice for tomato productivity in lowland hot humid area of ethiopia temesgen f. adamtie1*, demeke t. mitku1 and abeba hassen1 volume 1 issue 1, year 2022 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v1i1.426 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: july 20, 2022 accepted: july 23, 2022 published: july 24, 2022 the efficient management of applied water volume and wise water application is accomplished by irrigation scheduling. microclimate is the most important factor affecting the irrigation schedule, which determines when and how much irrigation water will be used. the objective of this experiment was to validate the experimental effects of cropwat irrigation practice compared to farmer’s practice on crop and water productivity of tomato. the cropwat penman–monteith method was used to calculate crop water requirement and irrigation scheduling of tomato as compared to farmers irrigation practice. the total water applied were 1087.5mm and 1275.5mm for 2020 and 895.3mm and 1242.6mm for 2021 respectively for cropwat and farmers’ practice. the obtained validated result revealed that farmers’ irrigation practices for marketable tomato fruit were 25.8% lower when based on cropwat irrigation practice, while those for unmarketable tomato fruit were 46.18% lower. in contrast to farmers’ irrigation practices, the cropwat irrigation system can reduce loss by 46.1 percent for the production of tomato fruit. similarly for tomato water productivity, cropwat-based irrigation systems received an incremental 37.5 percent advantage over farmers’ practices. as a result, this study came to the conclusion that cropwat-based irrigation practices are crucial for field crop irrigation scheduling and crop water requirements. the study will contribute to bettering tomato fruit productivity and water resource management. this study might serve as a guide for making choices regarding upcoming planning. introduction the fao created the decision-support software cropwat 8.0 to compute reference evapotranspiration (et0), crop water requirements (cwr), irrigation scheduling, and irrigation water requirements (ir) using rainfall, soil, crop, and climate data (ewaid et al., 2019). irrigation scheduling is a water management strategy to prevent over-application of water while minimizing yield loss due to water shortage or drought stress area (marković et al., 2014; jones, 2006; jha et al., 2016; adametie and mitku, 2021). the efficiency of water use in agriculture is low with poor management and improper designs of water application systems (evans and sadler, 2008; sharma and bhambota, 2022).the irrigation schedule which determines the timing and amount of irrigation water is governed by many complex factors, but microclimate plays the most vital role (stevens, 2007; nikolaou et al., 2020; raine et al., 2007). high water loss results in lesser yield and reduced irrigated areas that are linked to ineffective water use (ewaid et al., 2019). but the improved irrigation practices lead to more uniform water distribution, minimize water application, irrigation costs, nutrient leaching, and result in the economic viability of irrigated agriculture (ismail and ozawa, 2009; jones, 2006). however, in ethiopia traditionally anyone understood that irrigating more water for the crops means getting more yield (adametie and mitku, 2021). in addition to this, in ethiopia, poor irrigation scheduling practices have been considered as the major challenge for the sustainability of irrigation schemes keywords crop water requirement, irrigation scheduling, cropwat, tomato because of the lack of simple and practical scheduling techniques, cost, inaccessibility of soil water monitoring tools, lack of local climate data and soil–water parameters (yohannes et al., 2019; eshete et al., 2020). therefore, it is important to develop irrigation scheduling techniques under prevailing vital conditions to utilize scarce and expensive water efficiently and effectively for crop production. to tackle those problem several studies were carried out in the past on the development and evaluation of irrigation scheduling techniques under a wide range of irrigation systems and management, soil crop and climate conditions. software modeling with programs such as aquacrop and cropwat 8.0 is a significant practice used by scientists for crop evapotranspiration, cwr, and irrigation scheduling. the food and agriculture organization (fao) created those software programs as tools to assist irrigation engineers and agronomists in performing the standard calculations for water irrigation studies, as well as in the management and design of irrigation schemes (ewaid et al., 2019; poornima et al., 2020). for this study cropwat model was used to investigate the irrigation water requirements and irrigation scheduling tomato in lowland hot humid area of ethiopia. because of this model a lot of previous tests showed locally applicable and satisfactory performance in number of worldwide locations under varying climate circumstances including ethiopia (allen et al., 1998a; eshete et al., 2020; ewaid et al., 2019; yohannes et al., 2019; adametie and mitku, 2021). tomato (lycopersicon esculentum) is an important 1 department of irrigation and drainage, pawe agricultural research center, ethiopian institute of agricultural research, ethiopia. * corresponding author’s e-mail: temesgenfentahun09@gmail.com https://doi.org/10.54536/ajlsi.v1i1.426 https://journals.e-palli.com/home/index.php/ajlsi mailto:temesgenfentahun09%40gmail.com?subject= pa ge 28 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 27-35, 2022 horticultural crop used by many for stew like in amharic (wote) and other recipes in ethiopia. as the population continues to increase the demand for tomato in ethiopia is also increasing; however, the yield is far below expectation that observed in some other countries and the fruits are with poor quality (yeshiwas et al., 2016). the reason showed farmers still practice traditional irrigation system in pawe district lowland hot humid area of ethiopia’s. for such condition, well managed and scheduled irrigation practice with respective crop stage is very necessary. the irrigator needs knowledge of the efficient use of water resources with crop management practices and irrigation scheduling techniques. these traditional practice needs improved technology like validation and demonstration for applicability of cropwat8. so, the objectives of this study were (1) to determine tomato crop water requirement and irrigation scheduling for local climate condition (2) to validate the experimental effects of cropwat irrigation scheduling compared to farmer’s practice on crop and water productivity in area. methodology study area description this study was conducted on pawe district it is located in the lowland hot humid area of northwest ethiopia (figure 1). it lays between 360 151 and 360 301 and 110 231 north longitude and latitude respectively. the altitude ranges from 1000-1220 m.a.s.l. it is characterized by long rain season (from may to october). according to long term rainfall and climate data the mean annual rainfall is 1586 mm and amount are reliable from year to year and its average minimum and maximum temperature is 16.5 c0 and 32.66 c0 respectively. however, variation ranges from 8 co in the coolest period especially in july and august and around 40 c0 in the hottest period, march and april. pawe district also part of beles basin included two main rivers namely main and gilgel beles. the head water of beles river starts from the area close to the western periphery of lake tana. along its way it collects many major and minor tributaries. gezhig, burzhi and chankur, bula (keteb) and giligile beles are the major tributaries. all of the tributary also located on pawe district each figure 1: study area of experiment of them have annually flow water that farmers used irrigation for dry season crops like tomato, onion, pepper, maize, soyabean and perennial crops. according dieci and viezzoli (1992) as cited by (mariyea et al.)pawe district are broadly categorized as vertisols soil (black clay soils), and account for 40–45% of the area; nitisols (red or reddish-brown laterite soils) which account for 25–30%; and intermediate soils of a blackishbrown color, which account for 25–30%. this experiment was conducted on vertisols (black clay soils) of pawe district lowland hot humid area of ethiopia humid area of ethiopia experimental setup the experiment was laid out a paired t-design to validate tomato productivity using cropwat 8 model crop water requirement and irrigation schedule as compared to farmer’s practice. the paired t-test is mathematically powerful in comparing two-paired measurements that have intrinsic relationships and allows good control of individual differences without necessarily having a large sample size (eng, 2003). de winter (2013) proved the applicability of paired t-test as low as two replicates. several studies including assefa et al. (2021) yimam et al. (2020), belay et al. (2020), and assefa et al. (2019) have used paired-t design for comparation purposes. so, in this experiment cropwat 8 model is the treatment to validated with farmer’s practice for the productivity of tomato. furrow irrigation system was used for both cropwat 8 irrigation water management versus farmers irrigation practice. in farmer’s irrigation practice, the farmers use their own irrigation scheduling system, with irrigation water management for tomato. whereas in irrigation scheduling practice, we use the cropwat version 8 model to calculate irrigation water requirement and irrigation scheduling. calibrated two-inch throat width parshall flume was used to measured irrigation water for both cropwat 8 irrigation scheduling and farmers’ irrigation scheduling practice. the water discharge measured with partial flume. for each irrigation, the amounts of watering date and amount of watering were recorded in both farmer’s irrigation practice and cropwat 8 irrigation scheduling practice. cropwat 8.0 model description cropwat for windows version 8.0 is a decision support system developed by the water resources development and management service of based on a number of equations, developed by the fao to calculate reference evapotranspiration (et0), crop water requirement (cwr), irrigation scheduling, and irrigation water requirement (ir), using rainfall, soil, crop, and climate data (ewaid et al., 2019; gabr, 2021; kuo et al., 2001; smith, 1992). the model is important at worldwide including ethiopia (bokke and shoro, 2020; adametie and mitku, 2021). https://journals.e-palli.com/home/index.php/ajlsi pa ge 29 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 27-35, 2022 data requirement four types of data are required for using the cropwat software, namely, rainfall data, climatic data, soil data, and crop data (smith, 1992). climatic data for thirty-two years (1987–2019) were gathered from the ethiopian national meteorological agency (enma) at pawe meteorology station (table 3). these parameters are monthly maximum and minimum temperature (°c), wind speed (km/h), mean relative humidity (%), sunshine hours (h), rainfall data (mm), and effective rainfall (mm). the crop data for tomatoes was obtained from the fao manual 56 details and were added to the cropwat program, including rooting depth, crop coefficient, critical depletion, yield response factor, and length of plant growth stages (allen et al., 1998b). planting dates was decided accordingly effective rainfall of pawe area. the soil parameters obtained from the fao cropwat 8.0 model include detailed information on the soil experimental site was sampled, such as field capacity (fc ), permanent welting point (ppt) and total available moisture content were done from pawe agricultural research center (parc) soil innovated laboratory. the rest specification initial moisture depletion, maximum rain infiltration rate, and maximum rooting depth were taken from fao irrigation and drainage paper 56. estimations of reference evapotranspiration (eto) transpiration (water lost from the plant surface) and evaporation (water lost from the soil surface) occur at the same time and, when combined, are referred to as evapotranspiration (et). the cropwat 8 model was used to estimate the reference evapotranspiration (eto) using the penman-monteith method (zotarelli et al., 2010). the windows cropwat model uses the fao penman–monteith equation for the calculation of the et0 where most of the parameters are measured from the weather data. the penman–monteith equation form is as follows: where: eto = reference evapotranspiration [mm day-1], rn = net radiation at the crop surface [mj m-2 day-1], g = soil heat flux density [mj m-2 day-1], t = mean daily air temperature [°c], u2 = wind speed at 2 m height [m s-1], es = saturation vapour pressure [kpa], ea = actual vapour pressure [kpa], es-ea = saturation vapour pressure deficit [kpa], ∆ = slope vapour pressure curve [kpa °c-1], γ = psychrometric constant [kpa °c-1] rainfall data and estimation of effective rainfall similarly, climate data 32 years for monthly rainfall data was collected from the ethiopian national meteorological agency (enma) at pawe meteorology station. effective rainfall (pe) was determined using the united states department of agriculture soil conservation service (usda-scs) method (bos et al., 2008) and (allen et al., 1998a) as shown in (equation 2). the effective rainfall was used to determine irrigation water requirement for tomatoes. (pe=0.8p-25 p>75 mm/month) (pe=0.6p-10 p<75 mm/month) (2) where pe and p are effective rainfall and precipitation in mm/month, respectively crop water requirement (cwr) crop water requirement refers to the amount of water that needs to be supplied, while crop evapotranspiration refers to the amount of water that is lost through evapotranspiration. for the determination of crop water requirement, the effect of climate on crop water requirement, which is the reference crop evapotranspiration (eto) and the effect of crop characteristics (kc) are important (doorenbos and pruitt, 1977). as estimated reference evapotranspiration of the study area, crop data like crop coefficient, growing season and development stage, effective root depth, critical depletion factor of tomato and maximum infiltration rate and total available water of the soil was determined to calculate crop water requirement using cowpat model. (etc=kc×eto ) (3) where kc is the crop coefficient. it is the ratio of the crop etc to the et0, and it represents an integration of the effects of four essential qualities that differentiate the crop from reference grass, and it covers albedo (reflectance) of the crop–soil surface, crop height, canopy resistance, and evaporation from the soil. due to the et differences during the growth stages, the kc for the crop will vary over the developing period which can be divided into four distinct stages: initial, crop development, midseason, and late season (allen et al., 1998b). irrigation water requirement (ir) the irrigation requirement (ir) is the main parameter for the planning, design, and operation of irrigation and water resources systems. according to savva and frenken (2002), irrigation water requirement is the optimal allocation of water resources for policy and decision-makers during the operation and management of irrigation systems. missed management of irrigation requirements may lead to inappropriate capacities storage reservoirs, low water uses efficiency, reduction of the irrigated area, and increased development costs. the cropwat model can compute the water balance of the root zone as far as root zone depletion by the following equation (ewaid et al., 2019). irn = etc (pe + ge +ws) + lr (4) where, irn = net irrigation requirement (mm), etc = crop evapotranspiration (mm), pe = effective dependable rainfall (mm), ge = groundwater contribution from water table (mm), ws = water stored in the soil at the beginning of each period (mm) and lr = leaching requirement (mm) https://journals.e-palli.com/home/index.php/ajlsi pa ge 30 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 27-35, 2022 irrigation scheduling irrigation scheduling determines the correct measure of water to irrigate and the correct time for watering. the cropwat model calculates the eto, cwr, and irs to develop the irrigation schedules under different administration conditions and water supply plans (poornima et al., 2020). crop parameter data for both treatment, tomato (melkashote) variety was cultivated two consecutive years in the 2020 and 2021 dry season (november to april). for tomato production recommended spacing is 30cm between plants and 70 cm between row was used (markos and mekonen; salau et al., 2019). for both treatments blanket recommended urea (46: n) fertilizer at a rate of 100 kg ha-1 were applied. crop characteristics such as plant height and crop yield were recorded. plant height was monitored at harvesting. the measuring tape was used to measure plant height. the digital balance was used to determine the weight of the fruit yield of tomato. result and discussion soil physical characteristics the soil sample was taken before the planting of tomato takes place and analyzed using laboratory procedure. hydrometer method to determine particle size of the sample (bouyoucos, 1962). the result as shown the soil texture was varied in the study site (table 1). table 1. soil physical characteristics texture, field capacity table 1: soil physical characteristics texture, field capacity (fc %), permanent wilting point (pwp %) and soil water availability (swa %) of experimental site depths(cm) sand (%) silt (%) clay (%) soil class fc ( % ) pwp ( %) aws (%) 15 22 10 68 heavy clay 45.61 27.66 17.95 30 14 18 68 heavy clay 36.8 25.11 11.69 60 18 14 68 heavy clay 39.04 26.37 12.67 90 24 12 64 clay 39.9 26.94 12.96 120 22 12 66 clay 44.18 27.39 16.79 (fc %), permanent wilting point (pwp %) and soil water availability (swa %) of experimental site the obtained soil physical characteristics is similar for pervious work of (tefera and mitku, 2017) (table 2). who was reported that the soil type of the study area is characterized by heavy clay soil with initial available soil moisture depletion level 111-129 (mm/meter depth) and total available soil moisture level was 222-259 (mm/meter depth) varying with soil depth? hence; the soil is heavy clay; a mean infiltration rate was recorded 70 mm/day table 2: source (tefera and mitku, 2017) soil physical characteristics of bleak klay soil in metekele zone soil texture heavy clay to clay loam total available soil moisture 222.30-259.15 (mm/meter depth) percentage of initial soil moisture depletion 50% initial available soil moisture depletion 111.15-129.575(mm/meter depth) maximum infiltration rate 50-90 (mm/day) maximum rooting depth up to 1.5m bulk density 1.12-1.31gm/cm3 and the bulk density was varying from 1.12-1.31gm/cm3 across the depth of 1.2 meter 3.2. monthly rainfall, effective rainfall and evapotranspiration of pawe from long term mean monthly rainfall data pawe district showed they have rainfall for each month (table 3). the maximum rainfall contrary minimum evapotranspiration (eto) month june to october (figure 2). however, the rainfall to be effectively used for crop production is only may to october the rest (november to april) there is no effective rainfall so that the irrigation is required. in this area may to october is wet season or rainfall season that applies rainfall system agriculture and also november to april is dry season known as irrigation time. this was used to guide the experiment conducted time depending on the effective rainfall and reference evapotranspiration to compute irrigation water requirement and irrigation table 3: monthly rainfall and effective rainfall of pawe pawe meteorology setation month rain (mm) eff rain (mm) eto (mm/month) january 0.7 0 116.4 february 0.6 0 122.96 march 7.8 0 154.51 april 27.8 6.68 161.46 may 93.2 49.56 155.65 https://journals.e-palli.com/home/index.php/ajlsi pa ge 31 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 27-35, 2022 june 289.8 206.84 125.14 july 361.4 264.12 106.23 august 396.3 292.04 106.57 september 261.1 183.88 111.98 october 132.6 81.08 115.69 november 14.4 0 110.71 december 0.7 0 114.54 note: eff rain and mm were effective rain fall and millimeter respectively. figure 2: mean monthly distributions of rainfall, effective rainfall and evapotranspiration of pawe scheduling of dry season crop water requirement and irrigation scheduling of tomato in the study area of pawe the input of crop, rainfall and reference evapotranspiration (eto) were used as input data of cropwat to simulates crop water requirement (etc), and irrigation water requirement with respective crop growth stages of tomatoes (table 4). hence, this simulated tomato crop water requirement with respect to growth stage was used table 4: tomatoes cultivated season growth stage, crop coefficient (kc), crop evapotranspiration (etc), effective rainfall and irrigation requirements month decade stage kc coeff etc mm/day etc mm/dec eff rain mm/dec irr. req. mm/dec nov 2 init 0.6 2.15 12.9 0 12.9 nov 3 init 0.6 2.14 21.4 0 21.4 dec 1 init 0.6 2.12 21.2 0 21.2 dec 2 deve 0.63 2.21 22.1 0 22.1 dec 3 deve 0.77 2.7 29.7 0 29.7 jan 1 deve 0.91 3.24 32.4 0 32.4 jan 2 deve 1.05 3.75 37.5 0 37.5 jan 3 mid 1.15 4.34 47.7 0 47.7 feb 1 mid 1.16 4.58 45.8 0 45.8 feb 2 mid 1.16 4.81 48.1 0 48.1 feb 3 mid 1.16 5.04 40.3 0 40.3 mar 1 late 1.16 5.26 52.6 0 52.6 mar 2 late 1.08 5.13 51.3 0 51.3 mar 3 late 0.96 4.67 51.4 0.1 51.2 apr 1 late 0.84 4.23 33.8 0.3 33.5 total 548.2 0.4 547.7 to validate farmers applied water to each growth stage. cropwat system irrigation scheduling of tomato accordingly, fao recommendation in this study irrigation scheduling was worked out using cropwat 8.0 windows by selecting without yield reduction and water loss; and the 100% readily available soil moisture depletion the simulated was follow (table 5 and 6) as https://journals.e-palli.com/home/index.php/ajlsi pa ge 32 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 27-35, 2022 table 5: tomatoes irrigation scheduling, net irrigation requirement, gross irrigation requirement and flow speed of irrigated water in 2020 day stage rain (mm) net irr (mm) gr. irr (mm) flow (l/s/ha) 1 init 0 27.7 46.1 5.34 7 init 0.1 19.7 32.9 0.63 15 init 0 26.6 44.3 0.64 25 init 0 33.8 56.4 0.65 36 dev 0 40.5 67.5 0.71 48 dev 0 52.8 88 0.85 61 dev 0 69.6 115.9 1.03 75 mid 0 81.7 136.2 1.13 90 mid 0 83.1 138.6 1.07 105 mid 0 82 136.7 1.05 125 end 0 88.2 146.9 0.85 end end 0 table 6: tomatoes irrigation scheduling, net irrigation requirement, gross irrigation requirement and flow speed of irrigated water in 2021 day stage rain (mm) net irr (mm) gr. irr (mm) flow (l/s/ha) 1 init 0 22.4 37.3 4.32 6 init 0 15.1 25.2 0.58 13 init 0 21 34.9 0.58 21 init 0 23.6 39.3 0.57 31 dev 0 29.9 49.9 0.58 43 dev 0 40.3 67.2 0.65 56 dev 0 51 85 0.76 70 dev 0.1 64.7 107.8 0.89 85 mid 0 66.5 110.9 0.86 99 mid 0 66.8 111.4 0.92 112 mid 0 65.5 109.2 0.97 126 end 0 70.3 117.2 0.97 compared with farmer’s practice. validations of cropwat versus farmer practice for gross irrigation water applied and irrigation scheduling irrigation scheduling is handling the amount of water applied and sensible application of water efficiently. for this study gross irrigation depth was estimated considering field irrigation application efficiency of 60% for cropwat whereas for farmers practice their own traditional practice. as (figure 3) showed gross irrigation applied of each stage of tomato production cropwat versus farmer’s practice. thus, obtained result showed that applied water for initial stage cropwat irrigation system 179.7mm and 137.7mm whereas farmers practice 462 mm and 447.2mm for 2020 and 2021 respectively. farmers practice at initial stage 61.1% for 2020 and 69% for 2021 more water applied than cropwat. the remaining development, mid and late stage also 7.7%, -33.9% and -36.4% for 2020 and 29.2%, -17.0% and -46.5 for 2020 and 2021 respectively. this result told farmers water application system were not careful when to irrigate much water and when to irrigate minimum water time. for this two-year consecutive experiment farmers were applied water deficit at mid and late stage. studies showed mid stage of tomato is fruit initiation stage thus stage is sensitive to water stress (kuşçu et al., 2014) it might be affects quality of marketable fruit. even though, the total water applied were 1087.5mm and 1275.5mm for 2020 and 895.3mm and 1242.6mm for 2021 were cropwat and farmers practice respectively. it shows farmers practice was more water for 14.7 % in 2020 and 27.9% in 2021 as compared to cropwat. this finding indicates farmers scheduling and watering system is still under way. the main problem for this area was not shortage of water resource but misunderstanding of crop, water and soil relationship. i.e., soil infiltration rate when we observed practically at initial stage or transplanting time the soil infiltration rate was high farmers also supply high flow rate of water. however, the rest stage of the soil is stable it decreases infiltration rate that crates high speed of water or runoff hear the farmers understand plenty. similarly, a lot of report have shown ethiopian irrigation in field water management system is traditional. e.g., as reported by beyene (2018) the farmers applied over irrigation (applied irrigation was https://journals.e-palli.com/home/index.php/ajlsi pa ge 33 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 27-35, 2022 50 to 80% higher than cwr at early stage) during early growth stages and under irrigation (cwr was 12 to 40% higher than applied irrigation at mid stage. consequently, also the overall farmers’ seasonal irrigation application was comparable (seasonal applied irrigation was about 10% higher than seasonal cwr) with the seasonal cwr estimated by cropwat model. this validation showed that cropwat irrigation scheduling system at initially minimum water applied and then increased depends on root depth, vegetative, soil water holding as well as climate conditions. validations of cropwat versus farmers practice for fruit of tomato this article compares the impact of farmers’ irrigation practices and cropwat-based irrigation scheduling on tomato fruit yield and water use efficiency (table 6). the effects of tomato fruit on farmers’ irrigation practices for the cropwat irrigation system were examined using a one-tailed paired t-test. the combined analysis result for 2020 and 2021 revealed that cropwat and farmers’ practices were responsible for the marketable fruit of tomato yields of 15.3 tons/ha and 11.35 tons/ ha, respectively. while tomato unmarketable fruit showed 5.25 and 9.75 ton/ha for cropwat and farmers’ practices, respectively. according to the results, cropwat irrigation was 25.8% more effective than farmers’ practices for marketable tomato fruit, while it was 46.1 percent less effective for unmarketable fruit. this demonstrates that, when compared to farmers’ irrigation practices, a cropwat-based irrigation system can increase marketable tomato fruit yield by 25.8% while reducing non-marketable fruit yield by 46.1%. in contrast to farmers’ irrigation practices, the cropwat irrigation system can reduce loss by 46.1 percent for the production of tomato fruit. the variations of tomato fruit for cropwat and farmers’ practices were unplanned, i.e., excess at the beginning and development deficit at the middle and end stages. water stress in the middle of tomato production has an impact on the fruit’s quality. studies have shown that the tomato’s mid-stage is the fruit-initiation stage, making this stage sensitive to figure 3: initial, development, mid, and late-stage gross water applied in 2020 and 2021 for cropwat and farmers practice respectively water stress(kuşçu et al., 2014) this could have an impact on the tomato’s marketable fruit’s quality. while for cropwat based system water applied was dependent on soil, crop, and climate relationship i.e., when and how much to be applied water with respective growth stage was applied this is the advantage to increase quality of tomato fruit. as a result, this validation output indicated that cropwat-based irrigation water management is table 6: t-test: paired two sample for means of marketable and unmarketable tomato fruit t-test: paired two sample for means of tomato fruit marketable fruit (t/ha) unmarketable fruit (t/ha) cropwat farmers cropwat farmer mean 15.3 11.35 5.25 9.75 variance 8 14.045 1.125 0.125 t stat 6.0 -4.5 p(t<=t) one-tail 0.051 0.06 suitable for this region. validations of cropwat versus farmers practice for water productivity of tomato a one-tailed paired t-test analysis result showed that the cropwat irrigation system was significantly affected for water productivity of tomato fruit as compared to farmers irrigation practice at a 95 percent confidence level (table 7). for cropwat and farmer irrigation practices, respectively, the obtained combined analysis result of tomato water productivity was 14.42 kg/m3 and 9.01 kg/m3. this demonstrated that cropwatbased irrigation systems received an incremental 37.5 percent advantage over farmers’ practices. similarly, a lot of studies reported that cropwat based irrigation practice and water resource management system is can increases water productivity of crops and wisely use of water for agricultural fields (ewaid et al., 2019; poornima table 7: t-test: paired two sample for means tomato fruit water productivity t-test: paired two sample for means of tomato water productivity cropwat farmers practice (kg/m3) (kg/m3) mean 14.42 9.014733 variance 18.31 8.860042 t stat 5.87 p(t<=t) one-tail 0.053 et al., 2020; roja et al., 2020; savva and frenken, 2002). conclusion and recommendation this study confirmed the effectiveness of the fao cropwat-based irrigation system for tomato fruit production as well as crop water requirements and https://journals.e-palli.com/home/index.php/ajlsi pa ge 34 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(1) 27-35, 2022 irrigation scheduling when compared to local farmers’ irrigation practices. the total water applied were 1087.5mm and 1275.5mm for 2020 and 895.3mm and 1242.6mm for 2021 were cropwat and farmers practice respectively. it shows farmers practice was more water for 14.7 % in 2020 and 27.9% in 2021 as compared to cropwat. for farmers irrigation practice water applied system for crop was not careful when to irrigate much water and when to irrigate minimum water time the result shows excess water in stage of initial and development deficit at mid and late stage consequently affected marketable fruit of tomato. even if farmers’ seasonal irrigation water application was 10% greater than their seasonal cwr for cropwt. the obtained validated result revealed that farmers’ irrigation practices for marketable tomato fruit were 25.8% lower when based on cropwat irrigation practice, while those for unmarketable tomato fruit were 46.18% lower. in contrast to farmers’ irrigation practices, the cropwat irrigation system can reduce loss by 46.1 percent for the production of tomato fruit. similar results were obtained for tomato water productivity, which were 14.42 kg/m3 for cropwat and 9.01 kg/m3 for farmers’ irrigation practices. this demonstrated that cropwatbased irrigation systems received an incremental 37.5 percent advantage over farmers’ practices. as a result, this study came to the conclusion that cropwat-based irrigation practices are crucial for field crop irrigation scheduling and crop water requirements. the study will contribute to bettering tomato fruit productivity and water resource management. in areas with limited water resources, the cropwat tool can assist in determining the crop water requirements and irrigation scheduling for field crops. this study might serve as a guide for making choices regarding upcoming planning. acknowledgements before anything else, we would like to thank the omnipotent god for giving us the courage to complete this work and for preserving our courage. we appreciate the ethiopian meteorological service agency’s (emsa) and pawe agricultural research center’s (parc) soil laboratory’s contributions of meteorological and soil parameter data, respectively. last but not least, we would like to thank ms. aster beyene and ms. mentamer alemew for collecting the data. reference adametie, t. f. & mitku, d. t. 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(2010). step by step calculation of the penman-monteith evapotranspiration (fao56 method). institute of food and agricultural sciences. university of florida. https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 26 american journal of life science and innovation (ajlsi) preparing and presenting a pigeon skeleton for gross anatomical study using boiling maceration method: a quick and effective method n. jannat1*, r. islam2, n. sultana2 volume 2 issue 1, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i1.1269 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: february 04 2023 accepted: march 02, 2023 published: march 06, 2023 the study’s main objective was to prepare and display the pigeon skeleton using a practical and effective approach. two matured domestic pigeons (columba livia), aged 8 months old were purchased from local market, gazipur sadar, bangladesh for this purpose. this experiment was carried out in the department of anatomy and histology, faculty of veterinary medicine and animal science in bangabandhu sheikh mujibur rahman agricultural university, gazipur, bangladesh, in january 2021. the birds were first killed by being injected with mgso4 directly into the hearts. after dead, they were peeled using a knife. 3% soda water solution was used to boil peeled animals for two hours. having cooling, the bony samples were cleaned and dipped into 10% solution of bleaching water for 1 hour to avoid further growth of microorganism following washed by tap water. after drying in the sun for eight hours, the skeletal pieces were fully articulated for the skeletal frame. the whole processes were taken 2 days (33 hours). the boiling maceration method for preparation of skeleton is very effective because restrain the chance of losing small bones, requires less expenditure, time and labor. it is also visible that the bone’s original color was preserved. extracted bones were divided into two parts, axial and appendicular skeleton. axial skeleton comprises the bones of skull, vertebrae such as-cervical, thoracic, lumbar and sacral vertebrae (fused to form synsacrum) and coccygeal vertebrae or caudal vertebrae then ribs, costal cartilages and breast bone or keel bone. in contrast, the appendicular skeleton comprises the forelimb or wing bone like scapula, coracoid, clavicle, humerus, radius-ulna and manus. on the otherhand hindlimb or pelvic limb comprises of pelvic girdle, femur, tibio-fibula and feet bone (metatarsal and digits). in adult pigeon no developed tarsal bone. therefore, this is the most efficient way for quickly preparing and presenting skeleton for use in studies of the avian skeletal system. keywords skeleton, pigeon, effective, boiling maceration introduction skeletons serve as informative equipment in veterinary and medical education, exhibits in museums, hunting trophies, and an important part of scientific research materials (kempa, 2016). a skeleton is usually a rigid supportive or protective structure or framework of an organism that supports the rest of the body and facilitates movement (merrium-webster, 2021). the more delicate skeletal system of vertebrates is internal and is composed of mainly by connective tissues. this includes bone and the various fibrous substances that form the joints, connect bone to bone, bone to muscle, enclose muscle bundles and attach the internal organs to the supporting structure (sidnie, 2019). this skeleton is divided into two parts for study purposes: axial and appendicular. in which axial skeleton formed by the bones of skull, vertebral column, ribs and sternum. on the other hand, the main component of appendicular skeleton are limbs (fore and hind limbs) (ghosh, 2016). however, bird skeleton is less differing from the other vertebrates. birds have a lightweight skeleton made of mostly thin and hollow bones. the keel-shaped sternum (breastbone) is where the powerful flight muscles attach to the body. birds have a smaller total number of bones than mammals or reptiles. the significance of bird skeleton preparation to ornithology study and biodiversity preservation. avian skeleton preparation is basic need for gross anatomical study. the skeleton of birds is crucial for study on a variety of topics, including phylogenic studies, age and growth assessments, and functional morphology (bemis et al., 2004; burke and feduccia, 1997; olson 1973). skeleton are helpful for studying evolutionary morphology and identifying animals from archeological sites. skeleton preparation serves a precious opportunity to gain anatomical knowledge of bones, muscles, tendons, nerves, etc (takeshi, 2010). skeleton collections are necessary for various forensic applications, including identifying animal and bird carcasses that have been illegally stolen or bones that have been used as evidence in other crimes (olson, 2003). as a result, the necessity of bird skeleton preparation is increasing rapidly. for academic purposes different bird skeletons have also been prepared for anatomical and comparative studies. the main drawback of skeleton preparation is time consuming, with maceration often being the most time-consuming and difficult phase. skeleton preparation is always a drawn-out, laborious, and delicate procedure requiring numerous processes. several maceration methods, such as, burial maceration, cold water maceration, hot water maceration, insect maceration, chemical maceration, and enzymatic maceration are available (raghavan, 1964; gofur, 2010). veterinary educational institutions do not undertake skeleton preparation due to procedural 1 department of anatomy and histology, bangabandhu sheikh mujibur rahman agricultural university, gazipur, bangladesh 2 department of anatomy and histology, bangladesh agricultural university, mymensingh, bangladesh * corresponding author’s e-mail: njannat@bsmrau.edu.bd https://doi.org/10.54536/ajlsi.v2i1.1269 https://journals.e-palli.com/home/index.php/ajlsi pa ge 27 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 26-32, 2023 complexities (mahapatra et al., 2018). the main aim of this study was to describe the total time required for the skeleton preparation procedures as well as how quickly the axial and appendicular skeleton of bird species could be processed and assembled.the findings of this research will serve as a guide in the skeleton preparation of a bird species within a concise period of time and contribute to the knowledge for building up anatomy museum. through anatomy (osteology), one learns about the bones in anatomical and physiological ways. this present study was undertaken for better understanding of avian anatomy by preparation of skeleton in a quick and effective methods so that we can treat the bird species in better ways. materials and methods a total of 2 matured pigeons (columba livia), aged 8 months old, were purchased from local market in gazipur sadar and transported to the department of anatomy and histology, faculty of veterinary medicine and animal science, bangabandhu sheikh mujibur rahman agricultural university, gazipur, bangladesh. all the pigeon was possessed good health and devoid of any external deformities certified by the registered veterinarian. all kinds of precautions were taken during skeleton preparation such as use of rubber gloves during preparing a carcass and care was taken to avoid stick injuries according to baker et al. (2003). the materials used for skeleton preparation were: (1) wires of various gauges (2) drill machine (3) soda (sodium carbonate, na2co3) (4) 10% bleaching solution (5) wooden platform (6) good quality sticking materials (fevicol) (7) varnish. the methods involved in skeleton preparation were: (1) birds preparation (2) skinning, evisceration and defleshing of carcass (3) carcass maceration (4) sequential arrangement of bone and bone drying (5) construction of skeletal frame and bone varnishing. birds preparation before slaughter all the pigeons were kept 6hours fasting condition. then, mgso4 was injected directly into heart. the iacuc approved these euthanasia methods of killing animals for humane killing of animals and recommendations of the american veterinary medical association (avma) panel on euthanasia, 2000 and in accordance with humane euthanasia defined by the federal animal welfare act (54 fr 36112-36163) (source: iowa) skinning, evisceration and de-fleshing of carcass skin was removed by cutting between the skin and muscle with a scalpel while pulling the skin. then maximum amount of muscles, adipose tissue and all visceral organs were removed with the aid of scalpel, blades, forceps, scissors and knife up to the bones were visible. carcass maceration the bones of the pigeon were heated by using normal heater in 3% solution of soda water (sodium carbonate, na2co3) with anionic surfactant (detergent) to over 80ºc for 2 hours in a metal drum regarding to the concept from baker et al. (2003) and van cleave (2010). soda (sodium carbonate, na2co3) water solution was used for proper and complete digestion of muscles (gofur and khan, 2010). then it was kept 2 hours for cooling and after that the remaining fleshes, tissues and ligaments on the boiled bones were removed with knives. then all the bones were rinsed with clean tap water and dip into 10% bleaching water solution for 1 hour to prevent further growth of microorganisms (musa et at., 2015). sequential arrangement of bone and bone drying axial skeletal bones such as, skull, vertebrae, ribs and keel bone were arranged in sequential order and protected with the help of fine stainless steel wire. appendicular skeletal bones such as, forelimb and hindlimb bones were also placed in order in a steel plate. then all the bones were kept in sunlight for a period of 8 hours for complete sundry. construction of skeletal frame and bone varnishing the skeletal frame was constructed in a wooden stage of 12-inch length and 10-inch breadth. all the bones were articulated in a proper way to form a bird skeleton. construction of axial skeleton all the vertebrae such as, cervical, thoracic, lumbar and sacral vertebrae (fused to form synsacrum) and caudal vertebrae were articulated with the aid of fevicol glue in a sequential order. then, occipital bone of skull and cranial part of the atlas were drilled and tied by using stainless steel wire. all pair of ribs were attached with their corresponding vertebrae to sternum by using glue according to the concept of van cleave (2010). construction of appendicular skeleton the forelimb (wing) and hindlimb (leg) were articulated with the axial skeleton with the help of stainless steel wire. firstly, wing skeleton bones namely, scapula, coracoid, clavicle, humerus, radius-ulna and manus were articulated sequentially by using adhesive glue. then hindlimb bones namely, hip bone (consists of ilium, ischium and pin bone), femur, tibia -fibula and feet bone (metatarsal and digits) were tied up chronologically with the aid of fevicol glue. the skeletal framework was positioned in a wooden platform of required dimension according to the size of the animal for display and gross anatomical study. varnishing of bone was done by using combined mixture of thinner and varnish to give the bones shiny appearance. results the bones expelled from chemical maceration process https://journals.e-palli.com/home/index.php/ajlsi pa ge 28 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 26-32, 2023 table 1: steps involved with time requirement for preparation of pigeon skeleton. sl. no. steps involved for skeleton preparation time (hours) 1 birds preparation 7 hours 2 skinning, evisceration and de-fleshing of carcass 3 hours 3 carcass maceration 5 hours 4 sequential arrangement of bone and bone drying 10 hours 5 construction of skeletal frame and bone varnishing 8 hours comprised of the axial and appendicular skeleton. all the bones appeared whitish and intact. the axial skeleton comprised of the bones ofskull, vertebrae such ascervical, thoracic, lumbar and sacral vertebrae (fused to form synsacrum) and coccygeal vertebrae or caudal vertebrae, ribs, costal cartilages and breast bone or keel bone. on the other hand, the appendicular skeleton comprised of the forelimb or wing bone as like scapula, coracoid, clavicle, humerus, radius-ulna and manus, on the contrary hindlimb or leg bone comprises of femur, tibio-fibula and feet bone (metatarsal and digits). the time needed by each of the steps of pigeon skeleton preparation technique is shown in table 1. description of pigeon skeletal system there have been a number of modifications of the bones found in birds that enhance the ability to fly. axial skeleton this skeleton comprised of the bones ofskull, vertebrae such as-cervical, thoracic, lumbar and sacral vertebrae (fused to form synsacrum) and coccygeal vertebrae or caudal vertebrae, ribs, costal cartilages and breast bone or keel bone. skull the skull is divided into two components: 1) rounded cranium (neurocranium) and 2) conical facial region (viscerocranium) (figure 1). these two regions were easily identified by seeing two large orbits or openings into which the eyes fit. two very thin bones, the sphenoid and ethmoid bones, together form the very thin septum that separates these orbits. since the volume of the brain increases relatively little with respect to body size, smaller birds have a comparatively larger head than bigger species. bones of the cranium are: i) occipital bone (unpaired basioccipital bone, unpaired supraoccipital bone and exoccipital bones) ii) sphenoid bone (unpaired basisphenoid bone, laterosphenoid bone and unpaired parasphenoid bone) iii) squamosal bone iv) parietal bone v) frontal bone vi) ethmoid bone and viii) lacrimal bone bones of the face are: the bones of the facial portion of skull are –i) premaxillary bone ii) nasal bone iii) palatine bone iv) maxillary bone v) jugal/quadratojugal bone vi) vomer (usually unpaired) vii)pterygoid bone viii)quadrate bone ix) mandible (unpaired) and x) hyobranchial apparatus (unpaired). the conformation of the facial skeleton is influenced considerably by the shape and mobility of the beak. the rostral tip of the face is formed by the premaxillary bone having palatine process, frontal process and maxillary process (figure 1a). the nasal bone is bounded by the frontal and maxillary processes of the premaxillary bone. the palatine bone forms the continuation of the incomplete hard palate. the relatively small maxillary bone joins the premaxillary bone to form the upper beak’s short, caudal terminal portion. the mandible is a laterally flattened bone that only contributes to the vertical dimensions of the head. its six pairs of fused bones form an acutely angled, caudally open structure (figure 1b). the quadrate bone plays a key role in the movement of the maxillopalatine apparatus. it articulates with the mandible to form the principal joint of the lower jaw. vertebrae the vertebral column has 38 bones and is divided into five segments – the cervical vertebrae, the thoracic vertebrae, the lumbar vertebrae, the sacral vertebrae and the coccygeal vertebrae. the vertebral column is often described by way of the vertebral formula that is: c12, t7, l+s14 & c5 = 38 cervical vertebra the cervical vertebral column is typically s-shaped in avian species. there are 12 cervical vertebrae in the pigeon (figure 1d). the first cervical vertebra (atlas) is a small and ringshaped bone with a dorsal arch and a ventrally located body (figure 1e). a recess on the cranial surface of the body, the condyloid fossa forms the articular surface for the occipital condyle of the occipital bone. the dorsal surface of the body bears the articular surface for the dens of the axis. the second cervical vertebra (axis) is slightly larger than the atlas (figure 1f). its elongated body (corpus axis) articulates caudally with the third cervical vertebra. its cranial surface possesses a small process; the dens forms a joint with the atlas. on subsequent vertebral bodies and the articular surface is saddle-shaped. thoracic, lumbar, sacral and caudal vertebrae the seven thoracic vertebrae bear a pair of complete ribs consisting of a dorsal vertebral rib articulating with the vertebra and the ventral sternal rib, which in turn articulates with the breastbone (sternum). each vertebral https://journals.e-palli.com/home/index.php/ajlsi pa ge 29 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 26-32, 2023 rib bears a flat, backward-pointing spur, the uncinate process, which is characteristic of birds. total number of lumbar and sacral vertebrae are 14 in number. the seventh thoracic, lumbar, sacral and first caudal vertebrae are closely fused to form the synsacrum, to which the pelvic girdle is attached (figure 1g). posterior to the synsacrum is a series of free fused caudal vertebrae form the pygostyle, which supports the tail feathers (figure 1h). ribs there are seven pairs of ribs articulate with the thoracic vertebrae. the first, second do not articulate with the sternum. the third to the sixth have two segments, the vertebral segment articulate with thoracic vertebra, and sternal segment articulate with breast bone or sternum. all except the first and last have uncinate (meaning hooked or bent) processes that project backward over the outer surface of the next rib and connect to it by a ligament, which adds strength to the thoracic cavity (figure 1l). sternum the sternum is an extensive bone looks like quadrilateral plate exhibiting a large ventrally directed crest known as keel or carina, which serves as the bony surface for the origin of the major flight musclespectorals and supracoracoideus (figure 1m). figure 1: diagram of a pigeon skeleton. yellow border indicates the skull. (a) premaxillary bone, (b) mandible, (c) occipital bone, (d) cervical vertebrae, (e) atlas, (f) axis, (g) synsacrum, (h) pygostyle, (i) ilium, (j) ischium, (k) pubis, (l) ribs, (m) keel or breast bone, and (n) acetabulum. appendicular skeleton consists of forelimb or thoracic limb and hind limb or pelvic limb the forelimb or thoracic limb the skeleton of the forelimb or thoracic limb comprises the bones of the pectoral girdle and the bones of the wing. the fully developed avian pectoral girdle consists of thescapula, coracoid, clavicle, furcula or wishbone (figure 2). the scapula is sabre-shaped, narrow, thin and slightly curved, unlike the shoulder blade of other animals (figure 2.1). the glenoid process of the scapula laterally completes the articular surface for the head of the humerus. craniomedially the scapula is joined to the clavicle. the long caudal extremity is slightly curved and lies approximately parallel to the vertebral column, extending almost to the ilium. the coracoid bone is the strongest bone in the pectoral girdle and rod-shaped bone that connects the cranial border of the sternum with the shoulder joint (figure 2.3). the clavicle is a thin, curved rod like bone. its proximal part connects with the cranial extremity of coracoid and scapula. an osseous union joins the two clavicles to form the ‘wishbone’ (furcula) (figure 2.2). the bones of the wing consist of-humerus, radius-ulna and manus (carpus, metacarpus and digits). humerus, radius-ulna the humerus is a largest wing bone with an ovoid head for articulation with the scapula, coracoid and clavicle (figure 2a). the pneumatic foramen is located at the proximal end of the humerus allows the invasion of the clavicular air sac which pneumatizes the interior of this large bone. the forearm bone, ulna (figure 2b) is thicker and longer but radius (figure 2c) is thinner and shorter that lies laterally to the ulna. the large space between the ulna and radius is called the interosseus space. carpus, metacarpus, and digits (manus) the manus or hand, consists of the carpus, metacarpus and the digits. the carpus of an adult pigeon having two bones, namely-the ulnar and radial carpal bone articulate with distal end of ulna and radius bone respectively (figure 2d). the metacarpus is considered as a single bone that is produced by the union of three elements (alular, major and minor metacarpal bone). in the adult pigeon, these three elements are fused with the distal row of carpal bones, giving rise to the carpometacarpus (figure 2e). at the proximal end of the carpometacarpus, the carpal trochlea articulates with the carpal bones. the alular metacarpal bone bears the articular surface in figure 2: diagram of a pigeon skeleton showing the forelimb. (a) humerus, (b) ulna, (c) radius, (d) carpals, (e) metacarpals, (f) digits, (1) scapula, (2) clavicle, and (3) coracoid https://journals.e-palli.com/home/index.php/ajlsi pa ge 30 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 26-32, 2023 the distal end for the alular digit. the major metacarpal and the smaller minor metacarpal bone fused distally and extended towards the digits. in the distal extremity, each metacarpal bone bears a surface for articulation with the phalanges. so, recognizable digits are three (3) in number (figure 2f). pelvic limb or leg the pelvic limb is divided intopelvic girdle or hip bones and the leg. the pelvic girdle or hip bones consists of three bones ilium, ischium and pubis or pin bones (figure 1). the bones of the leg arefemur or thighbone, tibio-fibula and pes (tarsus, metatarsus and digits or toes) (figure 3). the pelvic girdle the ilium is large bone which fused to the synsacrum to provide strength and rigidity (figure 1i). the ischium is much smaller and is continuous with the ilium (figure 1j). an opening is present in ischium for the passes of sciatic nerve. the pubis is a narrow strip of bone that runs along the border of the ischeum to which it is joined for a short distance only. the free posterior end projects backward slightly beyond the ischeum to form the pin or pubic bones (figure 1k). the ilium, ischium and pubis which all meet at a deep concavity called the acetabulum, into which the head of the femur articulate (figure 1n). the leg consists of-femur or thighbone, tibiotarsus, fibula and pes (tarsus, metatarsus and digits or toes). femur the femur is a typical cylindrical shape long bone with a slight cranial curvature (figure 3a). the proximal extremity has a prominent head that articulate loosely into the acetabulum. the distal extremity has a deep pulley shaped articular surface for the patella (knee cap). two convex condyles, namely, lateral and medial, are present in the distal end of femur. the lateral surface of the lateral condyles articulates with the head of fibula. the tibia is a much longer bone than the fibula and is much thicker at the proximal end than it is at the distal end (figure 3c). the proximal row of tarsal bones is fused to the distal end of the tibia. for that reason, it is known as tibiotarsus bone. the fibula is greatly reduced bone with a slender spicule (needle-like) structure and has a flattened head for attachment to the proximal head of the tibia (figure 3b). pes (tarsus, metatarsus and digits or toes) there is no independent tarsus bone in the adult pigeon. the matured metatarsus is a long bone formed by the union of the second, third and fourth metatarsal bones (figure 3d). most pigeon have four digits or toes – three facing forwards (figure 3e) and one facing back (figure 3f). the basic number of phalanges on the toes are two, three, four, and five, respectively, one more than the number of the toe. each toe having one claw. so, number of claw are eight (8). claw is convex dorsally and concave ventrally (figure 3g). figure 3: diagram of a pigeon skeleton showing hindlimb. (a) femur, (b) fibula, (c) tibia, (d) metatarsal, (e) forward digits, (f) backward digits, and (g) claw discussion skeleton is the main framework of hard structures that supports and protects the soft structures of the body (getty, 1975). it is composed of bones, cartilage and ligaments which maintain the body’s shape amenability and locomotion (ghosh, 2006). skeleton preparation is one of the most important part for anatomical study. in this study, we reveled total duration for the preparation of pigeon skeleton took 2 days which is short period compared to the 18 days-2 hours both in pigeon and squirrel, 5 months-6 days-6 hours in deer, and 10 months-11 days-12 hours-30 minutes in crocodile (archana, 2018). now a day, veterinary educational institutions do not take responsibility for the preparation of skeleton due to procedural complication because different methods of skeleton preparation need different time (olson, 2003). oliveira (2018) claims that biological maceration utilising fly larvae and cold water did not produce a good outcome, leading to fragile bone. in this experiment we used 2 matured pigeon aged 8 months old. according to the concept of baker et al., (2003) all kinds of care and precautions were taken to avoid injuries before skeleton preparation. after sacrificing animals, we did not use any fixative solution because it may affect the bone tissue (takeshi, 2010). in order to quickly prepare the skeleton, skinning and defleshing was done as much as possible. then, 3% solution of soda water (sodium carbonate, na2co3) with anionic surfactant (detergent) was used for carcass maceration (baker et al., 2003 and van cleave, 2010). gofur and khan, (2010) stated that soda (sodium carbonate, na2co3) water solution is appropriate for proper and complete digestion of muscles. carcass maceration can be done by remaining the carcass in a pit for 1-2 months, according to the size of the animals and birds (raghavan, 1964). however, it is time consuming and have a chance to loose small bones. the odor and https://journals.e-palli.com/home/index.php/ajlsi pa ge 31 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(1) 26-32, 2023 discoloration of the skeleton are often the drawbacks of employing the fly larvae maceration method (auricchio & salomão, 2002). then the bones of pigeon were boiled in 3% solution of soda water for 2 hours because too much boiling can crack the hard tissue (takeshi, 2010). after boiling, removal of excess flesh as well as bleaching wash were done to avoid further decomposition of bone by microorganisms (gofur and khan, 2010). thereafter, the bones were dried completely by sun rays. afterwards, the bones were articulated sequentially to form a skeleton and this skeleton was positioned on a wooden stage with the help of stand and thick wire (figure 1). finally, the skeleton was labelled and housed in a display case (musa et al., 2015). the total number of bones evicted was 206. the axial and appendicular skeleton bones help in locomotion and protection of the brain, spinal cord and other internal organs of the body respectively (sturtz, 2012). in this experiment, all the extracted bones of the skeletal system were appeared whitish and intact as reported by kyle and jesse (2018). the structure of skeletal system of the pigeon is similar to previous descriptions on the domestic fowl (getty, 1975; konig, 2009). the axial skeleton comprised of the bones ofskull, vertebrae such as-cervical, thoracic, lumbar and sacral vertebrae (fused to form synsacrum) and coccygeal vertebrae or caudal vertebrae, ribs, costal cartilages and breast bone or keel bone. as opposed to, appendicular skeleton comprises of the bones of forelimb and hindlimb. forelimb or thoracic limb bones arescapula, coracoid, clavicle, humerus, radius-ulna, carpus, metacarpus and manus. hindlimb bones are pelvic girdle, femur, tibio-fibula, tarsal, metatarsal and toes. description of the bone are similar according to the concept from konig, (2009). in the present study, we followed the boiling maceration method for preparation of pigeon skeleton is swift and safe compared to the report of kyle, (2018), whose method involved burying of the dead animal for 60 days and handling of decomposed carcass using dangerous chemicals such as chlorine bleach and fumes, which is time consuming and not safe. conclusion our major goal was to prepare the skeleton in a practical and economical way. this approach is also simpler, less costly, and time-saving while maintaining the integrity of all the bones. this technique required a total of 33 hours, or 2 days, to prepare the skeleton of a pigeon. this is the shortest time ever for skeleton preparation and study. however, this might change depending on the size of the animals and birds. acknowledgement the authors are grateful to the department of anatomy and histology, faculty of veterinary medicine and animal science in bangabandhu sheikh mujibur rahman agricultural university, salna, gazipur, bangladesh for imparting the opportunity to conduct research to encourage avian skeleton learning among students of veterinary medicine and animal science. so, i would like to extend my gratitude to undergraduate students for their enormous work. references archana, m., satish, k.p., amarpal, & pawde, a.m.(2018). characterization of skeleton preparation in avian, mammalian, and reptiles species with empirical methodology. animal science reporter, 11(1), 01-09. auricchio, p. & salomão, m.g. 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(2010, february). how to prepare a chicken skeleton? http://scienceprojectideasforkids.com https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 1 american journal of life science and innovation (ajlsi) diversity and relative abundance of entomofauna of four ecologically different areas of chittagong university campus, bangladesh md. ikram ansar tuhin1, munira nasiruddin1, zannatul nayem1* volume 2 issue 2, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i2.1380 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: march 11, 2023 accepted: may 02, 2023 published: may 04, 2023 the present study was conducted to find the relative abundance and diversity of insects in the four areas of the chittagong university (cu) campus from january 2018 to december 2018. the entire study found six orders odonata, orthoptera, coleoptera, lepidoptera, diptera, and hymenoptera. three families under odonata, two under orthoptera, four under coleoptera, five under lepidoptera, five under diptera, and three under hymenoptera were collected from the four study spots. among them, fifteen species of odonata, seven species of orthoptera, seven species of coleoptera, thirty-five species of lepidoptera, five species of diptera, and four species of hymenopteran were identified. the highest number of insects (478) were collected in january 2018, whereas the lowest number of insects (404) were collected in august 2018. the highest abundance (1598) of insects was found in spot 1 (ground area) and the lowest (990) in spot 2 (hilly area). lepidoptera (2688) was the most dominant order in the four studied spots, followed by odonata (1453), orthoptera (505), coleoptera (223), diptera (202), and hymenoptera (186). during the study period, the highest species richness was observed in spot 3 (2.61 ± 0.01) and lowest in spot 2 (2.03 ± 0.008); the highest species diversity was observed in spot 1 (1.36 ± 0.02) and lowest in spot 2 (1.09 ± 0.01); and the highest species evenness was observed in spot 1 (0.53 ± 0.006) and lowest in spot 2 (0.47 ± 0.006). compared with the previous study, it can be concluded that the species diversity and abundance of odonata, orthoptera, and lepidoptera were increased, whereas the diversity and abundance of coleoptera and hymenoptera were decreased. the abundance and diversity of insects depended on seasonal fluctuation and ecological and environmental conditions. keywords entomofauna, ecological area, insect diversity, insect abundance, diversity indices introduction insects have been an immensely successful animal group, with possibly 2-10 million species on earth today. although evidence suggests that many insects died out at the end of the cretaceous, most have survived many geological events over the last few millions of years. this is now changing, with the human impact estimated to threaten the survival of a quarter of all insect species. in the agricultural ecosystem, biodiversity is important for food production and other ecological services, including the recycling of nutrients, regulation of microclimate and local hydrological processes, suppression of undesirable organisms, and detoxification of harmful chemicals. so, the task is now urgent to conserve this immense variety of life which is so vital to many ecosystem functions. threats are many and varied, with habitat destruction the worst threat, especially in the tropics where most insect diversity lives. other threats include invasive alien organisms, certain biological control practices, the use of pathogens, genetically modified crops, and global climate change. chittagong university (cu) campus is enriched with diverse insect fauna. the present investigation was undertaken to study the abundance and diversity of insects in four ecologically different sites of the cu campus, which were also widely separated by distance. the insects included in the study belonged to the orders odonata, orthoptera, coleoptera, lepidoptera, diptera and hymenoptera. several works on insect diversity and abundance abroad and in our country. amin et al. (2018) research on the functional and group abundance of insects on eggplant. in total 488 insects were collected from the eggplant field, which belonged to 20 species in 21 families and 10 orders. the abundance, richness and diversity of pest, predator, pollinator and other categories of insects differed significantly and the pest revealed the highest abundance and richness compared to others. rahman et al. (2017) studied the abundance and diversity of beneficial insect and spider species in the rice ecosystem in the sylhet region. many beneficial insect and spider species were found abundant at all growing stages in both rice fields. siregar et al. (2016) studied the diversity and abundance of insect pollinators in three different agricultural land uses, i.e., oil palm plantation, rubber plantation, and jungle-rubber in jambi, sumatera. a total of 497 individuals of insect pollinators were collected by them which belonged to 43 species in three orders (hymenoptera, diptera, and lepidoptera). siddiki (2015) studied the insect diversity and composition during the wet and dry seasons in three forest types of johor, malaysia. they focused on ten common insect orders. a total of 929 insects were found in the study of balakrishnan et al. (2014) on diversity of some insect fauna in different coastal habitats of tamil nadu, southeast coast of india. zou (2014) focused on two distinct taxa: ground beetles (coleoptera: carabidae) and geometrid moths (lepidoptera: geometridae) to study the diversity patterns along environmental gradients in the temperate forests of northern china. hong et al. 1 department of zoology, university of chittagong , bangladesh * corresponding author’s e-mail: zannatul.nayem29@gmail.com https://doi.org/10.54536/ajlsi.v2i2.1380 https://journals.e-palli.com/home/index.php/ajlsi pa ge 2 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 1-11, 2023 (2012) worked on insect diversity of mt. oseosan where a total 446 species of 108 families belonging to 11 orders were identified. park et al. (2012) surveyed insect diversity of yeonpyeong-do island in korea and the total of 209 species of 65 families under 10 orders were identified. aim and objectives 1. to study the morphological and structural characteristics of insect species from the four selected sampling sites in cu campus. 2. to investigate the abundance of identified species of the selected sampling sites during one year study period. 3. to investigate the variation in abundance among the species of the four selected spots. 4. to investigate the shanon-weiner diversity of the orders in the sampling sites. 5. to investigate community dominance of the identified species in the sampling sites. 6. to know and use fundamental concepts and information in areas of abundance and diversity of insects. methodology the present study was conducted in the cu campus, which is situated in close proximity of sitakunda hill forest. the area lies between about 22o27’30’’ and 22o29’0’’ north latitudes and 91o46’30’’ and 91o47’45’’ east longitudes. the campus is dominated by hills, valleys, creeks and streams, lakes, crop fields, and grass and fallow lands. this is the junction of hills and plains adorned with hilly streams. these features constitute a suitable habitat for insect diversity. approximately 72% of the total land is constituted by hillocks, which are 15-90 m high. the rest of the land is valley and/or plain. the campus consists of many secondary forests with woody trees, flowering plants, fruit plants, medicinal plants, herbs, and shrubs. the campus area has received great attention in various aspects of biodiversity and species richness, particularly for insects, birds, and wild mammals. during the study period, samplings were done for one year, from january-2018 to december-2018. a collection of data was done once in a month from four ecologically different areas in the four-terminal area (north, east, south and west) of cu campus (spot-1: garden area, spot-2: hilly forest area, spot-3: botanical garden with hilly stream, spot-4: pond area with agricultural crop field). figure-1 shows the four studied spot in a map. spot1 was located at the northern side of the campus. there was beautiful flower garden in front of the agricultural field which consisted of many flowering plants likegolap (rosa sinensis), gada (tagetes erecta), togor (tabernaemontena divaricata), hasna-hena (cestrum nacturnum), kamini (murraya paniculata), belly (jesminus sambac), herbs, shrubs, and few tall trees such asmahagoni (sweitenia mahagoni), teak (tectona grandis), neem (azadirachta indica), jarul (lagerstroemia speciosa), krishnochura (delonix regia), etc. plenty of small plants were available, especially mikania micrantha, asparagus racemosus, and so on. some wild flowering plants, including lantana camara and ficus benghalensis were also found in this semi-hilly area. spot2 was the hilly forest area on the campus’s eastern side. there was a dense growth of bush, tall and short trees, herbs, shrubs, and also wild flowering trees. many kinds of valuable trees such aseucalyptus (eucalyptus citriodora), jarul (lagerstroemia speciosa), bamboo (bambusa sp.), cycas (cycas pectinata), hortoki (terminalia chebula), polash (butea monosperma), nagessor (mesua nagassarium), teak (tectona grandis), debdaru (polytthia longifolia), krishnochura (delonix regia), garjon (dipterocarpus turbinatus), akashmoni (accacia racemosus) were seen throughout the entire area. wildflowers and grassland were also found in this hilly forest area. spot-3 was an ideal botanical garden established on the southern side beside a hilly stream with more than 10,000 plants of about 400 species comprising over 150 families of both exotic and indigenous species of timber, medicinal, fruits, spices, beverage, latex, orchids, novelty etc. have been preserved or are growing naturally and used as experimental materials. many kinds of flower plants such asjoba (hibiscus rosa-sinensis), chondromollika (chrysanthemum coronarium), golap (rosa sinensis), gada (tagetes erecta), togor (tabernaemontena divaricata), hasna-hena (cestrum nacturnum), rongon (ixora coccinea), bakul (mimusops elengi), gondhoraj (gardenia jasuninoides), kamini (murraya paniculata), dahlia (dahlia), belly (jesminus sambac), rojonigondha (polianthes tuberose) etc. were planted in the garden which increased the beauty of the garden. spot-4 was situated at the western and south-western parts of the campus area. the spot was occupied by wetland and crop fields. the rice crop fields were occupied with seasonal rice (oryza sativa) plants i.e., aus, aman and boro in the three stages i.e., nursery, transplanting and ripening. in this area, rice was extensively cultivated throughout the year. insects were collected once in a month from each sampling location mainly in the morning for one hour. sweeping process in each collection accomplished the collection of samples. insect sweeping net was used for this purpose. hand-picking collection was also followed for the small insects occupied in the leaf, grass as well as in the soil. the captured insects were chloroformed, killed, sorted, stretched, mounted, labeled, and preserved following general entomological procedures (borror et al. 1989). a total of 5257 insect specimens were collected during the study period. the insects were identified with the help of taxonomic keys and characters. firstly, specimens were identified up to family level. then each specimen was examined up to genus and species level by comparison with previously identified specimens preserved at the department of zoology, university of chittagong. the collected insect specimens were classified up to family following borror et al. (1989), richards and davies (1977), and then up to generic level. identification was made following bingham (1908) and talbot (1975) for lepidopterans, kirby (1914) for orthopterans and hymenopterans, maulik (1919) for coleopterans, fraser (1936) for odonates, van emden https://journals.e-palli.com/home/index.php/ajlsi pa ge 3 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 1-11, 2023 (1965) for dipterans, richards and davies (1977) for butterflies, dragonflies, damselflies and beetles and burton (1974). help from internet sources was also taken for identification. figure 2: the workflow figure 1: map of the study area showing the study spots shannon-weiner species diversity indices and community dominance were calculated to know the abundance and diversity status of the insect fauna of the cu campus area. https://journals.e-palli.com/home/index.php/ajlsi pa ge 4 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 1-11, 2023 result and discussion the present study deals with the faunistic study of the insect community in cu campus. the study of the abundance and diversity of insects helps to gather information on the statistics of entomofauna. sánchezbayo, f. and wyckhuys, k. a. g. (2019) studied the entomofauna and found that over 40% of insect species were threatened with extinction. their findings showed that habitat loss by conversion to intensive agriculture was the main driver of the decline, while agrochemical pollutants, invasive species and climate change were the additional causes. a total of 5257 insect specimens were collected during the study period. the collected specimens were primarily sorted into 6 orders. then they were identified into several different families within these orders. the orders are odonata, orthoptera, coleoptera, lepidoptera, diptera and hymenoptera. 3 families under order odonata, 2 families under order orthoptera, 4 families under order coleoptera, 5 families under order lepidoptera, 5 families under order diptera and 3 families under order hymenoptera were identified. for each order, families, genus and species a diagnosis is given. the collection included 73 species which are listed below: table 1: list of the insects collected during the study period order family scientific name common name odonata libellulidae orthetrum sabina (drury, 1770) green marsh hawk orthetrum pruinosum (burmeister, 1839) crimson-tailed marsh hawk neurothemis tullia (drury, 1773) pied paddy skimmer neurothemis fulvia (drury, 1773) fulvous forest skimmer rhyothemis variegata (linnaeus, 1763) common picturewing rhodothemis rufa (rambur, 1842) rufous marsh glider brachydiplax chalybea (brauer, 1868) rufous-backed marsh hawk crocothemis servilia (drury, 1770) scarlet skimmer brachythemis contaminata (fabricius, 1793) ditch jewel coenagrionidae ceriagrion coromandelianum (farbicius, 1798) coromandel marsh dart mortonagrion aborense (laidlaw, 1914) n/a agriocnemis lacteola (selys, 1877) milky dartlet platycnemididae onychargia atrocyana (selys, 1865) black marsh dart copera marginipes (rambur, 1842) yellow bush dart copera vittata (selys, 1863) blue bush dart orthoptera acrididae dissosteira carolina (linnaeus, 1758) road-duster oxya velox (fabricius, 1787) paddy field grasshopper oxya chinensis (thunberg, 1825) short horned grasshopper oxya hyla hyla (serville, 1831) rice grasshopper paratettix sp. (bolivar, 1887) creek pygmy grasshopper atractomorpha sp. (saussure, 1862) vegetable grasshopper gryllidae gryllus pennsylvanicus (burmeister, 1838) field cricket coleoptera chrysomelidae aulacophora frontalis (baly, 1888) pumpkin beetle aulacophora indica (gmelin, 1790) orange pumpkin beetle aspidomorpha sanctaecrucis (fabricius, 1792) golden tortoise beetle aspidomorpha miliaris (fabricius, 1775) green tortoise beetle coccinellidae menochilus sexmaculatus (fabricius, 1781) lady bird beetle carabidae harpalus distinguendus (duftschmid, 1812) ground beetle tenebrionidae eleodes hispilabris (say,1824) darkling beetle lepidoptera papilionidae papilio helenus (linnaeus, 1758) red helen papilio polytes (cramer, 1775) common mormon papilio demoleus (linnaeus, 1758) lemon butterfly papilio clytia (linnaeus, 1758) common mime graphium agamemnon (linnaeus, 1758) green-spotted triangle pieridae leptosia nina (fabricius, 1781) psyche eurema hecabe (linnaeus, 1758) common grass yellow https://journals.e-palli.com/home/index.php/ajlsi pa ge 5 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 1-11, 2023 catopsilia pomona (fabricius, 1775) lemon emigrant pieris canidia (evans,1912) indian cabbage white delias hyparete (wallace, 1867) painted jezebel cepora nerissa (fabricius, 1775) common gull appias libythea (swinhoe,1890) striped albatross nymphalidae athyma inara (westwood, 1850) himalayan colour sergeant junonia lemonias (linnaeus, 1758) lemon pansy junonia almana (linnaeus, 1758) peacock pansy junonia atlites (linnaeus, 1763) grey pansy junonia hierta (fabricius, 1798) yellow pansy junonia iphita (cramer, 1779) chocolate pansy ypthima baldus (fabricius, 1775) common five-ring lebadea martha (fabricius, 1787) knight mycalesis perseus (fabricius, 1798) common bushbrown polyura athamas (drury, 1770) common nawab hypolimnas bolina jacintha (drury, 1773) orienta great eggfly neptis hylas (moore, 1872) common sailor phalanta phalantha (drury, 1770) common leopard hypolimnas bolina bolina (linnaeus, 1758) sunda great eggfly parantica melaneus (cramer, 1775) chocolate tiger euploea core (cramer, 1780) common crow elymnias sp. (hubner, 1818) palmfly lycaenidae neozephyrus quercus (linnaeus, 1758) purple hairstreak castalius rosimon (fabricius, 1775) common pierrot spindasis sp. (tytler, 1915) rufous silverine hesperiidae notocrypta paralysos (fruhstorfer, 1911) banded demon baoris farri (moore, 1878) paintbrush swift telicota bambusae (moore, 1878) dark palm dart diptera sarcophagidae saracophaga sp. (meigen, 1826) true fly muscidae musca domestica (linnaeus, 1758) house fly drosophillidae drosophila melanogaster (meigen, 1830) common fruit fly tabanidae tabanus sp. (linnaeus, 1758) horse fly calliphoridae calliphora vicina (robineau-desvoidy, 1830) blue bottle blowfly hymenoptera sphecidae sphex lobatus (fabricius, 1775) digger wasp sceliphron violaceum (fabricius, 1775) fassorial wasp apidae xylocopa latipes (drury, 1773) tropical carpenter bee vespidae polistes sp. (latreille, 1802) paper nest wasp during the collection period, a total of 5257 insects were collected (table-2). january and april were the most dominant months. in january and april, 478 (9.10%) and 465 (8.85%) insects were collected where lepidoptera, odonata and orthoptera insects were mostly available. in february, march, july, november and december, 452 (8.60%), 454 (8.64%), 418 (7.95%), 430 (8.18%) and 416 (7.91%) the abundance was medium. in the month of august 404 (7.68%), september 412 (7.84%) and october 407 (7.74%) the abundance of the insects was lower. from 12 months observation (table-3) the highest abundance of insects was found in spot 1, medium abundance of insects was found at spot 2 and spot 4 and lowest abundance of insects was found in spot 3. from this study it is seen that the maximum numbers of insects were collected in the months of january (478), april (465) and may (462) when the temperature and weather condition was suitable and minimum number were collected in the months of august (404), october (407) and september (412) due to monsoon season. https://journals.e-palli.com/home/index.php/ajlsi pa ge 6 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 1-11, 2023 table 2: number of insects collected per order and month wise % composition of the collected insect orders during the study period. order ja n 20 18 fe b 20 18 mar 2018 apr 2018 may 2018 jun 2018 jul 2018 aug 2018 sep 2018 oct 2018 nov 2018 dec 2018 total % (m on th ) odonata 123 117 118 117 121 120 124 119 123 122 121 128 1453 27.64 o rt ho pt er a 56 43 35 44 45 46 39 36 39 42 45 35 505 9.61 c ol eo pt er a 27 21 17 20 20 13 16 17 18 17 20 17 223 4.24 le pi do pt er a 244 244 251 244 244 251 207 202 203 193 205 200 26 88 5 1. 13 diptera 16 13 15 23 17 17 12 14 16 14 25 20 202 3.84 h ym en op te ra 12 14 18 17 15 12 20 16 13 19 14 16 186 3.54 total 478 452 454 465 462 459 418 404 412 407 430 416 % (order) 9.10 8.60 8.64 8.85 8.79 8.73 7.95 7.68 7.84 7.74 8.18 7.91 table-3 shows the order wise distribution of the collected insects in the four study spots during the study period. butterflies (lepidoptera) were the most abundant group in spot 4 (834), spot 1 (762), spot 2 (655) and spot 3 (437). the lowest insect abundance order was hymenoptera which was found very few in number in different study spots, such as 62 from spot 4, 45 from spot 2, 43 from spot 3 and 36 from spot 1 respectively. amongst the orders, a total of 2688 lepidopterans, 1453 odonates, 505 orthopteran, 223 coleopterans, 202 dipterans, 186 hymenopterans were collected during the study period. of the total collected insects, maximum numbers of insects were collected from spot-1 and minimum numbers from spot-3. spot-1 had more insect species than were found at other sites. these differences could be the result of habitat and microhabitat differences among the sites. this spot consisted of several flower gardens, grassland and was also rich in small to big sized plants which were the natural breeding ground and food source of butterflies, bees, wasps and beetles which may explain the highest total number of species captured at this site. from the study, it was seen that lepidopteran, odonate, orthopteran and coleopteran species were most abundant in spot-1. thus, the large number of table 3: showing number of insects collected per order from the four spots and % composition of the collected insects during the study period. order spot 1 spot 2 spot 3 spot 4 total %(spot) odonata 431 313 322 387 1453 27.64 orthoptera 163 89 98 155 505 9.61 coleoptera 134 0 49 40 223 4.24 lepidoptera 762 655 437 834 2688 51.13 diptera 72 39 41 50 202 3.84 hymenoptera 36 45 43 62 186 3.54 total 1598 1141 990 1528 5257 % (order) 30.40 21.70 18.83 29.07 https://journals.e-palli.com/home/index.php/ajlsi pa ge 7 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 1-11, 2023 figure 1: percentage of the total insect found in each four spots lepidopteran and odonate insects was collected from spot-4 which was rich in grass areas. orthopteran and hymenopteran insects were mostly collected from spot-2 because of forest hilly areas. the large sized butterflies and other large sized insects were the inhabitants of forest and hilly areas and medium to small sized insects were the inhabitants of plain land and flower garden. the most significant characters were that the abundance of hymenopteran species very low in all spots and also in the study months. lepidopteran species were the most abundant insect order in almost all the months except july, august, september, october, november and december. hymenoptera is the lowest abundant insect order which was found in very low number except the month of march, april, july, and october. except hymenoptera, insects of all orders were found more or less in almost all the months. james (2011) while studying the diversity and abundance of insect fauna in rajshahi university campus illustrated that the highest number of insects was found in november 2008 followed by february 2009 and the lowest population of insects was found in january 2009. table – 4 showed the community dominance value with the dominant species found in each month in the studying spots.the community dominance value was 8.8% in the spot-1 in october, which was recorded as the highest value of the year. dissosteira carolina and ceriagrion coromandelianum were found in october. the second dominant species ceriagrion coromandelianum was used to calculate the community dominance value in the month october. in july, the most dominating insects were crocothemis servilia and aulacophora indica in the spot-1 and the community dominance value was 5.83%, which was the lowest value in the spot-1. the lowest value of community dominance indicated the diversity of species in the respective month. in spot-1, the most dominating species were oxya hyla hyla, crocothemis servilia and brachythemis contaminate. the second most dominating species was the copera spp. in spot-2, leptosia nina and papilio clytia were found in the month of august and the dominance value was 10.11%. the community dominance was low i.e., 8.41% in november. the most dominating insects were hypolimnas bolina jacinth and junonia iphita so, the most diversified month was november in spot-2. in spot-2, the most dominating species were athyma inara, pieris canidia and junonia atlites. the second most dominating species were junonia spp. and papilio spp. in spot-3, oxya hyla hyla and brachydiplax chalybea were found in the month of august and the dominance value was 12.79%. the community dominance was low i.e., 8.05% in january. the most dominating insect were copera marginipes and orthetrum pruinosum. so, the most diversified month was january in spot-3. in spot-3, the most dominating species was mortonagrion aborense. the second most dominating species were copera marginipes, rhyothemis variegate, copera vittata and brachythemis contaminate. in spot-4, oxya velox and mortonagrion aborense were found in the month of january and the dominance value was 8.61%. the community dominance was low i.e., 7.53% in june. the most dominating insects were catopsilia pomona and junonia almana. so, the most diversified month was january in spot-4. in spot-4, the most dominating species was ceriagrion coromandelianum. the second most dominating species were brachydiplax chalybea and orthetrum sabina. species richness varied from month to month. but throughout the year the species richness was highest in the spot 3 and lowest in the spot-2 (table-5). the highest species richness value was 2.66 found in spot 3 in november and lowest value was 1.97 which was also found in november in spot 2. the average highest species richness value was observed in the months of september and october (2.38 ± 0.14) and lowest value in january (2.31±0.13) and april (2.31 ± 0.11) during the study period. species richness value (average) fluctuated between 2.32 (±0.13) to 2.37 (±0.15) in the rest of the months (according to table-5). species diversity (h′) values in the twelve months study period in the four study areas was also given in table-5. the species diversity value was lowest in spot 2 throughout the year. the species diversity value was highest in the spot 1 in the month of january, february, march, june, july, september, november and december and in the spot 3 in the rest of the months. the highest species diversity was 1.45 in january which was found in spot 1 and in august which was found in spot 3. on the other hand, the lowest species diversity was 1.01 in march. the average highest species diversity value was observed in the month of october (1.31 ± 0.09) and the lowest value in the month of june (1.20 ± 0.05) during the entire study period. species diversity value (average) fluctuated between 1.21 (±0.08) to 1.29 (±0.13) in the rest of the months (according to table-5). species evenness values varied from month to month during the entire study period in the four study areas. the highest species evenness value was found in spot 1 in the months of january, february, march, june, september and december. the value was highest in july in spot 1 and 4 and in november in spot 1 and 3. the value was highest in the remaining months in spot 3. the lowest species evenness value was in spot 4 in the months of february https://journals.e-palli.com/home/index.php/ajlsi pa ge 8 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 1-11, 2023 table 4: monthly % community dominance (dc) of entomofauna of four ecologically different areas in cu campus collected from january 2018 to december 2018. spot-1 month most dominating species dc (%) jan oxya hyla hyla, rhodothemis rufa 7.33 feb agriocnemis lacteola, onychargia atrocyana 6.43 mar copera marginipes, rhyothemis variegata 6.57 apr brachythemis contaminate, ceriagrion coromandelianum 6.43 may oxya velox, brachydiplax chalybea 6.43 jun crocothemis servilia, copera vittata 6.57 jul crocothemis servilia, aulacophora indica 5.83 aug copera marginipes, agriocnemis lacteola 7.44 sep brachythemis contaminate, menochilus sexmaculatus 7.03 oct dissosteira carolina, ceriagrion coromandelianum 8.8 nov onychargia atrocyana, oxya hyla hyla 6.77 dec orthetrum sabina, menochilus sexmaculatus 7.09 spot-2 month most dominating species dc (%) jan athyma inara, junonia atlites 10 feb pieris canidia, junonia lemonias 9.47 mar pieris canidia, cepora nerissa 9.47 apr catopsilia pomona, graphium agamemnon 9.89 may junonia almanac, mycalesis perseus 9.38 jun athyma inara, hypolimnas bolina jacintha 9.38 jul junonia almanac, junonia atlites 9.18 aug leptosia nina, papilio clytia 10.11 sep catopsilia pomona, papilio helenus 9.57 oct graphium agamemnon, papilio demoleus 10 nov hypolimnas bolina jacintha, junonia iphita 8.41 dec leptosia nina, parantica melaneus 9 spot-3 month most dominating species dc (%) jan copera marginipes, orthetrum pruinosum 8.05 feb rhyothemis variegate, crocothemis servilia 8.64 mar brachythemis contaminate, mortonagrion aborense 8.43 apr tabanus sp., oxya velox 10.11 may rhodothemis rufa, ceriagrion coromandelianum 8.14 jun neurothemis fulvia, copera marginipes 8.75 jul ceriagrion coromandelianum, copera marginipes 10.47 aug oxya hyla hyla, brachydiplax chalybea 12.79 sep copera vittata, mortonagrion aborense 11.54 oct onychargia atrocyana oxya velox 11.69 nov rhyothemis variegate, mortonagrion aborense 11.84 dec brachythemis contaminate, copera vittata 11.11 spot-4 month most dominating species dc (%) jan oxya velox, mortonagrion aborense 8.61 feb onychargia atrocyana, ceriagrion coromandelianum 8.09 https://journals.e-palli.com/home/index.php/ajlsi pa ge 9 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 1-11, 2023 mar rhyothemis variegate, rhodothemis rufa 7.91 apr ceriagrion coromandelianum, copera vittata 7.59 may brachydiplax chalybea, crocothemis servilia 7.86 jun catopsilia pomona, junonia almana 7.53 jul eurema hecabe, paratettix sp. 7.89 aug papilio clytia, ceriagrion coromandelianum 8.33 sep oxya velox, brachydiplax chalybea 8.04 oct papilio demoleus, brachythemis contaminata 7.83 nov orthetrum sabina, copera marginipes 7.89 dec graphium agamemnon, orthetrum sabina 8.33 and may while it is lowest in spot 3 in december. the value was lowest in both spot 3 and 4 in june and spot 2 and 4 in may. the lowest value was found in spot 2 for the rest of the month. the highest value was 0.56 and the lowest value was 0.43, found in spot 2 in march. the average highest species evenness value was observed in october (0.52 ± 0.02) and the lowest value in the month of march (0.48 ± 0.03) during the entire study period. species evenness value (average) fluctuated between 0.49 (±0.03) to 0.51 (±0.04) in the rest of-the months (according to table-5). from figure-2, it has been seen that spot 3 had more species richness (2.61 ± 0.01) with low insect abundance (990) whereas spot 2 was less species rich (2.03 ± 0.008) with moderate insect abundance (1141). spot 1 was most diversified (1.36 ± 0.02) with high insect abundance (1598, 30.40%). on the contrary, spot 2 showed low species diversity (1.09 ± 0.01) with comparatively low insect abundance (1141, 21.70%). though the present study showed that the collected specimens were poorly distributed in all spots, the highest species evenness was found in the spot 1 (0.53 ± 0.006) with highest insect abundance (1598, 30.40%). species evenness was low in the spot 2 (0.47 ± 0.006) with low abundance (1141, 2170%). the environmental factor (both biotic and abiotic) like temperature, rainfall, humidity, vegetations and food sources directly affect the diversity and distribution of insect populations, which was also supported by morais et al. (1999), kittleson (2004), bispo and olivera (2007) and goldsmith (2007). the influence of humidity on density and diversity in the environment is likely to be an indirect effect operating via effects on availability. the differences could be the result of habitat and microhabitat differences among the sites. table 5: average (±se) monthly fluctuation in species richness (sr), species diversity (h′), and species evenness (e/j′) of the collected insects in four study spots. month jan feb mar apr may jun jul aug sep oct nov dec average sp ec ie s r ic hn es s ( sr ) spot 1 2.30 2.33 2.34 2.33 2.33 2.34 2.40 2.40 2.37 2.38 2.35 2.38 2.35±0.009 spot 2 2.05 2.02 2.02 2.04 2.02 2.02 2.01 2.05 2.06 2.05 1.97 2.00 2.03±0.008 spot 3 2.58 2.61 2.60 2.56 2.58 2.63 2.58 2.58 2.64 2.65 2.66 2.62 2.61±0.01 spot 4 2.29 2.34 2.33 2.31 2.32 2.31 2.43 2.46 2.43 2.43 2.43 2.46 2.38±0.02 av er ag e 2. 31 ± 0. 13 2. 33 ± 0. 14 2. 32 ± 0. 14 2. 31 ± 0. 11 2. 32 ± 0. 13 2. 33 ± 0. 14 2. 36 ± 0. 14 2. 37 ± 0. 15 2. 38 ± 0. 14 2. 38 ± 0. 14 2. 35 ± 0. 17 2. 37 ± 0. 15 sp ec ie s di ve rs ity (h ′) spot 1 1.45 1.37 1.35 1.34 1.38 1.29 1.32 1.28 1.36 1.36 1.44 1.38 1.36±0.02 spot 2 1.05 1.07 1.01 1.06 1.09 1.13 1.10 1.08 1.11 1.14 1.06 1.17 1.09±0.01 spot 3 1.30 1.30 1.27 1.42 1.42 1.18 1.25 1.45 1.29 1.43 1.44 1.22 1.33±0.03 spot 4 1.22 1.17 1.19 1.30 1.23 1.19 1.30 1.22 1.28 1.31 1.22 1.23 1.24±0.01 av er ag e 1. 26 ± 0. 09 1. 23 ± 0. 07 1. 21 ± 0. 08 1. 28 ± 0. 11 1. 28 ± 0. 11 1. 20 ± 0. 05 1. 25 ± 0. 07 1. 26 ± 0. 10 1. 26 ± 0. 05 1. 31 ± 0. 09 1. 29 ± 0. 13 1. 25 ± 0. 06 sp ec ie s e ve nn es s ( e /j ′) spot 1 0.56 0.53 0.52 0.51 0.53 0.50 0.51 0.50 0.52 0.53 0.56 0.53 0.53±0.006 spot 2 0.45 0.46 0.43 0.45 0.47 0.49 0.47 0.46 0.48 0.49 0.46 0.50 0.47±0.006 spot 3 0.50 0.50 0.49 0.54 0.55 0.46 0.48 0.56 0.50 0.55 0.56 0.47 0.51±0.01 spot 4 0.47 0.45 0.45 0.50 0.47 0.46 0.51 0.47 0.49 0.51 0.47 0.48 0.48±0.006 av er ag e 0. 50 ± 0. 03 0. 49 ± 0. 03 0. 48 ± 0. 03 0. 50 ± 0. 03 0. 51 ± 0. 03 0. 48 ± 0. 01 0. 50 ± 0. 01 0. 50 ± 0. 04 0. 50 ± 0. 02 0. 52 ± 0. 02 0. 51 ± 0. 04 0. 50 ± 0. 02 https://journals.e-palli.com/home/index.php/ajlsi pa ge 10 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 1-11, 2023 the climate condition of the campus is characterized by moderately high temperature, with plenty of sunshine in summer, moderate to excessive humidity, heavy rainfall in rainy season and charming cool dry weather in winter. during the study period temperature varied from 19o c in january to 28o c in may to september and 27o c in october to 20o c in december and mean annual rainfall was 225 mm. ecologically the campus is a tropical green forest area but the ecology has been suffering badly due to various biotic and abiotic factors and manmade causes. data of average monthly temperature, rainfall, humidity and wind speed for the entire study period were collected from the practical field study. the data is given below: in the present study, it was seen that some species were figure 2: spot-wise species richness (sr), species diversity (h), and species evenness (j) of the collected insects. table 6: meteorological data month average minimum temperature (˚c) average maximum temperature (˚c) average temperature (˚c) average rainfall/ precipitation (mm) wet days (>0.1 mm) relative humidity (%) average wind speed (mph) jan’18 13.7 26.4 20.0 6 2 58 1 feb’18 16.1 28.9 22.5 15 3 58 1 mar’18 20.4 31.6 26.0 50 5 65 2 apr’18 23.6 32.3 27.9 120 7 71 2 may’18 25.0 32.4 28.7 233 13 77 2 jun’18 25.3 30.8 28.0 578 22 83 2 jul’18 25.2 30.4 27.8 707 26 85 2 aug’18 25.2 30.5 27.8 517 25 86 2 sep’18 25.2 31.3 28.2 270 21 84 2 oct’18 24.0 31.2 27.6 205 8 78 1 nov’18 19.5 29.3 24.4 52 2 71 1 dec’18 15.1 26.8 20.9 9 1 68 1 mean 25.8 230.17 11.25 73.67 1.58 collected regularly while others were rare. these findings are consistent with many community studies, which show that a small number of species dominates the community, whilst the majority of species are relatively rare (el-moursy et al. 1999). five out of six insect orders showed seasonal variability. the only insect order hymenoptera did not show any significant seasonal variation in abundance. four broad patterns were distinct in the seasonal distribution of fauna. most of odonata and lepidoptera members were significantly higher during pre-summer (january to march, november, and december). orthoptera and coleoptera peaked during pre-summer and summer (march-may, november and december). diptera peaked during the monsoon period (april to june). the number of insects fluctuated in different months of the year due to several reasons. fluctuations in rainfall appeared to play a role in deciding the abundance of individual and overall fauna, along with other variables such as litter depth, litter moisture, humidity, and temperature that are directly or indirectly related to rainfall and insect population densities (wagner et al. 2003, vineesh et al. 2007). on the basis of the present observation and data collection, it can be said insect availability depends not only depended on seasonal fluctuation but also on suitable host plants. hence, their presence or absence, their abundance serves to monitor ecological changes in habitat thus warning about the deterioration of habitat. conclusion the present study indicates that the insect diversity is https://journals.e-palli.com/home/index.php/ajlsi pa ge 11 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 1-11, 2023 rich in the cu campus. moreover, insects play a vital role in the food chain and food web of any ecosystem. so, the present study is providing a clear checklist for environmentalists to understand the present condition of the ecosystem of the cu campus area. recommendation this research may help to make a further checklist of insect fauna of the cu campus. additionally, from the comparative analysis with the previous study it is distinguishable that whether the faunistic diversity increases or decreases as the university developed many infrastructures consequently the human settlement increases day by day. references amin, m.r., miah, m.s. and rahman, h. (2018). functional and group abundance of insects on eggplant. bangladesh j. agril. res., 43(4), 647-653. balakrishnan, s., srinivasan, m. and mohanraj, j. (2014). diversity of some insect fauna in different coastal habitat of tamil nadu, southeast coast of india. journal of asia-pacific biodiversity., 7(4), 408-414. bingham, c. t. (1908). the fauna of british india including ceylon and burma (butterflies) 2. taylor and francis, london, 8. borror, d. j., delong, d. m. and tripplehorn, c. a. (1989). an introduction to the study of insects. saunders college publishing. new york. 588-827. burton, j. (1974). the oxford book of insects. oxford university press, ely house, london. 208. el-morsey, a.a., gilbert, f., zalat, s. and el-hawagry, m.s. (1999). foraging behavior of anthacine flies (diptera:boombyliidae) in southern sinai, egypt. egyptian journal of biology, 1, 87-95. gleason, h.a. (1922). on the relation between species and area. ecol., 3, 156-162. fraser, f.c. 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(2015). abundance and diversity of insect fauna in four spots of chittagong university campus. j. biod. cons. biores. manang., 1(1), nasiruddin, m. and shiuli, f. a. (2017). entomofauna of four spots of chittagong university campus and their relative abundance and diversity. j. biodivers. conserv. bioresour. manag., 3(1). park, s., lim, h., hong, e.j. and jeon, y. (2012). survey on insect diversity of yeonpyeong-do island, korea. journal of korean nature, 5(1), 17-26. pielou, e.c. (1966). the measurement of diversity in different types of biological collections. journal of theoretical biology, 13, 131-144. rahman. m., maleque, m.a., uddin, m.s. and ahmed, j. (2017). abundance and diversity of beneficial insect and spider species on rice ecosystem in sylhet region. j. sylhet agril. univ., 4(1), 63-70. resh, v. h. and carde, h. t. (2003). lepidoptera; encyclopedia of insects. j. a. powell. academic press. 631-664. richards, o.w. and davies, r.g. (1977). imm’s general textbook of entomology. 2, 10th ed. classification and biology chapman and hall, london, england. 1354. sánchez-bayo, f. and wyckhuys, k. a. g. (2019). worldwide decline of the entomofauna: a review of its drivers. biological conservation., 232, 8-27. siddiki, a. (2015). insect diversity and composition during the wet and dry seasons in three forest types of johor, malaysia. m.s. thesis. faculty of science, technology and human development universiti tun hussein onn malaysia. siregar, e.h., atmowidi, t. and kahono, s. (2016). diversity and abundance of insect pollinators in different agricultural lands in jambi, sumatera. hayati journal of biosciences, 23(1), 13-17. talbot, g. (1975). the fauna of british india including ceylon and burma. butterflies, 1, first indian reprint edition. today and tomorrow’s printers and publishers, 600. van emden, f.i. 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(2014). insect diversity patterns along environmental gradients in the temperate forests of northern china. ph.d. thesis. university of london. https://journals.e-palli.com/home/index.php/ajlsi pa ge 1 pa ge 46 american journal of life science and innovation (ajlsi) microorganisms associated with tomato (lycopersicum esculentum) rot and effect of neem (azadirachta indica) extract in rot control in makurdi metropolis, benue state, nigeria igbe abi obed1, innocent okonkwo ogbonna2* volume 3 issue 2, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i2.3438 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: july 17, 2024 accepted: august 20, 2024 published: august 24, 2024 tomato (lycopersicum esculentum) is a vital agricultural crop globally, yet it is significantly affected by various microbial pathogens leading to post-harvest rot, which threatens food security and economic stability. this study aimed to identify the microorganisms associated with tomato rot in makurdi metropolis and evaluate the efficacy of neem (azadirachta indica) extract as a biocontrol agent. a total of 60 tomato fruits were sampled from the five major markets within the makurdi metropolis. they were cultured on nutrient agar, macconkey agar, potato dextrose agar, mannitol salt agar, and eosin methylene blue agar. the organisms identified were klebsiella sp, salmonella sp, proteus vulgaris, enterobacter sp, shigella sp, staphylococcus sp, bacillus sp, escherichia coli, rhizopus sp, mucor sp, aspergillus sp, and fusarium sp. neem extract was used for susceptibility tests on the bacterial isolates at 10%, 50%, and 100% concentration, and it was found most effective on some bacterial isolates at higher concentrations of 100% with wider zones of inhibitions. vegetative growth of the fungi on neem extract decreased with increase in concentrations. this finding proved the potentiality of plant extracts for controlling the fungal rot of tomato fruit. a pathogenicity test was conducted with readings taken at intervals of days and it was noticed that the microbial isolates were actually the cause of tomato rot. this study underscores the importance of identifying microbial threats to tomato crops and highlights neem extract as a promising natural alternative for managing tomato rot, promoting sustainable agricultural practices in makurdi metropolis and nigeria at large. keywords microorganisms, tomato, neem, susceptibility, zone of inhibition, bacteria isolates, food security introduction tomato (lycopersicum esculentum) is a widely cultivated and consumed vegetable, known for its nutritional value and economic importance. however, its high-water content makes it particularly susceptible to spoilage by various microorganisms, including bacteria and fungi (obeng, et al., 2018). the tomato fruit comprises the skin, pericarp and locular cavities. the locular cavities are filled with jelly-like parenchyma cells that surround the seeds. the cell walls are composed of alpha-cellulose, pectin, hemicelluloses and some protein (mautante & mala, 2024). it is rich in vitamins including vitamin a and vitamin c, carbohydrates, proteins, fats, fibres, potassium, and phytochemicals (talvas et al., 2010). it is rich in lycopene which has many beneficial health effects. tomato is a fruit that contains the seeds and ovary of a flowering plant (ugwu et al., 2014). it is known to be a very profitable crop that provides high returns for small scale farmers in most developing countries (lemma, et al., 2014). due to its nutritive value, taste, affordability, and accessibility, there has been an increase in demand by consumers (behravesh, et al., 2012). however, isolation and identification of microorganisms that are associated with spoilage of tomatoes have gained some research focus (akinyele, et al., 2020). in most developing countries, microbial infestation of tomatoes can occur during the harvesting period, post harvesting, handling, storage, transportation, and processing by customers (barth, et al., 2009 and yeboah et al., 2011). baiyewu et al.,2018 have also reported that another means of bacterial contamination is by exposing them on benches and baskets in the open markets for customers. the relative humidity (dew) during temporary storage of tomato fruits and nature of the storage room could play a great role in their contamination (pardaev, 2022). the proliferation of bacteria, especially in damaged tomatoes, could be considered more harmful when such contaminated tomatoes are consumed in improperly cooked food (valadez et al., 2019). several studies have been performed on bacteria associated with tomatoes and tomato products in several countries. a survey by ajayi and colleagues in the united states showed that clostridium sp., staphylococcus sp., and bacillus sp. were the predominant bacteria isolated from canned and raw tomatoes. in india, a study conducted on tomato puree showed the presence of klebsiella sp., proteus mirabilis, vibrio sp., and pseudomonas sp. (garg et al., 2017). in nigeria, wogu and ofuase isolated bacillus subtilis, klebsiella aerogenes, pseudomonas aeruginosa, salmonella typhi, proteus mirabilis, and staphylococcus aureus from spoiled tomatoes in benin city. a similar study also showed that the content of staphylococcus sp. (22.5%), bacillus sp. (20%) and escherichia coli (15%) in lagos state, nigeria 1 department of biotechnology, i. m. sechenov first moscow state medical university, moscow, russia 2 department of microbiology, joseph sarwuan tarka university, makurdi benue state, nigeria * corresponding author’s e-mail: ogbonna09@yahoo.com pa ge 47 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 46-50, 2024 statement of the problem azadirachta indica (commonly known as neem) is a tree in the mahogany family meliaceae. neem products are believed to be antifungal, antibacterial, antidiabetics, antiviral, contraceptive, and sedative. hence, it is particularly prescribed for treating skin diseases such as eczema, psoriasis, etc. (sharma and nupur, 2014). reports has shown that compounds from plant sources are moderately toxic and are suitable as fungicides. neem (azadirachta indica) extract has been studied for its antimicrobial properties, offering a potential natural solution for controlling tomato rot. the extract’s bioactive compounds can inhibit the growth of spoilagecausing microorganisms, thus extending the shelf life of tomatoes and reducing economic losses. by investigating the microorganisms associated with tomato rot and assessing the potential of neem extract as a biocontrol agent, this study aims to advance our understanding of disease dynamics in agricultural systems and contribute to the development of environmentally friendly strategies for managing tomato diseases. materials and methods tomato fruit samples were collected from the five biggest markets in the makurdi metropolis: modern market, wadata market, wurukum market, northbank market, and high-level market. each market sample was placed separately in a sterile plastic bag to prevent contamination. samples were immediately transported to the laboratory, of the department of microbiology, joseph sarwuan tarka university, makurdi, benue state. culturing, isolation, and identification of the major pathogenic organisms in the disease portion was carried out. all materials used were sterilised by appropriate methods to remove extraneous microbial contamination. glass wares were sterilised in a hot air oven at 1200c for one hour. in contrast, culture media were sterilized by autoclaving at 1210c for 15 minutes. inoculation loops were heated till red hot in a bunsen burner flame and allowed to cool before being used. work surface was identified routinely by cleaning with cotton wool impregnated with ethanol before the commencement and on completion of work. the aseptic technique was maintained throughout the cause of this work. preparation of stock sample and serial dilution the diseased portion was cut, crushed and fluids from the five different markets were squeezed into a conical flask labelled against each market. five test tubes containing 9ml of distilled water (dw) were set. 1ml of tomato juice from the conical flask was transferred into the first test tube labelled against the same market, giving a dilution of 10-1. this was mixed and 1ml was transferred to the next test tube (dw) marking its 10ml and giving it a dilution of 10-2. this process was continued till the 5th test tube giving a dilution of 10-5. this same process was carried out on the samples collected from all the markets. ethanolic extracts were prepared in the laboratory by the traditional method. one kilogram of fresh neem leaves (azadirachta indica) was collected from neem trees at joseph sarwuan tarka university, makurdi benue state. leaves were dried at room temperature and crushed in a mortar. combining the crushed leaf with ethanol and subsequently soaking the mixture overnight at room temperature. the next morning, the extract was strained through a miracloth, the liquid was kept in a water bath in the laboratory and allowed to evaporate to have a pure concentrated extract. microbiological analysis media were prepared according to the standard preparation protocol and the media used were nutrient agar, macconkey agar, potato dextrose agar (pda), mannitol salt agar (msa), and eosin methylene blue agar (emba). using a sterile pipette, aliquot of 0.1ml was taken from 10-5 dilution and was transferred into the duplicates plates of the media used i.e. nutrient agar, macconkey, pda, msa and emba. pour plate method of inoculation was used to inoculate on the media according to the markets they were collected from and on what symptoms or disease inferred from these markets. inoculated plates of the media were incubated at 370c for 24 hours for isolation of bacteria, and also at room temperature (25±20c) for 48–72 hours for isolation of fungi. representative colonies from all the media were sub-cultured on nutrient agar for pure isolate, which were later subjected to biochemical analysis for identification. observation of fungal characteristics and identification the fungal isolation on pda was identified by visual observation of their growth and microscopic examination using a drop of lactophenol cotton blue stain. the observed fungi were identified by comparison with diagram and keys documented by harrigan and mccance (1976) as reported by barton et al., 2006. distinguishing characteristics looked for included the general morphology and hyphae (septate and non-septate). the shape, colour and distribution patterns were also used in identification. the staining was done according to pelazar et al. (1999) as follows: a pin head of fungi growth was picked using needle. this was placed on a slide with a drop of lactophenol blue stain. a drop of distilled water was added. the slide was covered with a cover slip and observed under the microscope at high objective of x10 power magnifications. results and discussions table 1 below shows the mean of the total microbial load of tomatoes sold in different markets in the makurdi metropolis. this investigates the levels of microbial contamination present in tomatoes from various markets within the region. it measures and compares the total microbial load, including bacteria, fungi, and other microorganisms, to assess food safety and hygiene practices. the findings revealed significant differences pa ge 48 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 46-50, 2024 in microbial loads across different markets, highlighting potential health risks for consumers and the need for improved handling and storage practices to ensure the safety of tomatoes sold in these areas. table 1: mean of total microbial load of tomato sold in different markets in makurdi metropolis s/n samples tvc (cfu/g) tcc fungi 1 high level 163x105 120x105 148x105 2 wurukum 216x105 129x105 150x105 3 north bank 160x105 109x105 132x105 4 modern market 202x105 135x105 95x105 key: tvc = total viable count: tcc = total colony count: cfu/g = colony forming unit/g from the table, the bacterial count was maximum in nutrient agar with a total viable count ranging from 160 min. to 216 max., total colony count ranging from 109 min. to 135 max. and fungi count ranging from 95 min. to 150 max. the study on the morphological and microscopic features of fungi isolated from the tomato samples is represented in table 2. this focuses on identifying and characterizing the fungal species found in tomatoes. it examines both the physical characteristics (morphological features) such as the fungi’s colour, texture, and growth patterns, as well as their microscopic structures, including spore shape, size, and arrangement. this information helps in understanding the types of fungi present, and their potential impact on tomato quality and safety, and may provide insights into contamination sources and prevention methods. overall, it contributes to the knowledge of fungal diversity associated with tomatoes and its implications for food safety. table 2: morphological and microscopic features of fungi isolated from the tomato sample morphological characteristics microscopic examination suspected organism long hyphal growth which sporulated within two days to turn to black spores non-septate, branched mycelium with round-shaped sporangia rhizopus sp. white and woolly aerial growth that darkens as it sporulates non-septate hyphae with straight sporangiophore with many spherical spores mucor sp. pink fluffy and spreading colonies which are creamy around the edges. septate hyphae with sickle chlamydospores at the hyphae. fusarium sp. velvety filament white growth that sporulates into black powdery spores. long septate hyphae with conidiophore bearing brown spores aspergillus sp. table 3: microscopic and biochemical characteristics of the bacterial isolates gram reaction cat cit ure ind mr vp suspected organism -ve rod + + + klebsiella sp. -ve rod + + salmonella sp. -ve rod + + + + + proteus vulgaris -ve rod + + + enterobacter sp. -ve rod + shigella sp. +ve cocci + + staphylococcus sp. +ve rod + + + + bacillus sp. -ve rod + + + + escherichia coli key: '+' = positive result; '-' = negative result from the study, the microscopic and biochemical characteristics of the bacterial isolates which focus on identifying and characterizing bacterial strains isolated from the tomato samples are represented in table 3. the microscopic characteristics include the bacteria’s shape, size, arrangement, and staining properties., which helps in their classification. the biochemical characteristics involve various tests to determine metabolic activities, such as fermentation, enzyme production, and nutrient utilization. together, these analyses provide insights into the diversity, functionality, and potential pathogenicity of the bacterial isolates, contributing to a better understanding of their role in the ecosystem or their impact on health and disease. pa ge 49 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 46-50, 2024 table 4 and 5 summarizes the susceptibility test of neem extract in different concentrations on bacterial isolates using amoxicillin as control and the pathogenicity test of bacterial isolates on healthy tomatoes respectively. antibacterial effects of neem extract at various concentrations against specific bacterial isolates were identified and recorded. the effectiveness of neem extract is compared to that of amoxicillin, a commonly used antibiotic, serving as a control. the results indicate how different concentrations of neem extract inhibit bacterial growth, providing insights into its potential as a natural antimicrobial agent. the pathogenicity of isolated bacteria was evaluated by inoculating healthy tomato plants with these strains. the effects on tomatoes health, including disease symptoms and overall plant vigour, were observed. findings from this test help determine which bacterial isolates are harmful to tomatoes, contributing to understanding plant-bacterial interactions and potential agricultural impacts. table 4: susceptibility test of neem extract in different concentrations on bacterial isolates using amoxicillin as control bacterial isolates zone of inhibition per concentration (mm) 10% 50% 100% control (amoxycillin) klebsiella sp 12 15 salmonella sp 8 12 14 17 proteus vulgaris 6 11 15 enterobacter sp 11 13 17 19 shigella sp 11 16 18 21 staphylococcus sp 13 14 19 22 bacillus sp 8 13 18 escherichia coli 10 14 16 19 key: '-' shows no zone of inhibition table 5: pathogenicity test of bacterial isolates on healthy tomato bacterial isolates zone of pathogen spread per day (mm) day 1 day 2 day 3 day 4 day 5 day 6 day 7 klebsiella sp 3 5 9 13 15 salmonella sp 4 8 12 15 16 proteus vulgaris 2 6 11 13 16 18 enterobacter sp 3 7 13 15 17 20 shigella sp 4 8 10 14 15 19 staphylococcus sp 3 7 12 14 18 bacillus sp 2 6 9 12 15 17 escherichia coli 2 7 10 13 15 key: shows no zone of spoilage from the table, they were no or little infection on the first day and there were subsequent spread of infection and spoilage from the second day to maximum infection on the tenth day. conclusion this study was carried out to examine the tomato rot causing microorganisms and the effect of neem extract on rot control in the makurdi metropolis. the organisms identified were klebsiella sp, salmonella sp, proteus vulgaris, enterobacter sp, shigella sp, staphylococcus sp, bacillus sp, escherichia coli, aspergillus sp, rhizopus sp, mucor sp, and fusarium sp. neem extract was used to conduct susceptibility tests on the bacterial isolates and it was found most effective on some bacterial isolates at higher concentrations. a pathogenicity test was conducted with readings taken at interval of days and it was noticed that the microbial isolates were actually the cause of tomato rot. from the result of this study, it is found that tomato rot is caused by some microorganisms such as klebsiella sp, salmonella sp, proteus vulgaris, enterobacter sp, shigella sp, staphylococcus sp, bacillus sp, escherichia coli, aspergillus sp, rhizopus sp, mucor sp, and fusarium sp. and neem extract at 50% and 100% concentration can be used to control the rot caused by some of these organisms. therefore, the extracts are potentially simple environmentally safe alternative for use as botanical fungicides, and could be exploited to manage post-harvest diseases of tropical fruits effectively. neem extract, derived from the seeds, leaves, and bark of the neem can be used as natural pesticide due to its insecticidal properties. it can also be used in various traditional and modern medicines for their anti-inflammatory, antibacterial, antifungal, and antiviral properties, and as natural food preservatives. pa ge 50 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 46-50, 2024 references ajayi, a. (2013). nature of tomatoes microflora under storage. american journal of experimental agriculture, 3(1), 89–101. https://doi.org/10.9734/ ajea/2013/2177 akinyele, b. j., & akinkunmi, c. o. (2020). fungi associated with the spoilage of berry and their reaction to electromagnetic field. journal of yeast and fungal research, 3(4), 49–57. baiyewu, r. a., amusa, n. a., ayoola, o. a., & babalola, o. o. (2018). survey of the postharvest diseases and aflatoxin contamination of marketed pawpaw fruit (carica papaya l) in south western nigeria. african journal of agricultural research, 2(4), 178–181. barth, m., hankinson, t. r., zhuang, h., & breidt, f. (2009). compendium of the microbiological spoilage of foods and beverages. springer. barton, b. c., blaney, d., bidol, s. a., soliva, s., daly, e. r., & taylor, t. (2006). multistate outbreaks of salmonella typhimurium infections associated with consumption of restaurant tomatoes, usa. journal of epidemiology and infection, 9, 1-9. behravesh, c. b., blaney, d., medus, c., et al. (2012). multistate outbreak of salmonella serotype typhimurium infections associated with consumption of restaurant tomatoes, usa: hypothesis generation through case exposures in multiple restaurant clusters. epidemiology and infection, 140(11), 2053–2061. https:// doi.org/10.1017/s0950268811002895 garg, r. k., batav, n., silawat, n., & singh, r. k. (2017). isolation and identification of pathogenic microbes from tomato puree and their delineation of distinctness by molecular techniques. journal of applied biology & biotechnology, 1(4), 24–31. lemma, z., dawit, w., negari, m., chaka, a., selvaraj, t., & gebresenbet, g. (2014). identification of postharvest rotting microorganisms from tomato fruits (solanum esculentum mill.) in toke kutaye district of west shoa zone, ethiopia. journal of stored products and postharvest research, 5(3), 14–19. mautante, s. k., & mala, s. a. p. (2024). foliar application of sakurab (allium chinense g. don) extract on tomato (lycopersicon esculentum) production. american journal of agricultural science, engineering, and technology, 8(1), 39–47. https://doi.org/10.54536/ ajaset.v8i1.2604 obeng, f. a., gyasi, p. b., olu-taiwo, m., & ayehkumi, f. p. (2018). microbial assessment of tomatoes (lycopersicon esculentum) sold at some central markets in ghana. biomed research international, 2018, 6743826. https://doi.org/10.1155/2018/6743826 ogundipe, f., bamidele, f., adebayo-oyetoro, a., ogundipe, o., & tajudeen, o. (2012). incidence of bacteria with potential public health implications in raw lycopersicon esculentum (tomato) sold in lagos state, nigeria. nigerian food journal, 30(2), 106–113. https://doi.org/10.1016/s0189-7241(15)30043-6 pardaev, k. a. (2022). risk mitigation strategies in the tomato supply chain. american journal of agricultural science, engineering, and technology, 6(3), 118–123. https://doi.org/10.54536/ajaset.v6i3.863 pelazar, m. j., chan, e. c. s., & krieg, n. r. (1999). microbiology (5th ed.). mcgraw hill. popoola, l. (2015). poverty and economic viability of neem tree growing in nigeria. seminar presented at a workshop organized by central bank of nigeria, katsina state. sharma, d. d., & nupur, s. s. (2014). comparative study of different parts of azadirachta indica (neem) plant on the basis of antibacterial activity, phytochemical screening and its effect on rat pc-12 (pheochromocytoma) cell line. international journal of biotechnology, humanities, sports and allied fields, 2(7), 144–154. talvas, j., caris-veyrat, c., guy, l., rambeau, m., lyan, b., minet-quinard, r., et al. (2010). differential effects of lycopene consumed in tomato paste and lycopene in the form of a purified extract on target genes of cancer prostatic cells. american journal of clinical nutrition, 91(6), 1716-1724. https://doi.org/10.3945/ ajcn.2009.28785 ugwu, c. i., anyawu, u. m., ugwu, c. c. and okoro, j. n. (2015). isolation and detection of methicillin resistant staphylococci in healthy broilers in nsukka south – east nigeria. notulae scientia biologicae, 7(1), 20-25. valadez, a. m., schneider, k. r., & danyluk, m. d. (2019). outbreaks of foodborne disease associated with tomatoes (fshn 12-08). http://edis.ifas.ufl.edu/fs192 wogu, m. d., & ofuase, o. (2014). microorganisms responsible for the spoilage of tomato fruits, lycopersicum esculentum, sold in markets in benin city, southern nigeria. school of academics and journal of biosciences, 2(7), 459–466. yeboah, a. k. (2011). a survey on postharvest handling, preservation and processing methods of tomato (solanum lycopersicum) in the dormaa and tano south districts of the brong ahafo region of ghana (doctoral dissertation). http://ir.knust.edu.gh/ xmlui/handle/123456789/2079 pa ge 1 pa ge 37 american journal of life science and innovation (ajlsi) analysis of the temporal orientation of the supervisors of the specialization of physical education in baghdad mustafa hameed hussein1* volume 3 issue 1, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i1.2508 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: march 10, 2024 accepted: april 15, 2024 published: april 19, 2024 the educational process in any society constitutes an accumulation of knowledge resulting from the succession of times according to the ideologies of that society, taking into account its cognitive developments and the challenges it faces to defend its values and beliefs, through the development of promising educational programs that keep pace with the development of the present to assume its future status according to its glorious legacy, and to preserve this accumulation of knowledge across generations, the supervision and evaluation apparatus at the iraqi ministry of education constitutes its main task to evaluate the educational process and provide appropriate solutions to develop performance in the educational institution and for all scientific disciplines, including these specializations (supervisors of the specialization of physical education) who are responsible for the task of evaluating the sports educational process in iraq at the educational, administrative and technical levels within the educational institution. the research problem started from what is the temporal orientation of the supervisors of the specialization of physical education in baghdad so that we can evaluate their performance present and future in a way that suits the developments of society and achieve its goals, and the aim of the current research is to identify the temporal orientation of the supervisors of the specialization of physical education in baghdad, so it was necessary to build a measure of temporal orientation in the iraqi sports education environment first, the researcher has tested the significance of differences between the arithmetic averages of the temporal orientation of the research sample between (past, present and future) and the field research data revealed that there are significant differences in favor of both (past, present and future) present and future), which indicates the temporal direction of the examined sample towards the researcher recommended that supervisors should be educated on the subject of temporal orientation, and the process of linking these three eras (past – present – future) in constructive interaction with life to develop educational institutions, being directly responsible for evaluating the educational process in the field of sports activities. keywords analysis, temporal orientation, supervisors of specialization, physical education introduction philosophers have been interested in time since ancient times, and their orbit of interest revolved around the issue of the existence of time, whether it is abstract or related to human consciousness, there is no concept that casts a shadow over all movements and static, as time does in the human personality and life in general, and what it does have a serious impact, there is an important link between the concept of time and other concepts, including movement, place, causality, change and conscious action, just as there is a close relationship between the psychological, civilized, social and economic features of the (arab) human society, and between the time of living and the type and nature of its orientation time in general represents a new angle of view that deserves to be viewed from the arab production, it is a new entrance to it, characterized by it is known throughout human history that any nation, when it overcomes its situation and feels the imminent danger to its civilization, yearns for its past and heritage, and its originality inspires strength and inspires determination in order to awaken souls and send strength and solidity in the face of dangers, it was the ambition of man to determine the relationship of the arab man with his surroundings and himself and how he deals with the most important composite elements of his existence, namely the earth, time and movement, and it is known throughout human history that any nation always owning the corner of time, despite knowing the danger of this possession, because for a person, every possession is an increase in his possessions, except the possession of time. each of us has his own time, which is different from the others, it is not subject to measurement, and in order to be so, it must flow regularly, and time has different forms, first of all physical or natural time, which is the activity of the movement of the natural or astronomical world, that is, the time resulting from the movement of the planets, which is measured by means of measuring time known as hours, months, years, decades and centuries, and this time is shared by all people in terms of their understanding, their sense of it, and their perception of it, and the second is social time, which is the product of society from societies with all its beliefs and political conditions intellectual and social, and the third experimental time that is subject to human experience in his life, we may see it short or long, and we may see positive and negative, and that self-esteem, as we see with insight. because the educational process in any society is an 1 imamaladham university college, iraq * corresponding author’s e-mail: mustafa.hhameed.h@gmail.com pa ge 38 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 37-43, 2024 accumulation of knowledge resulting from the succession of times according to the ideologies of that society, taking into account its cognitive developments and the challenges it faces to defend its values and beliefs, through the development of promising educational programs that keep pace with the development of the present to assume its future status according to its glorious legacy. due to the important role that the supervisors of the sports education specialization play in evaluating the sports educational process in iraq at the educational, administrative and technical levels, we found it important to build a comprehensive framework about the variables of current research by designing a model that makes the educational process cherish its past and correct its mistakes at the present time and prepare for a bright future that generations can boast of. the problem of the research started from what is the chronological orientation of the supervisors of the specialization of physical education in baghdad so that we can evaluate their performance present and future in a way that suits the developments of society and achieves its goals. the current research aimed to identify the temporal orientation of the supervisors of the specialization of physical education in baghdad, so it was necessary to build a measure of temporal orientation in the environment of iraqi sports education first, the researcher identified the areas of his spatial research in the directorates of education in baghdad, and temporal for the period from 1/11/2023 to 31/1/2024, as well as the humanity represented by some supervisors of the specialization of physical education in baghdad. literature review the myth of gilgamesh provides a record, one of the oldest creations of the mind, of man’s rebellion against the power of time, always victorious, in his painstaking search for immortality, and escape from the current of time, and there is no doubt that the history of time – in general-is inseparable from the history of human thought, it is the theater of unlimited progress of man, and during successive eras man the circumstances and events around him, (walters, 1989). during the thousand years, scientists tried to study these periodic phenomena and find a relationship between them, because the many variables that occur in the manifestations of things due to the movement of astronomy made man call a universal name synonymous with the meaning of existence, called (time), hence many questions arose about the meaning of time and its relationship with existence and the creator of the universe, (dawnd, 2000; alsomaidaee et al., 2023). perhaps the existence of man in the world means his existence in time. it shows us as a movement emanating from the bowels of the past and passing through the present on its way to the future, that our life plans that we implement today imply the needs of tomorrow, as the past, present and future as interacting forces with each other are imprinted on the existence of the sense of time is not always practiced, of course, by people, but a certain amount of effort active activity is necessary for the successful integration of the past, present and future (berkow, 1994). after the past (the dominance of the past), events include sorrows and delights, because the past is a time that has passed away from people’s lives, it does not return on the one hand and is prone to forgetting on the other hand, the past is associated with goodness, youth and love sometimes or vice versa, each present is an extension of the past and is a precursor of the future, and the past may dominate the present until it is almost (suffocates), as there are people who are subject to the influences of their past and constantly feed their present with memories of the past so that it is he who guides them, (lam, 1976). there is no doubt it is through remembering (memory) that the individual’s connection with the past remains and is not interrupted once it is over, but remains, but when it is obvious that the individual cannot face the present, he may resort to situations in which he finds the fulfillment of his desires this is precisely a kind of escape towards the past of the individual, where he can live by imagination, in daydreaming as ordinary dreams, that is, he gets rid of the pressure of the present to satisfy his desires through imagination, (thomas, 1995; alubadi et al., 2020). as for the dimension of the present (the dominance of the present) means the events that occur in real time and the individual lives and interacts with them now, it is the bridge of communication and separation between the time dimensions, and the ground of conflict between the constants and variables of human psychological life, and it is the primary material with which we adapt our existence, it is people who or the future that threatens his safety, (goldrich, 1967). finally, after the future (the dominance of the future), an unknown world and events within the framework of the unseen, and therefore many have been motivated to speculate about it and access or predict its events and results. the awareness of the future has been accompanied by the development of man, but this development has compounded man’s bewilderment and surprise in front of the future, so efforts to see the future began, although human ignorance of it is the most important motivation for the continuation of life (kasteubam, 1985). the outlook on the future is at the core of the individual’s sense of time, because human nature originally begins to look forward to the outskirts of tomorrow with caution and anticipation, foresight and optimism, that is, an individual’s life in the present is usually directed towards the future, which gives him a goal for his actions, but some people who make his orientation mainly towards what is to come (the future) may be a desire for change, as a result of dissatisfaction with the present, or as a result of feeling that the future carries with it something different from the past, and the individual may allow the future to control his behavior, exaggerating expectation or failure to predict constitute a negative of adaptation, for their pa ge 39 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 37-43, 2024 mismatch with reality, which makes some of these people in a constant state of anxiety and preparing for dangers that have not yet arrived, drains from their psychological energies a lot, (david, 1996; alsomaidaee 2020). from the foregoing, we can say that the temporal orientation: (it is a space of trends and tendencies enhanced by the personal experience of the individual, some of them are occupied by the past and live in it, some are occupied by the present and live in it, and some are occupied by the future with optimism or pessimism for what the following days will be). materials and methods the researcher used the descriptive-analytical method to study the significance of the differences between the variables studied by collecting relevant data from the individuals of the research sample and analyzing them. sampling and data collection the current research community included the supervisors of the specialization of physical education in baghdad, and the research sample consisted of (105) supervisors, and table (1) shows the distribution of the members of table 1: shows the distribution of the sample members covered by the research s directorate survey sample sample construction sample application 1 baghdad/karkh 2 15 35 2 baghdad/rusafa 3 15 35 total 5 30 70 the sample covered by the research. metrics a research tool is “ the means or method by which a researcher can solve a problem, whatever those tools, (data or devices),” (allen, m.j & yen, w.m, 1979). the nature of the current research and its goals require the availability of a tool (scale) for the time orientation of the supervisors of the specialty of physical education in baghdad, and since the researcher was unable to obtain this scale previously prepared by other researchers in the field of sports-as far as he was aware-so the researcher built the scale according to the scientific steps:first: defining the concept of time orientation through reviewing some of the previous literature and studies that dealt with this concept, the researcher identified the concept, especially in the field of sports education, the temporal orientation is: (it is a space of trends and tendencies enhanced by the personal experience of the individual, some of them are occupied by the past and live in it, some are occupied by the present and live in it, and some are occupied by the future with optimism or pessimism for what the following days will be). second: determining the dimensions (axes) of the concept of time orientation the process of determining the basic considerations of the questionnaire preparation procedures has a great role in contributing to helping the researcher to choose the appropriate procedures for preparing the scale, so the researcher relied in determining the dimensions of the temporal orientation on studies and literature specialized in the temporal orientation about its concept and dimensions, and three dimensions of the temporal orientation were chosen that fit the work of iraqi mathematical education, namely, (gjesme, 1983):orientation towards the past (dominance of the past) it is the subordination of human behavior to the experiences of the past and not leaving them, attachment to parents, taking the line of isolation from society and self-closure, leading to the withdrawal of the individual from the present (reality) in favor of the dominance of the past. orientation towards the present (dominance of the present) is the subordination of behavior to the experiences of the coming present, manifested through its attachment to whims and acting according to its immediate sense, the withdrawal of the individual from the past and the future in favor of the dominance of the present. orientation towards the future (dominance of the future) it is the subordination of behavior in future prospects, determined by his insistence on achieving his ambitions and hopes, and the withdrawal of the individual from the past and present in favor of the dominance of the future. to verify the accuracy and correctness of these axes and their representation of the temporal orientation, the researcher presented these axes and the definition of the concept after describing each axis to (5) experts, and the researcher asked them to estimate the accuracy of the concept and the accuracy of its axes and their validity to measure the concept, and it was agreed on their validity and accuracy by 100% of the experts. third: preparing the paragraphs of the time orientation scale after reviewing some studies and arabic and foreign literature, and in light of the characterization of each axis, the researcher formulated the paragraphs of the scale between the style of verbal attitudes and the style pa ge 40 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 37-43, 2024 of self-assessment, where the style of verbal attitudes puts the respondent in situations similar to the present to real situations, and reduces the likelihood of random testing among the alternatives to the answer, and the adoption of a multiple choice method in the preparation of all paragraphs, as the scale consists of (21) paragraphs, each of which includes alternatives (a, b, c) so that each alternative represents one of the three time dimensions (past, present, future) respectively, the answer for each paragraph is by choosing either (a), (b) or (c), according to the application of the content of the paragraph to the respondent and expresses his answer for his time orientation, and with three graded alternatives to the answer (large, medium, few) and are given when correcting grades (3, 2, 1) respectively. logical analysis of paragraphs of the time orientation scale (validity of paragraphs) the concept of validity of paragraphs refers to the special inferences that the degrees of measurement come out of , in terms of their relevance, meaning and usefulness, which is the achievement of the truthfulness of the form and the meaning of collecting evidence that supports such inferences, (eble, r. l, 1972). based on this basis, after drafting the 21 paragraphs of the time orientation scale, the researcher presented them to (5) experts from sports management, testing and measurement specialists and asked them to study each of the paragraphs and give their opinions on their validity, and the experts discussed with the researcher about each paragraph in terms of the formulation of ideas and the content of the paragraphs, the researcher came out the scale in its initial form as described in appendix (1). psychometric (standard) characteristics of the time orientation scale (anastasia, 1982) indicates that “ paragraph analysis is the study that relies on statistical and empirical logical analysis of test units for the purpose of knowing their characteristics, deleting, modifying, replacing, adding, or rearranging these paragraphs in order to arrive at a consistent and truthful test that is appropriate in length and difficulty “. the characteristic (truthfulness) of a paragraph is one of the most important standard characteristics that should be ascertained in the construction of educational and psychological scales, since the truthfulness and stability of the scale largely depends on the characteristics of its paragraphs. the apparent truthfulness of the time-orientation scale it is one of the most important types of honesty in educational and psychological measures and indicates the extent to which the paragraph is related to the phenomenon to be measured, and the apparent honesty of the scale was achieved when they were presented in their initial form to a group of experts and a number of (5) experts to judge the validity of its paragraphs in measuring the phenomenon. treatment of internal consistency of timeorientation scale paragraphs for the purpose of checking the homogeneity of paragraphs, the internal consistency coefficient provides us with evidence of this “since the internal consistency coefficient is the correlation coefficient between the scores of each paragraph and the total score of the scale”. (horton, 1983), and the researcher used the simple correlation coefficient law (pearson) to extract the correlation relationship between each paragraph and the total score of the scale using the statistical bag (spss), and since all the paragraphs that were retained in the scale have their consistency coefficients in statistical significance, that is, the calculated values are significant below the significance level (0.05) so no paragraph fell, it can be said that the scale is homogeneous, and table (2) shows the coefficient of internal consistency of the paragraphs of the scale with the scale as a whole. table 2: shows the coefficient of internal consistency of the temporal orientation scale paragraphs domination of the past dominance of the present dominance of the future p pearson correlation p pearson correlation p pearson correlation 1 0.577* 1 0.198* 1 0.216* 2 0.170* 2 0.349* 2 0.391* 3 0.673* 3 0.531** 3 0.362* 4 0.605* 4 0.457* 4 0.311* 5 0.894* 5 0.318* 5 0.247* 6 0.878* 6 0.507** 6 0.315* 7 0.877** 7 0.502** 7 0.513** 8 0.162* 8 0.488* 8 0.210* 9 0.622* 9 0.264* 9 0.208* 10 0.433* 10 0.504** 10 0.193* 11 0.935** 11 0.456* 11 0.219* 12 0.583* 12 0.170* 12 0.231* pa ge 41 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 37-43, 2024 constancy of the measure of the temporal orientation means “ the constancy of the test to the consistency of the scores obtained by the same individuals themselves in different atmospheres, (eble, r. l, 1972). in order to find out the stability of the scale and the existence of several methods for calculating it, the researcher relied on the use of the stability analysis method with the alpha cronbach’s equation because it is one of the most used stability methods, as this method measures the internal homogeneity of the paragraphs of the scale, after the researcher unloaded the scores obtained from the (construction sample) of (30) individuals and calculated the scores of paragraphs and the total score for each respondent, the alpha cronbach’s equation was used, so the stability coefficient of the scale was (0.607), which is a stability coefficient good, according to foran. objectivity of the measure of the temporal orientation by distributing the scale to the experts to extract honesty and consistency, there was no problem or misunderstanding of the questionnaire paragraphs, the answers were clear and understandable, which indicates their objectivity, because objectivity “is the test that gives the same results no matter how different the correctors” (anastasia, 1982). applying the scale to the main sample the researcher proceeded to distribute the questionnaire form to the 70 members of the research sample (application sample). after completing the research steps, the researcher collected the questionnaire data from all the members of the research sample. two forms were excluded for violating the answer conditions and the remaining 68 forms were arranged in tables in preparation for statistical processing to display and analyze the results. statistical methods the statistical data was processed using the social sciences statistical bag (spss). results and discussions to find out the significance of the difference between the average time orientation according to its three dimensions and the theoretical (hypothetical) average of the scale using the test (t-test) for one sample, it turns out the following: the average time orientation scores of the research sample were greater than the theoretical average of the test, as the average time orientation scores were (47.91), while the theoretical average of this test was (42) with a standard deviation (5.479), and the calculated t-value of (72.107) was significant in statistical significance at the level of (0.001), as shown in table (3). 13 0.982** 13 0.177* 13 0.319* 14 0.163* 14 0.237* 14 0.489** 15 0.582* 15 0.599** 15 0.227* 16 0.845* 16 0.672** 16 0.187* 17 0.684* 17 0.230** 17 0.714** 18 0.768* 18 0.244* 18 0.301* 19 0.919** 19 0.205* 19 0.241* 20 0.926** 20 0.201* 20 0.333* 21 0.279* 21 0.207* 21 0.194* **. correlation is significant at the 0.01 level (2-tailed). *. correlation is significant at the 0.05 level (2-tailed). table 3: shows the arithmetic mean and standard deviation of the temporal orientation and by its dimensions s temporal orientation and its dimension sample size mean std. deviation theoretical mean calculated value (t) level of significance 1 temporal orientation 68 47.91 5.479 42 72.107 0.000 2 domination of the past 68 10.16 5.900 14 13.666 0.000 3 dominance of the present 68 19.43 7.042 14 22.748 0.000 4 dominance of the future 68 19.07 6.533 14 2.307 0.05 table (4) shows the analysis of the variance, through which the calculated value of (f) (40.944), which is significant at the significance level (0.001), means that there are significant differences between the orientations of the research sample towards (past, present and future), which is the first condition of (lsd test), and (leven statistical) achieved a value (1.709) at a significance level (0.184), which is not significant because it is greater than (0.05), this means homogeneity variability, which is the second condition of (lsd test). pa ge 42 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 37-43, 2024 table (5) shows the source of the discrepancy through the results of the (lsd test) on the studied sample, where the difference between (dominance of the past-dominance of the present) came with a value of (9.268 -*), which is significant at the level of significance (0.001) and in favor of the dominance of the present, while the difference between (dominance of the past – the dominance of the future (value-8.915 -*), which is significant at the level of significance (0.001) and in favor of the dominance of the future, this means that there are significant differences in favor of both (present and future), which indicates the temporal direction of the studied sample towards the present and the future at the expense of the past, and this is confirmed by the difference recorded between (dominance of the present – dominance of the future) at a value (0.353), which is non-significant at the level of significance (0.05), which means there are no significant differences between them. table 4: the variance analysis of the temporal orientation of the research sample shows source of variability sum of squares degrees of freedom mean square test value (f) level of significance between groups 3562.805 2 1781.403 40.944 0.000 within the groups 8527.677 196 43.509 total 12090.482 198 table 5: shows the source of variation (lsd test) in the research sample temporal orientation mean difference level of significance domination of the past dominance of the present -9.268-* 0.000 dominance of the future -8.915-* 0.000 dominance of the present dominance of the future 0.353 0.755 (*) sentiment is at an indicative level (0.05) conclusion the research achieved its first goal by building a time orientation scale specific to the iraqi sports environment, as well as getting acquainted with the time orientation of the supervisors of the sports education specialty in baghdad. it thus achieved its second goal, and the field research data revealed the presence of significant differences in favor of both (present and future), which indicates the time orientation of the sample studied towards the present and the future at the expense of the past, as it turned out that the majority of the sample members had a time orientation towards the future, and their percentage was (39.49%), followed by those in the rank who were oriented towards the present. their percentage was (37.67%), while the percentage of those who are oriented towards the past (22.69%). therefore, the supervisors of physical education specialization can be described as heading towards the future in their work based on the present to prepare their plans and develop the means and tools for their work. the researcher recommends that supervisors should be educated on the subject of temporal orientation, the process of linking these three times (past – present – future) in constructive interaction with life in educational institutions, being directly responsible for the evaluation of the educational process in the field of sports activities. references allen, m. j. , & yen, w. m. (1979). introduction to measurement theory. california: book cole. alsomaidaee, m. m. (2020). strategic crisis management in light of hidden economy activities in iraq/ diagnosis and treatment. tikrit journal of administration and economics sciences, 16(51 part 2). alsomaidaee, m. m., joumaa, b. a., & khalid, k. w. (2023). toxic workplace, mental health and employee well-being, the moderator role of paternalistic leadership, an empirical study. journal of applied business and technology, 4(2), 114-129. alubadi, a. s., khalel, o. a., & al-somaidaee, m. m. (2020). the impact of entrepreneurial orientation in enhancing the managers’ core competencies of charitable institutions in iraq. retrieved from researchgate website: https://www. researchgate. net/publication/342503229_the_impact_of_ entrepreneurial_orientation_in_enhancing_the_ managers’ _core_competencies_of_charitable_ institutions_in_iraqanastasia. (1982). psychological testing. 5th ed, new york: macmillan. berkow, d. (1994). a cross-cultural comparison of world views: american and fijian counseling students, counseling and values. david, s. (1996). business ethics. usa: mcgraw-hill companies , inc. dawnd, b. (2000). time orientation and temporal variattion a cross work groups: implications for group and organizational communication. western – journal – of communication. eble , r . l. (1972). essential of educational measurement. new york: paretic – hill . gjesme, t. (1983). motivation to approach (ts) and motivation to void failure (tf) at school scandinarain. pa ge 43 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 37-43, 2024 journall of education research, umt. goldrich, j. m. (1967). a study in time orientation: the relation between memory for past experience and orientation to the future. journal of personality and social psychology, 6(2), 216. horton, s. (1983). delivering industrial arts instruction to mildy hondicapped learners, career development for exceptional individuals. kasteubam, r. (1985). past revsur future orientation psychological for the elderly paper presented at the annual convehtion of the american psychological. lamn, h. (1976). sex and social cllass/ detemination of future orientation (time perspective) in a dolecents. journal of persunolity and social psychological, 34(3), 317326. thomass, h. (1995). the use of second other facher analysis in psychological research paper presentd at the annue metting of the south western research. walters, a. (1989). relationship between time orientation, knowledge of aids, and self-repovted sexal behavioral changes in college students. pa ge 1 pa ge 16 american journal of life science and innovation (ajlsi) investigating the influence of magnets in the growth of string bean (phaseolus vulgaris) plant john lloyd p. alarcon1*, jay c. cuesta1, mary rhose b. molejon1, jiezel d. paragsa1, nova mae q. ypon1 volume 3 issue 1, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i1.2450 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: january 20, 2024 accepted: february 18, 2024 published: february 23, 2024 magnetic treatment has potential as an agricultural tool, particularly for seed germination and seedling development. this study advances the knowledge of the effects of magnetic treatment on string beans (phaseolus vulgaris) plants. the plants subjected to magnetic treatment are more significant in size, height, and overall health. the t-test result shows a considerable difference between the control and experimental groups, which signifies the effectiveness of the magnetic treatment in the growth of the string bean (phaseolus vulgaris) plant. farmers are encouraged to explore employing magnets as a low-cost alternative to fertilizer, enabling increased plant development and healthy growth even without traditional fertilizers. economists may adopt magnetic treatment for its environmentally favorable properties and the transfer or continuing treatment of healthy, magnetically exposed seedlings. future researchers are encouraged to perform more studies to acquire more evidence on the efficacy of magnetic treatment. keywords magnetic treatment, seed germination, high plant yield, smart farming, alternative treatment introduction the magnetic field is one of the natural components of the earth. plants and other forms of life interact with magnetic fields daily. generally, the earth acts as a magnet with its south and north poles, and the natural effects of the magnetic field have been changing plant growth and yield around the globe (maffei, 2014). specifically, the electromagnetic spectrum of sunlight stimulates the growth of plants by the process of photosynthesis. the possible mechanism would be a change in the electrostatic balance of the plant system at the cell membrane level, as it is the primary site for action of any inhibition or enhancement of plant growth. magnetic treatments enhance seed vigor by influencing the biochemical processes, which stimulate the activity of proteins and enzymes. then, some studies reported that the magnetic field positively affected the number of flowers and yield, nutrient and water uptake, and increased seed germination and plant growth, which are benefits of the magnetic field’s higher strength. according to studies, magnetic fields can significantly affect seed germination, plant growth, development, and yield depending on the species and exposure characteristics, such as strength and duration, which can affect the growth pattern of the plants (da silva & dobránszki, 2016). the influence of the magnetic treatment on a string bean plant is the main objective of this experimental research. the study explicitly aimed to respond to these questions: i. what is the germination rate and mortality rate of the string bean (phaseolus vulgaris) plant? ii. what is the growth rate of the string bean plant (phaseolus vulgaris) in terms of the size of its leaves and length of growth? iii. is there any significant influence between the control and experimental group? literature review effects of magnetic treatment on plants sunflower seedlings were subjected to static magnetic fields with strengths ranging from 0 to 250 mt in stages of 50 mt for 1 to 4 hours. under laboratory germination tests, treatment of sunflower seeds in these magnetic fields accelerated germination and enhanced seedling length and dry weight. the best results came from applying 50 and 200 mt for two hours. seed coat membrane integrity was improved, cellular leakage was decreased, and electrical conductivity was decreased when seeds were exposed to magnetic fields. in the soil, treated seeds produced 1-month-old seedlings with statistically increased seedling dry weight, root length, root surface area, and root volume. alpha-amylase, dehydrogenase, and protease enzyme activity in the germination of seeds was considerably greater in treated seeds than controls. the quicker germination and early vigor of seedlings may be caused by the more significant enzyme activity in sunflower seeds treated with magnetic fields (vashisth & nagarajan, 2010). magnetic fields (mf) have recently been developed with positive effects on plant germination and growth. the goal is to ascertain the effects of magnetic treatment on the germination of the salvia officinalis l. species and calendula officinalis l. different seed groups were subjected to mf of 125 mt for varying lengths of time. the findings of chronic exposure at 125 mt were the greatest, with mgt significantly reduced compared to controls. the metrics measured for both seeds with treatment and pre-treatment were lower than the value matching the control (treatment 1-50) and markedly decreased (carbonell, florez, martinez, & montoya, 1 paramount school of arts, languages, management, and sciences, incorporated, philippines * corresponding author’s e-mail: alarcon.johnlloydpalmes@gmail.com pa ge 17 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 16-19, 2024 2012). researchers have found evidence that magnetic treatment can preserve crop production and water quality as well as quantity. this study thoroughly reviews studies conducted over the last 20 years on the impact of fields on plants. investigations are conducted into how magnetic fields (mf) affect several elements of plant development, seed germination, yield, quality, and water. the studies’ inconsistent findings and contradicting conclusions suggest that mf’s effect on plants may depend on the species and mf traits like intensity and exposure period (nyakane, marku, & sedibe 2019). in rio & rio’s (2013) study, both exposed and unexposed eggplants were discovered to have identical growth characteristics. additionally, it was shown that plants of okra, tomatoes, and eggplant exposed to emf had fewer insects and pests than plants that were not. further research on the beneficial impact of emf on the growth features of okra while considering other factors is advised. research paradigm care of the crops and waters them. the researcher also ensured that the crops received the right amount of sunlight. every day, the researcher checks the plants to monitor their growth so they may measure it along with the development of the plant’s leaves. statistical analysis statistical analysis is performed using the statistical package for social science (spss) software. the data obtained from the germination rate, mortality rate, sizes of leaves, and length of the plant are subjected to descriptive analysis using the central tendency, the mean score. as for the significance of observed differences, a t-test is utilized. results and discussion descriptive analysis of the growth of the control and experimental groups table 1 shows the bean (phaseolus vulgaris) plant’s growth under normal conditions and magnetic treatment. the unhealthy changes on the leaf and stem started to appear in the second week, especially in the control groups, followed by the experimental (with magnets) groups. at the end of the fourth week of observation, four samples died in the control group, while only one sample died in the experimental group. overall, 26 out of 30 in the control group survived, while 29 out of 30 stayed in the experimental group throughout the research observation. the result of the study implies that the bean plant in the observed growth has a significant growth rate compared to the bean plant samples in the control group. figure 1: the relationship between independent and dependent variables hypothesis of the study h0: there is no significant difference between the control (no magnets) and experimental (with magnets) groups of the string bean (phaseolus vulgaris) plants. materials and methods research design this paper is a quantitative study that utilized an experimental research design to investigate the influence of magnets in the growth of string beans (phaseolus vulgaris) plants. sample description string beans are one of the most widely grown vegetables in the philippines. it is a true legume and botanically more closely related to cowpea. a total of 60 samples of string beans (phaseolus vulgaris) plants were utilized: 30 samples with no magnetic treatment (control group) and 30 samples with magnetic treatment (experimental group). preparation and data gathering procedure the researchers gather essential materials, such as soil, seeds, seedling bags, and magnets, to plant the string bean seed. as the materials needed are prepared, the researchers start cultivating the soil and transferring it into each seedling bag. the researchers count the total number of seeds present and divide them equally into two groups, “with magnetic treatment” and “without magnetic treatment.” the researcher starts to plant the seeds in the corresponding seedling bags. there are 30 planted seeds with the magnetic treatment and 30 without the magnetic treatment. every day, the researcher takes table 1: germination rate and mortality rate of the string bean (phaseolus vulgaris) plants changes control experimental germination rate 26/30 (4th week) 29/30 (4thweek) unhealthy leaf 2/30 (2nd week) 2/30 (3rd week) unhealthy stem 1/30 (1st week) 2/30 (3rd week) plant death 4/30 1/30 seeds under magnetic treatment show 65 – 90 % of seed germination accompanied by high fresh and dry matter compared to seeds without magnetic treatment. it also revealed an increase in cellular metabolism in seeds under the experimental group. the magnetic field treatment accelerated seed germination, boosting seed metabolism (bezerra, ., 2023). in a similar study, the application of magnetic field treatment alters the magnetic character of plants, enhances germination and photosynthetic machinery, and affects the nutrient uptake and tissue production of the plant (ercan, 2022). descriptive analysis of the size of leaves and length of growths table 2 shows the control and experimental groups’ average plant height and size. the samples in the experimental group have more giant leaves (measured from margin to margin; width) than the sample in the pa ge 18 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 16-19, 2024 control group. the experiment group is 24 percent bigger. regarding plant growth, the average height after four weeks is 14.8 cm in the experimental group, 10% higher than the average height of the control group, 13.3 cm. the plant growth is measured from the base of the stem to the top of the plant. this implies that the leaves and growth of the bean plant exposed to the magnets grew more extensive and taller than those without magnets. table 2: size of leaves and length of growth of the string bean (phaseolus vulgaris) plants results size of leaves (cm) length of growth (cm) control experimental control experimental week 1 0.9 1.2 5.3 5.9 week 2 1.8 2.9 10.9 11.8 week 3 2.4 3.1 17.4 19.3 week 4 3.6 4.3 19.5 22.1 mean 2.2 2.9 13.3 14.8 fu (2012) discovered that the plant growth and leaf size indicators of plant health, samples exposed to magnets tend to grow faster, taller, and bigger than those in control groups. in the study of sarraf, kataria, taimourya, santos, menegatti, jain, ihtisham, & liu (2020), the application of pulsating and continuous magnetic fields at low to medium intensity has been shown to impact seed germination and development in some plant species positively. applying magnetic fields in plants has shown an increase in root and shoot growth, an increase in photosynthetic pigment content, and an increase in cell division, which leads to more extensive and taller plants. analysis of the statistical difference between the control and experimental group the table below shows the t-test on the effectiveness of the magnetic treatment in the growth of the bean (phaseolus vulgaris) plant between the control and experimental groups. the result revealed the value of p is 0.0025. the null hypothesis is rejected since it is less than the 0.05 level of significance. this implies a significant difference in the control and experimental groups regarding germination rate, plant growth and leaf size, and plant health. increasing the intensity of the magnetic fields yields significant physiological changes that positively affect the table 3: statistical comparison of control and experimental group using t-test comparison t-value p-value statistical significance decision control vs. experimental 4.05 0.0025 (p < .05) yes reject h0 germination, growth, and health of a plant, contributing to an increase in productivity than without exposure to the magnetic field. compared to traditional chemical methods, which can be expensive, harmful, and require precise application, using magnetic fields to stimulate germination is more straightforward, safer, and costeffective. (morillo-coronado, martínez-anzola, velandiadíaz, & morillo-coronado 2022). a study revealed that magnetic fields significantly boosted plant growth. seeds treated with magnetism had a higher germination rate and produced taller, more robust plants than the control group (fu, 2012). conclusions from the findings above, this study proved that magnetic treatments significantly impact the string bean plant. samples under magnetic field treatment are healthy compared to the samples in the control group. it also proved that there is a difference in the height of the plant and the size of the leaves in the experimental and control groups. magnetic treatment significantly influences the germination and growth of the string bean (phaseolus vulgaris) plant. using magnetic fields as a treatment for plant growth is a relatively new and emerging field with promising potential benefits. while the exact mechanisms are still being researched, studies suggest that magnetic fields can influence various aspects of plant physiology, leading to improved growth and yield. acknowledgments the authors thank the paramount school of arts, languages, management, and sciences incorporated faculty and staff for allowing us to conduct the study inside the school and for support throughout the process. the same gratitude is extended to the following people who played a significant role in the success of the study: clarisa alfeche, alben alfeche, lorena sanico, jonard sanico, welecita paragsa, jojie paragsa, minerva alburo, gerson alburo jr., naome quileste carlos quileste, and lovely rausch. references bezerra, e. a., carvalho, c. p. s., costa filho, r. n., silva, a. f. b., alam, m., sales m. v., dias, n. l., gonçalves, j. f. c., freitas, c. d. t., & ramos, m. v. (2023). static magnetic field promotes faster germination and increases germination rate of calotropis procera seeds stimulating cellular metabolism. biocatalysis and agricultural biotechnology, 49. carbonell, v. m., florez, m., & martinez, e. (2012). effect of magnetic field treatment of germination of medical plants (salvia officinalis, l. and calendula officinalis, l.). polish journal of environmental studies, 21(1), 57-63. da silva, j. a., & dobránszki, j. (2016). magnetic fields: how is plant growth and development impacted? protoplasma, 253(2), 231–248. pa ge 19 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 16-19, 2024 ercan, i., tombuloglu, h., alqahtani, n., alotaibi, b., bamhrez, m., alshumrani, r., ozcelik, s., & kayed, t. s. (2022). magnetic field effects on the magnetic properties, germination, chlorophyll fluorescence, and nutrient content of barley (hordeum vulgare l.). plant physiology and biochemistry, 170, 36-48. fu, e. (2012). the effects of magnetic fields on plant growth and health. young scientists journal, issue 11. maffei, m. e. (2014). magnetic field effects on plant growth, development, and evolution. frontiers in plant science, 5. morillo-coronado, a.; martínez-anzola, h.; velandiadíaz, j.; morillo-coronado, y. (2022). effects of static magnetic fields on onion (allium cepa l.) seed germination and early seedling growth. revista de ciencias agrícolas, 39(1), 30-41. nyakane, n. e., markus, e. d., & sedibe, m. m. (2019). the effects of magnetic fields on plants growth: a comprehensive review. international journal of food engineering, 5(1), 79-87. sarraf, m., kataria, s., taimourya, h., santos, l. o., menegatti, r. d., jain, m., ihtisham, m., liu, s. (2020). magnetic field (mf) applications in plants: an overview. plants, 9(9), 1139. vashisth, a., & nagarajan, s. (2010). effect on germination and early growth characteristics in sunflower (helianthus annuus) seeds exposed to static magnetic field. journal of plant physiology, 167(2), 149-156. pa ge 1 pa ge 8 american journal of life science and innovation (ajlsi) exploring the art of pedagogy in teaching linear programming to support novice performance at munkuye secondary school in nkeyema district: a hermeneutics phenomenological perspective mwambazi chrispine mulenga1*, francis simui2 volume 3 issue 1, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i1.2310 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: december 03, 2023 accepted: january 07, 2024 published: january 11, 2024 the study aimed to explore the art of pedagogy in teaching linear programming to support novice performance. the study was qualitative in nature and hermeneutic phenomenological design guided the study. the study had 16 participants who were selected through homogenous purposive sampling. data was produced through semi-structured interviews and focus group discussion schedules. then generated data was thematically analysed. findings of the study were that comprehensive pedagogy has been essential to secondary school students’ academic achievement and advancement. comprehensive pedagogy is typically demonstrated by the teaching strategies used to improve learning and the resources used in the teaching/learning process. however, most teachers were not employing appropriate methods in teaching linear programming hence poor pupils performance. freeman et al. (2014) supports the use of group work in the teaching/learning process since it encourages students’ social inclusion. make use of interactive techniques such as problem-solving workshops, case studies, and group discussions. participation is encouraged and newcomers can apply lp ideas realistically with the aid of interactive learning. on approaches to problem-solving, offer constructive criticism. beginners should apply lp techniques with plenty of possibilities to practice on a regular basis. keywords linear programming, learner performance, pedagogy ministry of education, curriculum development centre introduction the purpose of this study is to critically explore the art of pedagogy in teaching linear programming to support novice performance at munkuye secondary school in nkeyema district using hermeneutics phenomenology approach. i assert that the adequate information of lp as part of the pedagogical frame work significantly contributes to improved learner performance. this study offers appropriate pedagogic strategies employed in teaching l.p based on available educational references. teaching methods and concepts are based on both artistic and scientific principles. in order to stimulate learning environment, teachers employ art. therefore, the study of instruction and how pupils are taught material is known as pedagogy. the creation aids in the knowledge acquisition of students. howard (2021) applied the idea that pedagogy is similar to learning and teaching as an artistic endeavor. to put it simply, pedagogy is how educators teach linear programming theoretically and practically (howard gardner, 2021). in discourse, the term “pedagogy” has come to refer to instructional practices alone (pritchard & woollard, 2010). teaching, then, is the process of learning by experimentation and many approaches. since science and art are fields that are always changing, the term “pedagogy in education” is now used frequently. the value of pedagogy in instruction pedagogy has the capacity to fundamentally change the rules in the sphere of education. the tools and highlevel efforts needed to give pupils the fortitude to face the future can be found in a variety of pedagogies. one benefit of pedagogy learning is that it enhances education and boosts student performance, which benefits both teachers and learners. students find it difficult to comprehend the ideas of linear programming. to help in idea explanation, the instructor may use a strategy that considers the student’s developmental stage. moreover, in the event that the initial pedagogy fails, a teacher may try a second one (shumba, 1988). context the greek terms “agogos” (leader) and “paidos” (child) are the origin of the english word “pedagogy.” thus, pedagogy is the study of instruction and how students are taught stuff. it facilitates students’ acquisition of knowledge. the notion that pedagogy is comparable to learning and teaching as an artistic endeavor was put into practice by howard (2021). both artistic and scientific elements serve as the foundation for teaching concepts and processes. teachers use art to inspire pupils in an engaging learning environment. children pick up a variety of language and critical thinking abilities in school. teaching is an art, it instructs students and teachers must use the right tools and strategies. peyser (2006) defines pedagogy as the art or science of teaching and educational techniques. teachers’ philosophies influence pedagogy. to assist students 1 munkuye secondary school, nkeyema, zambia 2 institute of distance education, university of zambia, lusaka, zambia * corresponding author’s e-mail: mwambazicm@gmail.com pa ge 9 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 8-15, 2024 learn more effectively and develop higher-order thinking skills, well-thought-out pedagogy is required. the term “instrumental knowledge of pedagogy” is used to lead to scientific study. pedagogy has the capacity to fundamentally change the rules in the sphere of education. one benefit of pedagogy learning is that it enhances education and boosts student performance, which benefits both teachers and learners. a student-centered approach can raise the standard of instruction. it is possible for students to take full responsibility for their education. moreover, according to pedaste et al. (2015), pupils can grasp complex academic ideas at their individual speed. statement of the problem mathematical educators have put a lot of effort into identifying the primary causes of secondary school pupils’ poor linear programming ability. substandard rates of success in linear programming remain a concern despite all these admirable attempts. pedagogy help students improve their linear programming skills. we do not, however, know if students’ actual experiences with linear programming were consistent with educational instruction. this formed the foundation for the current study’s hypothesis. purpose of the study the study’s goal was to explore the art of pedagogy in munkuye secondary school’s linear programming instruction in order to enhance novice performance. research objectives the research objectives are: i. explore exploring the art of pedagogy in teaching linear programming to support novice performance. ii. describe appropriate pedagogic strategies to employ in teaching l.p to support novice performance. significant of the study more study data may be able to assist instructors in refining their methods for teaching linear programming. the study’s findings may be useful in developing inclusive education policy for educational institutions and the ministry of education. the study is significant for educators, students, and the country at large since it has the potential to improve comprehension and boost academic accomplishment. since economists and businesses add value to the nation, the data in this study might also be useful to them in their daily business dealings. methodology research design the study explored students’ lived experiences with their academic performance in linear programming using a hermeneutic phenomenology design. the study was qualitative in character, using methodologies and techniques of qualitative research under the given approach. this is due to its strict adherence to specific methods, like data analysis techniques and sample protocols (mulenga, 2015). additionally qualitative in nature, the study sought to evaluate people’s statements in order to look for supporting information, validate interpretations, and gauge the data’s internal consistency (white, 2018). furthermore, the research was qualitative in nature as it sought to offer a comprehensive comprehension of the low mathematics performance among senior secondary school students in a specific district (harrison, et al., 2017). thus, examine participant perspectives regarding the causes of low math learner performance. research site the current study was conducted at munkuye secondary school in nkeyema district of western province of zambia. target population, sample size and sampling procedure study population the target population of the study was grade twelve pupils who were at munkuye secondary school in 2023. sample size the study had 16 participants comprising 9 females and 7 males. sampling procedure the researcher used a homogenous purposive sample technique to choose study participants who could provide in-depth knowledge about the issue. according to denzin and lincoln (2017), this meant that participants in purposive sampling were selected based on their familiarity with the requested information. put differently, purposive sampling allows the researcher to use their judgment to choose the best volunteers to meet the study’s goals and provide answers to the research questions. according to makondo & makondo (2020), the purpose was to choose participants who will enable the researcher to gain phenomenon, rather than extrapolating findings to a broader population. research instrument, data generation and analysis procedure research instruments the instruments utilized to collect data from participants were semi-structured interview guides and focus group discussion guides, which encourage interaction and dialogue with the participants (wahyumi, 2012). the study findings are more credible when a range of techniques are used, as multiple points of view are used to collaborate and triangulate the facts. in-depth interviews extensive interviews were conducted with the intended audience. one-on-one interviews with participants were conducted while the researcher took notes. to obtain pa ge 10 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 8-15, 2024 insightful information on the research issue, in-depth interviews were employed. the use of this instrument has several benefits. among benefits are the following: in-depth interviews improved secrecy and privacy; participants had the opportunity to ask questions when they were unclear and the researcher had the opportunity to clarify; and the researcher was able to gather a wealth of insightful information. focus group discussions participants in the focus group talks were divided into groups of five. focus group talks promote dialogue and mutual understanding, particularly when tackling challenging topics. it makes it possible to examine both the parallels and contrasts in experiences and ways of thinking (lewis, 2010). this promotes shared perspectives and comprehension. according to moyle (2013), focus group conversations also have the advantage of allowing the researcher to listen to the participants, which empowers them in and of itself. in order to allow participants to go into further detail about topics that came up in the one-onone conversations, the researcher played a moderate role. the instruments utilized to collect data from participants encouraged communication and interaction with the participants (wahyumi, 2012). data analysis given that the study was entirely qualitative, themes were developed by grouping participant ideas that were comparable (mufalo and kabeta, 2019). the generated data was therefore subjected to a thematic analysis. ethical considerations in order to maintain their anonymity, the participants gave the researcher their consent. all participants were also given the assurance that the information collected would be handled strictly, with the utmost confidentiality, and used only for that reason. this was made possible by adhering to the ethical standards put forth by kimmel (2014), which include obtaining ethical clearance, getting participants’ agreement, ensuring their anonymity, and assigning them pseudonyms. as previously mentioned, kimmel’s (2014) highlighted ethical norms were fully taken into account. study findings and discussion the study revealed that pupils’ performance in linear programming was poor due to the methods teachers use. the revelations were established after sixteen (16) participants were interviewed to describe appropriate pedagogic strategies to employ in teaching l.p to support novice performance. as a result, eight (8) students claimed that linear programming was taught incorrectly, five (5) thought it was taught fairly, and three (3) said it was taught properly. this indicates that the pedagogic strategies used in teaching l.p to support novice performance in linear programming were not appropriate. an engaged, immersive, self-paced learning environment is facilitated by the use of pedagogy in instruction. however, it is only feasible when a progressive learning environment is constructed using the fundamentals of pedagogy. teachers can engage students’ interests and establish a forward-thinking learning environment through the use of pedagogy. by employing several instructional techniques, educators can assist learners in progressing at their own rate. however, in order to fully utilize pedagogy in the classroom, educators must employ creative approaches. it is cardinal to indicate that appropriate pedagogic strategies in teaching l.p can support novice performance in linear programming. this is because high quality teaching rests on teachers pedagogic. teachers should understanding the subjects they are teaching by knowing the structure and sequencing of concepts, developing factual knowledge essential to each subject and guiding their pupils into the different ways of knowing. it is also clear that when there is lack of appropriate pedagogy in teaching, pupil’s examination results are at risk. by using step-by-step problem solving, lp issues can be divided into more manageable phases. starting with simple examples and gradually introduce complexity (taha, 2016).thus, the issue of appropriate pedagogy by the teacher benefit the pupil and can enhance good performance, especially linear programming. consistent with these statements fletcher, (2018) issue that students have when learning linear programming teachers who lack creativity and understanding in the techniques they employ, results in subpar exam performance. teachers’ pedagogy was the theme that emerged during the discussions with participants. nama indicated in transcribed verbatim: “teachers who had appropriate pedagogy explained to pupils well and pupils got the concept without difficulties. the teacher who taught suitably made pupils to have an interest in the topic thereby inspiring the pupils to work extremely hard (nama, 10 .11. 2023). according to mwambazi et al. (2022), learner involvement is essential to solving the issue of subpar academic performance in linear programming. active participation is the appropriate approach instead of seeing them as just passengers (wang et al., 2015). similar opinions were expressed by mono, who claimed that meaningful learning is possible when pedagogy is prioritized. “where children understand the explanation of the topic, pupils tend to do better because they have understood and this produces a real desire for learning that should be at the heart of any school’s provision and needs proper teaching methods (mono,10 .11, 2023). all the sixteen (16) participants revealed that learnercentred teaching was the best teaching methodology that foster learning and enhance performance (mtitu, 2014). pupils who focus on what they are taught tend to do better and score more marks. this can only be done if teachers increase their efficiency and effectiveness in teaching. pa ge 11 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 8-15, 2024 wize commented that: “i did not understand linear programming concept because i could understand the way the teacher was explaining the topic. i just learn because i had to. it did not understand the topic; i had a lot of challenges in trying to understand. this was because my teacher failed to teach to help me understand” (wize, 18.11. 2023). by encouraging students to think critically and pay attention during lessons, student-centered techniques improve successful teaching. this method works well in the 21st-century learning environment, which demands more creative and interesting pedagogical approaches that improve conceptual retention, are learner-centered, and support better learning for students in the present (mtitu,2014). yet mungo stated that: “i did not understand anything in linear programming because the teacher was struggling in teaching the topic. i recall the teacher telling the class that don’t waste time answering any question on linear programming. this was so because, teachers lacked appropriate teaching approach (mungo, 25.11. 2023). ansah et al. (2020) discovered that subject-matter expertise of teachers has a major impact in their study. another theme that emerged was pupils’ confidence. taze commented that: “pupils lost confidence in teachers because they appeared to have no content. they were failing to help us understand questions better and apply the concept correctly as they were unable of employing a variety of strategies to retain high performance and good mark” (taze, 22 .11, 2023). the above assertion collaborates with watson (2016) who said that an effective teacher ought to have facts and skills. the skillset comprised computing and numerical abilities; conceptual structures, which were intricately, linked knowledge bases; and general strategies, which are protocols that direct the selection of skills. further, yote stated that: “i had difficulties in following what my teacher was teaching as the lessons were not interactive hence failing to understand the concepts. there was no student participation through discussions and problem-solving exercises. majority of the pupils in class listened to teacher talking (yote, 17.11. 2023). similar observations were made by smasse (2018), who noted that some math teachers continued to teach through lectures and that students were given tightly constructed sentences that they had to learn by heart and repeat back to the teacher when asked. another theme that emerged from the findings was, pedagogy for teaching in the classroom was not suitable. further, masisi stated that: “i had difficulties in understanding what my teacher was trying to teach as most of learners were lost in the process. the lesson was boring to follow. i left the class without getting anything. majority of the pupils in class listened to teacher talking (masisi, 19.11. 2023). these have an impact on students since they will be less adept at addressing problems (kamatchi & stanly 2021). all the sixteen (16) participants revealed that pedagogy for teaching and learning in the classroom involves teacher centred, an approach most teachers used to facilitate learning. using the effective pedagogical strategies in the classroom, teacher can assist students in achieving the learning objectives. by employing this tactic, students can collaborate, think, pair, and share-leveraging their combined knowledge and abilities to complete learning objectives (puteri, 2018). teaching linear programming can be approached in various ways to ensure students understand the concepts effectively. this can only be done if teachers increase their efficiency and effectiveness in teaching. sima stated that: “i did not understand anything. this was as a result of efficiency and effective teaching by my teacher. some pupils showed greater enthusiasm as they value the topic more and studied hard in order to deepen their knowledge” (sima, 30.11. 2023). the aforementioned results are consistent with the assertion made by (gunduz, 2015) that learners who actively participate in the process generate their own meaning and understanding of the subject matter. students should actively build their knowledge and comprehension from their experiences. instructors should facilitate learning by encouraging exploration, problem-solving, and hands-on activities (vygotsky, 1978). appropriate pedagogic strategies to employ in teaching l.p to support novice performance a 2014 study by njoroge on teaching strategies in secondary schools states that there has long been debate about the most effective ways to teach and understand math and science. the teaching approach and style were the main topics of discussion. one specific method that made some students hate the subject was instruction, which keeps them from enjoying their lessons and from benefiting from learning what they know on their own. moreover, the teacher-centered techniques of instruction that are commonly used are not innovative or enthusiastic to help students acquire the skills and knowledge that they need. thus, linear programming performance in secondary schools is deficient (shikuku, 2015). linear programming may not be a challenging topic if teachers can employ workable method that can provide meaningful learning opportunities for pupils to develop understanding of the topic through discovery. teaching linear programming (lp) effectively to novices requires employing pedagogic strategies that are clear, engaging, and practical. conceptual clarity through visual aids teachers should use visual aids, such as graphs, charts, and diagrams, to explain the fundamental concepts of lp. visual representations can enhance understanding and retention (hillier & lieberman, 2014). pa ge 12 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 8-15, 2024 conceptual clarity through visual aids enhances performance across various fields, including education, business, and communication. it can contribute to improved performance as it improves comprehension and retention. visual aids help in simplifying complex concepts, and remember (mayer and anderson 1992). facilitates learning and understanding visual aids, such as diagrams, charts, and info graphics, facilitate active learning. according to sundarararajan (2012), they captivate students by exhibiting material in a more charming and comprehensible. increased engagement visual aids capture attention and maintain engagement. in educational contexts, research by schwartz et al. (2009) highlights that visual aids stimulate interest and help maintain focus during learning. encourage critical thinking encourage novices to be creative when approaching lp problems. foster a problem-solving mind-set that involves exploring multiple solution paths. the effectiveness of these strategies may vary based on the learners’ backgrounds, interests, and prior knowledge. it’s essential to adapt and refine these strategies based on the specific needs of the novice learners (hillier & lieberman, 2014). constructivism certainly! pedagogy for teaching involves various strategies, approaches, and methods that educators use to facilitate effective learning. constructivism theory places a strong emphasis on how students actively create their knowledge and understanding from their experiences. educators facilitate learning by encouraging exploration, problem-solving, and hands-on activities (vygotsky, 1978). learners create their own meaning from the material, (gunduz, 2015). active participation is the appropriate approach instead of seeing them as just passengers (wang et al., 2015). in a similar vein, a social constructivist perspective on education sees teamwork as a novel approach to teaching and learning for both instructors and students (roya et al., 2015). according to social constructivist theory, social input has cognitive development. it highlights how knowledge is jointly produced and formed (eggen & kauchak, 2013). collaborative learning this approach involves students working together in groups to achieve learning goals. it fosters teamwork, communication skills, and peer learning (bruffee, 1999). multiple learners work together, like in small group instruction, and they all contribute and help each other learn (laal & laal, 2012). groups of students collaborate to solve problems, finish tasks, or produce products as part of the educational strategy known as collaborative learning (laal & laal, 2012). this kind of collaboration among learners provides valuable opportunities for individual learners especially those who may be lacking to learn to improve their quality of knowledge which in turn is likely to impact positively on the academic performance (smith & johnson, 2018). inquiry based learning inquiry-based learning investigates, and seeks answers. it promotes critical thinking, problem-solving, and curiosity (national research council, 2000). students address real-world problems, like in project-based learning, by asking questions and doing further research (tsafe, 2013). according to ball (2003) a teacher possessing strong mathematical pedagogical topic expertise can simplify mathematical concepts into less formal, abstract forms, making them understandable to pupils with varying cognitive abilities. by doing this, curiosity is piqued, causing students to appreciate the material that is being provided to them. additionally, because they are drawn in by the presentation, this may cause an attitude shift. hurrel (2013) further argued that in order for society to demand effective learning, quality instruction is both necessary and unavoidable. lee et al. (2019) assert that teachers possess outstanding pedagogical topic knowledge if they are able to instruct the lessons effectively. it is required of teachers to exhibit their understanding of this subject. constant and frequent feedback and remediation from the professors is also crucial. math performance is inevitably improved when students receive feedback and remediation. remediation helps students make up any inadequacies so they can compete on an equal footing with other students. instructors that employ interactive techniques get their students to participate actively. it has been demonstrated that project work, group projects, and discovery approaches are successful ways to teach and learn mathematics. when humor is used as a teaching approach, students perform better because the instruction is engaging and pleasant (ngussa and mbuta, 2017). in contrast, kolb (2014) discovered that students who were taught a problem-solving technique outperformed those who were taught a standard approach in linear programming. students were introduced to autonomous problem-solving strategies with the teacher serving as a facilitator. learner-centered teaching is what this sounds a lot like (mtitu, 2017). similarly, a study on creativity and problem-solving skills among higher secondary students was carried out by kumar, m. (2020). the study’s conclusions showed that higher secondary pupils have a high degree of problem-solving proficiency. in 2021, sathishkumar & prema conducted a study titled “problem solving ability and academic achievement on mathematics among ix standard students”. according to the study’s findings, girls and kids from rural regions were better at addressing problems. it was also found that students in government schools have lower problemsolving skills. these previously mentioned research show pa ge 13 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 8-15, 2024 that an individual’s capacity for problem-solving varies depending on their upbringing. the ability of teenagers at different times to solve problems is resolute by the facilities that are available to them. offering of various pedagogical theories and methods that educators can utilize in their teaching practices. pedagogy evolves, and new research continually contributes to refining these approaches. nonetheless, studies reveal that guided discovery, exposition, question-and-answer sessions, and group projects are the most often employed instructional strategies. conclusion incorrect delivery strategies for the lesson are the cause of students’ subpar performance. poor results are a result of most students not participating in the lesson’s delivery. consequently, educators must meet the needs of each student and ensure that none are left behind. it is important to promote group collaboration in adaptable thought processes and applications. ajogbeje and alonge (2012) assert that teacher competency is essential, stating that educators must exhibit proficiency in all subject areas’ pedagogical content understanding. in agreement with ajogbeje and alonge, lee et al. (2019) assert that teacher pedagogy content knowledge is fundamental since it focuses on how the teacher presents their teachings. facilitators possess outstanding pedagogy content knowledge if they are able to engage learners with their lectures. it is imperative to stress the critical significance that pedagogy plays. it was also found that the student teachers lacked instructional style, linear programming teaching skills, and subject-matter experience. teachers were not using creative teaching strategies to encourage learning linear programming. in particular, using pertinent word problems and incorporating hands-on experiments into math lessons can improve student performance and learning. suitable approaches must be used. recommendations based on the research findings therefore, the study recommended the following: i. in order to improve successful delivery, teachers should use proper teaching techniques, particularly for learner-centered activities where students have a key role. this should also include the materials and strategies for pupils to understand better (kaabo, 2019). ii. to enhance performance, the government, should hire content-based, trained math teachers. iii. to overcome pupils’ fear of mathematics, teachers should encourage them and help them develop a positive attitude toward the subject. iv in order to facilitate the shift from tangible to abstract mathematics, teachers should employ both virtual and concrete manipulatives in their lesson plans. mathematics instruction can benefit from both tangible and digital manipulatives. v. the mathematics curriculum has to be revised by the cdc (2013) to make it more manageable. vi. in order for instructors to exchange current teaching ideas, the district should arrange staff development workshops at the regional, district, cluster, and school levels through the province and district references abrami, p. c., bernard, r. m., borokhovski, e., waddington, d. i., wade, c. a., & persson, t. 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(2018). combining qualitative and quantitative research: semiotics, structuralism and content analysis. in r. nunkoo (ed.), handbook of research methods for tourism and hospitality management (pp. 373-383). edward elgar. https://doi.org/10.4337/9781785366284 pa ge 1 pa ge 28 american journal of life science and innovation (ajlsi) predictive value of rdw/plt for progression of covid-19 pneumonia: a potential biomarker for disease severity kang huang1, salwa m. imran2, wafa mohammad,3 pengfei liu4, yali chao5, suming zhang5* volume 3 issue 1, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i1.2441 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: february 02, 2024 accepted: march 15, 2024 published: march 19, 2024 identifying reliable predictors for covid-19 pneumonia progression is crucial for effective patient management. the purpose of this study is to evaluate the red cell distribution width to platelet ratio (rdw/plt) as a potential predictive biomarker for the development of moderate to severe covid-19 pneumonia. conducting a retrospective analysis from august 2021 to march 2023, we categorized patients with moderate covid-19 pneumonia at admission into different severity groups. the objective was to explore clinical and laboratory variables associated with disease progression. fifty-three patients initially diagnosed with moderate covid-19 were studied, of which 17 progressed to severe disease. univariate logistic regression analyzed various factors, including age, plt, rdw-sd, rdw/plt, ast, alb, crp, and il-6, evaluating their correlation (p < 0.05) with higher odds ratios of a poor prognosis. to ascertain these factors’ predictive power, receiver operating characteristic (roc) curve analysis was used. univariate logistic regression highlighted several factors associated with increased odds ratios for poor prognosis. notably, rdw/plt exhibited the highest predictive value (auc: 0.925, 95% ci 0.858–0.991) among single parameters in predicting the risk of covid-19 progression. this study underscores the potential of rdw/plt as an accessible and cost-effective biomarker for determining covid-19 pneumonia severity. the findings support its utility in risk stratification and clinical decisionmaking, offering valuable insights for effective patient management strategies. keywords covid-19, pneumonia, disease progression, biomarker, clinical decision-making introduction global health has been greatly impacted by the covid-19 pandemic, which has resulted in widespread illness and mortality (cases, 2020; fernandes et al., 2021). the persistent difficulties caused by the covid-19 pandemic highlight the vital need for strong prognostic markers that can enable early identification of patients at risk of severe disease progression; the search for trustworthy indicators to predict disease severity and progression in covid-19 patients remains a crucial focus as the scientific community steps up efforts to unravel the complex pathophysiology of sars-cov-2 infection (chen et al., 2020; sharma et al., 2020; sun et al., 2020). within the cohort of covid-19 patients initially presenting with moderate symptoms, a subset exhibited an alarming deterioration, progressing towards severe disease. notably, an intriguing pattern emerged upon meticulous analysis: those experiencing the severe progression manifested elevated levels of rdw/plt. this observation serves as the fulcrum for exploring the predictive value of rdw/plt in predicting covid-19 severity. rdw/plt, amalgamating red cell distribution width (rdw) and platelet count (plt) measurements, represents a composite metric derived from routine complete blood counts (pulgar-sánchez et al., 2021). the rdw captures the heterogeneity in red blood cell sizes, often indicative of underlying physiological perturbations, while plt reflects the platelet count critical for hemostasis and immune response regulation (ullah et al., 2020; wynants et al., 2020). these parameters, individually recognized for their diagnostic utility across various medical conditions, converge in rdw/ plt, offering a potentially novel prognostic avenue for covid-19. the exploration of rdw/plt as a predictive biomarker holds significant implications for clinical practice. should its predictive potential be validated, rdw/plt could emerge as a non-invasive, cost-effective, and readily available tool for risk stratification among covid-19 patients. its role in the early identification of individuals prone to rapid disease progression may revolutionize clinical decision-making, enabling timely interventions and tailored therapeutic strategies aimed at averting severe complications (albahri et al., 2020; borghesi et al., 2020; tian et al., 2020; ullah et al., 2020). moreover, the integration of rdw/plt into prognostic models may streamline resource allocation within healthcare systems, optimizing the allocation of limited resources toward high-risk patient cohorts. this research endeavor transcends mere association establishment by delving into the temporal dynamics and clinical correlations of rdw/plt alterations concerning covid-19 progression. understanding whether changes in rdw/plt precede the onset of severe symptoms or coincide with disease exacerbation remains a pivotal 1 department of emergency intensive care unit, affiliated hospital of xuzhou medical university, jiangsu, china 2 xuzhou medical university, jiangsu, china 3 king abdulaziz university hospital, jeddah, saudi arabia 4 shandong first medical university, shandong, china &department of anesthesiology, the affiliated hospital of xuzhou medical university, jiangsu, china 5 department of intensive care unit, affiliated hospital of xuzhou medical university, jiangsu, china * corresponding author’s e-mail: sumingzhang18@outlook.com pa ge 29 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 28-36, 2024 aspect to unravel for effective predictive utility. the nuances of rdw/plt kinetics throughout the disease course may provide critical insights into the trajectory of covid-19 and guide intervention strategies. additionally, investigating demographic variations and potential associations with comorbidities concerning rdw/plt elevations in covid-19 patients presents an intriguing avenue. determining whether specific patient subgroups exhibit distinct rdw/plt profiles could facilitate personalized risk assessment, tailored monitoring protocols, and targeted interventions. this pursuit aligns with the broader goal of precision medicine, aiming to tailor treatments based on individual patient characteristics and disease progression patterns. this study aims to scrutinize the red cell distribution width to platelet ratio (rdw/plt) as a potential harbinger of the progression of covid-19 pneumonia from moderate to severe stages. amid the diverse clinical manifestations and trajectories observed in covid-19 cases, identifying specific markers that herald a transition to severe illness holds paramount importance in clinical management and resource allocation. this study endeavors to bridge an essential knowledge gap in understanding covid-19 prognosis by exploring rdw/plt as a potential predictive biomarker. beyond establishing its association with disease progression, the comprehensive investigation aims to lay the groundwork for its potential clinical utility, stressing the necessity for robust validation and integration of rdw/plt into existing prognostic frameworks. literature review covid-19 pathophysiology and prognostic markers the clinical spectrum of covid-19, which is caused by sars-cov-2, is varied and ranges from mild respiratory symptoms to severe pneumonia and multiorgan dysfunction. finding trustworthy prognostic markers requires an understanding of the complex pathophysiological mechanisms guiding the course of the disease (huang et al., 2020; zhang et al., 2020). research into markers that reflect this dysregulation has been prompted by emerging evidence that points to a dysregulated immune response and an elevated inflammatory cascade as indicators of severe disease (levi et al., 2020; tay et al., 2020). red cell distribution width (rdw) and platelet count (plt) in disease evaluation rdw, traditionally a marker of red blood cell size heterogeneity, has garnered attention beyond its conventional utility. it has been linked to systemic inflammation and adverse outcomes in various pathological conditions (lippi & mattiuzzi, 2020; patel et al., 2015). in the context of covid-19, elevated rdw levels have been observed and correlated with disease severity, possibly reflecting underlying inflammatory processes and physiological stress (foy et al., 2020a; lippi & plebani, 2020). similarly, alterations in platelet count (plt) have been implicated in covid-19 pathogenesis, with thrombocytopenia often associated with severe disease and adverse clinical outcomes (althaus et al., 2021; xu et al., 2020). rdw/plt ratio as a potential biomarker the integration of rdw and plt into the rdw/plt ratio represents a composite marker that may hold significant prognostic value in covid-19. previous studies across various medical conditions have demonstrated the potential of rdw/plt as a prognostic indicator, reflecting systemic inflammation and disease severity (zhou et al., 2020; zorlu et al., 2012). applying this ratio specifically in covid-19 patients presents a novel approach to anticipate disease progression and severity. clinical studies investigating rdw/plt in covid-19 rdw/plt’s function in covid-19 prognosis has been specifically examined in recent clinical studies. as an illustration of its potential as a predictive marker for disease severity, a study by wang et al., 2020 found that severe covid-19 cases had noticeably higher rdw/plt ratios than moderate cases (wang et al., 2022). similarly, in a retrospective analysis, kilercik et al., 2021 found that in covid-19 patients admitted to intensive care units, high rdw/plt ratios were associated with unfavorable outcomes and severe disease (kilercik et al., 2021). mechanistic insights and future directions elucidating the mechanistic underpinnings of the association between elevated rdw/plt ratios and covid-19 severity remains a critical area for exploration. factors such as the inflammatory milieu, endothelial dysfunction, and potential interactions between platelets and red blood cells might contribute to alterations in rdw/plt ratios (henry, aggarwal, et al., 2020; lippi & favaloro, 2020). future research endeavors should focus on unravelling these mechanisms and conducting prospective trials to validate rdw/plt as a robust prognostic marker in covid-19. materials and methods study participants retrospective review of patient medical records from august 1, 2021, to march 1, 2023, at the third people’s hospital of yangzhou and the affiliated hospital of xuzhou medical university was part of the study. included were patients who had been diagnosed with covid-19 and initially classified as having a moderate illness. according to the world health organization’s interim guidelines, confirmation of covid-19 depended on positive results from high-throughput sequencing or real-time reverse transcription-polymerase chain reaction (rt-pcr) assays of nasal and pharyngeal swab specimens. following admission, patients were observed for at least 14 days to determine whether severe pneumonia had developed. pa ge 30 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 28-36, 2024 ethical approval the affiliated hospital of xuzhou medical university’s ethics committee approved the study protocol (protocol id: xyfy2022-kl034-01). data collection standardized data collection forms were used to gather information from electronic medical records about the following: demographics (age, gender), comorbidities (diabetes, hypertension, coronary heart disease), and laboratory parameters (such as white blood cell wbc, lymphocyte ly, eosinophil cell eos, platelet plt, mean platelet volume mpv, red cell distribution width rdw, red cell distribution width to platelet count ratio rdw/plt, aspartate aminotransferase alt, alanine aminotransferase alt, albumin alb, creatinine crea, c-reactive protein crp, and interleukin-6 il-6). patients were categorized into the progressive group (17 cases) and the stable group (36 cases) based on the development of severe illness within 3 days post-sample collection. patients already categorized as severe at admission were excluded from statistical analyses. clinical classification of covid-19 pneumonia based on the severity of their illness, patients with covid-19 pneumonia were divided into various clinical groups in this study. a decrease in oxygen saturation, aberrant chest imaging, or the manifestation of covid-19 symptoms without dyspnea were among the characteristics that classified the condition as mild. people who showed signs of a lower respiratory disease but were still able to maintain a sufficient oxygen saturation (spo2) of ≥94% on room air at sea level were classified as having a moderate illness. individuals classified as having a severe illness included those with heart rates under 94%, arterial partial pressure of oxygen to fraction of inspired oxygen (pao2/fio2) ratios less than 300 mm hg, elevated respiratory rates greater than 30 breaths per minute, or lung infiltrates greater than 50%. these categorizations were crucial in distinguishing and describing the different levels of severity among the covid-19 pneumonia cases in this investigation. statistical analysis all statistical analysis was performed on spss 26.0 and graphpad prism 8.0. the formats for descriptive statistics are mean ± sd, median (iqr), and n (%). the chi-square test or fisher’s exact test were used to analyze categorical variables, and the student’s t-test or mann-whitney u-test were used to analyze continuous variables. if the two-sided p value was less than 0.05, it was considered statistically significant. using single-variable logistic regression based on the likelihood ratio, risk factors were found, and these risk components were then combined to create a forest plot. the area under the curve (auc) was used to evaluate discrimination performance. results patient characteristics and group classification the rigorous application of inclusion criteria yielded a cohort of 64 individuals diagnosed with covid-19. following careful screening, 11 patients were excluded due to severe/critical covid-19 status at admission or being below 18 years of age, resulting in a final study population of 53 patients. among these, a subgroup of 17 patients experienced a distressing progression to severe pneumonia during their hospital stay, prompting a comparative evaluation to discern distinguishing clinical features, as shown in figure 1. figure 1: the flow diagram shows the study population enrollment pa ge 31 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 28-36, 2024 comparison between stable and progressive patients a thorough comparison of the clinical and demographic traits of patients who developed severe covid-19 and those who kept their condition stable is shown in table 1. interestingly, the mean age of patients in the progressive group was 7.88 years ± 14.66 years, which was significantly higher than the mean age of patients in the stable group (58.97 years ± 18.94) (p = 0.021). moreover, individuals with underlying coronary heart disease were notably more prevalent within the progressive group (41.2%) than in the stable group (2.8%) (p = 0.001). notably, various laboratory parameters manifested pronounced differences between the groups, with significantly higher levels of rdw-sd, rdw/plt, ast, crea, crp, and il-6 observed in the progressive cohort (all p < 0.05). table 1: characteristics between the stable and progressive patients characteristic stable group progressive group p gender, n (%) male 15 (41.7%) 11 (64.7%) 0.117 female 21 (58.3%) 6 (35.3%) age, years 58.97 ± 18.94 70.88 ± 14.66 0.021 times of vaccination 0 19 (52.8%) 8 (47.1%) 0.832 1 6 (16.7%) 4 (23.5%) 2 11 (30.6) 5 (29.4) comorbidities, n (%) diabetes 4 (11.1%) 4 (23.5%) 0.443 hypertension 12 (33.3%) 5 (29.4%) 0.775 coronary heart disease 1 (2.8%) 7 (41.2%) 0.001 laboratory findings wbc (×109/l) 4.57 ± 1.56 4.61 ± 1.81 0.929 ly (×109/l) 1.18 ± 0.53 0.89 ± 0.36 0.051 eos (×109/l) 0.01 (0.00-0.08) 0.00(0.00-0.00) 0.003 plt (×109 /l) 146.50 (124.25-193.25) 92.00 (66.50-106.5) 0.000 mpv (fl) 11.59 ± 1.13 11.98 ± 1.08 0.240 rdw-sd 40.58 ± 2.35 42.98 ± 2.15 0.001 rdw/plt (×10-2) 27.25 (20.43-33.60) 46.80 (38.95-64.95) 0.000 ast (u/l) 21.50 (18.50-34.00) 50.20 (29.80-88.40) 0.000 alt (u/l) 25.89 ± 21.85 38.25 ± 21.39 0.059 alb (g/l) 44.11 ± 3.37 38.91 ± 3.81 0.000 crea (umol/l) 68.50 (61.00-88.00) 90.00 (64-113) 0.038 crp (mg/ l) 22.92 (9.43-33.07) 58.00 (39.35-91.70) 0.000 il-6 (pg/ml) 21.85 (13.75-32.85) 81.50 (39.35-115.30) 0.000 predictors of progression from moderate to severe covid-19 univariate logistic regression analysis illuminated several potential predictors of disease progression, unveiling age, plt, rdw-sd, rdw/plt, ast, alb, crp, and il-6 as significantly associated with the transition to severe covid-19 pneumonia, as shown in table 2. the construction of a forest plot, which is shown in table 2: univariate logistic regression analysis independent high-risk factors for severity of covid-19 patients variables or (95%ci) p value age 1.05 (1.00-1.09) 0.029 eo 0.00 (0.00-455.76) 0.349 plt 0.94 (0.90-0.97) <0.001 rdw-sd 1.05 (1.02-1.08) 0.004 rdw/plt 1.21 (1.08-1.36) <0.001 ast 1.04 (1.02-1.07) 0.002 pa ge 32 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 28-36, 2024 figure 2, further underscored the prominence of rdw/ plt, il-6, crp, rdw-sd, age, and ast as prominent predictors in delineating disease severity. predictive value of rdw/plt intriguingly, receiver operating characteristic (roc) curve analysis highlighted the remarkable predictive capacity of rdw/plt in forecasting the risk of progression in covid-19 patients. with an impressive area under the curve (auc) of 0.925 (95% ci 0.858–0.991), rdw/plt emerged as the most robust single parameter. utilizing a cutoff value of 0.355, rdw/plt showcased a high sensitivity of 94.12% and specificity of 80.56% in predicting the risk of progression, which is shown in figure 3. alb 0.68 (0.54-0.84) <0.001 crea 1.02 (1.00-1.04) 0.052 crp 1.06 (1.03-1.10) <0.001 il-6 1.07 (1.03-1.12) <0.001 figure 2: forest plot figure 3: roc curve for the prediction of developing severe covid-19 pneumonia interpretation and implications these findings underscore the clinical relevance of specific hematological and demographic markers in predicting the trajectory of covid-19 pneumonia. notably, the identification of rdw/plt as a robust predictor of disease progression signifies its potential utility as an early prognostic marker, allowing for prompt interventions and focused clinical management. discussion according to the study’s findings, the red cell distribution width to platelet ratio (rdw/plt) may prove to be a useful biomarker for determining when covid-19 pneumonia will progress from a moderate to a severe stage (alballa & al-turaiki, 2021; ghahramani et al., 2020; yamada et al., 2020). since elevated rdw/plt values have been associated with disease progression, pa ge 33 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 28-36, 2024 physicians may be able to use them as a preemptive measure to identify patients who are more likely to develop severe illness in the future (alnor et al., 2020; gong et al., 2021). early detection could result in targeted therapy, attentive observation, and intervention strategies that could enhance patient outcomes (bonetti et al., 2020). the study’s findings indicate that the red cell distribution width to platelet ratio (rdw/plt) may be a valuable biomarker for predicting how covid-19 will progress. numerous studies have reported a relationship between rdw and disease severity for a range of medical conditions, including cancer, pulmonary disorders, and cardiovascular diseases (foy et al., 2020b). rdw is a measure of the heterogeneity in red blood cell size that is caused by oxidative stress, inflammatory reactions, and decreased erythropoiesis. within the framework of covid-19, the noted increase in rdw could be linked to the inflammatory reaction and subsequent cytokine storm, which could lead to modifications in red blood cell properties (liu et al., 2020; pongpirul et al., 2020). furthermore, the correlation between rdw/plt and the severity of the covid-19 virus is consistent with decreased platelet counts, which are frequently observed in cases of severe viral infections (ponti et al., 2020). this study’s findings align with previous investigations highlighting the role of rdw and platelet count as markers of systemic inflammation and hematological abnormalities (c. wang et al., 2020). elevated rdw has consistently been linked to adverse outcomes in diverse medical conditions, including cardiovascular and respiratory disorders (yuan et al., 2020). similarly, alterations in platelet count have been associated with inflammatory responses and endothelial dysfunction (li et al., 2020). this study contributes by emphasizing the utility of the composite rdw/plt ratio as a potential biomarker, specifically within the context of covid-19 pneumonia (ballaz et al., 2021). a plausible underlying mechanism for the observed association involves the systemic inflammation and oxidative stress triggered by sars-cov-2 infection (shiri et al., 2021). the virus-induced inflammatory response might lead to hematological changes, affecting red cell distribution and platelet activation (khosravi et al., 2021). the rdw/plt ratio could capture these underlying pathophysiological processes, serving as an indicator of disease severity (x. wang et al., 2020). furthermore, leveraging the rdw/plt ratio as a predictive tool may contribute to personalized approaches to managing covid-19. table 1 and table 2 in the current study elucidate demographic, clinical, and laboratory characteristics associated with the progression of covid-19 pneumonia. similar analyses in previous studies have often highlighted age, comorbidities, and specific laboratory parameters, such as inflammatory markers (e.g., crp, il6), as crucial predictors of disease severity (tjendra et al., 2020). however, the current study emphasizes the rdw/ plt ratio, which might have been less extensively in prior literature for covid-19 severity prediction. figure 2, displaying a forest plot of predictors, echoes findings from previous studies, often revealing multiple factors associated with disease severity. previous studies have reported diverse predictors, ranging from inflammatory markers to specific clinical parameters, consistently emphasizing the multifactorial nature of disease progression (krintus et al., 2014). figure 3 exhibits roc curve analysis, demonstrating the predictive performance of individual parameters for severe covid-19 pneumonia. prior research often presents roc analyses assessing the diagnostic or predictive accuracy of various markers, with auc values serving as indicators of predictive strength. in comparison to previous studies, the current study highlights the rdw/plt ratio as a potential strong predictor for disease progression (cai et al., 2019). the study highlights the potential of this readily available and cost-effective measure to aid risk classification and guide clinical decision-making (henry, de oliveira, et al., 2020). however, validation of these findings, exploring underlying mechanisms, and assessing the rdw/plt ratio’s value in larger patient cohorts necessitate further investigation. the prospective application of the rdw/ plt ratio in clinical practice holds promise for enhancing patient outcomes in covid-19 pneumonia. conclusion this study concludes that elevated rdw/plt values are linked to the advancement of illness, indicating the practicality of utilizing this readily available parameter in clinical settings. identifying patients at higher risk of developing severe illness early on could facilitate timely interventions, enhanced surveillance, and more effective allocation of healthcare resources. it provides initial evidence supporting the potential of the rdw/plt ratio as a prognostic biomarker in predicting the progression of covid-19 pneumonia from moderate to severe stages. strengths and limitations the strength of this study is that it investigates rdw/ plt as a potential predictor for covid-19 pneumonia progression using robust statistical analyses. clear inclusion criteria and focus on moderate covid-19 cases enhance specificity. the limitations are retrospective design and small, singlecenter sample size, which may introduce biases and limit generalizability. unaccounted confounding factors and the need for prospective validation in larger cohorts pose limitations. acknowledgements conception and design of the study: suming zhang. acquisition of data: suming zhang, pengfei liu, salwa mohammad imran. analysis and interpretation of data: suming zhang. drafting the article: salwa mohammad imran, wafa mohammad. revising it critically for pa ge 34 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 28-36, 2024 important intellectual content: suming zhang, salwa mohammad imran. final approval of the version to be submitted: all authors contributed to the final version. suming zhang takes responsibility for the paper as a whole. funding this work was supported by the wu jieping medical foundation’s special fund for clinical research under grant (320.6750.2021-08-3) and the xuzhou youth science and technology talent project under grant (kc2204). declaration of interest the authors report that there are no competing interests to declare. references albahri, o. s., al-obaidi, j. r., zaidan, a., albahri, a. s., zaidan, b., salih, m. m., qays, a., dawood, k. a., mohammed, r., & abdulkareem, k. h. 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(2020). clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet, 395(10229), 1054-1062. zorlu, a., bektasoglu, g., guven, f. m. k., dogan, o. t., gucuk, e., ege, m. r., altay, h., cınar, z., tandogan, i., & yilmaz, m. b. (2012). usefulness of admission red cell distribution width as a predictor of early mortality in patients with acute pulmonary embolism. the american journal of cardiology, 109(1), 128-134. pa ge 1 pa ge 25 american journal of life science and innovation (ajlsi) preparation and characterization of zinc oxide nanoparticles by co-precipitation method and evaluation of theirs antifungal activity in spore germination of dermatophytes hussein a. r. abbood1, salah s. zainalabden1 volume 3 issue 2, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i2.3183 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: june 20, 2024 accepted: july 24, 2024 published: july 27, 2024 this research study synthesized prepared zinc oxide nanoparticles using the co-precipitation method. the antifungal activities of these nanoparticles (zno nps) and their mode of action against trichophyton rubrum and microsporum canis were investigated using samples obtained from patients referred to consulting clinics in kirkuk city. these nano oxides were identified through xrd diffraction analysis, ftir measurement, and scanning electron microscopy (sem). the minimum inhibitory concentration (mic) and minimum fungicidal concentration (mfc) values were recorded as 1.330 and 4.530 µg/ml for trichophyton rubrum, and 0.333 and 10.67 μg/ml for microsporum canis, respectively. the antifungal effect of (zno nps) was similar to that of griseofulvin, while it exhibited a higher effect than ketoconazole. furthermore, (zno nps) demonstrated a significant (p < 0.05) inhibition effect on spore germination for all tested dermatophytes, although the extent of this effect varied depending on the fungal isolates. keywords dermatophyte, spore, nanoparticle, zno, antifungal introduction dermatophytes, a large group of fungi responsible for causing dermatophytosis or ringworm(bouchara &mignon, 2017). this group includes three main genera: trichophyton, microsporum, and epidermophyton spp (aljanabi, 2014). griseofulvin is considered one of the most widely used treatments for dermatophytosis(gupta et al., 2009) (barros et al., 2007), and therefore it was very important to increase its effectiveness through the development of nanoparticles(zili z et al., 2005). solid nanoparticles loaded with griseofulvin in the form of a gel showed very, very effective pharmacological capabilities against the pathogenic fungus(aggarwal &goind, 2013). microsporum canis zinc oxide nanoparticles (zno-nps) showed an effective effect against dermatophytes and show stronger inhibition compared to antifungals(ahmadpour et al.,2021) (el-diasty et al., 2013) many antifungals have shown resistance against skin fungi, which is considered a major challenge(elad et al., 1992) therefore, it was important to prepare drugs for pathogenic fungi that replace current medications. therefore, researchers have paid great attention to nanoparticles due to their high effectiveness due to their unique physical and chemical properties(vatsha b et al., 2013). many researches have indicated there are many different types of nanomaterials, including silver nanoparticles(abbood & a. h. s, 2020) and zinc nanoparticles(liu et al., 2009), which have shown antifungal and antibacterial activity. zinc oxide nanoparticles (zno nps) are known for their ability to create large surface areas and unique crystalline structures. these metal oxides are highly ionic(khaleel et al.,1999), unlike organic materials, possess exceptional durability, selectivity, and thermal resistance. incorporating zinc oxide into daily zinc supplements is common due to its vital role in maintaining human health. additionally, zno nps have shown compatibility with human cells (padmavathy &vijayaraghavan, 2008). the antimicrobial properties of zno bulk powder have been extensively studied, demonstrating its effectiveness against bacteria and fungi. in the agricultural industry, zinc compounds are primarily used as fungicides (bloom & markson., 2001) . materials and methods isolation and diagnosis of fungi samples were obtained from patients who were referred to consulting clinics in the city of kirkuk. the samples were collected either by scraping from the affected area or by cutting the affected part of the hair and nails. afterward, the samples were treated with 10% koh to analyze the keratinous material present. this analysis aimed to identify any highlights and spores. the isolated samples were then transferred to sterile petri dishes containing sabouraud’s dextrose agar (sda) medium, supplemented with 50 mg of the antibiotic chloramphenicol. this was done to facilitate the growth of fungi without any interference from bacteria and yeasts. subsequently, the isolated samples were sent to the mycology laboratory at the alqalam of the university. they were placed in an incubator set at a temperature of 27-30°c for a duration of two weeks. following the incubation period, the colonies were carefully tested (figure 1), and additional tests were conducted to confirm the diagnosis (table 1) (khan et al., 2021). 1 department of biology, college of education for pure sciences, kirkuk university, kirkuk, iraq * corresponding author’s e-mail: hussenabdulrazzaq@uokirkuk.edu.iq pa ge 26 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 25-32, 2024 inoculum preparation in light of the need to obtain reproducible and comparable results in antifungal susceptibility testing, it is important to standardize the inoculum. based on the clsi guidelines, the tested dermatophyte inoculum suspensions were made (wang et al., 2018). to induce sporulation, colonies grown on pda for 7-15 days were flooded with 10 ml of distilled water. the use of a sterile loop to mechanically break up the colonies yielded a suspension that contained some tiny fragments of hyphae and conidia. this mixture was then aliquoted into sterile tubes and allowed to settle at room temperature which facilitated sedimenting of larger particles. the suspension was then diluted to a final conidia concentration of 1-3 × 103 cells/ml using a haemocytometer according to clsi m38-a2. the dilution and preparation of stock solution of the antifungal agents in performing the comparison with (zno nps), commercially available antifungal reference powders of griseofulvin and ketoconazole were used to prepare stock solutions. another accurate way of measuring the antifungal powders was by using an analytical balance that measures up to 0. 0001g, according to the authors of the paper by (wang et al., 2018). deionized distilled water was employed for dissolving griseofulvin, ketoconazole, and (zno nps) to prepare stock solutions. to reach the final strength as required, further dilutions were made on the broth medium, to attain twice the strength. subsequently, a twofold serial dilution was performed to obtain the final concentration range of the drugs: it was found to be 0.04-19 µg/ml for ketoconazole and 0. 136-59 µg/ml for griseofulvin and 0.171-75 µg/ml for (zno nps). preparation of zinc oxide nanoparticles by coprecipitation method a solution of 0.4 molar aqueous zinc acetate (zn(ch3coo)2 2h2o) was created by dissolving 9.2 g of the compound in 100 milliliters of non-ionic water and allowing it to dissolve completely on a magnetic stirrer for 30 minutes. sodium hydroxide with a concentration of 1 molar was then added drop by drop until reaching a ph of 9. the solution was stirred at 70°c for 2 hours until a precipitate formed. the precipitate was filtered, washed with nonionic water and ethanol, and then dried at 80°c for 30 minutes. then, the precipitate was burned at 500°c for 5 hours and the process was further continued for desiccation of the precipitate at room temperature for overnight (khan et al., 2015) . zn(ch3coo)2·2h2o+ 2naoh →zno + 2ch3coona + 3h2o table 1: shows species and fungal species isolated from spices mold type hair penetration test growth at a temperature of 37°c diagnosis of large conidia diagnosis of small conidia rice test characteristics of fungal culture m.canis + fusiform, thick, rough-walled, with a curved or hooked end it is not found in abundance, and if it is found, it has a club shape yellow colony tapering at both ends; spindle-shaped t.rubrum + it takes the form of a cigar or a pen club-shaped to barrel-shaped, growing along the spindle filaments the colony gives off a red dye. below the plate, the conidia are club-shaped and the hyphae are tree-shaped figure 1: colony morphology of the a. t.rubrum and b. m.canis isolate pa ge 27 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 25-32, 2024 nanoparticle characterization techniques based on zinc oxide nanoparticles are being prepared fourier transform infrared spectroscopy (ftir) spectroscopy of infrared radiation was used to analyze ftir samples and the presence of functional groups and various features of the nanocomposite was studied (fadlelmoula et al., 2022). scanning electron microscope (sem) analysis it is applied for the average particle size analysis of the nanocomposite and the structure shape analysis. xrd was also employed to determine what elements are present in the sample. the results were taken and put down in pictures (xiong et al., 2020) x-ray diffraction analysis (xrd) analysis based on x-ray diffraction method is very popular technique for testing and determining the crystal structure of nanomaterials by using an xrd tool and it is a non-destructive way to characterize the physical properties, chemical composition and crystal structure, in addition atomic arrangement and thickness could be determine ( zhang et al., 2023). determination of mic values for dermatophytes the mic (zno-nps) and other antifungal drugs considered were assessed against the fungal strain by employing broth microdilution method. the method followed clinical laboratory standards institute (clsi) guidelines wherever possible, as described in filamentous fungi document m38-a2 (badiee et al., 2013). the minimum fungicidal concentration (mfc) when testing mic, the minimum fungicidal concentration (mfc) of zno nps against the dermatophytes studied was determined. for comparison, the bactericidal efficacy of griseofulvin and ketoconazole was also examined. samples from wells with no visible growth were then transferred to sda plates. additionally, positive controls (from growth control wells) and negative controls (from sterile control wells) were included in the experiment. the impact of biosynthesized zinc oxide nanoparticles (zno nps) on the germination of dermatophyte spores was assessed using as microscopic method to generate the conidia, the isolates were cultured on potato dextrose agar (pda) for 10-14 days, following the specific requirements of each dermatophyte genus. the resulting suspension contained both conidial and mycelial fragments. the spore count for each dermatophyte isolate was maintained at 40-60 spores per microscopic field. in order to interact with the zno nps, the spore suspension for each isolate was prepared, resulting in a concentration of 20-30 spores per microscopic field. the tubes containing the culture mixture were then placed in a shaking incubator at 35ºc for trichophyton spp. or at 30ºc for microsporum spp. to ensure even distribution of the zno nps. the germination of spores was studied through the daily microscope examination of the sample. the spore was deemed germinated once the length of the germ tube reached 1.5 times the diameter of the spore (plascencia‐ jatomea et al. ,2003). the antifungal drugs and sterile broth were used as substitutes also to the (zno nps) in the positive and negative controls. all the treatments were performed in three replications. the percentage of spore germination inhibition was determined with the help of the formula: % spore germination inhibition = sc ct/ sc × 100 *sc: average of spores germinated in control. *ct: average of spores germinated in treatment. results and discussion diagnosis of nano-zinc oxide by xrd the x-ray diffraction analysis was conducted to characterize the crystal structure and phase purity of the synthesized zno nanoparticles. figure (2) illustrates that the x-ray diffraction spectrum of the zinc oxide sample aligns with the standard spectrum of zinc oxide as per the database (zno jcpds 36-1451) (wirunchit et al., 2021). figure 2: x-ray diffraction (xrd) of silver nanoparticles ( zno nps ) pa ge 28 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 25-32, 2024 ftir measurement results for the prepared oxides the infrared spectra of zn(ch3coo)2.2h2o, which is the aqueous zinc acetate salt, are utilized in the preparation of nano-zinc oxide prior to conducting the reaction. this information is depicted in figure (3). the results obtained from the experiment lead us to the conclusion that the protein present in the reaction medium serves as a reducing agent and forms a protective layer around the biosynthesized zno nps, known as capping proteins. this capping protein plays a crucial role in preventing the undesirable agglomeration of particles in the medium, thereby ensuring the formation of highly stable zno nps(hadi&kadhim, 2019). one significant advantage of using capping protein over polymer and surfactant, which are commonly used as capping agents in the preparation of zno nps, is that it is cost-effective, safe, eco-friendly, and does not require any special conditions. additionally, capping protein acts as an anchoring layer for the transportation of drugs or genetic materials into human cells (hu et al., 2011), surpassing the capabilities of surfactant and polymer. moreover, the presence of a non-toxic protein cap enhances the uptake and retention of these materials inside human cells (rodríguez et al., 2013). figure 3: ftir spectrum of zno nps figure 4: scanning electron microscope (sem) of zno nps diagnosis of nano-zinc oxide by sem the scanning electron microscope (sem) was utilized to observe the morphological and structural properties of nanozinc oxide (zno). the nanometer range preparation of nanoparticles is evident in figure (4). the sem images revealed that certain nanoparticles were adequately dispersed, while the majority were found in agglomerated form. the agglomeration is attributed to both electrostatic effects and the presence of the aqueous suspension, which demonstrates the behavior of nanoparticle agglomeration. this finding aligns with previous studies on nanoparticle agglomeration (s zainal-abddeen et al., 2017) (gaikwad et al., 2015). determination of mic and mfc value against dermatophytes the antifungal efficacy of zno nps was studied against trichophyton rubrum and microsporum canis. griseofulvin and ketoconazole, commonly used antifungal medications, were employed as positive controls for comparison. the growth inhibition effect of zno nps on the mentioned fungi was determined through the broth microdilution method to establish the mic. the mic values for the tested substances are detailed in table 2. the findings indicated that zno nps exhibited antifungal properties against all tested dermatophytes within the range of 0.171-75 µg/ml, with varying susceptibility levels based on the fungal species. griseofulvin, with an mic range pa ge 29 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 25-32, 2024 of 0.136-59 µg/ml, only demonstrated antifungal activity against m. canis and t. rubrum, with mic values of approximately 10 µg/ml and 32 µg/ml, respectively. ketoconazole, having an mic ranging from 0.04 -19 µg/ ml, exhibited a comparable antifungal effect to that of zinc oxide nanoparticles (zno nps). statistical analysis indicated no significant variance between the two. the most effective mic of ketoconazole, at 0.6 µg/ml, was observed against m. canis. the biological activity of zinc oxide is attributed to the disruption of membrane lipids, which led to leakage of cell contents and ultimately cell death (xie et al.,2011) zinc oxide can also generate hydrogen peroxide and zn+2 ions, which cause cell death through a change in cellular metabolism. preliminary studies indicate that the antimicrobial properties of zno nps are due to the formation of free radicals on the nanoparticle surface. these free radicals are thought to damage lipids in the bacterial cell membrane, causing the membrane to leak and rupture (he et al., 2017) (reddy et al., 2007). table 2: comparative mic value of biosynthesized zno nps, griseofulvin and ketoconazole against t.rubrum and m.canis tested fungal strains mic/mean(µg/ml) zno nps 0.171-75 griseofulvin (0.136-59) pvalue (zno nps) 0.171-75 ketoconazole (0.04-19) pvalue t.rubrum 1.330 10 0.0439 1.330 1 >0.05 m.canis 0.333 32 <0.001 0.333 0.6 >0.05 the antifungal properties of zno nps and standard antifungal medications were evaluated concurrently with their minimum inhibitory concentration against the dermatophytes being studied. figures (5) and (6) show that the mfcs obtained are significantly higher than the mics. t.rubrum was found to be most susceptible to zno nps, with a minimum fungicidal concentration (mfc) of 4.530 µg/ml, compared to an mfc of 35 µg/ml for griseofulvin and 4 µg/ml for ketoconazole. for m.canis, the mfc of zno nps was 10.67 µg/ml, while the mfc of ketoconazole was 3 µg/ml. figure 5: sensitivity profile of t.rubrum to the antifungal agents figure 6: sensitivity profile of m.canis to the antifungal agents pa ge 30 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 25-32, 2024 effect of zno nps on spore germination of dermatophytes the impact of zno nps on the germination of spores from various dermatophytes is outlined in table (4) the study demonstrated that zno nps exhibited a notable and significant inhibitory effect on spore germination (p < 0.05) in all the dermatophytes tested. the impact of zno nps on fungal strains differed in magnitude, as indicated in table 4. additionally, the majority of germinated spores exhibited morphological transformations, including spore enlargement. these alterations align closely with the fungistatic properties of zno nps. consequently, it is postulated that the mechanism behind the fungistatic effect involves modifications in the osmotic balance and permeability of the spores (khalil et al., 2013). table 3: comparative mfc value of zno nps, griseofulvin and ketoconazole against t.rubrum and m.canis tested fungal strains mic/mean(µg/ml) zno nps 0.171-75 griseofulvin (0.136-59) pvalue (zno nps) 0.171-75 ketoconazole (0.04-19) pvalue t.rubrum 4.530 35 <0.001 4.530 4 >0.05 m.canis 10.67 >64 <0.001 10.67 3 >0.05 table 4: effects of zno nps on spore germination in the tested dermatophytes, alongside the reference antifungal drugs griseofulvin and ketoconazole tested fungal strains mean percentage of spore germination inhibition zno nps griseofulvin pvalue (zno nps) ketoconazole pvalue t.rubrum 98 0 <0.0001 98 0 <0.0001 m.canis 88 0 <0.0001 88 0 <0.0001 conclusion in the results obtained in this study and through the effectiveness of zno nps on two types of fungi and comparing them with antifungals, we can conclude that zno nps have a biological effectiveness more or similar to antifungals. acknowledgements the authors would like to thank the department of biology, college of education for pure sciences, kirkuk university, kirkukiraq. and alqalam university for the facilities they provided and for helping to enhance the quality of this work references abbood, a. h. s. h. a. 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(2022). nano-enhanced biolubricant in sustainable manufacturing: from processability to mechanisms. friction, 10(6), 803-841. https://doi. org/10.1007/s40544-022-0674-x. zili, z., sfar, s., & fessi, h. (2005). preparation and characterization of poly-ɛ-caprolactone nanoparticles containing griseofulvin. international journal of pharmaceutics, 294(1-2), 261-267. https://doi. org/10.1016/j.ijpharm.2005.01.020. pa ge 1 pa ge 69 american journal of life science and innovation (ajlsi) advancements in ichthyosis treatment in pediatric patients: evaluating the impact of immune system inhibitors targeting tnf-alpha, il-17a, il-4, and il-12 eiman hamza ahmed elsayed1*, rabab elsayed ali hassan khater2 volume 3 issue 1, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i1.2730 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: may 01, 2024 accepted: may 31, 2024 published: june 04, 2024 lamellar, x-linked recessive, and vulgaris ichthyosis represent rare genetic variants distinguished by abundant dry surface scales. the etiology of childhood ichthyosis involves a combination of acquired and inherited factors. children with ichthyosis have a th17dominant immune profile, similar to psoriasis, with a correlation between ichthyosis severity, erythema, trans epidermal water loss (tewl), and immunological markers. in order to examine the safety and effectiveness of immune system inhibitors that target tnf-alpha, il-17a, il-4, and il-12 in the treatment of pediatric ichthyosis, a retrospective research study was conducted on 30 pediatric patients aged 6-12 years at tertiary care pediatric dermatology hospitals in uae, for a time duration of 5 months. il-17-a is a key cytokine of ichthyosis, and its specific suppression is considered necessary in contrast to other cytokines. consequently, the choice of treatment was dictated by elements unique to the condition and the intensity of symptoms, focusing on using secukinumab as an il-17-a inhibitor. the findings showed that, following five months of treatment, patients receiving secukinumab showed positive outcomes, with their ichthyosis area severity index score declining by 45 to 50%. additionally, the proinflammatory rate of cytokines was significantly lower in this group of patients compared to the placebo-treated control group. the safety and efficacy of secukinumab compared to ixekizumab and brodalumab vs placebo were examined using t-tests, with statistical significance determined at p< 0.05. the present research study concludes that secukinumab effectively enhances skin texture and reduces scaling severity in ichthyosis, suggesting clinical relevance. the findings underscore secukinumab’s potential as a beneficial agent for various ichthyosis types, such as lamellar, x-linked, and vulgaris. the study’s comparison with placebo and other il-17a inhibitors like ixekizumab and brodalumab provides valuable insights into secukinumab’s efficacy and potential clinical uses. keywords ichthyosis, secukinumab, pediatrics, il-17, tnf-alpha, il-4, il-12 introduction lamellar ichthyosis, x-linked recessive ichthyosis, and ichthyosis vulgaris are among the rare genetic variations of this rare skin illness characterized by excessively dry surface scales. both acquired and inherited factors can contribute to the etiology of childhood ichthyosis. most cases of ichthyosis are caused by inherited ichthyosis vulgaris, caused by an inherited gene from one or both parents (pontello et al., 2020). another potential cause could be a genetic mutation that transpires in the womb, depriving the child’s skin of a vital protein known as filaggrin. adults with acquired ichthyosis vulgaris may develop the condition as a result of many medical conditions, including kidney failure, clofazimine, nicotinic acid deficiency, sarcoidosis, leprosy, and hiv (lilly & bunick, 2023). children’s ichthyosis is caused by epidermal development and metabolism anomalies, which result in severe skin dryness and scaling. mutations in particular genes involved in skin barrier function and cornification result in inherited forms of ichthyosis, such as autosomal recessive congenital ichthyosis (arci) and x-linked recessive ichthyosis (xli). impaired breakdown of cholesterol sulfate leads to hyperkeratosis and generalized brown scales in xli, which is associated with mutations in the steroid sulfatase (sts) gene. conversely, arci comprises several subtypes, such as congenital ichthyosiform erythroderma (cie) and lamellar ichthyosis (li), andpresents with with clinical symptoms , including rough scaling, erythroderma and collodion membrane after birth (joosten et al., 2022). skin cornification processes, such as desquamation, keratin synthesis, cornified envelope synthesis, and stratum corneum structure, are disrupted in ichthyosis. increased transepidermal water loss (tewl), a compromised skin barrier, and altered epidermal differentiation result from these disturbances. extensive skin scaling, hyperkeratosis, and inflammation are the hallmarks; erythroderma frequently follows. while acquired ichthyosis in children is linked to underlying illnesses or drugs that disrupt the establishment of the skin barrier, resulting in fishlike scales concentrated on the extremities, hereditary types such as xli and arci are essentially genetic in origin (dyer et al., 2013). studies on children with ichthyosis reveal a th17dominant immune profile with elevated expression of il17/tnfα-synergistic/additive genes. this immunological profile is comparable to that of the inflammatory 1 pediatric specialist in uae 2 dermatology specialist in uae * corresponding author’s e-mail: dr_eh2412@yahoo.com pa ge 70 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 69-74, 2024 condition psoriasis. the severity of ichthyosis, erythema, and transepidermal water loss (tewl) is significantly linked to immunological markers, suggesting a potential link between inflammation and skin barrier abnormalities. (czarnowicki et al., 2018; kim et al., 2022; paller, 2019). targeting tnf-alpha, il-17a, il-4, and il-12, immune system inhibitors have demonstrated potential in treating ichthyosis by treating immunological dysregulation and inflammation. while il-17a inhibitors may provide a specific treatment for the pathophysiology of ichthyosis, tnf-alpha inhibitors have been shown useful in treating inflammatory skin conditions. moreover, inhibitors that target il-4 and il-12 appear to have therapeutic applications (huangfu et al., 2023; paller, 2019; wang et al., 2021). drugs that inhibit factors related to the immune system, such as tnf-alpha, il-17a, il-4, and il-12, are often considered as potential treatments, including etanercept, adalimumab, infliximab (tnf-alpha inhibitors), secukinumab, ixekizumab, brodalumab (il-17a inhibitors), dupilumab (il-4 inhibitors) and ustekinumab (il12 and 23 inhibitors) (paller et al., 2017; peña-corona et al., 2023; zaenglein et al., 2021). the present research study aimed to improve the quality of life by examining the safety and efficacy of immune system inhibitors that target tnf-alpha, il-17a, il-4, and il-12 in pediatric ichthyosis treatment. literature review ichthyosis is a common skin ailment that impairs perspiration, causes itching, and causes recurrent infections. it also negatively affects the quality of life for those who have it. although diagnosis is hampered by diverse clinical presentation and inadequate phenotype– genotype association, the distinctive clinical symptoms represent a homeostatic attempt to restore the epidermal barrier. comprehending the molecular pathways is essential for creating novel therapy approaches that target the underlying causes of various ailments (gutiérrezcerrajero et al., 2023). ichthyoses and cornification disorders often require longterm retinoid treatment. however, it was found that longterm use of retinoids in ichthyoses may cause potential negative effects on the eyes, bones, heart, and psyche, requiring careful clinical considerations (zaenglein et al., 2021). based on the etiology of the skin disorder, gene therapy, and protein substitution are used as treatments for ichthyosis. it was identified that biological therapy has been effectively used to treat netherton syndrome and autosomal recessive congenital ichthyosis (joosten et al., 2022). in a pediatric patient with severe erythrodermic autosomal recessive congenital ichthyosis (arci), secukinumab was found to exhibit clinical efficacy and safety, resulting in a 48% reduction in the baseline score, a reduction in proinflammatory cytokines, and an abrogation of th17skewing (yogarajah et al., 2021). there is no particular treatment for ichthyosis; it is managed with systemic therapy, which includes uv radiation, moisturizing medicines, keratolytics, vitamin a derivatives, and therapeutic baths (takhtarova et al., 2021). alpha-hydroxy acids (ahas) like lactic, glycolic, or pyruvic acids are used to hydrate skin in children with ichthyosis by disintegrating corneocytes in the stratum corneum, improving moisture and decreasing thickness. particularly, lactic acid has demonstrated efficacy over petrolatum-based creams in treating ichthyosis vulgaris. it can be obtained as a 12% ammonium lactate lotion or compounded in a 5–10% concentration. ahas also help to improve desquamation and decrease corneocyte adhesion, which makes them useful for treating children’s ichthyosis symptoms (palmer & dunwell, 2022). salicylic acid, a keratolytic agent, is frequently used to treat ichthyosis because it softens keratin, loosens scaly, dry skin, and facilitates removal. although it is usually well tolerated, it may result in mild hives, irritation, rash, dryness, peeling, itching, redness, and skin swelling. adverse effects might be quite serious in certain instances. preparations of salicylic acid with concentrations ranging from 0.5% to 30% and 6%, particularly in pediatrics, are used to treat particular ailments of ichthyosis in children (cortés et al., 2020). vitamin b5 in dexpanthenol is a helpful treatment for children with ichthyosis vulgaris. it is a good alternative to use as a first-line therapy to enhance skin moisture and lessen dryness and scaling (cortés et al., 2020). children with ichthyosis are treated with emollient lotions such as dexeryl, which contains glycerol. based on studies, dexeryl improves skin condition by lessening the severity of xerosis and its associated symptoms. in 80% of treated youngsters, it is both effective and welltolerated. thick, scaly, dry, and cracked skin are the signs this treatment targets (blanchet‐bardon et al., 2012). propylene glycol is a useful treatment for several conditions that cause the skin to become scaled. because it keeps the skin free of scales and removes them from the skin, it is very effective in treating ichthyosis (hernándezmartin et al., 2013). congenital ichthyosis patients may benefit greatly from vitamin d treatment, as it has demonstrated a strong ability to ameliorate the skin condition of those afflicted. studies have demonstrated the high incidence of rickets and severe vitamin d insufficiency in children with congenital ichthyosis. short-term high-dose vitamin d treatment provided a notable clinical response in children with congenital ichthyosis. during the treatment, 400– 600 iu of cholecalciferol a day was advised after 60,000 iu of oral cholecalciferol was given daily for 10 days. the results showed that vitamin d treatment had a beneficial effect on the symptoms of congenital ichthyosis, as evidenced by the decrease in skin scaling and stiffness of the extremities (sethuraman et al., 2016). research revealed that congenital ichthyosis could be successfully treated when acitretin therapy was used to treat a newborn baby’s skin condition, even though pa ge 71 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 69-74, 2024 the baby’s skin condition had not improved with liquid vaseline treatment (gulasi, 2016). if congenital ichthyosis is treated, acitretin 0.5 mg/kg/ day should be administered initially and 1 mg/kg/day after two months. as the highest doses do not exceed 1 mg/kg/day, its short plasma half-life minimizes negative effects (zaenglein et al., 2021). immune system inhibitors such as etanercept (tnfalpha inhibitors) have no adverse effects when used to treat juvenile idiopathic arthritis and harlequin ichthyosis in children (baldo et al., 2021). on the other hand, studies indicated that adalimumab is also used to treat ichthyosis in children; it targets the cytokine tnf-alpha, which is linked to inflammation. it helps to improve skin conditions and manage symptoms (sticherling, 2020). it has been demonstrated that infliximab treatment is beneficial in lowering skin inflammation in children with ichthyosis, including netherton syndrome. studies have demonstrated that infliximab can improve skin lesions by reducing the expression of several inflammatory markers, such as tslp, il-6, and il-8. clinical investigations have shown that infliximab treatment significantly improves the skin signs of netherton syndrome, including itching, scaling, dry skin, and eczematous lesions (barbati et al., 2021; cicek et al., 2021; roda et al., 2017). il-17 a inhibitors secukinumab, which resembles the inflammatory profile of ichthyosis, have been demonstrated in clinical trials to be a safe and efficacious treatment for children with psoriasis. in particular, secukinumab resulted in a 48% decrease in the ichthyosisarea-severity-index score following a 6-month therapy term in a young patient with severe erythrodermic ichthyosis associated with abca12 deficiency in a therapeutic trial. treatment was well tolerated, and the patient’s blood cytokine profile consistently changed, showing a decrease in proinflammatory cytokines and a th17-skewing (albela et al., 2024; narbutt et al., 2023; yogarajah et al., 2021). in treating approximately 95% of patients with pediatric ichthyosis vulgaris, ixekizumab has demonstrated longterm safety and efficacy in decreasing dry scales. changes in profilagrin expression tackle the underlying pathology (pontone et al., 2022). dupilumab reduces inflammation and pruritus in patients with ichthyosis by inhibiting interleukin-4 and interleukin-13 signaling. studies indicate that netherton syndrome and bullous congenital ichthyosiform erythroderma bcie can be treated well and that managing ichthyosis in children may be possible (afshan & kelbel, 2019; almuhanna, alrashidi, et al., 2023; martin-garcía et al., 2023; olbrich et al., 2023). ustekinumab has been investigated for the treatment of ichthyosis in pediatric patients, particularly in the case of a 15-year-old boy with harlequin ichthyosis (hi). after one month of treatment, the off-label ustekinumab trial revealed a little improvement in erythema. ustekinumab was removed from the regimen after a year since it did not result in a meaningful response to treatment. however, ustekinumab may be a viable therapeutic option for other ichthyotic disorders (almuhanna, alasmari, et al., 2023). materials and methods study design the present research study was retrospective to examine the safety and efficacy of immune system inhibitors that target tnf-alpha, il-17a, il-4, and il-12 in pediatric ichthyosis treatment. study setting duration the current study was conducted on 30 pediatric patients (ages 6-12 years) diagnosed with different types of ichthyosis skin diseases at tertiary care pediatric dermatology hospitals in uae for 5 months from november to february 2024. participants inclusion criteria ➢ patients with ichthyosis between the ages of 6 and 12 who satisfied the study’s criteria and were verified by the patients’ database were included. ➢ subjects with a recent diagnosis or those with a previous diagnosis were included in the follow-up. ➢ clinical information included key ichthyosis conditions such as lamellar, x-linked recessive, and vulgaris ichthyosis. exclusion criteria ➢ the present research study eliminated patients who declined to participate or had missing data. intervention ➢ previous research studies found that tnf-alpha was elevated in netherton syndrome compared to psoriasis and atopic dermatitis, but it was still lower than in ichthyosis. together, tnf-alpha and il-17 activate genes and markers connected to il-17, so the main cytokine of the th17 pathway, il-17a, sharply increases during ichthyosis. idh-related th2 cytokine il-4 expression levels are comparable to controls in ichthyosis. furthermore, engaged in the immunological profile of ichthyosis, il-12 also impacts th1 immune responses. key cytokine of ichthyosis il-17-a inhibition ➢ il-17 a is the primary cytokine responsible for inducing an immune response by activating genes and infectious or inflammatory markers. ➢ the primary focus of this study was to inhibit il17-a in order to lessen the inflammatory and allergic symptoms associated with all three types of pediatric ichthyosis. ➢ il-17a inhibitors, including secukinumab, ixekizumab, and brodalumab, were administered to patients 6 to 12 years old, according to their body mass index, who had been diagnosed with multiple types of ichthyosis, such as vulgaris, lamellar, and x linked. pa ge 72 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 69-74, 2024 outcome measures ➢ in the result, it was found that after 5 months of therapy, the patients who were given secukinumab provided positive results, and the patient’s ichthyosis area severity index score decreased by 45 to 50 percent, and it significantly reduced the proinflammatory rate of cytokines against the control group, which was given with placebo. ➢ on the other hand, secukinumab is fda-approved safe and effective therapy even for severe erythrodermic ichthyosis. primary outcomes of secukinumab improvements in skin texture and a decrease in the production of scales were observed. furthermore, there was a noteworthy decrease in inflammation, as seen by a 45% improvement in the ichthyosis area severity index score within 6 months of treatment. secondary outcomes of secukinumab secukinumab is safe and effective in treating children with severe ichthyosis, significantly improving the severity of their symptoms. remarkably, the cytokine profile returns to normal after treatment, and proinflammatory cytokines drop. gastrointestinal problems, hypersensitivity reactions, and musculoskeletal pain have been linked to fewer side effects in conjunction with this good response. these results demonstrate the safety profile of secukinumab and suggest it as a viable therapy for pediatric patients with severe ichthyosis. statistical analysis t-tests were used to analyze associations between secukinumab’s effectiveness and safety profile and that of ixekizumab and brodalumab against placebo. employing an area severity index score assessment of secukinumab. at p< 0.05, statistical significance was determined. results and discussion treatment outcomes efficacy of secukinumab in comparison with other immune system inhibitors the efficacy of the il-17-a inhibitor, secukinumab, was evaluated by comparing its treatment outcomes with tnf-alpha, il-4, and il-12/13 inhibitors. given that il17-a is a key cytokine in various forms of ichthyosis, its targeted inhibition is deemed essential compared to other cytokines. therefore, treatment selection was based on disease-specific factors and symptom severity, prioritizing using secukinumab as an il-17-a inhibitor. secukinumab demonstrated rapid and effective symptom reduction, surpassing other il-17 inhibitors like ixekizumab and brodalumab. specifically, secukinumab achieved a 45% faster decrease in area severity index score and notable improvements in skin scaling and texture against the placebo-treated control group. these results underscore the efficiency of secukinumab as a treatment for ichthyosis, particularly in addressing symptom severity and enhancing skin quality. safety profile and adverse events in comparison with other il-17-a inhibitors secukinumab demonstrated high effectiveness and a lower incidence of hypersensitivity reactions and gastrointestinal issues than ixekizumab and brodalumab. however, it is important to note that brodalumab carries a risk of affecting mental health by potentially causing depression and generating suicidal ideation. additionally, there is a risk of malignancies in patients using ixekizumab and brodalumab for the treatment of ichthyosis. discussion childhood ichthyosis is a rare skin condition characterized by extremely dry surface scales. it is also known by the names lamellar ichthyosis, x-linked recessive ichthyosis, and ichthyosis vulgaris. a prenatal mutation or hereditary genes may impact the filaggrin protein in the skin. acquired ichthyosis vulgaris in adults can result from several different illnesses. research on children with ichthyosis reveals a th17dominant immune profile with elevated expression of il17/tnfα-synergistic/additive genes, similar to psoriasis. immune system inhibitors targeting tnf-alpha, il17a, il-4, and il-12 have shown potential in treating immunological dysregulation and inflammation. these drugs, including etanercept, adalimumab, infliximab, secukinumab, ixekizumab, brodalumab, dupilumab, and ustekinumab, may improve quality of life in pediatric ichthyosis treatment. long-term retinoid therapy is necessary for ichthyoses and cornification problems, although it can have detrimental consequences on the eyes, bones, heart, and brain. netherton syndrome and autosomal recessive congenital ichthyosis treatments include gene therapy and protein substitution. the present research study involved 30 pediatric patients diagnosed with ichthyosis skin diseases in uae from november to february 2024. participants included those aged 6-12 with recent or previous diagnoses. key ichthyosis conditions were included. the study found that tnf-alpha and il-17 activate genes and markers related to il-17, leading to increased th17a cytokine levels. idh-related th2 cytokine il-4 expression levels were comparable to controls. il-12 also impacts th1 immune responses. this research study focused on key cytokine il-17-a to reduce inflammatory and allergic symptoms in pediatric ichthyosis patients aged 6-12. il-17a inhibitors secukinumab, ixekizumab, and brodalumab were administered to patients with various ichthyosis types. after 5 months, patients with secukinumab showed positive results, decreased ichthyosis severity index scores by 45-50%, and significantly reduced cytokine proinflammatory rate. compared to il-17 inhibitors such as ixekizumab and brodalumab, secukinumab showed faster and more significant symptom reduction. secukinumab specifically produced a 45% quicker reduction in the area severity pa ge 73 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 69-74, 2024 index score and appreciable changes in the scale and texture of the skin. these outcomes highlight how well secukinumab treats ichthyosis, especially when improving skin quality and symptom severity. compared to ixekizumab and brodalumab, secukinumab showed great efficacy and a decreased rate of hypersensitivity responses and gastrointestinal problems against a control group. however, it is crucial to remember that brodalumab poses a risk of harming mental health, as it may lead to suicidal thoughts and sadness. furthermore, those treated for ichthyosis with ixekizumab and brodalumab run the risk of developing cancers. limitations ➢ the study has a limited sample size, which could affect the generalizability of the findings to a broader population. ➢ the cost of secukinumab and its accessibility to all patients, especially in regions with limited healthcare resources, could impact its applicability in real-world settings. conclusion in conclusion, this study demonstrated the ability to effectively improve the skin texture scaling area severity index via secukinumab, which may have clinical applications. these results highlight the potential of secukinumab to serve as an efficient and protective agent against all types of ichthyosis, including lamellar, x-linked, and vulgaris. this study provides valuable information regarding the effects of secukinumab relative to placebo and over the already studied outcomes of other il-17-a inhibitors, including ixekizumab and brodalumab, and its potential clinical applications. clinical implications the results obtained from the present research study could be valuable for clinical practitioners in public health care, particularly pediatricians, as they may consider incorporating secukinumab into the treatment regimen for ichthyosis, leading to improved area severity index scores and enhanced safety for pediatric patients. future directions ➢ conduct longitudinal research to evaluate the longterm effectiveness and safety profile of secukinumab in pediatric patients with ichthyosis. a minimum 12-month follow-up period should be a part of this study to assess the treatment’s long-term effectiveness and any possible side effects. ➢ recommended to examine if it would be advantageous to treat pediatric patients with ichthyosis with secukinumab in addition to other therapies like topical corticosteroids, emollients, or phototherapy. optimizing treatment outcomes using a combined strategy could result in synergistic effects. references afshan, t., & kelbel, t. (2019). m501 an adolescent patient with krt10 mutation-associated ichthyosis successfully treated with dupilumab. annals of allergy, asthma & immunology, 123(5), s139. albela, h., ingrid pao lin, t., lee, t. s., ooi, d., & leong, k. f. (2024). secukinumab therapy for paediatric patients with various phenotypes of congenital ichthyosis. clinical and experimental dermatology, llae082. almuhanna, n., alasmari, b. s., alhamazani, r., alkhezzi, s., alhomida, f. a., alasmari, b., & alshammari, r. (2023). assessing the use of ustekinumab in a pediatric patient with harlequin ichthyosis. cureus, 15(4). almuhanna, n., alrashidi, a. r., shaheen, e. a., alanazi, y., alfawzan, a., alharthi, r., & alhomida, f. a. (2023). assessing the use of dupilumab in a pediatric patient with bullous congenital ichthyosiform erythroderma. jaad case reports, 39, 17-20. baldo, f., brena, m., carbogno, s., minoia, f., lanni, s., guez, s., petaccia, a., agostoni, c., cimaz, r., & filocamo, g. (2021). juvenile idiopathic arthritis in harlequin ichthyosis, a rare combination or the clinical spectrum of the disease? report of a child treated with etanercept and review of the literature. pediatric rheumatology, 19(1), 80. barbati, f., giovannini, m., oranges, t., lodi, l., barni, s., novembre, e., baldo, e., cristofolini, m., stagi, s., & ricci, s. (2021). netherton syndrome in children: management and future perspectives. frontiers in pediatrics, 9, 645259. blanchet‐bardon, c., tadini, g., machado matos, m., & delarue, a. (2012). association of glycerol and paraffin in the treatment of ichthyosis in children: an international, multicentric, randomized, controlled, double‐blind study. journal of the european academy of dermatology and venereology, 26(8), 1014-1019. cicek, f., cekic, s., & kilic, s. s. (2021). infliximab therapy in an infant with netherton syndrome. pediatric dermatology, 38(3), 714-716. cortés, h., del prado-audelo, m. l., urbán-morlán, z., alcalá-alcalá, s., gonzález-torres, m., reyeshernández, o. d., gonzález-del carmen, m., & leyva-gómez, g. (2020). pharmacological treatments for cutaneous manifestations of inherited ichthyoses. archives of dermatological research, 312, 237-248. czarnowicki, t., he, h., leonard, a., malik, k., magidi, s., rangel, s., patel, k., ramsey, k., murphrey, m., & song, t. (2018). the major orphan forms of ichthyosis are characterized by systemic t-cell activation and th17/tc-17/th-22/tc-22 polarization in blood. journal of investigative dermatology, 138(10), 2157-2167. dyer, j. a., spraker, m., & williams, m. (2013). care of the newborn with ichthyosis. dermatologic therapy, 26(1), 1-15. gulasi, s. (2016). congenital ichthyosis: a case treated successfully with acitretin. iranian journal of pediatrics, 26(5). pa ge 74 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 69-74, 2024 gutiérrez-cerrajero, c., sprecher, e., paller, a. s., akiyama, m., mazereeuw-hautier, j., hernándezmartín, a., & gonzález-sarmiento, r. (2023). ichthyosis. nature reviews disease primers, 9(1), 2. hernández-martin, a., aranegui, b., martin-santiago, a., & garcia-doval, i. (2013). a systematic review of clinical trials of treatments for the congenital ichthyoses, excluding ichthyosis vulgaris. journal of the american academy of dermatology, 69(4), 544-549. e548. huangfu, l., li, r., huang, y., & wang, s. (2023). the il17 family in diseases: from bench to bedside. signal transduction and targeted therapy, 8(1), 402. joosten, m., clabbers, j., jonca, n., mazereeuw-hautier, j., & gostyński, a. (2022). new developments in the molecular treatment of ichthyosis: review of the literature. orphanet journal of rare diseases, 17(1), 269. kim, m., mikhaylov, d., rangel, s. m., pavel, a. b., he, h., renert-yuval, y., del duca, e., malik, k., huynh, t., & ibler, e. (2022). transcriptomic analysis of the major orphan ichthyosis subtypes reveals shared immune and barrier signatures. journal of investigative dermatology, 142(9), 2363-2374. e2318. lilly, e., & bunick, c. g. (2023). congenital ichthyosis: a practical clinical guide on current treatments and future perspectives. clinical, cosmetic and investigational dermatology, 2473-2479. martin-garcía, c., godoy, e., cabrera, a., cañueto, j., muñoz-bellido, f. j., perez-pazos, j., & dávila, i. (2023). report of two sisters with netherton syndrome successfully treated with dupilumab and review of the literature. international journal of immunopathology and pharmacology, 37, 03946320231172881. narbutt, j., niedźwiedź, m., lesiak, a., ceryn, j., & skibińska, m. (2023). secukinumab for the treatment of psoriasis in pediatrics: patient selection and acceptability. patient preference and adherence, 421-431. olbrich, h., sadik, c. d., ludwig, r. j., thaçi, d., & boch, k. (2023). dupilumab in inflammatory skin diseases: a systematic review. biomolecules, 13(4), 634. paller, a. s. (2019). profiling immune expression to consider repurposing therapeutics for the ichthyoses. journal of investigative dermatology, 139(3), 535-540. paller, a. s., renert-yuval, y., suprun, m., esaki, h., oliva, m., huynh, t. n., ungar, b., kunjravia, n., friedland, r., & peng, x. (2017). an il-17–dominant immune profile is shared across the major orphan forms of ichthyosis. journal of allergy and clinical immunology, 139(1), 152-165. palmer, v., & dunwell, p. (2022). a case of ichthyosis vulgaris and the use of 70% glycolic acid chemical peels for management. cureus, 14(9). peña-corona, s. i., echeverria, m. d. l. d. c., cortés, h., carmen, g.-d., & leyva-gómez, g. (2023). advances in the treatment of autosomal recessive congenital ichthyosis, a look towards the repositioning of drugs. frontiers in pharmacology, 14, 1274248. pontello, e., battajon, n., tormena, f., giovannini, m., buffo, m., mainini, n., visintin, g., favero, v., vendramin, s., & galeazzo, b. (2020). congenital ichthyosis in newborns. a case-series and review of the literature. european journal of pediatric dermatology, 30(2), 104-110. pontone, m., giovannini, m., filippeschi, c., oranges, t., pedaci, f. a., mori, f., barni, s., barbati, f., consonni, f., & indolfi, g. (2022). biological treatments for pediatric netherton syndrome. frontiers in pediatrics, 10, 1074243. roda, â., mendonça-sanches, m., travassos, a. r., soares-de-almeida, l., & metze, d. (2017). infliximab therapy for netherton syndrome: a case report. jaad case reports, 3(6), 550-552. sethuraman, g., marwaha, r. k., challa, a., yenamandra, v. k., ramakrishnan, l., thulkar, s., & sharma, v. k. (2016). vitamin d: a new promising therapy for congenital ichthyosis. pediatrics, 137(1). sticherling, m. (2020). dermatology part 2: ichthyoses and psoriasis. pediatric pharmacotherapy, 153-175. takhtarova, t. g., khismatullina, z. r., panova, l. d., & panova, a. n. (2021). ichthyosis (concept, pathohistology, clinical picture, treatment). vestnik dermatologii i venerologii, 97(3), 6-13. wang, m., zhai, x., li, j., guan, j., xu, s., li, y., & zhu, h. (2021). the role of cytokines in predicting the response and adverse events related to immune checkpoint inhibitors. frontiers in immunology, 12, 670391. yogarajah, j., gouveia, c., iype, j., häfliger, s., schaller, a., nuoffer, j.-m., fux, m., & gautschi, m. (2021). efficacy and safety of secukinumab for the treatment of severe abca12 deficiency‐related ichthyosis in a child. skin health and disease, 1(2), e25. zaenglein, a. l., levy, m. l., stefanko, n. s., benjamin, l. t., bruckner, a. l., choate, k., craiglow, b. g., digiovanna, j. j., eichenfield, l. f., & elias, p. (2021). consensus recommendations for the use of retinoids in ichthyosis and other disorders of cornification in children and adolescents. pediatric dermatology, 38(1), 164-180. pa ge 1 pa ge 39 american journal of life science and innovation (ajlsi) microbial inoculants in agriculture: a microbiological review from a nigerian perspective ewubare p. o.1*, omokaro g. o.2, ewansiha o. c.1, edosa j. o.1 volume 2 issue 3, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i3.2241 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: november 07, 2023 accepted: december 03, 2023 published: december 06, 2023 chemical compounds are absorbed by most crops from the soil. several synthetic fertilizers contain acid radicals, such as hydrochloride and sulfuric radicals, thus increasing soil acidity and adversely affecting soil and plant health. some plants can also absorb highly recalcitrant compounds. continuous consumption of such crops can lead to systematic disorders in humans. a reliable alternative to the use of chemical inputs is microbial inoculants, which can act as biofertilizers, bioherbicides, biopesticides, and biocontrol agents. microorganisms are capable of promoting plant growth, and controlling pests, diseases, and weeds. microbial inoculants are beneficial microorganisms applied either to the soil or the plant to improve productivity and crop health. these natural-based products are widely used to control pests and enhance soil and crop quality, thereby benefiting human health. microbial inoculants consist of a blend of microorganisms that work with the soil and its inhabitants to improve soil fertility and health, ultimately benefiting human health. they have the ability to minimize the negative impact of chemical inputs, thereby increasing the quantity and quality of farm produce. this review paper summarizes the effects of microbial inoculants on agricultural soil in nigeria by examining relevant works related to the topic. to achieve this, databases such as google scholar, frontier in microbiology, african journals online (ajol), scopus, web of science, sciencedirect, and directory of open access journals (doaj) were explored to identify studies on the effects of microbial inoculants on agricultural soil in nigeria. keywords nigeria, microbial inoculants, review, soil microbes, biofertilizer introduction soil microorganisms can enhance plant growth and safeguard soils from diseases and environmental stresses (glick, 2012). these microorganisms form partnerships with plants, promoting growth through beneficial traits such as providing nutrients via biological nitrogen fixation (bnf) and phosphate solubilization, as well as reducing stress through the regulation of 1-aminocyclopropane1-carboxylate deaminase expression and the production of phytohormones and siderophores, among other mechanisms (alori, 2016). introducing these helpful microbes into the soil and plants, a process known as inoculation, is a less harmful and more environmentally friendly approach compared to using chemical fertilizers. microbial inoculation technology is considered a sustainable agricultural practice, leading to reduced production costs. there is a growing trend in the use of symbiotic or free-living nitrogen-fixing bacteria in sustainable agricultural systems (koki & takayoshi, 2013). research conducted by omokaro et al. (2023) in delta state, nigeria, focusing on the perspective of smallholder farmers, it was noted that 47.6% of these farmers utilize chemical pesticides such as gammalin for the control of pests and diseases. utilizing inoculants is particularly appealing as it significantly decreases the need for chemical fertilizers and pesticides, and there is a rising availability of commercialized inoculants for various crops (babalola 2010; babalola & glick 2012). the use of microbial inoculants has proved to be a promising technology to obtain an increase in food production and a sustainable agricultural system. the effects of microbial inoculants on agricultural soils include the introduction of soil microbes such as bacteria and fungi. however, this paper reviews research on the effects of microbial inoculants in agricultural soils in nigeria and its impact on plants and soil. methodology a literature review was carried out to identify the relevant articles published. the earliest research publication concerning the review was found in 2006. google scholar, frontier in microbiology, african journals online (ajol), scopus, web of science, science direct and directory of open access journals (doaj) databases were explored to identify studies on organic amendment effect on soil fertility in nigeria using the following keywords in english; ‘microbial inoculants use in nigeria’, ‘effect of microbial inoculants in nigerian agriculture’, ‘effect of microbial inoculants in soil and plants in nigeria. a total of 150 articles were identified in the initial review but 20 articles were selected to be a good match for the review study based on the objective of the study and geographical location. figure 1 below shows the system search method. 1 department of soil science and land management, university of benin, p.m.b 1155, ugbowo, benin city, edo state, nigeria 2 institute of ecology, people’s friendship university of russia, miklukho maklaya 6, moscow, russia * corresponding author’s e-mail: paulewubare9@gmail.com pa ge 40 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 39-43, 2023 discussion microbial inoculants in agriculture microbial inoculants are formulations containing beneficial microorganisms crucial for maintaining sustainable agriculture in soil ecosystems. they serve as an eco-friendly option, potentially replacing chemical fertilizers and pesticides (babalola & glick, 2012). these inoculants consist of active microorganism strains that directly or indirectly enhance microbial activity, improving nutrient mobility in the soil. they can function as phyto-stimulants, bio-fertilizers, or microbial biocontrol agents, offering protection against various pathogens and acting as effective bio-herbicides (babalola, 2010). additionally, naturally occurring microbes obtained from the wild and applied to plants can also serve as bioherbicides (babalola, 2007; alori et al., 2017). given the growing global population and the consequent need to boost agricultural production, maintaining soil fertility is crucial. biofertilizers, comprising active microbes, present a viable technology to enhance food production without endangering human and environmental health. these biofertilizers encompass organisms that supply or facilitate the availability of different nutrients to plants, including nitrogen fixers, phosphorus solubilizers, potassium solubilizers, sulfur solubilizers, mycorrhiza, and more (pathak & kumar, 2016; alori et al., 2017). the effects of microbial inoculants in soil in the process of seed and soil inoculation, a large number of efficient and viable microbial cells are introduced into the soil to rapidly colonize the host rhizosphere. however, this introduction can significantly disrupt the balance of soil microbial communities (babalola, 2014). when using microbial inoculants, substantial quantities of microbial cells are introduced into the competitive soil environment. for instance, biocontrol pseudomonas inoculants that produce 2,4-diacetylphloroglucinol (phl) are introduced into the rhizosphere community in significant amounts, preventing the proliferation of specific fungal pathogens. azospirillum spp. produce relatively high levels of indole-3-acetic acid (iaa), which enhance the formation of plant lateral and adventitious roots, improving mineral and nutrient uptake. while introduced microbes may temporarily alter the equilibrium in the rhizosphere community, the bacterial community structure often rebounds due to ecosystem resilience. compounds like indole-3-acetamide, indole-3-pyruvate, indole-3-acetaldehyde, and 4-chloroindole-3-acetic acid have been found to exhibit auxin activity (roesti et al., 2006; olanrewaju et al., 2017). biological nitrogen fixation comprises both symbiotic nitrogen fixation and free-living nitrogen-fixing systems. symbiotic nitrogen-fixing genera include rhizobium, achromobacter, sinorhizobium, azoarcus, mesorhizobium, frankia, allorhizobium, bradyrhizobium, burkholderia, azorhizobium, and herbaspirillum (babalola, 2010). phosphorus (p) is a vital element essential for plant growth and development, second only to nitrogen. in soil, p exists in organic and inorganic forms that are not readily available to plants. however, several plant growth-promoting rhizobacteria (pgpr) have been identified to mobilize poorly available phosphorus through solubilization and mineralization. examples include pseudomonas spp., agrobacterium spp., bacillus circulans, azotobacter spp., bacillus spp., burkholderia spp., enterobacter spp., erwinia spp., kushneria spp., paenibacillus spp., ralstonia spp., rhizobium spp., rhodococcus spp., serratia spp., bradyrhizobium spp., salmonella spp., sinomonas spp., and thiobacillus spp. (azziz et al., 2012; alori et al., 2017). atmospheric nitrogen fixation and mineral solubilization, such as phosphorus (p), are direct mechanisms through which microbial inoculants exert their influence. in indirect growth promotion, microbial inoculants impact plants by inducing systemic resistance (isr) or systemic acquired resistance (sar), enhancing disease resistance (babalola, 2010). biotic and abiotic stress such as insect and nematode damage, drought or flood, presence of metals, chemicals (both organic and inorganic), ultraviolet light, extreme temperatures, mechanical wounding as well as fungal figure 1: flow chart showing the methodology of review of literature pa ge 41 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 39-43, 2023 and bacterial pathogens triggers increased production of ethylene in plants (ali et al., 2014). biocontrol pgpr that produces hcn can also synthesize some cell wall degrading enzymes or antibiotics. hcn can also act as an anti-fungi agent. hcn synthesized by pgpr is usually in small quantity, this ensures that the fungi do not develop resistance to the synthesized antifungal thereby enhancing the effectiveness of antifungal in soil (kundan et al., 2015; olanrewaju et al., 2017). table 1: some examples of the use of microbial inoculants in agriculture microbial location result and findings references rhizobium and bradyrhizobium strains ahmadu bello university, zaria, nigeria produced more shoot and nodule dry weight n’cho et al., (2015) rhizobacteria inoculant (nodumax legume inoculant) enugu state university of science and technology, nigeria increased leaf, stem and root dry weight of bambara groundnut. increased number of root nodules, fresh pods of bambara groundnut and soil total nitrogen content ikenganyia et al., (2017) bradyrhizobium ahmadu bello university, zaria, nigeria increased soil organic carbon, total nitrogen and carbon to nitrogen ratio omeke, 2017 arbuscular mycorrhizae fungi federal university of agriculture, abeokuta, ogun state, nigeria increased arbuscular mycorrhizae root colonization in soybean and maize yusif and hayatu, 2017 colletotrichum coccodes olabisi onabanjo university, ogun state control of velvetleaf (abutilon theophrasti) babalola, 2007 azospirillum and mazospiriflo-2 (azospirillum brasilense, strain) shanono, ibadan the combined inoculant reduced n accumulation in the shoot biomass of maize laditi et al., 2012 alternaria olabisi onabanjo university, ogun state weed control babalola, 2009 the effects of microbial inoculants on plants the success of microbial inoculation depends largely on the following: the plant species and cultivar, soil type, soil moisture and temperature conditions, the number of pathogens present in the soil around the plant and how the inoculants were prepared and applied (babalola et al., 2007). in nigeria, a study tested the effect of microbial inoculants and foliar fertilizers application on promiscuous soybean (tgx 1448-2e) under smallholder farmers’ conditions in the northern guinea savanna, of nigeria. three microbial inoculants; bradyrhizobium spp. (raca 6), arbuscular mycorrhizal fungi (rhizatech) and trichoderma harzianum (eco-t) and two foliar fertilizers; agroleaf high p and agolyser were used. grain yield was relatively increased by the application of raca 6 + tsp, raca 6 + agrolyzer and raca 6 + rhizatech + agrolyser compared to the control. the authors suggested that soybean co-inoculation with rhizobial or fungal inoculants and/or foliar fertilizers could lead to improved grain yields (ncho et al., 2013). a similar study was conducted for screening of 15 commercial and laboratory rhizobium inoculants in kadawa (sudan savanna) and samaru (northern guinea savanna) to identify effective and promising products on a promiscuous soybean genotype (tgx 1448-2e) and a malaysian genotype (samsoy-2). more so, an application of three commercial products and seven strains resulted in increased nodulation relative to the control in kadawa, while two commercial products and six strains increased nodulation, but only two strains resulted in significant increases in biomass and/or grain yield. the authors recommended 1495 mar, usda 4675, usda 110, tsbf 531 and tsbf 560 as effective inoculants to consider for improved grain yield in samaru (yusuf et al., 2012). more so, omeke, (2017) noted that, integration of inoculated soybean with rhizobium in the maizebased cropping systems in combination with n fertilizer application promotes higher carbon sequestration (stock) and soil n as compared to other treatments combination. a study was conducted in awka anambra state, nigeria to determine the suitability of decomposed rice husk, charcoal and coal to act as carrier materials for cowpea bradyrhizobia inoculants, using tal 209, tal 379 and tal 173 strains imported from us. the study showed decomposed rice husk as better carrier materials. a follow up pot experiment also confirmed the ability of bradyrhizobia in decomposed rice husk to nodulate, increase shoot dry matter and total n in cowpea in different soils relative to uninoculated controls (okereke and okeh, 2007). inoculation trials were also conducted on eutric cambisols (ec) and rhodic nitisols (rn) soils in a greenhouse study. soybean (tgx 1448-2e), cowpea (it90k-277-2) and groundnut (samnut 21) were used as test crops along with rhizobia inoculants (mar 1495, tsbf mixture, legumefix, histick and irj 2180a) to determine their response to soil type and ability to form pa ge 42 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(3) 39-43, 2023 symbiotic relationship with the crops. rhizobia strains mar 1495 and tsbf mixture showed similar ability to improve the productivity of soybean and groundnut and thus recommended for use as common inoculants for the two crops (aliyu et al., 2013). furthermore, the influence of rhizobia inoculation on n-fixation by cowpea (vigna unguiculata (l.) walp.), groundnut (arachis hypogaea l.) and bambara groundnut (vigna subterranea l.verdc.) was evaluated under field condition in maiduguri, sudano-sahelian zone of nigeria. strains isolated from the same crops the previous year were used to inoculate them the next year. the results showed rhizobia inoculation to increase the amount of nfixed by 46% over the control. cowpea fixed 42.68 kg n ha-1 while groundnut and bambara groundnut fixed 27.19 and 32.53 kg n ha-1, respectively. this indicates a higher potential of cowpea to alleviate soil nitrogen deficiency over the other legumes, particularly where the indigenous rhizobia nodulating the crops are inadequate (yakubu et al., 2010). conclusion in nigeria, the use of chemicals in agriculture can be avoided and thus they can be removed from human diets and minimize the reduction of environmental hazards. pest and weed control can be achieved by employing microbial inoculants as biocontrol agents and bio-herbicides. harnessing natural resources including beneficiary microorganisms is one of the most effective approaches to improving farm productivity and food quality in a sustainable way. microbial inoculant as part of agricultural practices will ensure healthy food security for the future population. several findings of microbial inoculants have been shown to improve soil properties, crops growth, biotic and abiotic stress and nutrient intake and further improve health living between microorganisms and plant in the root region. however, these findings are not restricted to nigeria alone and have necessitated the need to adopt microbial inoculants in other regions of the world. however, with the bulk of farmers in nigeria being unlettered and majority uneducated, it could be challenging to change their mindset and convince them of probable alternatives to the use of inoculants as a means of bio-fertilizers. references ali, m., cone, j. w., hendriks, w. h., & struik, p. c. 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(2017). influence of biochar application and amf inoculation on root colonization and selected soil chemical properties. annals of biological sciences, 5(1), 21-24. yusuf, a. a., jemo, m., nwoke, o. c., & abaidoo, r. c. (2012). evaluation of commercial and laboratory rhizobium inoculants on nodulation and yield of promiscuous soybean in the nigerian savanna. in m. k. a. adeboye, a. j. odofin, a. o. osunde, a. bala, & s. o. ojeniyi (eds.), soil resources management, global climate change and food security. proceedings of the 35th annual conference of the soil science society of nigeria (pp. 199 214). pa ge 1 pa ge 61 american journal of life science and innovation (ajlsi) revolutionizing pediatric dermatology: dupilumab’s impact on atopic dermatitis in kids eiman hamza ahmed elsayed1*, rabab elsayed ali hassan khater2 volume 3 issue 1, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i1.2696 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: may 01, 2024 accepted: may 31, 2024 published: june 04, 2024 eczema, or atopic dermatitis, is a common, non-communicable, immune-mediated, inflammatory skin illness mainly affecting children. it is a chronic condition. it causes mental health problems like anxiety, sadness, anxiety, depression, hyperactivity, and obesity. it is the first skin ailment and the fifteenth non-fatal disease. the purpose of the present research study was to evaluate the safety and effectiveness of biological treatment for atopic dermatitis using monoclonal antibodies against non-biological treatment, including antibiotics, immunosuppressants, demards, histamine antagonists and corticosteroids. patient information was gathered from fda-approved clinical trials. the comparative analysis found that biological therapies like dupilumab, omalizumab, and ustekinumab improve symptoms and disease control in atopic dermatitis. non-biological treatments may not be as effective. early antibiotic exposure can increase infection vulnerability. mild eczema infections require biological agents. topical corticosteroids can be combined with dupilumab for effective treatment of atopic dermatitis. studies show that dupilumab improves symptoms and quality of life, making it a better option than a placebo. fda recommends using biological agents over immunosuppressants in paediatrics for improved immune results. fda clinical trials showed that dupilumab is a safe and effective treatment for children with moderate-to-severe atopic dermatitis, reducing disease severity and improving quality of life in young ad patients. keywords atopic dermatitis, pediatrics, dupilumab, immunosuppressants, monoclonal antibodies corticosteroids introduction atopic dermatitis (ad), known as eczema, is a common, chronic, non-communicable, immune-mediated inflammatory skin condition that mostly affects children. it ranks first among all skin illnesses and 15th among nonfatal diseases (rusiñol & puig, 2024). it typically manifests within the first six months of life. it harms general health and is linked to a higher occurrence of mental health conditions and symptoms, including anxiety, depression, hyperactivity, sleep problems, asthma, hay fever, cardiovascular disease, stroke, and obesity (chovatiya & silverberg, 2019; nicholas et al., 2022; xu et al., 2019). it is caused by changes in the skin microbiotaimmune system hyperactivity, sensitivity to immunoglobulin e, neuro-inflammation (mandlik & mandlik, 2021), environmental variables, and hereditary predisposition. (grobe et al., 2019; mehta & fulmali, 2022). the intricate interactions between itch, inflammation, and barrier failure play a crucial role in this condition’s onset, progression, and chronicity, even though the pathophysiological mechanisms are yet unknown. skin inflammation that is aggravated by filaggrin abnormalities, intercellular lipids, and tight junctions is the hallmark of type 2 immune deviation. these abnormalities lead to barrier disruption in the skin, producing thymic stromal lymphopoietin, interleukin (il)-25, and il-33. subsequently, this inflammation intensifies the breakdown of the epidermal barrier by suppressing filaggrin expression in keratinocytes. moreover, various pruritogens and itch mediators generated during this inflammatory process can directly affect sensory nerves and induce itching (kim et al., 2019; nakahara et al., 2021). while in some children, food allergies are closely associated with ad, 30% of these children experience symptoms such as rash, itching, leaking, and dry, cracked skin. smaller kids show intolerance to some foods. red, weepy, crusty, itchy, and flaky skin patches that typically form in oval or circular forms are its defining feature (rusiñol & puig, 2024). age-related symptom variations include red rash in infants, thickened rash in children, and scaly rash in teens and adults. common 1 pediatric specialist in uae 2 dermatology specialist in uae * corresponding author’s e-mail: dr_eh2412@yahoo.com figure 1: conditions related to atopic dermatitis pa ge 62 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 61-68, 2024 features include extra skin folds and darkening (frazier & bhardwaj, 2020; soares et al., 2024). for patients with moderate to severe types of atopic dermatitis (ad), biological therapies are very important. these therapies concentrate on particular molecules involved in the inflammatory and immunological responses typical of ad. compared to more conventional treatments like corticosteroids or immunosuppressive medications, biological therapies like monoclonal antibodies like dupilumab and tralokinumab provide a more tailored approach by concentrating on particular molecular targets (deleanu & nedelea, 2019). new insights into the immunopathology of ad have made it possible to identify therapeutic molecular targets for innovative biological therapies. by modifying the immune response and inflammatory pathways, these treatments; including “il-4/il-13” inhibitors, jak inhibitors, and “il-13 inhibitors” have demonstrated notable success in treating ad. furthermore, the fda has approved biologics like dupilumab for treating moderate-to-severe ad, demonstrating their efficacy in enhancing patients’ quality of life and symptom relief (zhou et al., 2021). it provides a more focused and targeted approach. research has indicated that biological treatments are a useful means of alleviating symptoms. furthermore, biologics have demonstrated a quick start of action, offering immediate relief to patients who have not responded well to traditional therapy. clinical trials and real-world applications have provided strong evidence for the safety and effectiveness of biological therapy (caffarelli et al., 2023; montes-torres et al., 2015). the present research paper aimed to evaluate and compare the efficacy, optimal dosage, and frequency of administration of biological treatment such as monoclonal antibody dupilumab with standard treatment options, including antibiotics, corticosteroids, immunosuppressants such as cyclosporine, mycophenolate, tacrolimus, and azathioprine, histamines, and demards like methotrexate in the management of atopic dermatitis in pediatric patients. methods and clinical trials the research study on “revolutionizing pediatric dermatology: dupilumab’s impact on atopic dermatitis in kids” was conducted by employing the approved clinical trials studies of “united states-food & drug administration (fda)” among individuals of different ages, ethnicity, genders and health status. biological treatment options efficiency of biological treatment systemic non-biological therapies, such as oral corticosteroids, cyclosporine, methotrexate, azathioprine, and mycophenolic acid, effectively manage moderate to severe atopic dermatitis (paolino et al., 2023). nonbiological systemic therapy for atopic dermatitis may not be as effective at controlling symptoms and preventing exacerbations as biological treatments are (ferrucci et al., 2023). meanwhile, biological therapies, including dupilumab, omalizumab, and ustekinumab, offer a more targeted approach focusing on molecules linked to the immune response and inflammatory processes in ad. these biologics have shown a significant improvement in symptoms and quality of life for those with moderate to severe ad (chu et al., 2023) and disease control by targeting key pathways involved in ad pathogenesis, such as th2, th22, th17/il-23, and ige. monoclonal antibody dupilumab dupilumab obstructs the signalling pathways for interleukin 13 (il-13) and 4 (il-4). attaching to the alpha subunit of the interleukin-4 receptor (il-4rα), this method of action functions as a receptor antagonist. dupilumab specifically targets two important cytokines involved in asthma and atopic dermatitis inflammation: “il-4 and il-13” intracellular signalling. the simultaneous inhibition of “il-4 and il-13” signaling enhances skin barrier function, reduces type 2 inflammation, reduces itching, and improves skin lesions in people with atopic dermatitis. (kraft & worm, 2017). dupilumab significantly enhances clinical scores and symptoms in children with severe atopic dermatitis aged 6-11 years, improving their quality of life and smoother skin (berna-rico et al., 2023; cork et al., 2024). the recommended dosages and frequency of dupilumab for children with atopic dermatitis are weight-based and range from 200 mg to 300 mg for children weighing 15 to 30 kg, 30 to 40 kg, and 40 kg or more (berna-rico et al., 2023; cork et al., 2024; kamal et al., 2021). comparison with other treatment modalities antibiotics for atopic dermatitis since secondary skin infections frequently arise in ad patients, antibiotics, including cephalexin, clindamycin, doxycycline, azithromycin and amoxicillin/clavulanic acid, are frequently used to manage ad. in eczematous lesions of atopic patients, staphylococcus aureus colonization is common. it releases superantigens and exotoxins that prolong inflammatory reactions and worsen symptoms. patients with ad are often offered topical antibiotics such as gentamicin, fusidic acid, and mupirocin for mild to moderate secondary infections (neri, 2015). cephalexin is a first-generation cephalosporin used to treat staphylococcus aureus-related skin infections in atopic dermatitis, particularly in those with colonized staphylococcus aureus. its bactericidal activity supports its use in treating atopic dermatitis, prophylaxis, and minor procedures (class et al., 2022; rist et al., 2002). clindamycin a lincosamide antibiotic, is used to treat severe skin and pa ge 63 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 61-68, 2024 soft tissue infections caused by staphylococcus aureus. it works well against aerobic and anaerobic bacteria, especially in those with s. aureus colonization due to atopic dermatitis (coskey, 1978; mose et al., 2022). doxycycline a tetracycline antibiotic doxycycline, has been investigated for treating atopic dermatitis (ad). doxycycline may be able to treat ad since research indicates that it may include anti-inflammatory qualities and antibacterial benefits. doxycycline may have a role in regulating the immune response in allergy disorders such as ad. its use has been widely demonstrated to reduce mast cell histamine release and asthmatic patients’ levels of immunoglobulin e (ige) for skin disorders such as rosacea, acne, and perioral dermatitis (bohannon et al., 2020; hulme, 2023); it also functions by destabilizing nitric oxide synthase (nos), which can reduce inflammation and improve symptoms of atopic dermatitis by increasing degradation. it is frequently used to treat skin conditions such perioral dermatitis, acne, and rosacea. (bohannon et al., 2020). effectiveness and limitations children who were exposed to antibiotics in their early years had a higher chance of acquiring adverse events. irrational and direct use of broad-spectrum antibiotics increases susceptibility to infections and resistance rates, potentially leading to fatal diseases. the relative efficacy of antibiotics and biological treatment for pediatric atopic dermatitis depends on the particular circumstances and degree of the ailment. although they have a limited effect in treating the underlying inflammation of atopic dermatitis, antibiotics are frequently used to treat infections that may worsen the symptoms of atopic dermatitis. according to research, antibiotics may not significantly help children with minor eczema infections, and they may even increase skin sensitivity and resistance(choi et al., 2020; li et al., 2021; mubanga et al., 2021). corticosteroids in atopic dermatitis management depending on the severity of the ailment, different dosages and potencies of corticosteroids, such as hydrocortisone, fluticasone, betamethasone valerate, and tacrolimus, are frequently given to treat atopic dermatitis (drucker et al., 2018; stacey & mceleney, 2021). hydrocortisone hydrocortisone is frequently used for the treatment of atopic dermatitis in a variety of dosages and formulations. hydrocortisone comes in various formulations for atopic dermatitis, with concentrations ranging from 0.1% to 2.5%. these formulations include creams, lotions, gels, solutions, and ointments. lower potency hydrocortisone formulations “(class vi or vii), such as 1% cream” or ointment, are usually used for infants and children with atopic dermatitis. these formulations are administered twice a day to reduce inflammation and itch. low-potency steroids should be used for maintenance in cases of mild atopic dermatitis after intermediatepotency steroids (class iii, iv, and v) are taken for short periods to control flare-ups in severe atopic dermatitis cases in teenagers (haeck et al., 2011), while their excessive strength may result in negative side effects such as folliculitis, dermatitis, acne, atrophy of the skin, itchiness, changes in pigmentation, and inhibition of the hypothalamic-pituitary-adrenal axis (axon et al., 2021). fluticasone a corticosteroid, is available in several formulations and doses ranging from 0.05% to 2.5%. it is used in atopic dermatitis to reduce inflammation and symptoms by suppressing the release of inflammatory mediators (dhamija et al., 2024). on the other hand, a variety of negative side effects might occur, such as increased hair growth, dizziness, rash, itching, and redness of the treated area of skin. hives, breathing difficulties, swelling of the lips, tongue, or throat, skin pain, tenderness, or swelling, wounds that do not heal, severe skin irritation, or signs of systemic absorption such as weight gain, thinning or discolored skin, increased body hair, nausea, diarrhoea, fatigue, mood swings, menstrual changes, and sexual changes, as well as severe headaches, confusion, muscle weakness, vision abnormalities, heart symptoms, or serious allergic reactions are examples of serious side effects that can occur. (berth-jones et al., 2003; harvey et al., 2023). betamethasone valerate 0.1% twice daily for three to five days. the corticosteroid betamethasone can efficiently manage children’s eczema flare-ups, which enhances skin barrier function and reduces skin inflammatory responses (subramanian et al., 2018). topical & oral corticosteroids effectiveness in comparison with dupilumab-fda it has been shown that dupilumab (dupixent) can be used with or without topical corticosteroids in the treatment of disorders like atopic dermatitis based on the information from the sources that have been presented, including the fda clinical trials and reviews on the drug. dupilumab is a useful therapeutic option for moderate-to-severe atopic dermatitis since clinical trials have demonstrated that adding it to topical corticosteroids has significantly improved patient-reported symptoms and quality of life. according to a study, dupilumab is far more effective than a placebo in relieving the condition of individuals with atopic dermatitis when used in conjunction with topical corticosteroids. these trial studies have demonstrated the advantages of using dupilumab with topical corticosteroids, highlighting the drug’s efficacy in treating atopic dermatitis and minimizing the need for corticosteroids pa ge 64 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 61-68, 2024 alone. consequently, dupilumab combined with topical corticosteroids is a beneficial therapeutic strategy for diseases like atopic dermatitis, providing patients with better results and a higher quality of life, according to data from fda clinical trials and reviews (fda, 2017). immunosuppressant’s cyclosporine a calcineurin inhibitor, targets t cells specifically, inhibiting the transcription of the interleukin 2 gene, modifying the immune response, and lowering inflammation in diseases such as atopic dermatitis, typically administered at a dose of “5 mg/kg/day” and available in multiple forms, including intravenous formulations, oral solutions, and gelatin capsules (amber & tabassum, 2020; antti et al., 2021; megna et al., 2017; rajagopalan et al., 2022). mycophenolate based on numerous investigations and clinical trials, mycophenolate mofetil (mmf) has demonstrated promising outcomes in treating atopic dermatitis. studies showed that mmf is useful in treating moderate to severe ad among individuals who did not respond to conventional treatments; there was a notable improvement in the severity of the illness. mmf significantly reduced the scorad index, a gauge of the severity of ad, within a few weeks of treatment; it may be a useful substitute for people with severe or refractory types of ad because of fewer risks of causing adverse effects than other longterm therapies for the disease (phan & smith, 2020). azathioprine immunosuppressive drugs like azathioprine prevent the manufacture of purines, which impacts lymphocytes and helps regulate the immune system in atopic dermatitis (ad) by lowering inflammation and managing symptoms. the recommended daily dose of azathioprine for ad is 1-2.5 mg/kg, with adjustments based on individual patient response and tolerance. it can cause myelosuppression, increased risk of infection, and gastrointestinal problems. safety profile & clinical trials of immunosuppressants in comparison with dupilumab according to the information provided by the sources, including the fda approvals and clinical trials on the drug, dupilumab, also known as dupixent, has been the subject of substantial research spanning over “60 clinical trials” involving over “10,000” patients with various chronic conditions. for the treatment of disorders such as asthma, atopic dermatitis, chronic rhinosinusitis with nasal polyposis, and eosinophilic esophagitis, dupilumab, a completely human monoclonal antibody that inhibits interleukin-4 (il-4) and interleukin-13 (il-13) signaling has demonstrated noteworthy clinical advantages. dupilumab’s effectiveness in lowering type 2 inflammation, a major factor in many illnesses, has been demonstrated by these trials. specifically, the fda has approved dupilumab for kids with eosinophilic esophagitis aged one year and older, as well as kids with moderate-to-severe atopic dermatitis aged six months to five years. (fda, 2019). demards (disease-modifying anti-rheumatic drugs) methotrexate inhibits the immune system by inhibiting dna synthesis and cell division by blocking dihydrofolate reductase. for ad, methotrexate is administered at a starting dose of 10.3-2.6 mg/week, which can be changed according to the patient’s tolerance and reaction. weekly increases are possible, with maintenance dosages of about “11.7 mg” (lee et al., 2020; shah et al., 2018). comparison with biological agents like dupilumab on based on clinical trials in individuals with moderate to severe atopic dermatitis, dupilumab has been demonstrated to be more successful than methotrexate in reducing symptoms and quality of life. this conclusion is based on fda clinical trials and real-world analysis comparing the two medications. when compared to methotrexate, the trials showed that dupilumab improved atopic dermatitis symptoms and signs more, and there were fewer side effects and treatment discontinuations. for treating atopic dermatitis, dupilumab is therefore thought to be more effective and tolerable than methotrexate, according to data from fda clinical studies and real-world research (fda, 2017). histamines in atopic dermatitis treatment histamine antagonists, which block h1 and h4 receptors, reduce inflammation, nervous irritability, and itching to treat the symptoms of atopic dermatitis (ad). chronic dermatitis lesions lessen pruritus and inflammatory cytokines; in ad mice models, they have anti-inflammatory and anti-pruritic properties (albrecht & dittrich, 2015; ohsawa & hirasawa, 2014; schaper‐ gerhardt et al., 2020). histamine antagonist’s comparison with dupilumabfda clinical trials according to fda clinical trials, dupilumab is superior to histamine antagonists in treating disorders like atopic dermatitis and chronic spontaneous urticaria. atopic dermatitis patients with moderate-to-severe ad have shown notable improvements in their quality of life and a reduction in the severity of their condition after using dupilumab, an interleukin-4 receptor alpha antagonist. dupilumab, on the other hand, may provide a more thorough treatment of the disease’s underlying inflammatory processes than histamine antagonists, which target histamine receptors to relieve symptoms like pruritus. dupilumab is a better therapy choice than histamine antagonists for disorders including atopic dermatitis and chronic spontaneous urticaria because of its demonstrated ability to relieve signs and symptoms of ad with a tolerable safety profile (hon et al., 2021; clinical trials 2017). pa ge 65 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 61-68, 2024 tacrolimus as an immunosuppressant atopic dermatitis (ad) has found a valuable new therapy option in tacrolimus, a macrolide calcineurin inhibitor. it achieves its therapeutic effects through immune response modulation and inhibition of inflammatory processes linked to ad progression. in the treatment of moderate to severe ad, topical tacrolimus has demonstrated efficacy and safety, providing a non-corticosteroid option. research has shown that tacrolimus ointment (available in 0.03% and 0.1% concentrations) is more effective in treating ad than pimecrolimus and moderate corticosteroids. tacrolimus works by attaching itself to particular t cell receptors, which raises intracellular calcium levels and inhibits the transcription of cytokines such as “il-2, il-4, and il-5”. clinical studies have demonstrated topical tacrolimus’s effectiveness in lowering ad severity ratings and improving safety profiles with less systemic absorption (martins et al., 2015; umar et al., 2022). safety profile & clinical trials in comparison with dupilumab compared to tacrolimus, the fda clinical trials have yielded strong evidence for the safety and effectiveness of dupilumab in treating ailments such as atopic dermatitis. atopic dermatitis patients with moderate-to-severe ad have shown notable improvements in their quality of life and a reduction in the severity of their condition after using dupilumab, an interleukin-4 receptor alpha antagonist. dupilumab is a better therapy option than tacrolimus for ad patients due to its great efficacy in reducing symptoms and enhancing quality of life, as demonstrated by clinical trials. dupilumab is a better therapy choice than tacrolimus because of its focused mechanism of action and excellent safety profile (fda, 2019). summary of fda-approved clinical studies according to fda clinical trials, dupilumab has proven to be a highly effective and safe treatment for moderateto-severe atopic dermatitis in children. in children with atopic dermatitis, aged “6 to 11 years”, who are not sufficiently managed by traditional therapy such as topical corticosteroids and emollients, these trials have assessed the real-world effectiveness of dupilumab. research has indicated that dupilumab significantly improves overall quality of life, pruritus, sleep quality, and disease severity in young ad patients. more specifically, at week sixteen of treatment, a large percentage of patients showed statistically significant improvements in the “children’s dermatology life quality index (c-dlqi), sleep nrs (s-nrs), pruritus numerical rating scale (p-nrs), and eczema area severity index (easi) scores”. based on these results, young patients with moderate-to-severe atopic dermatitis can benefit from using dupilumab as a treatment option. it is a viable therapeutic alternative beyond topical medications (fda, 2019; kamphuis et al., 2022; napolitano et al., 2022). discussion eczema, or atopic dermatitis, is a chronic, noncommunicable, inflammatory skin ailment mainly affecting children. it ranks first among skin conditions and fifteenth among non-fatal diseases, and it can cause mental health problems. biological therapies like dupilumab, omalizumab, and ustekinumab target immune response and inflammatory processes in atopic dermatitis, improving symptoms, quality of life, and disease control. in contrast, nonbiological systemic therapy for atopic dermatitis may not be as effective in controlling symptoms and preventing exacerbations as biological treatments. early exposure to antibiotics can raise a child’s vulnerability to infections and resistance rates, which can result in life-threatening illnesses. the severity of the condition determines how well biological therapy and antibiotics work for pediatric atopic dermatitis. dupilumab and other biological agents are suggested for children with mild eczema infections. topical corticosteroids can be used alone or with dupilumab, a medication used to treat atopic dermatitis. clinical investigations demonstrate that augmenting corticosteroids with dupilumab greatly improves patients’ symptoms and quality of life. according to studies, dupilumab is a better treatment option for atopic dermatitis than a placebo, especially when used with topical corticosteroids. according to fda guidelines, biological agents are preferred in paediatrics over immunosuppressant including cyclosporine, mycophenolate, azathioprine and antimetabolite methotrexate because of their effectiveness in enhancing immune results rather than inhibiting the body’s immune system. it was evident from fda clinical trials that dupilumab is a safe and effective treatment for children with moderateto-severe atopic dermatitis, ages 6 to 11. studies reveal that in young ad patients, it considerably reduces the severity of the disease, pruritus, and general quality of life. in addition to conventional topical drugs, this treatment provides a good therapeutic alternative. challenges and considerations cost-effectiveness of dupilumab in comparison with non-biological treatments the analysis revealed that although the costs of dupilumab may be higher than those of non-biological treatments like topical medicines, it is related to more “quality-adjusted life years (qalys)” and good cost-effectiveness ratios. it suggests that compared to more affordable standard treatments like moisturizers and emollients, dupilumab adds value by significantly increasing health outcomes. according to the research, dupilumab presents a novel and efficacious therapeutic option for patients with moderate-to-severe atopic dermatitis, particularly when the effectiveness of conventional treatments is compromised. the severity of the ailment affects how pa ge 66 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 61-68, 2024 cost-effective dupilumab is compared to non-biological treatments; more severe patients are likely to benefit more from dupilumab in terms of better health and quality of life (anderson iii & szefler, 2019; sonya kahn et al., 2018). adherence to treatment regimens in pediatric patients in comparison to non-biological therapy, pediatric children with atopic dermatitis seem to adhere better to dupilumab treatment regimens. research has indicated that children with moderate-to-severe atopic dermatitis can experience improvements in disease severity, pruritus, sleep quality, and overall quality of life while using the completely human monoclonal antibody dupilumab. based on actual data from clinical trials, dupilumab significantly improves pediatric patients’ scores on the “eczema area severity index (easi), pruritus numerical rating scale (p-nrs), sleep nrs (s-nrs), and children’s dermatology life quality index (c-dlqi)”. dupilumab is well-tolerated and efficacious in juvenile populations, as evidenced by the high percentage of patients achieving “easi75” and the significant mean percentage reduction in p-nrs, s-nrs, and c-dlqi scores (napolitano et al., 2022; papp et al., 2021). conclusion potential advancements in biological therapies for atopic dermatitis targeted biological therapy dupilumab addresses treatment resistance and lowers the likelihood of relapse in patients with atopic dermatitis, improving treatment results and quality of life. this development in biological treatments is immensely beneficial in treating chronic inflammatory diseases like atopic dermatitis. importance of personalized treatment approaches optimizing results for children with disorders such as atopic dermatitis requires the use of personalized therapy techniques utilizing monoclonal antibodies, such as dupilumab. personalized treatment is a helpful therapeutic option for inadequately controlled atopic dermatitis, as studies in children aged 6-11 reveal significant improvements in disease severity, pruritus, sleep quality, and quality of life. recommendations ➢ real-world data on dupilumab’s effectiveness in diverse pediatric populations is needed to validate clinical trials and provide insights into its wider patient cohorts. ➢ examine the long-term safety and efficacy of dupilumab in treating pediatric patients with atopic dermatitis, as well as any possible negative effects and overall disease control. ➢ evaluate the effectiveness, safety, and tolerability of dupilumab compared to other biologic drugs in pediatric atopic dermatitis therapy, such as tralokinumab or omalizumab. limitations ➢ the severity of atopic dermatitis in children significantly influenced the response to dupilumab treatment, making it challenging to draw definitive conclusions. ➢ dupilumab treatment’s high cost and accessibility issues in certain healthcare settings caused hindrances in its widespread adoption and data availability in research studies. references albrecht, m., & dittrich, a. 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(2021). biological therapies for atopic dermatitis: a systematic review. dermatology, 237(4), 542-552. pa ge 1 pa ge 63 american journal of life science and innovation (ajlsi) evaluation of the antifungal effect of green synthesized metal oxide nanoparticles against plant pathogenic rhizoctonia species h. k. s. madusanka1*, a. g. b. aruggoda1, j. a. s. chathurika2, s. r. weerakoon3 volume 3 issue 2, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i2.4281 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: november 08, 2024 accepted: december 10, 2024 published: december 15, 2024 the current study successfully synthesized zinc oxide (zno), copper oxide (cuo), and iron oxide (feo) nanoparticles (nps) using cost-effective and environmentally friendly procedures. the synthesized nps were characterized by uv-vis spectroscopy and sem analysis. uv-vis spectroscopy revealed characteristic absorption peaks at 356 nm for zno nps, confirming their synthesis. sem analysis showed a heterogeneous distribution of nanoparticle sizes, with zno nps averaging 81 nm, cuo nps averaging 108 nm, and feo nps averaging 82 nm. the antifungal activity of the synthesized nanoparticles was evaluated at various concentrations using the poisoned food technique. the results indicated a dosedependent inhibition of mycelial growth by zno and cuo nps, with higher concentrations (500 and 1000 mg/l) showing significant inhibition compared to untreated rhizoctonia species. specifically, cuo nps exhibited mycelial growth inhibition percentages of 70.64% and 73.43% at 500 and 1000 mg/l, respectively, while zno nps showed inhibition percentages of 78.38% and 80.94% at the same concentrations. statistical analysis using one-way anova revealed significant differences among the treatment groups (p < 0.001). in contrast, feo nps did not exhibit a dose-dependent inhibition of mycelial growth but showed a minor, statistically insignificant promotion. among the tested nps, cuo nps at 1000 mg/l achieved the highest inhibition, followed by zno nps. the observed variations in mycelial inhibition by different nanoparticles at various concentrations underscore the complexity of nanoparticle-pathogen interactions and highlight the need for further research to optimize their antifungal efficacy. keywords antifungal activity, greensynthesized nanoparticles, poisoned food technique, rhizoctonia species introduction the rhizoctonia genus belongs to the basidiomycete phylum and consists of anamorphic fungi with different teleomorphs (moliszewska et al., 2023). rhizoctonia spp. is a significant plant pathogenic fungus responsible for diseases such as wilt and root rot. these diseases can manifest during the seedling stage as well as in the later stages of crop growth, leading to early infections that often result in plant death (jaiswal et al., 2014). r. solani produces numerous enzymes and phytotoxic substances that contribute to its pathogenicity, causing specific fungal disease symptoms in crops (mousa et al., 2024). the rhizoctonia genus comprises a vast and diverse group of soil-dwelling fungi, including binucleate rhizoctonia. while many species within this genus are plant pathogens and typically polyphagous, they exhibit distinct pathogenic preferences for specific plant species. despite their broad and varied host range, their affinity for certain plants is individually determined. among them, rhizoctonia solani is the most commonly reported and is recognized as a significant plant pathogen (moliszewska et al., 2023). the current research aims to investigate the antifungal potential of green-synthesized nanoparticles, including zno, cuo, and feo nps, against rhizoctonia species. the study will involve synthesizing the nanoparticles using green methods and characterizing their physicochemical properties. antifungal activity will be evaluated over a range of concentrations (0, 50, 100, 500, and 1000 mg/l) using the poison food technique to determine inhibition effects. literature review rhizoctonia spp cause significant damage to agricultural crops cuasing several diseases. sheath blight, caused by rhizoctonia solani, is a significant challenge for rice farmers, leading to yield losses ranging from 25% to 50% (chowdhury et al., 2019). rhizoctonia species are responsible for several diseases across different crops: soybeans suffer from aerial blight and web blight, tomatoes are susceptible to soil rot and fruit rot, peppers are affected by damping-off, and eggplants are prone to brown spot (tu et al., 1996). disease management for rhizoctonia spp is done by cultural practices and fungicides. cultural practices includes select cultivars that are resistant, tolerant, or less susceptible to rhizoctonia diseases, purchase certified disease-free seed or disease free transplants, crop rotation etc. although cultural approache is most essential control strategy, chemical management is still an important tool to decrease r. solani (tsror, 2010). as chemical management, applying pentachloronitrobenzene-based fungicides , tebuconazole, carbendazim, iprodione, and 1 department of agricultural and plantation engineering, the open university of sri lanka, nawala, nugegoda, sri lanka 2 department of urban bio-resources, university of sri jayawardanapura, ganagodawila, nugegoda, sri lanka 3 department of botany, the open university of sri lanka, nawala, nugegoda, sri lanka * corresponding author’s e-mail: hksma@ou.ac.lk pa ge 64 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 63-72, 2024 chlorothaloniland systemic fungicides such as carboxin, triadimefon, and thiophanate-methylis effective against rhizoctonia (karkee & mandal, 2020; chaube & pundhir, 2005). the growing concern over alternative approaches to chemical management of pathogenic fungi is closely linked to soil and environmental health. this includes issues such as increased pesticide residues in soil and water bodies, reduction in beneficial soil organisms, changes in soil physical and chemical properties, and pesticide resistance in pathogens (sharma et al., 2019). chemical control is the most widely used method to manage soilborne fungal pathogens. however, the negative environmental and health effects, disruption of natural ecosystem balance, and the emergence of fungal resistance to chemical pesticides highlight the need for sustainable and cost-effective alternatives. research has demonstrated that nanoparticles show promise as effective antimicrobial agents and biosensors for detecting plant pathogens, particularly against soilborne varieties, offering a comparatively environmentally friendly solution (dutta et al., 2023). nanoparticles smaller than 100 nm possess a higher surface area to volume ratio and enhanced reactivity, which enhances their suitability for widespread application in both human and plant pathology (jeevanandam et al., 2018). materials and methods biologic material and chemical reagents fresh samples of s. molesta and mimosa pigra were collected from uhana, ampara, sri lanka. high-purity zinc nitrate hexahydrate (zn(no3)2·6h2o) was procured from loba chemie pvt. ltd. extra pure analytical reagent grade cupric sulfate (cuso4·5h2o, minimum purity 99.5%) was acquired from sisco research laboratories pvt. ltd. sodium hydroxide pellets (naoh, minimum purity 98%) were sourced from loba chemie pvt. ltd. anhydrous ferric chloride of analytical research grade (fecl3) was obtained from himedia laboratories pvt. ltd. potato dextrose agar (pda) was purchased from himedia laboratories pvt. ltd. fusarium species were provided by the plant virus indexing centre in homagama, sri lanka. preparation of the mimosa pigra extract and synthesis of zno nps m. pigra leaves were thoroughly washed with tap water and rinsed with ddw to remove all debris. the washed leaves were then dehydrated at 42°c for 24 hours, ground, and sieved to remove large particles. eight grams of the dried leaf powder were mixed with 100 ml of ddw and stirred for 2 hours at 60°c and 400 rpm. the extract was centrifuged at 6000 rpm for 10 minutes, and the supernatant was filtered three times using whatman no. 1 filter paper. three grams of zinc nitrate hexahydrate (zn(no3)2·6h2o) were dissolved in 60 ml of the plant extract at room temperature and stirred for 1 hour. the solution was then heated in a thermal bath at 60°c for 12 hours to remove the water content. the resulting viscous paste was incubated at 100°c for 2 hours to form a crystalline powder-like material, which was subsequently calcined at 500°c for 2 hours. preparation of the salvinia molesta extract and synthesis of feo nps s. molesta was thoroughly washed and dehydrated at 42°c for 24 hours. the dried plants were then ground and sieved to obtain a fine powder. five grams of the powdered leaves were immersed in 80 ml of ddw and stirred for 1 hour at 60°c. the extract was filtered three times using whatman no. 1 filter paper. this leaf extract was mixed with a pre-prepared 0.1 m fecl3 solution in a 2:3 ratio. the ph of the mixture was raised to 10 by incrementally adding 1 m sodium hydroxide. the reaction mixture was left undisturbed for 24 hours to facilitate the synthesis of feo nps. subsequently, the mixture was centrifuged at 6000 rpm for 15 minutes, and the sediment was collected. the synthesized feo nps were washed trice with ddw and then calcined at 500°c for 2 hours. the resulting solid pellets were crushed using a mortar and pestle to obtain a fine powder-like structure. characterization techniques to verify the successful synthesis of zno, cuo, and feo nanoparticles, uv-visible (uv-vis) spectroscopy analysis was conducted with a resolution of 1 nm over a range of 190 nm to 800 nm using a quartz cuvette. the morphology of the synthesized nanoparticles was inspected using scanning electron microscopy (sem). the sem images were analyzed, and particle sizes were measured. in vitro evaluation of antifungal activity on mycelial development the antifungal activity of the nanoparticles against fusarium species was assessed using the poisoned food technique. potato dextrose agar (pda) medium was prepared with nanoparticle suspensions at concentrations of 0 (negative control), 50, 100, 500, and 1000 mg/l, and petri dishes (9 cm) were prepared with three replicates for each treatment. agar discs (8 mm in diameter) containing mycelia of fusarium species (7 days old, 28 ± 2°c) were then inoculated onto these plates. the plates were incubated at 28 ± 2°c until the fungal growth nearly covered the plate (9 cm). mycelial growth was measured as diameter (mm). the percentage inhibition was calculated using the following formula (1): inhibition percentage (%) = c-t/c ×100 (1) where “c” is the diameter of the fungal disc in the control, and “t” is the diameter of the fungal disc treated with different nanoparticle concentrations. analysis of variance (anova) was employed to determine significant differences between each mycelial growth treatment, with a 95% confidence interval (α = 0.05) applied. dunnett’s test was used to identify differences between the means of the inhibition percentage data pa ge 65 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 63-72, 2024 compared to the control group. duncan’s multiple range test (dmrt) was used to identify differences between the means of the inhibition percentage data for each treatment. the analysis was conducted using r statistical software (rstudio 2023.09.1+494). results and discussion the synthesized nanoparticles (zno, cuo, and feo) were characterized to confirm their successful synthesis and evaluate their properties. the uv-vis spectroscopy analysis revealed distinct absorption peaks, indicative of figure 1: uv-vis spectroscopy analysis of zno nps figure 2: sem images of zno nps at a scale of 200 nm figure 3: uv-vis spectroscopy analysis of cuo nps pa ge 66 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 63-72, 2024 the formation of nanoparticles. the morphology and size of the nanoparticles were further analyzed using sem. characterization of zno nps the green-synthesized zno nps were characterized using uv-vis spectroscopy with dmso as a reference. figure 1 shows a peak absorption at 356 nm, which preliminarily confirms the synthesis of zno nps. the consistent result was observed in a previous study of green synthesis of zno nps using an aqueous extract of figure 4: sem images of cuo nps at a scale of 10 µm figure 5: uv-vis spectroscopy analysis of feo nps figure 6: sem images of feo nps at a scale of 200 nm pa ge 67 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 63-72, 2024 epipremnum aureum leaves photocatalytic degradation of congo red having 326 as the maximum absorbance (razia sultana brishti et al., 2024). another study of the synthesis of zno nanoparticles using peels of passiflora foetida observed maximum absorbance at a wavelength ranging from 200 to 380 nm (khan et al., 2021). in a study on the green synthesis of zno-nps using juglans regia green husk aqueous extract, the biosynthesized zno-nps exhibited a prominent maximum absorbance band at 360 nm, as indicated by the uv-vis analysis results(dehghani & parinaz ghadam, 2023). collectively, these results affirm the successful production of zno nps in the current study. sem images in figure 2 reveal a heterogeneous distribution of shapes and sizes within the nanoscale range, with an average particle size of approximately 81 nm (35-164 nm). consistent results have been reported for green-synthesized zno nps with particle sizes of 30100 nm (thu et al., 2022), 79 nm (alyamani et al., 2021), and 75-90 nm (shanthi sathappan et al., 2021). characterization of cuo nps maximum absorbance in the uv-vis spectroscopy range of 190 nm to 450 nm was observed, with a peak at 224 nm, confirming the green synthesis of nanoparticles as shown in figure 3. similar results were reported in previous studies with absorption peaks at 265 nm (kumar et al., 2019), 220 nm (renuga et al., 2020), and 250–255 nm (kumar et al., 2017). the sem images illustrated in figure 04 reveal cuo nps with diverse shapes and sizes, averaging approximately 108 nm in diameter, and show a tendency to aggregate and form various structures. consistent results were also obtained in previous studies, reporting particle sizes of 18-106 nm (selvi et al., 2019), 197 nm (arushi saloki & daharwal, 2023), and 20-200 nm (manivannan rangasamy et al., 2023). characterization of feo nps uv-vis spectrometry indicated the formation of greensynthesized nanoparticles within the range of 200 to 600 nm, with a maximum absorbance at 300 nm, confirming the synthesis of feo nps. consistent results were also observed in previous studies, with maximum absorbance at 295–301 nm (karpagavinayagam & vedhi, 2019), 293 nm (andrade-zavaleta et al., 2022), and 230 and 290 nm (hussain et al., 2023). sem images in figure 6 show the shapes and sizes of the iron oxide nanoparticles, which exhibit a variety of nanoscale shapes and sizes, averaging around 82 nm (ranging from 41 to 140 nm), and tend to clump together and form multiple structures. similar results were also observed having particle sizes 30–100 nm (karpagavinayagam & vedhi, 2019), and 88 nm (aigbe & osibote, 2024). figure 7: rhizoctonia species mycelial growth inhibition test treated with nps synthesized by the green route: feo nps; (t1) 0 mg/l, (t2) 50 mg/l, (t3) 100 mg/l, (t4) 500 mg/l, (t5) 1000 mg/l figure 8: mycelium growth inhibition % of rhizoctonia species by different nanoparticles pa ge 68 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 63-72, 2024 evaluation of the antifungal effect of the nps the antifungal effect on mycelial growth of the synthesized nanoparticles was evaluated by amending the culture medium with different np concentrations and recording the fungus colony diameter over 7-9 days until the fungus nearly covered the plate (9 cm), with one nanoparticle tested at a time. all tested nps varied in their extent of inhibition of rhizoctonia species colony growth at different concentrations, with statistically significant differences calculated among the nanoparticles. as illustrated in figure 7, the inhibition of rhizoctonia species colony growth was observed. table 1: inhibition of colony growth of rhizoctonia species over control by poisoned food technique treated with zno nps concentration (mg/l) diameter (mm) colony growth inhibition % dmrt test result 0 80.03 ± 0.59 a 50 77.7 ± 4.64 2.91% a 100 78.9 ± 3.31 1.41% a 500 44.77 ± 7.17 44.06% b 1000 44.23 ± 8.21 44.73% b each value is mean of 3 replicates ± sd zno nps affected the mycelium growth at different concentrations, as tabulated in table 1. the one-way anova analysis demonstrated significant differences among the treatment groups, with a p-value (pr > f) of less than 0.001 and an f value of 35.14. this indicates that the variation in mycelial growth among the different nanoparticle concentrations is statistically significant. the control group (0 mg/l) exhibited a rhizoctonia mycelium growth diameter of 80.03 ± 0.59 mm. for the 50 and 100 mg/l concentrations, the mycelium growth diameters were 77.7 ± 4.64 mm and 78.9 ± 3.31 mm, respectively. according to dunnett’s test, there was no significant difference between the 0, 50, and 100 mg/l groups, despite inhibition percentages of 2.91% and 1.41% for the 50 and 100 mg/l concentrations, respectively. in contrast, the feo nps at higher concentrations (500 and 1000 mg/l) exhibited significant inhibition of mycelium growth compared to the control. the mycelium growth diameters for the 500 and 1000 mg/l concentrations were 44.77 ± 7.17 mm and 44.23 ± 8.21 mm, respectively. the inhibition percentages for these concentrations were substantially higher, at 44.06% for the 500 mg/l concentration and 44.73% for the 1000 mg/l concentration. these results indicate that higher concentrations of zno nps are more effective in inhibiting rhizoctonia mycelium growth. previous studies have also demonstrated the dosedependent effect of zno nps on the inhibition of rhizoctonia mycelium growth. for example, jannat et al. (2022) reported inhibition percentages of 17.1%, 31%, 40%, 48.7%, and 51.1% for r. solani at zno np concentrations of 0.05, 0.15, 0.25, 0.35, and 0.45 mg/ml, respectively. in another study, green-synthesized zno nps exhibited significant growth inhibition of r. solani, with inhibition percentages of 70.2 ± 0.5%, 61.4 ± 0.5%, 48.5 ± 0.5%, 1.2 ± 0.5%, and 0.6 ± 0.5% at concentrations of 1.0, 0.75, 0.5, 0.25, and 0.1 mg/ml, respectively (ali et al., 2022). those previous findings also support the dosedependent manner of zno nps inhibition of rhizoctonia mycelium growth. table 2: inhibition of colony growth of rhizoctonia species over control by poisoned food technique treated with cuo nps concentration (mg/l) diameter (mm) colony growth inhibition % dmrt test result 0 78.24 ± 1.25 a 50 73.38 ± 2.07 6.21% a 100 75.60 ± 3.58 3.37% a 500 22.97 ± 0.67 70.64% b 1000 20.79 ± 2.75 73.43% b each value is mean of 3 replicates ± sd the one-way anova analysis demonstrated significant differences among the treatment groups of different concentrations of cuo nps, with a p-value (pr > f) of less than 0.001 and an f value of 494.5. table 2 illustrates the dose-dependent effect of cuo nps on inhibiting the mycelium growth of rhizoctonia species. the control group exhibited a mycelium diameter of 78.24 ± 1.25 mm. at concentrations of 50 mg/l and 100 mg/l, the mycelium diameters were 73.38 ± 2.07 mm and 75.60 ± 3.58 mm, respectively, which were statistically similar to the control group (duncan’s multiple range test, dmrt), with growth inhibitions of 6.21% and 3.37%. in contrast, concentrations of 500 mg/l and 1000 mg/l resulted in mycelium diameters of 22.97 ± 0.67 mm and 20.79 ± 2.75 mm, respectively. these values were statistically similar to each other (dmrt) but significantly pa ge 69 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 63-72, 2024 different from the control group (dunnett’s test), showing inhibitions of 70.64% and 73.43%, respectively. these results indicate that higher concentrations of cuo nps are more effective in inhibiting rhizoctonia mycelium growth. previous study also suppor the findgs as at 150 ppm the cuo nps synthesized by chemical reduction method could inhibit the growth of r. solani by 62% (chowdhury et al., 2024). another study of green synthesized cuo nps in the concentrations of 50,100, and 200 ppm , maximum inhibition was recorded in 200ppm having 11.89% inhibition of r. solani(fetyan et al., 2024). similar study of cuo-nps biosynthesized from h. bacciferum leaf extract and antifungal activity for r. solani by 20, 50, 75, and 100 µg/ml they observed inhibition of mycelium growth 81.48% inhibition in 100 µg/ml concentration (hamdy et al., 2024). table 3: inhibition of colony growth of rhizoctonia species over control by poisoned food technique treated with feo nps concentration (mg/l) diameter (mm) colony growth inhibition % 0 80.53 ± 1.28 50 78.2 ± 3.54 2.89% 100 79.54 ± 0.63 1.23% 500 82.33 ± 2.06 -2.24% 1000 82.71 ± 0.60 -2.71% each value is mean of 3 replicates ± sd the one-way anova analysis indicated no significant differences in the mean radial growth of rhizoctonia mycelium treated with feo nps (f value: 2.81, pr > f > 0.05). the control and all treatments showed no significant differences, though minor inhibition and promotion zones were observed compared to the control. the maximum inhibition occurred at 50 mg/l, while the minimum inhibition was observed at 1000 mg/l. similar results were found in a study on the effects of feo nps on the mycelial growth of rot-causing fungi, where treatments with 0.1, 0.25, and 0.5 mg/ml resulted in mycelial growth of 12.00 ± 1.00 mm, 14.33 ± 0.57 mm, and 16.33 ± 0 mm, respectively, indicating higher concentrations led to increased growth rather than inhibition. many results found in the literature do not align with the observed data. for instance, a previous study reported significant inhibition of mycelium growth at various concentrations of feo nps. specifically, feo nps showed highly significant growth reduction, with the maximum growth inhibition (87.9%) observed at a concentration of 1.0 mg/ml (ali et al., 2020). another study investigated the growth inhibition percentages of different concentrations of iron oxide nanoparticles against two tested a. alternata strains. they observed mycelium inhibition at 29.85%, 47.24%, 55.95%, 65.74%, and 75.89% for concentrations of 50, 100, 200, 400, and 800 ppm, respectively (yassin et al., 2023). figure 8 illustrates that different concentrations of nanoparticles and various types of nanoparticles inhibit mycelium growth of rhizoctonia species in a dose-dependent manner. the maximum inhibition was observed with cuo nps at 500 and 1000 mg/l, followed by zno nps. the feo nps did not inhibit the rhizoctonia mycelium at higher concentrations, and instead promoted growth to a minor extent, although this increase was not statistically significant. the inhibition of mycelium growth by nanoparticles in a dose-dependent manner can be attributed to various mechanisms. for instance, nps may cause cell membrane damage, leading to increased membrane permeability. this disruption can induce responses within intracellular organelles, potentially altering dna and rna through mutation, affecting ion transport pathways, and disrupting protein functions (foldbjerg et al., 2015). smaller nanoparticles, as suggested by gliga et al. (2014), possess a larger surface area relative to their volume, facilitating easier penetration of fungal cell walls and membranes. this penetration can trigger responses within intracellular organelles. nanoparticles are also known to generate reactive oxygen species, which can further alter fungal membrane permeability. this alteration compromises cell structure and leads to osmotic imbalance, affecting fungal functions (kumari et al., 2019). studies have shown that silver nanoparticles inhibit spore germination in b. cinerea in a concentrationdependent manner (bayat et al., 2021). despite these insights, the mechanisms by which green-synthesized nanoparticles affect mycelium growth inhibition remain incompletely understood. further research is necessary to fully assess the potential impacts of nanomaterials on altering fungal growth dynamics. conclusion the current study successfully synthesized zno, cuo, and feo nanoparticles using a cost-effective and environmentally friendly procedure. the synthesized nanoparticles were characterized by uv-vis spectroscopy and sem analysis. the antifungal activity of the synthesized nanoparticles was evaluated at different concentrations using the poison food technique, testing one nanoparticle at a time. the results indicate that both zno and cuo nanoparticles exhibit a dose-dependent effect on mycelium growth inhibition, with higher concentrations (500 and 1000 mg/l) causing significant inhibition compared to the untreated rhizoctonia species. pa ge 70 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 63-72, 2024 in contrast, feo nanoparticles did not exhibit dosedependent inhibition of mycelium growth but caused minor promotion, which was not statistically significant. the study concludes that among zno, cuo, and feo nanoparticles, cuo nanoparticles at a concentration of 1000 mg/l demonstrated the maximum inhibition, followed by zno nanoparticles. the observed variations in mycelium inhibition by different nanoparticles at various concentrations highlight the complexity of nanoparticle interactions with fungal pathogens, underscoring the need for further research to optimize their antifungal efficacy. funding competitive research grant 2020, grant number 202003, the open university of sri lanka. declaration of generative ai and ai-assisted technologies in the writing process during the preparation of this work, the authors used chatgpt 3.5 to improve language and readability. after using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. references aigbe, u. o., & osibote, o. a. 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(2023). green synthesis, characterization, and antifungal efficiency of biogenic iron oxide nanoparticles. applied sciences, 13(17), 9942. https:// doi.org/10.3390/app13179942plications. journal of hazardous materials advances, 13, 100401–100401. https://doi.org/10.1016/j.hazadv.2024.100401 pa ge 1 pa ge 20 american journal of life science and innovation (ajlsi) post-operative complications following third molar extraction in qassim region saudi arabia: a retrospective study faris almutairi1*, faraj alotaiby1, abdullah alrashid2, nimran almodhaibri2 volume 3 issue 1, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i1.2440 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: january 30, 2024 accepted: march 02, 2024 published: march 06, 2024 third molar surgery is one of the most common dental procedures that can be done in the outpatient clinic. extraction complications can be very mild, like discomfort or mild pain, or severe, like a mandibular fracture. the study aims to investigate postoperative complications after a third molar extraction in the oral surgery dental clinic at qassim university. this retrospective study analyzed postoperative complication rates following third molar extractions performed at dental clinics in qassim, saudi arabia, between august 2022 and march 2023. descriptive statistics characterized the sample, and comparative analysis evaluated relationships between complications and factors like gender, age, and surgical aspects. ethical approval was granted, and sensitive patient information remained confidential. a total of 199 third morals were extracted from 70 patients with an average of 2.8 teeth per patient. more mandibular third molars were removed (55.8%) from the patients who participated compared to maxillary third molars extractions (44.2%). the results indicated that the rate of postoperative complications ranged from 2.5% to 64.7%. the most frequent postoperative complications were pain (64.7%), followed by swelling or edema (55.5%), trismus (32.8%), and dry socket (9.2%). this study’s postoperative complication rate of 10.4% following third molar surgery was consistent with other reports. younger patients and mandibular extractions presented higher complication risks. regional outcome data can guide clinicians locally to optimize preoperative planning and techniques, reducing risks for those undergoing this standard dental procedure. keywords third molar, tooth extraction, surgical procedures, complications, postoperative introduction impacted third molars, also known as wisdom teeth, are among the most prevalent dental abnormalities seen in oral surgery practices (miloro et al., 2004). according to various research studies examining different populations, approximately 16.7-68.6% have at least one impacted third molar (brown et al., 1982). the surgical removal of impacted or partially erupted third molars is among the most routinely performed outpatient dental procedures (visintini et al., 2007; woldenberg et al., 2007). while symptoms may vary depending on the individual, impacted third molars can lead to complications if left untreated, prompting their surgical extraction. this operation is commonly conducted in outpatient clinical settings to manage problems from impacted wisdom teeth (woldenberg et al., 2007). while third molar removal is typically uncomplicated, potential issues can occasionally emerge during the procedure or in the postoperative recovery phase (sayed et al., 2019). adverse outcomes range from mild, transient discomfort or pain to severe risks, like mandible fracture (chen et al., 2021; sayed et al., 2019). the reported complication incidence varies widely from 4% up to 30% or more of cases (bui et al., 2003), depending on surgery complexity, patient characteristics, and anatomic relationships of the impacted tooth (askar et al., 2019). pain and swelling are among the most prevalent issues after third molar surgery (bui et al., 2003), while mandible fracture is exceedingly rare (jerjes, swinson, et al., 2006). references indicate that many wisdom tooth operations proceed smoothly when handled professionally with care for individual variability. however, careful follow-up is prudent given the possibility of complications, though their severity and likelihood depend on various surgical and non-surgical influences (bouloux et al., 2007). patient age is one of the most significant factors associated with increased postoperative complications following third molar extraction (osunde & saheeb, 2015). in general, older patients have higher rates of complications compared to very young patients, who have much lower incidence (osunde & saheeb, 2015). smoking and other tobacco products also increase risk, primarily for developing local infections and dry sockets (rakhshan, 2018). high-level evidence is still required, but some research suggests that the frequency of complications may be slightly higher in females than in males, possibly due to hormonal factors involving oral birth control (benediktsdóttir et al., 2004). it has been demonstrated that careful pre-surgical planning, which includes the best surgical concepts and techniques, is essential for lowering the likelihood of unfavorable outcomes (benediktsdóttir et al., 2004; jerjes, el-maaytah et al., 2006; yuasa & sugiura, 2004). the body of research emphasizes how crucial it is to consider patient variability and use clinical and technical best practices to reduce complications (bouloux et al., 2007). 1 department of maxillofacial surgery and diagnostic sciences, college of dentistry, qassim university, buraidah, qassim, saudi arabia 2 college of dentistry, qassim university buraidah, qassim, saudi arabia * corresponding author’s e-mail: farisalmutairi558@outlook.com pa ge 21 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 20-27, 2024 surgical removal of impacted third molars is a standard outpatient procedure but can result in post-operative complications. local data on complication rates and trends after these extractions in qassin, saudi arabia, must be included. understanding regional outcomes may help optimize preoperative planning and surgery to reduce risks. the study evaluates complications following third molar complications at qassim university clinics to provide insights for improving local postoperative care and guidance for those undergoing this frequent dental operation in the region. materials and methods study design this retrospective study analyzed complications after wisdom tooth removal by reviewing past patient records from qassim, saudi arabia. the retrospective format provided an effective way to collect long-term data from many patients. however, complications were limited to those explicitly documented rather than directly observed. the records of patients who underwent third molar extraction between august 2022 and march 2023 were reviewed. ethical approval ethical approval was obtained from the local research ethics committee registration. no. h-04-q-001, qassim province. all patient demographics, history, and diagnosis information was kept strictly confidential. eligibility criteria inclusion criteria • patients who underwent extraction of one or more mandibular and maxillary third molars. • extractions performed at the oral surgery department of qassim university dental clinics between august 2022 and march 2023. • procedures carried out under local anesthesia/local infiltration on an outpatient basis. • patients who were over 18 years of age. • both male and female patients were included. • patients who had at least one post-operative followup visit to assess healing and complications. exclusion criteria • extractions done under general anesthesia • patients with concomitant systemic illness/diseases that could affect wound healing, such as diabetes, hiv/ aids, etc. • patients with pericoronitis, odontogenic infection, or facial space infection related to the targeted tooth. • patients requiring hospitalization or intravenous antibiotic therapy post-extraction. • pregnancy or lactation. • missing or incomplete medical records. • failure to attend scheduled postoperative follow-up visits. • extractions involving severe bone removal/impaction requiring flap surgery or osteotomy procedures • patients with a history of allergy or hypersensitivity to local anesthetic agents or antibiotics. surgical procedure an oral and maxillofacial surgeon worked in the dental clinics at qassim university to extract every third molar. the surgeon used a variety of methods under local anesthesia, depending on the location and extent of impaction of the tooth. this involved exposing the tooth by elevating the mucoperiosteal flap and retracting the lingual flap, if necessary. when necessary, elevators, forceps, and surgical drills were it breaks through bone, loosens tooth attachments, and extracts the impacted molars. after extraction, saline irrigation was used to clean and visualize the area. any remaining irregularities in the bone were cut away. after that, absorbable sutures were used to close the mucosal incisions or soft tissue flaps. patients were given analgesics and comprehensive wound care instructions after surgery. data collection data was collected by reviewing patients’ files for postoperative complications and then recalling them for follow-up in 1-week post-op appointments to record postoperative complications. all complications were recorded, including pain, swelling, edema, trismus, dry socket, infection, broken root, and any signs of nerve injury. data analysis data analysis was performed using spss statistics version 25 software from ibm. descriptive statistics included the mean and standard deviation for numerical variables and frequencies and percentages for categorical variables— comparative analyses employed chi-square and fisher’s exact tests as relevant. results were considered statistically significant at the p ≤ 0.05 level. results a total of 199 third morals were extracted from 70 patients with an average of 2.8 teeth per patient. there were more mandibular third molars extracted (55.8%) from the patients who participated compared to maxillary third molars extractions (44.2%) (fig. 1-2). the majority of patients were males (62.9%), while females comprised (37.1%) of the participants (fig. 3). the age ranged from 19 to 54 years with a mean (±sd) age of 28.2 ± 7.4 years. the most frequent age category among the participants was 25 to 29 years (35.7%), followed by 19 to 24 years (32.9%), then 30 years and older (31.4%) (fig. 4). [see table 1] table 1: demographic characteristics of patients and extracted third molars location characteristics n % age (mean ± sd) 28.2 ± 7.4 19 24 years 23 32.9% pa ge 22 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 20-27, 2024 table 2 presents the frequency and percentage of postoperative complications by patients and the total number of teeth extracted. concerning the total number of third molars extracted, the results indicated that the rate of postoperative complications ranged from 2.5% to 64.7%. the most frequent postoperative complications were pain (64.7%), followed by swelling or edema (55.5%), trismus (32.8%), and dry socket (9.2%). at the same time, infections, broken roots, and nerve injuries were found in less than 10% of third molar extraction (fig. 5). [see table 2] 25 29 years 25 35.7% 30 years and older 22 31.4% gender male 44 62.9% female 26 37.1% location of extracted third molars (n = 199) maxilla 88 44.2% mandible 111 55.8% extraction site (n = 199) 18 46 23.1% 28 42 21.1% 38 54 27.1% 48 57 28.6% figure 1: location of extracted third molars figure 3: patients’ gender figure 2: extraction site figure 4: patients’ age table 2: frequency and percentage of complications following third molar extraction complications n (%) n (%) patient (n = 70) tooth (n = 119) pain 44 (62.9 %) 77 (64.7 %) swelling / edema 38 (54.3 %) 66 (55.5 %) trismus 27 (38.6 %) 39 (32.8 %) pa ge 23 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 20-27, 2024 according to the previous results, the most frequent postoperative complications following third molar extraction were pain, swelling/edema, trismus, and dry socket. next, the relationship between these complications and patients’ gender, age, and the location of extracted third molars was studied. the results in table 3 indicated that most patients with pain complications were males (68.2%) compared to females (31.8%). however, the results showed no significant association between patients’ gender and pain complications (p = 0.230). similar results were revealed concerning swelling/ edema complications (60.5% vs 39.5%, p = 0.660) and trismus complications (74.1% vs. 25.9%, p = 0.0.124). on the other hand, results indicated more females with dry socket complications (54.5%) than males (45.5%). however, the results showed no significant association between patients’ gender and dry socket complications (p = 0.193) (fig. 6). [see table 3] dry socket 11 (15.7 %) 11 (9.2 %) infection 5 (7.1 %) 5 (4.2 %) broken root 3 (4.3 %) 3 (2.5 %) nerve injury 3 (4.3 %) 3 (2.5 %) figure 5: third molars extraction complications table 3: third molars extraction complications according to patients’ gender complications males females pvalue† pain no 14 (53.8 %) 12 (46.2 %) 0.230 yes 30 (68.2 %) 14 (31.8 %) swelling / edema no 21 (65.6 %) 11 (34.4 %) 0.660 yes 23 (60.5 %) 15 (39.5 %) trismus no 24 (55.8 %) 19 (44.2 %) 0.124 yes 20 (74.1 %) 7 (25.9 %) dry socket no 39 (66.1 %) 20 (33.9 %) 0.193 yes 5 (45.5 %) 6 (54.5 %) † chi-square test / fisher exact test figure 6: third molars extraction complications according to patients’ gender pa ge 24 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 20-27, 2024 table 4: third molars extraction complications according to patients’ age complications 19 24 years 25 29 years 30 years and older pvalue† pain no 6 (23.1 %) 10 (38.5 %) 10 (38.5 %) 0.379 yes 17 (38.6 %) 15 (34.1 %) 12 (27.3 %) swelling / edema no 5 (15.6 %) 15 (46.9 %) 12 (37.5 %) 0.018 yes 18 (47.4 %)* 10 (26.3 %) 10 (26.3 %) trismus no 10 (23.3 %) 19 (44.2 %) 14 (32.6 %) 0.067 yes 13 (48.1 %) 6 (22.2 %) 8 (29.6 %) dry socket no 18 (30.5 %) 22 (37.3 %) 19 (32.2 %) 0.618 5 (45.5 %) 3 (27.3 %) 3 (27.3 %) † chi-square test / fisher exact test figure 7: third molars extraction complications according to patients’ age table 5: third molars extraction complications according to location complications maxilla mandible pvalue† pain no 76 (62.3 %) 46 (37.7 %) < 0.001 yes 12 (15.6 %) 65 (84.4 %) swelling / edema no 73 (54.9 %) 60 (45.1 %) < 0.001 yes 15 (22.7 %) 51 (77.3 %) trismus no 86 (53.8 %) 74 (46.3 %) < 0.001 yes 2 (5.1 %) 37 (94.9 %) dry socket no 88 (46.8 %) 100 (53.2 %) 0.001 yes 0 (0 %) 11 (100 %) results in table 4 indicate a significant relationship between patients’ age and swelling/edema complications (p = 0.018). the results showed that around half of patients with swelling/edema complications were aged 19-24 years (47.4%), compared to (26.3%) in other age groups. in addition, the results showed a higher proportion of patients with pain (38.6%), dry socket (45.5%), and trismus (48.1%) complications among patients aged 1924 years compared to the other age groups; however, the results indicated no significant association between these complications and patients’ age (fig.7) [see table 4] a statistically significant relationship was observed between the location of extracted third molars and each pain, swelling/edema, trismus, and dry socket complication (p < 0.05) (table 5). the results indicated that the majority of trismus (94.9%), pain (84.4%), and swelling/edema (77.3%) complications corresponded to mandibular third molars compared to maxillary third molars extractions. moreover, the results showed that all dry socket complications corresponded to mandibular third molars. [see table 5] pa ge 25 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 20-27, 2024 discussion this study provides insight into complication rates after third molar surgery in qassim. pain and swelling were most common, while severe issues were rare. in this discussion, we compare our results to other studies and explore factors influencing trends. we also acknowledge limitations and recommend optimizations to reduce risks for patients undergoing this procedure in the region. the present study indicated that the range of different postoperative complications was 2.5% to 64.7%. fortunately, the majority of reported complications were minor and transitory. the current study focused on postoperative complications rather than intraoperative complications. a recent survey by azenha et al. reported an overall postoperative complication rate of 10.4% following third molar surgery. this is comparable to rates found in other similar studies, such as 9.8% reported by bui et al. and a slightly lower rate of 9.1% observed by muhonen et al. (azenha et al., 2014; bui et al., 2003). our study differed in finding most male patients, whereas this new study found more females. we also reported higher rates of more common issues like pain, swelling, and trismus as chief complications. in contrast, their overall lower complication rate of 10.4% included less prevalent issues like hemorrhage, fractures, and dry sockets as tops (azenha et al., 2014). the present study demonstrated that complications arose more frequently following the extraction of mandibular third molars than maxillary third molars. most other research that has examined complications associated with the removal of impacted wisdom teeth has likewise found that mandibular extraction sites are more prone to postoperative issues than maxillary locations (bui et al., 2003; sisk et al., 1986). according to existing research, dry sockets, infection, bleeding, and sensory issues caused by nerve damage are frequently reported postoperative complications following third molar surgery (bouloux et al., 2007; middlehurst et al., 1988). these published findings align with the results of the current study, which also observed dry sockets, infection, and sensory complications. the extraction of a third molar frequently causes expected but usually transient postoperative problems such as pain, swelling (edema), and restricted jaw opening (trismus). however, the pain from these procedures can last longer than the first week after surgery, and sometimes more care is needed, like dressing changes or antibiotics, which need to be given at a follow-up appointment (bouloux et al., 2007). according to studies, dry socket occurs in 0.3-26% of all extractions, with third molar extraction being the most common (bouloux et al., 2007; haug et al., 2005). following the extraction of mandibular third molars, specific controlled research has discovered an occurrence of up to 25%–30% (blum, 2002). various research has identified several risk factors associated with an increased likelihood of dry sockets occurring following third molar extraction. some of these risk factors include advanced age, female gender, oral contraceptive use, smoking, more incredible surgical trauma during the procedure, and a pre-existing condition known as pericoronitis (blum, 2002; haug et al., 2005). the study findings also align with the results of our current study results. the present study found a comparatively low incidence of dry sockets (9.2%); all cases happened following the extraction of mandibular third molars, and five occurred in patients aged 19–24. in this study, patients aged 19-24 had a higher incidence of dry sockets, which contradicts previous studies. in the literature, infections after surgery following third molar extraction have been documented at a prevalence of 0.8 to 4.2% (bouloux et al., 2007). similar to the current study, postoperative infections following third molar extraction were 4.2%. nerve injury to the lingual nerve or inferior alveolar nerve (ian) is one of the least desired side effects after third molar extraction (kushnerev & yates, 2015). because of its effect on speaking, mastication, swallowing, and social relations, it can be disastrous for sufferers (ziccardi & zuniga, 2007). in the current study, nerve injuries accounted for 4.3% of patients, and luckily, all of these injuries were temporary figure 7: third molars extraction complications according to patients’ age pa ge 26 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 20-27, 2024 and healed without any medical or surgical intervention. the depth of impaction and the proximity of molar roots to the alveolar canal are risk factors for injury to the inferior alveolar nerve (kang et al., 2020). root tip fractures are relatively common after third molar removal because of the high curvature of the roots and the presence of extra roots (kang et al., 2020). usually, the removal of the tips takes a little spare time to finish. deliberate preservation of the apical section of the roots may be required to avoid ian injury if preoperative imaging indicates a close relationship between the tooth root and the inferior alveolar nerve (meyer & bagheri, 2011). limitation one limitation of this study is that it needed to distinguish between simple dental extractions and more complex surgical extractions of third molars. this lack of procedure classification makes it difficult to compare postoperative complication rates based on the degree of difficulty. conclusion a spectrum of post-operative complications of the third molar extraction in the qassim region was documented in this study. the most commonly reported complications are pain and swelling, while the least common complications are root fracture and nerve injury. two-thirds of these complications were reported in patients younger than 30 years. lower third molars are associated with a higher complication prevalence than the upper third molars. references askar, h., di gianfilippo, r., ravida, a., tattan, m., majzoub, j., & wang, h. l. 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(2007). nerve injuries after third molar removal. oral and maxillofacial surgery clinics, 19(1), 105-115. pa ge 1 pa ge 44 american journal of life science and innovation (ajlsi) retroperitoneal müllerian cyst found incidentally during total laparoscopic hysterectomy: is there a role for mri in pre-operative evaluation? angela nyambura ngugi1*, joseph njagi1 volume 3 issue 1, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i1.2298 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: april 04, 2024 accepted: may 09, 2024 published: may 14, 2024 retroperitoneal cysts of müllerian origin are rare and often discovered incidentally during surgical procedures or imaging studies. we present a case of a retroperitoneal cyst found during a total laparoscopic hysterectomy performed for myoma removal. a 40-year-old female presented with symptomatic myomas and underwent pre-operative evaluation, including pelvic ultrasound, which revealed no abnormalities apart from the myomas. a cyst was incidentally discovered in the retroperitoneal space during a total laparoscopic hysterectomy procedure. the cyst was excised and sent for histological examination, which confirmed its müllerian origin. retroperitoneal cysts of müllerian origin are rare findings during tlh. this case report highlights the importance of thorough pre-operative evaluation and imaging studies and the significance of intraoperative awareness in identifying incidental findings. further research is warranted to determine the optimal diagnostic imaging strategies for such cysts. keywords retroperitoneal cyst, müllerian cyst, total laparoscopic hysterectomy, leiomyoma, incidental finding introduction retroperitoneal cysts of müllerian origin are rare findings, often discovered incidentally during surgical procedures or imaging studies. these cysts arise from remnants of müllerian duct remnants and can occur in various locations within the retroperitoneal space (santana gonzalez et al., 2021). uterine fibroids, also known as leiomyomas, are benign tumors originating from the smooth muscle cells of the uterus. although up to 70% of cases may be asymptomatic, they are sometimes associated with heavy menstrual bleeding, intermenstrual symptoms, pelvic pain, and pressure symptoms, necessitating treatment by medical therapies, interventional radiology, and surgical procedures (giuliani et al., 2020). total laparoscopic hysterectomy (tlh) is a minimally invasive surgical procedure frequently performed to manage symptomatic fibroids. while uterine fibroids are a common indication for surgical intervention, the discovery of an incidental retroperitoneal cyst during a tlh in a patient with uterine fibroids is a unique occurrence. incidental findings during tlh can pose diagnostic challenges and require careful consideration. although rare, retroperitoneal cysts of müllerian origin have been reported in the literature (yang et al., 2004). these cysts can present as an incidental discovery during surgery, often lacking pre-operative imaging clues (rivas et al., 2022). understanding such incidental findings’ characteristics, management, and implications is crucial for appropriate patient care. this case report presents the incidental discovery of a retroperitoneal cyst of müllerian origin during tlh performed in a patient with uterine fibroids. by highlighting this unique case, we aim to contribute to the existing literature and raise awareness about the importance of thorough pre-operative imaging and management of incidental retroperitoneal cyst findings during tlh. literature review müllerian cysts are uncommon findings that often present as incidental discoveries during imaging studies or surgical procedures. these cysts arise from remnants of the müllerian duct system (giuliani et al., 2020; wilson & bordoni, 2024). the müllerian ducts, also known as paramesonephric ducts, are paired ducts that originate from the intermediate mesoderm in the embryo (wilson & bordoni, 2024). during early fetal development, these ducts emerge alongside the urogenital ridge and run laterally. they terminate at the müllerian eminence within the primitive urogenital sinus. the müllerian ducts play a crucial role in the development of the female reproductive system (sugi et al., 2021). these paired structures emerge during early fetal life and contribute to the formation of several female reproductive organs, including the fallopian tubes, uterus, cervix, and the upper two-thirds of the vagina. in males, the müllerian ducts typically regress due to the influence of anti-müllerian hormone (amh) secreted by the developing testes (wilson & bordoni, 2024). however, remnants of these ducts such as müllerian cysts may persist in both sexes, leading to various clinical conditions. müllerian cysts result from focal incomplete regression of the müllerian ducts (sugi et al., 2021). areas where complete regression did not occur become localized cystic formations. these cysts can occur anywhere along the path of müllerian duct regression. retroperitoneal müllerian cysts are rare, 1 p.c.e.a tumutumu hospital, kenya * corresponding author’s e-mail: angiengugi75@gmail.com pa ge 45 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 44-47, 2024 with limited prevalence data (yang et al., 2004). they are typically discovered incidentally during imaging studies or surgery. the exact incidence remains uncertain due to their asymptomatic nature. when symptomatic, patients may experience abdominal pain or discomfort due to cyst enlargement or pressure symptoms related to adjacent structures such as the urinary tract (renzulli and candinas, 2009). imaging modalities that can be used in the detection of mullerian cysts include ultrasound, ct and mri (stefanopol et al., 2022; yacoub et al., 2021; yang et al., 2004). ultrasound can visualize müllerian cysts as hypoechoic lesions. however, ultrasound may not always provide detailed anatomical information compared to mri. ultrasound-guided aspiration can be performed for diagnostic purposes (yacoub et al., 2021). on ct scan mullerian cysts may appear as well-defined hypoattenuating cystic masses. mri however provides superior soft tissue characterization and multiplanar imaging capabilities for accurate pre-operative assessment. mri characteristic features for mullerian cysts include well-defined, unilocular cystic lesions, hypointense signal on t1-weighted images and hyperintense signal on t2weighted images (renzulli & candinas, 2009). pelvic mri remains the imaging modality of choice for diagnosing müllerian cysts, allowing accurate assessment and guiding clinical management (yacoub et al., 2021). treatment options for müllerian cysts include observation and monitoring for asymptomatic cysts with regular follow-up imaging to exclude any other conditions (johan et al., 2020). surgical excision is indicated for symptomatic or large cysts. pre-operative mri guides surgical planning and should be carried out whenever possible (johan et al., 2020). complete cyst excision is usually the goal intraoperatively (yacoub et al., 2021). although rare, complications of müllerian cysts may include infection, hemorrhage, mass effect and transition to malignancy (zhu et al., 2023). methodology case presentation a 40-year-old african female, para 1+0, presented to pcea tumutumu hospital in nyeri, kenya, with a ten-year history of intermenstrual bleeding and heavy and prolonged periods associated with dysmenorrhea and intermittent lower abdominal pain radiating to the back. in 2013, she was diagnosed with uterine fibroids through a pelvic ultrasound scan and had been managed conservatively for her symptoms. however, she recently visited the gynaecology outpatient clinic with persistent lower abdominal pain and irregular periods. a repeat ultrasound scan revealed two intramural hypoechoic masses measuring 96.74x95.75x115.52 mm and 26.44x17.51x36.71 mm, respectively, along with a pedunculated fibroid in the left adnexal region. the decision was made to proceed with tlh. laboratory investigation the patient was admitted on 17th may 2023, and routine pre-operative tests were conducted, including full blood count, urea, electrolytes, creatinine, random blood sugar, and group crossmatch. the only abnormal findings were a microcytic anemia with hb of 7.5 g/dl (ref 11.5-16.5 g/dl) and mcv of 67 fl (ref 75-100 fl), due to chronic blood loss. serum tumor marker tests were not carried out. results the tlh procedure was performed under general anesthesia by consultant obstetrician/gynecologist and laparascopic surgeon dr njagi. intraoperatively, the leiomyomas were noted to have fatty degeneration. after the removal of the uterus, a retroperitoneal cyst of size 40mm by 5mm was incidentally noted (figure 1a). macroscopic examination revealed a white encapsulated lesion adherent to the left lower abdominal wall. the cystic lesion was filled with brownish fluid (figure 1b). the lesion was surgically excised as a whole and sent for histopathological analysis. the whole surgical procedure took a total of 2 hours and 50 minutes with a total intraoperative blood loss of about 50cc. the post-operative stay was uneventful, with the patient experiencing mild pain. vitals remained stable, and a physical examination indicated normal bowel sounds. post-operative medication consisted of paracetamol 1g iv tds for 3 days and ceftriaxone 1g iv twice daily (bd) for 3 days. upon discharge, the patient was a b figure 1: a retroperitoneal cyst of size 40mm by 5mm was incidentally noted (a). the cystic lesion was filled with brownish fluid pa ge 46 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 44-47, 2024 figure 2: histopathological analysis of the excised retroperitoneal cyst showed a thick-walled multilocular cyst lined by benign ciliated epithelium prescribed oral metronidazole 400mg tds, cefuroxime 500mg bd, aclosara sp (aceclofenac/paracetamol and serratiopeptidase 100mg/325mg/15mg, sara remedies) po tds for 5 days, and ifas (iron and folic acid) 2 tabs po bd for 1 month. histopathological analysis of the excised retroperitoneal cyst showed a thick-walled multilocular cyst lined by benign ciliated epithelium (figure 2). adjacent reactive nodes were noted attached to the external wall. the features were consistent with a cyst of müllerian origin, further supporting the incidental finding during tlh. the cyst wall consisted of a lining epithelium resembling the müllerian epithelium, surrounded by fibrous tissue. no atypical cells or malignancy was identified. during a follow-up visit in the gynecology clinic scheduled two weeks post-surgery the patient had no complaints and the physical exam was unremarkable. the surgical wound was healing well. we explained the histology results to the patient. discussion the incidental discovery of a retroperitoneal cyst of müllerian origin during a tlh in a patient with uterine fibroids presents an intriguing case and raises several important considerations. retroperitoneal cysts are often misdiagnosed pre-operatively (johan et al., 2020). in our case, the patient presented with a history of metrorrhagia, dysmenorrhea, and lower abdominal pain radiating to the back. these symptoms, along with the presence of uterine fibroids on ultrasound imaging, prompted further investigation and the decision to proceed with tlh. the intraoperative discovery of a retroperitoneal cyst, unrelated to the uterine fibroids, highlights the importance of appropriate pre-operative imaging and evaluation to detect such lesions early. additionally, thorough exploration during surgical interventions is important, even in cases where the primary pathology is well-defined. pre-operative ultrasound imaging may not reliably detect retroperitoneal cysts of müllerian origin, as seen in our case where the cyst was not visualized on the preoperative ultrasound scan (yacoub et al., 2021). magnetic resonance imaging (mri) provides a sophisticated method of distinguishing mullerian anomalies from one another, characterizing the degree of defect severity, and evaluating for concomitant urogenital anomalies noninvasively and without radiation exposure, with superior soft-tissue delineation and availability of advanced functional sequences (udayakumar et al., 2023). mri is also a commonly used tool for evaluation of pelvic malignancies. mullerian cysts may occasionally have malignant transformations that would only be picked by careful imaging using mri, as has been shown by occasional case reports (xiao et al., 2022; zhu et al., 2023). further studies may be done to determine the utility of pre-operative mri imaging in tlh to facilitate the identification of pathology. histopathological analysis confirmed the cyst to be of müllerian origin, characterized by a lining epithelium resembling the müllerian epithelium, surrounded by fibrous tissue. the absence of atypical cells or malignancy supported the benign nature of the cyst. it is essential to differentiate retroperitoneal cysts of müllerian origin from other retroperitoneal cystic lesions, such as pa ge 47 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 44-47, 2024 lymphatic cysts or cystic teratomas, to guide appropriate management and prevent unnecessary interventions. the retroperitoneal cysts of müllerian origin are believed to be embryological remnants of the müllerian duct system (santana gonzalez et al., 2021). these remnants can give rise to various cystic structures in the retroperitoneal space, presenting a diagnostic challenge due to their rarity and diverse clinical presentations. managing retroperitoneal cysts of müllerian origin largely depends on the clinical presentation, size, and symptoms associated with the cyst. asymptomatic cysts can be managed conservatively with regular monitoring. however, in cases where the cyst is symptomatic, surgical excision is warranted (renzulli and candinas, 2009). the incidental discovery of retroperitoneal cysts during tlh presents a unique opportunity for their complete removal, minimizing the risk of future complications or recurrence (stefanopol et al., 2022). the importance of appropriate post-operative care, including pain management and prophylactic antibiotics, cannot be overstated to ensure optimal recovery. this case report emphasizes the need for thorough preoperative evaluation and intraoperative vigilance during tlh. the incidental discovery of retroperitoneal cysts of müllerian origin during tlh highlights the importance of considering these rare entities in the differential diagnosis of retroperitoneal cystic lesions. further research is warranted to explore the prevalence, clinical characteristics, and optimal management strategies for retroperitoneal cysts of müllerian origin, particularly when encountered during tlh. enhanced awareness among clinicians and gynaecological surgeons regarding possible incidental findings during tlh can aid in early detection, appropriate management, and improved patient outcomes. conclusion the incidental discovery of a retroperitoneal cyst of müllerian origin during tlh in a patient with uterine fibroids highlights the diagnostic challenge and importance of thorough intraoperative exploration. this case underscores the need for vigilance and consideration of rare entities in the differential diagnosis of retroperitoneal cystic lesions encountered during tlh. further research is necessary to advance our understanding of retroperitoneal cysts of müllerian origin and optimize pre-operative detection using mri. references giuliani, e., as-sanie, s., & marsh, e. e. (2020). epidemiology and management of uterine fibroids. international journal of gynecology & obstetrics, 149, 3–9. https://doi.org/10.1002/ijgo.13102 johan, s., hassan, m. f., hayati, f., azizan, n., payus, a. o., & edwin see, u. h. (2020). huge retroperitoneal cyst masquerading as ovarian tumour: a case report. frontiers in surgery, 7. renzulli, p., & candinas, d. (2009). symptomatic retroperitoneal cyst: a diagnostic challenge. ann r coll surg engl, 91, w9–w11. https://doi. org/10.1308/147870809x400877 rivas, a. g., epelman, m., ellsworth, p. i., podberesky, d. j., & gould, s. w. (2022). magnetic resonance imaging of müllerian anomalies in girls: concepts and controversies. pediatric radiology, 52, 200–216. https:// doi.org/10.1007/s00247-021-05089-6 santana gonzalez, l., rota, i. a., artibani, m., morotti, m., hu, z., wietek, n., ... ahmed, a. a. (2021). mechanistic drivers of müllerian duct development and differentiation into the oviduct. frontiers in cell and developmental biology, 9. stefanopol, i. a., baroiu, l., neagu, a.-i., danila, d. m., nechifor, a., miulescu, m., ... tatu, a. l. (2022). clinical, imaging, histological and surgical aspects regarding giant paraovarian cysts: a systematic review. therapeutics and clinical risk management, 18, 513–522. https://doi.org/10.2147/tcrm.s361476 sugi, m. d., penna, r., jha, p., pōder, l., behr, s. c., courtier, j., ... choi, h. h. (2021). müllerian duct anomalies: role in fertility and pregnancy. radiographics, 41, 1857–1875. https://doi.org/10.1148/rg.2021210022 udayakumar, n., smith, e., boone, a., & porter, k. k. (2023). a common path: magnetic resonance imaging of müllerian and wolffian duct anomalies. current urology reports, 24, 1–9. https://doi. org/10.1007/s11934-022-01138-1 wilson, d., & bordoni, b. (2024). embryology, mullerian ducts (paramesonephric ducts). in statpearls. statpearls publishing. xiao, x., ma, r., shi, l., wang, c., chen, j., lu, y., ... fu, f. (2022). case report: stage iiic primary malignant mixed müllerian tumor of the fallopian tube: a case of 5-year disease-free survival after cytoreductive surgery combined with peritoneal resection and adjuvant chemotherapy with paclitaxel plus carboplatin. frontiers in oncology, 12. yacoub, j. h., clark, j. a., paal, e. e., & manning, m. a. (2021). approach to cystic lesions in the abdomen and pelvis, with radiologic-pathologic correlation. radiographics, 41, 1368–1386. https:// doi.org/10.1148/rg.2021200207 yang, d. m., jung, d. h., kim, h., kang, j. h., kim, s. h., kim, j. h., & hwang, h. y. (2004). retroperitoneal cystic masses: ct, clinical, and pathologic findings and literature review. radiographics, 24, 1353–1365. https://doi.org/10.1148/rg.245045017 zhu, x., peng, c., huang, y., & zhou, y. (2023). uterine cervical müllerian adenosarcoma possibly arising from ovarian cystadenofibroma: a case report and review of the literature. frontiers in oncology, 12. pa ge 1 pa ge 1 american journal of life science and innovation (ajlsi) spectrum of adolescent marijuana use in secondary schools at sagamu, south-west nigeria: factors, determinants and scope ejiroghene oluwafunke okorosobo1, ademola mufutau amosu1, aloysius obinna ikwuka2*, francis chigozie udeh2 volume 3 issue 2, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i2.2993 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: may 22, 2024 accepted: june 25, 2024 published: june 28, 2024 adolescents’ use of marijuana is a challenge of public health concern, and this also relates to several communities in nigeria. the use of marijuana and other substances has been reported among the youths. however, the contributing factors to its consumption among these consumers are yet to be fully documented in some nigerian states. this research aimed to investigate the spectrum of marijuana use among adolescents in secondary schools at sagamu. the study employed a descriptive, cross-sectional design. a multi-stage sampling technique was used to select 388 respondents. a semi-structured questionnaire with cronbach’s alpha of 0.72 was employed. the predisposing, reinforcing, and enabling factors were measured on an 18-point rating scale. pearson’s correlation and linear regression analysis were conducted to give statistical responses to the research hypotheses using spss version 23. the response rate in this research was 98%. the mean age of the respondents was 15.46±1.37years. 56.2% were females, 74.5% were christians, and 84% were yoruba. 4.6% of the respondents have an uneducated dad, 3.9% have an uneducated mum, 70.6% have a self-employed dad, and 78.1% have a self-employed mum. 92.5% of the respondents have a good knowledge of marijuana use. 77.6% have a positive attitude towards marijuana use. 48.7% of the respondents could be influenced by the reinforcing and enabling factors, and there was a 7.2% prevalence rate of marijuana use. there was a significant association between peer influence and marijuana use (r=0.105; r2=0.11; p=0.39), but none between respondents’ predisposing factors and marijuana use (p>0.05). peer influence was the only significant determinant of marijuana use among the respondents. peer education from homes, schools, and governments is therefore recommended to promote the non-use of marijuana and other substances among adolescents. keywords spectrum, adolescent, marijuana use, secondary schools, sagamu, south-west nigeria, factors, determinants, scope introduction adolescent substance use involves the use of illegal drugs, cigarette smoking, alcohol consumption, and misuse of prescription drugs among adolescents, whom theworld health organization (who) described as people aged between 10 to 19 years and make up 20% of the global population (united nations children’s emergency fund (unicef, 2022). substance use burden increases among adolescents and young adults, with an unequal distribution across age groups, countries, and epidemiological parameters (global burden of disease collaborative network, 2015). the use of marijuana poses a danger to adolescents, which ripple with long-term consequences for the general population. the age of initiation is the mid-teens in many developed countries (united nations office of drugs and crime [unodc], 2021; who, 2016). in the united states of america, 75.6% of high school students were reported to have used addictive substances, including marijuana, cigarettes, alcohol, or cocaine. in addition, 90% of the us population is initiated into substance use before the age of eighteen, of which 25% become addicted (center on addiction and substance abuse [casa], 2011). apart from marijuana, other substances such as alcohol, tobacco, illicit drugs, e.g., cocaine, heroin, and opiates which are commonly abused, have harmful effects on the brain and other vital organs, e.g., lungs (baysah, 2023; udeh, 2023a; udeh, 2023b). cystic fibrosis is a genetic disease affecting mainly the lungs (ikwuka, 2023a). other affected organs include the pancreas, liver, kidneys, and intestine. clinical features of cystic fibrosis include dyspnea, cough with sputum, sinusitis, poor growth, fatty stool, finger and toe clubbing, etc (ikwuka, 2023a). cough with sputum can be complicated by blood in the sputum (hemoptysis), which can lead to anemia (ikwuka, 2023e; musa, 2023). marijuana use has been linked to mental health disorders. different researchers are still investigating the links between marijuana use and metabolic disorders, although some studies have reported no significant association between marijuana use and metabolic disorders. among emerging adults, current marijuana users were 54% less likely than never to present with metabolic syndrome. current (aor 0.49; 95% ci, 0.25-0.97) and past (aor 0.61; 95% ci, 0.40-0.91) middle-aged adult marijuana users were less likely to have metabolic syndrome than never users (vidot, 2016). metabolic disorders, e.g. hypertension, adiposity, diabetes mellitus and dyslipidemia, collectively known as metabolic syndrome diseases (msds) are diseases related to one another (ikwuka, 2015; ikwuka, 2017a; ikwuka, 2017c; ikwuka, 1 school of public and allied health, babcock university, ilisan-remo, nigeria 2 college of medicine and health sciences, american international university west africa, banjul, the gambia * corresponding author’s e-mail: aloysiussweet@yahoo.com pa ge 2 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 1-12, 2024 2023c; ikwuka, 2023f; ikwuka, 2024; virstyuk, 2016). different studies have shown that msds are associated with asymptomatic hyperuricemia, systemic immune inflammatory processes, and fibrogenesis all of which can lead to nephropathy (ikwuka, 2017d; ikwuka, 2017e; ikwuka, 2018c; ikwuka, 2018d; ikwuka, 2019a; ikwuka, 2019c; ikwuka, 2022; ikwuka, 2023d; virstyuk, 2017a; virstyuk, 2018a; virstyuk, 2019; virstyuk, 2021a; virstyuk, 2021b). substance use disorder is an expensive and preventable public health problem. the assessment of the annual cost of substance use disorder in united states hospitals showed that a total of 13.2 billion dollars was the annual estimated attributable medical cost for substance use disorders (peterson, 2021). a single cause (determinant) cannot initiate the use of marijuana among adolescents, rather it is a complex of predisposing, reinforcing, and enabling factors. there is a multilevel complexity as these factors are further composed of individual determinants as described and used in constructing the research instrument for this study. the predisposing factors include age, gender, ethnicity, parents’ socio-economic status, knowledge, and attitude. reinforcing factors include family and/or siblings’ use of marijuana; and peer influence. the enabling factors include easy access, availability, cost of marijuana, and the school environment. physiological, genetic, and psychological determinants are also associated with substance use. these determinants are strongly influenced and the key influencers include family, social networks, and peer pressure, with best friend use reported to influence the most (english, 2023). certain substances used and abused are derived from agricultural products e.g. beer from wheat and barley, hallucinogens from certain plants, tobacco from dried tobacco leaves, and marijuana from varieties of cannabis plants such as the cannabis sativa, cannabis ruderalis, and cannabis indica (gloss, 2015). the agricultural source of marijuana therefore makes it easy to procure for recreational use. marijuana plant contains numerous cannabinoids including δ−9-tetrahydrocannabinol (the principal cannabinoid) and cannabidiol (world health organization [who], 2024; national center for complementary and integrative health, 2018). cannabis usage is associated with oxidative stress. linked to the induction of oxidative stress are major free radicals. among these major free radicals, superoxide anions, hydroxyl radicals, and hydroperoxyl radicals are of physiological significance. a non-radical of physiological significance is hydrogen peroxide (ama, 2023; ekechi, 2023a; ikwuka, 2023b; uche, 2023). marijuana use has been reported to lead to addiction, poor concentration, poor academic performance, thefts, organized crime, violence, mental illnesses, injuries, infections, and behavioral disorders in the involved individuals (centers for disease control and prevention [cdc], 2023; national center for drug abuse statistics [ncdas], 2024). other substances including tobacco, alcohol, and illicit drugs like opiates, heroin, and cocaine are also popular and toxic (baysah, 2023; udeh, 2023a; udeh, 2023b). interestingly, marijuana and individual cannabinoids have been reported to help manage the side effects of cancer and cancer therapies, but no research has shown that they can cure cancer (national academies of sciences, engineering, and medicine, 2017). substance use burden increases in adolescence and young adulthood (center on addiction and substance abuse, 2011). globally, an estimated 181.8 million individuals who were between 15 and 64 years old used non-medical marijuana in 2013. marijuana was the most commonly used hard drug among youths in 2019, and in 2021 it became the most cultivated and trafficked drug worldwide (united nations office of drugs and crime [unodc], 2021). in 2023, among teenagers in the united states of america (usa), marijuana was the second most abused substance after alcohol (national center for drug abuse statistics, 2024). there were 70.46 million users in the usa, 61.99 million users in asia, 53.6 million users in africa, 29.47 million users in europe, and 3.4 million users in oceania (statista, 2023). irrespective of age, 37.7% of women in developed countries/regions like the usa, west and central europe (european monitoring center for drugs and drug addiction [emcdda], 2023), australia, and new zealand consume marijuana, while only 10.5% of women in africa and asia use it (statista, 2023). in a national survey in nigeria, the northwest region has the highest number of marijuana users (akannam, 2008). it is also published from a high school students survey that 65% of the students used drugs to be happy with their friends (abudu, 2008). in sagamu, the people call marijuana other names like hemp, “ganja”, “igbó”, “pot”, “káyá”, “wee-wee”, “ojà”, gbáná and abana. it was also reported that 76% of secondary school students in sagamu used illicit drugs, and 49.3% used them for calmness and sleep (ojieabu, 2017), in the form of selfmedication (oriavwote, 2022). metabolic syndrome diseases (msds) are interrelated diseases with very high economic costs, morbidity, and mortality rates, thus requiring the search for new and effective treatment options (ikwuka, 2024). treatment optimization in msd patients using a combination of hmg-coa and sglt-2 inhibitors, and a2rb (at1) has resultant clinical effectiveness as indicated by marked improvements in metabolic functions of the heart, liver, pancreas, and kidney (ikwuka, 2017b; ikwuka, 2018a; ikwuka, 2018b; ikwuka, 2021; virstyuk, 2017b; virstyuk, 2018b; virstyuk, 2018c; ikwuka, 2024). in addition, glucagon-like peptide 1 receptor agonists (glp-1 ras) e.g. liraglutide have been found to improve the efficacy of treatment and clinical course of type 2 diabetes mellitus and hypertension in patients with such comorbidities (ikwuka, 2019b). it has also been reported that coconut water has hepatorenal protective functions against alloxan-induced type 1 diabetes mellitus (ekechi, 2023b). pa ge 3 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 1-12, 2024 with the increased prevalence of marijuana use among adolescents and the high incidence of implications on health and the economy, evaluating the spectrum of marijuana use among adolescents would be very important. it will also be important to analyze the association between predisposing factors, reinforcing factors, enabling factors, and marijuana use. an efficient approach to prevent adolescent marijuana use would be very beneficial to both public health and the economy. therefore, as part of the sustainable development goals (sdg), this study would give clearer insights into the determinants and scope of marijuana use among adolescents and provide answers to strengthen the prevention of its use among adolescents especially those in secondary schools. in addition, this study will expose where further research can be done, and suggest areas where laws, policies, and interventions can be developed and implemented. materials and methods study area the study was conducted at sagamu, the headquarters of sagamu local government area (lga) in ogun state, south-west nigeria. with a land area of 68.03 km2, it is surrounded by odogbolu lga in the east, lagos state in the south, ikenne lga in the north, and obafemi owode lga in the west. the major economic engagement of the locals is kolanut farming. study population the study population comprised adolescents between the ages of 11 and 19 who attended secondary schools located within the study area. sample size the cochran formula for descriptive, cross-sectional, qualitative study was used: sample size for large population >10,000 = n = (z2 pq)/d2 z = standard normal deviate (at 5% type 1 error) = 1.96 p = prevalence of drug use in nigeria = 65% (abudu, 2008) q = complementary proportion equivalent to = 1-p = 1-0.65 = 0.35 d = degree of accuracy desired (absolute precision) = 5% = 0.05 n = (1.962×0.65×0.35)/0.052 = 359.57 ~ 360 with the inclusion of a 10% non-response rate, 396 students were later expected to be recruited for the study. sample size justification the cochran formula was appropriate for this descriptive, cross-sectional, qualitative study. a cross-sectional study could be qualitative or quantitative, with specific formulas for each, and in situations where the study population is less or greater than 10,000 (udeh, 2023c). the appropriate formula was used with a study population above 10,000, thereby justifying the sample size. inclusion and exclusion criteria respondents included students between the ages of 11 and 19 who consented to participate voluntarily. students outside the age range who did not consent to participate voluntarily were excluded. study design all the secondary schools in sagamu were listed. the engaged secondary schools were selected using simple random sampling by balloting. a proportionate sampling technique was used to derive the number of participants per school, and a systematic sampling technique was used to get the number of students needed from each school. structured, self-administered questionnaires were presented to the respondents and the sections included the socio-demographic characteristics, knowledge of marijuana use, attitude towards marijuana use, and associated factors (reinforcing and enabling) for marijuana use. the questionnaire was validated by face validity, item validity, and construct validity. the questionnaire was confirmed reliable after a pilot study was conducted with 10% of the sample size among students outside the study area. also, the cronbach alpha analysis was 0.72. data analysis participants responded to the questions on knowledge with a “yes” or “no”. the attitude, reinforcing, and enabling factors were responded to, using a four-point likert scale. the likert scale rating was calculated by multiplying the highest point by the number of variables. the hypotheses were tested with pearson correlation and linear regression analysis at p-value<0.05. data was analyzed using statistical package for social sciences (spss) version 23. ethical considerations the babcock university health research ethical committee (buhrec) granted permission to conduct this study. the ministry of education at sagamu, and the secondary schools involved also permitted the conduct of this study. each participant individually consented willingly, and all data extracted were kept confidential. results and discussions eight questionnaires were invalid as the participants did not fill in the parameters asked, giving a 98% response rate. the results are presented below in tables and figures. table 1: socio-demographic characteristics of the respondents (n=388) socio-demographic variables considered respondents in this study (n=388) frequency(n) percentage (%) age (in years) 11-13 19 4.9 pa ge 4 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 1-12, 2024 a majority (76.0%) of the students were between ages 1416, with the mean age of respondents being 15.46±1.37 years. 218 (56.2%) were female, and the majority (74.5%) were christians. the students were somewhat evenly distributed among the classes. however, the lowest number was in senior secondary school one (ss1) or grade 10. 84.0% were yorubas. approximately 39.4% of the respondents’ fathers had secondary school education and less than half (45.4%) of their mothers had secondary school education. predisposing factors to marijuana use among the respondents virtually all (96.1%) of the respondents have heard of table 2: respondents’ knowledge about marijuana use (n=388) socio-demographic variables considered respondents in this study (n=388) frequency(n) percentage (%) have you heard about marijuana before? yes 373 96.1 no 15 3.9 my knowledge about marijuana is from family members 22 5.7 friend 54 13.9 14-16 295 76.0 17-19 74 19.1 mean age (x̄) = 15.46±1.37 gender male 170 43.8 female 218 56.2 religion christianity 289 74.5 islam 96 24.7 traditional 3 0.8 class ss1 (grade 10) 110 28.4 ss2 (grade 11) 138 35.6 ss3 (grade 12) 140 36.1 ethnicity yoruba 326 84.0 igbo 45 11.6 hausa 7 1.8 others 10 2.6 father’s educational level no formal education 18 4.6 elementary school 30 7.7 secondary school 153 39.4 hnd/university 129 33.2 postgraduate 58 14.9 mother’s educational level no formal education 15 3.9 elementary school 34 8.8 secondary school 176 45.4 hnd/university 110 28.4 postgraduate 53 13.7 pa ge 5 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 1-12, 2024 marijuana use before the study. more than half (52.8%) of the respondents knew about marijuana from school, while only 5.7% knew about marijuana from family members. a majority (96.1%) of the respondents knew that marijuana is an illicit drug and 81.4% of them knew that marijuana is used to treat mental disorders like anxiety when administered at a low dose. a majority (95.6%) of the respondents knew that marijuana is harmful to health. 93.3% of the respondents knew that addiction is an effect of marijuana use. concerning forms of marijuana use, cigarette (33.0%) and powder (29.4%) forms were the most popularly known forms among the respondents. other forms were known by just a few number of the respondents viz liquid (13.4%), eating/chewing (10.7%), and inhalable vapor (13.4%). media 107 27.6 school 205 52.8 marijuana is an illicit drug. yes 373 96.1 no 15 3.9 marijuana is used to treat mental disorders like anxiety at a low dose. yes 316 81.4 no 72 18.6 using marijuana is harmful to health. yes 371 95.6 no 17 4.4 addiction is an effect of marijuana use. yes 362 93.3 no 26 6.7 *forms of marijuana use cigarette 275 33.0 powder 245 29.4 liquid 112 13.4 eating/chewing 89 10.7 inhalable vapor 112 13.4 *marijuana used can result to anxiety 97 18.4 poor judgment 71 13.5 death 85 16.1 increased appetite 93 17.6 cancer 181 34.3 note: * indicates questions with multiple response table 3: respondents’ attitude towards marijuana use (n=388) s/no. items sa a d sd n (%) n (%) n (%) n (%) 1 my family and friends use it, therefore, i should do so too. 9(2.3) 8(2.1) 87(22.4) 284(73.2) 2 i will openly discuss about marijuana use among adolescents. 100(25.8) 161(41.5) 65(16.8) 62(15.9) 3 i believe marijuana boosts self-confidence. 34(8.8) 64(16.5) 108(27.8) 182(46.9) 4 i would likely use marijuana because it helps the brain to function better. 5(1.3) 8(2.1) 89(22.9) 286(73.7) 5 i will make use of small quantities of marijuana since it is not dangerous. 7(1.8) 12(3.1) 94(24.2) 275(70.9) 6 i do not like my friends using marijuana. 189(51.3) 88(22.7) 36(9.3) 65(16.7) note: sa (strongly agree, 3); a (agree, 2); d (disagree, 1); sd (strongly disagree, 0). likert rating scale = 3×6 = 18 pa ge 6 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 1-12, 2024 most (73.2%) of the respondents strongly disagreed that they should use marijuana because their family and friends use it. 67.3% of the respondents either strongly agreed or agreed that they can openly discuss marijuana use among adolescents. 27.8% of the respondents disagreed that marijuana boosts self-confidence whereas 46.9% strongly disagreed. a majority (73.7%) of the respondents strongly disagreed that they would use marijuana because it helps the brain to function better. most (70.9%) of the respondents strongly disagreed that they would make use of small quantities of marijuana since it is not dangerous. more than half (51.3%) of the respondents strongly agreed that they do not like their friends to use marijuana. reinforcing and enabling factors for marijuana use among the respondents table 4: respondents’ reinforcing and enabling factors for marijuana use (n=388) s/no. items sa a d sd n (%) n (%) n (%) n (%) 1 peer influence is not a consideration in adolescents’ marijuana use 44(11.3) 75(19.3) 119(30.7) 150(38.7) 2 family and siblings’ use of marijuana may encourage adolescents to use it 79(20.4) 103(26.5) 66(17.0) 140(36.1) 3 easy access to marijuana may aid its use 92(23.7) 148(38.1) 64(16.5) 84(21.7) 4 marijuana’s low cost enables one to easily access the substance 81(20.9) 127(32.7) 92(23.7) 88(22.7) 5 the school environment supports adolescents’ marijuana use 14(3.6) 18(4.6) 75(19.3) 281(72.5) 6 enforcing substance use regulations and policies will reduce marijuana use 149(28.4) 132(34.0) 43(11.1) 64(26.5) note: items 1 and 2 are reinforcing factors; items 3-6 are enabling factors; sa (strongly agree, 3); a (agree, 2); d (disagree, 1); sd (strongly disagree, 0). likert rating scale = 3×6 = 18 table 5: summary of descriptive statistics of variables in the study variables maximum points on a scale of measure respondents in this study (n=388) prevalence (%) x̄ (s.e.) ±sd attitude towards marijuana use 18 13.98(0.13) 2.52 77.6 reinforcing and enabling factors 18 8.77(0.16) 3.20 48.7 38.7% strongly disagreed and 30.7% only disagreed that peer influence is not a consideration in adolescents’ marijuana use. few (20.4%) of the respondents strongly agreed that family and siblings’ use of marijuana may encourage adolescents to use marijuana. approximately 23.7% of the respondents strongly agreed that easy access to marijuana aids its use. the respondents almost equally agreed and disagreed that the low cost of marijuana enables one to easily access the substance. most (72.4%) of the respondents strongly disagreed that the school environment supports adolescents’ marijuana use. few (28.4%) of the respondents strongly agreed that enforcing substance use regulations and policies will reduce marijuana use. the respondents’ attitude towards marijuana use measured on an 18-point rating scale showed that the respondents’ mean score was 13.98±2.52, translating to an attitudinal prevalence of 77.6% which means that only 77.6% of the respondents have a positive attitude towards marijuana use (see table 5). in addition, on an 18-point rating scale, the reinforcing and enabling factors for marijuana use among the respondents had a mean score of 8.77±3.20 which means that these factors can influence 48.7% of the respondents (see table 5). table 6: table 6: relationship between predisposing factors (level of knowledge and attitude), reinforcing factors (peer influence and family influence), and enabling factors with marijuana use among the respondents hypotheses factors variables marijuana use; (n=388) r p-value 1 predisposing level of knowledge 0.14 0.79** attitude towards marijuana use -0.05 0.30** 2 reinforcing peer influence 0.11 0.03* family influence -0.29 0.57** pa ge 7 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 1-12, 2024 test of hypotheses pearson correlation and linear regression analysis were conducted at a 0.05 level of significance. at p≤0.05, the null hypothesis was rejected and the alternate hypothesis was accepted. on the other hand, with p>0.05, the null hypothesis was accepted and the alternate hypothesis was rejected. hypothesis 1 h0: there is no significant relationship between predisposing factors and marijuana use among adolescents. h1: there is a significant relationship between predisposing factors and marijuana use among adolescents. result as seen in table 6, the respondents’ predisposing factors (knowledge [r=0.14; p=0.79], and attitude [r=-0.05; p=0.30]) had a p-value>0.05, thus confirming h0 and rejecting h1. therefore, there is no significant relationship between predisposing factors and marijuana use among adolescents. hypothesis 2 h0: there is no significant relationship between reinforcing factors (peer influence and family influence) and marijuana use among adolescents. h1: there is a significant relationship between reinforcing factors (peer influence and family influence) and marijuana use among adolescents. result as seen in tables 6 and 7, peer influence has p<0.05 while family influence has p>0.05. this means that there is a significant relationship between peer influence and marijuana use. on the other hand, there is no significant relationship between family influence and marijuana use. hypothesis 3 h0: there is no significant relationship between enabling factors and marijuana use among adolescents. h1: there is a significant relationship between enabling factors and marijuana use among adolescents. result table 6 shows that easy access to marijuana, affordability of marijuana, school support for non-use of marijuana, and policies on marijuana use, all had a p-value>0.05, thus confirming h0 and rejecting h1. therefore, there is no significant relationship between enabling factors and marijuana use among adolescents. discussion adolescents in high school tend to be exposed to various vices like alcohol abuse, illicit drug use, smoking, prostitution, etc by their peers. to shy away from segregation or avoid being termed weak, some of them bend to peer influence and get engaged in these bad habits. in this study, the spectrum of adolescent marijuana use including associated factors, determinants and scope among adolescents attending secondary schools in sagamu, south-west nigeria were evaluated. this study shares similar socio-demographic characteristics with that of another study (almahdi, 2018). the similarities are that most of the respondents were 14-16 years old, and female respondents were more than males. sagamu is the headquarters of sagamu local government area (lga) in ogun state, southwest nigeria. south-west nigeria is a geopolitical zone, occupied and dominated by the yoruba tribe. this fact explains why most (84.0%) of the respondents were yorubas. less than half of the parents had university degree or postgraduate qualification. fathers achieved this feat as 48.1% of them either had a first degree or a postgraduate degree, as against 42.1% of the mothers. the data obtained on the predisposing factors (knowledge and attitude towards marijuana use) indicated that a majority (92.5%) of the respondents have a high basic knowledge of marijuana use and 77.6% of them have a positive attitude towards marijuana use. this result on knowledge is similar to the finding of another study which reported that most of the school-going adolescents in ramotswa, botswana are aware of substance use and abuse (gotsang, 2017). however, unlike this study which showed that the highest number of the students first learned about marijuana in school, most of the students in ramotswa learned about marijuana from television. table 7: association between peer influence and marijuana use among the respondents variable marijuana use respondents in this study (n=388) peer influence r r2 b βeta f p-value 0.105 0.011 0.245 0.245 4.311 0.039 3 enabling easy access to marijuana -0.09 0.69** affordability of marijuana -0.02 0.59** school support for non-use 0.06 0.18** policies on marijuana use -0.03 0.55** note: * = statistically significant; **= not statistically significant pa ge 8 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 1-12, 2024 an international prospective study on the knowledge and attitude of adolescents to marijuana use also showed that 20% of the respondents admitted not being knowledgeable about marijuana while the remaining 80% only had partial knowledge of marijuana (jacobs, 2021). as only a few of them could identify the forms of marijuana; therefore, the level of knowledge does not translate to respondents using marijuana. it was also reported that 40% of secondary school students in oyo state, southwest nigeria had positive attitude towards substance use (ajibola, 2018) which is lower than the 77.6% reported in this study. the high values reported on knowledge and attitude in this study could be attributed to the measures the schools are currently taking to enlighten their students on substance use and its adverse effects. the reinforcing and enabling factors were evaluated to likely influence 48.7% of the respondents in this study. the reinforcing factors made up of peer influence and family influence are similar to the variables evaluated by another study on the assessment of alcohol and substance use among undergraduates in southwest nigeria (adekeye, 2012). the answers on the enabling factors including easy access to marijuana, its affordability, school support for non-use of marijuana, and policies on marijuana use were distributed almost evenly among the respondents. this divided opinion explains why the reinforcing and enabling factors could influence almost half (48.7%) of the respondents. the prevalence rate of marijuana use among the respondents was low as approximately 30 (7.2%) out of the 388 agreed to have either used or are currently using marijuana. a different study reported a higher prevalence rate and found that 65% of high school students in nigeria use substances like tobacco, alcohol, cannabis, etc. (abudu, 2008). this nationwide analysis showed that students use these drugs for a good time with their pals (abudu, 2008). a similar study in sagamu, this study area, reported a 76% prevalence rate among secondary school students (ojieabu, 2017), and a 17.4% prevalence rate of substance use among students in ramotswa, botswana has also been reported (gotsang, 2017). another study reported 2% prevalence rate of marijuana use among school-going adolescents in eight sub-saharan african countries: benin, ghana, liberia, mauritius, mozambique, namibia, seychelles, and tanzania (kugbey, 2023). the test of hypothesis 1 in this study revealed that there is no significant relationship between predisposing factors (knowledge and attitude) and marijuana use among adolescents. this means that knowledge and attitude are not significant determinants of marijuana use, a hypothesis supported by the findings of another study (duru, 2017) which stated that predisposing factors do not influence substance use. however, another study has reported that there is a significant relationship between knowledge and marijuana use (kugbey, 2023). adolescence comes with various challenges. a child acquires knowledge as he grows. however, due to the curious nature of adolescents, they tend to indulge in some acts they categorically know have implications. this explains why the high knowledge and positive attitude do not significantly determine marijuana use by the respondents. the test of hypothesis 2 showed that peer influence is a significant determinant of marijuana use (p<0.05), whereas family influence is not (p>0.05). the findings on reinforcing factors in this study are almost similar to that of another study which stated that both parental use and peer influence have no significant contribution (p>0.05) to student’s use of alcohol and other substances including marijuana (adekeye, 2015). as adolescents progress to secondary school, they spend more time with their friends at school as their time at home reduces. this idea gives insight to why peer influence could significantly determine marijuana use and family influence could not. strict training by a parent that uses marijuana may likely make an adolescent not use marijuana, but once there is an interaction with friends that use it, such an adolescent may choose to have a taste of it due to adolescent curiosity. finally, the test of hypothesis 3 in this study showed that the enabling factors are not significant determinants of marijuana use among the respondents. prospects for further research will be to recommend that sociodemographic variables like age, gender, religion, family size, and family income be statistically analyzed to identify their significant relationships with marijuana use. conclusion peer influence was the only significant determinant of marijuana use among adolescents attending secondary schools in sagamu, south-west nigeria. other determinants e.g. knowledge, attitude, family or siblings’ use of marijuana, easy access to marijuana, low cost of marijuana, school support for non-use of marijuana, and policies on marijuana use are not significant determinants of marijuana use among adolescents. the scope of marijuana use in this study includes the prevalence rate of marijuana use, which was low (7.2%), and the purpose of marijuana use, which was mainly for exam success. even though the variables that make up the predisposing, reinforcing (except peer influence), and enabling factors have no significant relationship with marijuana use; nevertheless, 48.7% is a huge figure to ignore. therefore, families, schools, and governments must contribute their quota in reducing marijuana use among adolescents. acknowledgments special thanks to all secondary school students in sagamu, south-west nigeria who voluntarily consented to participate in this study after making an informed decision. their assistance in conducting this research is highly appreciated. references abudu, r. v. 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(2017)a. diagnostic and prognostic markers of the diabetes mellitus type 2 course in connection with essential arterial hypertension taking into account the kidney function. precarpathian journal pulse, 8(44), 53-62. virstyuk, n. h., ikwuka, a. o., losyuk, l. v., kobrynska, o. ya., & markiv, h. d. (2017)b. dapagliflozin utility in patients with diabetes mellitus type 2 and essential hypertensive disease. actual problems of modern medicine, 4(60)1, 76-79. available online: http://www. umsa.edu.ua/journal2stat4_2017_eng.html virstyuk, n. g., & ikwuka, a. o. (2018)a. features of asymptomatic hyperuricemia in patients with diabetes mellitus type 2 and concomitant essential arterial hypertension. clinical and experimental pathology, 1(63), 22-26. https://doi.org/10.24061/1727-4338. xvii.1.63.2018.5. virstyuk, n. g., ikwuka, a. o., & didushko, o. m. 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(2021)a. asymptomatic hyperuricemia and functional state of the kidneys in patients with essential arterial hypertension and concomitant diabetes mellitus type 2. european journal of clinical medicine, 2(3), 100-104. https://doi. org/10.24018/clinicmed.2021.2.3.65. virstyuk, n. h., ikwuka, a. o., luchko, o. r., & kocherzhat, o. i. (2021)b. peculiarities of renal insufficiency in patients with diabetes mellitus type 2 and arterial hypertension. materials of scientific-practical conference with international participation “achievements and prospects of experimental and clinical endocrinology” twentieth danilevsky readings, p. 86-87. world health organization alcohol, drugs and addictive behaviours unit. (2016). the health and social effects of non-medical cannabis use. cannabis. available online: https://www.who.int/substance_abuse/ publications/cannabis_report/en/index4.html world health organization alcohol, drugs and addictive behaviours unit. (2024). cannabis. available online: https://www.who.int/teams/mental-healthand-substance-use/alcohol-drugs-and-addictivebehaviours/drugs-psychoactive/cannabis pa ge 1 pa ge 33 american journal of life science and innovation (ajlsi) impact of sdg3 in the prevention of physical and mental effects of infertility observed in nigerian women: a scoping review tayyibat ayoola egbewole1, ncube f.1, aloysius obinna ikwuka2* volume 3 issue 2, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i2.3139 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: june 27, 2024 accepted: july 31, 2024 published: august 03, 2024 infertility significantly impacts both the physical and emotional well-being of individuals and is particularly prevalent in lowand middle-income countries like nigeria. since many causes of infertility are avoidable, there is potential for public health initiatives and sustainable development goal 3 (sdg3) to play a role in health education and prevention. this scoping review aimed to assess existing literature for a better understanding of the factors relevant to analyzing how sdg3 might influence nigerian women’s ability to mitigate the physical and mental effects of infertility. exhaustive searches were conducted across electronic databases including scopus, web of science, jstor, cinahl, medline, and pubmed. the initial review commenced in october 2023, with the final search concluded in march 2024. identified papers underwent screening using the prisma extension for scoping review flow diagrams. after removing duplicates and screening titles and abstracts, 1,340 papers were identified, with 169 reports evaluated for inclusion. ultimately, 46 papers met the inclusion criteria for this scoping review. data analysis revealed various factors contributing to the burden of infertility among nigerian women, encompassing medical conditions, modifiable risk factors, environmental influences, physical and psychological stressors, and policyrelated considerations. socio-economic factors were also implicated in infertility within this population. however, significant gaps in knowledge persist, as no substantial findings were uncovered regarding the true causes across these categories. therefore, addressing the burden of infertility among nigerian women requires a multi-faceted approach including interventions aligned with sdg3, public awareness campaigns, health screening initiatives, utilization of assisted reproductive technologies, provision of physical and psychological wellness services, as well as counseling support. keywords impact, sustainable development goal 3, sdg3, prevention, physical effects, mental effects, infertility, nigeria, women, scoping review introduction the united nations implemented quality health and wellness as one of the 17 sustainable development goals in 2015, intending to guarantee robust livelihoods and advocate wellness for all, regardless of age (united nations, 2015). sustainable development targets emphasize living a healthy lifestyle, with indicators tracking progress across countries (ritchie, 2018). sdg3 comprises thirteen targets and twenty-eight indicators to monitor progress. these targets focus on reducing maternal and child mortality, combating diseases, enhancing psychological well-being, deterring substance misuse, diminishing road-related accidents, and granting widespread entry to medical care and birth control. sdg3 additionally strives to attain comprehensive health protection and impartial entry to medical care for all sexes, which endeavors to guarantee communal availability of family planning as well as sexual and reproductive health amenities (united nations, 2017). several studies have explored the frequency of infertility in underdeveloped and developing countries, such as nigeria, and its potential impacts on the physical and mental wellness of affected individuals. gametes are ova and sperm cells that are haploid and have one copy of each type of chromosome i.e. 1–22 x or 1–22 y (ikwuka, 2023a). the world health organization (who) defines clinical infertility “as the inability to give birth to a healthy baby after at least one year of regular, unprotected sexual activity” (who, 2020). estimates vary, suggesting that globally, between 48.5 million and 186 million couples may be affected by primary or secondary infertility (starrs, 2018), depending on the measurement method (inhorn, 2015; mascarenhas, 2012). primary infertility refers to the inability to conceive or start the development of a child. one significant factor contributing to secondary infertility is infections of the reproductive tract (kaufman, 1999), which are more prevalent than primary infertility (starrs, 2018) and describes the inability to become pregnant even after previous attempts (bowa, 2012). autoimmune antibodies produced by the female partner against sperm cells from the male partner have been implicated as a cause of female infertility. other immune reactions include antibodies to clotting factors, which result in both thrombosis and hemorrhage; and hemolytic reactions during transfusion of blood products (ikwuka, 2023e). oxidative stress has also been associated with infertility. linked to the induction of oxidative stress are major free radicals. among these major free radicals, superoxide anions, hydroxyl radicals, and hydroperoxyl radicals are of physiological significance. a non-radical of physiological significance is hydrogen peroxide (ama, 1 faculty of health/public health institute, liverpool john moores university, liverpool, united kingdom 2 college of medicine and health sciences, american international university west africa, banjul, the gambia * corresponding author’s e-mail: aloysiussweet@yahoo.com pa ge 34 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 33-45, 2024 2023; baysah, 2023; ekechi, 2023a; ikwuka, 2023b). the influence and effects of hormonal imbalance in females leading to infertility in such females have also been reported in different studies (aliu-ayo, 2023a; aliu-ayo, 2023b). parenthood is often socially and culturally expected in many societies, and infertility’s physical and psychological effects on individuals’ well-being and health have been extensively researched in underdeveloped and developing countries (nahar, 2022). many forms of stigmatization are experienced by those who do not fulfil the societal norm which demands having offspring. this can result in stress, unstable marriages, verbal abuse, violence against partners, separation, and exclusion. a thorough definition of fertility awareness considers behaviorally influencing variables at every stage of life, such as cognitive, social, environmental, and developmental aspects (mccurdy, 2015). it is widely acknowledged that a lack of knowledge about fertility hurts views and actions concerning sexual and reproductive health (srh) throughout the course of one’s life. these behaviors include not using family planning (fp) methods, delaying the application of fp methods after giving birth or after an abortion, stopping the use of fp methods, using ineffective fp methods, getting pregnant unintentionally, and having false beliefs about being infertile (dyer, 2002). diabetes mellitus and arterial hypertension remain two of the most common diseases in the world. today, diabetes mellitus (dm) ranks third in the overall structure of morbidity and mortality after cardiovascular diseases and oncological diseases (virstyuk, 2021a). in addition, the interplay, role and effects of metabolic syndrome diseases on male fertility are still being investigated by different researchers. metabolic disorders, e.g. hypertension, adiposity, diabetes mellitus and dyslipidemia, collectively known as metabolic syndrome diseases (msds) are diseases related to one another (ikwuka, 2015; ikwuka, 2017a; ikwuka, 2017c; ikwuka, 2023c; ikwuka, 2023f; ikwuka, 2024; virstyuk, 2016). different studies have shown that msds are associated with asymptomatic hyperuricemia, systemic immune inflammatory processes, and fibrogenesis all of which can lead to nephropathy (ikwuka, 2017d; ikwuka, 2017e; ikwuka, 2018c; ikwuka, 2018d; ikwuka, 2019a; ikwuka, 2019c; ikwuka, 2022; ikwuka, 2023d; virstyuk, 2017a; virstyuk, 2018a; virstyuk, 2019; virstyuk, 2021a; virstyuk, 2021b). governments and sexual reproductive health right (srhr) organizations still overlook infertility despite several other delicate srhr issues being addressed, including abortion, teenage srhr, and comprehensive sexuality education (ombelet, 2011). inaccurate prevalence numbers on infertility contribute to its invisibility and undermine justifications for action, including offering complete reproductive care, preventing and de-stigmatizing infertility-related disorders (ombelet, 2011). furthermore, little is known about current initiatives to address infertility in nations with low and moderate incomes, such as nigeria (kroes, 2019). men’s involvement in infertility management and counseling is encouraged by the availability of assisted reproductive technologies (arts), which also improve gender relations and raises awareness of female infertility (inhorn, 2015). however, the greatest obstacle to expanding access to arts in nations like nigeria will continue to be the high cost of these treatments (starrs, 2018). many couples wait until they need treatment. as a result, additional initiatives are required to lessen the stigma associated with infertility and the detrimental consequences it has on the lives of those who are struggling with it. since avoidable factors account for a large share of infertility; public health, and sustainable development can contribute to reduce the burden (van der poel, 2012). with over 220 million inhabitants, nigeria is the most populous country in africa. there has yet to be evidence that the nation’s 2.5% annual population growth is causing its financial and socio-economic needs, particularly in healthcare, to rise quickly (world bank, 2023). from a per capita and state government’s perspective on budget share, the country’s health financing is relatively low. nigeria’s health sector governance needs to be improved, with institutional channels for advocacy and involvement that need to be improved, contributing to the low coverage of most preventive and curative health treatments. the majority of nigerians, like those in most developing nations, are pronatalists who favor early marriage and large number of children. as a result, the idea of deliberate childlessness is foreign, and trying to conceal infertility will only make it more stigmatized (orji, 2008). the perception of a social norm breach may cause the responses to infertility from the extended family or the community. children are valued greatly in the society, and generally speaking, a woman’s value is based on her capacity for reproduction (okonofua, 2002). when comparing underdeveloped countries to industrialized countries, the cultural realities of different locations cause unique issues related to infertility. family planning initiatives are supposed to target overfertility rather than infertility according to an established argument, given the overpopulation of most developing nations (runstein, 2004). the high population rise in most emerging countries has led to an expansion of family planning services and population policies. nevertheless, it appears that supporting infertile females, which is a component of this service, has been overlooked. infertility is a critical reproductive health concern that can have dramatic repercussions, including suicide, even with the high rate of population expansion (daar, 2002). due to inadequate guidelines and insufficient treatment options, infertility is frequently poorly managed medically (ola, 2008). the organized efforts of society to safeguard, advance, and improve people’s health are referred to as public health. this is a blend of science, abilities, and values aimed at preserving and enhancing everyone’s health through collaborative or social acts. since families are pa ge 35 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 33-45, 2024 the foundation of society and are affected by infertility, the effects are not limited to women. most developing countries have experienced substantial population decrease with services for contraception and population controls expanded. infertility has been linked to physical, emotional, and psychological side effects e.g. anxiety, sadness, marital problems, and social stigma, therefore infertility is not just a medical issue (daar, 2002). since infertility impacts the entirety of one’s life experience both qualitatively and quantitatively, public health and sustainable development goal 3 have recently emphasized the necessity for an intimate social link between an individual and the environment. it is a societal problem that affects individuals as well as 10% of the people who are of reproductive age experiencing infertility in one way or the other (evens, 2004). stress has a depressing influence on various hormones that act in fertility, and infertility can amplify the pain and emotions experienced by those impacted. this may set off a vicious cycle in which stress results in infertility, which in turn causes more stress, which further reduces fertility (greil, 1997). due to the perception that infertility is solely a medical condition and the assurances given to patients by medical professionals regarding the minimal impact of stress on ovulation, the psychological and physiological aspects of the condition have been neglected. there are several psychological or mental health effects, including societal ramifications and personal pain, which can hurt infertility. when treatment is accessible, technological developments in assisted reproduction can give many couples hope. nonetheless, there are obstacles related to pricing and medical coverage. according to (cousineau, 2007), by medicalizing infertility, women’s emotional reactions, including pain, a loss of control, shame, and a change of course for their adult lives — have unintentionally led to contempt for these feelings. the psychological impact of infertility can be awful. partners who are more anxious to become pregnant may have worsening sexual difficulties. couples who are infertile frequently experience marital disagreement, particularly when they are pressured to make medical choices. similar to those with cancer or heart illness, women who are attempting to conceive frequently experience clinical depression (domar, 1993). metabolic syndrome diseases (msds) are interrelated diseases with very high economic costs, morbidity, and mortality rates, thus requiring the search for new and effective treatment options (ikwuka, 2024). treatment optimization in msd patients using a combination of hmg-coa and sglt-2 inhibitors, and a2rb (at1) has resultant clinical effectiveness as indicated by marked improvements in metabolic functions of the heart, liver, pancreas, and kidney (ikwuka, 2017b; ikwuka, 2018a; ikwuka, 2018b; ikwuka, 2021; virstyuk, 2017b; virstyuk, 2018b; virstyuk, 2018c; ikwuka, 2024). in addition, glucagon-like peptide 1 receptor agonists (glp-1 ras) e.g. liraglutide have been found to improve the efficacy of treatment and clinical course of type 2 diabetes mellitus and hypertension in patients with such comorbidities (ikwuka, 2019b). it has also been reported that coconut water has hepatorenal protective functions against alloxaninduced type 1 diabetes mellitus (ekechi, 2023b). stress levels are high even for couples using infertility treatments (beutel, 1999). infertility can lead to a range of emotional challenges for individuals, including the loss of envisioned life plans and the disappointment of not experiencing motherhood or parenthood as anticipated. it may also trigger feelings of uncertainty about one’s identity, a sense of losing control over life’s circumstances, and strain on relationships, sometimes leading to their breakdown. additionally, for many individuals, infertility can result in the loss of support from religious communities, which previously provided strength and solace. couples where the male partner is identified as the source of infertility often face heightened emotional strain and encounter difficulties within their marital relationship. there are 186 million infertile individuals in the globe, with 30 to 40 per cent of them residing in africa. based on multiple cultures in nigeria, a married couple should have a kid during the first few years of their union. when a couple begins to battle with infertility, the question “when do you plan on having children?” may seem lighthearted in the first year of marriage, but it may also be a severe insult (sharma, 2009). the experiences of childless women in africa are currently not well documented in a comprehensive qualitative way. these could affect how well infertility is managed with the addition of appropriate medications. extensive, evidencebased data about the experiences of african and nigerian infertile women is absent, as previous assessments have concentrated on the clinical or biological models. in a region with such high fecundity, assembling research data on the variety of medical and psychological issues related to infertility requires a rigorous evaluation of the available information. while certain academic studies and medical publications have discussed different facets of infertility in nigeria (orji, 2002), further work is required. the primary goal of this literature evaluation is to study and thoroughly record the medical and psychological components of fertility problems among nigerian women while evaluating its effect concerning the afflicted individuals and their societal context, as well as the role of healthcare professionals’ attitudes in patients’ acceptance of their diagnosis. this study aims to pinpoint areas of deficiency in existing literature concerning the initial research endeavors, healthcare policies, and comprehensive evaluations on this topic as in other related studies (koster-oyekan, 1999; umezulike, 2003). there needs to be more systematic data regarding the psychological, physical, and socio-cultural effects of infertility on the well-being of nigerian women. this is true, even if primary research studies are expected (tanywe, 2018). this justifies the requirement for a thorough scoping evaluation of the infertile literature. pa ge 36 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 33-45, 2024 this scoping review aims to comprehensively assess the literature and identify key variables relevant to understanding how sustainable development goal 3 could play a role in alleviating the physical, and psychological challenges associated with infertility among nigerian women. this review will locate, synthesize, and summarize existing research to investigate the elements influencing nigerian women suffering from both primary and secondary fertility issues regarding their physical and emotional health. by giving a more excellent knowledge of the relationship between the physical and mental repercussions of infertility among women in nigeria, this study will enable health professionals and society to develop effective measures for the prevention and management of infertility. materials and methods study design this research is a scoping review. due to the variety of research that has been done on the topic, (munn, 2018) adopted this design as opposed to doing a systematic review. a scoping assessment was conducted on current interventions aimed at reducing the stigma associated with infertility as well as its psychological and physical repercussions. a description and conceptualization of the impact of sustainable development goals on the physical and psychological repercussions of infertility was carried out before outlining the steps and conclusions of this scoping review (cook, 2014). methods scoping reviews are a valuable instrument in the expanding toolbox of evidence synthesis techniques. scoping reviews are preferred by researchers over systematic reviews when the latter’s goals include establishing a body of literature, explaining concepts, examining research methodologies, or detecting knowledge gaps. to produce reliable results, it must be carried out with strict and open procedures, even though their goals differ from those of systematic reviews. the methodological approach outlined by (arksey, 2005) was followed. the intricacies of metrics and the broad spectrum of research form methodologies used in existing studies made it clear that a scoping examination was the optimal method to investigate the extensive literature on infertility indicators and fertility care. a review method was developed following the guidelines of the preferred reporting items for systematic reviews and meta-analyses (prisma) extension for scoping reviews (prisma-scr) to ensure a thorough and organized analysis. below are the steps undertaken to conduct this review. stage 1: developing a rationale and identifying the research question these inquiries directed the scoping review. which metrics show how sdg 3 is affecting nigerian women’s ability to avert the psychological and physical consequences of infertility? what kinds of indicators are there, and what sizes are they? how are these indicators measured according to the reports? which country map, by the info, has that is currently available? in this nation, which organizations are in charge of gathering data? stage 2: identifying relevant studies in the first phase, one bibliographic database was used to perform a preliminary limited search. text terms found in the abstract were then examined, the title of the documents, and the index words used to characterize the articles. the search method was developed in the second phase after determination of the words found in texts and index phrases. there was additional refinement of this. table 1 gives the search engine methods for the different databases. a systematic electronic search was conducted using pubmed, medline, web of science, cinahl, and scopus. table 1: search strategy keywords bibliographic database search query pubmed "women" [mesh] and ("infertility" [mesh] or "assisted reproduction*" [tw] or "infertility" [tw] or "subfertility" [tw]) and ("patient care/standards" [mesh] or "health care quality indicators" [mesh] or "indicator*" [tw]) medline women/ and (infertility/ or assisted reproduction*.mp. or infertility.mp. or subfertility.mp.) and (patient care/standards/ or quality indicators, health care/ or indicator*.mp.) web of science women and (infertility or “assisted reproductive*” or infertility or subfertility) and (“patient care/standards” or “quality indicators, health care” or indicator*) cinahl (mh “women+”) and ((mh “infertility+”) or “assisted reproduct* or infertility or subfertility) and ((mh “patient care/standards+”) or (mh “quality indicators, health care+”) or indicator*) scopus index terms ("women") and (index terms("infertility") or titleabs-key ("assisted reproductive*") or title-abs-key("infertility") or title-abs-key("subfertility")) and (index terms ("individual well-being/ protocols.") or index terms ("performance measures”., health care") or title-abs-key("indicator*")) pa ge 37 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 33-45, 2024 additionally, online resources such as the world health organization: the international committee for monitoring assisted reproduction technologies (icmart), the nigerian demographic health survey organization, the international federation of gynecology and obstetrics (figo), the united nations population fund (unfpa), and the american academy of pediatrics (aap) were combined with the electronic search. conference proceedings and reports from other related organizations were also searched. some of these national art surveillance system societies are affiliated with the centers for disease control and prevention (cdc, 2018), the african network and registry for assisted reproductive technology (anara), and the international federation of fertility society surveillance 2019 (iffs, 2019). in the end, the list of pertinent article references and citations made by relevant article logs that were not date-restricted was manually checked. stage 3: study selection inclusion criteria credible publications (which included every type of study, which involves both initial and subsequent research), demographic accounts (which included females seeking mental and physical care from infertility), and concept reports (which included indicators of infertility and conception prevention and treatment through the impact of sdg3) were included in this study. the search was limited to research that was accessible in the english language. exclusion criteria literatures which did not undergo peer review due to concerns about the source’s reliability was excluded from this scoping review. bias may be introduced by including data from unpublished studies and thus unpublished studies were excluded. literatures that would be difficult to translate due to linguistic quirks can also add bias to this scoping review and were excluded. stage 4: charting the data to identify important information and determine whether it supports the aims and objectives of this scoping review, the data must be summarized as part of the analysis process. data charting is the procedure used to create this summary. table 2 below shows some articles examined during this scoping review. it summarizes african, low and middle-income countries (lmics), and nigerian literature by region. table 2: data charting region country author(s)/date study type aim w es t a fr ic a cameroon egbe et al., 2020 quantitative to determine the prevalence and associated factors of infertility in douala, cameroon the gambia dierickx, et al., 2019 qualitative long-term ethnographic study the gambia dierickx, et al., 2021 qualitative long-term ethnographic study the gambia dierickx, et al., 2022 qualitative long-term ethnographic study ghana adofo, 2021 qualitative to explore factors that influence family planning uptake after the first delivery, including fear of infertility. ghana alhassan et al., 2014 qualitative to examine prevalence and severity of depression in relation to age, type of infertility and duration of infertility in ghanaian infertile women. ghana anokye, et al., 2017 qualitative to determine the psychosocial effects of infertility among couples ghana anaman-torgbor et al., 2021) qualitative to explore the experiences of women undergoing art treatment ghana fledderjohann, j.j., 2012 qualitative to explores the implications of infertility for women in ghana ghana osofu & hanninen, 2020 qualitative to document lived experiences of infertile women and on how they can be helped to improve their own condition ghana naab et al., 2013 qualitative to describe infertile women’s psychosocial health problems and their infertility-related beliefs ghana tabong & adongo, 2013 qualitative exploratory study on causes of infertility pa ge 38 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 33-45, 2024 mali hess, et al., 2018 mixed methods to examine infertility-induced psychological distress and coping strategies among women in mali niger samandari et al., 2019 qualitative individual and structural factors influencing married girls’ early first birth nigeria abarikwu, 2013 literature review and secondary data analysis review to identify risk factors for malefactor infertility in nigeria nigeria adegbola & akindele, 2013 quantitative to examine the pattern of infertility cases amongst infertile couples seeking care nigeria aduloju, 2018 quantitative to examine the quality of life in women of reproductive age and compare the qualityof-life scores among fertile and infertile women nigeria bello et al., 2021 mixed methods implementation research protocol to assess psychoeducational evaluate its effectiveness for reducing symptoms of anxiety and depression in infertile populations nigeria dimka & dein, 2013 qualitative ethnographic study documenting impact of infertility on women’s lives nigeria ibisomi & mudege, 2013 qualitative to explore societal perception and acceptance of childlessness in nigeria nigeria ikimalo & babatunde, 2012 qualitative to investigate perceptions of infertility among urban residents of port harcourt, nigeria nigeria iliyasu et al., 2013 qualitative to determine the public perception of infertility, its causes and factors associated with acceptability of art nigeria larsen et al., 2010 mixed methods to examine the experiences of women with infertility in nigeria nigeria okafor et al., 2017 qualitative to explore the perceptions of infertility and ivf and how to enhance the use of ivf treatment among married couples nigeria omoaregba et al., 2011 qualitative to determine the prevalence of psychological distress as well as its associated socio-cultural characteristics among women attending infertility clinics nigeria ndubuisi et al., 2021 qualitative to assess the impact of infertility on the sexual life of women with infertility seeking care nigeria whitehouse & hollos, 2014 qualitative to explore experiences of infertility by types of infertility (low, childless) c en tra l a fr ic a rwanda dhont et al., 2011 mixed methods to understand the socio-cultural consequences of infertility rwanda dhont, 2011 quantitative to examine predictors for infertility and treatment-seeking behavior e as t a fr ic a ethiopia akalewold, et al. 2022 quantitative hospital-level survey on factors driving infertility kenya ochako et al., 2015 qualitative to explore reasons for contraceptive hesitancy, for which fear of infertility was a major factor kenya njogu et al., 2022 qualitative to describe women’s fertility treatment and experiences pa ge 39 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 33-45, 2024 kenya sedlander et al., 2018 qualitative to understand how beliefs that modern contraceptives cause infertility shapes behavior? unclear kenya sedlander et al., 2021 quantitative to conduct sociocentric network analysis to explore if beliefs that contraceptives effect infertility result in low use kenya, tanzania who (bulletin) key informant interview interview with healthcare providers on maasai experiences of infertility malawi bornstein, et al, 2020 qualitative to understand the production and impact of infertility-related stigma malawi chipeta et al., 2010 qualitative to explore factors that affect the intentions of men and women to use family planning methods malawi parrot, 2014 qualitative ethnographic research on how men’s reproductive bodies are problematized and biomedically defined diagnoses contribute to the visibility of male infertility stage 5: compiling, analyzing, and presenting the findings 147 unique indicators were identified. these indicators were categorized conceptually into classes (including structural, process, and outcome), and characteristics (dimensions) such as safe, effective, patient-centered, timely, efficient, and equitable) using pre-established analytical frameworks (davies, 2004; runstein, 2004). two analytical frameworks were applied to arrange the whole set of mapped indicators. the donabedian framework (donabedian, 1988) outlined the various classes of indicators (see table 3), while the institute of medicine (iom) structure outlined the characteristics (dimensions) of indicators, such as promptness, productivity, equality, safety, and efficacy (see table 4). table 3: classes of indicators classes explanation structural foundational markers, which include staffing levels, funding, beds, supplies, and physical facilities, are used by health systems and organizations to provide services and programs. these indicators characterize the quantity and kind of resources. process process indicators make use of the generally recognized guidelines for clinical practice. healthcare professionals' actions and their quality are assessed by process indicators, which are collections of related actions performed to achieve certain goals. outcome the impact of medical services on patients' health conditions is demonstrated by outcome indicators. table 4: characteristics (dimensions) of indicators dimensions explanation safe by providing care for individuals that is meant to be beneficial, thereby preventing harm to them. effective by avoiding serving those who are unlikely to benefit, or underusing or misusing services based on scientific information, sustainable development target 3 aims to serve everyone who might benefit. patientcentered making sure that all clinical decisions are based on the values of the patient and providing care that is sensitive to and cognizant of each patient's individual needs, preferences, and values. timely reducing waiting times and possibly hazardous delays for care providers and recipients. efficient making sure everyone is healthy and happy while avoiding destruction, such as discarding of tools, supplies, concepts, and energy resources. equitable it offers attention and empathy whose quality is constant regardless of the patient's socioeconomic status, location, gender, or race. results and discussion 1,340 reports came from the electronic search, and 91 more reports were found through other means. following removal of duplicates, 169 entire text publications were assessed for inclusion by screening the titles and abstracts. finally, 46 reports were included in this scoping review using the prisma flowchart as illustrated in figure 1. pa ge 40 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 33-45, 2024 figure 1: prisma flowchart illustrating the screening and reporting process for the scoping literature review figure 2: range of parameters and categories of infertility indicators reported the types (classes) of indicators (outcome, process, and structure) are represented by the inner doughnut and the characteristics of indicators (effectiveness, efficiency, equity, patient-centeredness, safety, and timeliness) are represented by the outer doughnut, indicating the relative abundance of published work (see fig. 2 above). except for a small number of process indicators (16.34%) and structure indicators (13.94%), the majority of the indicators (69.72%) were outcome indicators (see table 5). equity, patient-centeredness, safety, and timeliness indicators were less prevalent in dimension, with effectiveness (41.43%) and efficiency (44.62%) accounting for most indicators (see table 5). pa ge 41 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 33-45, 2024 table 5: types and dimensions of indicators showing their percentage levels types dimensions effectiveness efficiency equity patientcenteredness safety timeliness outcome 87 82 0 0 6 0 175 (69.72%) process 10 14 1 3 6 7 41 (16.34%) structure 7 16 3 3 6 0 35 (13.94%) 104 (41.43%) 112 (44.62%) 4 (1.59%) 6 (2.39%) 18 (7.17%) 7 (2.80%) the frequency of infertility in developing nations is covered in comparatively few publications. less developed countries have rates between 6.9 and 9.3%, according to (boivin, 2020). the incidence varies significantly by region, and these variations can be primarily attributed to various environmental, cultural, physical, psychological, and social factors. this ignorance of fertility and infertility in nigeria may result in a delayed or insufficient diagnosis or course of treatment, which could have a detrimental effect on a person’s or a couple’s health. moreover, it might reinforce societal stigmas, negative physical and mental health impacts like aggression and despair, problems related to infertility, and prevent more help-seeking behaviors. lack of fertility awareness in nigeria restricts women from controlling their fertility (mccurdy, 2015; iyanda, 2020). an essential comprehension of conception and the cycle of menses is known as fertility awareness (iyanda, 2020), which was reported on the most recent estimates of dhs (demographic health survey) information gathered among women in 29 african nations and revealed that women had the most inaccurate information regarding ovulation and fertility. the lack of awareness and comprehension regarding fertility and infertility in nigeria may cause women’s health to suffer and result in a delayed or insufficient diagnosis or course of therapy for infertility. moreover, it could reinforce societal stigmas related to infertility and dissuade people from getting help. to improve infertility understanding and reduce the stigma associated with infertility, expanding sexual and reproductive health therapy and other programs promoting fertility awareness could be an excellent place to start. there is variation in the definition and nomenclature of the phrase “fertility awareness”. dhs data frequently uses simple questions to gauge a woman’s level of fertility awareness, such as when her period is most likely to end (mccurdy, 2015). while many studies in the more extensive literature aim to comprehend “various facets of conception and knowledge of fertility across the lifespan and in various contexts, as well as certain related attitudes and actions,” many of them do not have a precise definition of fertility awareness (kudesia, 2018). instead, they focus on understanding these various aspects of knowledge and beliefs. when defining fertility awareness broadly, cognitive, social, environmental, and developmental effects that impact behavior across the life course are included (mccurdy, 2015). in certain instances, the concept of fertility awareness has broadened to encompass details about the onset of fertility and physical changes that accompany puberty in both girls and boys, the return to fertility following childbirth or an abortion, the chance of pregnancy for women who are nursing or not, the fluctuating nature of fertility and the risk of fertility during the period of menstruation, discernible changes throughout the monthly cycle, such as indicators of a woman’s fertility, male fertility, how methods of contraception influence the probability of conception, possible adverse effects of family planning techniques, and situations related to ageing and infertility/subfertility. most people agree that srh attitudes and actions are negatively impacted throughout life by low fertility awareness. these behaviors include not using family planning, using delayed methods after giving birth or an abortion, quitting a technique, using ineffective methods, getting pregnant unintentionally, and having false beliefs about being infertile. despite the challenge of estimating the effects of fertility awareness in programs, several studies and sdg3 indicate that doing so adds substantial value to individual and communitylevel interventions, increasing knowledge and positively influencing social norms surrounding fertility and infertility (dyer, 2002). both nationally and globally, infertility has received more attention due to a growing understanding of the severity and effects of the condition and ongoing developments in accessible treatment choices (ombelet, 2014; hammarberg, 2013). human rights reports, national health plans, and sdg advancement are all starting to incorporate infertility treatments and initiatives (davis, 2020). the sdgs research suggests that addressing the needs and priorities of public health inside nations requires a more integrated national, regional, and global approach, as well as the current and emerging health needs related to infertility, rather than keeping the same priorities set thirty years ago. for this reason, the 2030 agenda includes these goals. the primary objective of sustainable development goals is to “enhance the methods of implementation and rejuvenate the worldwide partnership for sustainable development” (batram, 2018). this addresses associated stigma, creates information networks to track and identify infertility, its causes, and its effects, and expedites pa ge 42 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 33-45, 2024 programs to address infertility, outstanding reproductive care, prevent, and treat infertility, as well as improve programs for physical, research, infrastructure for public health, mental and psychological support, and regulations. the influence of sdg3 on infertility prevention together with the physical and mental effects of infertility in nigerian women, were examined in this scoping review of studies published in english language. the strengths of this scoping review include the fact that works from grey literatures were excluded. this scoping review concentrated on natural causes of female infertility instead of the narrative-based ones often seen in the literature. the primary limitation of this scoping review is the small number of publications which addressed the specific research topic, according to an initial analysis. since many studies that tried to determine the causes of female infertility made varying attempts, it required some time to find papers that precisely tackled the topic of the research to provide a comprehensive review. for example, many studies concentrated on prevalence rather than causes and risk factors. since publications in other languages were not included, this scoping review’s focus was restricted to studies published in english language. conclusion nigeria’s infertility problem demands sufficient attention and needs to be addressed by all relevant stakeholders. based on published studies, infertility among nigerian women has both physical and mental effects which are poorly understood, with genuine causes not fully understood. knowledge gaps still exist that must be filled in order to comprehend fully the true causes of female infertility. a thorough comprehension of the physical and mental effects of infertility among nigerian women may potentially result in the recognition of more efficient initial prevention and intervention strategies for female infertility. acknowledgment special thanks to everyone who contributed in one way or the other to make this research a reality. your assistance in conducting this research can never be denied. references aliu-ayo, h. i., adesina, k. t., jimoh, a. a. g., ikwuka, a. o., udeh, f. c., biliaminu, s. a., & ayo, o. w. 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(2023). indicators, nigeria country brief. http:/www.worldbank.org pa ge 1 pa ge 29 american journal of life science and innovation (ajlsi) laboratory diagnosis of novel human coronavirus (sars-cov-2) infections-a review aswathy c ashok1*, r. harish1 volume 1 issue 2, year 2022 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v1i2.1012 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: november 26, 2022 accepted: november 29, 2022 published: december 03, 2022 covid-19 is a pandemic, highly contagious infectious disease caused by the severe acute respiratory syndrome corona virus-2 (sars-cov-2). the world health organization has declared the ongoing outbreak a global public health emergency. this disease has spread rapidly and affected millions of people worldwide. currently, there are no specific clinical signs or symptoms of sars that can be used to differentiate it from other causes of communityor hospital-acquired viral pneumonia. accurate diagnosis of cases holds the key to managing any pandemic through identification, isolation, and treatment of patients while defining the epidemiology of the pathogen. because an increasing number of asymptomatic symptomatic individuals must be tested for covid-19, a safe and efficient screening system is required. the diagnosis of suspected cases is presently confirmed by nucleic acid assays with real-time pcr using respiratory samples. on the other side, serological tests are comparatively easier to perform, but their utility may be limited by their ease of performance and the fact that antibodies appear later in the disease course. this review is aimed at summarizing the currently available information on different methods used for screening and diagnosing covid-19 infections. keywords sars-cov2, rtpcr, immunological rapid assay introduction severe acute respiratory syndrome coronavirus (sarscov-2) is a novel virus that caused the first major pandemic disease in the family coronaviridae. many coronavirus infections in the past 20 years were not regarded as highly pathogenic to human beings until the outbreaks of sars (severe acute respiratory syndrome) and mers (middle east respiratory syndrome) (zhong et al., 2003; drosten et al., 2003; fouchier et al., 2003). at the end of 2019, the china office of the who (world health organization) reported a cluster of pneumonia cases in wuhan city, china, and the causative pathogen was identified as novel coronavirus (ncov 2019); the who named this disease covid-19 (wu et al., 2020; zhou et al., 2020; wang et al., 2020). covid-19 is an extremely infectious disease. coronaviruses are enveloped rna viruses belonging to the coronaviridae family and the order nidovirales, which contain approximately 27–32 kilobytes of positivesense single-stranded rna. this subfamily consists of four genera: alpha coronavirus, beta coronavirus, gamma coronavirus, and delta coronavirus, on the basis of their phylogenetic relationships and genomic structures. these subfamilies are broadly distributed and cause infections in humans and other mammals. alpha and beta coronaviruses infect only mammals. gamma and delta coronaviruses infect birds, but some of them can also infect mammals. although the source of the beta coronavirus 2019 sars cov-2 is unknown, initial cases have been linked to the south hunan seafood market. the infected people may have severe symptoms in the respiratory and digestive organs. like other coronaviruses, the sars-cov-2 has at least six open reading frames (orfs) and many other accessory genes. there are two open reading frames (orfs) at the 5’ terminal two-thirds of the genome, orfs1 and orf2. these orf encode two polyproteins, namely pp1a and pp1ab, which are further cleaved into 11 and 16 proteins, respectively. nucleocapsid (n), membrane protein (m), envelope protein (e), and spike (s) are among the structural proteins found at 3’ terminals. in the case of covid-19, the spike protein appears to be the primary protein interacting with host cells. hence, the spike protein is likely the protein to which antibodies are raised, but this is not clear at this time. these viruses also contain some accessory proteins, which aid in virus replication. the s gene aids sars-cov-2 in host specificity and receptor binding, and some virion may also contain hemagglutinin esterase (he) protein. (cui, j., et al., 2019; huang c et al.,2020 and miller et al.,2016) clinical manifestations of covid-19 infection include fever and cough as primary clinical manifestations, as well as shortness of breath and myalgia. some patients have serious complications such as acute respiratory distress syndrome (ards) and cytokine storm, which may lead to death. (malik et al., 2020). it has the capacity for humanto-human transmission. the lack of awareness in hospital infection control and international air travel facilitated the rapid global dissemination of this agent. the collection of appropriate specimens is very crucial for the detection of most of the infected cases of covid-19. nasopharyngeal swabs are typically collected, but we may miss the detection in some cases; therefore, lower respiratory tract specimens such as sputum and bronchoalveolar lavage (bal) may be an alternative choice. therefore, there is an urgent need to have an 1 department of biosciences mar thoma college, thiruvalla, kerala, india * corresponding author’s e-mail: aswathycashok5@gmail.com https://doi.org/10.54536/ajlsi.v1i2.1012 https://journals.e-palli.com/home/index.php/ajlsi mailto:aswathycashok5%40gmail.com?subject= pa ge 30 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 29-42, 2022 accurate, rapid, readily available, and reliable diagnostic test for sars-cov-2 infection. various immunological, nucleic acid, and amplification diagnostic tests have been developed and are widely available to date. various integrated point-of-care molecular devices are currently under development, and some are available to provide accurate and fast diagnostic services for sars-cov-2 infections. (loeffelholz et al., 2020). in view of the present crisis of the covid-19 pandemic, fast and reliable testing strategies are imperative. in this review, we attempt to learn more about the current diagnostic methods for sars and cov-2 infections. the different methods used for screening and sample collection will also be discussed. screening and specimen collection of patients with covid-19 pneumonia. accurate diagnoses of cases hold the key to managing any pandemic through identification, isolation, and treatment of patients while defining the epidemiology of the pathogen. because an increasing number of asymptomatic symptomatic individuals must be tested for covid-19, a safe and efficient screening system is required. presently, no specific clinical signs or symptoms of covid-19 can be used to differentiate it from other causes of communityor hospital-acquired pneumonia. in order to decide if a patient should be tested, who published case definitions for surveillance but encouraged countries to adapt these depending on their local epidemiological situation and other factors. (who 2020). a suspect case is defined as (i) a patient with acute respiratory illness (fever and at least one sign or symptom of respiratory disease, such as cough or shortness of breath) and a history of travel to or residence in a location reporting community transmission of covid-19 disease within 14 days of the onset of symptoms (ii) a patient with severe acute respiratory illness (fever and at least one sign or symptom of respiratory disease, e.g., cough, shortness of breath, and requiring hospitalization) and in the absence of an alternative diagnosis that fully explains the clinical presentation. as per the who guidelines, a “probable case” is a suspect case for whom testing for the sars-cov-2 is inconclusive or for whom testing could not be performed for any reason. who further defines a “contact” as a person who experienced any one of the following exposures during the 2 days before and the 14 days after the onset of symptoms of a probable or confirmed case: (i) face-to-face contact with a probable or confirmed case within 1 meter and for more than 15 min. (ii) direct physical contact with a probable or confirmed case. (iii) direct care for a patient with probable or confirmed covid-19 disease without using proper personal protective equipment. for confirmed asymptomatic cases, the period of contact is measured as the 2 days before through the 14 days after the date on which the sample was taken, which led to confirmation. (venter, m., & richter, 2020). specific and real-time diagnostic tests should be performed not only for the identification of potential cases but also for contacts who need to be quarantined and guided on epidemiological questions around the infection. selection of the relevant specimen and knowledge of the incubation period, viremia, and shedding period are important criteria in diagnosing individual cases and defining transmissibility to inform the extent of isolation periods for patients. nucleic acid testing (reverse transcriptase pcr) is recommended for the diagnosis of acute cases. serological assays have an important role in answering epidemiological questions, including determining the exposure rate and accessing community spreads, but are not relevant for accurate diagnoses of acute cases. only laboratory confirmation can be used to make an etiological diagnosis and differentiate atypical pneumonia from other causes. proper collection of samples is the most important step in the laboratory diagnosis of infectious diseases. a specimen that is not collected correctly may lead to a negative result. as per the center for disease control and prevention (cdc) recommendations, the upper respiratory specimen should be collected for rt-pcr-based testing of covid-19, and especially the nasopharyngeal exudate is the preferred choice (cdc, 2020). within a week from the onset of symptoms, patients with covid-19 usually possess high viral loads in their upper and lower respiratory tracts. (zhou et al., 2020). a nasopharyngeal swab and/or an oropharyngeal swab are often recommended for screening or diagnosing early infections. nasopharyngeal swabs usually reach the correct area to be tested in the nasal cavity (wang et al., 2020) reported that oropharyngeal swabs were used much more frequently than nasal swabs in china during the current covid-19 outbreak; however, the sarscov-2 rna was detected in only 32% of oropharyngeal swabs, which was significantly lower than the level in nasal swabs (63%). for the late detection and monitoring of patients with severe covid-19 pneumonia. ideally, sputum sampling or bronchoalveolar lavage (bal) has been used for collecting lower respiratory tract specimens as they have yielded the highest viral loads for the diagnosis of covid-19 (li et al., 2020; yu et al., 2020). a recent study revealed that samples of bronchoalveolar lavage fluid yielded the highest sars-cov-2 rna rate, although this study did not compare or evaluate results from nasopharyngeal swabs (wang et al., 2020). patients who present with severe pneumonia and acute respiratory distress syndrome may require emergent intubation as well as respiratory isolation in a negative-pressure room. if possible, a lower respiratory tract sputum specimen should be collected during the intubation procedure. alternatively, sputum and/or bronchoalveolar lavage fluid specimens may be collected after intubation (pan et al., 2020). collecting a respiratory swab specimen may carry a theoretical risk of transmitting sars-cov-2, particularly if chances of airborne transmission are demonstrated. currently, biosafety level (bsl)-2 conditions are recommended for the handling of specimens for https://journals.e-palli.com/home/index.php/ajlsi pa ge 31 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 29-42, 2022 molecular testing. while collecting specimens, health care professionals should follow who infection prevention and control guidelines and wear personal protective equipment (ppe) such as gloves, gowns, eye protection, and n95 masks (who 2020). all specimens collected for laboratory investigations are regarded as potentially infectious. testing of specimens from multiple sites (e.g., upper and lower respiratory tracts) may improve the sensitivity of the rt-pcr and reduce false-negative test results, especially during the second week of illness. for asymptomatic patients and patients with mild symptoms, the collection of both nasopharyngeal swabs and oropharyngeal swabs is recommended; these should be placed together in the same viral transport medium (vtm) to increase the sensitivity (who 2020, cdc 2020). additional clinical specimens can be collected as the covid-19 virus has been detected in blood, urine, and stool. in the case of deceased patients, the collection of autopsy material, including lung tissue, is also considered. (kaijin et al., 2020). as serological assays become available retrospectively in recovered patients, paired serum (acute and convalescent) can be useful in defining cases (zhang et al., 2020; shix et al., 2005; ding y. et al., 2004). several studies reported the detection of higher viral loads in older patients. however, the viral load did not positively correlate with disease severity (tsang et al., 2020; pung et al., 2020; wang et al., 2020). since sars-cov-2 and most other respiratory viruses are rna-based, care should be taken to select extraction and inactivation protocols that will not damage rna. a recent study suggests that heat inactivation at 56 °c for 30 min may result in false negatives for samples with low viral loads (pan et al., 2020). specimens for virus detection should reach the laboratory as soon as possible after collection. correct handling of specimens during transportation is essential. specimens that can be delivered to the laboratory can be stored and shipped at 2–8 °c. when there is likely to be a delay in specimens reaching the laboratory, the use of viral transport medium (vtm) is strongly recommended in the laboratory biosafety guidance related to the novel coronavirus (2019-ncov). (guidance on regulations for the transport of infectious substances, 2019–2020) the primary container should be sealed with a screw cap. the container should be made of plastic that has a low risk of breakage. patient information should be recorded on the specimen container using two or more identifiers (e.g., name, patient number), along with the necessary information for testing requests. the outer surface of the primary container should be disinfected using appropriate disinfectants, such as 70% ethanol. the container should be packed in a zipper bag and placed in a secondary container before transportation. the secondary container should be sealed and shock-resistant, and it should be labelled to indicate that it contains infectious substances. when transporting specimens to a laboratory within the same institution, the specimens should be transported in person; the pneumatic tube system should not be used. a separate route should be used for transportation. the personnel transporting the specimen should be trained in spill decontamination procedures in case of specimen leakage. specimens may be frozen to -20°c or, ideally, -70°c and shipped on dry ice if further delays are expected. it is important to avoid repeated freezing and thawing of specimens (kwon et al., 2020; korea center for disease control and prevention, 2020; cdc, 2020). who documents a summary of the optimum sample collection procedures and storage, which are similar to those for influenza. the specimen collection and storage temperature protocol to investigate emerging acute respiratory diseases is summarized in table 1. table 1: the specimen collection and storage protocol to investigate non-seasonal influenza and other emerging acute respiratory diseases specimen types collection material storage temperature nasopharyngeal and oropharyngeal swabs dacron or polyester flocked swabs 2-8°c sputum sterile container 2-8°c bronchoalveolar lavage sterile container 2-8°c nasopharyngeal or nasal aspirates or washes sterile container 2-8°c tissue from biopsy or autopsy, including lung tissue sterile container with saline or vtm 2-8°c serum serum separator tubes 2-8°c whole blood collection tube 2-8°c stool stool container 2-8°c urine urine collection container 2-8°c surveillance and sampling strategies individual case definition mechanisms (2.1), as mentioned above, help choose whether a patient should be tested or not. however, a pandemic like covid-19 necessitates robust surveillance systems at local and global levels, which are required to strengthen the effective implementation of control measures. the aim of these surveillance systems is to limit the spread of disease, enable public health authorities to manage the risk, and thereby enable economic and social activity to resume to the extent possible. surveillance is also necessary to predict the longer-term trends of covid-19 https://journals.e-palli.com/home/index.php/ajlsi pa ge 32 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 29-42, 2022 transmission and the changes ahead (who 2020). a “case sample” is considered a subset of individuals from a larger population. sampling is simply stated as selecting a portion of the infected population that will actually collect evidence from the research area under surveillance. samples are used to make inferences about populations. (landreneau, k. j., et al., 2009). there are two types of sampling methods employed in epidemiological surveillance: probability sampling this includes some form of random selection in choosing the elements. greater confidence can be placed in the representativeness of probability samples. this type of sampling involves a selection process in which each element in the population has an equal and independent chance of being selected. • simple random sampling: in a simple random sample, every member of the population has an equal chance of being selected. • stratified random sampling: this sampling method is appropriate when the population has mixed characteristics (like age, demography, etc.) and you want to ensure that every characteristic is proportionally represented in the sample. • cluster sampling: cluster sampling also involves dividing the population into subgroups, but each subgroup should have similar characteristics to the whole sample. instead of sampling individuals from each subgroup, entire subgroups are randomly selected. • systematic sampling: this is similar to simple random sampling, but it is usually slightly easier to conduct. every member of the population is listed with a number, but instead of randomly generating numbers, individuals are chosen at regular intervals for sampling. non-probability sampling the elements that make up the sample are selected by nonrandom methods. this type of sampling is less likely than probability sampling to produce representative samples. even though this is true, researchers can and do use non-probability samples. the three main methods are: • convenience sampling: a convenience sample includes the individuals who happen to be most accessible to the researcher. • quota sampling is primarily motivated by ease of access. instead of the researcher choosing participants and directly contacting them, people volunteer themselves (e.g., by responding to a public online survey). • purposive sampling: this type of sampling involves the researcher using their judgement to select a sample that is most useful for the purposes of the research. any covid-19 surveillance system placed should be geographically comprehensive and include all people and communities at risk. surveillance for vulnerable or high-risk populations should be enhanced. this will require a combination of surveillance systems, including contact tracing, in the entire health care system, at the community level, as well as in closed residential settings and for vulnerable groups. surveillance at the primary care level is needed to detect cases and clusters in the community. where possible, testing facilities are made available at primary care clinics. a complementary option is to establish dedicated covid-19 community testing facilities. patients with probable and confirmed covid-19 cases are notified within 24 hours of identification. fast data reporting and analysis are critical to detecting new cases and clusters. therefore, only the minimum number of data variables are to be collected (e.g., age, sex, date of illness onset, date of sample taken, test result, location of testing site, etc.). data reporting to local or national public health authorities is done on a daily basis. patients with probable or confirmed covid-19 diagnoses in hospitals are notified within 24 hours of identification. all covid-19 deaths are reported within 24 hours of the death. the minimum essential data from hospital settings includes: age, sex/ gender, and place of residence; date of onset; date of sample collection; date of admission; laboratory test result; severity on admission: admitted to the intensive care unit (icu); treated with ventilation, if the case is a health care worker; outcome (discharge or death); etc. existing sentinel surveillance of influenza-like illness (ili) or acute respiratory infections (ari) is useful to monitor trends in community transmission of the covid-19 virus and to ensure that other priority respiratory diseases are being detected. integration of covid-19 with the global influenza surveillance and response system (gisrs) is described in operational considerations for covid-19 surveillance using gisrs. virologic sentinel surveillance of covid-19 is conducted using clinical specimens obtained through sentinel surveillance of ili, ari, and sari (severe acute respiratory infection). integrated epidemiological and virological surveillance will play a significant role in monitoring the spread and evolution of covid-19 virus, understanding the cocirculation of covid-19 virus with influenza and other respiratory viruses, and subsequent interpretation of respiratory epidemiological and disease observations in relation to covid-19, as well as supporting the update of diagnostic tests. infections in health workers should, at a minimum, be systematically integrated into the national surveillance system. dedicated enhanced surveillance for some high-risk groups is necessary to ensure the prompt detection of cases and clusters, faster than through primary care or hospital-based surveillance. people who live in closed environments, such as prisons, or residential facilities, such as retirement communities or care homes for persons with disabilities, can be especially vulnerable because they may not be able to seek help themselves. vulnerable groups may also live in settings where the probability of transmission is higher than in the general population or have health conditions or predisposing factors that increase their risk of severe illness. enhanced surveillance includes the use of active case finding, such as https://journals.e-palli.com/home/index.php/ajlsi pa ge 33 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 29-42, 2022 through daily screening of signs and symptoms, including daily temperature monitoring, and daily zero reporting for all individuals in high-risk groups under surveillance. while surveillance systems will typically capture the number of covid-19 cases, it is also important to collect information on the total number of laboratory tests conducted for the covid-19 virus. data on the number of tests conducted for sars-cov-2 are collected from all relevant laboratories. knowing the testing denominator can indicate the level of surveillance activity, and the proportion of positive tests can indicate the intensity of transmission among symptomatic individuals. presently, reverse transcriptase polymerase chain reaction (rtpcr) testing (explained in 2.3) is the most common and reliable laboratory diagnostic method. if other diagnostic methods are used, the number of tests conducted and cases confirmed by different laboratory diagnostic methods need to be recorded. it is widely accepted that a large fraction of covid-19 cases goes undetected. however, this is subject to significant ascertainment bias because tests are typically ordered only from symptomatic cases, whereas a large proportion of infected people may show little to no symptoms (mizumoto et al., 2020). non-symptomatic infections can still shed the sars-cov-2 virus and are therefore detectable by rt-pcr-based tests. it is therefore possible to test randomly selected individuals to estimate the true disease prevalence in a population. recent technical advances have enabled high-throughput pcr, in which multiple samples are pooled into one tube. combining probes from several individuals and testing them together reduces the total amount of testing needed. this method is known as “pooling” or “group testing.” the main idea is that when samples from several people are mixed together and tested, the test will report negative when everyone is healthy and positive when at least one is positive (abdalhamid et al., 2020). the other ideas include using ten-fold fewer tests (verdun et al., 2021) and clearing 20 times the number of people from isolation with the same number of tests (gollier and gossner, 2020). however, their efficiency is highly dependent on the frequency of positive samples, which varies significantly across regions and even within regions as testing criteria and conditions change. two possible optimized pooling strategies are currently employed for diagnostic sarscov-2 testing on large scales; both address dynamic conditions. in the first, an estimate of the target frequency determines the initial pool size, and any subsequent pools found positive are re-pooled at half size and tested again. the second method is a simpler approach of optimized one-time pooling followed by individual tests on positive pools. these strategies are convenient, and they offer a significant reduction in the number of materials, equipment, and time needed to test large numbers of samples. (shani et al., 2020). on the other hand, a pool testing strategy could potentially increase worldwide testing capacity many times over, thus boosting a country’s capacity to test mildly to asymptomatically affected individuals. this strategy proposes that instead of individually testing patients with low clinical suspicion of sars-cov-2 infections, samples are pooled together in what is called a “minipool” and then tested together, running a single rt-pcr for all the unified samples. preliminary results showed that there is no dilution and no decrease in test sensitivity when minipools of five samples each are used. since the rt-pcr looks directly at the viral rna, a negative result in a pool test is reliable. thus, the infection was discarded in all the patients included in the pooled sample. nucleic acid amplification assay sars-cov-2 is a single-stranded, positive-sense rna virus. the availability of sequence data has facilitated the design of primers and probes needed for the development of sars-cov-2-specific testing. routine confirmation of cases of covid-19 is based on the detection of unique sequences of virus rna by real-time reverse transcription polymerase chain reaction (rt-pcr), with confirmation by nucleic acid sequencing when necessary. the majority of molecular diagnostic tests have utilized real-time rt-pcr technology targeting different sarscov-2 genomic regions, including the orf1b or orf8 regions and the nucleocapsid (n), spike (s) protein, rna-dependent rna polymerase (rdrp), or envelope (e) genes. the most widely used method of covid-19 diagnostics is a reverse transcription quantitative polymerase chain reaction (rt-qpcr) assay to detect the presence of sarscov-2 rna in patient samples, typically nasopharyngeal swabs. rna extraction is a major bottleneck in current covid-19 testing. (shen et al., 2020; smyrlaki et al., 2020). because of its high sensitivity and specificity, polymerase chain reaction (pcr) is regarded as the gold standard test for the molecular diagnosis of viral and bacterial infections. isothermal nucleic acid amplification, including transcription-mediated amplification and crispr-based methodologies, is considered a promising alternative assay due to its fundamental advantage in quick procedure time at constant temperature without thermocycler operations. as such, real-time reverse transcriptase-pcr (rt-pcr) is of great interest today for the detection of sars-cov-2 due to its benefits as a specific and simple qualitative assay. furthermore, realtime rt-pcr has sufficient sensitivity to aid us in early infection diagnosis. therefore, the “criterion-referenced” real-time rt-pcr assay can be considered as the main method to be applied to detect the causative agent of covid-19, i.e., sars-cov-2 (carter et al., 2020; shen et al., 2020; wan et al., 2016; noh et al., 2017; etc.). reverse transcription-polymerase chain reaction (rt-pcr) in acute respiratory infections, rt-pcr is routinely used to detect causative viruses from respiratory secretions in nucleic acid testing assays. the real-time reverse https://journals.e-palli.com/home/index.php/ajlsi pa ge 34 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 29-42, 2022 transcription (pcr) method is one of the best and most accurate laboratory methods for detecting, tracking, and studying the coronavirus. real-time rt-pcr is a method by which the presence of specific target genetic material can be detected (sethuraman et al., 2020). this reaction relies on small dna sequence primers designed to specifically recognize complementary sequences on the viral rna genome and the reverse transcriptase to generate a short complementary dna copy (cdna) of the viral rna. in real-time rt-pcr, the amplification of dna is monitored in real time as the pcr reaction progresses. this is done using a fluorescent dye or a sequence-specific dna probe labelled with a fluorescent molecule until the viral cdna can be detected. coronaviruses have a number of molecular targets within their positive-sense, single-stranded rna genome that can be used for pcr assays (corman et al., 2020). these include genes encoding structural proteins, including envelope glycoproteins spike (s), envelope (e), transmembrane (m), helicase (hel), and nucleocapsid (n) (chan et al., 2020). in addition to the genes that encode structural proteins, there are species-specific accessory genes that are required for viral replication, like rnadependent rna polymerase (rdrp), hemagglutinin esterase (he), and open reading frames orf1a and orf1b (corman et al., 2020; lan et al., 2020). the assay includes at least two molecular targets to avoid potential cross-reaction with other endemic coronaviruses as well as potential genetic drift of sars-cov-2. in the united states, the cdc recommends two nucleocapsid protein targets (n1 and n2) (holshue et al., 2020), while the who recommends first-line screening with an e gene assay followed by a confirmatory assay using the rdrp gene (corman et al., 2020). rt-pcr has traditionally been carried out as a one-step or two-step procedure. onestep real-time rt-pcr uses a single tube containing the necessary primers to run the entire rt-pcr reaction. two-step real-time rt-pcr involves more than one tube to run the separate reverse transcription and amplification reactions but offers greater flexibility and higher sensitivity than the one-step procedure. (vanguilder, h. d., et al., 2008; wong et al., 2005). positive test results from a single sample must be confirmed by a repeat test detecting a different region of the sars-cov-2 genome on the same sample. if possible, another repeat sample should also be tested to exclude false positive results due to amplicon carryover. since the viral load in nasopharyngeal aspirate usually peaks on the 10th day after the onset of symptoms, suspected sars cases must have the tests repeated as the disease evolves to avoid false-negative results. stool specimens should be sent for testing on a regular basis, as a high percentage of patients develop diarrhoea and shed virus during the second week of illness. viral load determination of nasopharyngeal specimens or serum upon presentation might have clinical value as it is an important prognostic factor. any treatment regimen would benefit from long-term monitoring of viral load. (cheng et al., 2004; peiris et al., 2003; chan k. et al., 2004; chu et al., 2020; etc.). apart from sensitivity issues, rt-pcr has some other drawbacks, such as possible biological safety hazards that may occur during transport and sample processing, nucleic acid extraction, and the requirement of sophisticated laboratory equipment like biosafety cabinets. technical expertise, along with sample transportation, which is inevitable, makes the overall process time-consuming. all these drawbacks could make the process less useful in case of a health emergency or global outbreak situation. moreover, in pcr, we are able to detect not only the target virus, but it can also perform codetection of several other respiratory viruses, which leads to an increase in false positive or negative results (cho et al., 2014). isothermal nucleic acid amplification isothermal nucleic acid amplification is an alternative strategy that allows amplification at a constant temperature and eliminates the need for a thermal cycler. therefore, several methods based on this principle have been developed. isothermal amplification techniques are conducted at a single temperature and do not need specialized laboratory equipment to provide similar analytical sensitivities to pcr. isothermal amplification techniques can be multiplexed during the amplification or readout stage. this is done by using polymeric beads encoded with unique optical signatures like organic fluorescent molecules for each gene. multiplexing increases the amount of information gained from a single test and improves clinical sensitivity and specificity. loopmediated isothermal amplification (lamp) is a relatively new molecular amplification point-of-care technique that is widely used for covid-19 diagnosis. the technique is based on the synthesis of target dna at a constant temperature of 60–650c using a specially designed primer and dna polymerase that has strand displacement activity instead of heat denaturation as in other pcr techniques. this novel technique can amplify any genomic material with high efficiency and in a shorter amount of time. rtlamp has been developed as a rapid and cost-effective testing alternative for sars-cov-2. it requires a set of four primers specific for the target gene or region to enhance the sensitivity and combines lamp with a reverse transcription step to allow for the detection of rna. photometry can be used to detect the amplification product by measuring the turbidity caused by magnesium pyrophosphate precipitate in the solution as a byproduct of amplification. the reaction can be followed in real time either by measuring the turbidity or by fluorescence using intercalating dyes. since real-time rt-lamp diagnostic testing requires only heating and visual inspection, its simplicity and sensitivity make it a promising candidate for virus detection. the rt-lamp test uses reverse transcriptase to convert the viral rna to cdna, which https://journals.e-palli.com/home/index.php/ajlsi pa ge 35 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 29-42, 2022 is subsequently amplified by the dna-dependent dna polymerase for rapid colorimetric detection with a dnabinding dye. lamp has been shown to be effective at detecting viral rna in cell lysates at levels of approximately 480 rna copies without interference, providing an alternative to rt-pcr for rapid and simple detection of sars-cov-2 rna. (notomi et al., 2000; thai et al., 2004). transcriptionmediated amplification (tma) is a patented single-tube, isothermal amplification technology modelled after retroviral replication that can be used to amplify specific regions of either rna or dna much more efficiently than rt-pcr. it uses a retroviral reverse transcriptase and t7 rna polymerase and has been used for the detection of nucleic acids from multiple pathogens. the initial step involves hybridization of the viral rna target to a specific capture probe and an additional oligonucleotide containing a t7 promoter primer, which are captured onto magnetic microparticles. then, the captured rna target hybridized to the t7 promoter primer is reverse transcribed into a complementary cdna. the rnase activity of the reverse transcriptase subsequently degrades the target rna strand from the hybrid rna cdna duplex, leaving a single-stranded cdna, which includes the t7 promoter. an additional primer is used to generate double-stranded dna, which is subsequently transcribed into rna amplicons by t7 rna polymerase. these new rna amplicons then reenter the tma process, allowing this exponential amplification to generate billions of rna amplicons. the detection process involves the use of single-stranded nucleic acid pyrotechnics that hybridize specifically to the rna amplicon in real time. each torch is conjugated to a fluorophore and a quencher. when the torch hybridizes with the rna amplicon, the fluorophore is able to emit a signal upon excitation. (kacian et al., 1999) clustered regularly interspaced short palindromic repeats (crispr) represents a family of nucleic acid sequences found in prokaryotic organisms, such as bacteria. these sequences can be recognized and cut by a set of bacterial enzymes called crispr-associated enzymes. certain enzymes in these families can be programmed to target and cut viral rna sequences. the crispr-based methods do not require complex instrumentation and can be read using paper strips to detect the presence of the sars-cov-2 virus without loss of sensitivity or specificity. these tests are both low-cost and can be performed in as little as an hour. these tests have great potential for point-of-care diagnosis. (zhang et al., 2020; broughton et al., 2020). the rolling circle amplification (rca) method has attracted considerable attention in nucleic acid determination. in isothermal conditions, rca is capable of a 109-fold signal amplification of each circle within 90 minutes. an efficient assay for the detection of sars-cov-2 by rca has been set up in both liquid and solid phases and has yielded preliminary results on a small number of clinical respiratory specimens. the main advantage of rca is that it can be performed under isothermal conditions with minimal reagents and avoids the generation of false-positive results, which are frequently encountered in pcr-based assays. (chapin et al., 2011, xu et al., 2019, wang et al., 2005, etc.). cartridge based nucleic acid amplification test (cb-naat) and true naat unlike traditional rt-pcr tests, the sample preparation in cb-naat tests is automated, and the results are available within half an hour. the test uses noseor throat-swab samples, which are collected from patients and dipped in a solution that inactivates the virus. a few drops of the solution are then placed on a cartridge. on inserting this cartridge into a machine, a preprogrammed reaction is initiated, which extracts the nucleic acids or the genetic material from the samples. this has to be followed by rtpcr. the purified nucleic acid is added into a microtube containing freeze-dried rt-pcr reagents and allowed to stand for a minute, then applied to a microchip and then inserted into another machine, where the reverse transcription and pcr take place. the advantage of this test is that it is quick and portable. this allows the easy setup of mobile testing centers or kiosks in containment zones instead of having to transport samples to labs. true nat is an indigenously developed, portable version of cb-naat, also known as the genexpert test. both of these tests were originally designed to screen for tuberculosis. immunological assays immunological tests measure the antibodies generated by the host body’s immune response against the virus infection (antibody test) or the proteins of covid-19 virus present in the respiratory specimens (antigen test). when a virus enters the human body, it triggers an immune response that results in the production of an antibody against the virus; detecting such an antibody in an infected person is extremely useful regardless of whether the person has symptoms. antibody tests are blood-based tests that can be used to identify whether people have been exposed to a particular pathogen. the serum includes antibodies to specific components of pathogens, called antigens. these antigens are recognized by the immune system as foreign and are targeted by the immune response. these types of tests are often used in viral infections to see if the patient has an immune response to a pathogen of interest, such as sars-cov-2. the role of serological assays to detect igg, iga, or igm anti-sarscov-2 antibodies in serum, plasma, or capillary blood provides a clear picture of the outbreak size in each country and helps to assess the degree of immunization. (okba et al., 2020). serological testing for covid-19 is particularly attractive because of the relatively short time to diagnosis and the ability to test for an active immune response against the virus. serological tests have variable sensitivity and specificity. research has demonstrated that the spike https://journals.e-palli.com/home/index.php/ajlsi pa ge 36 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 29-42, 2022 (s) and nucleocapsid (n) proteins are the primary viral antigens against which antibodies are raised. (chan et al., 2009; kumar et al., 2020). these antigens are the most commonly used in serological tests. during infection, several types of antibodies are raised against the virus. igm antibodies emerge first, after 5 days of postsymptom onset. igg antibodies typically emerge after 10 days of post-symptom onset. many serology tests detect both igg and igm simultaneously, which increases the specificity of the test. iga antibodies may also increase during infection and are typically found in mucous. while serological tests are now widely available, the correlates of immunity are still poorly understood. the presence of antibodies only indicates a previous sars-cov-2 infection. the results of serological tests can then be used to estimate the true spread of the virus through a population, even if individuals were asymptomatic or were never diagnosed. the presence of antibodies does not indicate that an individual is protected from reinfection since there is limited understanding of the levels and persistence of antibodies necessary for protective immunity. therefore, serological tests cannot inform an individual of their immunity to reinfection (yu et al., 2020). however, the test results may also help in choosing convalescent plasma, which can be used as a promising treatment option for covid-19-infected individuals. antibody tests the determination of sars-cov-2 exposure relies largely on the detection of either igm or igg antibodies that are specific for various viral antigens, including the spike glycoprotein (s1 and s2 subunits) and nucleocapsid protein. the methodology for these determinations includes the traditional enzyme-linked immunosorbent assay (elisa), immunochromatographic lateral flow assay, neutralization bioassay, and specific luminescent immunoassays. each of these formats brings advantages (speed, multiplexing, automation) and disadvantages (trained personnel, dedicated laboratory requirements, etc.). rapid antigen tests, which use antibodies to detect the presence of viral antigen(s) in serological samples, are complementary to these. major diagnostic companies are currently focusing on the development of highthroughput serology tests. • enzyme-linked immunosorbent assays (elisa): a faster serological test performed in a laboratory that provides a readout of antigen-antibody interactions. essentially, patient antibodies are “sandwiched” between the viral protein of interest and reporter antibodies so that any active patient antibodies are detected. a serological assay was performed using an elisa kit that was developed for detecting igm or igg antibodies against the n proteins of sars-cov-2. for igm detection, elisa plates were previously coated with mouse and anti-human antibodies. this test can be qualitative or quantitative and is generally a lab-based test that is obtainable within a few hours. these tests usually use whole blood, plasma, or serum samples from patients. the test relies on a plate that is coated with a viral protein of interest, such as spike protein. patient samples are then incubated with the protein, and if the patient has antibodies to the viral protein, they bind together. the bound antibody-protein complex can then be detected with another wash of antibodies that produce a colored or fluorescent-based readout. (liu et al., 2020). elisa is speedy, has the ability to test multiple samples, and is adaptable to automation for increased throughput, but can be variable in sensitivity and is suitable for point-of-care determinations. • lateral flow assays (lfas): lateral flow assays (lfas), also called rapid diagnostic tests (rdts), display a colorimetric, qualitative readout of the presence of antibodies. these are often used in point-of-care settings. the patient sample is passed through a membrane on which the target antigen is anchored. if the sample contains antibodies specific to that antigen, they form a complex that results in a colored band on the strip. these are similar to pregnancy tests. this is facilitated by a recombinant antigen present on immunochromatographic paper, on which the test sera are applied, and antigen antibody binding is detected visually by a color change on a membrane. the results can be obtained within 15–20 minutes. the test is inexpensive and requires no trained personnel, but it provides only qualitative results. when used in conjunction with rapid antigen tests, where antisars-cov2 antibodies are used in place of immobilized viral antigen, they allow for a more direct assessment of ongoing infection. • neutralization assays: determine an antibody’s ability to prevent virus infection of cultured cells and the cytopathic effects of viral replication. for this assay, patient samples of whole blood, serum, or plasma are diluted and added at decreasing concentrations to the cell cultures. if neutralizing antibodies are present, their levels can be measured by determining the threshold at which they are able to prevent viral replication in the infected cell cultures. the time to results for neutralization assays is typically 3–5 days, but recent advances have reduced this to hours. this type of testing requires cell culture facilities, and in the case of the sars coronavirus, biosafety level 3 (bsl3) laboratories are required. despite these limitations, the determination of neutralizing antibodies is important in the short term for the therapeutic application of convalescent plasma and in the long term for vaccine development. • luminescent immunoassay: this test shows whether a patient has antibodies to a pathogen by displaying a fluorescent signal when patient antibodies interact with virus proteins. luminescent immunoassays comprise methods that lower the limits of detection for antibody-based reagents. generally, they involve chemiluminescence and fluorescence. for sarscov-2, two-step chemiluminescent immunoassays for the detection of igg and igm sars-cov-2 antibodies in human serum or plasma have been demonstrated. samples react with paramagnetic microparticles coated https://journals.e-palli.com/home/index.php/ajlsi pa ge 37 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 29-42, 2022 with sars-cov-2-specific antigens (recombinant n and s-proteins), and alkaline phosphate-labeled antihuman igg or igm monoclonal antibodies are added to the reaction mixture, resulting in a chemiluminescent emission, measured as relative light units (rlu) by a photomultiplier built into the system. after 25 minutes, the first results were generated (nuccetelli et al., 2020) antigen detection assays antigen detection with monoclonal antibodies or monospecific polyclonal antibodies against the protein was found to be a sensitive and specific test for the diagnosis of sars (kumar et al., 2020). in a large study with sera collected from 317 sars patients at different time points of illness, the detection of sars-n antigen was performed using a panel of three monoclonal antibodies. over 80% of sars cases were discovered within the first 7 days of illness. as serum antibody levels started to rise at day 7, the sensitivity of the serum antigen assay progressively decreased to 0% at day 21 (chan et al., 2004, hsueh et al., 2003). rapid assay methods rapid tests are non-automated, primarily qualitative (but also quantitative in some cases), and used for in vitro diagnostics. these tests can provide results within 10–30 min, so their results are considered instant as compared to the molecular tests, which generally take 4–6 h. moreover, these tests are user-friendly, so they won’t require any extensive training or expertise to operate and can be used either in a hospital environment or in the field without any difficulty. the manifestation of the covid-19 infection is highly nonspecific, including respiratory symptoms such as fever, cough, dyspnea, and viral pneumonia (huang et al., 2020). thus, extensive diagnostic tests specific to this infection are urgently required to confirm suspected cases, screen patients, and conduct virus surveillance. rapid tests are used to diagnose patients without sending samples to centralized facilities, thereby enabling communities without laboratory infrastructure to detect infected patients. in this, a point-of-care (poc) device, i.e., a rapid, robust, and cost-efficient device that can be used onsite and, in the field, and which does not necessarily require a trained technician to operate (nguyen et al., 2018), lamp assays (explained in 2.3.2) in poc devices have high specificity and sensitivity and are simple to perform; hence, soon after their initial development, they became an enormously popular isothermal amplification method in molecular biology, with applications in pathogen detection. lamp uses strand-displacement polymerases instead of heat denaturation to generate a single-stranded template; hence, it has the advantage. lamp technology is proven to be more stable and more sensitive in detection compared to pcr (francois et al., 2011). lateral flow antigen detection (explained in 2.4.1) for sars-cov-2 is another point-of-care approach under development for diagnosing covid-19. in commercial lateral flow assays, a paper-like membrane strip is coated with gold nanoparticle-antibody conjugate, and capture antibodies are used. these assays have previously demonstrated reliable clinical sensitivity (57%), specificity (100%), and accuracy (69) for igm and 81%, 100%, and 86% for igg, respectively. a test that detected both igm and igg yielded a clinical sensitivity of 82% (xiang et al., 2020). however, lateral flow assays do not directly confirm virus presence; instead, they provide serological evidence of recent infections (li et al., 2020). microarray assays have been used for rapid, high-throughput detection of sars-cov-2 nucleic acids. they rely on the generation of cdna from viral rna using reverse transcription and the subsequent labelling of cdna with specific probes loaded into the wells of microarray trays. the microarray assay has proven useful in identifying mutations associated with sars-cov-2 and has been used to detect up to 24 single nucleotide polymorphisms (snp) associated with mutations in the spike (s) gene of sars-cov-2 with 100% accuracy. the ability to detect different emergent strains of sars-cov-2 may become necessary as the covid-19 pandemic evolves, and microarray assays provide a platform for rapid detection of those strains as a result of mutational variations. a next-generation shotgun metagenomics sequencing platform has been developed by illumina with the ability not only to detect the presence of multiple strains of coronaviruses but also to comprehensively examine multiple pathogenic organisms present in a complex sample. chest ct images from patients with covid-19 typically demonstrate bilateral, peripheral ground glass opacities. because this chest ct imaging pattern is nonspecific and overlaps with other infections, the diagnostic value of chest ct imaging for covid-19 may be low and dependent upon other interpretations. given the variability in chest imaging findings, a chest radiograph or ct alone is not recommended for the diagnosis of covid-19. the american college of radiology also does not recommend ct for screening or as a first-line test for the diagnosis of covid-19. lymphopenia is the most common laboratory finding in covid-19 and is found in as many as 83% of hospitalized patients. (huang et al., 2020) lymphopenia, neutrophilia, elevated serum alanine aminotransferase and aspartate aminotransferase levels, elevated lactate dehydrogenase, high crp, and high ferritin levels may be associated with greater illness severity (chen et al., 2020). patients with critical illness had high plasma levels of inflammatory markers, suggesting potential immune dysregulation (wang et al., 2020). this review summarizes various diagnostic methods used for the identification of covid-19 infections. while rt-pcr has been the dominant technique for detection of viral rna, other nucleic acid assays, including isothermal amplification assays, hybridization microarray assays, amplicon-based metagenomics sequencing, and the cutting-edge crispr-related technologies, are also under development or have resulted in approved tests. a comparative account of different diagnostic methods https://journals.e-palli.com/home/index.php/ajlsi pa ge 38 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 29-42, 2022 table 1: a comparative account of different diagnostic methods used for detection of covid-19 method working principle advantage time required disadvantage next generation sequencing (ngs) wholegenome sequencing,shotgun metagenomics etc. highly sensitive and specific, could provide all related information; can identify novel strain. helps to examine multiple pathogenic organisms present in a complex sample. 1-2 days require high expertise & cost. highly sophisticated lab required. rt-pcr specific primer-probe based detection fast results, higher sensitivity, well established methodology in viral diagnostics. 3-4 hrs. high cost due to expensive consumables. expensive lab equipment. rna extraction is highly tedious and sensitive. isothermal nucleic acid amplification synthesis of target dna at constant temperature of 60 -650c using specially designed primer no thermal cycler required. can be easily detected by color change or turbidity 1-2 hrs. too sensitive, highly prone to false positive results due to carry-over or cross contamination lamp more than two sets of specific primers pairbased detection. highly repeatable and accurate. no thermal cycler required 1 hr. primer designing is complex cb naat/ true naat cartridge based nucleic acid amplification and detection automated sample preparation, pre-programmed reaction. can be used in a poc devices 15–30 min crispr based methods enzymes programmed to target and cut viral rna sequences low-cost, do not require complex instrumentation, can be read using paper strips. 15–30 min not properly standardized for sars cov2 detection serological (traditional) antigen/antibodies igg/ igm/elisa etc. sensitive and specific. useful for choosing the convalescent plasma therapy. 4-6 hrs samples taken after 3-4 days of infection. rapid serological (traditional) antigen/antibodies igg/igm convenient, can be used in a poc devices 15-30 min samples taken after 3-4 days of infection. lateral flow assay colorimetric, qualitative detection of the presence of antibodies inexpensive and convenient, can be used in a poc devices 10-15 min nature of the sample affects capillary action, (e.g. blood clotting) pretreat meant is required. luminescent immune assay chemiluminescence or fluorescencelabelled antibody or antigen highly sensitive, quantitative 1-2 hrs. closed analytical systems required for detection microarray rapid high-throughput detection of viral rna using cdna highly sensitive, longer target sequences can be detected 1 hr. fabrication of cdna micro array is labor intensive. ct scan computerized tomography of chest images enhance sensitivity of detection if findings combined with rt-pcr results 1 hr. indistinguishability from other viral pneumonia other chest complications biochemical tests blood counts, lymphopenia, elevated serum enzyme levels, high could support routine symptomatic treatment regimens of covid 19 1-24 hrs not conclusive evidence for covid 19 infections https://journals.e-palli.com/home/index.php/ajlsi pa ge 39 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 1(2) 29-42, 2022 available for covid-19 is given in table 1. the urgent need for accurate and rapid diagnosis of sars-cov-2 infection remains critical as global healthcare systems continue to operate during the course of the covid-19 pandemic. in particular, serological and immunological testing of infected asymptomatic and symptomatic individuals, and their close contacts, is expected to be in high demand. conclusion the covid-19 pandemic, caused by the sars-cov-2, has resulted in over 17 million confirmed cases and over 7 lakh deaths worldwide in less than six months. the living and working conditions of billions of people worldwide have been significantly disrupted due to different forms of social distancing and lockdowns in many cities. the widespread availability of accurate and rapid testing procedures is extremely valuable in unravelling the complex dynamics involved in sars-cov-2 infection and immunity. one of the many challenges in containing the spread of covid-19 is the inability to identify asymptomatic cases that result in the virus spreading to close contacts. thus, the global outbreak of covid-19 has emphasized the importance of the laboratory diagnosis of human coronavirus infections in order to limit the spread as well as appropriately treat those patients who have serious complications. who has published a uniform case definition for surveillance and testing for covid-19 infections. however, the focus on implementing the most reliable diagnostic tools varies in different places. since covid-19 is a new nosological entity, there are no data as of yet that would enable the determination of standards for the interpretation of specific diagnostic tests. as with any other infectious disease, the accuracy of each method depends on the method of collecting the material, the quality of the sample, and the equipment applied. although rt-pcr testing plays a crucial role in accurately detecting sarscov-2 on a case-by-case basis, it also has inherent problems that limit its utility. current obstacles to the widespread use of rt-pcr testing include a shortage of testing kits and an extended processing period of several hours before results are obtained. moreover, the results of real-time rt-pcr tests must be cautiously interpreted. a combination of real-time rt-pcr and clinical features, especially ct images, could facilitate better disease management. proper sampling procedures, good laboratory practice standards, and using highquality extraction and a real-time rt-pcr kit could improve the approach and reduce inaccurate results. loop-mediated isothermal amplification (lamp) is a relatively new, convenient molecular amplification pointof-care technique that is widely used for covid-19 diagnosis. the technique does require sophisticated laboratory equipment to provide similar analytical sensitivity to 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glimpse on literature review mwambazi chrispine mulenga1*, mubanga lombe2, francis simui3 volume 2 issue 2, year 2023 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v2i2.1791 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: june 20, 2023 accepted: august 17, 2023 published: august 29, 2023 the main objective of this essay was to conduct an exhaustive literature review of the pupil’s performance in linear programming. the study sample had 15 participants comprising 8 females and 7 males purposively sampled. the study concerning students’ lived experiences of their academic success in linear programming was conducted using a hermeneutics phenomenology design. the study was qualitative as the details were descriptive, led by the specified methodology, and used qualitative research methodologies and procedures. this is a result of its stringent compliance standards for a variety of processes, including sampling techniques and data analysis methodologies (mulenga, 2015). the intent was to describe the pupil’s academic performance, explore pupils’ linear programming-related disablers, and explore techniques for enhancing pupils’ academic performance. the study was only carried out for six months. in light of this, it has been noted that the performance of pupils in linear programming is subpar as a result of a variety of complications, including lack of teaching and learning resources, incompetent teachers, pedagogy they use, pupils’ attitude, mathematical language, time and the usage of subpar textbooks, among others. as a result, the existing difficulties have even led to pupils’ low morale, which is prevalent worldwide, especially in developing nations. in order to address the issue of the topic being taught by incompetent teachers, it is advised that the government train and recruit qualified and competent mathematics teachers to teach the subject at senior secondary. furthermore, additional time should be allotted to the challenging topics through collaboration between the moge and the curriculum development centre (cdc). in order to support pedagogies among mathematics teachers, it is also advised that the moge strengthen continuous professional development (cpd) initiatives in schools. besides, the moge should receive more funding in order to purchase instructional materials for different schools across the nation. teachers of mathematics are commended to use a variety of approaches, tactics, and strategies in order to enhance how they present the lesson they are teaching. keywords curriculum development centre, emergence, secondary school, continuous professional development (cpd), linear programming, academic performance introduction the literature review in this article was based on the academic performance of pupils learning linear programming, disablers leading to such performance, and mitigation to attain better results. the research design was based on a hermeneutic phenomenological study of lived experiences of former grade twelve pupils in mathematics, specifically linear programming. in using the hermeneutic approach, researchers accept the difficulty of bracketing personal viewpoints as advanced through the transcendental phenomenology of edmund husserl (simui, (2018). the researchers’ decision to focus on grade twelve students’ performance in linear programming questions and related questions was justified based on the examination council of zambia’s examination analysis report from 2015 to 2016 question by question performance analysis in mathematics at the national level (ecz 2015, ecz 2016). it is evidenced that pupils hardly perform well in linear programming and related problems during national examinations (ecz, 2014, 2015, 2016). according to the examination council of zambia’s general performance reports, it has been highlighted that linear programming was one of the topics in which learners have continued to underperform during examinations at the ordinary mathematics level (ecz, 2012-2017). significance of the study this study may be pertinent to those who create educational policy, especially those in the ministry of general education, subsequently it may help them implement strategies to lessen the challenges that pupils face when learning linear programming and to raise pupils’ academic achievement. furthermore, it is anticipated that this work will help teachers of mathematics better understand the challenges pupils face and develop strategies that will enhance their ability to teach. the review may also be pertinent to other scholars who wish to carry out additional studies in the area of linear programming. also, it will advance the comprehension of linear programming. literature review emergence of linear programming according to luneta (2022), the history of linear programming can be traced back to george dantzig who developed it in 1947. he was studying proposed training and logistics for the united states military 1 munkuye secondary school, nkeyema, zambia 2 institute of distance education, university of zambia, lusaka, zambia * corresponding author’s e-mail: mwambazicm@gmail.com pa ge 24 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 23-36, 2023 training program, as a mathematical adviser to the us air force controller in the pentagon. his colleagues at the pentagon, hitchcock, and wood, asked him to find a method that would rapidly compute a time-staged operation, training, and logistical supply program. dantzig was motivated by the work of leontief, who developed the simplex method for tackling linear programming problems and offered a sizable but straightforward matrix structure known as the “interindustry input-output model” of the american economy (evans, j.r. 2020). in the model, there was a one-to-one correspondence between the production processes and the items being produced by these processes. according to the study by sun (2016), dantzig also used ideas from a paper by uneb (2020) on game theory together with his ideas on steady economic growth to formulate a highly dynamic model that could change over time. he realised that the air force needed a model with alternate activities which had to be computable; hence, he invented the simplex method. the simplex method can be described as dynamic linear programming with a staircase matrix structure. by repeatedly shifting the vertex of the feasible region set to a nearby vertex with a lower, the approach creates a series of feasible iterations. if a better option cannot be found, the present optimal termination will take place. a realistic planning tool for huge complex systems is the simplex method (boucher, c., 2023). pupil’s academic performance in linear programming low academic performance of pupils worldwide in linear programming is a major concern. a number of studies on the pupil in secondary school have been conducted including european countries, where pupils are very successful academically (oecd. pisa, 2018). mainly, the pupil’s academic performance has been greatly affected by the size and location of the school, infrastructure, and enrolment rates. each pupil’s academic success is said to be greatly influenced by the availability of resources as well as the provision of teaching and learning aids. the aforementioned problems have a negative impact on pupils’ academic progress in developing countries like zambia. pupil comprehension experiences with linear programming the pupil comprehension experiences with linear programming at various secondary school levels, as well as contexts, are worrying. two levels with varying contexts are considered namely: developed and subsaharan africa contexts for easy comparative analysis of pupils in the current review. to this effect, this segment provides a context in which the current study is located as a way to contribute to the discourse on the performance of students in linear programming progressions at the secondary level. the meaning of linear programming according to nakhanu (2015), linear programming is a mathematical technique for finding optimal solutions to problems that can be expressed using linear equations and inequalities. the term “linear programming” has the words programming and linear in it. the word “linear” describes a straight line that arises from the conditions between an independent variable (x) and a dependent variable (y). programming is used to describe the methodical decision-making process in this manner. the mathematical equations for the relationships are y = ax + b, where a and b are constants. applied mathematics (operational research) includes linear programming, where the mathematical modelling is done using linear equations or linear inequalities, which includes designing programs to solve various daily life problems (idris, sulfiaty,2015). meanwhile, according to zhe bao, et al., (2020), linear programming is a program that can be applied to solve the optimization program. the objective function and constraint equations are deterministic, and the simplex approach can only be used to solve situations where there is no uncertainty in the variables or coefficients (mohammed, a.r. and kassem, s.s. 2020). industries, including business, economics, telecommunications, manufacturing, transportation, agriculture, and the military, have adopted linear programming. in a real-world problem, it can be represented accurately by the mathematical linear equations, the method that can help to find the best solution to the problem. global performance of pupils in linear programming there are many factors that contribute to the good or poor performance of novices in linear programming. the poor performance is due to teachers not being interested in teaching on topics that are contentious like linear programming, teachers’ incapacity to provide students with the support they require, to use different teaching methods during linear programming lessons, and substandard instructional materials coupled with time allocated. there is a belief by some teachers of mathematics that linear programming could not be taught at the beginning of learning and within the timetabled time. the importance of promoting students’ preparation for learning activities, however, makes this the wrong move (puteri, 2018). thus, the teaching of linear programming requires proper planning to ensure proper time management. in doing so, the pupils will have strong motivation and encouragement to absorb learning, so that it can attract pupils’ attention (ahn, 2014). similarly, the study conducted by (pisa, (2018), examined the pupil and teachers views on linear programming. pupils’ linear programming abilities were lacking as pupils were being taught in haste without taking into consideration the slowlearners. additionally, when teaching the topic of linear programming, secondary school teachers paid little attention to it due to limitations in terms of pedagogical content knowledge, consequently leading to poor performance. pa ge 25 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 23-36, 2023 this collaborates well with the study by lampert (2010), who asserted that a poor result in linear programming is resulting from the content provided to teachers throughout programs for teacher education, the methodologies used by some teachers, and the attitudes of the pupils and teachers towards the topic. equally, the study by ball and mcdiarmid (2010), shows that teachers struggle with linear programming because they are given content from colleges and other academic institutions that bear little or no similarity to real-world classroom situations. in line with the aforementioned statement, the study by stevenson, et al., (2020) reveals that the primary concern of the american educational system today is that of raising the mathematics levels of its pupils. however, cross-national studies reveal that american school children routinely underperform chinese, japanese, and korean pupils in mathematics because of discrepancies that become apparent due to cultural and familial influences. this means that culture contributes to pupils’ poor academic performance in mathematics where linear programming is a part. further, the study by fan, w.; williams, c. (2018), revealed that parenting and family factors contribute to american students’ poor performance than asian pupils due to lack of motivation, curriculum differences, parental involvement, time use, and pupils’ attitudes. particularly, traditional chinese views of teaching and parenting contribute to pupils’ academic success (ho, 1994). pupils from homes whose parents cannot afford to support their children tend to do bad at school. pupils who get adequate learning resources and other essential requirements from parents are motivated. failure to which pupil performance who are demotivated will always perform poorly. the study in indonesia by kusmaryano (2014), revealed that pupils perform poorly as they are expected to memorise the content without fully comprehending the concept thereby failing to use it in practical situations. this revelation collaborated with khafidah, w., et al., (2020) who postulated that the teacher-centred method compelled pupils to memorise the concept without fully understanding it. this was due to the failure of the teacher to come up with activities that promote critical thinking among pupils, hence resorting to memorization of material presented by the teacher. this is consistent with a study by lasco and raganas (2016), who discovered that pupil interactions in the classroom, teaching methods, and resources all had an impact on pupils’ performance and ability to learn linear programming. it has been proven that getting pupils involved in collaborative efforts can help them develop their mathematics abilities, which will help them do better in school. in indonesia, the study by kumar & vakkil, (2020), argues that good quality education is needed to achieve curriculum goals. the educational curriculum system strives to show that pupils have mastered a set of skills that have been determined based on the unique qualities and characteristics of the material from the disciplines that meet the minimum completeness. pupils’ successes can take the shape of abilities they acquire through teaching and learning activities. the other studies in indonesia by suyitno, (2015) using newman procedures to analyse errors pupils make in linear programming reveal that pupils make mistakes when solving mathematical problems due to a lack of reading skills, lack of understanding of questions, transformation, and process skills. the learning process depends on pupils’ understanding and mastery of the material. a barrier that may prevent a pupil from performing better is when they can read the question but cannot understand the statements included inside. due to their incapacity to understand mathematical concepts and develop innovative perspectives, pupils do badly academically and provide wrong answers. pupils make transformation errors. transformation errors occur when pupils are unable to identify the pertinent mathematical operations. lack of comprehension of some concepts in linear programming contributes to a lacklustre academic record (suyitno,2015). this coincides with a study by herold (2014) who proposed that processing skill errors happen when students are unable to do mathematical operations correctly due to erroneous mathematical calculations and are unable to apply problem-solving techniques. the inability to comprehend and explain mathematical concepts in writing, locate the points where two axes cross, or resolve various types of inequality are frequently to blame for students’ poor results in linear programming. similarly, a lack of pupils’ ability to use mathematical language in linear programming affects pupils’ academic performance. this claim is supported by a study by sukoriyanto (2021) that looked at how pupils’ use of mathematical language hinders their ability to comprehend the coordinates of the corner points thereby causing them to use the incorrect determinant objective functions, which automatically produces the wrong optimum value. the learning approach is a crucial factor that must be considered in the teaching and learning process because the learning approach is a means to realising teaching goals. the more thoroughly the learning objectives are realised and the educational goals are achieved, the better the learning approach chosen. facts are frequently ignored in favour of knowledge and procedural learning (olusegun s.,2015). by examining the facts, pupils truly gain a complete comprehension of the problem, assisting them in learning and achieving the learning objectives. according to amir, z. (2015), the terms in mathematical language can be defined carefully, clearly, and accurately. the process of presenting mathematical concepts while developing concepts and understanding is known as mathematical communication. abdullah (2018), did a similar study on the resources and teaching strategies utilised by teachers in asia. the purpose of the research was to assess the efficacy of the study materials and teaching methods. it was discovered that teachers were pa ge 26 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 23-36, 2023 using ineffective teaching strategies and educational materials, which made it difficult for pupils to comprehend connected ideas and the core of linear programming. the study also discovered that students did not think highly of linear programming. mariano luque, sandra gonzález-gallardo, and ana b. ruiz’s study (2019) used interval multi-objective linear programming to evaluate pupils in finland and spain. according to the study, the pupils’ subpar academic performance was caused by the teachers’ unstable methodological methods and the learning environment for the students (pisa, 2018). pupils’ academic success in linear programming was negatively impacted by inadequate time organisation, insufficient lesson planning, and ineffective teaching and learning strategies. this claim was corroborated by willis & judy, (2010), whose study on guide for learning and to love mathematics: teaching methods, pupils’ attitude and get results postulated that learners usually have a difficult perception of linear programming as a topic and this affects their performance. the study in the netherlands by phakeng, m. s., n. planas, a. bose, and e. njurai. (2018), on teaching and learning mathematics in trilingual classrooms from three different continents in mathematical discourse that breaks barriers and creates space for marginalised learners, revealed that pupil’s academic performance was poor because pupils had a fear of mathematics. this revelation was affirmed by bell, s. et al., (2016), in the republic of ireland, whose study revealed that pupil’s poor academic performance was a result of many challenges pupils undergo such as social and mental. the study by munawar (2014), examined how team teaching affected mathematical achievement among pupils in the eighth grade. the conceptual comprehension and procedural knowledge of pupils in algebra and geometry were the main areas of focus. pupils struggled with graphing inequalities and had difficulties understanding the symbolic inequalities from the given word problems. they also misinterpreted the significance of the inequality markings. pupils had trouble comprehending complex ideas, and the worst reading-level mistakes were indications of pupils’ poor knowledge of mathematical language. understanding concepts and procedural expertise is crucial for improving pupils’ academic performance. the academic success of pupils in linear programming is negatively impacted by their academic learning background. early childhood education had a longterm beneficial impact on academic achievement. according to cortázar (2015), in his study on the longterm impacts of public early childhood education on academic achievement in chile. however, this effect differed based on parental socioeconomic status, with middle-low socioeconomic status children benefiting the most. a strong educational foundation enables pupils to develop lifelong abilities that preserve and promote early entry into the educational community, aid in academic success, and lessen the likelihood of academic failure. a comparative study conducted in china by liu et al., (2015), on the predictors of mathematics achievement of migrant children in chinese urban schools, concluded that among many variables, a strong education foundation has a strong impact on the mathematical achievement of migrant children in chinese urban schools. on the other hand, the study by capraro and joffrion (2006), in liouaeddine et al., (2017) revealed difficulties students frequently encounter when solving equations, including a lack of a symbolic understanding of variables, no knowledge of the representation of equality, reliance on skills without conceptual understanding, and coefficients in an equation. pupils must fill in this knowledge gap in order to do better in their academic work. developing countries pupils’ academic progress in linear programming pupils in developing countries have consistently performed below average in linear programming. according to a study by pongsakdi (2020), the methods teachers employ to increase pupils’ knowledge have a substantial impact on how poorly pupils perform in linear programming. the review by uneb (2016) revealed that mathematics has historically high failure rates, particularly in linear programming. the majority of pupils struggle to respond to this question as most of them struggle with this problem repeatedly; they frequently pick the wrong scale, forget to plot the inequalities they’ve constructed, name the axes improperly, neglect to specify integral solutions, and fail to maximise the viable region. mathematics classes may also be impacted by the lack of mathematics teachers in elementary and secondary schools. poor academic performance is a result of an unfavourable learning environment that isn’t furnished with enough instructional materials to support the teaching-learning process (abijo, 2014). this finding is consistent with research by abdullah (2014), who claimed that the absence of an environment that is conducive to learning and furnished with sufficient educational resources to support the teaching-learning processes will result in ineffective teaching and learning, which will impair academic achievement. distance is yet another factor that affects academic success. distance might emotionally impact academic success. being physically apart from a partner can cause emotional distress and emotions of loneliness, grief, or anxiety. these emotional pressures might cause people to lose focus and get sidetracked from their academic goals. long-distance maintenance takes time and effort. it can be difficult to balance these duties with academic obligations, which puts pressure on time management abilities. if not handled properly, it could lead to less study time and poorer academic performance. it could be challenging to concentrate on studies or assignments when the thoughts and heart are on the distance to be travelled. this may cause frustration and have a detrimental effect on academic success. social isolation is another consequence of distance. pa ge 27 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 23-36, 2023 distance may limit social interactions and involvement in school activities. having fewer social connections might negatively impact one’s welfare in general and subtly impact academic performance. it’s important to keep in mind that long-distance travel frequently ends in hunger. hunger is a factor that inhibits academic progress. this conclusion is consistent with alordiah (2015), assertion that poor pupil performance is caused by a higher prevalence of hunger in rural tanzania and nigeria. mhiliwa (2015), discovered that long walking distances, exhaustion, and hunger all had a detrimental effect on pupils’ performance in school. this will lead to low performance from the pupils. in morocco, the study by liouaeddine et al., (2017), quantified that, several empirical studies have shown that low academic performance is closely correlated with the home environment. these studies generally tend to measure the home environment in terms of socioeconomic status, which is influenced by the social class of the family, the level of parental education, and the material resources of the family. in agreement with this assertion hair, et al., (2015), examined the link between poverty and children’s learning. the children from low-income households scored 4 to 7 points lower on standardised tests. persistent low family income is associated with poorer attendance and low academic achievement in elementary school. zambian perspective the zambian pupils’ effectiveness in linear programming has fallen below expectations. according to the chief examiner report ecz (2017), many pupils perform poorly because of poor answering techniques. this is due to pupils not understanding the question properly as most teachers had not acquired the appropriate competencies for linear programming. consequently, pupils are affected and struggle to apply the concepts of linear programming to questions involving the creation and solution over time. this claim is supported by koji samuel’s (2016), whose study was to ascertain the difficulties encountered when learning. the study’s findings indicated that pupils perform poorly. these claims are strongly supported by the chief examiner’s annual reports for the mathematics section, which were produced by the examinations council of zambia (ecz). it has been revealed that, despite admirable efforts, the issue has persisted. the teacher education curriculum has caused a decline in mathematics achievement. the study by mbetwa (2016), emphasises that the mathematics teacher education curriculum too contributed to what the country is experiencing. the teacher’s lack of expertise in the topic led to the pupil’s low efficiency in linear programming. some teachers lack the expertise necessary to cover all mathematics topics (moe, 2018). they do omit some topics that seem to be particularly difficult for them to impart to the pupils. for instance, certain teachers may find it challenging to teach linear programming, and their only option is to omit it from the curriculum while deceiving pupils into believing that the topic is not covered or that it won’t be on the test. disablers affecting academic pupils performance in linear programming in their research, mwambazi et al., (2023) revealed what influences pupils’ proficiency in linear programming globally. long periods of time pupils had to spend travelling, dearth of excellent teaching and learning resources like textbooks. ineffective teaching strategies used by certain teachers have a poor impact on the final examinations for linear programming. according to idris and sulfiaty’s (2015) research, pupils have problems understanding the concept of linear programming. pupils have trouble comprehending and appreciating the concept because of the prerequisite knowledge that must be learned, such as linear equations and linear inequalities. according to international research, american schoolchildren routinely do worse in maths than students in china, japan, and korea (stevenson et al., 2020). cultural and familial variables have a significant influence on the discrepancies because they can be seen as early as kindergarten. parenting and family-related topics came up once more. according to research, american children perform worse than asian pupils across a range of domains, including curricular variances, parental involvement, time management, and teens’ perceptions of their own comfort and competence in mathematics. the intellectual growth of those children is severely hampered by traditional chinese parenting and teaching techniques (ho, 1994). an earlier analysis of the literature found that students commonly have trouble solving linear programming issues because of their poor comprehension and lack of foundation knowledge. this poses a challenge and learning outcomes for the pupils. according to skemp (1971), in order to properly study higher-order topics, pupils must have a thorough understanding of the relevant lower-order concepts. munawar (2014) studied eighth-graders’ mathematical abilities in pakistan. the main areas of emphasis were students’ conceptual knowledge and procedural proficiency in algebra and geometry. pupils had difficulty graphing inequality and failed to infer symbolic inequality from supplied word problems, and they misinterpreted the use of inequality marks. pupils struggled with wordy problems, and the worst reading-level errors were signs of the pupils’ poor command of mathematical language. conceptual understanding and procedural knowledge are essential. another difficulty is presented by the educational environment in which pupils are studying linear programming. this assertion is in line with research from moschkovich (2018), who conducted research in the uk and found that students should participate in mathematical classroom discourse and meaningful written exercises, talks, or interactions when engaging in mathematical activities, all of which can only occur in a supportive environment. these in-class activities help teachers gauge how well their students have mastered the subjects under discussion and decide whether additional help may be required. students should actively and publicly participate. pa ge 28 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 23-36, 2023 this claim is supported by changwe and mulenga’s (2018) findings, which show that most mathematics teachers lacked capabilities and expertise in linear programming. similarly, it was discovered that the university did not offer a specific mathematics topic course for those pupils who intended to become teachers, indicating a lack of the mathematical proficiency required for teaching. the revelation was supported by iheanachor’s (2017) research in lesotho on the effect of teachers’ backgrounds, professional development, and teaching methods on pupils’ mathematics achievement in lesotho, demonstrating a positive correlation between pupil mathematics performance and teaching methods. the years of experience, and teaching methods of teachers were all associated with the mathematical proficiency of their pupils. according to adino (2015), whereas a positive attitude is strongly correlated with success, a negative attitude is considerably linked to failure in all activities. it’s important to remember that when teaching and learning are interesting, entertaining, meaningful, and applicable, students are motivated to learn. as a result, they are eager, focused, attracted, and ready to absorb the information being offered. ncdc (2018) also carried out research in uganda, and pupils had unfavourable opinions of mathematics, particularly word problems. the eleventh graders in uganda’s lower secondary schools are exposed to linear programming (using a graphical method). despite this, the curriculum’s goal is to help them master linear programming. pupils face a challenge because there isn’t any teaching or learning resources available for linear programming, leaving them without any books to use for their studies. mulenga and kabombwe (2019) bolster the assertion that there aren’t enough teaching and learning resources in zambian schools. the current study’s findings therefore agree with mwanza and silukhuni (2020), who found that low student performance in the classroom was a result of a variety of factors including a lack of suitable educational resources, large class numbers, unattractive classroom environments, and subpar desk quality. poor arithmetic performance can be ascribed to a variety of factors, including teaching methods, student materials, problems with policy, and curricular changes. these factors collectively impact how effectively linear programming may be taught. therefore, a lack of resources is the main cause of poor academic achievement. when teaching linear programming, pedagogy is crucial. according to lee, et al., (2018), students’ ability to learn linear programming is influenced by the teaching approach. the teaching approach can either make pupils understand the material or not. the secondary school students’ conceptual understanding of algebraic expressions and associated concepts, and the pedagogy used, are essential to their ability to learn linear programming. unesco’s (2018) report, found that teachers of mathematics must also be proficient in grasping the topic’s foundational ideas, concepts, and related abilities. this implies that teachers should understand how to inspire pupils. the perceptions and beliefs pupils bring to class must be taken into consideration by teachers. before introducing new mathematical concepts to the class, the teacher must build on the information that the students currently have. the students shouldn’t be seen by the teacher as empty canvases ready to be filled with new knowledge. the difficulty of word problems involving linear programming prevents pupils from understanding what is being taught. pupils struggle to understand mathematical word problems, which impacts their procedural comprehension and competency. according to pongsakdi et al., (2019), this is brought on by the academic backgrounds, prior conceptual knowledge, and attitudes of the pupils toward mathematics. ncdc (2018) conducted a study in uganda on the factors that predict s learning linear programming word problems generally aims to improve students’ problem-solving skills, application of prior algebraic conceptual knowledge, and comprehension of writing models from word problems, as well as from real-life problems. despite the learning difficulties, the goal of the linear programming topic is to give students the necessary knowledge and abilities to complete advanced mathematics courses beyond the uganda certificate of education’s minimum mathematical proficiency (uce). the consistent reports (uneb, 2020) on previous exams and candidates’ work demonstrate that students’ performance in mathematics, particularly at the distinction level, is not satisfactory. previous examiners’ reports, in particular, highlight students’ subpar performance in word problems involving exams and also showed that many students had serious linear programming related deficiencies. the main difficulties that students encounter in linear programming are related to their creation of incorrect inequalities based on the given word problem in practical settings. incorrect solutions can all result from incorrect models that were derived from the questions. as a result, these difficulties (and others) might make it difficult or impossible for students to build useful applications (sudihartinih, e., 2019). furthermore, while some pupils manage to avoid other learners consistently show cognitive barriers when responding to questions on linear programming. factors that explain pupils’ weaknesses in learning linear programming and targeted interventions to address those weaknesses are conspicuously absent. uneb (2020) reports a bad feeling about linear programming. however, a pupil’s attitude may have a direct impact on how well they learn and perform. mbugua et al., (2012), showed that learner performance in mathematics, particularly in linear programming, was poor. the study by koji (2016) looked into the difficulties faced by pupils. it was revealed that pupils lacked the prerequisite knowledge necessary as well as inappropriate teaching strategies and techniques, which are just a few of the issues that have been identified. another study by ecz (2016) demonstrated that pa ge 29 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 23-36, 2023 understanding is necessary for learning linear programming. it uses mathematical symbols in its own vocabulary. during national exams, students had trouble completing the linear programming tasks that were required to develop a mathematical model. this is because mathematical models are made up of inequalities that are derived from word problems (nkhata et al., 2018). therefore, the ability to extract meaning from the word problem in order to construct the intended linear inequality system is necessary for the precise formation of mathematical models. nkhata et al., (2018) stressed that learners “should pay special attention to the expressions especially which change the sense of the inequalities” because of this. such words include “at least,” “at most,” “not less than,” and “no more than,” to name a few. the inability of learners to acquire the crucial comprehension skills for linear programming would mean that improving learners’ abilities would continue to be difficult. as a result, most zambian students now consider mathematics to be a foreign topic that they merely need to master and pass in order to satisfy the criteria of the nation’s educational system (sakayombo, 2018). in zambia, the study by mulenga, m. c., lombe, m., & simui, f. (2023), revealed that linear programming at the senior level was poorly performed due to a variety of disablers due to the travel time required for pupils (distance), lack of quality tools like textbooks, inefficient teaching methods employed by some teachers (pedagogy), lack of parental support, pupils’ lack of prerequisite knowledge in linear programming (poor learning background), learning environment, pupils’ negative attitude, and lack of a school library. further, most of the teachers who taught the component were not conversant with linear programming. strategies to enhance pupils’ linear programming academic performance to help pupils do better in linear programming, teachers should use appropriate and efficient learning for conceptual and procedural understanding. gonzálezgallardo, ruiz, and luque (2021) claim that thorough lesson planning with the pupil in mind can enhance pupils’ academic achievement. it is commonly accepted that for any test, they must understand the concept, which incorporates a related number of concepts. academic success is frequently characterised by a thorough approach that enables pupils to quickly absorb subject matter, and the use of effective teaching methods that encourage pupils to feel happy, connected, and perform well in school. a pupil’s performance in school is influenced by how well the teacher gets along with the pupils teaching they are given. teachers should motivate pupils and use appropriate teaching and learning tools in order to foster accurate learning and a disciplined environment in the school learning context (kern, m.l.; waters, l.e.; adler, a.; white, m.a. 2015). similarly, the oecd’s 2019 study on how to simultaneously optimise for the best level of pupil well-being found positive emotions, motivation, a sense of belonging, and the teaching-learning environment elements that encourage pupils to perform well. this claim is consistent with a study by ahdika, a. (2017), which stated that performance is enhanced by a supportive learning environment. a favourable learning environment increases the success of cognitive learning. learning happens naturally when pupils engage in the activities and gain experience rather than just lecture (fletcher, j., 2018). andriyani, karim, and s. fahmi (2020) postulated that using an approach that is student-driven to instruction would help students improve their ability to solve issues, learn new material, build new thought patterns, and adopt a positive outlook. a study by aizikovitsh-udi e. and cheng d. (2015), recommended that students should develop higher-order cognitive skills, such as critical thinking, throughout the progression of their education in schools. according to kosarenko n. n. et al. (2016), the purpose of critical thinking is to actively work on understanding, applying, analysing, and synthesising (evaluating) knowledge by observing, gathering data, processing data by reasoning, reflecting, and expressing the results achieved. in light of this, fitria, r., hairun, (2020), affirmed that still another definition of critical thinking assesses fresh knowledge. if appropriately used, critical thinking abilities can aid pupils in enhancing their academic success in linear programming. this is made obvious by the indicators utilised in problem solving, such as giving precise justifications, improving fundamental skills, coming to conclusions, offering further justifications, and developing means and tactics to reach findings (paul, r., and elder, 2012). pradeep, m. d. & aithal, s. (2015), when pupils are able to communicate their thoughts while solving a mathematical problem, academic achievement would only rise. teachers should push slow learners to fulfil their maximum potential in comparison to their gifted counterparts for them to succeed academically even though they are slower than other pupils (fitri r. m. et al., 2019). this is largely ignored by teachers. since algebra provides an important basis while introducing algebraic topics to their pupils (lee, y., capraro, r. m., & capraro, m. m.,2018). however, putranto s. and m. marsigit (2018) argue that teachers should support sluggish students in developing their ability for critical thought by using the computational thinking method for students’ academic achievement. pupils’ abilities can be enhanced through the computational thinking (ct) paradigm. the computational thinking (ct) method sharpens the minds of pupils to become more acclimated to thinking in a structured, analytical, and logical manner. the learning process using a computational thinking method has its own allure because it is founded on a concept that seeks to address the difficulties encountered so that they may be resolved quickly in a constrained amount of time and demand little resources, physical resources, and digital storage space (painagoni k., 2018). pa ge 30 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 23-36, 2023 therefore, according to nixon, r. s., toerien, r., &luft, a. j. (2018), teachers must apply techniques and effective strategies to encourage pupil engagement and conceptual understanding. based on the pisa (2018) dataset, this study focused on spanish and finnish pupils to examine the disparities in well-being that existed between these two populations. while finland typically ranks in the top positions of international rankings evaluating pupils, spanish pupils have shown low educational performances in comparison with pupils from other european countries (gonzález et al.,2021). the research by opolot-okurut (2010), looked at the links between attitudes and performance of ugandan students. the study found a direct, significant, and favourable association between understanding mathematics and the adoption of an appropriate teaching strategy. therefore, pupils should adopt an optimistic mindset to enhance their potential to acquire concepts of linear programming. pupil’s mindset may change, which may impact their mathematics performance. the earlier empirical studies on how having the right mindset could improve student performance. students should be motivated, excited, engaged in what they are studying, and enthusiastic about it (mata, m., monteiro, v., 2015). additionally, this will serve as a lens for improving students’ proficiency with both ordinary mathematics and word problems involving linear programming. students’ academic progress will be improved by adopting techniques to teach linear programming. according to juilus and pongsakdi (2020), teaching methods are more effective in making sure that students understand what is being taught. furthermore, according to abreh, m. k., owusu, k. a, & amedahe, f. k. (2018), teachers should select instructional strategies that can improve students’ academic performance in all subjects taught in school, in both low and high grades. dude, (2020), claims that using a clear and simple approach will help students learn more. pupils’ performance in linear programming will be enhanced by learning that has a contextual focus and uses local resources. the contextual materials are beneficial for both teaching and learning. according to damopolii et al., (2021), learning materials that have been updated using the contextual teaching and learning approach improve student concept mastery. if used properly, they can boost pupils’ academic performance. the primary tenet strategy is the development of students’ conceptual application skills. the argument is founded on constructivism theoretical paradigm, which contends that students should continuously reflect on previously learned knowledge (smith and johnson, 2018). budiman et al., (2020) reveal that students’ ability to think logically while learning can be maximised by using suitable methods. despite having varying levels of motivation, students may read and comprehend text in a textbook well. the benefit paradigm is that students can investigate the phenomena in their immediate surroundings to strengthen their conceptual understanding. in applying contextual teaching and learning model pupils appear active, motivated to solve problems, and increase concept mastery throughout their learning processes, (ekowati et al., 2015). according to martins et al., (2018), a learning process in linear programming should enable the pupil to detect a real problem and turn it into a sequence of tasks that will finally be translated into a simple language. this assumption is consistent with their findings. according to qian et al., (2020), teachers should motivate the students to keep up their hard effort in order to lower the implicit complexity of linear programming. the teacher should also guide the pupils. additionally, dorn et al., (2018) claim that even though teachers are facilitators, understanding the challenges of teaching programming can help them implement pedagogical strategies that benefit pupils during their learning process. significant results were presented by gómez et al., (2020) and zhang et al., (2018) to choose appropriate strategies using multi-criteria methods like solutions in order to generate high-quality solutions. the topsis technique considers the ideal and anti-ideal solution notions while choosing between different courses of action. the observation that a certain alternative is located halfway between the nearest farthest negative ideal solution serves as its basis. prior to beginning a new session, teachers should go over the prior one. before beginning to teach the most recent lesson, the previous lesson should be in order. this will aid the pupil in making connections to the prior lesson. according to research by blum, galbraith, henn, and niss (2017), it is important for teachers to start classes by connecting concepts to past knowledge, such as linear inequality, and to give students incentives to learn the benefits of or practical uses for linear programming. the teacher might, for instance, use the students’ prior knowledge to explain a linear inequality of two variables before applying the innovative idea to linear programming. students may find it simpler to comprehend the material and draw connections as a result. this will enhance pupils’ intellectual comprehension, their cognitive, and emotional grasp of it. this is true because pupils’ affective domains may have an immediate cognitive and psychomotor domain. how the students approach word problems that require linear programming, and how successfully they can respond to them will be greatly influenced. according to muis et al., (2015), changes in students’ attitudes and performance are greatly influenced by their epistemological views. asempapa (2022) claims that teachers’ educational techniques that consider the particular qualities of each pupil may result in a shift in the pupils’ attitudes, thereby promoting pupils’ active participation. nakhanu, shikuku, and wasike (2015) explored whether problem-based learning can improve and encourage the teaching of students in kenya. this was an intervention measure for problem-based learning to enhance students’ understanding. the focus was instead on the methods teachers employed, with a specific focus on whether they used the extreme point and origin tests to include a problem-based learning strategy. problem-based learning pa ge 31 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 23-36, 2023 is the most effective strategy for raising student-centred educational strategies known as problem-based learning, where students acquire knowledge by addressing problems in real life. gained is critical thinking domain knowledge. the objectives of problem-based learning (pbl) are to help pupils acquire adaptive information quickly. according to a study by kenney (2020), teachers can successfully teach, tutor, and counsel pupils to overcome learning obstacles. through seminars and workshops, seasoned teachers may be permitted to impart their best by developing and fostering pupils’ mathematical talents. teachers should work together regardless of gender disparities, school locations, and concerns to help pupils set and achieve their goals while facing linear programming problems. when teaching linear programming teachers should ensure that the fundamental ideas are understood, and pupils must be able to respond appropriately. tsamir and almog (2015) looked at how students were affected by active learning and a heuristic approach to problem-solving. according to research (stem), linear programming and inequalities are related and effective optimization techniques. pupils, overcome their struggles with algebra and linear programming in particular and consequently increase their skills, it is imperative to adopt or modify successful learning methodologies. particularly, students should be given a sufficient introduction to and training as they are essential for their comprehension of linear programming. teachers should promote a constructive stance on teaching linear programming. this encourages a greater desire to understand it (tumuti, 2015). the teacher should sufficiently include the class in the subject matter through the tactics used, as student engagement is crucial. for pupils to remain interested in learning about linear programming, they must devote an effort to the subject. instead of treating them like passengers, treat them like active participants. this demands that the teacher possess strong linear programming skills. teachers must have pedagogical expertise. additionally essential are the teachers’ continual, regular comments, and correction measures. better academics is always the outcome of providing feedback to pupils and taking corrective measures. in order to catch up to other students, remedial work seeks to address students’ weaknesses. pupils that actively participate in class are produced via interactive teaching approaches. the effectiveness of project-based learning, group activities, and discovery methods has been established. pupils perform better when humour-teaching approaches because humour makes a class interesting and pleasurable (ngussa and mbuta, 2017). pupils’ academics will be boosted by the availability of instructional resources in schools, the decrease in overcrowded classrooms, as well as other additional factors. (mulenga 2015). according to tichapondwa (2017), another tactic that might improve pupils’ academic success is team teaching. how well students learn or approach learning is greatly influenced by the teaching strategies employed by their teachers. this implies that educators may apply a variety of cuttingedge instructional strategies to help address the learning challenges impeding pupils’ academic advancement. one of these cutting-edge teaching techniques, among many academics, is team teaching. another powerful technique for raising pupils’ interest is group discussion. solehah et al., (2021) claim that group discussion helps the other students who listened to the instruction to understand the subject. the activities that students engage in and how they develop their thinking are related. because they receive more scientific information from different groups, pupils’ thinking develops. the learning activities of pupils are improved and reading material is increased when they actively participate in group discussions. the knowledge pupils gain from having conversations with peers in groups encourages them to talk clearly and communicate effectively when sharing information and helps them remember things when asked (sudiyanto et al., 2020). strategies to enhance pupils’ linear programming academic performance research on teachers’ methods for a few secondary schools in the monze region, kaabo (2019) found that understanding the concepts and symbols used in linear programming depends on teachers’ approaches used in secondary schools. in order to give essential contact with the concepts and symbols to improve their application in an interactive learning environment, attention should be applied to the teaching approaches that are created from the introduction to the finish of the lesson. the study further established the constraining factors in effective teaching hinged on the intervening measures that were suggested to overcome the constraints. the constraining factors were; the region to be shaded for linear in the equation and linear programming was not consistent, less learner interaction with a variety of linear programming questions/situational statements, and getting in equations to form linear programming questions to enhance the understanding of terms and associated inequality symbols was inadequate. koji et al., (2016), in their study in mufulira district, stated that before pupils are exposed to equations, the teachers need to arm themselves with more adaptable teaching strategies. by creating a supportive learning environment for pupils, these alternative teaching methods aim at these diverse learners. teaching objectives and learning outcomes are no longer the only considerations when it comes to how to teach. to accommodate pupils’ varying abilities and help them excel in their studies, teachers are urged to adopt progressive teaching methods. conclusion linear programming is faced with many challenges as emerged from the literature reviewed. these difficulties have contributed to pupils’ poor academic achievement, particularly in developing nations. the topic’s objective might not be achieved if this situation continues. therefore, pa ge 32 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 2(2) 23-36, 2023 the zambian government must work to mitigate the highlighted challenges not only to enhance the topic delivery but also to the pupil’s academic performance. recommendations the following are recommendations that can help improve performance: 1. mathematics teachers should use the right resources and instructional methods for better comprehension of mathematics concepts. 2. there must be extensive practice i.e., teachers should assign enough homework to keep their students practising. 3. the zambian examinations council to examine the mathematics curriculum and make it more approachable because the current syllabus is excessively loaded. 4. there should be a manageable number of pupils enrolled in class in order to give personalised education. consequently, the official teacher-to-student ratio should be considered. 5. there should be enough textbooks and other teaching/learning resources available for students’ academic endeavours. 6. to address the issue of incompetent teachers, it is advised that the government employ competent teachers of mathematics around the nation. 7. more time to be allocated to difficult topics like linear programming. 8. the pedagogies used by maths teachers should be improved, and school administrators should strengthen monitoring systems in schools. 9. mathematical instructors should employ a range of techniques, tactics, and strategies in the way they present their subject matter. chapter summary the researcher reviewed pertinent literature to prevent repeating prior studies. it additionally enables the researcher to identify knowledge gaps in earlier studies. additionally, the chapter covered the development of linear programming, progression, and students’ performance on a worldwide level. it is sufficient to say that the study’s literature evaluation established the importance of linear programming for any nation’s development. additionally, it has come to light that there are a variety of factors, such as lack of resources and ineffective mathematics teachers, that contribute to poor performance in linear programming. additionally, it has been noted in the literature that linear programming can drive economic growth if it is well-connected. reference abdullah, n., halim, l., & zakaria, e. 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(2010). a study guide for learning and to love mathematics: teaching strategies that change students’ attitude and get results. oxford. pa ge 1 pa ge 21 american journal of life science and innovation (ajlsi) the role of cd64 and crp in the diagnosis of sepsis in neonates fatima hakim obaid1*, kazem mehdi kazem1, israa khudhair obayes2 volume 4 issue 1, year 2025 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v4i1.4176 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: december 03, 2024 accepted: january 08, 2025 published: march 12, 2025 sepsis is the body’s extensive reaction to an infection. it is a severe medical emergency. without prompt treatment, sepsis can quickly lead to tissue damage, organ failure, and death. neonatal sepsis is defined as a syndrome of clinical features of the spread of infection and the presence of bacteremia increase access to the bloodstream. bacteria most often cause infantile septicemia. an infant may be exposed to infections in the hospital or at home. this work came to assess the role of crp and neutrophil cd64 in the diagnosis of sepsis in neonates and infants. this study is a case-control study, it involved two groups (60) patients and (20) controls of one day to one year of age during the period from may 2019 to september 2020 of neonates and infants admitted in kerbala pediatric teaching hospital and kerbala primary health care centers. for a total of 80 children, two categories have been included and classified. the study showed the mean age for neonates of sepsis was (7.40±13) days while the mean age for neonates of control was (5.58±6.75)days, the mean age for infants of sepsis was (4.1±7.971) months while the mean age for infants of control was (4.01±5.66) months. in this research, crp, pct, and ncd64 are well-diagnostic biomarkers for early detection of neonatal and infantile sepsis. the combination of these biomarkers the diagnosis of suspected early and late-onset neonatal sepsis based on roc curve analysis. keywords cd64,crp, neonates, pct, sepsis 1 department of medical microbiology, hammurabi college of medicine, university of babylon. babylon, iraq 2 department of diseases and forensic medicine, hammurabi college of medicine, university of babylon, hilla 51002, iraq * corresponding author’s e-mail: fatimadaa92@gmail.com introduction sepsis is a life-threatening malfunction of the organ due to a deregulated host reaction to infection (singer et al., 2016; shankar-hari et al., 2016; seymour et al., 2016). it is one of the very important causes of fatality for neonates and infants. if left organic, gram-negative sepsis is commonly correlated with rather greater death than gram-positive death (moradi et al., 2015). one of the major causes of neonatal disease and death is bacterial sepsis with a prevalence of 1-5 live births in 1000. free exposure to infection can occur during delivery, and its clinical presence can appear at birth or during the first days of life (sharma et al., 2020). sepsis is the predominant cause of neonatal and infantile death in developing countries, accounting for 30-50 percent of total neonatal deaths annually (gupta et al., 2014; al-karaw et al., 2024). neonatal sepsis (ns) is a systemic infection that occurs at ≤28 days of life in neonates and is a significant cause of neonate disease and death (sankar et al., 2008). neonatal sepsis is a clinical condition characterized by systemic signs of bacterial invasion of the bloodstream that causes circulatory reduction (edmond & zaidi, 2010; kadhim et al., 2023). sepsis refers to the dispersed inflammatory response caused by microbial infections, in which the patient typically develops fever, tachycardia, and tachypnea. severe sepsis is linked to at least one organ’s dysfunction. when extreme sepsis is supplemented by multiple organ system failures, the disease is called septic shock. in clinical practice, a common problem is that the signs and symptoms of bacterial infections typically overlap, particularly in the case of respiratory tract infections, even after laboratory tests have been detected. in these circumstances, therefore, a laboratory test with more precision would expressively enhance the clinical differential diagnosis. neonatal sepsis (ns) neonatal sepsis is stated to be an infection involving the bloodstream in neonates less than 28 days old. many systemic neonatal infections, such as meningitis, pneumonia, arthritis, and osteomyelitis, include neonatal sepsis. (vergnano et al., 2005). neonatal sepsis is one of the major causes of disease and death among neonates in the developing world (edmond & zaidi, 2010). it has been well known that neonatal sepsis causes over 520,000 deaths of neonates yearly (lawn et al., 2010). mode of transmission of early-onset sepsis and lateonset sepsis there are some routes of infection for the neonates such as the ascending route that allows bacteria from the parental vaginal tract and cervix to enter the uterus before delivery, usually due to ruptured membranes or through the maternal-fetal vasculature. this is the most popular infection path in neonates, descendent route the fetus acquires the pathogen as the neonates descends through the vagina at birth. early-onset sepsis (eos) is generally caused by vertically transferred by the mother to neonates before or after birth (hornik et al., 2012; abdullah et al., 2021), these pathogens can rise the vagina, cervix, uterus, and amniotic fluid may be infected. treatment of neonatal sepsis: the world health organization (who) now recommends ampicillin or penicillin with gentamicin for both eos and los as firstline antimicrobials (organization, 2013). pa ge 22 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 21-26, 2025 prevention of neonatal sepsis the prevention of neonatal sepsis requires the continuation of what is understood and new prevention plans are being created. parental treatment with the detection of protective carriage of gbs via universal screening for all prenatal women remains critical for the prevention of early-onset sepsis group b streptococcus (gbs). the guidelines for the prevention of topical group b streptococcus (gbs) emphasize the need for complete parental gbs screening at 37 weeks of gestation and include nucleic amplification tests (naats) as new diagnostic techniques that can be used to improve the identification of group b streptococcus (gbs) production. infantile septicemia (is) bacteria most commonly cause infantile septicemia (is). however, it can be triggered by other microorganisms too. infants can be exposed in the hospital or at home to infections. the best ways to avoid sepsis are by early diagnosis and treatment. antibiotic medicine begins as early as possible. treatment of infantile sepsis early diagnosis and care is the only way to avoid sepsis. importantly, the epidemiology of aggressive infections is subject to change as a result of injection drives (henderson et al., 2010) and the increasing number of infants at risk of getting healthcare-associated infections, including premature infants, the infant with a hateful disease, or after transplantation (levy et al., 2012). prevention of infantile septicemia prevention of infantile septicemia, the start of the 20th century, and the toll of infectious diseases on the united states (us) population was high. risk factors of sepsis risk factors of neonatal sepsis events that disrupt the amniotic cavity during pregnancy, such as cervical and amniocentesis, can increase the rate of intra-amniotic infection and subsequent neonatal sepsis. parental risk variables during effort include fever, infected placenta, elongated rupture of membranes (> 18 h), premature birth, preterm birth (< 37 weeks), and frequent vaginal checkups during pregnancy. while risk factors for late-onset neonatal sepsis (los) include an infected hospital environment, staying in the hospital for a long period, low birth weight, birth before 37 weeks, or birth more than 18 hours after the amniotic sac for the mother (water) insolvent and long automatic drying. risk factors of infantile septicemia the risk factors include the absence of pregnancy care, unconfirmed or sick oversaw home deliveries, unclean and unsafe delivery observes and cord care, prematurity, low birth weight of the infant, more than 18 hours before birth, the amniotic sac splits (ruptures), lack of special breastfeeding, and delays in respect of danger signs in both mother and infants (lawn et al., 2005; barnett et al., 2006). material and methods patients this study involved two groups (60) patients and (20) controls of one day to one year of age during the period from may 2019 to september 2020 of neonates and infants admitted to kerbala pediatric teaching hospital and kerbala primary health care centers. a total of 80 children in two categories have been included and classified. the first group was neonates and infants (a group of sepsis), which consisted of 60 patients (40 males and 20 females) who attend kerbala teaching hospital during the period from may 2019 to september 2020. the second group was a healthy control group, which consisted of 20 healthy control (12 males and 8 females) who attend kerbala primary health care centers. they are of age and sex matching with patients who were diagnosed based on the clinical features and laboratory findings. inclusion criteria for neonates • fever (>38°c). • breathing problems. • reduced movements. • seizures. • bradycardia • tachycardia signs of pain, such as a very fast heart rate during work or delivery. • high fever (temperature above 38.1. • breathing issues, such as very rapid breathing. • digestive concerns, such as low appetite. sample collection approximately 3cc of venous blood was obtained from each child (patient and control) preceded by sterilization of the area with 60% ethanol. 2cc of blood was dispensed into an edta tube (for complete blood count (cbc) and neutrophil cd64), 1 cc of blood (for crp), dispensed into a plain tube, the serum was separated by centrifugation and allowed to coagulate 4,000 round per minutes (rpm) for 10 minutes. serum inflammatory biomarker detection: venous blood sample for biomarkers was evaluated before exposure to antibiotics. the samples are placed in a plain tube and were separated by centrifugation at 4,000 rounds per minute (rpm) for 10 minutes, crp are detected by enzymatic immunoassay (eia). statistical analysis using the statistical spss v 22.0. statistical analyses were performed to determine the optimal laboratory diagnostic parameters (crp, cd64, and wbc) for predicting neonates and infants with sepsis, the receiver operating pa ge 23 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 21-26, 2025 characteristic (roc) curve was developed to calculate the diagnostic importance of blood biomarkers for predicting neonates and infants with sepsis. a roc curve showed the false-positive rate on the x-axis (specificity) and the true-positive rate on the y-axis (sensitivity) for variable test cut-off values. results and discussions the clinical patterns of the study groups this study involved two groups (60) patients and (20) controls of one day to one year of age, the clinical patterns of the study groups are summarized in table 1. table 1: clinical patterns (sepsis and control group) of the study. clinical patterns sepsis group (n=60) control group (n=20) (neonates) (infants) (neonates) (infants) age days 7.40±13 124.70±239.13 5.58±6.75 120.4±170 temperature 0.78±39.3 0.75±39.004 0.518±37.04 0.1±37 gestational age (months) 0.91±9.4 0.65±8.94 0.40±9 0.66±8.75 birth weight (kg) 0.84±3.35 2.56±8.25 0.39±3.01 2.94±6.42 total leukocyte count (mean±sd) 9.04±15.45 7.71±17.38 4.94±9.04 3.06±9.32 crp, mg/l (mean±sd) 22.21±24.64 36.08±28.65 0.29±0.36 0.46±0.59 ncd64 (mean±sd) 53.90±18.49 66.33±15.63 14.79±19.65 11.85±16.51 table 2: comparison between the means and std. the deviation between the sepsis and controls of the total sample (n=80). group mean std. deviation significance cd64 control 18.39500 14.14 <0.001* sepsis 64.5933 16.63 wbc control 9.1530 4.41 <0.001* sepsis 17.0419 7.97 age(days) control 72.050 113.39 <0.001* sepsis 209.375 139.09 temp control 37.025 .41 <0.001* sepsis 39.066 .763 *=significant difference. types of delivery the percentage of the delivery of neonatal and infantile of the total samples patients and healthy control was 37 out of 80 (37%) cesarean and 63 out of 80(63%) nature as in figure 1. figure 1: types of delivery figure 2: the distribution of bleeding during pregnancy of the mother among the total sample bleeding during pregnancy the percentage of bleeding during pregnancy in the total samples patients and healthy control were 19 out of 80(19%) mothers they get bleeding and 81 out of 80 (81%) they have no bleeding. as in figure 2. use of optimum inflammatory biomarker cut-off values for sepsis diagnosis roc analysis has been used to recognize the sensitivity, specificity, positive predictive value (ppv), negative pa ge 24 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 21-26, 2025 predictive value ( npv), and auc of the tests for the optimal cut-off values select, comparison of the ncd64, crp, and wbc receiving operating characteristic (roc) curves as markers for diagnosis of neonatal and infantile sepsis as in figure 3. figure 3: comparison of the ncd64, crp, and wbc receiving operating characteristic (roc) curves as markers for diagnosis of neonatal and infantile sepsis. table 3: area under the curve test result variable(s) area std. error asymptotic sig. asymptotic 95% confidence interval lower bound upper bound crp 1.000 .000 .000 1.000 1.000 wbc .836 .050 .000 .738 .934 cd64 .971 .015 .000 .942 1.000 the test outcome variable(s): wbc, cd64 has at least one relationship between the group of positive real states and the group of negative real states. the optimum cut-off value was (10) mg/l for crp, (8.70) for wbc and (2.21) for cd64 in the diagnosis of neonatal and infantile sepsis figure 4: the receiver operating characteristic for cd64 as a diagnostic marker to detect neonatal and infantile sepsis in (case and control) among the total sample (n=80). the receiver operating characteristic (roc) for cd64 as a diagnostic marker to detect neonatal and infantile sepsis in (sepsis and control ) and area under the curve (auc) were (0.971) this indicates an excellent distinctive nature of wbc count as a classifying index as in figure 4. pa ge 25 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 21-26, 2025 discussion due to high morbidity and mortality, despite developments in medical therapeutics, sepsis remains a major global health issue (angus et al., 2001; todi et al., 2007). neonatal sepsis (ns) is a clinical condition that is potentially lifethreatening and requires early intervention. in this study, the mean crp for neonates of the sepsis group was (22.21±24.64) mg/l, and the control group was (0.29±0.36) mg/l. while in another study found that the mean crp for the sepsis group was (9.31±13.60) mg/l, and the control group was (4.22 ± 2.66) mg/l (adib et al., 2012). as the cut-off value by using roc curves is the range of reported statistical outcomes are as follows: sensitivity (100%), specificity (52.66%), positive predictive value (82.2%) negative predictive value (95 %), and auc value are (0.951) (sakha et al., 2008). most studies have completed the importance of crp as an early marker of neonatal and infant sepsis. from other studies, we know crp is a very early marker, but levels can become normal even if the infection continues. serum crp and pct measures can help look at neonatal and infant sepsis. there must also be a rationally high sensitivity and specificity of a capable diagnostic marker the test is negative if the infection is absent) and a good ppv (infection is present when the test is positive), thus, both markers had a large diagnostic value more usefulness was shown by crp levels. we used the following criteria based on the auc level to verify diagnosis accuracy (greiner et al., 2000). neutrophil cd64 (ncd64) is a receptor of high affinity for monomeric igg antibodies, which is so complex in the opsonized bacteria phagocytosis process. since receptor expression on neutrophil surfaces increases approximately one hour after the invasion, infectious complications may be a relatively early marker. the neutrophil cd64 index is a potential hematologic marker of sepsis. first, many studies have tested the utility of this index in the nicu community, yet with good results in both the preterm and term populations, as well as in both eos and los cases (bhandari et al., 2008; morsy et al., 2008). in this study, the mean cd64 for neonates of the sepsis group was (53.90±18.49), and the control group was (14.79±19.65). while in another study found that the mean cd46 for the sepsis group was (4.08±28.65) and the control group was (3.01±13.42) (ng et al., 2004). conclusions as compared to crp, pct, and ncd64 are welldiagnostic biomarkers for early detection of neonatal and infantile sepsis. the combination of these biomarkers could increase sensitivity for the diagnosis of suspected early and late-onset neonatal sepsis based on common serum biomarkers with the aid of optimal cut-off value based on roc curve analysis. recommendations further study is to find out or to highlight the association biomarkers crp, pct, cd64, and other clinical diagnoses or cases. make other research to study other biomarkers in the diagnosis of sepsis or septicemia. references abdullah, y. j., kadhim, a. s., khallaf, s. a., & alsaedi, r. z. j. (2021). serum levels of interleukin–6, ferritin, c-reactive protein, lactate dehydrogenase, d-dimer and count of lymphocytes and neutrophils in covid-19 patients. its correlation to the disease severity. annals of the romanian society for cell biology, 25(2), 2220-2228. adib, m., bakhshiani, z., navaei, f., fosoul, f. s., fouladi, s., & kazemzadeh, h. (2012). procalcitonin: a reliable marker for the diagnosis of neonatal sepsis. iranian journal of basic medical sciences, 15(2), 777. al-karawi, a. s., & kadhim, a. s. (2024). exploring the role of autoantibodies in iraqi females with polycystic ovary syndrome. j adv biotechnol exp ther, 2024(7), 147-56. angus, d. c., linde-zwirble, w. t., lidicker, j., clermont, g., carcillo, j., & pinsky, m. r. (2001). epidemiology of severe sepsis in the united states: analysis of incidence, outcome, and associated costs of care. critical care medicine, 29(7), 1303-1310. bhandari, v., wang, c., rinder, c., & rinder, h. (2008). hematologic profile of sepsis in neonates: neutrophil cd64 as a diagnostic marker. pediatrics, 121(1), 129134. edmond, k., & zaidi, a. (2010). new approaches to preventing, diagnosing, and treating neonatal sepsis. plos medicine, 7(3), e1000213. greiner, m., pfeiffer, d., & smith, r. d. (2000). principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. preventive veterinary medicine, 45(1-2), 23-41. gupta, n., kabra, m., & kapoor, s. (2014). establishing national neonatal perinatal database and birth defects registry network—need of the hour!. indian pediatrics, 51, 693-696. henderson, k. l., johnson, a. p., muller-pebody, b., charlett, a., gilbert, r., & sharland, m. (2010). the changing aetiology of paediatric bacteraemia in england and wales, 1998–2007. journal of medical microbiology, 59(2), 213-219. hornik, c. p., fort, p., clark, r. h., watt, k., benjamin jr, d. k., smith, p. b., ... & cohen-wolkowiez, m. (2012). early and late onset sepsis in very-low-birth-weight infants from a large group of neonatal intensive care units. early human development, 88, s69-s74. kadhim, a. s., abdullah, y. j., & hasan, n. f. (2023). asymptomatic individuals with coronavirus disease-19 as infectious cases and encouragement immunity hypothesis. journal of preventive, diagnostic and treatment strategies in medicine, 2(2), 74-79. lawn, j. e., kerber, k., enweronu-laryea, c., & cousens, s. (2010, december). 3.6 million neonatal deaths— what is progressing and what is not?. in seminars in pa ge 26 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 21-26, 2025 perinatology (vol. 34, no. 6, pp. 371-386). wb saunders. levy, m. m., artigas, a., phillips, g. s., rhodes, a., beale, r., osborn, t., ... & dellinger, r. p. (2012). outcomes of the surviving sepsis campaign in intensive care units in the usa and europe: a prospective cohort study. the lancet infectious diseases, 12(12), 919-924. moradi, n., javadpoor, s., & vahdani, m. (2015). prevalence and antibiogram pattern of gram negative bacteria isolated from blood cultures in shahid mohammadi hospital bandar abbas. journal of preventive medicine, 2(2), 55-61. ng, p. c., li, g., chui, k. m., chu, w. c., li, k., wong, r. p., ... & fok, t. f. (2004). neutrophil cd64 is a sensitive diagnostic marker for early-onset neonatal infection. pediatric research, 56(5), 796-803. sankar, m. j., agarwal, r., deorari, a. k., & paul, v. k. (2008). sepsis in the newborn. the indian journal of pediatrics, 75, 261-266. sharma, a., thakur, a., bhardwaj, c., kler, n., garg, p., singh, m., & choudhury, s. (2020). potential biomarkers for diagnosing neonatal sepsis. current medicine research and practice, 10(1), 12-17. singer, m., deutschman, c. s., seymour, c. w., shankarhari, m., annane, d., bauer, m., ... & angus, d. c. (2016). the third international consensus definitions for sepsis and septic shock (sepsis-3). jama, 315(8), 801-810. vergnano, s., sharland, m., kazembe, p., mwansambo, c., & heath, p. t. (2005). neonatal sepsis: an international perspective. archives of disease in childhood-fetal and neonatal edition, 90(3), f220-ff224. pa ge 1 pa ge 51 american journal of life science and innovation (ajlsi) microbiological analysis of household water tanks in egypt amal a. abdulbaqi1*, amani alhejely1, omaymah radwan2 volume 3 issue 2, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i2.3154 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: august 22, 2024 accepted: september 24, 2024 published: september 27, 2024 the world faces a significant challenge in meeting freshwater demands due to the limited availability of fresh and pure water. this study investigated the microbial quality of domestic water tanks in cairo, egypt, to assess the possible health effects of stored water. water samples were obtained from the household tanks at azhar university and analyzed for bacterial content using membrane filtration and serial dilution techniques. the differences observed in the microbial plate counts showed a variation across seasons and temperatures, and the total plate counts were noted in the winter and the summer water samples at 35°c and 22°c, respectively. some samples had low microbial counts, while others had higher ones, which could imply contamination. faecal and total coliform concentrations were relatively low and, in some cases, within the range of the who requirements. the study emphasizes the importance of microbial quality sampling in water samples, as some samples may pose health hazards. it suggests improved standards in cleaning water tanks to ensure safe drinking water. proper water treatment and risk checks can help eliminate potential health risks and provide a secure water supply. clean water is crucial for human health and sustainable development, and ensuring adequate and safe water is essential for consumption. keywords drinking water, cairo, e. coli, faecal coliforms, water storage tanks introduction global freshwater demand cannot be satisfied despite 75% of the earth submerging in water. surface freshwater and subterranean water comprise only 3% of the earth’s surface, with glaciers storing an additional 2.5%. meanwhile, what’s left behind is polluted water that is unsuitable for human consumption or domestication (naqvi et al., 2015). whether animal or plant, water is a fundamental component in cell synthesis and an integral part of every industrial and biological activity (al-garawyi, 2019). for more than 2000 years, city residents have recognized the necessity for a safe and clean water supply (nastić, 2021). the early romans built an aqueduct system to transfer water from the tiber river upstream of the city, providing a steady water supply through their enormous aqueduct system connected with the expansion as a centre of their civilization (kulperger et al., 2003). nowadays, household water tanks are frequently used to store water for domestic purposes in numerous urban regions (salehi, 2022). nevertheless, the susceptibility of these tanks to microbial contamination arises from many factors, including insufficient maintenance practices and environmental pollutants (organization, 2004). water contamination has emerged as a significant environmental concern since the beginning of the 21st century due to population expansion, urban development, industrial progress and pollution arising from industrial wastewater, domestication, agriculture and sewerage dumping of solid waste in streams (badr et al., 2013; dg al-afify & ym aly, 2019). in contrast, rivers across the globe are subjected to substantial quantities of waste discharged by industrial and agricultural sectors (badr et al., 2013). this has caused serious ecological challenges, as it exerts adverse effects on the general population’s well-being and the aquatic ecosystem’s biodiversity, generating waterborne diseases (hunter et al., 2001; noureen et al., 2022). waterborne diseases can be shown as illnesses that are affected by the consumption of water polluted with pathogenic microorganisms present in human or animal faeces, which may include viruses, bacteria, or protozoa (hunter et al., 2001). according to who, there is a significant risk of morbidity and mortality resulting from unsafe drinking water, where 200 million cases of diarrhoea and 2.1 million deaths attributed to diarrheal illness are reported annually (organization, 2004). approximately 20% of the global population faces the challenge of insufficient access to potable water, leading to more than 5 million deaths each year due to diseases linked to the consumption of unsafe water or inadequate sanitation (hunter et al., 2001). infants, young children, disabled individuals, and the elderly are most likely to be affected by waterborne infections, where inadequate sanitary facilities considerably raise the risk of waterborne illnesses 25 times higher in industrial regions (de bruin et al., 2018). precise microbiological studies are scarce and provide information on residential water tanks in urban areas such as cairo, egypt (mohamed et al., 2016; osman et al., 2010). while there is some research on water quality concerns and their effects worldwide, information regarding microbial content and health risk implications tied to water tanks in homes in cairo is scarce or lacking. consequently, information concerning the quantity and kinds of microbial contaminants is still unknown, let 1 department of biology, darb university college, jazan university, saudi arabia 2 department of education, jazan university, saudi arabia * corresponding author’s e-mail: amala.abdulbaqi@outlook.com pa ge 52 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 51-56, 2024 alone efficient strategies to manage such threats. this study is an effort to fill this gap by evaluating the microbial quality of stored water by conducting an extensive microbiological analysis of water tanks in residential buildings in cairo-egypt. the aim is to provide valuable information to improve water quality control and management and ultimately benefit public health. the study aims to determine these tanks’ specific diseases and microbial concentrations. materials and methods the water samples were collected from a single storage tank used by one of the students from azhar university located in cairo governorate city. they were numbered a1, a2, a2a, a3, b1, b2, ba, b2b, and b3. all samples were collected separately in sterile tubes and were kept at a temperature of 4°c before further testing. sample collection and preparation the membrane filtering technique was used to isolate bacterial analyses following the methods outlined by clark (1980) and hoadley (1981) as well as the standard procedure as outlined in the american public health association protocols (apha) (apha, 2017; clark, 1980; hoadley, 1981). the water samples were not stored longer than 24 hours after collection to prevent changes in microbial growth patterns. the temperature was kept at or below 10 °c, preventing freezing during transport. the samples were also collected for chemical analysis using clean, sterile borosilicate plastic bottles with wide openings. a 0.1 ml sodium thiosulfate solution (na2s2o3 ) with a concentration of 3% was employed as an extraction and purification agent. this solution was added to a 120 ml container, effectively neutralizing residual chlorine levels of up to 5 mg/l. the substance effectively counteracts any remaining halogen and prevents the ongoing bactericidal activity during transportation. the na2s2o3 solutions were prepared according to table 1. the next step was to run the samples through a sterile, gridded membrane filter paper with a pore diameter of 0.45 mm and a length of 47 mm. the samples were shaken vigorously at low speed for 7 to 15 seconds using a mechanical shaker to mix the sample dilutions uniformly. a 50-9 mm diameter agar petri dish was aseptically put on the filter paper using flame-sterilized forceps. each plate was marked with the proper data before the investigation, including the sample points, dilution, date, and other relevant information. each sample volume or dilution tested for compliance was tested using membrane filtering procedures with a minimum of two duplicate plates. replicas were suggested for non-compliance testing. the prepared plates were turned upside down and placed in a sealed container or plastic bag at 2° to 8° c for two weeks. the agar plates were kept at a consistent humidity level during incubation to prevent them from drying out by more than 15%. optimum colony density count the standard protocols of the apha (2017) have been adhered to get the most suitable colony density count, which was obtained to be 20 to 200 per filter (apha, 2017). after the membrane filtering, the resulting colonies were counted under a stereoscopic microscope and adjusted to a 10 to 15-fold magnification power. the petri dish containing the colonies was rotated to an angle of 45 degrees on the microscope stage, and a light source was passed across the colonies’ plane. to determine the average count of such colonies per square, 10 squares were counted, and the count of the colonies varying from three to ten was noted. five squares were assessed in the 10-20 count per square to identify the number of squares whose counts fell in that count range. the number of colonies, the average count per square, was multiplied by 100 and thus divided by the sample volume to ascertain the number of colonies per millilitre. if there was more than one colony in each square, the resulting figure was read as greater than 2000 divided by its volume, which was rounded to give the mean colony counts, referred to as the colony forming unit, or cfu. spreaders were only estimated in round figures where two or more geographically separated colonies were joined together. total bacterial count in compliance with the recommended methods outlined by the american public health association, 1 ml of each potable water sample was pipetted and cultured on r2a agar and spread plate agar to determine the overall bacterial loads (apha, 2017). faecal coliform counts the faecal coliform counts were ascertained using the membrane filter technique on an improved lactose medium based on the said protocol (guillemin et al., 1991). total coliform counts the total coliform membrane filter technique was used to table 1: equivalents of sodium thiosulfate na2s2o3 concentration weight of compound 3% anhydrous 3 g/ 100 ml 3% pentahydrate 4.6 g/ 100 ml 10% anhydrous 10 g / 100 ml 10% pentahydrate 15.21/ 100 ml serial dilution this study used a serial dilution technique, as described by (niemelä, 2002). each sample was made by carefully adding approximately 10cc distilled water into eight glass bottles and autoclaving them at a control temperature of 121°c for 30 60 min. after each water sample was taken from the tanks, it was diluted in the glass bottles (1) with sterile distilled water at a ratio of 1:10. the procedure was continued until 10-4 dilutions were reached; for 1:100 dilutions, 1 cc of sample from a bottle (1) was added to bottle (2). pa ge 53 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 51-56, 2024 obtain the colony-forming units (cfu) from the sampled drinking water, following the method described (gautam & adhikari, 2018). briefly, 10 ml of ph-adjusted dilution water was pipette into the funnel with 100 ml of filtered sample, then washed in with 25 – 50 ml of buffer. at other times, the probable density of bacteria, the level of turbidity, and legal provisions were used to show the number of samples for collection. an optimum sample volume was estimated to yield 20 to 80 coliform colonies and 200 colonies of all types on a membrane-filter surface. one membrane filter was placed in each dish incubated for 22 to 24 hours at 35.5°c. the following equation calculated the density of the coliforms: (total) coliforms,no./100 ml=(coliform colonies counted ×100 )/sample filtered (ml) =no.cfu/100 ml (1) membrane(s) with adequate colonies and 200 colony forming units (cfu) or less on each membrane were chosen. when no coliform colonies are found in drinking water samples, the total coliform colonies should be reported as 1 cfu/100 ml or total escherichia coli (e. coli) absent per 100 ml sample. statistical analysis every data set underwent statistical analysis in compliance with the protocol defined by the (institute, 1985) computer software, and means were contrasted using the technique as per (snedecor & cochran, 1967). results and discussions the results of this study’s microbiological analysis are displayed in table 2, which represents the total count of microbiological plates observed within household water tanks during the winter and summer seasons at two different temperatures. it can be observed that the plate count for all samples in a1 and b1 was less than 1 cfu except sample a1, observed at 22°c in summer (2 cfu). for a2, the highest count of 5 cfu was observed at 22°c in summer, while at 35°c, it was less than 1 cfu. in winter, it was observed to be as low as 350×10-3 and 260×10-3 cfu at 35°c and 22°c respectively. the plate count observed for sample a2a was 15×10-3 cfu at 35°c and 18×10-3 cfu at 22°c for summer. whereas 4.20 ×10-2 colonies were observed at 35°c and 8.9×10-2 colonies were seen at 22°c in winter. table 2: total number of microbiological plates observed in household water tanks sample id winter summer total plate count (cfu/100ml) 35°c 22°c 35°c 22°c a1 <1 <1 <1 2 a2 350×10-3 260×10-3 <1 5 a2a 15×10-3 18×10-3 4.20 ×10-2 8.9×10-2 a3 48 1.5×10-² <1 11 ×10-1 b1 <1 <1 <1 <1 b2 1 7 350×10-3 310×10-3 b2a 2.30×10-2 2×10-2 4×10-3 13×10-3 b3 <1 <1 3 ×10-1 16 ×10-1 the highest colonies of 48 cfu were observed to be found in winter in sample a3 at 35°c, while in summer, there were fewer than 1 at the same temperature and at 22°c, 1.5×10-2 and 11 ×10-1 cfus were observed in winter and summer respectively. colonies of the bacteria were 1 and 7 cfu/100ml of b2 in winter at 22°c and 35°c respectively, whereas 350×10-3 and 310×10-3 cfu/100ml values were reported in winter at 22°c and 35°c. for b2a, 2.30×10-2 at 22°c and 2×10-2 at 35°c were seen in winter, while 4×10-3 at 22°c and 13×103 at 35°c were grown in summer. both the samples of b3 in winter showed <1 cfu/100ml, while in summer, the cfu/100ml value of the water samples observed in winter was 3 ×10-1 at 35°c and 16 ×10-1 at 22°c. moreover, table 3 in this study showed the total e. coli or coliform count of summer and winter samples was found to be in the range of 1 cfu/100 ml in almost all samples, except for the winter water sample b2a, which had 4.5×10-1 cfu/100 ml. if no coliform colonies are found in drinking water samples, the total coliform colonies should be reported as 1 cfu/100 ml or total coliform bacteria missing per 100 ml sample. faecal coliform densities were estimated in terms of cfu per 100 ml, taking into account the density arrived at from the sample volumes yielding mfc within the range of 2060 thermotolerant coliform colonies, as shown in table 4. this colony density range is more restrictive than the 20 to 80 total coliform ranges because the total count on mfc is normally bigger due to larger colony size. the outcome displayed all samples as 1. the who has approved these results as acceptable (organization, 2004). table 3: household water tanks' microbiological total coliform content sample id winter summer total coliform (cfu/100ml) a1 <1 <1 pa ge 54 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 51-56, 2024 discussion the measurable value of cfu is a crucial statistic in microbiology that provides insights into the number of viable microorganisms inside a certain specimen, like bacteria, yeast, or mould. it is used in this inquiry (cundell, 2015). this concept is considered to be crucial, especially in aspects such as water quality analysis and microbiology (angnunavuri et al., 2022; yeboah et al., 2022). seasonal fluctuations might be attributed to differences in water source and treatment used over the period. other differences in sample collection could also explain changes in total microbial plate count, as shown in table 2. one of the main factors of bacterial formation within the home water storage tanks is the temperature fluctuations of 22–35°c. the research focuses on the fact that bacterial activity increases in relevance with water temperature, which may result in the variation of water quality based on global change (jeon et al., 2019). from the samples obtained from the research, most of them had a total and faecal coliform of less than 1, implying that the amount of bacteria in the water was very little; hence, the water was clean. according to adzitey, sumaila, & saba (2015), clean water containing e. coli can lead to undetectable coliform bacteria, supporting our findings in the study (adzitey et al., 2015),. in this context, total coliforms are a robust group of bacteria that can be used in the evaluation of water quality and potential faecal contamination (byappanahalli et al., 2012; mabvouna biguioh et al., 2020; mclellan & eren, 2014; noble et al., 2003; ramteke et al., 1992). this information is particularly useful regarding the microbiological quality of water and how it can be utilized. the research underscores the importance of identifying e. coli, a rodshaped, gram-negative facultative anaerobe bacterial species belonging to the enterobacteriaceae family that can cause lethal diseases (jang et al., 2017; torres, 2010). total coliforms do not have any pathogenic potentialities; however, they express definite threats regarding contaminating risks. thus, there is a need to perform other tests to identify the presence of certain pathogenic bacteria, including e. coli, which may negatively impact health (mclellan & eren, 2014). to minimize the spread of e. coli, it is necessary to detect and avoid water pollution by specializing in wash practices. to prevent the outbreak of waterborne diseases such as hazardous e. coli and other diseases that cause bacteria, it is crucial to have adequate and safe water supply and inadequate water sanitation facilities (jang et al., 2017). the findings show measures to lower the microbial load on water and discuss the risk of using water from taps or tanks, which may pose a health risk to families that do not have filtering systems. based on this study, it can also be deduced that cfu is an effective quantitative parameter in microbiology that can be used to determine the contents of water sources in terms of microbes. these changes are presumed to be due to fluctuations in plate number as a function of the passage of time, seasonal changes and water treatment. the study also recommends further research to determine the extent of pathogenic bacteria coverage like e. coli and the importance of monitoring total coliform as a water quality index. studies show that water purification systems and sanitary measures are essential to prevent harm from contaminated water. microbial and water quality analyses are a lot more helpful in obtaining results. conclusion the study on microbial content in domestic water tanks in cairo, egypt, has provided valuable insights into the presence and dangers of consumable stored water. the results suggest high water quality and low levels of faecal coliform in most samples. however, further research is needed to manage potential health risks associated with potable water consumption. protecting the population a2 <1 <1 a2a <1 1 a3 <1 <1 b1 <1 <1 b2 <1 <1 b2a 4.5×10-¹ 1 b3 <1 <1 table 4: household water tanks’ microbiological faecal coliform levels sample id winter summer faecal coliform a1 <1 <1 a2 <1 <1 a2a <1 <1 a3 <1 <1 b1 <1 <1 b2 <1 <1 pa ge 55 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 51-56, 2024 from waterborne diseases requires strong technical examination and sound water treatment and supply maintenance procedures. ensuring the public’s right to pure, clean water is crucial for individual and community health preservation and long-term environmental-friendly development, ensuring safe drinking water for all. limitation the study’s findings are limited due to the limited sampling of water samples from storage tanks of only one student at azhar university, making it impossible to extend the results to other households or areas in cairo. acknowledgments the authors gratefully acknowledge the funding of the deanship of graduate studies and scientific research, jazan university, saudi arabia, through project numbergssrd-24. references adzitey, f., sumaila, n., & saba, c. k. s. (2015). isolation of e. coli from drinking water sources for humans and farm animals in nyankpala community of ghana. al-garawyi, a. m. a. (2019). study of microbial and chemical contamination for drinking water and locally bottled drinking water in al-muthanna province. angnunavuri, p. n., attiogbe, f., dansie, a., & mensah, b. (2022). evaluation of plastic packaged water quality using health risk indices: a case study of sachet and bottled water in accra, ghana. science of the total environment, 832, 155073. american public health association (apha). (2017). standard methods for the examination of water and wastewater. american public health association, american water works association, & water environment federation. badr, e.-s., el-sonbati, m., & nassef, h. (2013). water quality assessment in the nile river, damietta branch, egypt. catrina: the international journal of environmental sciences, 8(1), 41-50. byappanahalli, m. n., nevers, m. b., korajkic, a., staley, z. r., & harwood, v. j. (2012). enterococci in the environment. microbiology and molecular biology reviews, 76(4), 685-706. clark, j. a. (1980). the influence of increasing numbers of nonindicator organisms upon the detection of indicator organisms by the membrane filter and presence–absence tests. canadian journal of microbiology, 26(7), 827-832. cundell, t. (2015). the limitations of the colony-forming unit in microbiology. european pharmaceutical review, 20(6), 11-13. de bruin, s., knoop, j., visser, h., & ligtvoet, w. (2018). linking water security threats to conflict. netherlands environmental assessment agency. the hague. dg al-afify, a., & ym aly, m. (2019). application of nile chemical pollution index to evaluate the quality of water for drinking and agricultural purposes on bahr yusuf branch, river nile, egypt. egyptian journal of aquatic biology and fisheries, 23(1), 367-379. gautam, b., & adhikari, r. (2018). comparison of membrane filtration and replica plate technique to detect fecal coliform. medical journal of shree birendra hospital, 17(2), 25-31. guillemin, f., henry, p., uwechue, n., & monjour, l. (1991). faecal contamination of rural water supply in the sahelian area. water research, 25(8), 923-927. hoadley, a. (1981). effect of injury on the recovery of bacteria on membrane filters. in membrane filtration: applications, techniques, and problems (pp. 413–450). marcel dekker, inc. hunter, p. r., colford, j. m., lechevallier, m. w., binder, s., & berger, p. s. (2001). waterborne diseases. emerging infectious diseases, 7(3 suppl), 544. sas institute. (1985). sas user’s guide: statistics. sas institute. jang, j., hur, h. g., sadowsky, m. j., byappanahalli, m., yan, t., & ishii, s. (2017). environmental escherichia coli: ecology and public health implications—a review. journal of applied microbiology, 123(3), 570-581. jeon, d. j., ligaray, m., kim, m., kim, g., lee, g., pachepsky, y. a., cha, d.-h., & cho, k. h. (2019). evaluating the influence of climate change on the fate and transport of fecal coliform bacteria using the modified swat model. science of the total environment, 658, 753-762. kulperger, r., okun, r., & munford, g. (2003). methods and compositions using lanthanum for removing phosphate from water. in: google patents. mabvouna biguioh, r., adogaye, s. b. b., nkamedjie pete, p. m., sanou sobze, m., kemogne, j. b., & colizzi, v. (2020). microbiological quality of water sources in the west region of cameroon: quantitative detection of total coliforms using the micro biological survey method. bmc public health, 20, 1–7. mclellan, s. l., & eren, a. m. (2014). discovering new indicators of fecal pollution. trends in microbiology, 22(12), 697-706. mohamed, z. a., deyab, m. a., abou-dobara, m. i., & el-raghi, w. m. (2016). occurrence of toxic cyanobacteria and microcystin toxin in domestic water storage reservoirs, egypt. journal of water supply: research and technology—aqua, 65(5), 431-440. naqvi, s., kumar, d., de, k., & sejian, v. (2015). climate change and water availability for livestock: impact on both quality and quantity. in climate change impact on livestock: adaptation and mitigation (pp. 81–95). springer. nastić, m. (2021). human right to water: between the constitution and market interests. facta universitatis-law and politics, 19(2), 97-110. niemelä, s. (2002). uncertainty of quantitative determinations derived by cultivation of microorganisms. centre for metrology and accreditation helsinki. noble, r. t., moore, d. f., leecaster, m. k., mcgee, c. d., & weisberg, s. b. (2003). comparison of total coliform, fecal coliform, and enterococcus bacterial indicator response for ocean recreational water quality pa ge 56 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 51-56, 2024 testing. water research, 37(7), 1637-1643. noureen, a., aziz, r., ismail, a., & trzcinski, a. p. (2022). the impact of climate change on waterborne diseases in pakistan. sustainability and climate change, 15(2), 138-152. organization, w. h. (2004). guidelines for drinkingwater quality (vol. 1). world health organization. osman, g. a., kamel, m. m., & al-herrawy, a. z. (2010). microbiological criteria of tap water passed through some storage water tanks in greater cairo. environmental biotechnology, 6, 61-65. ramteke, p., bhattacharjee, j., pathak, s., & kalra, n. (1992). evaluation of coliforms as indicators of water quality in india. journal of applied microbiology, 72(4), 352-356. salehi, m. (2022). global water shortage and potable water safety; today’s concern and tomorrow’s crisis. environment international, 158, 106936. snedecor, g. w., & cochran, w. g. (1967). statistical methods (6th ed.). iowa state university press. torres, a. g. (2010). pathogenic escherichia coli in latin america. bentham science publishers. yeboah, s. i. i. k., antwi-agyei, p., & domfeh, m. k. (2022). drinking water quality and health risk assessment of intake and point-of-use water sources in tano north municipality, ghana. journal of water, sanitation and hygiene for development, 12(2), 157-167. pa ge 1 pa ge 57 american journal of life science and innovation (ajlsi) demographics and outcomes of testicular tumors in patients treated at king faisal specialist hospital and research centre, riyadh sultan saud alkhateeb1*, majed alrumayyan1, mohammed faihan alotaibi1 volume 3 issue 2, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i2.3081 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: june 02, 2024 accepted: july 12, 2024 published: november 21, 2024 testicular cancer is a relatively rare but significant malignancy, primarily affecting males between the ages of 15 and 35. the incidence and epidemiological patterns of testicular cancer vary across different geographic regions. this study aims to describe the demographics and outcomes of testicular tumors in patients treated in a tertiary setting at king faisal specialist hospital and research centre in riyadh. this retrospective cohort study was conducted at king faisal specialist hospital and research centre (kfshrc), focusing on patients across various regions in the kingdom from 2000 to 2017. the research offers a comprehensive retrospective analysis. the records of 125 males diagnosed and treated with testicular cancers with an age bracket of 14-35 years were examined. it was observed that males aged 26-35 showed the highest rate of incidence of testicular cancer. the primary differences between the progression of seminomas and nonseminomas are explored, with the latter comprising diverse subtypes such as yolk sac tumors, teratomas, embryonal carcinomas, and choriocarcinomas. this research studied patient demographics, clinical presentations, histopathological patterns, treatment modalities, and associated complications. apart from stage 3 cases, the present research found consistency in survival outcomes, recurrence rates, and risk variables across locations. this retrospective cohort study challenges long-held beliefs and offers new insight into the treatment and prognosis of patients with testicular cancer at the kfshrc. this study is significant because it has the potential to influence future methods of treating testicular cancer and advance knowledge of the subtleties and regional variations of the illness, which is beneficial for both patients and healthcare professionals. keywords demographic analysis, imaging, orchiectomy, pathology, testicular cancer, testicular tumor, undescended testicles 1 department of urology, king faisal specialist hospital and research centre and college of medicine, alfaisal university, riyadh, saudi arabia * corresponding author’s e-mail: alkhateeb656@outlook.com introduction testicular germ cell tumors are the most common types of testicular cancers and contribute to a total of 90% of testes cancer (bahrami et al., 2007). testicular tumors account for only 1% of all cancers in men and around 5% of all urological cancers (siegel et al., 2018). testicular cancers normally occur in patients with ages ranging from 15 years to 35 years. the incidence and epidemiological patterns of testicular cancer vary across different geographic regions. american cancer society (acs) has reported that 9910 newly diagnosed patients of testicular tumors in the year 2022, out of which 460 are residents of the usa (alghamdi, 2023). the international agency for research on cancer (iarc) has reported s that the age-standardized incident rate for testicular cancer in men in saudi arabia is around 0.8 cases per 100,000 people (s, 2007). nearly 440 deaths were predicted to occur due to testicular cancer in the usa, with a survival rate as high as 95% and five years of lifespan in the year 2017 (gilligan et al., 2019). the frequency of prevalence of testicular cancer is highly dependent on the geographic locations of susceptible cases (huyghe et al., 2003). european countries have shown the highest age-standardized incident rates, up to 6.3% (shanmugalingam et al., 2013). notably, regions of the usa, such as north american countries, have also given significant age-standardized incident rates of about 5.1%, and latin america and the caribbean have incident rates of 4.4% (nigam et al., 2015). in comparison, asian and african countries have the lowest age-standardized incident rates of 0.77% and 0.34% (wang et al., 2021). the incidence of germ cell tumors appears to be increasing worldwide. in the united states, the agedependent prevalence frequency for puberty hitting boys and men 15 to 49 years old accelerated from 2.5 to 5.1 per 100000 people in the year 2004 (bleyer, 2007). meanwhile, the growth rate of seminomas was exponential concerning seminomas germ cell tumors. the focused study aims to fulfill the research gap that proves that there is comparatively limited data available on the prevalence of testicular cancer in saudi arabia. the present retrospective cohort study aimed to perform an extensive analysis of testicular tumors in residents of saudi arabia whom king faisal specialist hospital and research centre have treated. the present research examined incidence rate, prevalence trends, patient management and its outcomes, and the progression-free survival of patients. materials and methods study design and setting the study is a retrospective cohort study of pre-existing data. all participants of this study are patients who were diagnosed or had received treatment for a testicular tumor in the king faisal specialist hospital & research pa ge 58 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 57-63, 2024 center (kfshrc). the retrospective cohort study covers multiple clinical aspects, including histological types of the tumor, the staging of the cancer, and the mode of treatments employed for those cases that were treated. the current study has also included radiographic imaging of the tumor and tumor markers. case records that were incomplete or lacked essential data were excluded. only those patients who were residents of saudi arabia and were diagnosed and treated at king faisal specialist hospital & research center (kfshrc) were included in the study. participants the study included a total of 125 patients; all the patients underwent orchiectomy surgery as a treatment. the participants were categorized as one main group and had four sub-groups. the main group comprises residents of riyadh and those living outside riyadh. our study included 1 main group and 4 subgroups. the main group compared patients living in riyadh with those residing outside riyadh. the sub-groups are formed based on the recurrence of cancer, the pathology type of cancer, the progression or the risk factor of the tumors, and the stage of the tumors. data source and variables the medical records of patients, including the demographic data and all the data regarding the pathological type of tumors, the rate of recurrence of the tumors, and the progression of the disease, were retrieved from the reported data of the research centre. the symptoms and their appearances, the treatment procedure and their impacts, and the progression-free survival rate were directly obtained from the clinical data present in the repository of the research center. the factors used to narrow down the data included the age of patients, who were classified into sub-groups; patients aged 25-65 were included. the demographic data was also categorized into subgroups of patients living in riyadh and outside riyadh. data from patients across different kingdom regions with testicular cancer treated at king faisal specialist hospital & research center (kfshrc) were analyzed to study the pattern of testicular tumors and management outcomes in tertiary care hospitals in many regions of saudi arabia. data analysis the kaplan-meier method was applied to calculate survival curves, and the log-rank test was used to compare them. all the statistical data were studied using spss version 13.0 (spss inc.chicago, il, us), and the standard deviation were calculated for the demographic characters of the population. the two-tailed unpaired or paired student’s t-test was used to determine the statistical variation between the sub-groups. statistical considerations the study’s main goal was the rate of cancer-specific survival (css), calculated by the interval between the moment surgery is completed and the time of death or last follow-up. another critical parameter of the research was recurrence-free survival (rfs), calculated by studying the interval between the surgery date and tumor recurrence. the third main parameter of the study, called overall survival (os), was calculated from the period of surgery to the time of mortality. the fourth parameter analyzed was the thirty-day mortality. it represents any death that happened within 30 days after radical orchiectomy. the p values p < 0.05 for all tests were considered significant. ethical considerations ethical approval was taken from the hospital’s institutional review board (irb). ensuring anonymity and secrecy is the top concern when discussing and publishing our results. participants’ private information is kept private, protecting their right to privacy and following the protocol according to ethical guidelines. results and discussions analysis of survival curve analysis the study used a chi-square homogeneity test to find the difference between the survival rate of patients living in riyadh and those residing outside riyadh. the relation between these variables was non-significant 1, x2 (1, n = 125) = 0.97, p=0.32, and the survival curve showed no difference in overall survival. nonetheless, there was a difference in survival between seminoma and nsgt; the test was marginally significant at x2 (1, n = 125) = 3.14, p=0.07. moreover, the overall survival between patients with undescended and normal testis showed no difference, and the test was not significant x2 (1, n = 125) = 0.25, p=0.6. finally, survival rate by stage presentation showed no difference in the overall testicular cancer stage groups 1a-2c presentation except for group 3, which had the worst prognosis, and the test was marginally significant x2 (6, n = 125) = 11.68, p=0.06. patients characteristics the present research included a total of 125 patients who were diagnosed and treated at king faisal specialist hospital & research center (kfshrc) between 20102017. all these patients suffered from two different pathologies, including non-seminomatous germ cell tumors (nsgct) and seminomatous germ cell tumors. patient characteristics are shown in the figures below. the median age was 33 (range: 28–40 yr). figure 1 shows that the highest ratio of patients affected by testicular cancer lies in the age bracket of 26-35 years. one reason behind this could be the presence of seminomatous germ cell tumors, which commonly affect men in their thirties and above. the second highest number of patients noted was 0-25 years old; these patients are more likely to be affected by non-seminomatous germ cell tumors. 9.60% of patients were 46-55, while patients in their late fifties and early sixties constituted only 4%. patients in their late sixties were countable as they accounted for less than 1% of the whole selected population. pa ge 59 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 57-63, 2024 figure 1: age ratio of patients affected by testicular cancer. types of pathologies seventy-three out of a total of 125 patients were affected by non-seminomatous germ cell tumors (nsgct). of those 73 patients, nearly 54 were affected by mixed germ cell tumors. it was also seen that almost 52 recorded cases were affected by seminomas. the line graph in figure 3 shows the survival probability of patients affected by the non-seminomatous germ cell tumor (nsgct) and seminomatous germ cell tumor. seminoma survival rates are higher than other testicular germ cell tumor forms, as indicated by the curve representing this tumor type. the high survival rate for seminomas is because, compared to other forms of testicular germ cell tumors, seminomas usually show less aggressive behavior. the survival probability declines with time, as seen by the curves for each of the three groups. the decline in survival probability is because some cancer patients still pass away after receiving treatment. the degree of overall survival decreases over time. figure 2 shows an examination of survival curves reveals a noteworthy distinction in overall survival between seminoma and other germ cell cancers. demographic characteristics figure 2: survival curves between seminoma and other germ cell cancers. analysis of patients’ demographic data showed that nearly 50% of included patients were residents of riyadh. almost 12% belonged to the northern province, while nearly 10% of the cases were reported from the eastern province. jawf and tabuk showed the lowest cases, i.e., only 0.8%. figure 4 depicts a difference in survival between the two groups: the group living in riyadh has a higher probability of survival than those living in other regions. this high survival probability could be due to several factors, such as healthcare access or lifestyle differences. the likelihood of survival is high in the first few years following surgery, but it then starts to decline. according to the survival curve study, figure 3 shows no overall survival difference between riyadh and other places. most patients, i.e., 29.6% included patients, were diagnosed and treated at stage 1a, and 8.8% were diagnosed at stage. at stage 3, the worst progression was seen, and nearly 25.6% patients of total patients suffered through it. this worst progression can be due to the aggressive nature of non-seminomatous germ cell tumors (nsgct). at the end of the study, 6(4.8%) patients had recurrence, 4(3.2%) passed away due to disease, and 115(92%) were in remission. risk factorcryptorchidism in the case of patients with testicular cancer, undescended testis appeared to be a prominent risk factor. the graph in figure 5 shows that the probability of survival is lower for people who have been affected by an undescended testis condition than for people who have not been exposed to any such risk factor. patients with any history of undescended testis are likely to develop testicular cancer. figure 5 shows no discernible difference in overall pa ge 60 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 57-63, 2024 figure 3: survival difference between riyadh and other places figure 4: stage presentation of tumor figure 5: survival curve analysis shows no significant difference in overall survival between patients with descending testicles and those without. survival between patients with descending testicles and those without, according to survival curve analysis. discussion testicular cancer is a malignant tumor associated with the testicles, which are the primary components of the male genitals. testicles are primarily responsible for the production of sperm; they also regulate hormonal balance as they are responsible for the release of testosterone. testicular cancer usually occurs between the ages of 15 and 35. testicular cancer is common in young men, and it can be easily diagnosed by self-examination of the testicles and the tumor can be cured by surgery if diagnosed early (altunkurek, 2020). the degree of incidence of testicular cancer highly depends upon factors such as demographic locations, age, multiple types of pathology, and congenital disabilities such as cryptorchidism, which act as major risk factors (yazici et al., 2023). the reported cases of testicular cancers globally are approximately 78400, making it the 20th most prevalent cancer (safiri et al., 2023). testicular cancer showed an interesting trend in terms of demographic locations; it was seen that more than 62 nations worldwide were affected (bray et al., 2013), with the highest rate in west, north, and south europe, oceania, and north america. the continents that are less developed, such as asia and africa, showed the lowest rate (giona, 2022). the rate of incidence of testicular cancer in saudi arabia has been accelerated in the last decades (abomelha, 2017). as per the saudi cancer registry (scr), in 2017, the total reported cases reached the figure of 8850 new cases, and the death toll reached an approximate number of 410 (baird et al., 2018). in europe, the survival rate shown by testicular cancer is 95% per person, which to date is the highest recorded survival rate for any cancer occurring in men (znaor et al., 2020). there are many risk factors contributing to testicular cancer in men, such as smoking, undescended testis, family history, and infertility. the major risk posing factor is cryptorchidism, or the condition of having an undescended testicle. patients with this condition are more likely to get testicular cancer compared to others (ergül et al., 2024). patients having genetic conditions such as klinefelter syndrome can raise a person’s chance pa ge 61 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 57-63, 2024 of acquiring testicular cancer (swerdlow et al., 2006). in case of genetic mutation such as those in the kitlg gene also increases the risk of developing testicular cancer (pyle et al., 2024). it can relapse in men who have been affected previously by testicular cancer in either testicle. the study has shown that the age bracket mostly affected by testicular cancer ranges from 15-35, so men at this age are most prone (franco et al., 2023). one of the main reasons why european and american nations have shown the highest rate of incidence for testicular cancer is the fact that white men are more prone to this cancer compared to other ethnicities (ghazarian et al., 2015). environmental factors also play a role in the prevalence of testicular cancer. studies have shown that exposure to toxins such as endocrine-disrupting chemicals (edcs) during pregnancy and the early stages of life increases the risk factor of acquiring testicular cancer (cannarella et al., 2023). seminomas and nonseminomas (nsgcts) are the primary germ cell tumors, accounting for most testicular malignancies. because nsgcts grow more quickly and are generally more aggressive, postoperative care is frequently needed (siddiqui et al., 2020). seminomas may not require further procedures since they grow more slowly and are less aggressive (bray et al., 2006). to diagnose and treat both, the first step is usually orchiectomy (testicle removal). orchiectomy is a surgical method and is known as the gold standard for treating testicular cancer. post-orchiectomy treatment plans are tailored for the specific stage of the tumor identified after surgery (stephenson et al., 2019). our study assessed the survival rate regarding disease recurrence, pathology type, patients with risk factors, and stage presentation between patients living in riyadh, treated at kfshrc, and patients from different kingdom regions. we retrospectively went through our patient’s records from 2010-2017. subsequently, our follow-up with the patients post-orchiectomy included radiological images and tumor markers (chavarriaga et al., 2023). the study showed that the frequency of incidence of testicular cancer showed a significant relation with the age of the patients, as most of the studied patients were in the age group of 26-35 years. our retrospective cohort study found no statistically significant variations in overall survival between patients residing in and outside of riyadh, indicating that geographic location may not be a relevant factor in the prognosis of seminoma and nonseminoma germ cell tumors (gcts). furthermore, the assessed risk factors affected neither the seminoma nor the nsgct groups’ overall survival. the results revealed that patients with pure seminomas had improved overall survival rates than those with pure nsgcts, which suggests that nonseminoma variations were naturally more aggressive (fero et al., 2021). stage 3 presentations showed noticeably worse survival rates, underscoring the significance of early discovery and care, while stage 1a–2c gcts showed similar survival outcomes (abdul-muhsin et al., 2021). one of the main reasons no significant difference was seen in patients living inside riyadh and receiving the best treatment at king faisal specialist hospital & research center (kfshrc) and outside riyadh is that patients across the kingdom were able to receive timely diagnosis and treatment. the lack of correlation between the risk factors under study and survival highlights the need for more research into more comprehensive risk profiles that affect the progression of tumors. another explanation is that risk factors mentioned in the study are not associated with a worse prognosis. therefore, neither the treatment regimen nor the follow-up should be changed for patients with testicular cancer and for patients who have risk factors. furthermore, our findings converge with previous studies showing that seminoma had better survival overall than nsgct. at the end of the study, out of 125 patients, 115(92%) were in remission, 4 passed away and 6 had recurrence. these findings show that testicular cancer has a good prognosis if treated properly (cassell et al., 2020). our finding that pure seminomas have a higher survival rate than nsgcts corresponds with previous studies and highlights the intrinsic aggressiveness of nonseminoma variations. this knowledge can inform strategies for targeted treatment and risk stratification. in our tumors staging sub-group, it was seen that group 2c had 4 patients only, making the results significant. although our study was conducted at kfshrc, we did not limit ourselves to the samples of patients treated at kfshrc only; we included patients from other tertiary hospitals across the kingdom. the number of testicular cancer patients in the kingdom has been rising recently; during our study, we analyzed 125 cases from 2010 to 2017. even though our sample size is small compared to the entire population, it can still serve as a starting point for more extensive research that can be carried out soon. as per our best knowledge and extensive research, we can surely state that there is not enough research conducted in saudi arabia on testicular cancer. we selected king faisal specialist hospital & research center (kfshrc) as our referral hospital because it is the primary national referral cancer center, receiving about 22% of all cancer cases nationwide. our results provide new insight into the kfshrc’s strategy regarding the pattern of tumor progression and treatment outcomes for patients with testicular cancer. furthermore, this will contribute to the body of knowledge regarding the management and surveillance of testicular cancer, namely gcts, in saudi arabia. future directions despite the sample size constraints, the study establishes the foundation for additional research on testicular cancer in saudi arabia. we must evaluate risk profiles more extensively, as the study indicates that some risk factors and survival are unrelated. understanding the intrinsic aggressiveness of nonseminoma variants should be useful in developing strategies for focused treatment and risk classification. research on tumor progression and pa ge 62 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 57-63, 2024 treatment outcomes is essential as the number of instances of testicular cancer in the kingdom rises. the correlations between various parameters reviewed in this retrospective provide light on trends and results at a national referral center, which helps with the care and surveillance of testicular cancer in saudi arabia, particularly germ cell tumors. additional comprehensive study is necessary to enhance comprehension and direct advancements in diagnosis, therapy, and patient management. conclusion this retrospective research challenges geographical assumptions regarding testicular cancer in riyadh and surrounding areas. no significant differences in stage were found between riyadh and residents living outside riyadh. it also revealed that factors such as genetics, smoking, and family history did not affect survival in seminoma or nonseminoma germ cell tumors. seminoma showed a higher overall survival, indicating differences in treatment response. it is important to detect testicular cancer early, as stage 3 presentations correlated with the lowest survival. although this study was conducted in riyadh, future research should involve diverse tertiary care facilities nationwide. finally, this study challenges geographical assumptions and opens avenues 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(2006). cancer incidence and mortality in men with klinefelter syndrome: a cohort study. the journal of urology, 175(4), 1364-1365. wang, s.-c., chang, n.-w., chen, w.-j., yang, m.h., chen, s.-l., & sung, w.-w. (2021). trends of testicular cancer mortality-to-incidence ratios in relation to health expenditure: an ecological study of 54 countries. international journal of environmental research and public health, 18(4), 1546. yazici, s., del biondo, d., napodano, g., grillo, m., calace, f. p., prezioso, d., crocetto, f., & barone, b. (2023). risk factors for testicular cancer: environment, genes and infections—is it all? medicina, 59(4), 724. znaor, a., skakkebæk, n. e., rajpert de meyts, e., laversanne, m., kuliš, t., gurney, j., sarfati, d., mcglynn, k. a., & bray, f. (2020). testicular cancer incidence predictions in europe 2010–2035: a rising burden despite population ageing. international journal of cancer, 147(3), 820-828. pa ge 1 pa ge 1 american journal of life science and innovation (ajlsi) effects of elevated co2 and light intensity on growth, yield, and nutritional quality of tomato (solanum lycopersicumsolanum lycopersicum) in controlled environment agriculture systems a. a. y. amarasinghe1*, k. p. g. d. m. polwaththa1, d. m. suratissa2 volume 4 issue 1, year 2025 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v4i1.4353 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: january 01, 2025 accepted: february 04, 2025 published: february 10, 2025 controlled environment agriculture (cea) is an improved cultivation system that promotes plant development by optimizing environmental factors such as carbon dioxide (co2) concentration and light intensity. this study considered the interactive effects of enhanced levels of co2 (400-1000 ppm) and varying light intensities (200-600 µmol/m2/s) on growth characters, yield, and nutritional composition of tomato (solanum lycopersicum). under hydroponics conditions, a factorial experiment was conducted in a climate-controlled greenhouse and plant physiological responses, productivity, and fruit quality were assessed. higher level of co2 and light significantly increased plant height, leaf area, chlorophyll content, photosynthetic rate, and yield. the highest yield (6.86 kg/plant) was recorded at an 800 ppm co2 and 600 µmol/m2/s light intensity regime, while peak values for fruit weight and dry matter concentration impetuous at 1000 ppm co2. nutrition content of fruit showed varied response for co2-light interaction. with increased co2 and light intensity, the highest lycopene content attained was 12.69 mg/100 g at 1000 ppm co2 and 600 µmol/ m2/s light. conversely, increasing co2 concentrations push vitamin c and protein content lower, probably due to nutrient dilution effects driven by biomass growth. these results highlight the need to pair co2 enrichment with light supplementation in optimizing the productivity-nutrition quality balance. an optimal combination of co2 and light intensity on the range of 800-1000 ppm co2 and 600 µmol/m2/s offers useful understandings for greenhouse and vertical farming systems. future studies must investigate long term effects on post-harvest quality and economic feasibility to keep improving co2-light management strategies in cea systems. keywords co2 enrichment, controlled environmental agriculture, light intensity, nutritional quality, tomato yield 1 department of export agriculture, faculty of agricultural sciences, sabaragamuwa university of sri lanka, sri lanka 2 faculty of science, university of colombo, sri lanka * corresponding author’s e-mail: rathna.agric2@gmail.com introduction controlled environment agriculture (cea) consists of fast-developing technologies that allow the maximization of plant growth by conscientiously manipulating environmental influences such as temperature, humidity, light intensity, and carbon dioxide concentration. artificial intelligence, internet of things, and robotics are being integrated with cea in precision agriculture as scalable solutions for global food security and for sustainable agricultural methods (polwaththa et al., 2024). medium temperature (21-250c) available in the most growing areas are favorable for tomato growth and flowering. however, the production of better yield and yield traits require comparatively low temperature (10-200c) during fruit setting in some areas (nur et al., 2022). among all these factors, co2 enrichment and light supplementation take center stage in the improvement of plant productivity and resource-use efficiency. the cultivation of tomato (solanum lycopersicum) in controlled environments has gained prominence due to its economic importance and sensitivity to environmental factors (wang et al., 2021). however, while co2 enrichment proves beneficial in photosynthesis and biomass accumulation, its combined effect along with light intensity on yield and the nutritional quality of fruits remains questionable and one big area of research (zhao et al., 2022). the interaction of these two factors is of great importance for optimizing cea strategies to achieve high yield and high-quality production. higher levels of co2 have been studied for their effects on photosynthesis, stomatal conductance, and carbon allocation in plants. previous studies showed that co2 concentrations between 600-1000 ppm can hence promote 20-40% growth and fruit yield in tomatoes by enhancing the efficiency of carbon fixation and reducing photorespiration (jin et al., 2023). conversely, while higher co2 induces increased fruit biomass, it may modify the compositions of essential elements due to the nutrient dilution effect in which higher carbohydrate accumulation translates into lower concentrations of minerals and proteins (taub et al., 2008). in addition to co2, light intensity plays a big part in tomato yield and fruit quality. the photosynthetic efficiency and biomass accumulation dearly depend on the photosynthetically active radiation (par) available. studies are showing light intensity of about 400-600 µmol m²/s significantly enhanced lycopene biosynthesis, vitamin c concentration, and sugar accumulation in tomatoes (hao et al., 2021). also, higher light intensity might counteract some undesirable effects of co2 enrichment by enhancing the production of secondary metabolites and curtailing the nutrient dilution (zhang et al., 2023). while there has been increasing interest in ceabased co2 enrichment and light management, few researches have been conducted into their interaction pa ge 2 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 25-32, 2024 affecting growth and nutrient composition of the crop, particularly tomato (polwaththa & amarasinghe, 2024). thus, this study intends to address this knowledge gap through assessing the interactive effects of elevated co2 and light intensity on tomato plant physiology, yield performance, and fruit nutritional composition in order to provide understandings to greenhouse operators or vertical farm managers trying to optimize co2 and light supplementation strategies in sustainable tomato production. to achieve this, the study was conducted to study how the interaction between elevated co2 concentrations ranging from 400 to 1000 ppm and light intensities of 200 to 600 µmol/m²/s affect tomato growth, yield, and fruit nutritional composition. materials and methods pilot study a preliminary pilot experiment was performed in sri lanka to identify the range of most appropriate co2 and light intensity for the growth of tomatoes in advance of the main experiment. in this pilot trial, five co2 concentrations, 200, 600, 1000, 1400, and 1800 ppm, and four light intensities, 200, 400, 600 and 800 µmol/ m2/s, were tested in controlled conditions to investigate plant growth responses, physiological parameters, and symptoms of possible stress. under these conditions, the plants grew for six weeks, recording plant height, leaf area, chlorophyll content, photosynthesis rate, and stomatal conductance. it has been concluded from this experiment that levels above 1000 ppm of co2 do not contribute much to increasing photosynthesis, whereas at 1400 and 1800 ppm, some indications of stomatal closure and hence low transpiration use efficiency were evident. while higher light intensities of 600 µmol/ m²/s increased photosynthetic efficiency, excessive co2 beyond 1000 ppm, under such a light level, resulted in chlorophyll degradation and reduced the stomatal conductance, indicating possible co2 saturation effects. based on the results from the pilot experiment, co2 levels from 400 ppm up to 1000 ppm and light intensities from 200 µmol/m2/s up to 600 µmol/m2/s were chosen to narrow down the ranges for application in the main experiment, hence ensuring optimum physiological performance without causing stress to the tomato plants. main experiment experimental design the effects of elevated co2 and different light intensities upon growth, yield, and nutritional composition of the tomato (solanum lycopersicum) in a controlled environment agriculture (cea) system were examined in this study. since photosynthesis occurs under the direct influence of both co2 and light intensity, the effects of their interaction were focused on rather than individual responses. therefore, a randomized complete block design (rcbd) in a factorial arrangement was carried out to express interaction effects of co2 and light intensity. the treatments consisted of four levels of co2 concentration (400 ppm, 600 ppm, 800 ppm, and 1000 ppm) and three levels of light intensity (200 µmol/ m2/s, 400 µmol/m2/s, and 600 µmol/m2/s). each design treatment was instantiated in this experiment for 10 replications thus making 120 experimental units. each treatment consisted of an individual tomato plant grown in well-controlled environments. greenhouse setup and environmental control the setup was carried out in a climate-controlled greenhouse containing an automated co2 enrichment system and full-spectrum led lighting to achieve the targeted levels of co2 and light. a hydroponic cultivation method was used to ensure a steady supply of nutrients for all experimental units. infrared gas analysts (irga) monitored and regulated co2 concentrations every 2 hours to maintain continuous levels of co2, which were delivered by the automated diffusion system integrated into the greenhouse ventilation. the intensity of the light was controlled through programmable led lamps, which were programmed to provide 16 hours of light and 8 hours of darkness a day. quantum light sensors placed at the canopy level ensured that the target par intensity was met for each treatment. temperature and humidity were controlled by employing air conditioning and automated misting systems, keeping the range of 25 ± 20c and 65% ± 5% relative humidity. these environmental parameters mostly eliminated possible external environmental variations, permitting the differences in plant performances to be directly attributed to co2 and light intensity treatments. plant materials and growing conditions a hybrid tomato cultivar was selected for the present study because of its high yield potential, long fruiting period, and response to the modification of environmental conditions. seeds were sown into peat-based seedlings trays under controlled conditions and were transplanted 21 days later into hydroponic system integrated with fertigation for supplying a nutrient solution at constant intervals. the nutrient solution supplied to the plants contained macronutrients like nitrogen, phosphorus, and potassium and essential micronutrients like magnesium, calcium, and iron. ec of 2.5 ms/cm and ph values ranging from 5.8 to 6.2 were maintained in the solution to encourage maximum uptake of nutrients. data collection and measurements growth and physiological parameters to evaluate the effect of co2 and light intensity on plant growth, morphological and physiological traits were recorded at 30, 60, and 90 days after transplanting (dat). plant height (cm) was noted using measuring tape while leaf area (cm2) was determined using an automated leaf area meter. the chlorophyll content was measured using the spad chlorophyll meter, which gives a relative index of chlorophyll concentration. photosynthetic rate (mol h2o/m2/s) was measured with pa ge 3 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 25-32, 2024 a handheld photosynthesis system (li-6400xt, li-cor biosciences) by measuring net co2 assimilation under a standardized set of conditions. stomatal conductance (mol h2o/m2/s) was measured with the same instrument to determine the plant’s ability to limit water loss under different co2 levels. yield and productivity measurements after harvest (90 dat), total yield per plant (kg/plant) was recorded by weighing all fruits from each plant using a precision digital scale. individual fruit weights (g) were determined by finding the average of ten randomly weighed fruits per plant. dry matter content (%) was determined by drying an aliquot of tomato fruit at 700c for 48 hours and measuring the dry-to-fresh weight ratio. a digital refractometer (atago pal-1; japan) was used to ascertain the sugar content (0brix) by measuring the soluble solids concentration of fruit juices. these yieldrelated measurements gave understanding as to the impact of co2 and light intensity on total tomato production. nutritional composition analysis in the present study, the quality of tomatoes was assessed in terms of vitamin c, lycopene, and protein contents by different determination methods. vitamin c content was assayed by hplc using a c18 column. the lycopene content was assayed by spectrophotometry at 502 nm after extraction with hexane and acetone. protein content was analyzed by means of the kjeldahl method, which quantifies total nitrogen as an indicator of protein concentration. all these biochemical analyses were done in triplicate to ensure accuracy and repeatability. these nutritional analyses gave enough evidence on how enrichment with co2 and light intensity could affect the health-related properties of the tomatoes. statistical analysis data were analyzed using two-way analysis of variance (anova) in sas with the objective of explaining the interaction effects of co2 enrichment and light intensity on growth, yield, and nutritional value of tomato. in the present study, the factorial anova model was examined considering the interaction between the four co2 levels of 400, 600, 800, and 1000 ppm and three light intensities, i.e., 200, 400, and 600 µmol/m2/s. duncan’s multiple range test (dmrt) was applied at p ≤ 0.05 to differentiate the means for statistical differences in treatments. thus, it has been utilized to compare various treatment combinations in terms of identifying the optimal levels of co2 and light that would most enhance the growth of tomato and fruit quality. there were 10 replicates for each combination of treatments in order to achieve enough statistical power and ensure reliability of the outcome. results and discussion growth and physiological responses table 1: growth and physiological responses under different co2 and light levels 1 light intensity (µmol/ m²/s) plant height (cm) leaf area (cm2) chlorophyll content (spad index) photosynthetic rate (µmol co2/m²/s) stomatal conductance (mol h2o/ m²/s) 400 200 101.39 ± 13.07 g1 275.52 ± 51.92 g 44.26 ± 4.79 f 10.64 ± 1.87 f 0.30 ± 0.11 a 400 400 112.38 ± 11.78 f 293.98 ± 46.92 g 47.85 ± 3.98 ef 16.73 ± 2.31 e 0.21 ± 0.11 abc 400 600 121.69 ± 11.01 f 343.67 ± 49.20 f 50.35 ± 4.54 e 18.87 ± 1.87 d 0.14 ± 0.09 cd 600 200 116.45 ± 13.22 f 297.93 ± 38.72 g 49.97 ± 4.51 e 16.08 ± 1.49 e 0.26 ± 0.14 ab 600 400 137.03 ± 11.98 e 387.87 ± 26.68 de 54.53 ± 3.80 d 19.32 ± 2.64 d 0.15 ± 0.15 bcd 600 600 141.49 ± 10.67 de 370.13 ± 41.58 ef 56.47 ± 4.94 d 22.07 ± 2.17 bc 0.23 ± 0.06 abc 800 200 141.19 ± 13.30 de 364.51 ± 42.06 ef 55.98 ± 4.19 d 16.67 ± 2.29 e 0.23 ± 0.06 abc 800 400 150.03 ± 9.43 d 417.79 ± 40.98 cd 57.26 ± 5.13 cd 20.40 ± 2.32 cd 0.13 ± 0.12 cd 800 600 164.59 ± 13.33 c 431.45 ± 36.08 bc 63.67 ± 3.27 ab 26.52 ± 2.11 a 0.08 ± 0.15 d 1000 200 171.96 ± 10.38 bc 442.45 ± 53.71 bc 60.65 ± 5.67 bc 20.58 ± 1.93 cd 0.14 ± 0.11 cd 1000 400 179.89 ± 13.04 b 461.30 ± 37.08 b 63.78 ± 3.10 ab 23.30 ± 2.19 b 0.05 ± 0.14 d 1000 600 197.99 ± 11.96 a 488.52 ± 44.66 a 67.04 ± 5.11 a 26.22 ± 2.47 a 0.13 ± 0.10 cd means followed by the same small letters in the same column are not significantly different at 5% level in duncan’s multiple range test. the results in table 1 shows that high co2 concentration and higher light intensity had a significant influence on plant height and leaf area. tomato plants cultivated at 1000 ppm co2 and 600 µmol/m2/s light intensity yielded the highest plant height (197.99 ± 11.96 cm) and leaf area (488.52 ± 44.66 cm2), whereas the lowest was at 400 ppm co2 and 200 µmol/m2/s (101.39 ± 13.07 cm, 275.52 ± 51.92 cm2). the increased plant height development and leaf growth can be attributed to increased cellular enlargement and biomass accumulation, as high co2 enhances the carbon input into photosynthesis, thus pa ge 4 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 25-32, 2024 enhancing plant development (zhang et al., 2023). additionally, the increased light intensity also enhances more carbon assimilation and light energy uptake, thus supporting more leaf growth and expansion. chlorophyll content also increased with rising co2 and light, maximally 67.04 ± 5.11 spad index at 1000 ppm co2 at 600 µmol/m2/s. chlorophyll content was minimum at 400 ppm co2 at 200 µmol/m2/s light (44.26 ± 4.79 spad index). increased content of chlorophyll under increased co2 concentration results from optimization of nitrogen use efficiency, promoting chlorophyll biosynthesis (jin et al., 2023). high light intensity also triggers photoreceptors, causing expression of genes involved in chlorophyll biosynthesis, leading to increased accumulation of photosynthetic pigments (huang et al., 2021). the same has been found in spinach (spinacia oleracea), where increased co2 facilitated the preservation of chlorophyll, thereby increasing the efficiency of photosynthesis (zhao et al., 2022). excessive light in the absence of supplemental co2 at its optimal concentration induces photooxidative stress, however, which degrades chlorophyll, as seen in strawberry (fragaria ananassa) (jin et al., 2023). photosynthetic rate was substantially enhanced by co2 enrichment and increased light intensities, with the maximum of 26.52 ± 2.11 µmol co2/m2/s at 800 ppm co2 and 600 µmol/m2/s light intensity. photosynthetic activity plateaued at 1000 ppm co2 (26.22 ± 2.47 µmol co2/m2/s), indicating a co2 saturation point at which any additional increase in co2 does not augment photosynthesis. this is due to the fact that rubisco, the enzyme for carbon fixation, is saturated under high co2 levels, thus limiting further improvement in photosynthetic performance (wang et al., 2021). similar trends have also been reported in pepper (capsicum annuum), where the photosynthesis rates increased with co2 enrichment up to an optimal level, beyond which photorespiration and stomatal limitations restricted further improvement (wang et al., 2021). correspondingly, studies of wheat (triticum aestivum) have shown that co2 saturation effects reduce stomatal conductance and thereby limit co2 diffusion to the mesophyll cells (taub et al., 2008). there was a reduction in stomatal conductance with the increase in co2 levels, with the lowest values recorded at 1000 ppm co2 under 400 and 600 µmol/m2/s light intensity (0.05 ± 0.14 and 0.13 ± 0.10 mol h2o/m2/s, respectively). the reduction in stomatal conductance can be attributed to co2 induced stomatal closure, lowering guard cell turgor pressure and resulting in partial stomatal closure and reduced water loss via transpiration (zhang et al., 2023). while this can increase water-use efficiency, prolonged stomatal closure can prevent evaporative cooling, thus enhancing the likelihood of leaf overheating when high light is applied. comparable findings have been reported in cabbage (brassica oleracea) as well, where reduced stomatal conductance under high co2 helped in water conservation but at the same time increased its susceptibility to heat stress (huang et al., 2021). additionally, in soybean (glycine max), stomatal conductance reduction at high co2 levels increased water-use efficiency and thus rendered the plants droughttolerant (zhao et al., 2022). yield and productivity responses the data indicated (table 2) that co2 concentration of higher levels and greater light intensity increased tomato yields. the highest yield (6.86 kg/plant) was achieved at 800 ppm co2 and 600 µmol/m2/s light intensity, while the lowest (4.59 kg/plant) was recorded at 400 ppm co2 and 200 µmol/m2/s light intensity. the enhanced production at elevated co2 can be due to increased photosynthesis and carbon assimilation, as usually reported in previous research on tomato and other crops (hao et al., 2021; jin et al., 2023). the similar results were obtained in wheat and rice, as elevated co2 concentration enhanced biomass accumulation and grain yield (zhu et al., 2020). at 1000 ppm co2 concentration, yield did not rise above the 800 ppm level, and this could indicate a saturation effect. some studies show that at very high levels of co2 concentration, there can be a process of photosynthetic down-regulation with declining returns in yield increase (ainsworth & rogers, 2007). furthermore, although the co2 concentration is increased but with reduced light, photosynthetic efficiency could not be optimized (zhang et al., 2023). table 2: yield and productivity under different co2 and light levels co2 level (ppm) light intensity (µmol/m²/s) yield (kg/ plant) fruit weight (g) dry matter (% fw) sugar content (obrix) 400 200 4.59 ± 0.87 c1 136.59 ± 12.80 e 7.41 ± 1.72 f 6.08 ± 0.95 g 400 400 5.30 ± 1.27 bc 148.03 ± 13.03 e 9.27 ± 1.65 de 6.69 ± 0.71 fg 400 600 6.01 ± 1.50 ab 155.26 ± 7.64 d 10.18 ± 1.32 d 8.49 ± 1.04 cd 600 200 5.68 ± 1.35 abc 155.68 ± 12.59 d 8.21 ± 1.40 ef 6.71 ± 1.00 fg 600 400 5.46 ± 1.14 bc 158.23 ± 11.73 cd 9.33 ± 1.24 de 7.71 ± 0.84 de 600 600 5.96 ± 1.11 ab 159.20 ± 10.19 cd 10.33 ± 1.06 cd 9.03 ± 0.95 bc 800 200 6.30 ± 0.82 ab 166.80 ± 8.43 bc 9.93 ± 1.41 d 7.12 ± 0.95 ef 800 400 5.74 ± 1.21 abc 167.97 ± 12.80 bc 10.42 ± 1.56 cd 7.89 ± 0.80 de 800 600 6.86 ± 1.21 a 166.65 ± 7.28 bc 12.47 ± 1.69 ab 9.97 ± 0.35 a pa ge 5 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 25-32, 2024 1000 200 6.75 ± 1.24 a 174.30 ± 12.01 ab 11.65 ± 1.28 bc 7.78 ± 0.97 de 1000 400 6.80 ± 1.19 a 179.29 ± 12.65 a 12.28 ± 1.38 ab 8.88 ± 0.94 c 1000 600 6.69 ± 1.23 a 182.47 ± 11.31 a 13.40 ± 1.42 a 9.80 ± 0.86 ab 1 means followed by the same small letters in the same column are not significantly different at 5% level in duncan’s multiple range test, fwfresh weight. both co2 and light intensity had a very significant impact on fruit weight, with the maximum fruit weight (182.47 g) at 1000 ppm co2 and 600 µmol/m2/s light intensity and minimum (136.59 g) at 400 ppm co2 and 200 µmol/ m2/s light intensity. this is consistent with the work of taub et al. (2008), who noted that co2 enrichment maximizes fruit size in crops because of the enhanced accumulation of carbohydrates. dry matter content also followed the same pattern, with the highest (13.40% fw) being achieved at 1000 ppm co2 and light intensity of 600 µmol/m2/s. this indicates that high levels of co2 not only increased the fruit size but also allowed more dry matter to accumulate, which could be the consequence of greater storage of carbohydrates in the fruit tissues. the same was observed in strawberries and bell peppers wherein the firmness of fruit and dry matter was enhanced by co2 enrichment (wang et al., 2021). sugar content (0brix) was significantly influenced by the two factors of co2 and light intensity; the highest value, 9.970 brix, was recorded under the conditions of 800 ppm co2 combined with 600 µmol/m2/s. this result agreed with other works in proving that high co2 increases the sugar accumulation in tomato fruits through the increased fixation of carbon and synthesis of carbohydrates (li et al., 2019). however, sugar content at 1000 ppm co2 did not keep increasing, perhaps due to the co2induced dilution effect in which too much carbohydrate accumulation may result in a relative decrease in soluble sugar concentration. similar reports were given on lettuce and cucumber where sugar contents increased with co2 enrichment but did not keep increasing at very high levels (zhao et al., 2022). meanwhile, light intensity significantly affected sugar accumulation: fruits developed under a higher light level-600 µmol/m2/s-had significantly higher 0brix values than fruits under low light. this agreed with reports that enhanced light intensity increases the activity of the enzymes in sugar metabolism, hence improving the sweet taste in fruit (hao et al., 2021). the findings indicate that co2 enrichment and light intensity significantly impact tomato yield and productivity and that the optimal combination is 800 -1000 ppm co2 and 600 µmol/m2/s light intensity. the results agree with findings in controlled environment agriculture (cea) systems where the same co2 and light optimization led to enhanced crop productivity and improved fruit quality (zhang et al., 2023). however, supra-optimal co2 concentrations in excess of 1000 ppm did not generate further yield and quality enhancements and suggests that cea systems have to strike a balance between co2 enrichment and the optimal amount of light concentration. nevertheless, co2-induced carbohydrate storage also has to be closely regulated to avoid deleterious effects of nutrient dilution. these findings have excellent applicability to greenhouse and vertical farm operations since they demonstrate that co2 enrichment and supplemental light optimization can maximize tomato productivity and quality in closed systems. the long-term sustainable effects of co2 and light optimization on post-harvest quality and profitability of tomatoes in commercial cea production systems can be further investigated. nutritional composition and fruit quality table 3: nutritional composition and fruit quality under different co2 and light levels co2 level (ppm) light intensity (µmol/m²/s) vitamin c (mg/ 100g) lycopene (mg/ 100g) protein content (% fw) 400 200 19.50 ± 2.42 abc1 8.51 ± 2.53 cd 1.13 ± 0.20 ef 400 400 20.72 ± 2.95 ab 8.73 ± 1.74 cd 1.55 ± 0.16 b 400 600 21.96 ± 2.80 a 11.01 ± 2.01 abc 1.93 ± 0.21 a 600 200 19.58 ± 3.01 ab 8.38 ± 2.79 d 1.00 ± 0.22 f 600 400 19.97 ± 2.63 ab 9.60 ± 2.07 bcd 1.33 ± 0.22 cd 600 600 20.65 ± 2.94 ab 9.37 ± 1.94 bcd 1.63 ± 0.14 b 800 200 19.32 ± 2.31 abc 9.06 ± 2.26 bcd 0.73 ± 0.23 g 800 400 18.61 ± 2.70 bcd 9.54 ± 2.03 bcd 1.25 ± 0.18 de 800 600 19.27 ± 3.14 abc 11.53 ± 2.46 ab 1.48 ± 0.25 bc 1000 200 16.79 ± 2.40 cd 9.40 ± 2.63 bcd 0.55 ± 0.22 g 1000 400 16.20 ± 2.44 d 9.59 ± 2.54 bcd 1.00 ± 0.25 f 1000 600 18.36 ± 2.72 bcd 12.69 ± 1.98 a 1.31 ± 0.18cde pa ge 6 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 25-32, 2024 1 means followed by the same small letters in the same column are not significantly different at 5% level in duncan’s multiple range test, fwfresh weight. table 3 shows that the nutritional composition and fruit quality of tomato with elevated co2 and light intensity. vitamin c level in tomato fruit was significantly influenced by light intensity and co2 interaction. the highest vitamin c content (21.96 ± 2.80 mg/100g) was observed at 400 ppm co2 with 600 µmol/m2/s light intensity, and the lowest (16.20 ± 2.44 mg/100g) was observed at 1000 ppm co2 with 400 µmol/m2/s light intensity. vitamin c or ascorbic acid is significantly impacted by light intensity since it takes part in antioxidant defense and photoprotective responses in plants (shivashankara et al., 2013). vitamin c biosynthesis is promoted with intense light by initiating metabolic processes involved in antioxidant biosynthesis and photosynthetic carbon fixation (laxman et al., 2014). nevertheless, co2 enhancement above 600 ppm reduced the concentration of vitamin c, in agreement with previous studies on lettuce (lactuca sativa) that had increased co2 inducing reductions in ascorbic acid levels (mattson et al., 2022). similar was the trend in bell pepper (capsicum annuum), where high levels of co2 lowered the content of vitamin c even though it augmented fruit yield (dong et al., 2018). high co2 decline is due to dilution effects, which are the result of enhanced biomass gain, as well as modification in carbohydrate metabolism (rajashekar, 2018). lycopene content was also affected by co2 level and light intensity, with the highest concentration at 1000 ppm co2 with 600 µmol/m2/s light intensity at 12.69 ± 1.98 mg/100g. the lowest lycopene levels were at 400 ppm co2 with 200 µmol/m2/s light intensity at 8.51 ± 2.53 mg/100g. lycopene is a carotenoid pigment responsible for the red coloration in tomatoes and is highly dependent upon light exposure and co2 assimilation. increased light intensity enhances lycopene biosynthesis by upregulating phytoene synthase and carotenoid biosynthetic genes (verma et al., 2024). previous studies in tomatoes and strawberries (fragaria ananassa) have shown that high light intensity positively correlates with lycopene accumulation due to increased photosynthetic electron transport activity (dannehl et al., 2021). the increase in lycopene content under high co2 levels (800-1000 ppm) agrees with previous reports in cucumber (cucumis sativus) and watermelon (citrullus lanatus), where high co2 enhanced carotenoid biosynthesis through increased carbon fixation (ren et al., 2014). however, at very high co2 enrichment levels (>1000 ppm), lycopene content may be adversely affected by changes in phytoene desaturation processes (talebi et al., 2024). the protein content of tomato fruits is significantly different due to the interaction between co2 and light treatments. the highest protein content of 1.93 ± 0.21% fw was recorded at 400 ppm co2 with 600 µmol/m2/s light intensity, while the lowest, 0.55 ± 0.22% fw, was recorded under 1000 ppm co2 combined with 200 µmol/m2/s light intensity. in plants, protein biosynthesis is intimately associated with nitrogen assimilation, which shows a close interaction with co2 fertilization and light energy availability. plants under high co2 enhance carbohydrate accumulation; however, this quite frequently causes the opposite effect, a decrease in nitrogen uptake and protein synthesis, by downregulating nitrate reductase activity (loladze et al., 2019). similar reductions in protein concentration have been reported in wheat and rice grown under elevated co2, which further supports the hypothesis that co2 induced carbohydrate accumulation dilutes protein levels in plant tissues (pimenta et al., 2023). on the other hand, light intensity is one of the most important environmental factors affecting amino acid metabolism and protein synthesis. high light intensities enhance photosynthetic efficiency and nitrogen assimilation, which in turn increase protein content (pan et al., 2019). there is an interactive effect of light and co2, in that the dual increase of both factors has indeed improved biomass production, probably linked to nutrient dilution effects mainly under extreme levels of co2. conclusions this work has proved that high co2 levels and increased light intensity have a great effect on the growth, yield, and nutritional composition of tomatoes in cea systems. maximum plant height, leaf area, chlorophyll content, photosynthetic rate, and yield were observed under the optimum combination of 800-1000 ppm co2 and 600 µmol/m2/s, while beyond 1000 ppm co2 saturation resulted in diminishing returns. nutritional quality also varied; while lycopene was enhanced with increasing co2 and light, the vitamin c and protein contents declined under high co2 due to nutrient dilution effects. such findings have drawn attention to a balanced use of co2 and light to assure optimum productivity without loss of quality in the produced fruits in tomato. these findings also have practical application for both greenhouse and vertical farming systems: the optimal level of co2 ranges from 800-1000 ppm with the light intensity of 600 µmol/m2/s for their best performance. long-term effects on post-harvest quality and economic viability for commercial tomato production should be further researched. references ainsworth, e. a., & rogers, a. 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(2008). effects of elevated co2 on photosynthetic rate and stomatal conductance in wheat. global change biology, 14(3), 565-575. https://doi.org/10.1111/j.13652486.2007.01511.x verma, k., sinha, p. g., & mathur, v. (2024). temporal impact of elevated co2 and temperature on solanum lycopersicum l.: insights into growth, phenology, ultrastructure, nutritional quality, and metabolite composition. journal of crop health, 12(3), 57-69. https://doi.org/10.1007/s10343-024-01057-w wang, s., zhang, l., & chen, h. (2021). impact of co2 enrichment on nutritional composition and antioxidant properties of greenhouse-grown strawberries. scientia horticulturae, 278, 109876. zhang, l., wang, x., & li, j. (2023). co2 enrichment strategies in plant factories: effects on crop yield, fruit quality, and economic feasibility. agricultural and food chemistry, 71(5), 3217-3229. zhao, y., huang, j., & liu, p. 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(2020). rising co2 concentrations enhance biomass production but reduce grain protein content in wheat and rice. nature climate change, 10(9), 863-868. pa ge 1 pa ge 8 american journal of life science and innovation (ajlsi) examining early communication profiles in preschool children with autism spectrum disorders in dubai, u.a.e.: a comparative analysis of cognitive, language and symbolic play abilities rajeshree singhania1* volume 4 issue 1, year 2025 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v4i1.2645 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: march 13, 2024 accepted: april 20, 2024 published: february 20, 2025 effective communication skills in young children encompass a diverse range of abilities crucial for early autism spectrum disorder (asd) identification. this study aims to analyse communication skills in 36 preschool children (ages 1.6 to 4.6 years) diagnosed with autism in dubai, employing the early communication assessment (eca) tool developed by coupe o’kane and goldbart (1998). the eca evaluates 13 areas of early communicative functioning categorised into 3 to 5 developmental stages, ranging from reflexive to intentional conventional. communication abilities were assessed, and the 5 eca levels were condensed into 3 for comparative analysis. key communicative areas, such as joint referencing, social organisation, turn-taking, and imitation, were identified as significantly delayed in this cohort. conversely, affective behavior, audio-visual behaviors, and physical production of sound exhibited relatively lesser impact. contrary to common assumptions, poor eye contact was not the predominant indicator of communication challenges. comparative analyses were conducted between communication skills and perceptive cognitive abilities, language proficiency, and symbolic play. no substantial correlation was found between perceptive cognitive abilities and communication skills. however, a significant association was observed between communication abilities, language proficiency, and symbolic play. these findings underscore the complexity of early communication profiles in preschool children with asd in dubai, emphasising the need for comprehensive assessment and targeted interventions beyond conventional indicators. keywords imitation, joint referencing, language, social organisation, symbolic play, turn-taking introduction autism spectrum disorder is a common neurodevelopmental disorder which prevails from early on in life. it is characterised by difficulty in speech, communication, language, and social interactions and restricted and repetitive patterns of behaviours, interests or activities, which may vary in individuals (campisi et al., 2018). since it is a spectrum disorder, each person’s symptoms severity and manifestation will differ substantially, resulting in various clinical presentations(lord et al., 2020). over the past few decades, the prevalence of asd has increased considerably, drawing the attention of researchers, clinicians, and policymakers worldwide. just under 1% of people globally are thought to have autism, while estimates are higher in high-income nations(lord et al., 2020). there has been a discernible rise in the prevalence of asd worldwide, making it a major public health concern. numerous studies have consistently shown an increased trend in the prevalence of asd, even though variances in reported rates are influenced by cultural, environmental, and diagnostic factors (salari et al., 2022). the global prevalence of asd has increased four to five times, with a higher prevalence in boys than in girls. the average prevalence in asia, europe, and north america is 1%(durkin & wolfe, 2020). in the united states, the prevalence of asd among 8-year-old children was 1.59 in 2014 and 1.54 in 2016, while in italy, it was 1.15% (narzisi et al., 2020). in asia, it was 3.9%; in the arab countries around the gulf, it was 0.14 to 2.9% (qiu et al., 2020). as a result of increased awareness and improved diagnostic techniques, the centers for disease control and prevention (cdc) reports that new estimates in the united states indicate that roughly 1 in 44 children have received an asd diagnosis (prevention, 2024). the fact that prevalence rates differ throughout nations and areas emphasises the necessity of having an adequate understanding of the disorder’s worldwide effects. asd is characterised by difficulties in social communication, restricted behaviors, and lack of understanding and use of gestures. it can also lead to a restricted range of interests, reliance on sameness, and repetitive activities. however, the diagnostic criteria for asd have evolved, with the fifth edition of the dsm5 consolidating several distinct disorders into a single term, asd, acknowledging the diverse presentation and individual variability in symptomatology(sharma et al., 2018; tsai et al., 2020). many first-contact carers and physicians in dubai focus on these while ignoring others, such as joint referencing and turn-taking. this may delay the diagnosis of autism and thus hinder effective early intervention. early communication is transparent with overt intentions, but in children with asd, they remain opaque (fuller & kaiser, 2020). however, communication is a broad concept that involves multiple components, both lingual and para-lingual (maljaars et al., 1 singhania clinic & therapy centre dmcc, dubai, united arab emirates * corresponding author’s e-mail: rajeshris@gmail.com pa ge 9 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 8-20, 2025 2011). autism causes a reduction in the frequency and diversity of early communication forms, which results in a wide range of clinical images and challenging diagnoses, particularly in children under the age of four (bradshaw et al., 2021). for instance, not all children have poor eye contact or dislike being cuddled. communication skills include joint attention, social referencing, mutual gaze, imitation, communication intentions, turn-taking and social interaction, social organisation, communication in context, and audio-visual behaviour (chung, 2020). literature review joint attention/social referencing babies learn to make eye contact, or “look,” at their caregiver as early as two hours after birth. infants begin to display their need to share other people’s attention between the ages of 9 and 14 months, showing that they wish to join points of reference (such as the light or fan). in addition to pointing out the objects, children simultaneously glance at other individuals to see if they share their interest in the same subject. one could refer to this urge as social attention (mundy, 2023; mundy & bullen, 2022). social referencing is similar to the notion of shared attention and is defined as a process of emotional communication in which one’s perception of another individual’s interpretation is used to form one’s understanding of the event (cornew et al., 2012). four abilities (lawson et al., 2018)have been proposed, including the ability to discriminate emotional expressions on the caregiver’s face, recognise their meaning, respond to them emotionally, and finally, use them to regulate one’s behaviour. the first level is already observable in a 6-month-old baby (berger & ingersoll, 2013). imitation imitation is crucial in understanding others and is the earliest form of social-communicative interaction (berger & ingersoll, 2013) . imitation starts with adults attributing imitation to the infant’s undifferentiated behaviour, then imitating the baby, stimulating the child to copy. imitation then progresses from sporadic imitation through immediate and systematic copying to deferred imitation, which is not automatic but involves cognitive processes, and the child chooses what to copy (mcauliffe et al., 2020). a child’s decision about what to imitate and cognitive processes play a key role in the process of imitation, which is essential to language development. adults begin by assuming that a baby’s actions are imitations and move from intermittent to delayed imitation. the inability of people with asd to pay attention to and form an internal representation of behavior makes it difficult for them to mimic it. they display “parasitic” aberrant imitation, which is predictive of children with autism having poor expressive abilities (mcauliffe et al., 2020; mundy & bullen, 2022). mutual gaze it is proposed that mutual gazing is a basic type of human communication behavior that provides a foundation for other communication modes. the adult and youngster look at each other for extended periods. there is a great deal of face-to-face and eye-to-eye communication during these times. gaze coupling, a turn-taking exchange that mimics later gaze patterns observed in mature discourse, modifies the reflexive gaze (wiklund, 2023). intentional communication occurs in the setting of mutual gaze, which develops into mutual orientation or attention. the behavior consists of looking, making an interested facial expression, and positioning oneself with movement and posture. throughout early infancy, people with asd exhibit disruption of mutual gaze, regardless of their intellectual ability. one of the main areas of difficulty associated with asd is mutual gazing (rollins et al., 2021; wiklund, 2023). social organisation various organisational strategies are used for communication. these are needed to initiate, maintain, or terminate communication (baron-cohen, 1988). strategies required to initiate communication include eye contact, proximity, and vocalisation. maintaining communication needs strategies like question/answer and statement and reply. breaking off or terminating a communication entails strategies like moving away or breaking eye contact (erturk et al., 2021). skills acquired through infancy through interpersonal interactions underpin the skills needed for conversational and discourse management. individuals with asd lack strategies to organise communication. it is probably more evident in high-functioning verbal individuals capable of conversation (loukusa, 2021). turn-taking and social interaction turn-taking, seen in social interaction or dialogue, starts with the adult leaving space for children to fill their turn with sounds or actions. the adult utilises the child’s existing repertoire of behaviours by imitating them. this produces reciprocation behaviours in the child. these reciprocal behaviors are imitations of various adult and altered and novel responses. hence, the adult allows the child to take turns, but the child also gives reinforcing feedback to maintain and change adult responses (ramey et al., 1978). individuals with asd have difficulty taking turns at a very early age, both in vocal interactions and physical tasks (baren-cohen, 1988). communicative intentions intention is the speaker’s motivation for communication, often expressed through gestures and vocalisations. early communication, such as proto-imperative and protodeclarative, involves using another person to obtain something or draw attention. by 9 months, infants can express intention using gestures and vocalisations. other behaviors include gaining attention, drawing attention, and regulating social contact. communication intentions include greeting, protesting, rejecting, naming, and pa ge 10 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 8-20, 2025 informing. individuals with asd lack communication intentions due to impairment in perception and mentalising ability (schütz et al., 2020). comprehension in context autism disrupts the normal development of language, leading to delayed and deviant language development. the primary reason for learning a language is communication, which is the primary reason for communication. neonatal infants are biologically tuned to person-mediated events, responding more to speech than non-speech sounds. in the first six months, infants start to read signals and expressions of others’ behavior, gradually developing into a highly developed area of expertise. individuals with asd have difficulty in early communicative behaviors, affecting their language development, socialisation, and self-expression(elbeltagi et al., 2023). language problems asd often affect communication rather than language, with around half of all children failing to develop functional speech. spoken language ability in asd ranges from muteness to apparent facility, with a persistent impairment in communicative use and understanding of complex concepts. autism-related speech is nonproductive, echolalic, pedantic, and monotone, with limited use for sharing experiences or conversing. early communication trajectory demonstrates a relationship to language acquisition, and studies on symbolic play in children with asd vary. some children show cognitive abilities in symbolic play, while others show less interest and ability(vogindroukas et al., 2022). asd greatly impacts preschool-aged children’s communication abilities; therefore, early identification and intervention are essential. these children frequently display distinctive communication profiles, including delayed language learning, unusual language use, and trouble interacting with others on a reciprocal basis. early indications of asd usually show up in toddlerhood, when parents and other caregivers notice changes in their child’s communication style. these include a lack of interest in social relationships, restricted gesture use, delayed or absent language milestones, and issues with shared attention. this study aimed to investigate children with autism and their communication profiles. to ascertain which components of preschoolers’ communication are more likely to be impacted, the study examined various aspects of their communication. in addition to inadequate eye contact and aversion to making physical contact, other communication issues were investigated. the relationship between communication and children’s cognition, symbolic play, and language development was explored. materials and methods study participants the study included children aged from 1.6 to 4.6 years diagnosed with autism spectrum disorder (asd) at a dubai clinic between 1998 and 2008. diagnoses were made based on dsm iv criteria, considering communication, cognition, symbolic play, and language assessments. cases with incomplete assessments were excluded, resulting in the analysis of 36 children. early communication assessment (eca) communication abilities were assessed using the early communication assessment (eca) developed by coupe o’kane and goldbart (1998). the eca comprises 13 areas of early communicative functioning, which are categorised into five levels: reflexive, reactive, pre-intentional, primitive intentional, and intentional conventional levels. the 13 regions include affective communication, mutual gaze, social organization, joint reference, turn-taking and social interaction, comprehension in context, communicative intentions, cognitive roles, first meaning, imitation, auditory and visual behaviors, vocal production, and physical production. the 5 levels were condensed into 3 for comparison purposes: group 1 (not developed), group 2 (growing), and group 3 (developed). each child’s ability was assessed concerning these groups for the 13 areas. cognitive abilities perceptual cognitive abilities were evaluated using the munich functional developmental test, specifically focusing on concepts like object permanence, copying, matching, and sorting. this individually administered test assessed gross motor, fine motor, perceptual, cognitive, language expression, comprehension, social skills, and self-reliance abilities. language skills the reynell developmental language scales (reynell, 1978) were employed to measure oral expressive and receptive language skills in children aged 1 to 7. language categorised into severe delay, moderate delay, mild delay, or no delay was determined based on the difference from chronological age. symbolic play the lowe-costello play test assessed the symbolic play (lowe & costello, 1998), focusing on early concept formation symbolisation. they utilised four sets of toys in free play, which were scored according to guidelines, and the raw score was converted to play age in months. play age was categorised into severe delay, moderate delay, mild delay, or no delay based on the difference from chronological age. data analysis demographic data, symbolic playtest results, language age, non-verbal development, and communication assessments were comprehensively analysed. descriptive statistics and comparative analyses were performed to discern patterns and relationships among variables. pa ge 11 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 8-20, 2025 results and discussion the study investigated the relationship between communication, language development, and non-verbal cognitive and perceptual development in children with asd. the total number of children investigated was 36, of which 25 were boys and 11 were girls. table 1 shows the distribution of children in the study, ranging from 1.6 to 4.6 years. the largest age group had 13 children aged 1.6 to 2 years, while the smallest group contained 2-4 children aged 4.1 to 4.6 years. the study also categorises children into three main groups: asians, arabs, and europeans. the majority of the children are of asian nationality (22 children), followed by arabs (10 children) and a smaller representation of europeans (4 children). table 1: age distribution and nationality profile in the cohort age bands (in years) number of children 1.6 to 2 13 2.1 to 2.6 3 2.7 to 3 10 3.1 to 3.6 4 3.7 to 4 4 4.1 to 4.6 2 nationality profile in the cohort nationality number of children asians 22 arabs 10 european 4 table 2: the difference between chronological age and ability in symbolic play in months difference between the chronological age and the play age in months number of children severe delay a difference of more than 12 months 17 moderate delay a difference of 6 to 12 months 12 mild delay difference of less than 6 months 3 no delay 0 missing data 4 symbolic play the study used the lowe-costello play test to evaluate the pretend play abilities of children with asd. the results showed that 17 children had a play age more than 12 months behind their chronological age, indicating a significant delay in their play skills. 12 children had a play age 6-12 months behind their chronological age, showing some delay, while 3 children had a play age less than 6 months behind their chronological age, showing minimal delay. no children showed no delay, and data was unavailable for 4 children. symbolic and most delayed communication skills showed no significant difference between the delayed and not delayed groups, as shown in table 3. table 3: symbolic play and communication symbolic play most delay communication skills p value symbolic play social organisation 1.246 symbolic play joint referencing 1.000 symbolic play imitation 0.4016 symbolic play turn-taking 0.0003* symbolic skills and most delayed of communication skills showed no significant difference between the delayed and not delayed group hence were not correlated. pa ge 12 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 8-20, 2025 cognitive delay non-verbal cognitive and perceptual development was measured using the munich functional development test. the difference between the chronological and cognitive ages was calculated and tabulated. severe delay was found in 13 children, while no delay was seen in 11 children in their chronological and cognitive age, as seen in table 4. table 4: the difference between the chronological age of each child with cognitive-perceptual development in months difference between the chronological age and the cognitive age in months number of children severe delay a difference of more than 12 months 13 moderate delay a difference of 6 to 12 months 9 mild delay difference of less than 6 months 3 no delay 11 the study found no significant correlation between perceptual cognition and communication areas in children with asd. the children struggled with joint referencing, social organisation, turn-taking, and imitation. however, their development was not significantly linked to their non-verbal cognitive skills. this suggests that despite having typical cognitive abilities, they still had significant delays in specific communication areas. language delay the child’s language was assessed in their mother tongue on the reynell language development test. the difference between the chronological and language ages was calculated and tabulated. results showed that 25 children had a language age 18 months behind their chronological age, 8 had a 12-17 month delay, 2 had a 3-12 month delay, and 1 had no delay. most children had some language delay, with severe delay affecting over twothirds of them. table 6: the difference between the chronological age of each child with language age in months difference between the chronological age and the language age in months number of children severe delaya difference of more than 18 months 25 moderate delay a difference of 12 to 17 months 8 mild delay a difference of 3 to 12 months 2 no delay 1 table 5: perceptual cognition versus communication perceptual cognition most delay communication skills p value perceptual cognition social organisation 0.0913* perceptual cognition joint referencing 0.0179* perceptual cognition imitation 0.2322 perceptual cognition turn-taking 0.7795 perceptual cognition and most delayed of communication skills showed significant difference between the delayed and not delayed group hence were not correlated. table 7: language and communication language most delay communication skills p value language social organisation 0.4 language joint referencing 0.1 language imitation 0.2 language turn-taking 0.025* language skills and most delayed of communication skills showed no significant difference between the delayed and not delayed group hence were not correlated. pa ge 13 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 8-20, 2025 the figure shows that language and most delayed communication skills showed no significant difference between the delayed and the most delayed groups. analysis of communication to facilitate analysis, the levels in each communicative area were divided into three categories once each child’s abilities were determined to be one of the three to five levels: in group 1, the child was at level 1 or lower, meaning that the communication area was not developed. in group 2, the child was at levels 2 to 3 (when there were five levels in that region) and level 2 (when there were three levels in that area), indicating that the communication area was developing. in group 3, the child reached the highest level (either 3 or 5) in that communication area, indicating that the communication area had been developed. table 8: analysis of each of the 13 communication abilities on the eca in the cohort communication area not developed developing developed missing data communication in context 7 18 6 5 communicative intentions 6 14 7 8 cognitive roles 5 14 6 11 meaning 5 9 4 18 mutual gaze 6 23 1 6 social organisation 18 8 4 6 joint reference 18 9 2 7 turn-taking 13 10 7 6 imitation 15 9 4 8 affective communication 0 16 20 0 audio-visual behaviour 0 14 13 9 physical production 0 13 8 15 *a lack of information in that area in the records causes discrepancies in the numbers. figure 1: analysis of each of the 13 communication abilities on the eca in the cohort as developed, developing, or not developed the communicative areas that were most delayed or affected were joint referencing, social organisation, turntaking, and imitation, as seen in table 9. table 9: further analysis of each of the most delayed of the 13 communication abilities on the eca in the cohort communicative areas developed not developed joint referencing 2 18 social organisation 4 18 turn-taking 7 13 imitation 4 15 pa ge 14 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 8-20, 2025 the communicative areas that were least affected were affective behaviour, audio-visual behaviours, and physical production, as seen in table 10. figure 2: further analysis of each of the most delayed of the 13 communication abilities on the eca in the cohort figure 6: further analysis of each of the least delayed of the 13 communication abilities on the eca in the cohort – group c table 10: further analysis of each of the least delayed of the 13 communication abilities on the eca in the cohort – group c communicative areas not developed developing developed affective behaviour 0 16 20 audio-visual behaviour 0 14 13 physical production 0 13 8 those communicative areas that were moderately affected were – communicative intentions, communication in context, mutual gaze, and cognitive roles, as seen in table 11. table 11: further analysis of each of the moderately delayed 13 communication abilities on the eca in the cohort – group b communicative areas not developed developing developed communicative intentions 6 14 7 communication in context 7 18 6 mutual gaze 6 23 1 cognitive roles 5 14 6 physical production 0 13 8 pa ge 15 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 8-20, 2025 figure 4: further analysis of each of the moderately delayed 13 communication abilities on the eca in the cohort – group b the total number of ‘not developed’ and “developed” levels in groups a, b, and c were scrutinised and compared, as shown in table 12. the table compares the communication abilities of both parameters in three groups (a, b, c) based on eca. group a has the most affected communicative areas, group b has moderately affected areas, and group c has the least affected areas. table 12: comparison of ‘not developed’ and ‘developed’ communication abilities in groups a, b, c communication area group a (most affected) group b (moderately affected) group c (least affected) total 'not developed' levels 64 29 0 total 'developed' levels 17 24 41 comparing communication to language development and non-verbal cognitive and perceptual development communication in children with normal or mildly delayed non-verbal cognitive and perceptual development on the mft was investigated. table 13 presents a comprehensive analysis of communication levels in 12 children with normal to near normal cognitive and perceptual development categorised into three groups (a, b, c) based on their communication abilities. the results show that the most affected group has not developed communication levels, while the moderately affected group has not developed communication levels but is showing progress. the least affected group has not developed communication levels but has reached the developing stage. table 13: number of ‘developed,’ ‘developing,’ and ‘not developed’ communication abilities in group a, b, and c in the 12 children with normal to near normal cognitive perceptual ability on the mft group a communicative areas group b communicative areas group c communicative areas number of children with ‘not developed’ levels 6 3 2 number of children with ‘developing’ levels 2 5 6 number of children with ‘developed’ levels 0 0 0 table 14: comparison of ‘not developed’ and ‘developing’ communication levels in children of varying nonverbal cognitive perceptual development cognitive development communicative areas group a (no to mild delay) group b (moderate delay) group c (severe delay) 'not developed' levels group a 6 3 2 the communication in children of different non-verbal, cognitive, and perceptual development groups was accessed. there were 3 groups – the severely delayed, the moderately delayed, and mildly delayed + the no delay. table 14 compares ‘not developed’ and ‘developing’ communication levels in children with autism across various non-verbal, cognitive, and perceptual development groups. the study found that group a has the highest number of ‘not developed’ levels, while group c has a substantially less number. developing levels are more prominent in groups b and c, indicating progress in communication abilities despite cognitive delays. pa ge 16 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 8-20, 2025 group b 5 3 4 group c 6 6 5 'developing' levels group a 2 5 6 group b 2 5 4 group c 0 0 0 communication among children of different language development groups was accessed as well. there were 3 groups – the severely delayed, the moderately delayed, and mildly delayed + the no delay. table 15 compares ‘not developed’ and ‘developing’ communication levels in children with autism across different language development groups. severe language delay (group c) has the highest number of ‘not developed’ levels. groups a and b show more pronounced ‘developing’ levels, indicating progress in communication abilities despite language delays. group a, representing no to mild language delay, has no children with ‘not developed’ communication levels. table 15: comparison of ‘not developed’ and ‘developing’ communication levels in children of varying language development language development communicative areas group a (no to mild delay) group b (moderate delay) group c (severe delay) 'not developed' levels group a 0 0 0 group b 2 1 1 group c 16 10 10 'developing' levels group a 0 6 6 group b 4 4 5 group c 1 1 1 communication and language delay are related. the number of non-developers is highest in all 3 with severe language delay. group a had the highest number of nondevelopers and had no developing communicators in the severe language delay group. analysis of communication the ec categorised communication skills into three groups: underdeveloped, developing, and developed. the analysis found that the most affected communication areas were social organisation, joint referencing, turntaking, and imitation. the least affected areas were affective communication, audio-visual behavior, and physical production. moderately affected areas were communication intentions, context, mutual gaze, and cognitive roles. the study used a fisher chi-square test to assess the relationship between a child’s asd diagnosis and communication skills, as seen in figure 5. figure 5: analysis of communication p value from fishers’ chi-square test pa ge 17 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 8-20, 2025 the study results reveal that mutual gaze is a communication area where children with asd show some developmental delay but to a lesser extent than other social communication skills. children with asd show more difficulty with mutual gaze than expressing emotions or using gestures but less difficulty compared to skills like initiating shared focus or taking turns in conversation. figure 6: analysis of communicationmutual gaze vs. least and most delayed communicative skillsp-value figure 7: communication vs language, symbolic play, and perceptual cognition no significant difference was found between the developed and not-developed groups regarding the most delayed skills as they were summed up and compared to the developed and not-developed aspects of symbolic language play. however, a significant difference was found between the developed and not developed groups regarding the most delayed skills and perceptual cognition, as seen in figure 7. discussion in this analysis of a preschool cohort with asd at a dubai clinic, substantial results were gathered. boys dominated the cohort. asians were the most common race, followed by arabs and europeans. most children showed delays in language and symbolic play, with most showing significant delays, while about half demonstrated normal to mild cognitive-perceptual abilities. most cases showed marked delays in language development, and different areas of communication showed varying degrees of growth. in particular, the least developed behaviors were joint reference, social organisation, turn-taking, and imitation; in contrast, the most developed behaviors were reciprocal gaze, comprehension in context, communication aims, and cognitive roles. the two most advanced communication behaviors were found to be affective and audio-visual. crucially, language and symbolic play correlate strongly with the development of all communication components, although perceptual-cognitive capacity did not show any significant correlation. most of the pre-school children with asd who came to the clinic were boys with ages ranging from 1.6 to 2 pa ge 18 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 8-20, 2025 years and then from 2 years 7 months to 3 years. the sex ratio corresponds to that found by other studies. various studies consistently observed a male-to-female ratio of 4:1 in classical autism, a trend also reflected in relatives exhibiting a broader autistic phenotype (loomes et al., 2017; maenner, 2021). studies with expanded definitions indicated a linear variation in gender ratio with iq, approaching 2:1 in individuals with severe cognitive impairment. however, cognitively capable girls faced challenges in diagnosis due to milder social impairments compared to boys (doi et al., 2022; mcquaid et al., 2021). however, according to a study, there is no significant correlation between gender, race and severity of asd. it is reported that girls are better at masking their asd symptoms and imposing delayed diagnosis (napolitano et al., 2022). autism, though a highly heritable disorder, predisposing loci on the sex chromosomes have not been found. a genetic mechanism hypothesises that an imprinted x-linked gene(s) influences the threshold for phenotypic expression of many autistic characteristics and is thus protective (may et al., 2019). the gene is expressed only on the x-chromosome inherited from the father, raising the threshold for phenotypic expression. it is normally silenced when transmitted maternally. because only females have a paternal x chromosome, the threshold for phenotypic expression is higher in them than in males (ferri et al., 2018). the ``extreme male brain’’ theory suggests that the behaviours seen in autism are an exaggeration of typical sex differences. evidence supports female superiority in humans in the ability to read nonverbal signals, specific language-related skills, and theory of mind. prenatal and neonatal testosterone exposures impact social development and are risk factors for autism spectrum conditions (van eijk & zietsch, 2021). most of the children in the study belonged to the asian ethnicity, mirroring the demographic distribution of dubai, where asians constituted the largest group, followed by arabs and europeans/americans. however, race showed no correlation with the severity of autism. additionally, nearly half of the children exhibited normal to mild delays in cognitive perceptual ability, indicating that their non-verbal skills mostly fell within the normal range. this aligns with findings from other studies suggesting that children with autism tend to excel in tasks involving perceptual-motor organisation and rote memory skills (sodano, 2022). a study discovered a substantial connection between iq and asd, especially among people with above-average intellect. significant cognitive deficits are present in high-iq individuals with asd, especially in the areas of social cognition, verbal working memory, and visual pattern recognition (rommelse et al., 2015). in children with autistic spectrum disorder, symbolic or representational play exhibited considerable delay, with the majority experiencing severe delays. however, 50 percent of the children demonstrated some evidence of symbolic play, with three showing only mild delays. language delay was prevalent among nearly all children, with most experiencing delays ranging from 12 to 18 months. this is in line with the findings of other studies as well. a study found that children with asd have significant impairments in their spontaneous expressive language abilities, with syntax and narrative skills increasing with age. pragmatic skills improved from toddler to preschool but remained stable. longitudinal data showed significant improvements in observation of spontaneous expressive language (osel) syntax scores over time (thomas et al., 2021). the least developed aspects of communication, including joint reference, social organisation, turn-taking, and imitation (group a), were most affected by autism. conversely, affective behavior, audio-visual behavior, and sound production (group c) were the best-developed aspects. another study found that an initial imitation deficit in toddlers with asd at age 2 improved gradually until age 5. these skills were linked to cognitive and language abilities, and addressing early imitation deficits, joint reference, and social organisational skills could improve the broader competence of young children (pittet et al., 2022). these communication aspects demonstrated varying degrees of development, emphasising the heterogeneous nature of communication difficulties in children with autism. additionally, communication challenges were strongly associated with language development, with group a showing a clearer relationship to language delay than groups b and c. however, communication difficulties were not significantly related to non-verbal ability, indicating that even the most able children faced similar challenges in certain aspects of communication, particularly those in group a. another study examined language profiles in 104 children with asd and found that nonverbal cognition and joint attention significantly predicted receptive and expressive language scores, suggesting the importance of multiple assessment methods and joint attention as an intervention target (nevill et al., 2019). the study indicates that while assessing young children with asd, several aspects of early communication should be kept in mind. poor eye contact and dislike of cuddling may not be seen in all these children. the most sensitive indicators were poor joint referencing, turn-taking, social organisation, and imitation. conclusion in conclusion, the study evaluates the communication abilities of children with asd and reveals distinct patterns across different cognitive and language development levels. the study categorised children into three groups (a, b, c) with varying levels of nonverbal cognitive, perceptual, or language development. group a showed the most complications, including joint reference, social organisation, taking and imitation. communication difficulties in group a are strongly associated with language 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(2023). gaze behavior. in speech and interaction of preadolescents with autism spectrum disorder: focus on prosody, disfluencies and comprehension problems (pp. 95–115). springer. pa ge 1 pa ge 6 american journal of life science and innovation (ajlsi) antioxidant activities of selected milk teas and fruit teas using 2,2-diphenyl-1picrylhydrazyl (dpph) assay perez, jeanine therese v.1, perez, lyx jady fy n.1, padroncillo, femma joy a.1, parreño, kirk andrew v.1, policarpio, marti angela j.1, porras, alliah rose b.1, salamat, adriel amadei s.1, sararaña, rachel joy s.1, secapuri, lyle josh c.1, tacluyan, ann marie heather t.1, tosoc, daiel dwight g.1,vito, joel b. jr.1, perez, jose jr. g.1, palmos, r.j.1, salanio, r.1, leong-on, ma. socorro g.1* volume 4 issue 2, year 2025 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v4i2.5050 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: june 02, 2025 accepted: july 08, 2025 published: october 11, 2025 milk teas and fruit teas are popular as a refreshment, but their percent antioxidant properties need to be determined. in this study, the antioxidant activities of selected commercialized milk teas (matcha, okinawa, and winter melon) and fruit teas (green apple, kiwi, and lychee) were determined using the 2,2-diphenyl-1-picrylhydrazyl (dpph) assay in terms of % scavenging activity. in that assay, the plates with milk teas and fruit teas were incubated in the dark and covered with aluminum foil for 30 minutes at room temperature before being measured for absorbance at 570 nm with a microplate reader. they were kept out of the light until they were evaluated for analysis. within 30 minutes of incubation, the discoloration from purple to yellow was observed. three (3) trials with three (3) replicates were carried out with negative control (water with sugar and creamer for milk tea and water with sugar for fruit tea) and positive control (vitamin c). using one-way anova, the matcha (mean = 62.9, sd = 2.6) okinawa (mean = 46.4, sd = 5.9), and winter melon (mean = 61.6, sd = 2.8) milkteas have anti-oxidant activities when compared to the negative control (mean = 0.0, sd = 0.0) (p < .05) both matcha and winter melon have the best antioxidant activities, but not comparable to the positive control, vitamin c (mean = 98.7; sd = 0.62) (p < .05). while the green apple fruit tea (mean = 95.5; sd = 2.4) kiwi fruit tea (mean = 97.3, sd = 1.5), and lychee fruit tea (mean = 94.4; sd = 1.3) (p > .05) have comparable antioxidant activities when compared to the negative control (mean sd = 0.0). kiwi fruit tea has the best antioxidant activity and comparable to vitamin c (mean = 98.7; sd = 0.62). this study scientifically established that both matcha and winter melon commercial milk teas have antioxidant activities of 61.6 to 62.9%. while the commercial kiwi, green apple, and lychee fruit tea ranged from 94.4 to 97.3%. these antioxidant activities can be useful in preventing diseases brought by free radicals, which can be verified by conducting more studies. keywords 2, 2-diphenyl-1-picrylhydrazyl (dpph) assay, antioxidant activity, fruit teas, milk teas 1 medical laboratory science, college of pharmacy and medical technology, university of san agustin, iloilo city, philippines * corresponding author’s e-mail: usa.edu.ph@usa.edu.ph introduction the scientific community is very interested in antioxidants and their effects in a variety of sectors, including food engineering, medicine, and pharmacy (munteanu & apetrei, 2021). according to xiao et al. (2020), reactive oxygen species (ros) have the potential to harm biological macromolecules, resulting in oxidative stressrelated diseases, lipid, protein, and dna damage, as well as cell aging. an imbalance between the production and neutralization of oxidants leads to oxidative stress. oxidative stress causes a variety of illnesses and disorders, including cancer, chronic renal disease, neurological diseases, cardiovascular diseases (cvds), and chronic obstructive lung disease (sharma et al., 2022). oxidative stress could be avoided using antioxidants. the antioxidant properties of several types of tea have been recognized. as a beverage, milk teas grew in popularity (ong et al., 2021). by experimenting with different creamers, flavors, and sweeteners, it opened the door for tea lovers to enter the industry (bastasa et al., 2022). on the other hand, fruit teas gained popularity as cool alternatives. a range of fruits, herbal infusions, and occasionally even boba pearls for texture are frequently included. these beverages transcended tradition and became a phenomenon in society and culture. millennials and generation z filipinos become much more interested in milk tea (national nutrition council, 2020). matcha tea, one of the milk teas, was examined for its nutritional makeup and demonstrated antioxidant qualities (koláčková et al., 2019). high concentrations of catechins, including egcg, which have antiinflammatory and antioxidant properties, were found in matcha. there are numerous possible health advantages of matcha green tea. compared to other green tea kinds, it had higher quantities of phenolic acids, quercetin, rutin, theanine, and chlorophyll (kochman et al., 2020). additionally, general studies on matcha tea indicated that it had positive effects on anti-tumorigenesis, cardiometabolic health, and cognitive function (sokary et al., 2023). on the other hand, the primary constituents of okinawa milk tea are often tapioca pearls, black tea, milk, and kokuto, a unique okinawan brown sugar (saiful, 2022). in the zaiyar et al. study. using the dpph assay, black tea showed high antioxidant contents in 2020. using dpph, the abts test (2,2′-azino-bis 3-ethylbenzothiazoline-6pa ge 7 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 6-10, 2025 sulfonic), and frap (ferric reducing antioxidant power), stobiecka et al. (2022) found that cow milk, the most widely used type of milk worldwide, had the lowest antioxidant potential when compared to other animal milk. in the kinjo et al. (2019) study, kokuto, a non-centrifuged cane sugar, showed antioxidant and anti-stress properties. tapioca pearls are made from cassava (manihot esculenta). zekarias et al. (2019) stated that it was thought to be a treatment for a number of ailments, such as diabetes, celiac disease, bone and neurological health, cardiovascular disease, allergens and prostate issues, git issues, and blood pressure issues. winter melon (benincasa hispida), was evaluated as a nutrient-dense vegetable in alsaadi and abass’s (2020) study because it was a good source of natural sugars, amino acids, organic acids, minerals, and vitamins. according to the pharmacological investigations, the plant had a wide range of pharmacological properties, such as analgesic, antioxidant, anti-inflammatory, and central nervous system effects. antibacterial, antidiabetic, nephroprotective, diuretic, and hypolipidemic properties. triterpenes, phenolics, sterols, glycosides, and other functionally significant bioactives and medicines are abundant in it. using water-immersing restraint stress (wrs) and indometacin [indo]-induced gastric mucosal damage in rats, as well as dpph to investigate the antioxidation effect and free radical scavenging of watermelon seed methanol extract, winter melon seed extract demonstrated an improved correlation between dependent dpph concentration and pyloric association action (purohit et al., 2019). fruit tea is another type of tea offered in milk tea establishments. dried fruit, flowers, or leaves are combined to make fruit teas. antioxidant qualities are also present in the fruits used to make these teas. green apples (malus domestica) have been shown in laboratory experiments to have a potent antioxidant impact that lowers cholesterol, lipid oxidation, and the growth of cancer cells. quercetin, catechin, fluorine, and chlorogenic acid are among the many phytochemicals found in apples that have strong antioxidant properties (aksoy & ötleş, 2022). conversely, kiwi (actinidia deliciosa) fruits are incredibly rich in vitamin c and also contain a variety of other nutrients, such as nutritionally significant amounts of dietary fiber, potassium, vitamin e, and folate, as well as several bioactive substances, such as a variety of antioxidants, phytonutrients, and enzymes, that contribute to their metabolic and functional advantages. a rising body of evidence from human intervention research was drawing special attention to the role that kiwi fruit plays in digestive health (richardson et al., 2018). additionally, phytochemical components with anticancer, antibacterial, antioxidative, and antiglycating effects have been discovered in lychee (litchi chinensis sonn.). in addition to improving insulin resistance, lychee seed, lychee seed extracts, and related compounds have been shown to have promising antihyperglycemic properties, including lipid regulation, neuroprotection, antiinflammatory, anti-neurotoxic, and renoprotective effects (zhang et al., 2021). to completely grasp the potential of these many teas and tastes, more research is needed, as the total health impact depends on individual circumstances and consumption habits. the antioxidant qualities of several plant extracts have been the subject of numerous investigations. nevertheless, no research had been done on the antioxidant content of commercial milk teas or fruit teas, which were becoming more and more popular. thus, this investigation of the antioxidants found in particular fruit and milk teas has been carried out. this study aimed to determine the antioxidant activities of the selected milk teas (matcha, okinawa, and winter melon) and fruit teas (green apple, kiwi, and lychee) using a 2,2-diphenyl-1-picrylhydrazyl (dpph) assay in terms of % scavenging activity. the study also determined the best milk tea and fruit tea that exhibited the highest antioxidant, and they were compared to the positive control used (ascorbic acid). this study established the antioxidant activities of one commercial brand of milk teas (matcha, okinawa, and winter melon) and fruit teas (green apple, kiwi, and lychee). this may benefit drinkers of these milk teas and fruit tea materials and methods only a post-test experimental design was used in this study. using the dpph assay, various milk and fruit teas were screened for antioxidants. the ethics committee of a private university in iloilo city reviewed this study, which was carried out there. this research was verified by a licensed chemist. in order to ensure dependability, this study used three replicates with three trials. every instrument had the proper calibration. acquisition of milk tea and fruit tea the selected milk and fruit teas were acquired from a local seller in iloilo city dpph assay three distinct flavored milk teas, three distinct flavored fruit teas, as well as positive and negative controls, were evaluated for antioxidant activity in this study. 180 μl of dpph solution and 20 μl of the commercially available fruit teas (green apple, kiwi, and lychee) and milk teas (matcha, okinawa, and winter melon) were added to each well of a 96-well plate. the stock solution was created by dissolving 0.00591 g of dpph (0.3 mm) in 50 ml of methanol. another stock solution was made with 50 ml of methanol and 0.5 g of ascorbic acid (10 mg/ml). in the wells of the 96-well plate, 180 μl of dpph solution was mixed with 20 μl of distilled water with creamer and sugar (a negative control for milk teas). to prevent false positive results in the antioxidant activity of the samples, a non-dairy creamer was employed in the production of negative control for milk teas from the local vendor. additionally, 180 μl of dpph solution and pa ge 8 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 6-10, 2025 table 1: antioxidants of selected milk teas and fruit teas using a 2,2-diphenyl-1-picrylhydrazyl (dpph) assay through % scavenging activity milk teas and fruit teas % scavenging activity mean sd matcha with tea 62.9c 2.6 okinawa with tea 46.4b 5.9 winter melon with tea 61.6c 2.8 negative control for milk teas (distilled water with creamer and sugar) 0.0a 0.0 green apple with tea 95.5d 2.4 kiwi fruit with tea 97.3d,e 1.5 lychee with tea 94.4c 1.3 pure tea 91.5b 0.5 negative control for fruit teas (water with sugar) 0.0a 0.0 positive control (ascorbic acid) 98.7e 0.62 note. significant if p-value <0.05 20 μl of distilled water with sugar (the negative control for fruit teas) were poured into the 96-well plate’s wells. the positive controls were 180 μl of dpph solution and 20 μl of ascorbic acid (10 mg/ml) that were dispersed in the 96-well plate’s wells. a microplate reader was used to measure the absorbance at 570 nm after the plate had been incubated for 30 minutes at room temperature in the dark while covered with aluminum foil (suarez et al., 2021). the materials are kept out of the light until they are assessed for analysis since dpph is photosensitive (lim et al., 2023). the purple-to-yellow staining was noticed within 30 minutes of incubation. three replicates and three trials were used to conduct the test. the following formula was used to calculate the chosen milk teas’ and fruit teas’ dpph radical scavenging activity. the plate was incubated in the dark, covered with aluminum foil, for 30 minutes at room temperature before being measured for absorbance at 570 nm with a microplate reader (suarez et al., 2021). dpph is photosensitive; the materials are kept out of light until they are evaluated for analysis (lim et al., 2023). within 30 minutes of incubation, the discoloration of the purpleto-yellow color was observed. the test was carried out in three replicates with three trials. the dpph radical scavenging activity of the selected milk teas and fruit teas was determined using the formula: % dpph radical scavenging activity = {(absorbance of negative control absorbance of sample)/ (absorbance of negative control)}x100 waste disposal segregation and proper garbage disposal procedures were adhered to. for local rubbish collection, general waste was disposed away in the appropriate bins. chemical wastes were divided into different containers and categorized based on their content. ascorbic acid and dpph were categorized as organic halogenated trash and were electronically and physically recorded with the appropriate waste codes before being submitted to the pollution control officer. statistical analysis in this study, the mean and standard deviation were computed, and comparisons were performed using oneway analysis of variance (anova) followed by tukey’s honest significant (p ≤ 0.05). results and discussion in contrast to the negative control (mean sd = 0.0), table 1 demonstrates that the milk teas made from matcha (mean = 62.9, sd = 2.6), okinawa (mean = 46.4, sd = 5.9), and winter melon (mean = 61.6, sd = 2.8) exhibit antioxidant properties. = 0. winter melon and matcha both exhibit the highest levels of antioxidant activity, however they are not on par with ascorbic acid, the positive control. in contrast to the negative control (mean sd = 0.0), the antioxidant activities of the green apple fruit tea (mean = 95.5; sd = 2.4), kiwi fruit tea (mean = 97.3, sd = 1.5), and lychee fruit tea (mean = 94.4; sd = 1.3) are comparable. the greatest antioxidant activity, on par with vitamin c, is seen in kiwi fruit tea (mean = 98.7; sd = 0.62). discussions there was antioxidant activity in every flavor of milk tea, matcha, okinawa, winter melon, and fruit tea, as well as in green apple, kiwi, and lychee. this is because flavonoids, a subgroup of polyphenols, are present. a wide range of fruits and plant-based flavorings include them. much research has focused on their ability to scavenge free radicals and reduce oxidative stress. flavonoids have the ability to neutralize free radicals such as dpph radicals and give hydrogen atoms or electrons (kumar & pandey, 2013). because fruits, even when powdered, are rich in flavonoids, fruit teas have higher antioxidant activity than milk teas. our study’s results demonstrate this, since pa ge 9 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 6-10, 2025 while both displayed the highest antioxidant activity within their respective groups, kiwi fruit with tea had a higher antioxidant activity than matcha with tea. the concentration of antioxidant chemicals during drying procedures may be the reason for the release of flavonoids from the cell’s fruits (nunes et al., 2016). catechins are abundant in tea, which was used as one of the flavorings. tea can also help reduce inflammation and cancer (musial et al., 2020).the findings indicated that matcha tea had the highest percentage of scavenging activity among the milk teas. this is consistent with a comparable study that was previously carried out in which matcha tea’s nutritional composition was examined and antioxidant qualities were found (koláčková et al., 2019). high concentrations of catechins, including egcg, are found in matcha (kochman et al., 2020). out of all the fruit teas examined, kiwi fruit had the highest antioxidant activity. according to a related study about the fruit’s potential medical benefits, the kiwi fruit itself has significant levels of phenolic acid, with protocatechuic acid having the highest concentration (zhang et al., 2021). one kind of phytochemical that is frequently referred to as a secondary metabolite of plants is phenolic acid, a nonnutritive substance that may improve human cell function (yoo et al., 2018). numerous bioactivities, including antitumor, anti-inflammatory, antioxidant, hypoglycemic, and hypolipidemic effects, are also attributed to them. the total antioxidant qualities of the fruit tea are enhanced by the inclusion of tea, which contains phytochemical qualities of its own (bag et al., 2022). ascorbic acid, which is the positive control of this study, appears in the body in the form of ascorbate, which is recognized for its powerful antioxidant and anti-inflammatory characteristics. it is clear that the radical anion monodehydroascorbate largely reacts with radicals, while ascorbic acid functions mostly as a donor of single hydrogen atoms (njus et al., 2020). all of these characteristics work together to provide ascorbic acid with exceptional antioxidant benefits that are higher compared to milk teas and fruit teas, which makes it ideal to use as a positive control. so far, this study is the first attempt to determine the antioxidant of the commercial of selected milk tea and fruit teas. thus, future researchers could try a different approach to address the limitations of our study, which includes selected samples of milk teas (matcha, okinawa, and winter melon) and fruit teas (green apple, kiwi, and lychee) along with pure tea. in addition to our analysis of the data, this study was limited to using two negative controls specific to milk teas and fruit teas. distilled water with creamer and sugar was utilized as a negative control for milk teas. distilled water and sugar were utilized as negative controls for fruit teas. this adds to our growing research body, further enriching our understanding of antioxidant activities of commercial milk teas (matcha, okinawa, and winter melon) and fruit teas (green apple, kiwi, and lychee). conclusion the study demonstrated the antioxidant activity of the selected milk teas (matcha, okinawa, and winter melon) and fruit teas (green apple, kiwi, and lychee) in terms of % scavenging activity. the best milk tea and fruit tea that exhibited antioxidant activity were matcha and winter melon and kiwi, for fruit tea, respectively. they can be useful in preventing diseases like cancer, inflammatory and cardiovascular disorders, and other conditions brought on by free radicals, subject to further studies. it is recommended to explore other health benefits of milk teas and fruit teas aside from their antioxidant properties, and may also opt to use vitamin e as a positive control. references aksoy, i. s., & ötleş, s. 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(2020). antioxidant and antiglycation properties of seventeen fruit teas obtained from one manufacturer. applied sciences, 10(15), 5195. https://doi.org/10.3390/app10155195 pa ge 1 pa ge 48 american journal of life science and innovation (ajlsi) prevalence of medications errors and factors of harmful errors in neonates and paediatrics at tertiary and paediatric hospitals: a multicenter study murooj shukry1*, mohammed hamed alotaibi1, manal bajammal1, nissreen althaqafi2, saleh alghamdi3 volume 3 issue 1, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i1.2729 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: april 07, 2024 accepted: may 14, 2024 published: may 21, 2024 medication errors (me) are human mistakes that may occur during drug use, prescribing administration or monitoring. this study aimed to determine the prevalence of mes among pediatric patients receiving tertiary treatment in pediatric speciality hospitals and describe their patterns and causes. three institutions participated in a multicenter retrospective analysis. reports of all mes involving paediatrics aged ≤13 years who were hospitalised or visited the emergency department or outpatient clinics were reviewed 1 january 2019 31 december 2019. the national coordinating council for medication error reporting and prevention classification was used to categorise mes and to define prme (category ci) and patient–harming mes (category ei). a retrospective study was conducted with a total of 318 mes reported, with an overall prevalence of 0.9 per 1,000 patients. the prevalence was markedly lower in the cht (0.35 / 1,000 patients) than in kamc and kfsh&rc (1.73 and 3.5). the highest rate of mes was observed in admitted wards (14.69 / 1,000 patients). most commonly, mes occurred at the prescribing stage (81.1%), were caused by a physician (83.3%), and were discovered by the pharmacist (85.5%). improper medication dosage (34.0%), frequency (14.2%), and duration (10.4%) were the three most common forms of mes. paediatric inpatient treatment has a significant frequency of mes, accounting for 24% of hospitalisations. the me may not get to the patient if independent double-check prescriptions, accurate medication reconciliation of new prescriptions against the previously prescribed, and improved medicine labelling and packaging techniques are used. keywords medication error, neonatal, prescribing error, prevalence, pediatric, saudi arabia, factors introduction medications are active substances used to treat, prevent, and diagnose diseases and constitute a valuable tool for modern medicine. however, improper use of medications can cause harmful consequences to the patient, leading to death (hussain et al., 2014). from the public health view, medications and pharmaceutical substances are authorised in local markets in perspective to help improve the population’s health and reduce disease rates and related morbidity and mortality (laatikainen et al., 2022). from this point, medications’ positive therapeutic and or preventive effect is only judged under optimal safety and effectiveness conditions of usage (maaskant et al., 2015). medication errors “mes” constitute a circumstance where medication is administered outside the recognised safety and effectiveness usage conditions. they are defined as human mistakes occurring in hospitals and represent an issue of high priority in healthcare systems globally (tajik, 2020). they may occur due to misunderstanding during handoffs as the healthcare providers may not be well affiliated across borders (alqenae et al., 2020). these errors may occur during the drug’s use, prescribing, administration, or monitoring, thus involving all healthcare professionals (abuelsoud, 2019). they may result in increased illness severity, other adverse effects, or death and are associated with unnecessary healthcare costs (khaemba, 2014). medication errors account for 84.2% of all medical errors, estimated to cause 98,000 hospital deaths and more than one million injuries annually, costing up to $ 29 billion globally. consequently, medical errors rank as one of the top causes of death in the united states besides car accidents, breast cancer, and acquired immunodeficiency syndrome (a truter, 2017; khaemba, 2014; krzyzaniak & bajorek, 2016). medication errors involve errors due to human or system factors. a study indicates (escrivá gracia et al., 2019) medication-related occurrences cause approximately 2 per 1000 hospital fatalities and 6-12% of hospital admissions, respectively. human-related errors are a lack of performance and/ or compliance with procedures or protocols. many factors including misunderstanding, inaccurate copying, lack of knowledge, heavy workload, misjudgment, use of nonstandard abbreviations, lack of information about the patient, and insufficient or shortage of staff (rodziewicz et al., 2022; thomas et al., 2019). besides health professional factors, patient-related factors may also underlie me, such as patients’ lack of understanding of their treatment and administration errors, related to prescription and incorrect dosages (valencia quintero et al., 2021). the risk of human errors increases with an increased workload, stress, sleep deprivation, drugs with similar and complex names, and handling of medications by non-pharmacists, besides non-ergonomically designed 1 pharmaceutical care division, king faisal specialist hospital and research centre, jeddah, saudi arabia 2 saudi medication safety center, king abdulaziz medical city-national guard health affairs, jeddah, saudi arabia 3 pharmaceutical care division, children hospital, taif, saudi arabia * corresponding author’s e-mail: muroojshukry143@outlook.com pa ge 49 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 48-60, 2024 information systems (khayam, 2015). a review based in iran revealed that me was the most common type of medical error, with prevalence ranging from (10-80%) (vaziri et al., 2019). suppose mes constitute a global concern for causing severe medical consequences. in that case, they should be more seriously considered for infants and children, especially in developing countries where this age category represents a large proportion of the population (hmedat et al., 2017). mes are prevalent in picus and nicus, affecting medication orders and patient days (alghamdi et al., 2019). while prescribing safety has become very clear in adults, the focus has been less on newborns, infants, children, and adolescents, as clinical trials are insufficient for children due to cost and liability problems and regulations that impede such studies (al-jeraisy et al., 2011). moreover, the prevalence of me in pediatric patients is three times higher than in adults (brennanbourdon et al., 2020). the available evidence suggests that mes among pediatric patients represent a greater risk of death than adults, owing to additional factors that put children at higher risk from mes, such as age and weight differences, in addition to other physiological features (al-jeraisy et al., 2011; fernández-llamazares et al., 2012; impicciatore et al., 2001; kaushal et al., 2001). further, mes have been reported in various pediatric settings with an incidence rate of 1 in 6.4 medication orders (dedefo et al., 2016). these involved multiple medications, and several risk factors have been identified in the literature (antonucci & porcella, 2014; khayam, 2015). such observations make the prevention of mes a priority for health systems and a clinical necessity, integrated under the broad mission of protecting patients. consequently, several efforts are undertaken to reduce drug error rates, using multiple human and technological tools, both at the healthcare provider’s and patient’s levels (dedefo et al., 2016; devine eb, 2005; fernández-llamazares et al., 2012; hussain et al., 2014; ross et al., 2000). among these strategic actions is monitoring mes, which has become an essential tool to improve the safety and efficacy of medication dispensation systems by analysing the risk in case errors are reported. the use of computerised provider order entry (cpoe) was shown to reduce the prevalence of me highly (liu et al., 2023). error reporting systems depend on the ability of physicians, pharmacists, and nurses to commit error detection and reporting protocols (khaemba, 2014). as determined in one study, nurses were less likely to report me as they feared the consequences that followed it. physicians were more likely to report the me. these considerations are of higher importance in the pediatric population. as such, we designed this study to provide insight into the extent and determinants of mes among neonates and children in tertiary and pediatric hospitals in the western region of saudi arabia. the following objectives were explored: • to estimate the yearly prevalence of mes among pediatric and neonatal patients in different types of hospital settings. • to compare the prevalence of mes between the different hospitals (tertiary and paediatrics) and different departments to determine the settings with the highest risk of mes. • to describe the pattern and categorisation of mes based on the reported data; • to investigate patient and healthcare professionalrelated factors associated with mes and to analyse the predictors of the patient -reaching mes (prme) and patient-harming mes (phme). materials and methods study design three referral hospitals in the western region of saudi arabia-king abdulaziz, medical city national guard (kamcjed, a 751-bed hospital), children’s hospital of taif (cht), and king faisal specialist hospital and research centre jeddah (kfshrc, a 380-bed hospitalwere the subjects of a multicenter retrospective study. data was gathered for this investigation from 1 january 2019 31 december 2019. there were no increased patient hazards or interventions in the research. initially, the study involved all eight tertiary and pediatric hospitals in the western region. subsequently, a systematic sampling method was used to include all cases of mes reporting over the study period from all participating centres. however, due to feasibility challenges, cluster sampling was used to select three centres depending on the authors’ convenience and data accessibility. ethical considerations the protocol conformed with the declaration of helsinki’s ethical guidelines for using human subjects’ data and material in medical research. institutional review board provided the ethical approval for kfsh with irb study number 202068, kaimarc with irb study number rj20/173/j, and an official approval letter from taif’s director of health affairs. eligibility criteria the inclusion criteria for this study were as follows: a) the study included all mes reports declared in the participating centres from 1 january 2019 31 december 2019. b) only me reports concerning neonates and pediatric patients under 13 years admitted or visited the outpatient clinics or emergency were included. the exclusion criteria were: a) patients over 13 years were excluded from the study. b) oncology and haematology neonates and paediatrics units were excluded. c) patients with incomplete data were excluded from the study. data collection a structured, precoded excel form was used to collect all relevant data from both sources, i.e., the me report and patient file. the form was designed by authors based on pa ge 50 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 48-60, 2024 the crucial me report content and the literature review of relevant studies. the excel form collected the following data: 1. me-related data including date, location (hospital, department), a person who reported the me, stage of error (prescribing, transcription, dispensing, administration, monitoring), type of mes (incorrect dose, incorrect medication, incorrect route, expired medication, missed medication, and incorrect time), and medicines in the cause (name, indication, dose, route, higher alert [yes/no], dosage form). further, the severity of mes was classified using the national coordinating council for medication error reporting and prevention (ncc merp) categorisation (merp, 1998) 2. patient-related factors, including age, gender, weight, medical history, chronic disease, mental illness (yes/no), consciousness status on the day of the me (conscious/ unconscious), number of medications prescribed on the day of the me, length of hospital stay, presence of a caregiver and relation to the child (mother, father) 3. data relating to the healthcare professional in the cause of the me, including speciality (pharmacist, physician, nurse), grade (senior, junior), position (temporary, contract worker, permanent). 4. there is a predefined list of 8 contributing factors: missing clinical information, lack of double-checking, and lack of staff education and training. besides, data regarding the number of admissions by participating centres and units were collected on a separate sheet and used as a denominator in calculating me prevalence, as indicated later. procedure the researchers conducted the data collection onsite. me reports, including electronic and handwritten reports, were retrieved from the participating centres’ me reporting system. the corresponding patients’ files were reviewed to extract further relevant data not available in the information. data were entered directly using the predesigned excel form, which resulted in 3 separate databases, one for each centre. the principal investigator and coauthors reviewed the three partial databases for accuracy and completeness and then merged them into one final database. the final database was coded to respect the patient’s confidentiality before being shared for statistical analysis. study outcomes the study defined one primary outcome and two secondary outcomes. the primary outcome consisted of the prevalence of mes, which 1,000 patients computed regarding the total number of admissions and/or visits in each centre over the study period. the secondary outcomes consisted of patient-reaching me (prme), defined as a me of ncc merp category ci. further, patientharming me (phme) was also a secondary outcome, expressed as a me of ncc merp category ei. statistical methods the database was edited and imported to spss version 21 for statistical analysis. the results of the descriptive statistics that were carried out to present the patterns of mes and associated factors were presented as frequency and percentage. using cross-tabulations, chi-square and fisher’s exact tests were used to analyse patientreaching mes (prme). on the contrary, the independent risk factors of prme were analysed by a technique known as multivariate binary logistic regression. with a confidence interval (ci) of 95%, results were presented as odds ratio (od). a p-value < 0.05 is considered statistically significant for the study. results and discussions prevalence of neonates and pediatric medication errors by department and centre the three participating centres reported 318 mes in total, figure 1: prevalence of medication errors by department and centres in three tertiary care hospitals of western saudi arabia, 2019 pa ge 51 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 48-60, 2024 including 135 (kfsh), 93 (cht), and 90 (kamc), and an overall prevalence of 0.9 per 1,000 patients. compared to the two tertiary care hospitals, kamc and kfsh, the rate was lower at the children’s hospital (0.35 per 1,000 patients) than at both facilities (figure 1). the prevalence of mes varied significantly amongst hospitals. at the departmental level, inpatient wards had the most significant rates of mes (14.69 per 1,000 patients), whereas emergency departments had the lowest rates (0.14). patient-related factors of me table 1 illustrates the demographic and clinical features of individuals with mes. the age distribution of the 318 patients revealed two main age groups: 1 month-2 years (37.7%) and 6-12 years (35.5%), with 57.2% of the patients being male. 60.4% of the patients in the study had comorbidities, and 7.2% had mental disabilities. the patient was unconscious in 13.8% of the instances, was taking four or more prescriptions in 48.1% of the cases, and had no carer present in 30.2% of the cases when the medication mistakes happened. table 1: patient-related factors of medication errors parameter category frequency percentage center chat 93 29.2 kamc 90 28.3 kfsh 135 42.5 patient’s age * 0-30 days 9 2.8 31 days – 2 years 120 37.7 2-6 years 72 22.6 6-12 years 113 35.5 gender * male 182 57.2 female 132 41.5 weight * <5 kg 1 .3 510 kg 132 41.5 1120 kg 101 31.8 >20 kg 79 24.8 comorbidity‡ any 192 60.4 cancer 39 12.3 hematological disease 35 11.0 mental disability 23 7.2 other chronic condition 184 57.9 consciousness state * conscious 270 84.9 unconscious 44 13.8 number of medications 13 86 27.0 46 153 48.1 7+ 73 23.0 data not available 6 1.9 presence of caregiver no 96 30.2 yes 215 67.6 data not available 1 2.2 * because of missing data, not all values sum up to the total ‡ a patient may have more than one comorbidity patterns and characteristics of mes most commonly, mes occurred at the prescribing stage (81.1%), were caused by a physician (83.3%), and were discovered by the pharmacist (85.5%). premature medicine dose (34.0%), incorrect frequency (14.2%), and duration (10.4%) were the most common types of mes, and cumulative mistakes (two or more errors in the same report) were discovered in 5.0% of instances (table 2). the most common medication classes involved in mes were antibiotics (25.5%), followed by supplements (9.4%) and corticosteroids (7.2%). high-alert medications were involved in 17.3% of the cases. the most common routes of administration were oral (49.7%) and iv (40.3%). other medication characteristics are depicted in table 3. of the total mes, 47 (14.8%) reached the patient (prmes rate=14.8%, ncc merp class cf), among which only 2 (0.6%) were harmful and required intervention (phmes rate=0.6%, ncc merp class ef) (table 4). pa ge 52 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 48-60, 2024 table 2: characterising patterns of medication errors error characteristic category frequency percentage location general ward 139 43.7 outpatient clinics 89 28.0 ed 34 10.7 pediatric icu 31 9.7 neonatal icu 23 7.2 nursery 2 0.6 stage prescribing 258 81.1 transcription 3 0.9 dispensing 33 10.4 administration 20 6.3 monitoring 4 1.3 type improper dose 108 34.0 wrong frequency 45 14.2 wrong duration 33 10.4 wrong strength 24 7.5 wrong medication 23 7.2 wrong rate of infusion 20 6.3 wrong route 15 4.7 duplication 15 4.7 wrong dosage form 9 2.8 contraindicationknown allergy 7 2.2 delayed dispensation 5 1.6 expired medication 4 1.3 monitoring error 3 0.9 wrong patient 1 0.3 other § 6 1.9 cumulative error no (only one medication error) 302 95.0 yes (2 or more) 16 5.0 days from prescription same day 305 95.9 one day or more later 13 4.1 person who discovered the error pharmacist 272 85.5 physician 2 0.6 nurse 43 13.5 patient 1 0.3 professional in cause nurse 11 3.5 pharmacist 35 11.0 physician 265 83.3 data missing 7 2.2 position junior 74 23.3 senior 56 17.6 data not available 188 59.1 § include damaged medication (3), wrong instruction (2 cases), and trade name in prescription (1) pa ge 53 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 48-60, 2024 table 3: characteristics of the involved medication parameter category frequency percentage class antibiotic 81 25.5 supplements (vitamins, minerals, etc.) 30 9.4 corticosteroids 23 7.2 antihypertensive 20 6.3 electrolyte 18 5.7 analgesic 15 4.7 anticonvulsant antiepileptic 13 4.1 laxative 10 3.1 anti vomiting 8 2.5 psychotropics 8 2.5 other § 92 28.9 route of administration oral 158 49.7 iv 128 40.3 im 7 2.2 subcutaneous 9 2.8 topical / local 16 5.0 other 7 2.2 offlabel use no 307 96.5 yes 11 3.5 offlabel dose no 313 98.4 yes 5 1.6 offlabel route no 317 99.7 yes 1 0.3 high alert no 263 82.7 yes 55 17.3 § include 29 other medication classes involving 1 (0.3%) and 7 (2.2%) cases table 4: distribution of medication errors according to the national coordinating council for medication error reporting and prevention (ncc merp) classification ncc merp category n % the error did not reach a patient 271 85.2 a: circumstances or events that have the capacity to cause the error. 42 13.2 b: an error occurred, but the error did not reach the patient 229 72.0 error reached the patient 47 14.8 c: an error occurred that reached the patient but did not cause harm. 35 11.0 d: an error occurred that reached the patient and required monitoring to confirm that it resulted in no harm to the patient and/or required intervention to preclude harm. 10 3.1 e: an error occurred that may have contributed to or resulted in temporary harm to the patient and required intervention. 1 0.3 f: an error occurred that may have contributed to or resulted in temporary harm to the patient and required initial or prolonged hospitalisation. 1 0.3 factors contributing to mes a lack of staff education and training regarding me was identified in most cases (90.2%). the next three most frequently identified factors included an absence of double-checking (66.8%), missing clinical information of the patient (48.1%), and missing drug information (32.0%) (figure 2). pa ge 54 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 48-60, 2024 factors associated with prme prmes occurred at the same frequency in the three hospitals (p = 0.241). prmes rate was significantly higher in icu (31.5% vs. up to 13.5%, p = 0.001) and in the case of unconscious patients (38.6% vs. 11.1%, p < 0.001) and absence of caregiver (29.2% vs. 8.8%, p < 0.001), compared to their counterparts respectively; and the rate increased with the number of prescribed medications (p = 0.008). expectedly, the later the stage of me occurrence, the more likely it reached the patient (p < 0.001), and prmes occurred less likely in mes occurring on the day of prescription of the involved medication (13.1% vs. 53.8%, p < 0.001). further, mes caused by nurses were more likely to be prmes (72.2% vs. up to 34.3%, p < 0.001), whereas those discovered by a pharmacist were less likely to be prmes (10.3% vs. 39.5% or more, p < 0.001). high-alert medications were associated with a higher prme rate (32.7% vs. 11.1%, p < 0.001). three among the eight contributing factors increased the prmes’ risk significantly, including drug name, label, filling, and package problem (39.5% vs. 11.4%, p < 0.001), staffing shortage and workload (29.4% vs. 13.0%, p = 0.011), and lack of doublechecking (20.3% vs. 3.8%, p < 0.001) (table 5). figure 2: contributing factors in medication errors table 5: factors associated with medication errors reaching the patient factor category error reached the patient p-value no yes n % n % center/department factors center cht 83 89.2 10 10.8 kamc 78 86.7 12 13.3 kfsh 110 81.5 25 18.5 .241 hospital type children 83 89.2 10 10.8 tertiary 187 83.6 37 16.4 .193 department general ward 122 86.5 19 13.5 icu 37 68.5 17 31.5 ed 33 97.1 1 2.9 outpatient 79 88.8 10 11.2 .001* patient-related factors age 030 days 5 55.6 4 44.4 31 days 2 years 100 83.3 20 16.7 2-6 years 64 88.9 8 11.1 6-12 years 98 86.7 15 13.3 .057 gender male 159 87.4 23 12.6 female 108 81.8 24 18.2 .277 pa ge 55 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 48-60, 2024 weight <5 kg 0 0.0 1 100.0 5 10 kg 111 84.1 21 15.9 1120 kg 86 85.1 15 14.9 >20 kg 69 87.3 10 12.7 .107 cancer no 237 84.9 42 15.1 yes 34 87.2 5 12.8 1.000f haematological disease no 236 84.9 42 15.1 yes 30 85.7 5 14.3 1.000f mental disability no 248 85.5 42 14.5 yes 18 78.3 5 21.7 .362f other chronic condition no 112 87.5 16 12.5 yes 153 83.2 31 16.8 .291 consciousness state conscious 240 88.9 30 11.1 unconscious 27 61.4 17 38.6 <.001* number of medications 13 78 90.7 8 9.3 46 133 86.9 20 13.1 7+ 54 74.0 19 26.0 .008* presence of caregiver no 68 70.8 28 29.2 yes 196 91.2 19 8.8 <.001* factor related to the medication error stage prescribing 235 91.1 23 8.9 transcription 3 100.0 0 0.0 dispensing 24 72.2 9 27.3 administration 7 35.0 13 65.0 monitoring 2 50.0 2 50.0 <.001* cumulative error no 259 85.8 43 14.2 yes (2 or more) 12 75.0 4 25.0 .270 days from prescription same day 265 68.9 40 13.1 one day or more later 6 46.2 7 53.8 <.001* person who discovered the error pharmacist 244 89.7 28 10.3 physician 0 0.0 2 100.0 nurse 26 60.5 17 39.5 patient 1 100.0 0 0.0 <.001* professional in cause nurse 3 27.3 8 72.7 pharmacist 23 65.7 12 34.3 physician 240 90.6 25 9.4 <.001* position junior 67 90.5 7 9.5 senior 46 82.1 10 17.9 data not available 151 84.4 28 15.6 .332 factors associated with the medication route of administration oral 136 86.1 22 13.9 parenteral (iv, im, s/c) 121 84.0 23 16.0 topical / local 14 87.5 2 12.5 .852 high alert no 234 89.0 29 11.0 yes 37 67.3 18 32.7 <.001* medication duration temporary 188 85.5 32 14.5 chronic 78 83.9 15 16.1 .720 pa ge 56 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 48-60, 2024 contributing factors factor 1 no 146 88.0 20 12.0 yes 125 82.2 27 17.8 .151 factor 2 no 187 86.6 29 13.4 yes 84 82.4 18 17.6 .322 factor 3 no 246 86.3 39 13.7 yes 24 75.0 8 25.0 .088 factor 4 no 248 88.6 32 11.4 yes 23 60.5 15 39.5 <.001* factor 5 no 247 87.0 37 13.0 yes 24 70.6 10 29.4 .011* factor 6 no 28 90.3 3 9.7 yes 242 84.6 44 15.4 .396 factor 7 no 260 85.8 43 14.2 yes 11 73.3 4 26.7 .252f factor 8 no 101 96.2 4 3.8 yes 169 79.7 43 20.3 <.001* factor 1: clinical information missing; factor 2: drug information missing; factor 3: lookalike and sound-alike medication; factor 4: drug name, label, filling, and package problem; factor 5: staffing shortage and workload problem; factor 6: lack of staff education and training problem factor 7: patient education problem; factor 8: no double-check. the test used: f fisher’s exact test; otherwise, the chisquare test was used. * statistically significant result inappropriate drug labelling or packaging (or=3.13, p=0.042) and a lack of double-checking (or=5.49, p=0.016) were independently linked with the incidence of prme, according to multivariate regression analysis (table 6). table 6: predictors of medication errors reaching the patient predictor level or 95%ci p-value department general ward ref .193 icu 0.35 0.07 1.65 .183 ed 0.37 0.04 3.31 .374 outpatient 2.01 0.65 6.26 .227 consciousness state conscious ref unconscious 2.95 0.67 13.07 .155 number of medications 13 ref .970 46 0.90 0.31 2.65 .852 7+ 0.99 0.27 3.71 .994 presence of caregiver no ref yes 0.39 0.13 1.16 .090 days from prescription same day ref one day or more later 12.43 2.15 71.70 .005* professional in cause nurse 7.71 1.37 43.49 .021 pharmacist 1.08 0.34 3.46 .892 physician ref .061 high alert no ref yes 2.46 0.95 6.38 .064 factor 4 no ref yes 3.13 1.04 9.41 .042* pa ge 57 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 48-60, 2024 factor 5 no ref yes 1.55 0.50 4.87 .450 factor 8 no ref yes 5.49 1.37 21.94 .016* multivariate binary logistic regression. dependent factor: patient-reaching medication error (yes). or: odds ratio; 95% ci: 95% confidence interval ref: category used as a reference in the calculation of or factor 4: drug name, label, filling, and package problem factor 5: staffing shortage and workload problem factor 8: no double check * statistically significant result discussion mes in the pediatric population constitute a significant health issue. they comprise a high risk of iatrogenic injury, which exposes increased morbidity, mortality, health expenditure, and legal issues. in the current multicenter investigation, we examined the trends and characteristics of all mes reports from the two tertiary hospitals and the children’s hospital in saudi arabia’s western area over the course of a year. findings showed that me prevalence ranged between 0.35 and 3.5 /1,000 patients, depending on the hospital, and between 0.14 and 14.7 / 1,000 patients, depending on the department. most mes consisted of prescribing errors made by doctors and reported by the pharmacist. nevertheless, 14.8% of the mes reached the patient was predicted by several risk factors, notably earlier prescription date, absence of doublechecking, and inappropriate drug labelling or package, with a 3 to 12fold risk of occurrence. on the other hand, the rate of phmes was very low (0.6%), not enabling inferential analysis. the prevalence of mes – rates and calculation methods various calculation methods have been used to estimate the prevalence of mes in literature, making the comparative analysis difficult across different studies and settings. the prevalence of mes was determined in the current study by considering all hospital admissions or visits during the same period, depending on whether the me occurred in an inpatient or outpatient environment. this calculation method adjusts for the patient flow in the given hospital or department while disregarding the number of medication orders. a systematic review by alghamdi et al. found that, in every 100 medication orders, approximately 15 mes were reported in pediatric intensive care units and up to 78 in neonatal icu (santesteban et al., 2015). prescription mistakes were observed in between 5.7% and 35.2% of prescriptions or medicine orders for newborn care, according to three distinct studies, according to a second comprehensive analysis by santesteban et al.. another study that assessed the efficacy of a pharmacy-led intervention to reduce mes in the pediatric ward of a maternity and children’s hospital, focusing on prescribing errors, found that an intervention was necessary for 10% of the medical orders (jennifer l costello 1, 2007). the rates reported in the previously mentioned studies, including the two systematic reviews, can be considered consistent with each other, highlighting an alarming frequency of mes. these figures seem to be less reflected when using the number of hospital admissions as a denominator, such as in the present study. another calculation method that is reported in the literature uses the patient’s parameter as the denominator. in the comprehensive systematic review by alghamdi et al., four studies-two in the picu and two in the nicu-used this calculating approach and found that the prevalence rates of me were, respectively, 6.4 to 9.1 and 4 to 35.1 per 1,000 patient days (santesteban et al., 2015). compared to the 5.1 to 40.1 per 1,000 hospital admissions rates found in in-patient departments in the present study, the prevalence rates reported in alghamdi et el. systematic reviews are likely to be considerably high; this is by assuming that patient days are higher than the raw number of hospital admissions. although such a comparison is inaccurate due to dissimilar calculation methods, this appearing discrepancy may indicate a probable underreporting of mes in our study population. in an intriguing method, costello et al. demonstrated that underreporting of mes is a significant limitation of the voluntary reporting strategy in an exciting approach. a pharmacist-led intervention would considerably enhance reporting of mes while decreasing the severity of the errors reported (dedefo et al., 2016). other calculation methods were used in reporting mes. for example, a study by morriss et al. reported mes prevalence in nicu regarding medication doses and found 69.5 mes per 1,000 doses (izadpanah et al., 2018); such a method may be more applicable for dispensing errors. based on nurses‘ self-reports, another study from iran estimated the number of mes involving nurses during the past month in pediatric emergency wards. the authors found that nurses caused an average of ~42 cases of mes in the studied month (emily beth devine 1 & kerm henriksen 1, 2005). along with this variation in calculating techniques, alghamdi et al. also emphasised a serious lack of uniformity in the definition of mes used by various research, with several of them employing definitions that were created locally (santesteban et al., 2015). such heterogeneity in mes’ definition and reporting systems and the calculation method of mes prevalence pa ge 58 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 48-60, 2024 hinders the comparisons between the different studies and settings. this indicates the need for a standardised practice using evidence-based, homogenous strategies. types and patterns of mes the current investigation showed doctors were the most frequently implicated in mes, with prescription mistakes accounting for over 80% of cases. second, nurses were engaged in 13.5% of the instances, particularly while giving medicine, which accounted for 10.4% of all mes. hence, the study revealed that the most common type of mes was improper medication dose, strength, frequency, or duration, probably indicating the challenging aspect of the prescribing practice in the pediatric population. given the frequency of prescribing errors in the pediatric population, several authors were concerned about studying this particular type of me separately in pediatric settings. further, the authors reported that dosing errors were the most commonly reported subtype (santesteban et al., 2015). a study from south africa consistently showed that the leading cause of me in children was dose error, especially in intensive care, as this category of patients requires an individual description of drugs according to age, body, and weight. medicines that increased the risk of error were antibiotics, sedatives, analgesics, bronchodilators, and cardiovascular medications (khayam, 2015). a saudi study in the general pediatric ward and picu of king abdulaziz medical city, riyadh, found that 56% of the medication orders contained prescribing errors, and 79% were classified as harmful. additionally, dose and frequency errors accounted for approximately 50% of the cases, and an overdose was detected in 18.7% of the prescribing errors. conversely, a study conducted in neonatal care settings in the tertiary care hospital of south india showed significantly higher rates of administration errors reported in some of the included studies, accounting for up to 77% of mes (b prakadeesh bharathi, 2020). in contrast, other studies reported relatively higher rates of drug preparation errors (morriss et al., 2009). in sum, mes can happen at any point throughout the administration of a medicine, from prescription to administration and followup, and drug interactions can even cause them. thus, all healthcare professionals are responsible for detecting and prospectively managing the factors causing mes when dealing with a patient (abuelsoud, 2019). identification and prevention of mes risk the identification of the associated risk factors constitutes a critical step in the prevention of mes. as revealed in the current study, among the predominantly identified factors is the lack of staff education and training regarding mes, found in 90.2% of the cases. this was followed by an absence of doublechecking by the involved healthcare professional, accounting for two-thirds of the cases. both issues can be corrected using education and awareness interventions targeting the concerned health professionals. several factors for mes are highlighted in the literature, such as multiple medications and extended stays in the hospital, which increase the risk of both mes and drug interactions (antonucci & porcella, 2014). further, neonatal care settings, notably nicu, are reportedly more exposed to mes than other pediatric settings (b prakadeesh bharathi, 2020). another study in us revealed that physicians were had an increased likelihood of major medical errors due to poor well-being, burn-out and low work unit safety grades (tawfik et al., 2018). on the other hand, the current study has the particularity to have analysed factors associated with prme. finding such variables may be crucial for lowering the incidence and severity of mes and related morbidity and mortality. among these factors, we highlight the patient’s consciousness status and the caregiver’s presence or absence. the clinical significance of these two patientrelated factors reflects the potential contribution of the patients or their caregivers in managing the risks of mes. the adjusted regression model highlighted three other factors: the absence of doublechecking, which was demonstrated to increase the risk of prme by 5.5 times in an independent fashion, and inadequate drug labelling and packaging, which increased the same risk by 3.1 times. this demonstrates the importance of enhancing the standards of routine practice among health professionals regarding medication use. prevention of medication errors should be set in health authorities and institutions. of the several efforts undertaken to reduce me rates, a significant focus is on using information technology to mitigate the human factor most commonly underlying mes (dedefo et al., 2016; fernández-llamazares et al., 2012; khaemba, 2014). another encouraged strategy is to increase the number of clinical pharmacists and enhance their training, as these constitute physicians’ backup in ensuring the medical prescription’s safety and adequacy (devine eb, 2005). reports showed that the role of clinical pharmacists in reducing mes is prominent, owing to their crucial role in identifying issues related to the safe use of drugs and the potential health problems that arise from the misuse (dedefo et al., 2016). from a larger perspective, reducing mes requires setting high-level caution at all stages of the mediation process, such as packaging, labelling, description, copying, dispensing, and administration in the internal and external patient settings. particular attention should be drawn to managing home medication, which increases the risk of human error by involving a third party, including parents and caregivers. hence, physicians should assess the family environment before prescribing for children to ensure correct administration (hussain et al., 2014). strengths and limitations the present study is limited by the reliance on a voluntary me reporting system, resulting in under-reporting and underestimating me prevalence rates. the study also overlooked potential measures for assessing and improving pa ge 59 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(1) 48-60, 2024 me reporting practises, which may differ between centres and departments. further, the retrospective design made collecting relevant data a challenging task, notably the assessment of the contributing factors. nevertheless, the present study has the advantage of being conducted in three referral centres and including all mes reported in the study year. it supports the good external validity of the findings and provides a reliable baseline figure for further interventional studies in any of the three included centres. conclusion paediatric inpatient care has a significant frequency of mes, especially in tertiary care facilities, which account for 24% of hospital admissions. implementing a systematic doublecheck of prescriptions, including recent and older ones, and improving drug labelling and packaging methods may prevent many mes. the adequate education of health professionals and caregivers regarding mes should be considered a high-priority tool to improve medication safety among pediatric patients. further corrective measures and awareness-raising campaigns should be implemented in all referral hospitals to modify the risk factors and improve medication practice at all stages. abbreviations children’s hospital in taif (cht), confidence interval (ci), emergency department (ed), factor (f), intramuscular (im), intravenous (iv), intensive care unit (icu), institutional review board (irb), kingdom of saudi arabia (ksa), king abdul-aziz medical city (kamc), king faisal specialist hospital and research center (kfsh&rc), medication error (me), the national coordinating council for medication error reporting and prevention (nccmerp), neonatal intensive care unit (nicu), odds ratio (or), patient harm medication error (ph-me), pediatric intensive care unit (picu), patient reaching medication error (pr-me), subcutaneous (s/c), statistical package for the social sciences (spss) funding source the author (s) received no financial support for the research, authorship, and/or publication of this article. ethical approval was obtained from the institutional review board of kfsh, kaimarc and an official approval letter from the director of health affairs in taif. acknowledgement quality department staff at king faisal specialist hospital and research center-jeddah, king abdul-aziz medical city-jeddah and children’s hospital-taif. presented at ashp mid-year on december 7th, 2021 and at pediatric pharmacy association on may 4th, 2022. references a truter, n. s. a. j. c. m. 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[medication errors in pediatrics]. andes pediatr, 92(2), 288-297. https://doi. org/10.32641/andespediatr.v92i2.1357 (errores de medicación en pediatría.) vaziri, s., fakouri, f., mirzaei, m., afsharian, m., azizi, m., & arab-zozani, m. (2019). prevalence of medical errors in iran: a systematic review and meta-analysis. bmc health services research, 19(1), 622. https://doi. org/10.1186/s12913-019-4464-8 pa ge 1 pa ge 11 american journal of life science and innovation (ajlsi) heavy metal analysis in crassostrea iredaleicrassostrea iredalei (slipper oyster) from dumangas and barotac nuevo, iloilo, philippines carisma g. m. s.1, campania n. f.1, casipe b. j.1, chua a. d. t.1, consolacion j. r. i.1, daylo d. k. s.1, dela cruz a. p. j.1, escultero h. j. r.1, fajardo o. m. g.1, falsis h. i. d.1, felecio j. r. b.1, blancaflor j. m.1, palmos r. j. p.1, visitacion r.1, leong-on m. s.1* volume 4 issue 2, year 2025 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v4i2.5047 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: april 20, 2025 accepted: may 22, 2025 published: october 11, 2025 it is relevant to analyze the concentrations of heavy metals in c. iredalei. this study was conducted to analyze the concentrations of the heavy metal contents, such as copper (cu), lead (pb), and zinc (zn) in c. iredalei from dumangas and barotac nuevo, iloilo, philippines. the samples of c. iredalei were analyzed using flame atomic spectrophotometry, and results were compared to regulatory limits set by the international shellfish safety and sanitation standards. in the town of dumangas, pb (mean = 0.01mg/l; sd = 0.11;) cu (10.44 mg/l; sd = 2.95); and zn (mean = 3.35 mg/l; sd = 0.13) were within the accepted values set by the regulatory limits of the international shellfish safety and sanitation standard. the samples of c. iredalei were taken from march 31st and april 7th, 2023. on the other hand, in the town of barotac nuevo, pb (mean = -0.09, mg/l s = 0.02); cu (mean = 10.14 mg/l, sd = 0.95; and zn (mean = 3.21 mg/l; sd = 0.10) were within were within the accepted values set by the regulatory limits of the international shellfish safety and sanitation standard, too. this implies that the c. iredalei samples from farms in towns dumangas and barotac, iloilo, philippines, were safe for consumption and do not pose threats concerning heavy metal intoxication. keywords crassostrea iredalei, heavy metal determination, slipper oyster, spectrophotometry 1 college of pharmacy and medical technology, university of san agustin, iloilo city, philippines * corresponding author’s e-mail: mleongon@usa.edu.ph introduction a popular delicacy that is widely cultivated in the philippines for human consumption is the slipper oyster, or crassostrea iredalei (monaya et al., 2022). therefore, encouraging a sustainable economy will raise the fishing industry’s and its related sectors’ economic value (rustia et al., 2023). it is a popular item throughout asia due to its inexpensive cost, especially among the poor (pakingking et al., 2022). one essential food source that may be found all across the coastal region is c. iredalei. the nation produced 40,736.89 metric tons (mt) of c in 2021. western visayas contributed 10,886.16 mt, the secondhighest amount among the other regions (department of agriculture’s bureau of fisheries and aquatic resources, 2022). meat production for domestic use is the primary objective of oyster farming. by 2020, it is anticipated that oyster production alone will be worth php 1.552 billion. nutrition, food security, and the battle against poverty depend on them because they are the primary source of food and money for poor fishermen (lebata-romos et al., 2023). situated in the center of the philippines, the western visayas region is one of the main suppliers of oysters. more farms are being established in the western visayas region as a result of the need for extra sources of income brought on by small-scale fishermen’s diminishing catch. however, contamination in aquatic biological systems has become much more common in recent years due to mining and agricultural activities (noman et al., 2022). heavy metals in coastal and estuarine ecosystems, along with other environmental pollutants, have raised concerns in environmental conservation (wang et al., 2022). oysters are filter feeders that can collect pathogens from surrounding waters, potentially causing foodborne diseases in consumers (pakingking et al., 2022). persistently polluted organisms absorb heavy metals, which can cause severe illnesses such food poisoning, liver damage, cardiovascular abnormalities, and even death (anandkumar et al., 2018). food contamination by toxic heavy metals is a worldwide environmental risk that impacts hundreds of millions of people. their toxicity results in a broad range of harmful biological effects (balali-mood et al., 2021). oysters are valued as delicacy, but their fitness for human consumption should be monitored, especially as they are believed to be hyperaccumulators of a number of dangerous metals, such as cadmium, copper, and zinc (wang & weng, 2018). cellular processes such as growth, proliferation, differentiation, damage repair, and apoptosis are all hampered by heavy metals. through the production of reactive oxygen species (ros), oxidative stress, enzyme inactivation, and weakened antioxidant defense, these metals cause toxicity. high levels of exposure to heavy metals, especially lead and mercury, can cause serious side effects such as kidney failure, bloody diarrhea, and colic in the abdomen (tsai et al., 2017). due to equipment problems, the bureau of fisheries and aquatic resources (bfar) has not performed heavy metal analyses in the past four years, saving them for cases of oyster exportation outside of the country (r. chua, personal communication, january 10, 2024). therefore, it pa ge 12 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 11-14, 2025 seems sense to investigate the levels of heavy metals in c. iredalei in dumangas and barotac nuevo, iloilo. the purpose of this study was to examine the levels of heavy metals in c. iredalei from barotac nuevo and dumangas, iloilo. in particular, this study was able to characterize the many types of heavy metals that are present in c, including lead (pb), copper (cu), and zinc (zn). c iredalei, from the oyster farms at barotac nuevo and dumangas, iloilo, during the last week of march and first week of april 2024. additionally, the study measured the amounts of cu, pb, and zn in c. iredalei at the time of collection and determined the heavy metal concentration in c. iredalei with regard to the international safety and sanitation standard for shellfish. according to the study’s findings, it was safe to eat c. iredalei from the municipalities of dumangas and barotac nuevo. in the last week of march and the first week of april 2024. the pb, cu, and zn levels were within the permissible range established by the international shellfish safety and sanitation standards. materials and methods research design the occurrence of heavy metals like lead (pb), copper (cu), and zinc (zn) in oysters from dumangas and barotac nuevo, iloilo, was investigated using a descriptive quantitative design. additionally, it determined that the concentrations of certain heavy metals were within permissible bounds. research setting oyster farms in dumangas and barotac nuevo provided the data for this investigation. the laboratory of a private institution in iloilo city was used to examine the heavy metals. ethical considerations this study was approved by the ethics committee of a private university in iloilo city. data collection procedures collection of c. iredalei: for two weeks, c. iredalei samples were taken every week from two locations: dumangas and barotac nuevo. preparation of samples: in accordance with the techniques of azevedo et al. (2019), pakingking et al. (2022), and villafuerte et al. (2022), the c. iredalei were taken out of their shells using a knife. during transportation, the meat and intravenous fluid were gathered in a zippered storage bag and kept on ice. a mortar and pestle were then used to defrost and homogenize the samples. after being moved to their individual crucibles, the homogenates were weighed at 25 grams apiece. after being dried at 105 °c in a memmert drying oven, the oyster samples were moved to a muffle furnace and heated for at least 24 hours at an increasing temperature of 450 °c to 500 °c. a desiccator was then used to chill the samples. two milliliters of nitric acid were added to each oyster sample, which was then heated on a hot plate until it dried. to get carbon-free ash, this process was carried out at least twice. the samples were cooked on a hot plate with ten milliliters of 1n hydrochloric acid until all of the ashes had been dissolved. following a 2.5 millimeter pore size whatman grade 5 filter, the materials were moved to a 25 milliliter volumetric flask. detection of heavy metals: a flame atomic absorption spectrophotometer (agilent aa55 my1546001) was used to measure the levels of cu, pb, and zn in the filtered digests. the stock standard solution was diluted to a concentration of 1000 micrograms per milliliter (µg/ml) or parts per million (ppm) in order to create all of the heavy metal standard solutions. the stock solution was then further diluted into concentrations of 0.300, 0.500, 0.700, 1.000, and 1.500 µg/ml to create the working standard solutions. every result was expressed in parts per million or milligrams per liter. analytical grade substances were used in this investigation. a certified chemist examined and verified the study. five (5) replicates of each of the two (2) sets of samples were examined. interpretation of results: the mean and standard deviation were used to determine whether the results fell within the range of the international shellfish safety and sanitation standards, which are regulatory limits established by the food and drug administration and the european union. results and discussions the results showed that the zn (mean = 3.35 mg/l; sd = 0.13), cu (mean=10.44 mg/l; sd = 2.95), and pb (mean = 0.01 mg/l; sd = 0.11;) in the municipality of dumangas were all within the acceptable levels established by the international shellfish safety and sanitation standard (table 1). the c. iredalei samples were taken from march 31 and april 7, 2023. on the other hand, pb (mean = -0.09 mg/l, s=0.02), cu (mean = 10.14 mg/l, sd = 0.95), and zn (mean = 3.21 mg/l, sd = 0.10) were all within the permitted limits established by the international shellfish safety and sanitation standard in the town of barotac nuevo. table 1: concentrations of heavy metals in c. iredalei collected from dumangas, iloilo, philippines heavy metal accepted value concentration (mg/l) mean sd interpretation pb 1.5 0.01 0.12 within accepted values cu 100 10.44 3.23 within accepted values zn 150 3.35 0.14 within accepted values pa ge 13 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 11-14, 2025 table 2: concentrations of heavy metals in c. iredalei collected from barotac nuevo, iloilo, philippines heavy metal accepted value concentration (mg/l) mean sd interpretation pb 1.5 -0.09 0.02 within accepted values cu 100 10.14 1.04 within accepted values zn 150 3.31 0.11 within accepted values discussion zinc, copper, and lead in c. iredalei that were gathered from the municipalities of dumangas and barotac nuevo, iloilo met the the international shellfish safety and sanitation standard’s permissible limits. these heavy metals’ comparatively low levels in c. iredalei can be ascribed to the sampling locations’ seclusion from mining operations and industrial industries. the farms where the c. iredalei were gathered and isolated from other rivers in the area. according to noman et al. (2022), contemporary mining and farming methods have the potential to contaminate aquatic biological systems, which in turn may affect the levels of heavy metals in aquatic invertebrates like c. iredalei. zinc and copper were the most prevalent metals found because of cu and zn-based agricultural products that are widely used in nearby shrimp and fish-rearing ponds. the levels of these heavy metals were found to be consistent with the findings of pakingking et al. (2022) in their examination of slipper oyster farming areas of the cogon and palina rivers and cabugao bay (in roxas city and the municipality of ivisan, respectively, capiz province, western visayas, philippines). concentrations of lead, cadmium, mercury, and chromium, meanwhile, were below the legal thresholds established by the us and the eu. the food and drug administration. additionally, research by ochoa et al. (2013) found that the concentrations of heavy metals in oysters cultivated in catalonia, spain’s ebro delta were largely similar to those found in earlier studies conducted in various unpolluted areas. this suggests that the higher exchange rate with marine water in bays where oysters are grown results in a decrease in metal levels. since zinc and copper are regarded as necessary nutrients for the typical growth and development of c. iredalei, the comparatively greater quantities of these elements are to be expected (ochoa et al., 2013). according to pan and wang (2009), aquatic invertebrates contain copper, and the bioaccumulation of this element might rise in direct proportion to the size of the animal. natural cofactors in enzymatic processes within cells are zn and cu, which occur as ions (zn2+ and cu2+), and high levels of these metals are obtained by c. iredalei through their diet. because pb has been frequently linked to human poisonings, periodic monitoring of harmful metals in oysters should continue even though the concentration was below permissible levels (balali-mood et al., 2021). according to this study, c. iredalei can be safely consumed because heavy metals like lead, copper, and zinc were within permissible bounds. however, ongoing observation is necessary. only the quantity and interpretation of lead, copper, and zinc from c.iredalei samples collected between march 31 and april 7, 2024, from the municipalities of dumangas and barotac viejo, iloilo, are covered by the findings. conclusion as of the last week of march and the first week of april 2024, the levels of lead, copper, and zinc in c. iredalei from dumangas and barotac nuevo were within the permissible range established by the international shellfish safety and sanitation standard. in terms of heavy metal intoxication during those months, the c. iredalei samples were safe to eat. it is advised that heavy metals, such as cu, pb, and zn, be continuously monitored in c. iredalei, not just in dumangas and barotac nuevo, iloilo, but across the region. measuring the concentrations of chromium, iron, and cadmium might also help future studies build on the current findings. references anandkumar, a., nagarajan, r., prabakaran, k., bing, c. h., & rajendran, r. 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(2022). bioaccumulation and health risk assessment of heavy metals in slipper-shaped oysters (magallana bilineata) from barotac nuevo and dumangas, iloilo [bachelor’s thesis]. university of san agustin. pa ge 1 pa ge 15 american journal of life science and innovation (ajlsi) phytochemical analyses and cytotoxicity activity using artemia salinaartemia salina (brine shrimp) lethality assay of the caladium bicolorcaladium bicolor and alocasia sanderianaalocasia sanderiana ethanolic leaf extract barnuevo, a. e.1, abkilan, m. j. m.1, agan, f. a. a. s.1, almoete, m. a. b.1, bastian, j. a. l.1, brigola, s. d. d.1, celiz, m. a. f. p.1, contreras, e. b. d.1, cuizon, b. l. a.1, dalaga, e. e. a.1, perez, j. g.1, jamili, j. j.1, penaverde, f. v.1, leong-on, m. s.1* volume 4 issue 2, year 2025 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v4i2.5052 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: april 12, 2025 accepted: may 16, 2025 published: october 14, 2025 even though a lot of research has previously been done on both c. bicolor and a. anderiana, more has to be done to assess the phytochemicals and cytotoxic potential (lc50 value) of their leaves. this work sought to identify the phytochemicals and cytotoxic activity of crude ethanolic leaf extracts of c. bicolor and a. anderiana using the a. salina (brine shrimp) lethality assay. during the phytochemical analysis, the leaves were boiled in distilled water and mixed with various reagents. the colors were examined to determine whether the various phytochemicals were present or not. a. salina cysts in saltwater were permitted to hatch in order to perform the cytotoxicity. the ten (10) nauplii were moved to various crude ethanolic leaf extract concentrations. the sixth (6th) hour death of nauplii was used to calculate the percent mortality rate. according to the results, the leaves of c. bicolor tested positive for flavonoids, terpenoids, tannins, and saponins. on the other hand, c. bicolor leaves tested negative for alkaloids and steroids. the presence of cardiac glycosides, terpenoids, and tannins in a. anderiana leaves was found to be positive. the leaves of a. anderiana, however, tested negative for alkaloids, steroids, flavonoids, and saponins. on the sixth (6th) hour, the lc50 value of a. anderiana crude ethanolic leaf extract was 4,712.4 μg/ml, whereas that of c. bicolor was 874,028.4 μg/ml. in the sixth hour, neither extract was cytotoxic. the phytochemicals and cytotoxic levels of the crude ethanolic leaf extracts of c. bicolor and a. anderiana were determined in this study. the findings can be applied to future research on the anti-inflammatory, antibacterial, antioxidant, and anti-diabetic bioactivities of c. bicolor and a. anderiana, which may yield non-cytotoxic medications. keywords alocasia sanderiana, brine shrimp lethality assay, caladium bicolor, cytotoxicity, phytochemical analyses 1 college of pharmacy and medical technology, university of san agustin, iloilo city, philippines * corresponding author’s e-mail: mleongon@usa.edu.ph introduction over the years, medicinal plants has provided a notable edge over a variety of ailments. phytochemicals from medicinal plants were thought to be safe, bioactive, and biodegradable. among many other pharmacologically significant activities, laboratorybased in vitro investigations have connected these substances to antioxidant, anti-inflammatory, and antidiabetic qualities (mapfumari et al., 2022). even at low concentrations, phytochemicals found in medicinal plants have demonstrated varying degrees of efficacy against bacteria, making them a viable reservoir of therapeutic characteristics (maharaj et al., 2022). in order to identify different classes of phytoconstituents present in different parts of the plant for the purpose of drug discovery, phytochemical screening is a scientific process that involves analysis, examination, extraction, and experimentation. the active components can then be taken for further study and research (sharma et al., 2022). apart from phytochemical analysis, which was employed as a preliminary test to screen for the therapeutic properties of plants, cytotoxicity assays were employed to determine the capacity of particular chemicals or mediator cells to consume living cells. the cytotoxicity test employed in this study is the brine shrimp lethality test (bslt). the toxicity test is based on the number of brine shrimp that die after being exposed to varying concentrations because brine shrimp are cytotoxic to a variety of chemicals and natural compounds. in traditional medicine, c. bicolor and a. anderiana are frequently used to cure a variety of conditions, such as jaundice, boils, snake bites, and hyperglycemia (arbain et al., 2022). however, according to research by ezea (2022), c. bicolor, also referred to as “ede umuagbara,” is a wild cocoyam found in southeast nigeria. numerous species of alocasia have been found to have medicinal uses, including acute toxicity tests and anti-inflammatory, anti-diabetic, antihyperglycemic, antioxidant, antibacterial, antiparasitic, and anti-cancer effects (arbain et al., 2022). despite the fact that both c. bicolor and a. anderiana have been the subject of numerous investigations, more research is required to assess the phytochemicals’ cytotoxicity. nevertheless, there was a gap in determining the extracts’ lethal dose. only the widely recognized phytochemicals have been the subject of the few research that have been conducted on both plants for the phytochemical analysis. the presence of other phytochemical kinds in the plants used in this investigation has not yet been investigated. the study sought to identify the phytochemicals and cytotoxicity activities using the brine shrimp lethality assay of c. bicolor and a. anderiana. the study’s specific goal was to identify the phytochemicals such as tannins, saponins, terpenoids, flavonoids, steroids, and alkaloids pa ge 16 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 15-19, 2025 found in the leaf extracts of c. bicolor and a. anderiana. the study also determined the degree of cytotoxicity of c. bicolor and a. sanderiana, as well as the median lethal concentration (lc50) of their crude ethanolic leaf extracts on the sixth hours. the results of the phytochemical analyses and cytotoxicity activity are useful to potential pharmaceutical researchers that will use the plant-derived constituents of c. bicolor and a. sanderiana for future bioactivity screening. materials and methods research design the phytochemical analyses of the two plants’ crude ethanolic leaf extracts were described using a descriptive study design. however, an experimental design was employed to use the various amounts of crude ethanolic leaf extracts to evaluate the degrees of cytotoxicity. research setting at a private institution in iloilo city, the phytochemical and cytotoxicity investigations were carried out utilizing the phytochemical screening (color test) and brine shrimp lethality assay. ethical considerations the accredited ethics review committee of a private university had reviewed the research. data collection procedures plant identification and collection: the leaves of c. bicolor (figure 1.) and a. sanderiana (figure 2) were gathered from buhang jaro in iloilo city and balabag in pavia, figure 1: photograph of c. bicolor plant figure 2: photograph of a. sanderiana plant respectively. for accurate identification, the department of agriculture identified the plant samples. the mature leaves that were gathered were pest-free and devoid of any dry or browning areas (leong-on, 2022b). phytochemical analyses using conventional protocols, tannins, saponins, terpenoids, cardiac glycosides, flavonoids, steroids, and alkaloids were screened. leong-on (2020) mentioned the works of tariq et al. (2012) and tiwari et al. (2011), and all of the processes were taken from those works (leongon, 2022c). test for tannins (ferric chloride test) a 0.5g of dry leaves were boiled for three minutes in a test tube with 20 ml of distilled water. after cooling, the mixture was filtered. three milliliters of filtrate were then mixed with five drops of 0.1% ferric chloride. the presence of tannins was detected by the development of a brownish-green to blue-black coloring. test for terpenoids (salkowski test) a 0.5g of dried plant material was dissolved in 10 ml of distilled water. after cooling, the mixture was filtered. a layer was created by adding 0.75 ml of concentrated sulfuric acid after 1.25 ml of extract and 0.5 ml of chloroform had been combined. the presence of terpenoids was detected by the interface turning reddish-brown. test for flavonoids (aluminum chloride colorimetric technique) a 0.5 gram of dried plant material was added to 20 milliliters of distilled water and brought to a boil. after cooling, the mixture was filtered. three drops of a 1% aluminum solution were added to three milliliters of filtrate. flavonoids were detected by the emergence of a yellow tint. test for steroids (salkowski test) one milligram of crude plant extract was dissolved in ten milliliters of chloroform. the test tube was then filled by the sides with an equivalent volume of pure sulfuric acid. the test tube’s top layer glowing red and the sulfuric acid layer changing yellow with green fluorescence were signs that steroids were present. test for alkaloids (wagner’s test) a0.5g of dried plant material was cooked in 10 ml of distilled water. after cooling, the mixture was filtered. next, three milliliters of filtrate were mixed with three drops of wagner’s reagent. a white precipitate appeared, indicating the presence of alkaloids. extraction of plant compounds each plant’s fresh leaves were cleaned, dried, and then pulverized into a powder. specimen vials were then used to keep the powdered leaves. the extractant pa ge 17 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 15-19, 2025 employed was ninety-five percent (95%) ethanol at room temperature. the extractant was poured until it was one inch higher than the plant material’s overall height. to prevent the plant material from oxidizing, a black cloth was placed over the container holding the mixture. for 48–72 hours, the extract was homogenized, and it was stirred every 8–12 hours. a fresh cloth or filter paper was then used to filter the extract. evaporation using a rotary evaporator set at 40–45 degrees celsius was used to dry and remove the extractant. after that, half of the extract was put on a petri dish and covered with gauze to dry it (leong-on, 2022a). before being utilized for a cytotoxicity assay and a test for steroids, this was left for three to four days. cytotoxic analyses using brine shrimp lethality assay to ascertain the plants’ potential for cytotoxicity, they were analyzed using the brine shrimp lethality assay. compared to more complex and costly in-vivo and invitro experiments, this bioassay was able to detect a wide range of bioactivity that was present in the extract. preparation of seawater thirty-eight grams (38 g) of rock salt was dissolved in 1 l of distilled water. hatching of brine shrimps for this technique, brine shrimp eggs, also known as artemia salina leach, were gathered. the prepared seawater was poured into a shallow rectangular dish, filling it 34% of the way. the jar was filled with fifty milligrams (50 mg) of brine shrimp cysts. black cartolina paper was used to darken a section of the container. for two days, the nauplii cysts were let to hatch in the seawater tank and grow into nauplii-like adults. the nauplii without eggshells were gathered in the lit section of the tiny tank after the freshly born shrimp were attracted to a light source. to make the fresh, pure saltwater more visible, these were pipetted and filtered. for every sample concentration, ten (10) nauplii were employed (suryawanshi et al., 2020; leong-on, 2022). preparation of test samples every sample concentration was measured using small container cups. after adding 100 mg of the sample to 5 ml of the produced saltwater, various concentrations of crude ethanolic leaf extracts, ranging from 0 to 10 mg, were added to the cups. one drop of seawater was added to the container for 0 mg. each addition contained 1 mg of the plant material, and the solution was utilized for the remaining concentrations (leong-on, 2022). cytotoxicity assay five milliliters of prepared saltwater were added to the cup with the dry extract and swirled. by pouring a drop of seawater to a white spoon and counting the brine shrimp nauplii until there were ten, ten were added to each cup containing the plant extract. after removing superfluous water, two drops containing ten brine shrimp nauplii were introduced to the cup containing the ten nauplii together with five milliliters of seawater and extract. their food, 5 ml of saltwater, was mixed with 3 mg of measured yeast in a different container. a single drop of the suspension was added to each cup. the light was used to preserve these cups. the nauplii were fed using the yeast solution. after six hours, the number of dead shrimp was counted under a magnifying lens, and the percentage of mortality was then calculated. recording of observations every six hours, the quantity of dead brine shrimp was tallied, and the subsequent observations were contrasted with the test control. the brine shrimp’s mortality percentage was determined by counting the number of dead shrimp each hour (kale et al., 2019). % mortality = (number of dead nauplii)/(number of live nauplii) using brine shrimp lethality assays of c. bicolor and a. sanderiana crude ethanolic leaf extracts, this study created three (3) trials and five (5) replicates to guarantee the validity and reliability of the procedures to ascertain the phytochemical and cytotoxicity analyses. the expert validated the results. statistical tool after calculating the percent mortality (%), the median lethal concentration (lc50) was determined six hours later using the cytotoxicity bioassay instrument. the lc50 value was found using the probit analysis. waste disposal toxic materials or residues were disposed of in receptacles designated for hazardous chemical wastes and tightly sealed to prevent leaks. dry infectious garbage was placed in a red container, while wet infectious waste was placed in a yellow bin (nandy et al., 2022). results and discussions the results indicated that the presence of tannins with a brownish-green solution and saponins with a hazy appearance was detected. there were also flavonoids with a light yellow solution, and terpenoids with a light red-brown solution. nevertheless, c. bicolor tested negative in the test for alkaloids, which had a clear, light brown solution, or steroids, which had a dark green solution (table 1). regarding a. sanderiana leaves, it showed positive results for the presence of terpenoids with a light red solution, and tannins with a brownish-green solution. with a clear, light brown solution devoid of foam, however, it tested negative for saponins. a clear, purple solution was produced, and the results were negative for flavonoids as well. alkaloids have a clear, light brown solution and steroids have a dark green solution, hence those tests are negative (table 1). pa ge 18 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 15-19, 2025 table 2: cytotoxicity of crude ethanolic of c. bicolor and a. sanderiana leaf extract using brine shrimp (a. salina) assay concentrations (mg/5ml) % mortality of a. salinaa. salina in c. bicolorc. bicolor (mean+sd) % mortality of a. salinaa. salina in a. a. sanderianasanderiana (mean+sd) 0 0.0 + 0.00 1.6 + 0.08 1 1.0 + 0.00 2.2 + 0.06 2 1.0 + 0.00 2.2 + 0.12 3 1.0 + 0.00 3.2 + 0.18 4 1.2 + 0.45 3.0 + 0.10 5 1.4 + 0.55 3.6 + 0.03 6 1.2 + 0.45 3.8 + 0.12 7 1.4 + 0.55 3.2 + 0.12 8 1.2 + 0.45 3.6 + 0.08 9 1.4 + 0.55 3.8 + 0.06 10 1.6 + 0.55 4.2 + 0.06 lc 50 874,028.4 µg/ml 4,712.4 µg/ml both c. bicolor and a. sanderiana have saponins and tannins. these compounds from wrightia tinctoria, euphorbia hirta, thespesia populnea, and cassia alata were demonstrated efficacy against bacillus subtilis and pseudomonas aeruginosa at a dose of 0.5 µg (rji et al., 2019). both c. bicolor and a. sanderiana contain terpenoids. these compounds are known to possess antiviral, antifungal, antimicrobial, and antiparasitic qualities. they also have anti-inflammatory, antiviral, and antioxidant lower the risk of vascular diseases (ullah, 2020) and cancer chemopreventive effects (ramteke et al., 2021). both c. bicolor and a. sanderiana have been found to contain cardiac glycosides. this compound had been shown to increase cardiac output. it was discovered that c. bicolor contains flavonoids. flavonoids are effective in preventing the growth of tumor cells and inducing the activation of several vital detoxification enzymes. the cytotoxicity of plants varies according to their constituent parts and the extractant used. when the lc50 value was 500–1000 μg/ml, the cytotoxic activity was deemed mild. when the lc50 result falls between 100 and 500 μg/ml, it indicates moderate toxicity; when it falls between 0 and 100 μg/ml, it indicates strong toxicity. when the lc50 value exceeds 1000 μg/ml, it is classified as non-cytotoxic (nguta et al., 2013). strongly poisonous plants may provide new scaffolding for the development of anti-cancer medications. conversely, those with low toxicity might be excellent candidates for the creation of food supplements, nutraceuticals, or herbal medications. c. bicolor exhibited less cytotoxic activity than a. sanderiana. however, both crude ethanolic leaf extracts were non-cytotoxic because their lc50 values were greater than 1000 μg/ml. non-cytotoxic medications may be derived from plants such as c. bicolor and a. sanderiana. when compared to chemically manufactured drugs, natural products may have fewer adverse effects or be non-cytotoxic, which could explain their growing popularity. the study’s results were accurate only when the crude ethanolic leaf extracts of c. bicolor and a. sanderiana death rate was calculated at the sixth hour and lc50 values were determined. the mature leaves were gathered from san isidro, jaro, and balabag, pavia, iloilo city, philippines, at 6:00 in the morning. this investigation served as the foundation for additional bioactivity testing of crude ethanolic leaf extracts of a. sanderiana and c. bicolor. conclusions the phytochemicals found in c. bicolor leaves included flavonoids, terpenoids, cardiac glycosides, tannins, and saponins; alkaloids and steroids did not test positive. tannins,and terpenoids are found in a. sanderiana leaves. alkaloids, steroids, flavonoids, and saponins all table 1: phytochemical analyses of the c. bicolor and a. sanderiana leaves plant tannins saponins terpenoids flavonoids steroids alkaloids c. bicolor positive positive positive positive negative negative a. sanderiana positive negative positive negative negative negative negative control negative negative negative negative negative negative the c. bicolor’s lc50 value was 874,028.4 μg/ml whereas the a. sanderiana was 4,712.4 μg/ml (table 1), according to a. salina nauplii death rates. at the sixth hour, both the ethanolic leaf extracts were not cytotoxic. pa ge 19 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 15-19, 2025 had negative results. the lc50 value for c. bicolor was 874,028.4 μg/ml but the value for a. sanderiana was 4,712.4 μg/ml,. this outcome makes it clear that on the sixth (6th) hour, neither the crude ethanolic leaf extract of c. bicolor nor a. sanderiana was cytotoxic. the study’s findings can be applied to future research on c. bicolor and a. sanderiana’s various bioactivities, including their antiinflammatory, antioxidant, antibacterial, and anti-diabetic properties. references arbain, d., sinaga, l. m. r., taher, m., susanti, d., zakaria, z. a., & khotib, j. (2022). traditional uses, phytochemistry and biological activities of alocasia species: a systematic review. front pharmacol., 24(13), 849704. https://doi.org/10.3389/fphar.2022.849704. ezeaa, i. b. (2022). evaluating the potential of wild cocoyam (caladium bicolor) for citric acid production in a submerged culture of aspergillus niger. bulletin of the national research centre, 46(84), 1-6. https://doi. org/10.1186/s42269-022-00776-2 leong-on, m. s. g. (2022). module on cytotoxicity screening of plant extract using brine shrimp (artemia salina) assay. university of san agustin. leong-on, m. s. g. (2022a). module on extraction of plant compounds. university of san agustin. leong-on, m. s. g. (2022b). module on plant identification, authentication, and collection. university of san agustin. leong-on, m. s. g. (2022c). module on phytochemical analyses of plant extract. university of san agustin. maharaj, a., naidoo, y., dewir, y. h., & rihan, h. (2022) phytochemical screening, and antibacterial and antioxidant activities of mangifera indica l. leaves. horticulturae, 8, 909. https://doi.org/10.3390/ horticulturae8100909 mapfumari, s., nogbou, n. d., musyoki, a., gololo, s., mothibe, m., & bassey, k. (2022). phytochemical screening, antioxidant and antibacterial properties of extracts of viscum continuum e. mey. ex sprague, a south african mistletoe. plants, 11(16). https://doi. org/10.3390/plants11162094. nandy, s., fortunato, e., & martins, r. (2022). green economy and waste management: an inevitable plan for materials science. progress in natural science: materials international, 32(1), 1–9. https://doi.org/10.1016/j. pnsc.2022.01.001 ramteke, a. m., ganatra, s., gour, p., jasudkar, d. p., & tulankar, a. l. (2021). anti-cancer inhibition studies of naturally occurring terpenes using in vitro technique. journal of physics, 1913(1), 012081. https:// doi.org/10.1088/1742-6596/1913/1/012081 raji, p., samrot, a. v., keerthana, d., & karisna, s. (2019). antibacterial activity of alkaloids, flavonoids, saponins and tannins mediated green synthesised silver nanoparticles against pseudomonas aeruginosa and bacillus subtilis. j clust sci, 30, 881–895. sharma, t., pandey, b., shrestha, b. k., koju, g. m., thusa, r., & karki, n. (2020). phytochemical screening of medicinal plants and study of the effect of phytoconstituents in seed germination. tribhuvan university journal, 35(2), 1–11. https://doi. org/10.3126/tuj.v35i2.36183 ullah, a., munir, s., badshah, s. l., khan, n., ghani, l., poulson, b. g., emwas, a. h., & jaremko, m. (2020). important flavonoids and their role as a therapeutic agents. pubmed central, 25(22), 5243. https://doi. org/10.3390/molecules25225243 pa ge 1 pa ge 1 american journal of life science and innovation (ajlsi) in vitroin vitro antihelmintic activity of the hydroethanolic leaf extracts of avicennia marinaavicennia marina (white mangroves) on eudrilus eugeniaeeudrilus eugeniae (african nightcrawlers) malacad1, magallanes1, felizarte1, fenete1, guillermo1, jaspe1, jordan1, legada1, losañes1, marañon1, esteva1, palmos1, barrido1, leong-on1* volume 4 issue 2, year 2025 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v4i2.5052 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: april 12, 2025 accepted: may 16, 2025 published: june 25, 2025 research is needed to determine the avicennia marina hydroethanolic leaf extract is effective against adult e. eugeniae, despite research showing that it can eliminate helminths such as pheretima posthuma. the purpose of this study was to ascertain the hydroethanolic leaf extracts of a. marina antihelmintic effectiveness against adult e. eugeniae in vitro. using e. eugenie adults (6–8 cm in length and 0.2–0.3 cm in breadth), the percentage vermicidal activity of three concentrations of dried crude hydroethanolic leaf extract of a. marina, 25, 50, and 75 mg/ml of distilled water, was assessed every three hours for 12 hours. five replicates, three trials, distilled water as a negative control, and mebendazole as a positive control were all examined. according to the findings, the hydroethanolic leaf extracts of a. marina have shown antihelmintic efficacy against e. eugenie adults in vitro at doses of 50 mg/ml and 75 mg/ml utilizing kruskal-wallis and lsd on the 3, 6, 9, and 12 hours, with a p-value of less than .05. in both concentrations, 50 and 75mg/ml, until the 12-hour exposure to the extract, there was no discernible difference between them. in vitro antihelmintic activity was best demonstrated at 50 mg/ml for 3 hours (mean = 91.1; sd = 26.6), and the in vitro antihelminthic activity was significantly superior to that of the positive control for the same duration (mean = 2.22; sd = 8.61). the results scientifically demonstrated the antihelmintic efficacy of hydroethanolic leaf extract of a. marina against e. eugeniae and provided the foundation for testing against ascaris, trichuris trichiura, and hookworms. keywords antihelmintic, avicennia marina, eudrilus eugeniae, hydroethanolic extract, vermicidal activity 1 medical laboratory science, college of pharmacy and medical technology, university of san agustin, iloilo city, philippines * corresponding author’s e-mail: mleongon@usa.edu.ph introduction according to estimates, 1.5 billion people, or 24% of the world’s population, are affected by helminth infections, making it one of the most common infections in the world (world health organization, 2023). it is especially common in third-world countries, where poor sanitation and hygiene create the perfect conditions for parasitic helminth infection and extensive transmission. between 33.8% to 75.9% of filipinos were afflicted with soiltransmitted helminth (sth) infections in the philippines, with schoolchildren being the most susceptible (mationg et al., 2021). the roundworms ascaris, trichuris trichiura, and hookworms are the most frequent causes. numerous health problems, such as stomach pain, diarrhea, loss of blood and protein, rectal prolapse, and physical and cognitive growth retardation, can be caused by significant helminthic infections in humans (cdc, 2022). over the past few decades, numerous anthelmintic studies have been developed as a result of the increased incidence and prevalence of helminthic illnesses. using roundworm species that are morphologically similar to parasitic species is one strategy. ogen et al. (2017) proposed using the common soil earthworm pheretima posthuma as test subjects because of its morphological similarity to intestinal roundworms. in the philippines, e. eugeniae (african nightcrawlers) are especially common and make excellent subjects for anthelmintic research. additionally, they are helminths, which and intestinal roundworms have comparable anatomy (pueblos et al., 2015; asiimwe et al., 2023; hogade et al., 2014). commercial medications such as albendazole, mebendazole, levamisole, and pyrantel pamoate were frequently used to treat helminth infections in humans (moser et al., 2017). despite their effectiveness, these medications have a number of drawbacks when used over extended periods of time. in their research, kotze et al. (2019) observed that helminth parasites have a propensity to become resistant to some drugs following prolonged therapy. agricultural research, which focused on natural chemicals with anthelmintic action, was therefore very interested in novel natural alternatives. other plants with comparable phytochemical components, including leucaena leucocephala (lead tree), carica papaya (pawpaw), and cayriata auriculata (ear-leafed cayratia), have been the subject of numerous studies and have been shown to be effective anthelmintic agents (kancherla et al., 2019; widaad et al., 2022). in particular, the synergistic anthelmintic effects of flavonoids and tannins mostly functioned by making the nematode cuticle stiffer, which inhibited growth and encouraged death (greiffer et al., 2022). the anthelmintic activity of a. marina is one of the plants that can be investigated. this kind of mangrove can be found in rivers, streams, coastal regions, and the intertidal zones of seabeds. for coastal barangays, it was a natural defense, particularly in remote, impoverished pa ge 2 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 1-5, 2025 areas. the effectiveness of a. marina tree extracts against helminths, including the earthworm pheretima posthuma, has been the subject of a limited number of studies (kondepudi, 2018). furthermore, previous research had shown that phytochemicals like phenolic compounds, alkaloids, flavonoids, sugars, carotenoids, aliphatic alcohols, amino acids, hydrocarbons, fatty acids, tannins, saponins, and terpenes were present (wilfred et al., 2023). in support of this, mughal et al. (2016) also found that a. marina contains phytochemicals proteins, amino acids, flavonoids, phenols, carbohydrates, and alkaloids. accordingly, the phytochemical substances saponins, flavonoids, and tannins were nematicidal chemicals according to research by wilfred et al. (2023) and mughal et al. (2016); d’addabbo et al. (2022). the use of a. marina leaf extract hydroethanolic against adult e. eugeniae has not been studied, despite the fact that it has been scientifically demonstrated to kill helminths like p. posthuma. the overall goal of this work was to ascertain the hydroethanolic leaf extracts of a.marina’s antihelmintic effectiveness against e. eugeniae in vitro. the study’s specific goal was to ascertain the hydroethanolic leaf extracts of a.marina’s in vitro antihelmintic activity against adults of e. eugeniae using varying concentrations (25, 50, and 75 mg/ml) and time (3, 6, 9, and 12 hours) in terms of percent vermicidal activity (va) in comparison to the negative control, distilled water and the positive control, mebendazole (25 mg/ml). in terms of percent vermicidal, the study identified the optimal concentrations (25, 50, and 75 mg/ml) and times (3, 6, 9, and 12 hours) that demonstrated the hydroethanolic leaf extracts of a. marina’s in vitro anthelmintic activity against e. eugeniae adults. the study’s findings gave a. marina’s vermicidal action against e.eugeniae a scientific foundation. materials and methods a quantitative post-test, fully randomized approach was employed in the investigation. the % vermicidal (mortality) effects of a.marina at three different doses (25, 50, and 75 mg/ml) against e. eugeniae adults were evaluated. the study was reviewed by a private university’s recognized ethics review committee. in addition, the study received biosafety and ethics clearance. e. eugeniae were gathered from a vermicomposting region in antique province, a. marina were gathered from the riverbanks in dumangas, iloilo. the test was carried out in antique between february and april of 2024. identification of samples the pictures of trees, fruits, and plant leaves (figure 1) were sent to the department of environment and natural resources for verification. this sample’s taxonomic classification is as follows: kingdom: plantae phylum: tracheophyta class: magnoliopsia order: lamiales family: acanthaceae genus: avicennia species name: marina common name: api-api or white/gray mangrove figure 1: images of the plant a. marina: a. yellowish leaf abaxial surface; b. edge of the leaf (adaxial surface); c. arrangement of leaves (opposite); d. roots (pneumatophores that resemble pencils); e. trunk of a tree (lenticels and smooth bark); f. the crown of a tree figure 2: photograph of a. marina whole plant collection of plant material a. marina leaves were gathered in dumangas, iloilo. samples of plants were gathered 100 meters from the road. the leaves were picked between 6:00 and 7:00 a.m. leaves that were not budding or decaying were used. leaf samples were free of insect bites, trauma, and irregularities. collection was avoided within 3 days after rainfall in the area. identification and collection of e. eugeniae a veterinarian identified the worm. a veterinarian verified the authenticity of e. eugeniae worms that were gathered from vermicomposting facilities in the province of antique. according to the research of akande et al. (2018), the earthworms were measured to be 6–8 cm long and 0.2–0.3 cm wide. when the earthworms were collected, they were alive and moving. following the pa ge 3 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 1-5, 2025 confirmation of the roundworm species, the e. eugeniae worms were cleaned of dirt and debris using a standard saline solution. preparation of a. marinaa. marina extract following collecting, the leaves were carefully cleaned with tap water and then rinsed with distilled water. the leaves were allowed to air dry at room temperature for an hour without exposure to sunlight in a screen bag. the leaves were allowed to air dry before being ground into a fine powder in a blender. one gram of powdered leaves was mixed with five milliliters of 95% ethanol for a duration of seventy-two hours in order to homogenize the leaves. after that, a muslin cloth was used to filter the homogenized leaves. the ethanol was then extracted from the filtrate using a rotary evaporator set to 45 °c. the filtrate was dried in an oven set to 45 °c in order to recover either the powdered or sticky form.to be sure there was no ethanol in the extract, a flame test was conducted. an anthelmintic test was performed on the dried, crude hydroethanolic extract against adult e. eugeniae. ibrahim et al. (2022) modified this process and used it in their studies. antihelmintic assay preparation the kondepudi (2018) method was used to perform the anthelmintic assay. the ethanolic leaf extract was made into a stock solution at varying doses (25, 50, and 75 mg/ ml). a positive control solution containing 25 mg/ml mebendazole and a negative control solution consisting of distilled water were made. each disposable plastic petri dish with a perforated lid and a 5.9-inch diameter was filled with three adult e. eugeniae. each treatment’s 5 ml solution is used to soak them. in accordance with the experiment carried out by bestari et al. (2020), the percentage vermicidal of the helminth was measured at 3-hour intervals for 12 hours. validity and reliability a certified medical technologist verified the study’s findings. to ensure the study’s reliability, three concentrations—25 mg/ml, 50 mg/ml, and 75 mg/ ml—were used in five replicates with three trials. to guarantee the precision of the tests conducted, the devices were also appropriately calibrated. waste disposal the adult e. eugeniae samples, both dead and alive, were disposed of appropriately by filling the petri plates with a 10% hypochlorite solution and securing the arrangement with tape. to guarantee the organisms’ total demise, the sample was submerged for a whole day. after being put in the biohazard bag, the petri plates were disposed of in the proper garbage receptacle. statistical analysis the mean and sd of the results were displayed. using varying quantities of a.marina hydroethanolic leaf extract, the study compared the mean results of vermicidal counts of e. eugeniae taken every three hours for 12 hours using the kruskal-wallis test. statistical significance was defined as a p-value of 0.05. to determine whether there were any notable variations or differences between the treatments, a post-hoc fisher’s least significant difference (lsd) test was also employed. results and discussions table 1 demonstrates that a. marina hydroethanolic leaf extracts at 50 and 75 mg/ml demonstrated antihelmintic efficacy against e. eugeniae. vermicidal activity (va) showed p< 0.05 for e. eugeniae adults on the 3, 6, 9, and 12 hours using kruskal-wallis and lsd. the antihelmintic effects of the 50 mg/ml and 75 mg/5ml doses of a. marina extract did not significantly change from the 3 to the 12 hour. there was no antihelmintic effect from the 25 mg/ ml. 50 mg/ml was the optimal dose and duration that demonstrated in vitro antihelmintic action for the three hours of exposure (mean = 91.1; sd = 26.6), and it was noticeably superior to the positive control during that period (mean = 2.22; sd = 8.61). table 1: anthelmintic activity of a. marina hydroethanolic leaf extract against e. eugeniae taken at different hours using different concentrations in terms of % vermicidal activity(va) treatments time (hours) 3 6 9 12 %va %va %va %va m sd m sd m sd m sd 25 mg/ml a8.89 15.4 a20a 24.6 a33.3a 30.9 a46.7a 35.2 50 mg/ml b91.1a 26.6 b93.3a 25.8 c95.6a 17.2 c97.8a 8.61 75 mg/ml b91.1 a 26.6 b93.3 a 25.8 c93.3 a 25.8 c100 a 0.0 negative-control (distilled water) a0.0 a 0.0 a0.0 a 0.0 a0.0 a 0.0 a15.6b 30.5 positive-control mebendazole) 25 mg/ml a2.22 a 8.61 a6.67 a 13.8 b51.1 ab 39.6 b84.4ab 27.8 note: letters before the numbers mean comparison with the treatments; while letter after number means comparison among the hours pa ge 4 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 1-5, 2025 discussions significant anthelmintic activity was demonstrated by the hydroethanolic leaf extract of a. marina at concentrations of 50 and 75 mg/ml in terms of percent vermicidal. significant anthelmintic action was demonstrated by concentrations of 50 mg/ml and 75 mg/ml in terms of percent vermicidal, with the peak activity occurring after 12 hours. our results suggest that the phytochemical compounds present in a. marina, including alkaloids, flavonoids, sugars, carotenoids, aliphatic alcohols, amino acids, hydrocarbons, fatty acids, phenolic compounds, tannins, saponins, and terpenes, may have contributed to the anthelmintic activity (wilfred et al., 2023; mughal et al., 2016). greiffer et al. (2022) found that flavonoids and tannins particularly inhibit helminth proliferation and cause helminth mortality. in addition to a. marina, several plants were reported to possess these chemicals, including l. leucocephala (widaad et al., 2022). because of its physical similarity to diseased helminths, e. eugeniae was used in the study as a reference for creating synthetic anthelmintic treatments. notably, the strongest anthelmintic activity against e. eugeniae was obtained at concentrations of 50 mg/ml and 75 mg/ml. the results of kondepudi’s (2018) investigation are consistent with the observed trend, which shows that higher concentrations exhibit stronger anthelmintic activity. the highest level of anthelmintic activity of a. marina was similarly seen at a concentration of 75 mg/ml to p. posthuma. as demonstrated by the notable impacts on mortality employing varying doses of hydroethanolic leaf extracts of a. marina, the results gave a scientific foundation for the vermicidal actions of a.marina against e. eugeniae. for future research on helminths or organisms that share a biological morphology with african nightcrawler worms, such as hookworms, trichuris trichiura, and roundworms (ascaris suum), a concentration of 50 mg/ml may be utilized as a reference. given the persistently high incidence of helminth infections in the philippines, the experimental findings are positive about the best use of readily available plants with anthelmintic qualities. the results show that natural products are reliable and strong enough to serve as a pointer to other possible sources of helminth infection treatment. this is especially true for higher concentration therapies. furthermore, by placing more significance and focus on the product’s source, this may also have positive economic effects. last but not least, our research may help address the ongoing concern about the spread of drug-resistant parasites (fissiha & kinde, 2021) by introducing a novel antidote that significantly slows the development of undesired resistance. only the hydroethanolic leaf extract of a. marina at concentrations of 25 mg/ml, 50 mg/ml, and 75 mg/ ml and the anthelmintic assay method produced these results. different results can be obtained by using a different extractant, concentration, technique, or part of the plant. conclusion comparing the extract concentrations at 50 mg/ml and 75 mg/ml to the negative control (distilled water), there was notable antihelmintic activity based on the percentage of vermicidal activity, from 3 to 12 hours. on the 3rd hour, the optimal concentration and duration of in vitro anthelmintic action were 50 mg/ml. on the 3rd hour, the extract doses of 50 mg/ml and 75 mg/ml exhibited similar vermicidal effectiveness to the positive control, mebendazole. the results scientifically demonstrated the antihelmintic efficacy of hydroethanolic leaf extract of a. marina against e. eugeniae and provided the foundation for testing against ascaris, trichuris trichiura, and hookworms. since the study demonstrated the plant’s effectiveness against e. eugenia in vitro, it is advised that a study on in vitro human helminths be carried out utilizing the crude ethanolic extract of a. marina leaves. the phytochemical of a. marina leaves should be used to identify or isolate the particular components that give it its anthelmintic effects. references akande, a. a., aboaba, s. a., & flamini, g. 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(2023, january 18). soil-transmitted helminth infections. https://www.who. int/news-room/fact-sheets/detail/soil-transmittedhelminth-infections pa ge 1 pa ge 27 american journal of life science and innovation (ajlsi) impact of bulky & liposomal secukinumab on lymphocytes of psoriatic patients, to determine the improvement in bioavailability of secukinumab nanolipo gazala layas1*, mohammad isreb2, nader ghaderi3, fanila shahzad1, pouria akhbari4, diana anderson1, andrew wright3, mojgan najafzadeh1 volume 4 issue 1, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v4i1.3268 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: june 24, 2024 accepted: july 30, 2024 published: june 15, 2025 psoriasis is an immune-mediated disease affecting the skin and joints. biologic treatments are often used in moderate to severe cases, administered as a monotherapy or in combination with other treatments. liposomes are versatile and can enhance drug retention, reducing systemic side effects, and are used for therapy and research. this research study aimed to investigate the geno toxicity of secukinumab, an ideal biologic treatment for psoriasis (a human igg1k antibody, anti-il17a), in bulk and liposome nanoparticles on the lymphocytes of psoriatic patients in comparison with healthy persons. geno toxicity of secukinumab in bulk and liposomal form was evaluated and compared by using the comet and micronucleus assays. from assays, it was demonstrated that secukinumab in both forms did not exhibit geno toxicity and reduced dna damage following the treatment of psoriatic patient lymphocytes and healthy individual’s lymphocytes with secukinumab bulk and liposomes format. secukinumab used (2.1 and 2.8 µg/ml) with two different concentrations, and effectively decreased dna damage induced by h2o2 in both groups to almost the negative control level. secukinumab bulk and liposome form markedly reduced the h2o2-induced damage and proficiently diminished its adverse effects both in the comet (p<0.0001) and micronucleus as-says (p<0.01). overall, secukinumab in both forms showed anti-genotoxic and protective effects by expressing its potential to reduce dna damage produced by oxidative stress and it was observed that it would not induce any further damage in the lymphocytes of healthy individuals and patients. keywords geno toxicity, micronucleus assay, oxidative stress, psoriasis, secukinumab introduction psoriasis was initially characterized as a dermatologic disorder primarily affecting epidermal keratinocytes. however, recent studies have shed light on its immunemediated pathogenesis, making it one of the most prevalent immune-mediated conditions (kadam et al., 2010). psoriasis is a chronic autoimmune disease characterized by systemic manifestations and inflammatory changes that predominantly affect the skin and joints. the disease can significantly compromise the overall quality of life of those affected. hyper proliferating body patches are primarily caused by autoimmune illness, a disorder marked by an overactive immune system. psoriasis is a multifaceted disease, and its pathogenesis is not yet fully understood. despite the availability of several treatment options, psoriasis remains a challenging disease to manage. therefore, further research is necessary to understand the disease’s pathogenesis and develop more effective treatments to improve patients’ quality of life (rendon & schäkel, 2018). throughout the world population, up to 2.5% of psoriasis vulgaris (chronic skin disorder) prevalence rate was reported (elder, 2018). various psoriatic clinical phenotypes are highly regarded as chronic plaque (psoriasis vulgaris) with an accountancy of 90% cases (griffiths & barker, 2007). genetically mutated individuals are believed to have high rate of susceptibility of psoriasis vulgaris through environmental factors, as a result of which adaptive and innate immune pathways activates inflammatory responses. lesions are highly visible, directing to a substantial proportion of physical, emotional, social and psychological disability in the patients (nestle et al., 1994; chen et al., 2018). the cause of psoriasis is still unknown. according to a study, erythrocyte-superoxide dismutase activity was lower in psoriasis patients and blood levels of malondialdehyde and nitric oxide end products were greater. the pathogenesis of psoriasis is not fully elucidated; three factors are responsible for the disease pathogenicity including; t-cells, tumor necrosis factor-alpha (tnfα), and dendritic cells (matsuzaki & umemura, 2018) . while its major clinical findings are the presence of psoriatic patches on epidermal layer and deep down histologically to the dermis, which revealed that it is not restricted to the epidermal layer. the main member of the il-17 group, il-17, interacts with several immune cell types and keratinocytes in the dermal layer of the skin, making psoriasis more pathogenic. there are six components of il 17 (a-f). consequently, the human t cell has number of subsets, and one of these cells is th17, which produces the cytokine il-17. the research findings from animal 1 school of life sciences, university of bradford, bradford, bd7 1dp, uk 2 school of pharmacy, university of bradford, richmond road, bradford, bd7 1dp, uk 3 bradford teaching hospitals nhs foundation trust, st luke’s hospital, little horton lane, bd5 0na, uk 4 institute of biomedical and clinical science, college of medicine and health, university of exeter, ex2 5dw, uk * corresponding author’s e-mail: layas698@outlook.com pa ge 28 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 27-35, 2025 knockout models proposed that il-17 and th17 play a major role to cause the pathogenesis of this disease. by stimulating keratinocyte multiplication in the epidermis, th17 cytokines il-17, il-21, and il-22, which are released by activated t cells, the adaptive immune system is maintained and psoriatic inflammation is sustained. il-17a has been found to be used in the treatment of moderate to severe psoriasis plaque with an improved efficacy. the inhibitory mechanism of il-17a works swiftly and induces sustained responses with a significant safety assurance profile. through the clinical severity of the pathological condition, treating parameters of psoriasis are determined, which are based on numerous factors, for instance the dimensions of affected area, the pasi grades, and the life quality index of dermatology. mild conditions can be addressed with topical therapies, whereas moderate to severe cases may necessitate phototherapy or systemic therapy, comprising biological or non-biological interventions or a combination of both. secukinumab, also known as ain457, is a biological medication that functions as an antiil17a monoclonal antibody (mab). it is employed to treat moderate and severe psoriasis, as well as psoriatic arthritis (wagener et al., 2013). chinese hamster ovary (cho) cells were used to manufacture secukinumab, an igg1/k-class property (molden et al., 2021). secukinumab has been shown to have a superiority over other biological treatments in terms of achieving severity index pasi 75 (bagel et al., 2021). also, it has the ability to sustain its effectiveness over time contrasted to additional biological treatments (bissonnette et al., 2018). neutralized and binding aspects of the cytokine interleukin-17a (il17a) are responsible to improve the secukinumab work efficiency for the treatment of psoriasis. secukinumab is a fully human mono-clonal antibody that selectively binds and neutralizes the pro-inflammatory cytokine inter-leukin-17a (il-17a31009130) without producing any effect on il-17f and th17 cells (berg et al., 2021). additionally, it has no direct impact on the th1 cascade. current studies have revealed that increased t-cell abnormalities and oxidative stress (os) are central parameters that lead to the pathogenesis of psoriasis. reactive oxygen species (ros) suppress regulatory t lymphocytes’ (treg) ability to reduce inflammation while promoting the growth and specialization of th1/ th17/th22 cells. successive secretions of inflammatory cytokines, such as tumor necrosis factor-alpha (tnf-α), vascular endothelial growth factor (vegf), interleukin (il)-17, il-22, and interferon-gamma (ifn-γ), stimulate keratinocyte number increasing and angiogenesis (lai et al., 2018; ciążyńska et al., 2021). liposomes are vesicles that contain several thin layered membrane or shells comprised of a phospholipid bilayer covering an insignificant amount of aqueous liquid, used for biotechnological and biomedical roles. on the basis of preparation and its use, they are different in size ranging from several specific nanometers to hundred micrometers (lombardo & kiselev, 2022). bio-logically active compounds of liposomes, such as genetic materials, chelating agents, proteins, enzymes, peptides, vaccines, hormones and antimicrobial agents have been evaluated for patient use. because of its superior mechanical, optical, and electrical capabilities, quantum mechanics, and nanoscale volumesurface area ratio, liposomes are becoming more and more common in sophisticated technology (sercombe et al., 2015; nsairat et al., 2022). because they increase the flow of drugs, they also aid in reducing treatment side effects. this study aimed to determine that how liposomes and secukinumab bulk form affected dna damage in peripheral blood cells from both healthy people and psoriasis sufferers by using the comet assay and micronucleus (cbmn) assay. additionally, the study aimed to determine the best concentration of figure 1: pathogenesis of psoriasis, the cytokines were activated in psoriasis and their further cascades in the skin. interleukin (il)-23 produced by various il-23producing cells induces il-17 production from various il-17-producing cells such as t-helper (th)17, il-17 producing cd8+ t cells (tc17), and innate lymphoid cells type 3 (ilc3). il-17 stimulates keratinocytes to produce inflammatory cytokines/chemokines, which further activate il-23-producing cells and recruit il-17-producing cells and neutrophils pa ge 29 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 27-35, 2025 secukinumab liposome to prepare topically for treating psoriasis in the future. the cellular response to the bulk and liposome forms was investigated and the optimal dose for bulk and liposome forms was obtained. in the presented study, the geno toxicity assays with different end-points were used to assess the impact of secukinumab bulk and liposome forms on lymphocytes from psoriasis patients contrasted to healthy controls (yamanaka, 2021). materials and methods materials secukinumab was obtained from novartis pharmaceuticals (cosentyx). 1, 2-dipalmitoyl-sn-glycero-3-phosphorac-(1-glycerol) sodium salt (dppg), 1,2-dioleoyl-snglycero-3-phsphoethanolamine (dope), purity >99%, cholesterol (chol), methanol, dichloromethane (dcm) were obtained from sigma-aldrich company ltd. (uk). all other reagents used were derived from sigma aldrich u.k. unless otherwise stated. for the preparation of secukinumab solutions, drugs were in distilled water (150mg/ml). a 75μg/ml stock solution was prepared. the prefilled secukinumab syringes (150 mg) were used in this study to produce the liposome format of this biological agent and investigate its comparison with bulk form. the compound was diluted to a suitable concentration for treating the cells in vitro. characterization and production of peptide-loaded liposome liposomes were prepared by using the bangham process, which involved dissolving methanol and dichloromethane (dcm) and methanol (3:1 v/v) with1,2dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt dppg, and (2 mg), 1,2-dioleoyl-sn-glycero3-phsphoethanolamine dope, and cholesterol (2:2:1 molar ratio). the solution was transmitted to a rotary evaporator flask and at 40 °c the respective organic solvent mixture and after six hours of evaporation, the mixture was rehydrated with 10ml distilled water. after that, the films were placed within 1ml of secukinumab for 1 h at 60 °c by employing a bath sonicator (150 w), then the sample exposed through freeze-thaw cycles at -20°c and thawing at 60°c, and then diluted with 75 μg/ml of distilled water. centrifuging the finished mixture at 22,000 x g for 30 minutes at 4 °c, helped to guarantee purification and eliminate any remaining non-encapsulated medicines. the modest size and poly disparity index of the liposome formulations were evaluated by means of dynamic light scattering (dls) with zetasizer zs-90 and model zen 3600. in triplicate form, all measurements were performed. measures were found to be < 150 nanometers. ethical approval the study on the genetic and environmental effects on lymphocytes from different illness situations, such as inflammatory, malignant, and precancerous ones, employing different genetic endpoints, has been authorized by the leeds east research ethics committee (rec) (rec reference number: 12/yh/0464). ethical approval has been given by the subcommittee for ethics in research with human subjects at the university of bradford (reference no.: 0405/8). the research support and governance office bradford teaching hospital nhs foundation granted the re da number: 1202. blood sample collection blood samples were collected from 40 individuals (20 healthy and 20 patients) using the vein-puncture technique, after obtaining consent and following safety measures. basic information was gathered from individuals about health and lifestyle factors using a questionnaire, as shown in table. 1, while healthy individual’s data was presented in table. 2. blood was collected from a patient with moderate psoriasis under systemic treatment; non were taking any biological treatment. table 1: list of patients’ confounding factors c on fo un di ng fa ct or s c at eg or ie s fr eq ue nc ie s (n = 20 ) pe rc en ta ge (n = 20 ) age 21-40 6 30 41-60 7 35 60-80 7 35 gender male 9 45 female 11 55 ethnicity asian 3 15 caucasian 17 85 smoking history ex-smoker 5 25 non-smoker 15 75 table 2: list of healthy controls confounding factors c on fo un di ng fa ct or s c at eg or ie s fr eq ue nc ie s (n = 20 ) pe rc en ta ge (n = 20 age 20-35 14 70 36-50 6 30 gender male 10 50 female 10 50 ethnicity asian 10 50 caucasian 7 35 african 1 5 arab 2 10 smoking history non smoker 19 95 ex-smoker 1 5 cell viability determination cell viability was determined using the cell counting kit 8 (cck8) from vwr, uk. blood samples were added with the chemicals in eppendorf tubes as described earlier for pa ge 30 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 27-35, 2025 the comet assay treatment step and rpmi-1640 medium for the preparation of cell culture. after that tubes were subjected to centrifugation at 3000 rpm (705 g) and the remained precipitates were discarded, and the mass of cells were suspended. following the manufacturer’s procedure, the experimental strategy was carried out and the kit’s contents were applied to the cell suspension. cell viability of ≥80% was considered for use in all experiments. cell treatment and the comet assay complete blood samples from 20 psoriasis patients and 20 healthy persons were collected and supplemented with 10% dmso and rpmi medium and quickly stored at -80 °c. five groups were selected for treatment, in order to conduct the experiment, including an un-treated group (nc) containing the blood sample and rpmi 1640 medium. a positive control containing media, blood, and a total of 10μl, 75μm/ml hydrogen peroxide (h2o2), and two treatment groups with different doses of “secukinumab (2.8µg /ml, 2.1µg/ml) including or excluding h2o2”, and 1000μl of treatment volume were used in the experiment. it was then “centrifuged at 3000 rpm” and incubated at 37°c (tice et al., 2000; najafzadeh & anderson, 2016). using a computer terminal komet 6 software and kinetic imaging (andor technology ltd, belfast) and fluorescence microscope (20x magnification) equipped with ccd camera, 100 cells from each treatment group were fixed using comet assay parameters such as olive tail moment and percentage tail dna. cytokinesis block micronucleus assay (cbmn) fresh blood sample (350 μl) and 130μl of phytohaemagglutinin (pha) were added to 25cm3 vented cap corning flasks containing “4.5 ml rpmi-1640 medium augmented with 1% of penicillin-streptomycin (aminoglycoside antibiotic) and 15% fetal bovine serum (fbs)”. followed by incubation for 24h at room temperature i.e., 37⁰c in the existence of 5% co2 and then the test chemicals secukinumab bulk and liposome, h2o2 were added. for the untreated negative control plain basic medium was used, and as positive controls mitomycin c (mmc) (0.4μm) and h2o2, (75μm) were used. in each flask 30μl of cytochalasin b (cyt b) (1mg/ml) was added at 44h and cultured under the same conditions for another 20h (fenech et al., 2016). using a 40x microscope to score 1000 cells per treatment group, including the nuclear division index and the percentage of binucleated cells, the frequency of micro nuclei (mni) was ascertained (fenech, 2007). the ndi was calculated using the equation eq. a.1: ndi = m1 + 2(m2) + 3(m3) / n where, m1 = mononucleated cells, m2= binucleated cells, m3 = multinucleated cells, n = total number of viable cells scored (fenech et al., 2016). statistical analysis for the determination of significant values, data was analyzed through one-way anova (tofind the difference between results obtained from research) and t-tests to determine the significant values. a value p= <0.05 was considered statistically significant. graph pad prism 8 was used for statistical data analysis. results and discussion results when secukinumab liposomes were tem-analyzed, homogeneous, spherical bio particles with sizes between 100 and 200 nm were discovered. figure 2: (a) tem analysis of the aqueous dispersion of secukinumab, (b) tem imaging the liposomal form of secukinumab figure 3: cytotoxicity of secukinumab on human peripheral lymphocytes after 24 hrs exposure to different concentrations (2.1, 2.8, and 3.751 μg/ml) by using cck8 pa ge 31 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 27-35, 2025 the proliferation rate was computed by dividing the total number of counted cells by the number of living or viable cells. a cell counting kit was used to check the integrity and viability of the lymphocytes, as shown in figure 3. the effects of secukinumab and h2o2 on the lymphocytes from healthy the responses of bulk and liposome forms of secukinumab on lymphocytes of dna from healthy and psoriatic patients treated with h2o2 using “olive tail moment” (otm), standard errors and significance were displayed in the figure 4. using one-way anova, the study showed that h2o2, treatments significantly increased dna damage in cells by increasing otm **p ≤ 0.01in psoriatic and ***p≤0.001 in healthy individuals in comparison to untreated cells. however, secukinumab treatments in the two different forms with or without h2o2 were not significant (ns). in comparison with the positive control treatments, a remarkable reduction in the dna damage was observed after the addition of secukinumab ***p≤0.001 for the bulk in patients without the h2o2, and **p ≤ 0.01 for all the other treatments, as shown in figure 4. figure 4: the effects of secukinumab and h2o2, on the lymphocytes from healthy and patient groups measuring otm (* com-paring the treatments to the without treatments or negative control or nc, ns (non-significant) comparing the groups with different treatments to the nc group, + comparing the groups with different treatments to the positive control or pc) ( n=20 in the healthy control group and 20 in the patient group), ), n/c stands for the negative control, p/c; positive control, bulk; secukinumab bulk, liposome (liposome form of secukinumab), empty; liposomes with no drug, hydrogen peroxide (h2o2) results presented in figure 4 illustrate the responses of bulk and liposome forms of secukinumab on lymphocyte dna from healthy and psoriatic individuals treated with h2o2 using tail dna %, standard errors se and significance. dna damage observed in the patient group was higher compared to the healthy as predicted. the pc revealed a significant rise in the tail dna control cells, ****p ≤ 0.0001. in comparison to the nc, secukinumab treatments—liposomal and bulk, with or without h2o2, were not very important. when compared to pc therapies, the inclusion of secukinumab ++++p ≤ 0.000, considerably reduced the damage, as shown in figure 5. figure 5: the effects of secukinumab and h2o2 on the lymphocytes from 20 healthy and 20 patient groups measuring tail dna% (* comparing the treatments to the nc, ns (non-significant) comparing the treatments to the nc, + comparing the treat-ments to the pc) ( n=20 in the healthy control group and 20 in the patient group), ), n/c stands for the negative control, p/c; positive control, bulk; secukinumab bulk, liposome (liposome form of secukinumab), empty; liposomes with no drug, hydrogen peroxide (h2o2). pa ge 32 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 27-35, 2025 cytokinesis block micronucleus assay (cbmn) the micronucleus assay reveals higher levels of mnis in patients who are not receiving therapy, making it a dependable and appropriate approach for examining dna damage at chromosomal levels. secukinumab 2.1µg/ml in bulk and liposome forms has shown some attenuation of the consequences caused by h2o2, as illustrated in figure 6. figure 6: the average of mni in binc scored per 1000 cells from 5 healthy individuals and 5 psoriasis patients, n=1000. data are expressed as means ± standard error (se).seven treatment groups included the negative control, two positive control groups (0.4µm mmc) (75µм of h2o2), 2.1µg/ml of secukinumab, 2.1µg/ml of secukinumab with h2o2 and the liposome form of both (*** represents p< 0.0001, **p<0.001, *p<0.02, *p<0.018, ns=not significant). ( n=5 in the healthy control group and 5in the patient group), ), n/c stands for the negative control, p/c; positive control, bulk; secukinumab bulk, liposome (liposome form of secukinumab), empty; liposomes with no drug, hydrogen peroxide (h2o2) discussion secukinumab currently is one of the best choices with proper efficacy on psoriasis conditions, even on the increased level of il-23 and jak inhibitors. however, in the case of secukinumab failure, combined therapy would be ideal (damiani et al., 2022). also, secukinumab is a well-known biological therapy considered the most cost-effective bio-logical agent (schweikert et al., 2020) . moreover, a range of side effects linked with these biological compounds, such as paradoxical reactions (prs), which are the result of worsening of immunemediated inflammatory disease (miyagawa, 2022) and increased eosinophilic disorders (bridgewood et al.,, 2022). it is therefore desirable to reduce the con-centration of the antibody in the blood and increase its residency in the psoriatic dermal layer by encapsulation in liposomes. using the comet and cbmn assays, the study examined the effects of “secukinumab bulk and liposome forms” on human peripheral lymphocytes from 20 psoriasis patients and 20 healthy persons. the research examined secukinumab’s possible protective effects on h2o2-induced dna damage in lymphocytes, demonstrating the anti-oxidant properties of both forms and damage that was similar to that of patients who were not treated. our results showed that secukinumab significantly de-creased the percentage of dna damage in lymphocytes in the comet assay (+++ p < 0.001). both groups had considerable levels of dna damage, according to the comet assay, but secukinumab dramatically reduced the effects of h2o2 in both liposomal and bulk forms. the investigation, which used both versions of secukinumab, supplemented with or without h2o2, to treat patient groups and healthy individuals, did not find any statistically significant differences in dna damage. empty liposomes had no significant damage on the cell, which shows that it in itself is safe on cells. according to the study, secukinumab may be able to both protect and lessen the damage that h2o2 causes to dna in both healthy people and psoriasis patients. oxidative stress-induced synthesis and amplification of pro-inflammatory cytokines can be inhibited by the anti-il-17 drug secukinumab (zou & meng, 2021). increased reactive oxygen species (ros) damage dna, lipids, and proteins, as well as signaling molecules and other physiological activities, leading to diseases through oxidative stress. when compared to the negative control, the oxidative stress therapy secukinumab did not significantly damage dna in lymphocytes from either patient group or from healthy individuals. the outcomes were very similar to those of the negative control. this suggests that the concentration of 2.1μg/ml of secukinumab is nongenotoxic to the cells in both groups. hydrogen peroxide is an oxidative stress inducer compound which causes significant amounts of oxidative stress-related dna damage in peripheral lymphocyte cells (najafzadeh et al., 2009; stanić et al., 2016). our results are in accordance with these studies as h2o2 significantly induced dna damage in both subjective groups; healthy individuals and psoriasis patients. when compared to the positive control alone, the administration of various pa ge 33 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 27-35, 2025 forms of secukinumab, such as liposome and bulk form, dramatically decreased dna damage caused by h2o2. secukinumab, with antioxidant defense playing a part, reduced damage to a level comparable to the untreated control group. the psoriasis area and severity index and oxidative stress indicators were found to be positively correlated. using cck8, the study examined the cytotoxic effects of secukinumab on healthy peripheral lymphocytes, and the results showed that these cells were cytotoxic in a concentration-dependent manner (lin & huang, 2016). it was observed that, increased concentration of secukinumab (3.75µg/ml) was found to be cytotoxic, while the two lower concentrations of the drugs were tested 2.1µg/ml and 2.8µg/ml, had increased cell survival rates of over 75%. the cbmn assay is an effective test to study the capability of genotoxic agents to cause various clastogenic (chromosome breakage) and an eugenic effects (causing daughter cells to have an abnormal number of chromosomes during cell division) (fenech, 2009). it was utilized in the current study to describe the effects of secukinumab on healthy individuals versus psoriasis patients. also, to determine the protective potential of the drug against h2o2-induced geno toxicity. this assay assesses several parameters, including micronuclei (mni), which form during anaphase and indicate chromosomal remains or lost chromosomes generated during nuclear division. to enhance the sensitivity and reliability of the assay, cytokinesis is blocked using cytochalasin b, which facilitates the accumulation of binc. the presence of mni in binc only reflects damage induced after treatment, reducing the possibility of scoring pre-existing damage. this approach effectively determines the effects of the test chemicals. by implementing this methodology, the sensitivity and reliability of the assay are increased, and the effects of the test chemicals are accurately determined (magdolenova et al., 2012; co-operation ofe, 2010). in comparison with the untreated cells, no effects on mni frequency were reported when the lymphocytes of healthy patients receiving both kinds of secukinumab treatment. “the mni frequency in lymphocytes(***p<0.001) was considerably elevated by the mmc and 75 μm h2o2, yet secukinumab (bulk or liposome co-supplemented with h2o2) exhibited significant reduction in the number of mni in lymphocytes (*p<0.05)”. “it was manifested that liposomal secukinumab caused a significant reduction in the number of mnis in patients (***p<0.001 as compared to un-treated cells). moreover, the inclusion of mmc and 75 μm h2o2 revealed a significant increase (***p<0.001) in the mni number in lymphocyte count in comparison to untreated cells”. this all confirms the safety of secukinumab in both forms, bulk and liposome, on lymphocytes. this could be the first step in using the liposome form as a topical treatment. therefore, the results demonstrate that all the treatment sets from both investigative groups have shown the binc % and the ndi within the normal ranges: a typical ndi value represents a successful division. evaluation of the assays shows that treatment of healthy and patient groups with secukinumab in both forms did not induce a significant number of mni in binc at basal levels. however, when 2.1μg/ml of the drug was co-treated with h2o2, secukinumab provided substantial protection against h2o2-induced damage and reduced the frequency of mni in healthy binc. a significantly raised number of mni were observed in mono nc of the patient group compared to healthy individuals, which indicates pre-existence of dna and chromosomal damage due to the disease state, medications or other confounding factors. secukinumab 2.1μg/ml re-duced the frequency of mni in mononc of the patient group at a basal level. however, the results were not statistically significant. the consistency of the data suggests that con-founding factors were not an issue. limitations ➢ due to differing degrees of safety and efficacy, standardizing liposomal and bulk secukinumab formulations and dosages is essential to maintaining similar results throughout investigations. ➢ the liposomal and bulk medication secukinumab may protect against oxidative stress, but more research is necessary to assess any possible off-target effects and determine the treatment’s overall risk-benefit profile. conclusion the aim of this study was to investigate the effect of secukinumab, in both liposomal and non-liposomal forms, on dna damage in healthy individuals and psoriasis patients. despite higher levels of damage in the sick group compared to the healthy group, secukinumab significantly reduced h2o2-induced dna damage in the presence of h2o2, attenuating its harmful effects. the results were consistent across the comet and micronucleus assays, with secukinumab 2.1μg/ml demonstrating enhanced efficacy against h2o2-mediated dna damage. according to the study, both types of secukinumab have the ability to protect against oxidative stress caused by h2o2, which may help with repair without having a major negative impact on geno toxicity. furthermore, the liposomal form of the drug was found to be safe for localized treatment via subcutaneous injection or transdermal drug delivery preparation. however, additional investigations are required to elucidate other possible molecular mechanisms involved. in conclusion, this study demonstrated the ability to effectively deliver secukinumab via liposomes, which may have clinical applications. these results highlight the potential of secukinumab to serve as a protective agent against oxidative stress-induced dna dam-age. overall, this study provides valuable insight into the effects of secukinumab on dna damage and its potential clinical applications. pa ge 34 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 27-35, 2025 recommendations ➢ extensive research ought to appraise secukinumab’s enduring impacts on oxidative stress and geno toxicity in order to appraise its resilience and possible unfavorable consequences in the long run. ➢ large-scale clinical trials involving human participants are required for further research studies to evaluate secukinumab’s efficacy, safety, and tolerability in a range of patient populations. acknowledgement authors are thankful to university of bradford, for their continued support in this research work. authors contribution all authors contributed to the study conception and design. g. layas & m. isreb n. ghaderi & fanila: conceptualization, methodology including ethica approval, samples collections and statistical analysis; p. akhbari, d. andreson: analytical interpretation; a. wright & m. najafzadeh: research discussion and review. abbreviations cho chinese hamster ovary cells chol cholesterol cbmn cytokinesis-block micronucleus dcm methanol dichloromethane dope dioleoyl phsphoethanolamine dppg 1,2-dipalmitoyl-sn-glycerol sodium salt il-17 interleukin 17 mab monoclonal antibody psai psoriasis area and severity index th t-helper cells tnfa tumor necrosis factor-alpha rpmi mammalian cell culture media/ roswell park memorial institute references bagel, j., blauvelt, a., nia, j., hashim, p., patekar, m., de vera, a. 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(2021). literature overview of the il-17 inhibition from psoriasis to covid-19. journal of inflammation research, 14, 5611–5621. pa ge 1 pa ge 13 american journal of life science and innovation (ajlsi) evaluating the efficacy of proximal femoral nail antirotation with augmentation in managing osteoporotic intertrochanteric fractures: a comprehensive review ramadan mohamed elsaid ahmed1* volume 3 issue 2, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v3i2.2759 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: may 25, 2024 accepted: june 29, 2024 published: july 03, 2024 addressing intertrochanteric fractures in those with impaired bone quality using orthopedic surgery is a huge challenge. this review assesses the effectiveness of proximal femoral nail antirotation (pfna) with augmentation, cement being the best augmentation compared to others, in healing these fractures. comparative analyses show that pfna is significantly better than pfn, with reduced operative times, blood loss and complications, especially in high-risk elderly patients. this review compiles data from 15 articles which aims to provide an overall picture of the present evidence on the use of pfna technique supplemented by the application of osteoporotic intertrochanteric fractures. augmentation methods, such as cement augmentation, have come to the forefront as important adjuncts to increase stability and effectiveness in pfna procedures. research has demonstrated that adapting the cement augmentation technique helps to restore load-bearing capacity, lower implant failure rate and to better the clinical results in total joint arthroplasty. pfna with cement augmentation effectively treats intertrochanteric fractures in osteoporosis hips, providing satisfactory results and potential for further clinical development. this review brings to the forefront the necessity of stabilizing the osteodensification and the proper cement augmentation, all of which aid in enhancing stability, occasioning less complications and leading to better postoperative outcomes. the future development in pfna techniques and the trending advancement of augmentation strategies would allow the field of surgery to benefit from an even more optimized way of dealing with these fractures in the clinical setting which is both promising and favorable. keywords augmentation, cement, fracture, osteoporotic intertrochanteric fractures, pfna introduction osteoporosis is a prevalent disease caused by the low density of bone mass, poor bone microarchitecture and fragility fractures. osteoporosis has been a major concern worldwide, affecting 200 million people (noh et al., 2020). the high rate of global morbidity osteoporosis is emphasized by its incidence in the population size of the majority of regions, estimating 18.3% of the entire world population, with a gender disparity showing a higher incidence rate in females with 23.1%, as opposed to men with 11.7% (salari et al., 2021). such figures favor the importance of developing health systems that take into consideration gender-specific factors, more especially the hormonal changes during and after menopause in women. witnessing a similar situation in china where adults aged 40 and above reveal a considerably high incidence of osteoporosis in men at 5.0% and women at 20.6% as well as a rather high occurrence of vertebral fractures in males at 10.5% and females at 9.7%, the pattern also follows at the global level (wang et al., 2021). the figures from china not only reflect the extensive gender discrepancy in osteoporosis prevalence, but also remind of the worldwide challenge of osteoporosis management. surprisingly, though, only a small fraction of patients with osteoporosis or patients who have suffered from a fracture are being properly treated in china, implying to the existence of wide gap of treatment. proximal femoral nail antirotation (pfna) pfna is a cutting-edge orthopedic implant designed for managing proximal femoral fractures, which are commonly seen in the geriatric population with osteoporosis as fractures predominantly occur at the intertrochanteric site (sachin, 2019). the intramedullary fixation gadget has changed shock management progressions by creating extreme stability and rotational control that is of core significance in the management of fracture cases (bizzoca et al., 2023). notably, this pfn has a threaded titanium alloy at its proximal end. the design of the medical screw, the helical blade, is decisive in enhancing the primary stability of the damaged area by engaging tightly to the cancellous bone from the neck to the head of the femur (wang et al., 2020). this links with the bone amalgamation within the bone readily reduces the chances of implant migration as a result of complications that if not addressed would be a setback in the treatment especially in the event of osteoporotic bone where screw fixation may not be adequate. furthermore, the pfna comes at the cutting edge of technology as its features allows for customization of the implant in line with patient anatomy and various fracture patterns. doctors can design the implant in a way that exactly fits each case by accurately controlling the implant configuration according to the need of each case, to enhance stability and provoke better healing (ledet et al., 2018; meng et al., 2023). the architecture of 1 rashid hospital trauma centre, 315 umm hurair, dubai, united arab emirates * corresponding author’s e-mail: rmdanelsaid@yahoo.com pa ge 14 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 13-24, 2024 the pfna is designed and developed very smoothly to take on the pressing forces that are experienced by the body during weight-bearing (augat & von rüden, 2020). its novel generation eliminates the stress concentration close to the fractured part of the femoral shaft and by the consequence, protects the implant’s integrity and prevents its possible failure. as many of its effects are due to structural integrity, the pfna enables early stages of mobilization and rehabilitation that could be invaluable for functional recovery and independence induction in patients. through its function of rendering the bone firm in position and by promoting wound endogenous healing, the pfna aids in completing the recovery process faster and improves the patient’s general situation (yu et al., 2020). osteoporotic intertrochanteric fractures & pfna osteoporotic fractures are linked to decreased quality of life, greater morbidity and in some cases of pelvic, spine and hip fractures, higher chances of death (gold et al., 2019). the fracture mostly affects the regions of the hip, spine, and distal radius, with hip fractures, especially intertrochanteric fractures, being the most serious due to their higher rates of morbidity, disability, and mortality (bishnoi et al., 2018). however, the management of osteoporotic intertrochanteric fractures which causes bone demineralization in the elderly requires new advancement in orthopedic surgical methods to produce efficient treatment results. intertrochanteric fractures near the proximal end of the femur, one of the most common types of osteoporotic fractures, interfere with many daily tasks such as walking and performing other weight-bearing exercises. this can greatly reduce a patient’s quality of life (maffulli & aicale, 2022). however, the issues that occur while treating these fractures are caused by compromised bone quality of osteoporosis that usually result in problems with the achievement and maintenance of a fixation which is stable (chen et al., 2022). the usual fixation techniques are very helpful in dealing with general bone damage, but when a problem arises in the context of osteoporotic bone the approach is limited due to the lack of bone mass and the specific changes in biomechanical properties. the pfna system transformed orthopedic surgery through the development of an advanced method for stabilizing and managing fractures in the proximal femur, increasing stability and aiding the natural bone healing process (yu et al., 2020). this particular implant, was designed unmistakably to face to the problems caused by osteoporotic bone disease (patil, 2019). among its attributes that heighten the rigidness of the implant fixation the pfna stands out. the operation of the pfna system in antirotation is another remarkable feature that targets the high rate of the rotation of the femoral head and neck cost as an enormous failure of the conventional fixation systems (chen et al., 2020). the pfna system specifically addresses these drawbacks as it offers an internal alignment device that promotes the mechanical stability of the fracture site, thereby enabling the patient to start rehabilitation earlier and decreasing the risk of potential problems (zheng et al., 2021). through the use of augmentation, the optimal observance of the moderation principle is reached. as bone cement and a strong ligation structure strengthen the damaged area, they become an invaluable tool in the modern treatment of intertrochanteric fractures in osteoporosis (ni et al., 2022). this context demonstrates the urgent need for pioneering and innovative surgical solutions like pfna system to be developed in order to prevent the onset of osteoporotic fractures in an aging global population. moreover, the pfna system provides an essential addition to the therapy of these types of bone fractures as they proceed towards the next level. implants (pin, screws, plates, bone cements) are used to increase weight-bearing capacity, to reinforce the fixation, and to provide additional support to the osteoporotic bone (hollensteiner et al., 2019). pfna with hyperextension is developed with biomechanical principles of fracture fixation in mind. thus, the pfna unit helps in bringing about a steady healing environment within the bone which enhances the chance of bone healing. the pfna design targets to reduce the strain on the bone, distribute uneven loads, and minimize the risk of the head of the femur twisting and collapsing, which are known complications in the healed intertrochanteric fracture in osteoporotic condition (inginshetty, 2019; lakhmania, 2020). the clinical data showed that the pfna system was highly effective in managing fractures in the intertrochanteric region of the bone associated with osteoporosis (su et al., 2022). studies have proven better results regarding shorter operating times, less bleeding and quick healing of operated areas (xu et al., 2018). what is more, the amplified treatment has been demonstrated to considerably lower the risk of implant failure and secondary procedures, solutions which bring in more risks in the treatment of osteoporotic fracture. the general surgeon, patient screening, and technical part of pfna implantation involve some certain things to be considered so as to get all the benefits from the pfna system. on top of the healing benefits of any other surgical intervention, the specific pfna system has its own risks and complications, which stresses the need for all clinicians to know the indications, contraindications, and surgical principles for an optimal procedure. augmentation augmentation in orthopedic surgery is supplementary to bone-implant interfaces, parallelly improving stability and better treatment outcomes. it is very appropriate where the bone scenarios are challenging i.e. osteoporotic fractures. this approach bases its actions on several strategies directed at enhancing the fixation knowing and reducing the chance of implant loss. once the bone cement is injected, it comes around the implant, usually polymethyl methacrylate (pmma), that it has been part of the fractured site (soleymani eil bakhtiari et al., 2021). its pa ge 15 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 13-24, 2024 role here is to seal the gaps within the porous bone as well as deliver stronger and better connection or an anchor for the implant. the ability of cement augmentation to enhance the contact area of the bone-implant and transfer the loads as well as provide biomechanical stability, which is vital for fracture healing success, is emphasized by a way of hauling up the bone-implant contact area (weiser et al., 2018). further the concept of augmentation extends beyond just the use of extra grafts and pins, where additional fixation elements like cerclage wires or cables are also employed to strengthen the construct and minimize the stress concentrations (thakur, 2022). these adjunctive stabilization techniques supplement the implant and even out the stress it faces. this may help to reduce the risk of implant migration failure. the use of these enrichment techniques in orthopedic surgery is extensive to enhance the outcome in various procedures, such as the fixation of fracture, joint arthroplasty, and spinal fusion (schuetze et al., 2019). one of the major functions of augmentation is to reinforce implant stability and to merge boneimplant integration that in turn will raise the chances of early mobilization and rehabilitation leading to the quick recovery of the patients with an improved outcome (tolstunov et al., 2019). specifically, augmentation is a proven auxiliary treatment in orthopedic surgery providing biomechanical reinforcement, improving the general condition of fractures and thus offering complex clinical care. this review intends to report on the impact of employing the proximal femoral nail antirotation (pfna) alongside the augmentation therapies in the care of osteoporotic intertrochanteric fractures. this review aims to analyze the current literature representing a combination of clinical studies and articles that will determine effectiveness and safety of pfna augmentation compared to standard fixation techniques. primarily, the review will focus on the outcomes such as rate of healing of the fractures, stability of the implants, complications after surgery and patient’s satisfaction with the recovery progress. moreover, the examination reveals the possible pros and cons of merging the augmentation procedure with the pfna technique employed in the treatment of osteoporotic intertrochanteric fractures, taking into account bone quality, the implant positioning, and surgical method. this review aims to summarize the positive impact of pfna in bone augmentation, aiming to improve treatment results and patient lives for femoral neck fractures. methodology search strategy the table 1 below shows the methods used in search strategies to identify corresponding studies of the searched literature to the research topic. the strategy includes a set of keywords and terms relevant to pfna, osteoporotic fractures, augmentation techniques, and surgical fixation methods, as they are intended to provide the broadest coverage of the journals containing the research on this topic. table 1: search strategy s. no search strategies 1. (“proximal femoral nail antirotation”) and (“pfna”) and (“osteoporotic fractures”) and (“osteoporotic intertrochanteric fractures”) and (“treatment approaches to osteoporosis”) 2. (“osteoporosis management strategies”) and (“augmentation techniques”) and (“bone cement augmentation”) and (“surgical fixation methods”) and (“cement augmentation femur fractures”) (“biomechanical studies on augmentation”) inclusion and exclusion criteria the table 2 provides the details of inclusion and exclusion criteria that are used to filter out the relevant studies with a focus on the role of proximal femoral nail antirotation (pfna) and augmentation in the management of osteoporotic intertrochanteric fractures. studies that met the desired criteria, dealt with the topic within the specified timeframe, and utilized the defined keywords as themes were included for analysis. table 2: inclusion and exclusion criteria inclusion criteria exclusion criteria studies that focuses on the use of proximal femoral nail antirotation (pfna) and augmentation in the management of osteoporotic intertrochanteric fractures. studies that do not focuses on the use of proximal femoral nail antirotation (pfna) and augmentation in the management of osteoporotic intertrochanteric fractures. studies published within the last six years (2018-2023) have been included. studies older than six years have been excluded. studies that are in relation with the keywords from the title were included. studies that are not in relation with the keywords from the title were excluded. pa ge 16 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 13-24, 2024 article selection the table 3 below specifies criteria for article inclusion in the review and elucidates studies that assess the effectiveness of proximal femoral nail antirotation (pfna) and augmentation techniques in treating osteoporotic intertrochanteric fractures. the research covers several works published in different journals and various years. it analyzes both surgical strategies and their consequences, focusing on pfna and general augmentation. table 3: article selection s.no author journal year findings 1. schuetze, konrad md; eickhoff, alexander md; röderer, goetz md; gebhard, florian md; richter, peter h. md journal of orthopaedic trauma 2019 the article offers insights into the efficacy of augmentation techniques in orthopedic surgery for managing osteoporotic fractures, addressing challenges related to mechanical failure and exploring diverse augmentation materials and strategies. 2. dr. vinod nair, dr. shubhanshu gupta, dr. anant krishna, dr. amol patil and dr. avinash kumar international journal of orthopaedics sciences 2019 the study compared the effectiveness of proximal femoral nail (pfn) and proximal femoral nail antirotation (pfna) in stabilizing unstable intertrochanteric fractures, finding pfna significantly reduced operative duration, blood loss, and hospital stays. 3. mittal, ankit; gill, simrat p.s.; kumar, dinesh; singh, jasveer; kumar, harish; rajput, ajay mamc journal of medical sciences 2021 this study compared the effectiveness of pfna and pfn in treating intertrochanteric fractures. despite similar functional outcomes upon fracture union, pfna exhibited fewer complications and shorter operative times, indicating potential benefits for osteoporotic, elderly patients. 4. avate shivaji, gaonkar kiran l., vatsa madhuranjan, kumari lipi, patil sudhir research journal of pharmacy and technology 2023 the study evaluated the functional outcomes of pfn a-ii implants for intertrochanteric and sub trochanteric femur fractures, revealing their stability, biomechanical acceptability, and minimal complications. 5. noratep kulachote, paphon sangasoongsong, norachart sirisreetreerux, kulapat chulsomlee, sorawut thamyongkit, siwadol wongsak, geriatric orthopaedic surgery & rehabilitation 2020 the study assessed outcomes of elderly intertrochanteric fracture patients treated with pfna, with and without cement augmentation, finding higher return rates to prefracture ambulatory level with cement augmentation, suggesting its efficacy in improving functional outcomes in highrisk geriatric patients. 6. hu s, du s, xiong w, chen s, song h, chang s chinese journal of reparative and reconstructive surgery 2021 the study evaluated the effectiveness of proximal femoral nail anti-rotation (pfna) in treating high plane intertrochanteric femur fractures, finding satisfactory outcomes, good fracture reduction quality, and prevention of internal fixation failure. 7. alexander m. keppler, daniel pfeufer, fabian kau, christoph linhart, christian zeckey, carl neuerburg, wolfgang böcker, christian kammerlander injury 2021 the study evaluated the impact of cement augmentation of the proximal femoral nail antirotation (pfna) on postoperative load capacity in older adult proximal femur fracture patients, revealing a significant increase in early mobilization, especially in frail patients with poor bone quality. 8. liqin zheng, xinmin chen, yongze zheng, xingpeng he, jingxiong wu & ziling lin bmc musculoskeletal disorders 2021 a study using finite element analysis compared cement augmented pfna in stable and unstable intertrochanteric fractures, finding that cement augmentation improves cutout resistance, especially in unstable fractures, suggesting potential clinical benefits. pa ge 17 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 13-24, 2024 results and discussion efficacy and management of pfna with augmentation the efficacy of pfna with augmentation in managing osteoporotic intertrochanteric fractures has been the focus of relatively more interest and research within the orthopedic surgery as of late. with the help of an extensive literature review, it becomes apparent that the techniques of augmentation, particularly the use of cement augmentation, are an essential part of enhancing the efficiency of pfna and patients’ outcomes. osteoporotic intertrochanteric fracture is a special challenge because of its compromised bone quality and hence a high risk of instrumentation failure (rozell et al., 2019). pfna is generally preferred intramedullary fixation device it does this through its central anchoring 9. konrad schuetze, s. ehinger, a. eickhoff, c. dehner, f. gebhard & p. h. richter archives of orthopaedic and trauma surgery 2021 the study demonstrates the safety and efficacy of cement augmentation of the pfna blade, effectively preventing mechanical failure without increasing complications or mortality. careful decision-making and preparation are emphasized to manage potential blood pressure changes during the procedure. 10. navin kumar singh, vijay sharma, vivek trikha, shiva gamanagatti, amit roy, avtar singh balawat, aravindh palaniswamy, amrut raje diwakar journal of clinical orthopaedics and trauma 2019 in a prospective study comparing pfna-ii and dhs in stable intertrochanteric fractures in elderly patients, comparable functional and radiological outcomes were found, indicating that pfna-ii may offer advantages in high-risk elderly patients, including shorter surgical time and reduced blood loss. 11. ong-art phruetthiphat, teerapat tutaworn, yanin plumarom, thipachart punyaratabandhu, chaisiri chaichankul, and warat tassanawipas journal of clinical case studies 2019 in a preliminary study on elderly patients with unstable intertrochanteric fractures, cementaugmented pfna demonstrated no mechanical failures, suggesting its potential as a safe alternative for managing these fractures. 12. eleonora piccirilli, ida cariati, matteo primavera, rebecca triolo, elena gasbarra & umberto tarantino bmc musculoskeletal disorders 2022 a literature review on augmentation techniques for osteoporotic fragility fractures found promising outcomes in stability and strength compared to non-augmented fractures. the review recommends considering augmentation techniques for elderly patients with comminuted fractures and poor bone quality. 13. stefano marcia, mario muto, joshua a. hirsch, ronil v. chandra, nicole carter, paola crivelli, emanuele piras & luca saba neuroradiology 2018 a narrative review on vertebral augmentation for osteoporotic vertebral compression fractures (vcfs) highlights its safety and effectiveness in reducing pain and disability, despite ongoing debate. the review indicates favorable outcomes with low rates of serious complications and no increased risk of subsequent fractures. 14. leonardo stramazzo, salvatore ratano, francesco monachino, davide pavan, giuseppe rovere, lawrence camarda journal of clinical orthopaedics and trauma 2021 a systematic review emphasizes the safety and effectiveness of cement augmentation in stabilizing trochanteric hip fractures, leading to satisfactory mobility outcomes and minimizing complications. cement augmentation is identified as a valuable method for enhancing fixation in osteoporotic hip fractures. 15. an sermon, ivan zderic, roberto khatchadourian, simon scherrer, matthias knobe, karl stoffel, boyko gueorguiev journal of biomechanics 2021 the study found that bone cement augmentation significantly improves the cut-out resistance of pfna and tfna head elements in cadaveric femoral heads with poor bone quality, suggesting its potential as a valuable treatment option in these cases. pa ge 18 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 13-24, 2024 in the proximal femoral canal (patil, 2019). in the case of osteoporotic bone, on the other hand, the conventional fixation techniques may not be sufficient enough to guarantee enough stability throughout the healing period. this is the place where the augmentation techniques play an important role, which is to reinforce the construction and impede any mechanical failure (schuetze et al., 2019). several studies have examined the efficacy of pfna in augmentation, with a stronger interest on cement augmentation than in other bone graft augmentations (piccirilli et al., 2022). the studies in this area clearly show these strategies to add stability and reduce shortcomings such as instrumentation cutout or implant failure. achieving this goal is possible as pfna in combination with cement bonding allows for improved implant fixation and better load distribution, thus reducing the risk of implant failure (guo et al., 2022). the pfna and augmentation is quite prominent in this context as they are capable of improving the outcome in the postoperative situation. lower operative time, less amount of bleeding and days of hospital stay are documented in patients having cement augmentation rather enforced with the conventional pfna fixation (kulachote et al., 2020). this simply means less time spent in hospital, fewer resources used, and more positive feedback after discharge. such outcomes not only help the hospital to cut costs, but also improve patient outcomes, which in turn contributes to their faster recovery. furthermore, along with pfna, augmentation helps to promote early ambulation of the elderly patients with osteoporotic fractures; thereby, this is very important (piccirilli et al., 2022). cement augmentation, as one of the augmentation techniques, was successful in restoring postoperative load capacity, allowing patients to bear weight and engage in rehab early. early ambulation not only shortens the rehabilitation cycle but also minimizes the rate of post-surgery problems like deep vein thrombosis and pressure ulcers caused due to long isolation (wang et al., 2023). the evidence undoubtedly confirms that for the treatment of osteoporotic fractures of the femur that involves the intertrochanteric region, a pfna osteosynthesis combined with augmentation technique is the best option. techniques of augmentation, including bone cement augmentation, ensure a good stability of fracture but reduce the risk of postoperative morbidity and accelerate the patient recovery, result in positive clinical outcomes (chiapasco & casentini, 2018). nevertheless, it should be noted that despite improvements in the augmentation strategies, one should also mind the need for more research to suitably optimize them and make sure that their long-term outcomes are good enough, especially concerning implant survival and healing fractures. in spite of this, the augmented pfna remains a very effective method of managing the osteoporotic intertrochanteric fractures which should be employed not only in postoperative but in long-term patient’s care. the research confirmed that bone cement reinforcement could increase the shear strength of the pfna and tfna heads in human femoral heads with poor bone quality. this finding showed the possible use of bone cement cementation in these situations. the comprehension of literature also reveals that those patients did better in terms of stability and strength after fracture repair which they previously did not have by being augmented compared to non-augmented patients. the review suggested the use of supplemental techniques for elderly patients with comminuted fractures and bad bone stock (sermon et al., 2021). moreover, the examination provides evidence concerning the safety and efficiency of strengthening the pfna delivery system by augmenting the blade with cement, however, risks and complications are not increased and morality remains as it was (schuetze et al., 2021). typically, attention is paid towards the careful decision-making and early planning to allow proper management of potential effects on blood pressure during the procedure. the evidence presented indicates that the use of addition techniques, especially cement augmentation, plays a leading role in influencing the effectiveness of the pfna procedure. interestingly, the outcome of the procedure can be greatly improved in conditions where poor bone quality is detected. comparative analysis of pfna with other instruments comparative study between proximal femoral nail antirotation (pfna) and proximal femoral nail (pfn) are essential for the selection of suitable treatment techniques for intertrochanteric fractures, particularly the osteoporotic and elderly individuals (nair et al., 2019). the analysis of influential studies in connection with pfna indicates that pfna has unique benefits of better stability, outcomes, and complication risk compared to the pfn. in trials wherein pfna and pfn were compared, pfna more often turned out to be the option most preferred for managing intertrochanteric fractures, particularly among patients with osteoporotic bone quality (mittal et al., 2021). while both pfna and pfn provide similar functional results after fracture union, the additional advantages of superior biomechanical support and fixation failure prevention contribute to choosing pfna over pfn in addressing the patients. not only does pfna reduce the operative duration as well as its incumbent morbidity, but it also has a shorter period of convalescence in comparison to pfn (duramaz & i̇lter, 2019). several comparative studies revealed that the operative time for pfna was shorter, which was attributed to the design features of pfna as it is known to be easy to insert and has many advanced features. besides reduced bleeding inside the operating room anesthesia time will be minimized and patients will have improved outcomes not being into such exposure for a long time (patil, 2019). apart from this, pfna is associated with a lower rate of complications than in the case of pfn and therefore, the superiority of pfna is obvious in the management of intertrochanteric fractures pa ge 19 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 13-24, 2024 (mittal et al., 2021). there are potential complications related to the implant’s migration, cutout, and secondary fractures, but the frequency of these events is lower with pfna that exhibits better biomechanical properties and stability of its fixation. this is notably the case for elderly patients whose osteoporosis puts them at heightened risk of the fixation breakdown and the subsequent revision surgery. the results of the prospective study that compared the outcomes between proximal femoral nail antirotation (pfna-ii) and dynamic hip screw (dhs) for stable intertrochanteric fractures among elderly patients were similar with respect to functions and radiology images. the results imply pfna-ii is quite effective procedure, especially for elderly patients in high-risk group with less surgical time and blood loss volume (singh et al., 2019). these outcomes bring emphasis to pfna-ii as a prime preference in such group of patients with benefit of simple yet direct implants that cope with skeletal complications and sophisticate patient’s outcomes. the research carried out a particular pfna-ii implants, used for intertrochanteric and sub-trochanteric femoral fractures, for the ability of stability, biomechanical acceptability, and low complications. this outcome shows that such pfna-ii implants with augmentation equally enable the reduction of lateral fracture union in high-risk elderly individuals, which usually leads to the recovery of acceptable functional outcomes (avate et al., 2023). functional results of augmentation in conjunction with pfna-ii implants the pfna-ii implants evaluation for intertrochanteric fractures indicates not only their intrinsic stability and biomechanical compatibility but also stresses the significance of utilized augmentations for optimum recovery. augmentation, mainly with cement, gives more strength to the pfna-ii fixations by improving fixation and resistance to a variety of complications (ni et al., 2022). cement augmentation plays a vital role together with pfna-ii implant, the added lateral stability and biomechanical support for the osteoporotic bone help prevent prosthesis migration. this increase in the concrete content side by side with the cement augments the surgeons’ ability to achieve a stronger implant stronger anchorage with femoral heads utilizing the load distribution for the prevention of implant migration, cutout, and secondary fractures. this is particularly meaningful in elderly patients with impaired bone quality in a high-risk geriatric category, where conventional fixation methods may lead to fractures that are incapable to endure physiological stresses (singaram & naidoo, 2019). some studies show that cementing as well as augmentation can produce satisfactory functional outcomes and adequate fracture reduction when used concurrently with pfna-ii implants (yang et al., 2020). the addition of cement to the implant is not only beneficial to the biomechanical characteristics of the implant but also assists the motion process of the patient and accelerates the whole healing process, thus, lead to the better patient satisfaction and faster recovery. on the one side the absence of complications involving pfna-ii implant may also have been facilitated by use of cement augmentation which implies even greater reliability of this option for fracture fixation in high-risk geriatric patients. the additional stability provided by cement augmentation mitigates complications like implant migration, breakout and secondary fractures resulting in improved clinical outcomes and reduced number of revision surgeries. advantages and disadvantages of cement augmentation cement augmentation is one of the main techniques used in the management of intertrochanteric fractures associated with osteoporosis, an especially common choice in cases where the poor bone quality hampers the obtainment of a stable fixation. cement augmentation helps to increase the fixation through the stimulation of bone interface, which limits the movement and subsequently boosts the clinical outcomes (weiser et al., 2018). among the many advantages that cement augmentation presents are the fast mechanical support it brings. adding bone cement to fill the gaps between the implant and surrounding bone eliminates the effect of dynamic forces, increasing the stability and durability of the implant, and as a result, it will be less susceptible to loosening or displacement. this is very much evident in osteoporosis bone tissue, during which the bone healing is very much dependent on the after fixation of the bone (winkler et al., 2018). cement augmentation of a fracture site creates more equal distribution of the load, disappearing the potential stress concentration at bone-implant interface and thus promoting better healing outcomes. given that, bone regeneration is the means by which this is achieved and results in better implant fixation and reduced rates of implant failure. it provides for earlier mobilization and weight-bearing which was proven to be beneficial for patients recovering and rehabilitation. conversely, reinforcement of surrounding cement is not without possible risks either. in individuals with osteoporosis, cement augmentation of internal fixation in hip fractures improves stability and lowers the chance of implant failure. its safety and effectiveness in stabilizing trochanteric fractures are demonstrated by this systematic review, providing this susceptible group with a viable therapy alternative (stramazzo et al., 2021). one of the possible damaging effects is cement leakage that arises when cement extravasation into the surrounding tissues and causes problems ranging from embolism to nerve injury and pulmonary complications (yousuf et al., 2021). precise technique and the full employment of recommended procedures can give certain guarantees to operate without any leakage of cement and any of the associated problems. the association of use of cement augmentation to the proximal femoral nailing pa ge 20 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 13-24, 2024 anti-rotational (pfna) device is a latest development in treatment options of osteoporotic intertrochanteric fractures, especially in elderly people with non-optimal bone quality. literature has regularly shown this induction of the load capacity to postoperative state which has the influence of the fixation within the femoral head, increasing the resistance to physiological loads and lesser probability of failure in fixation. in addition, cement augmentation aids in preventing the fragility endpoints, most frequently seen with osteoporotic intertrochanteric fractures. through putting cement into the femoral head voids, surgeons increase implant stability and hence the probability of failure or implant migration lowering. through the use of this, not only the treatment of fractures is improved but also revision surgical needs are reduced, therefore decreasing health costs as well as the patients’ suffering. the utilization of cement augmentation does not imply any major increase in the complication rates or mortality, clearly indicating the safety and efficiency of such method as a surgical support in the course of the osteoporotic intertrochanteric fracture management. the figures establish that cement augmentation has performed well while the rate of complications like infection, cement leakage, and pulmonary embolism has minimal positive effects. the above mentioned points emphasize the use of cement augmentation to exclude it out of the surgical intervention of the osteoporotic intertrochanteric fractures which might be very harmful to geriatric patients (roth et al., 2021; ulusoy et al., 2018; yu et al., 2023). making sure that thorough preoperative planning and good technique are performed as much as possible is essential to minimize risk of these complications. since cement augmentation will most likely eliminate reliance on adjusting construction for future revision if fixation fails or nonunion, it could affect the patient’s success rate (schlundt et al., 2018). these risks still exist; however, then them, the advantages of a cement-augmented nail compensate them, above all for those geriatric patients who are prone to fractures and who have poor bone quality. future strategies & advancements in pfna technology pfna in the future together with further advancements will be instrumental in shifting management of osteoporotic intertrochanteric fractures to a whole new level, offering new pathways for better outcomes. the biomechanical development has been at the edge of the latest technology of the implant design refinement, researchers will always strive to work better under the unique challenges posed by osteoporotic bone (chandra & pandey, 2021). in so doing, the above-mentioned process would include improving implant topography, material constitution, and surface properties so as to ensure better bone integration and more satisfactory bone repair. moreover, the trend is to develop new strategies of pfna augmentation incomparable with the fixation. scientists are testing new tissue substitutes like bioresorbable polymers as well as biologics, which enhances implant incorporation and final outcomes thereby improve long-term results (sharma et al., 2021). these advancements demonstrate the ability for the creation of even more stable types of fixation as well as reducing the risk of fixation failure in the generally high-risk of the geriatric patients who also suffer from compromised bone quality. with mi (minimally invasive) technique being a promising fundamental element of pfna technology, this is another remarkable area that needs to be explored (villena gonzales et al., 2019). technology advances in percutaneous insertion techniques as well as guided navigation systems are intended to decrease surgical trauma, reduce soft tissue damage and facilitate speedy recovery while maintaining the highest possible quality of reduction and fixation of the fracture (karkenny et al., 2019). this means that these innovations could transform the surgical practice with better patient experiences, reduced recovery time, and greater operational end result. moreover, the smart implant technology integration carries with it the advantages of not only monitoring in real-time implant performance, but also patient recovery status (gaobotse et al., 2022). the data generated through the use of sensors and wireless communication systems could provide critical information concerning the cyclic load and fracture healing trajectories that would be used to tailor personalized care for the individual patient and to optimize the treatment plan. additionally, research on biological enhancement options, like growth factors, stem cells or bone substitutes, are likely to explore other strategies are effectively to improve the healing of fractures and implants integration (perez et al., 2018). preclinical trials and clinical trials should be conducted to understand the safety and efficacy of the combination of novel treatment modalities with the pfna fixation. at the end of the day, these innovations greatly affect the quality of care and ultimate results of patient outcomes. much good can come from individualized treatment plans designed to accommodate given patient peculiarities and features of their particular fracture (moore, 2019). such an approach can help to control complications and optimize treatment outcomes. the new advanced operating techniques and minimally invasive approaches are the major aspects that guarantee enhanced patient experience characterized by the shortening of postoperative pains, early recovery and mobilization of a patient. with the innovations that are yet to be fully implemented, a new future awaits the treatment of osteoporotic intertrochanteric fractures. it will heavily rely on more efficient and precise approaches that will cater for the individual’s needs. results the trend to examine the effectiveness of proximal femoral nail antirotation (pfna) with augmentation as a method for treatment of osteoporotic intertrochanteric pa ge 21 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 3(2) 13-24, 2024 fractures can be seen as an especially strong trend in recent orthopedic surgery (jin et al., 2021; zheng et al., 2021). however, during extensive literature review, it soon comes apparent that allocation techniques, particularly cement augmentation, has been largely adopted as in to boosting the effectiveness of pfna and to better the patients’ prospects. the problem of intertrochanteric fracture in osteoporosis is particularly complicated because of loss of bone quality, an effect that raises the risk of instrumentation failure. among others, pfna is a preferred intramedullary fixation as its central anchoring of the proximal femoral canal makes it possible to overcome the problem of giving the fixation the desired amount of stability in the cases of osteoporotic bone (patil, 2019). precautionary augmentations, thus, rationalizes stress reinforcement and also reduces risks of failure. although pfna and augmentation approaches have shown indisputable positive results, the research and development still require continued development of these modalities to guarantee long-term success. though the procedure has proved its effectiveness in regard to stability and its inherent complication reduction, future research should thus be directed to improving cement filling and the development of more advanced cement substitutes that could minimize the risk of leakage (yee et al., 2020). besides, we also need study designs comparing different types of augmentations and implant styles in order to choose the most effective strategy for the treatments of osteoporotic intertrochanteric fractures. in addition, evaluating the effectiveness of biological augmentation approaches, such as growth factors or stem cells, in promoting the integration of both the healing of fractures and implants, are worth studying before trials during which preclinical and clinical trials will be implemented. clinicians can utilize research results to optimize therapeutic measures and improve patient outcomes in proximal femoral fracture fractures. introduction of pfna with augmentation in the site of osteoporotic intertrochanteric fractures will have a profound effect on the health care system and better outcomes for the patients. the augmentation techniques, generally the cement augmentation, provide stability better, faster surgeries, and shorter hospitalization time instead of traditional fixation methods. while such innovative technologies contribute to better cost management of the healthcare facilities, they also yield the improved patient experience and faster recovery outcomes. in addition, the employment of these augmentation techniques makes it possible for ambulation earlier and for partial weight-bearing, which is necessary for reducing the complications of postoperative and ensuring the fracture heals properly (chirayath et al., 2024). by using pfna together with enhancement in clinical practice, surgeons can have an osteoporotic intertrochanteric fractures adequate treatment and a good patient outcome. there is the effectiveness of pfna in conjunction with augmentation discovered in managing the trochanteric fractures. so, this progress contributes some stability and additional support of bones injured by osteoporosis in orthopedic surgery. the employment of constructing procedures especially using cement augmentation techniques at their core has proven to be the crucial aspects greatly targeted at achieving stability and achieving reduced risk of complication common with initial fixation methods. nonetheless, for augmentation to be old and trusted, more scientific exploration has to be done such as on the most effective strategies as well as for the longterm success. exploration of these areas of inquiry helps clinicians develop better treatment methods and enhance patient outcomes by providing more effective things in the management of osteoporotic intertrochanteric fractures which, in turn, will improve quality of life and be beneficial to patients in care. conclusion this review underscores the credibility of proximal femoral nail antirotation (pfna) with augmentation, which significantly boost the chances of this approach being favored for the management of osteoporotic intertrochanteric fractures. the comparative research justifies the use of pfna instead of the known proximal femoral nail (pfn) especially in the high risk elderly patients. cement incorporation will be the undeniable support throughout the postoperative period, as research shows the high load capacity and implant stability. in short, cement augmentation along with pfna technique based on the results of 15 concerns included in this review provides a reliable way to treat fractures produced due to osteoporosis in the intertrochanteric area of the femur. recommendation future study should examine the long-term effects of pfna with cement augmentation in osteoporotic intertrochanteric fractures, as well as comparing its efficacy and safety to alternative stabilizing procedures in randomized controlled trials, in order to optimize treatment 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(2021). cement augmentation of the proximal femoral nail antirotation for the treatment of two intertrochanteric fractures-a comparative finite element study. bmc musculoskeletal disorders, 22, 1-13. pa ge 1 pa ge 20 american journal of life science and innovation (ajlsi) ethnobotanical assessment of medicinal plants used by the higaonon tribe in barangay langag, esperanza, agusan del sur jean rose altrecha abejo1*, meljian t. demetillo2 volume 4 issue 2, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v4i2.6217 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: september 25, 2025 accepted: october 30, 2025 published: december 02, 2025 indigenous medicinal plants are harnessed by the higaonon tribe, enriching their cultural heritage and advocating sustainable healthcare practices through the utilization of their therapeutic properties. this study aims to document and explore the traditional medicinal plant knowledge and practices of the higaonon tribe in the northeastern part of mindanao, philippines. the study was conducted in barangay langag, esperanza, agusan del sur, among elderly villagers, traditional healers, and herbalists. a total of 13 plant species belonging to 11 families were identified and documented, along with their local names, plant parts used, methods of preparation and administration, and medicinal uses. the most used plant parts were leaves, followed by bark, roots, and rhizomes. the plants were used to treat various ailments such as fever, stomachache, cough, diarrhea, hypertension, wounds, and others. the lamiaceae family provided the highest number of medicinal plant species in the study. this research contributes to the documentation of traditional medicinal knowledge, supports future pharmacological research, and aids in the conservation and sustainable use of medicinal plants. keywords ethnobotanical, herbalists, medicinal plants, traditional healers introduction plants have been utilized as medicines for ages, primarily as traditional preparations. it is estimated that around 85% of traditional medicines used for basic healthcare globally are plant-based (niazi & monib, 2024). despite the availability of conventional or mainstream medicine, a significant number of individuals, especially those residing in rural areas, still opt for traditional medicine as their preferred approach to treat various ailments (“prevalence and socio-demographic variations in traditional medicine use: a study from east nile locality, sudan,” 2024). the world health organization has estimated that around 80% of the population in developing countries relies on traditional medicine. tribal communities, for instance, prefer medicinal herbs because they are more affordable than expensive pharmaceuticals. these communities have inherited the ancestral knowledge of indigenous healthcare practices (jana et al., 2021). traditional healers impart their indigenous wisdom to patients, sharing knowledge about the preparation of home-based medicinal plant remedies like decoctions and poultices, which are vital for essential healthcare (who 2002). additionally, local inhabitants assert that medicinal plants possess the ability to address ailments and expedite wound healing. the significance of medicinal plants lies in their potential to assist in the treatment of wounds and the prevention of diseases. apart from being cost-effective, traditional medicine is also believed to have fewer harmful side effects that could potentially be detrimental to one’s well-being (“herbal and traditional medicines pharmacovigilance for holistic treatment,” 2023). the transmission of this traditional knowledge occurs orally within kinship networks, but its preservation is at risk due to waning interest among younger generations. additionally, the gradual integration of western medicine and the process of modernization further exacerbate the threat of this knowledge becoming extinct (aziz et al., 2018). the documentation of medicinal plants utilized in traditional and complementary medicine holds great significance, as it serves not only to safeguard cultural practices but also to facilitate the exchange of knowledge among communities and countries, thereby forming a network of shared wisdom (miguéis et al. 2019). however, the documentation of medicinal plants remains incomplete in many countries. indigenous peoples and rural communities possess traditional knowledge about the uses of these plants, but their existence is endangered by various cultural, social, and economic factors. unfortunately, only a fraction of this valuable information has been thoroughly and systematically recorded (who, 1993). various regions across the globe have seen the undertaking of ethnobotanical studies aimed at documenting the traditional knowledge held by indigenous communities regarding medicinal plants. furthermore, ethnobotanical studies yield valuable information that serves as a foundation for subsequent phytochemical and pharmacological research. in the philippines, several research studies have been carried out to evaluate the ethobotanical knowledge of diverse ethnic groups. these studies include investigations among the tagabawa tribe in davao del sur (waay-juico et al., 2018), the mamanwa tribe of surigao del norte and agusan del norte (nuneza et al., 2021), and the manobo tribe in davao occidental 1 city college of bayugan, bayugan city agusan del sur, 8503, philippines 2 biology department, caraga state university, philippines * corresponding author’s e-mail: philippinesjeanrose.abejo@deped.gov.ph pa ge 21 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 20-27, 2025 (cabugatan et al., 2022). through these studies, numerous plant species with promising medicinal properties have been identified, opening avenues for further exploration of their pharmacological activities. however, only few studies have been published (olowa et al., 2012; buenavista et al., 2022) on the medicinal plants used by higaonon tribe in mindanao and there are still no studies recorded in agusan del sur. the higaonon tribe, one of the 110 indigenous peoples in the philippines recognized by the national commission on indigenous peoples. they are predominantly found in the northeastern part of mindanao, particularly in the provinces of agusan del norte, agusan del sur, bukidnon, and misamis oriental. like many other indigenous communities, the higaonon have their own unique culture and traditions, including their use of traditional medicine. the use of traditional medicine has been an integral part of their culture for thousands of years. also, traditional medicine remains their primary form of healthcare. however, no extensive research of local higaonon tribe’s medical knowledge and practices has been done in the area. thus, the goal of this research was to uncover and document traditional medicinal plant knowledge and practices of higaonon tribe in the area. the obtained data were utilized to direct subsequent pharmacological investigations and to safeguard the conservation of medicinal plants and indigenous knowledge. moreover, the documentation of these indigenous plants and associated ethnobotanical knowledge can be used as basis for developing management plant for conservation and sustainable use. objectives of the study this study tries to investigate the medicinal uses of indigenous plants to human and to document native medicinal plants available at brgy. langag, esperanza, agusan del sur. specifically, the study sought to shed light on the following research objectives: 1. identify and classify the medicinal plants used by higaonon tribe in langag, esperanza, agusan del sur 2. determine the plant sources, plant habit, and plant part used; 3. determine the mode of preparation and application of each medicinal plants; and 4. determine the common ailments, diseases treated of each identified medicinal plan. expected output of the study 1. a comprehensive documentation of the medicinal plants used by the higaonon tribe in brgy langag, esperanza agusan del sur. 2. a list of the identified plants. 3. a report that can be used to validate the ethnomedicinal practices of the higaonon tribe and promote their conservation. significance of the study this study will contribute to the preservation of traditional knowledge of medicinal plants and promote their use in primary healthcare. the documentation of the traditional medicinal practices of the higaonon tribe will also enhance the understanding of the importance of biodiversity conservation in the philippines. benefitting the study are the various sectors as follows: medical practitioners this study benefits health professionals as it provides valuable insights into traditional medicinal practices and the potential of medicinal plants. the findings can contribute to the development of new drugs, herbal remedies, and alternative treatments. healthcare professionals can incorporate this knowledge into their practice and improve patient care. local community the study can contribute to improving the health and well-being of the local community by validating and preserving their traditional medicinal practices. it can help community members access alternative and locally available healthcare options. educators the study can be used by educators to promote cultural diversity and appreciation among students, fostering an understanding of indigenous knowledge systems and traditional healing practices. future researchers the study can contribute to the existing body of knowledge in ethnobotany, traditional medicine, and indigenous practices. future researchers can build upon this study’s findings, expanding understanding of medicinal plants and their traditional uses. materials and methods study site the study was conducted among elderly villagers, traditional healers, and herbalists in barangay langag, esperanza agusan del sur. esperanza using geographical coordinates of 8°41′n 125°39′e. (figure 1) a 1st class municipality in the province of agusan del sur, philippines. according to the 2020 census, it has a population of 59,353 people (esperanza, agusan del sur wikipedia, 2021). the ethnic group in this area is higaonon and banwaon speaking. pa ge 22 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 20-27, 2025 figure 1: the study site – langag, esperanza agusan del sur data collection and selection of study participants fieldwork for this study will be conducted between april and may 2024. prior to the conduct of study, approval and in accordance with the guidelines provided by the indigenous peoples mandatory representative (ipmr) and the chieftain of the higaonon tribe was obtained. the participants were purposively selected, comprising herb sellers, traditional health practitioners, farmers, and individuals with indigenous knowledge. key informants of 20 were composed of 11 females and 9 males with an age range from 40-65 years old. each informant verbally consented in advance and voluntarily before the interviews began. they were given the assurance that the information would be used solely for academic reasons because knowledge is a natural resource for the local population. after explaining the purpose of our study, the researcher took note of the gender, age, and marital status of every respondent. the respondent’s location, degree of education, and method of learning about medicinal plants, their uses, plant parts used, diseases treated, the preparation and administration methods, and the conservation status (availability of the plants) were also noted by the researcher. ethnobotanical information was obtained from participants using a semi-structured questionnaire. the questionnaire was prepared in english. however, to facilitate efficient communication, informal discussion with herb sellers and traditional health practitioners was held in cebuano, the local language in these local government areas. identification of medicinal plants preliminary identification of the specimens was documented by taking photographs. characteristics were noted for accurate classification, then referred to the website used to identify its common name, scientific name through the book “amazing healing plants” by jc kurian and website stuartchange.com and other internet sources. data analysis with descriptive statistical methods using percentages, the study tried to analyze and summarize data on the reported medicinal and wild edible plants and associated indigenous knowledge. results and discussion the study documented the utilization of 13 plant species by the higaonon tribes residing in brgy. langag, esperanza agusan del sur, which were found to belong to 13 genera across 11 families. table 1: list of medicinal plants used for the treatment of various ailments common among the higaonon tribe of brgy. langag, esperanza agusan del sur family scientific name common name uses plant parts use methods of preparation and administration apiaceae apium graveolens hilbas fever, body pain. stomach leaves poultice, applied to the body to relieve soreness and inflammation and kept in place with a cloth. asteracea blumeabalsamifera gabon body pain leaves poultice, applied to the body to relieve soreness and inflammation and kept in place with a cloth. pa ge 23 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 20-27, 2025 caricaceae carica papaya kapayas dengue leaves leaves are soaked in hot water and the infusion (1 cup) is taken three times daily lamiaceae vitex negundo lagundi fever, stomachache, cough and body pains leaves leaves are soaked in hot water and the infusion (1 cup) is taken three times daily lamiaceae coeusblumei benth mayana cough, fever leaves leaves are soaked in hot water and the infusion (1 cup) is taken three times daily lamiaceae menthaalberti sennen karabo cough leaves leaves are soaked in hot water and the infusion (1 cup) is taken three times daily moraceae ficus benjamina linn balete sprain bark poultice, applied to the body to relieve soreness and inflammation and kept in place with a cloth. myrtaceae psidium guajava bayabas ulcer, diarrhea, wounds leaves chewed, also fresh leaves are boiled in water and a cup of the decoction is taken as tea three times a day, fresh leaves are crushed and used as woundndressing daily piperaceae piper betle buyo flatulence, stomachache leaves leaves are boiled in water and ½ cup of the decoction is taken three times a day poaceae cymbopogon tanglad hypertension leaves leaves are soaked in hot water and the infusion (1 cup) is taken three times daily rutaceae citrus maxima buongon cold fever leaves infusion or decoction of leaves (1 cup) is taken orally as tea three times a day sapotaceae chrysophyllum cainito kaimito vomiting, stomachache, cough and cold leaves leaves are soaked in hot water and the infusion (1 cup) is taken three times daily zingiberaceae curcumalonga linn dulaw (yellow) swelling rhizome or roots crushed and used as wound dressing daily the plants were categorized based on their local names, the parts used, preparation methods, and medicinal properties. among the identified families, lamiaceae contained three plant species. in contrast, the remaining 10 families, namely poaceae, myrtaceae, zingiberaceae, apiaceae, rutaceae, asteracea, caricaceae, moraceae, sapotaceae, and piperaceae, each contributed one species (refer to table 1). the indigenous people of the higaonon tribe and traditional healers residing in brgy. langag, esperanza agusan del sur, employs a variety of plants available in their surroundings, utilizing different plant parts such as leaves (84%), roots (8%), and bark (8%), which aligns with the practices observed in this study. the leaves, including newly sprouted and young leaves, were the most utilized plant part, with 11 species involved. additionally, roots were used in one plant species, and rhizomes/roots in another. based on the responses and observations, the prevalent ailments treated with medicinal plants in brgy. langag includes stomachache, fever, cough, diarrhea, hypertension, and wounds. given limited access to modern healthcare, a significant portion of the population in brgy. langag continues to rely on traditional healthcare practices, although some individuals combine traditional and contemporary medicine approaches. the lamiaceae family emerged as a prominent source of medicinal plant species in the present study. this botanical family encompasses numerous plants that serve as functional food sources, yield oils, and possess medicinal properties. it also encompasses a significant number of flowering plants belonging to the mint family. ethnobotanical, pharmacological, and phytochemical investigations have revealed that lamiaceae exhibits a wide range of activities, such as antioxidants, antihyperlipidemic, vasorelaxant, and thrombolytic effects, both in vitro and in vivo. these mechanisms contribute to various aspects of cardiovascular diseases (cvd), including stroke, heart attack, and others, as pa ge 24 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 20-27, 2025 highlighted in a recent study by chakrabartty et al. (2022). for the poaceae family, certain plant species within it have been found to have therapeutic applications in treating hypertension, diabetes, inflammation, parasitic infestations, and ulcers, as well as possessing diuretic and antioxidant properties, as indicated in a study by fatima et al. (2018). in the case of the myrtaceae family, several of its species have been utilized as spices and have a longstanding history in folk medicine for treating various ailments. multiple studies have highlighted their antimicrobial, anti-inflammatory, and antioxidant activities. as for the zingiberaceae family, almost every part of these plants is utilized as a source of food, natural dyes, and traditional medicine. their rhizomes are recognized for their medicinal, pharmacological, and nutritional properties, as explored in the research by rachkeeree et al. (2018). the apiaceae or umbelliferae family, which includes plants like celery, carrot, and parsley, is primarily known for its aromatic nature and characteristic hollow stems. many plants within this family serve as condiments or vegetables, while some also possess medicinal properties, as noted in the study by tshikalange et al. (2017). on the other hand, the rutaceae family comprises medicinal plants used in the treatment of pain, dermatitis, rheumatism, and other inflammatory conditions, albeit with limited use due to potential toxicity. over centuries, the members of the asteraceae family have been incorporated into both dietary and medicinal practices, as explored by rolnik and olas (2021). the caricaceae family is globally recognized for its therapeutic and nutritional properties, with various parts of the plant being utilized for their therapeutic applications since ancient times, as highlighted in the research by singh et al. (2020). figure 2: the different parts of plants used against ailents common among the higaonon tribe of brgy. langag, esperanza agusan del sur figure 3: mode of preparation of remedies used in the treatment of ailments common among the higaonon tribe of brgy. langag, esperanza the moraceae family encompasses plants whose fruits, roots, and leaves are utilized in traditional medicine to address a range of ailments, including gastrointestinal issues (such as colic, indigestion, loss of appetite, and diarrhea), respiratory conditions (like sore throats, coughs, and bronchial problems), cardiovascular pa ge 25 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 20-27, 2025 disorders, and as anti-inflammatory and antispasmodic remedies, as discussed in the study conducted by mawa et al. (2013). sapotaceae is valued for its external application in treating skin diseases, rheumatism, headaches, chronic constipation, piles, hemorrhoids, and is sometimes employed as an emetic and galactagogue, as outlined by chakradhari et al. (2019). piperaceae, found in many countries, is utilized for its potential to address diverse ailments such as cancer, urinary disorders, fever, stomachaches, and to improve appetite, as reported in the research by eyob et al. (2018). identified medicinal plants used by higaonon tribe figure 3: medicinal plants used by higaonon tribe based on the findings from the interviews with the respondents, it was revealed that leaves were the most frequently utilized part of the plant in their therapeutic practices, followed by bark, roots, and rhizomes. according to liu et al. (2023), alkaloids are a class of chemical substances that are synthesized and accumulated in leaves through the process of photosynthesis. the active ingredients found in most herbal preparations include tannins, coumarins, flavonoids, essential oils, and inulins. unlike medications that necessitate the use of whole plants, which can lead to uprooting, the utilization of leaves helps conserve the plants, as leaves are abundant, easily gathered, and capable of regeneration. the higaonon tribe in brgy. langag, esperanza agusan del sur employed various plant parts in the preparation of medicinal plants, such as concoction, decoction, extraction, infusion, crushing, and soaking of leaves. the most common method of preparation reported involved infusion (42.9%), decoction (21.4%), poultice (21.4%), then crushed and direct eating (7.1%) indicating both pa ge 26 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(2) 20-27, 2025 internal and external applications. conclusion thirteen medicinal plants have been documented, which demonstrate significant efficacy in treating stomachache, fever, cough, diarrhea, hypertension, and arthritis. the leaves of these plants are commonly utilized through decoction, poultice, crushing, and infusion, with oral administration being the preferred method. the higaonon tribe firmly believes in the remarkable effectiveness of these herbal remedies, often witnessing complete recovery within two to three days. notably, there is no uniformity in the preparation and dosage practices. these traditional medicinal plants play a crucial role in providing accessible treatment in the absence of immediate access to hospitals and modern healthcare facilities. in conclusion, this study expands our understanding of the medicinal plants utilized by the indigenous higaonon tribe in brgy. langag, esperanza agusan del sur. the findings demonstrate that most of the documented medicinal plants possess multiple therapeutic properties. furthermore, certain plants identified in this study exhibit similarities in terms of their uses and application methods. as such, these findings serve as valuable baseline data for future research endeavors, including pharmacological investigations, as well as the preservation of both medicinal plants and local knowledge to mitigate the risk of extinction. additionally, this research will serve as a valuable resource for other researchers who are interested in pursuing further investigations into ethnomedicinal plants. the data collected will aid local management in formulating policies for conservation, reproduction, and advocacy to ensure the sustainability of these plants. future researchers can utilize this data to conduct phytochemical screenings and other laboratory tests on selected medicinal and wild edible plants, as well as develop strategic plans for conservation. moreover, efforts to strengthen dissemination and information campaigns regarding the uses of these plants will be emphasized. references arquion, r. d., galanida, c. c., villamor, b. and aguilar, h. t. 2015. ethnobotanical study of indigenous plants used by local people of agusan del sur, philippines. asia pacific higher education research journal, 2(2). asiimwe, s., namukobe, j., byamukama, r., & imalingat, b. 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(2019). informant consensus: a new approach for identifying potentially effective medicinal plants. in plants and indigenous medicine and diet (pp. 91-112). routledge. tshikalange, t. e., modishane, d. c., & tabit, f. t. (2017, january 2). antimicrobial, antioxidant, and cytotoxicity properties of selected wild edible fruits of traditional medicinal plants. journal of herbs, spices & medicinal plants, 23(1), 68–76. https://doi.org/10.1 080/10496475.2016.1261387 waay-juico, m., cortuna, g., evangelista, s., gatal, r., licuanan, c., & tapia, f. (2018, january 5). ethnobotanical practices of tagabawa tribe on selected medicinal plants at barangay jose rizal, sta. cruz, davao del sur, philippines. journal of complementary and alternative medical research, 4(3), 1–12. https://doi.org/10.9734/jocamr/2017/38301 world health organization (1993). international union for conservation of nature and natural resources, world wide fund for nature. in guidelines on the conservation of medicinal plants. gland: international union for conservation of nature and natural resources. available at: https://apps.who.int/iris/ handle/10665/41651. world health organization (2002). regional strategy for traditional medicine in the western pacific. who regional office for the western pacific. available at, https:// apps.who.int/iris/handle/10665/206953. pa ge 1 pa ge 36 american journal of life science and innovation (ajlsi) malaria prevalence and associated risk factors among pregnant women attending the prenatal care unit of the ntoum departmental hospital (ndh), northwest gabon bourdettes meye1*, hilaire moundounga kenguele2, ludjer mpiga ekambou3, strédice maguinga guitouka4 , robert eya’ama mve5, kevin alame emane1, joseph privat ondo1, louis clément obame engonga1 volume 4 issue 1, year 2024 issn: 2833-1397 (online) doi: https://doi.org/10.54536/ajlsi.v4i1.4680 https://journals.e-palli.com/home/index.php/ajlsi article information abstract received: march 02, 2025 accepted: april 10, 2025 published: june 15, 2025 malaria during pregnancy is a significant public health problem in endemic countries. this study aimed to determine the prevalence of malaria and associated risk factors among pregnant women attending the departmental hospital of ntoum (northwest gabon) for prenatal care. a cross-sectional survey was conducted for 6 months (july to december 2024), among pregnant women attending the prenatal care unit of the ntoum departmental hospital (ndh). pregnant women selected for the study were asked to complete a structured questionnaire. at the end of the interview, a nurse drew a blood sample in an edta tube. malaria was detected by thick blood smear using the who method. a p-value < 0.05 was considered statistically significant. blood samples were collected from 202 pregnant women. malaria was diagnosed in 23.3%. patients aged between 20 and 30 years (53.2%), with a gestation of 4 to 6 months (46.8%) and living in the first district of ntoum city (70.2%) were the most infected with the disease. most of these women had completed secondary education (87.2%) and were unemployed (68.1%). fever and headache were the most frequently reported symptoms, accounting for 51.1% and 42.5% of cases, respectively. women who slept under an insecticide-treated net or used insecticide spray were less prone to malaria infection; but, the difference was not significant (p˃0.05) in contrast, pregnant women who reported episodes of fever were significantly less likely to test positive to malaria (or = 0.24 [0.09 0.61]; p =0.002). among the women who tested positive, 83% had a low parasitemia. plasmodium infection in the komo mondah department is high. the development of a malaria vaccine would make it possible to immunize all participants and thus reduce the spread of malaria worldwide. keywords gabon, malaria, ntoum, pregnant women, prevalence introduction worldwide, malaria is a major public health problem (organization, 2023). in 2023, nearly 263 million cases and 597,000 deaths were recorded across the globe, with africa remaining the continent most affected by the disease, accounting for 94% of malaria cases and 95% of deaths (organization, 2024). pregnant women and children under 5 are the most vulnerable group to the disease (organization, 2019). the who’s 2021 world malaria report highlighted that one in three pregnancies is exposed to a malaria infection in the african region, and that the continent’s west zone has the highest rate of exposure to malaria (40%), followed by the central zone (39%) and the east and southern zone (22%) (organization, 2022). every year, nearly 25 million pregnant women are confronted with the consequences of malaria, and the risk of infestation is greatest during the first trimester of pregnancy (steketee et al., 2001). according to one study, a fifth (20%) of all stillbirths and 11% of all neonatal deaths are the result of this disease (obossou et al., 2024). gabon, like most african countries, is aiming to achieve universal health coverage. in this central african country crossed by the equator, the incidence of malaria and its lethality fell in 2022 (oms gabon, 2023). more than one household in five (21%) owns at least one insecticide-treated mosquito net (itn), and the proportion of households with an itn is higher in rural compared to urban areas (29% versus 21%) (edsg, 2022). faced with the rapid spread of mosquitoes worldwide, the use of insecticides has become widespread (fu & guanli, 2024). just over half (51%) of the population of the communes of libreville, owendo and akanda use aerosol insecticides, and a quarter (25%) of these households appreciate spiral fumigants (pamba et al., 2021). in the southeast of the country, 47% of people in franceville city use indoor insecticide spraying, compared to 21.1% in moanda city (kenguele et al., 2022; mabiala et al., 2021). according to several publications, not sleeping under an insecticide-treated net, nor spraying one’s home with residual aerosol insecticides, contribute to the spread of malaria. likewise, being unemployed, living in unhealthy neighborhoods and having a low level of education are risk factors associated with malaria (okiring et al., 2019; rek et al., 2020; ramharter et al., 2007). 1 department of biochemistry, laboratory of research in biochemistry (larebio), university of sciences and techniques of masuku (ustm) franceville, gabon 2 department of biology, laboratory of molecular and cellular biology (labmc), university of science and techniques of masuku (ustm), franceville, gabon 3 ntoum departmental hospital (hdn), ntoum, gabon 4 nkembo specialist hospital, libreville, gabon 5 university hospital, jeanne ebori foundation (chufje), libreville, gabon * corresponding author’s e-mail: meyebourdettes@yahoo.fr pa ge 37 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 36-44, 2025 the detection, identification and quantification of malaria parasites are strategies advocated by the who to combat malaria worldwide (organization, 2020) effectively. microscopic observation of parasites using thin and thick blood smears remains the reference method for adapting therapeutic management (organization, 2014). this parasitological technique is sometimes combined with a rapid diagnostic test (rdt) based on immunochromatography (al-quhaiti et al., 2022; bah et al., 2020). the third demographic and health survey in gabon indicated that the estuary province, which includes seven communes, had a high prevalence of malaria infection among pregnant women (edsg, 2022). to the best of our knowledge, no study of gestational malaria has been carried out in the commune of ntoum. in order to address this problem, new reliable and accurate data on malaria prevalence and associated factors among pregnant women living in this commune are needed. this study will provide updated information on the extent of malaria infection in this vulnerable group residing in north-west gabon, in the komo mondah department. material and methods study setting and location a prospective, observational study was conducted at ntoum hospital for 6 months, from july to december 2024. ntoum hospital is the only public health facility in the commune of ntoum, capital of the komo mondah department. the hospital operates every day of the week, day and night. the town of ntoum is 34 km away from the capital city libreville, and is geolocated at 0° 17′ 18″ north and 9° 46′ 0″ east. it is a rural community estimated in 2012 to have 11,813 inhabitants (dgs, 2015). the region’s relief is made up of flooded areas and numerous rivers. the vegetation is characterized by riparian forests, located in swampy areas, marked by a predominance of mangroves, forests under fallow land located mainly along the main roads and dense evergreen rainforest located inland. the locality’s hot, humid conditions favor the life cycle of anopheles mosquitoes, the main vectors of malaria in gabon (mbouloungou et al., 2019; agyekum et al., 2021). in the commune of ntoum, malaria transmission is intense and permanent (meye et al., 2024). study population and inclusion criteria the study population consisted of all pregnant women who had given informed consent to participate in the study and who, during the study period, had attended a prenatal consultation at ntoum hospital. all pregnant women who had been screened for malaria during their antenatal visit were included in the study, and those who were not screened for malaria were excluded from the study. data and biological material collection pregnant women who signed the informed consent form were included in the study and interviewed by a midwife. the self-administered questionnaire asked about the respondent’s socio-demographic characteristics (age, level of education, place of residence, occupation, etc.), obstetrical factors (gestational age), malaria preventive measures (use of insecticide-impregnated mosquito nets, use of insecticide spray) and malaria symptoms. at the end of the interview, the pregnant woman was taken to the sampling room, where a nurse performed a venipuncture on an edta tube. study approval and ethical considerations the study was approved by the ndh general management. all procedures contributing to this project comply with the ethical standards of the relevant national and institutional committees on human experimentation, and with the declaration of helsinki of 1975, as revised in 2008. statistical analysis the data collected were entered into a database using microsoft excel version 2016. all statistical analyses were performed using r software version 1.4.2. descriptive statistics were used in the analysis of sociodemographic characteristics, obstetric history, prevalence of malaria infection and preventive measures for malaria infection, and variables were presented as frequencies and percentages. odds ratio and pearson’s chi square test used to determine the degree of association between the disease and independent variables. a p-value < 0.05 was considered statistically significant. biological analysis and measurements a microscopic blood test was carried out as a standard screening test on all participants. parasitological examinations of the thick blood smears were carried out by a trained laboratory technician. briefly, after identifying the end of an object slide, 5 µl of whole blood was pipetted into the center of the slide using a calibrated pipette. using the corner of another clean, degreased slide, the drop of blood was spread evenly to form a circle approximately 12 mm in diameter. a hairdryer was then used to heat the spread. after cooling, the entire preparation was covered for 10 minutes with a 10% solution of giemsa. the dye was then removed with a gentle trickle of tap water, and the slide dried at room temperature. slides were read using a light microscope (leica dm500), and 200 microscopic fields were systematically examined for malaria hematozoa using a 100x immersion objective. these were counted alongside leukocytes. the number of parasites was reported per 1 microliter of blood (based on 8,000 leukocytes per microliter of blood). results and discussion results sociodemographic and obstetric characteristics, clinical symptoms, and preventive measures of study participants two hundred and two (202) survey forms were completed pa ge 38 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 36-44, 2025 and used for data analysis, indicating a participation rate equal to 100%. at the time of inclusion, the average age of the participants was 27.5 years with an age range between 15 and 44 years. patients aged 20 to 30 were the most represented (n = 117, 58%), followed by those over 30 (n = 57, 28.2%). the main clinical signs reported in the survey were headaches (n =71, 35.1%), cough (n =38, 18.8%), diarrhea (n =22, 10.8%) and general fever (n =58, 28.7%). most participants (n =156, 77.2%) lived in the first district of neighboring ntoum commune. the remainder lived in the second district (n=24, 11.8%) and surroundings (andem, kango, libreville and cocobeach) (n=22, 11%). more than two-third (n=138, 68.3%) of participants had completed secondary school, and a few hold a university degree (n=37, 18.3%).. a large number (139, 68.8%) were unemployed.. the majority of respondents (n= 105, 52%) were in their second trimester of gestation, 24.7% were carrying a first-trimester pregnancy and 23.3% a third-trimester pregnancy. the survey revealed that more than half the participants did not sleep under an itn (52%) or use a mosquito repellent (58.4%) (table 1). table 1: sociodemographic and obstetric characteristics, clinical symptoms, and malaria preventive measures, of pregnant women attending the ntoum departmental hospital measured variables (n) (%) age (years) < 20 28 (13,8) 20 – 30 117 (58) >30 57 (28,2) respiratory signs dyspnea 03 (1,5) suffocation 09 (4,5) cough 38 (18,8) choking/fever 03 (1,5) none 149 (73,7) neurological signs dizziness 46 (22,7) headache 71 (35,1) asthenia 1 (0,6) none 84 (41,6) digestive signs diarrhea 22 (10,8) vomiting 21 (10,4) epigastralgia 12 (6,1) vomiting / headache 2 (1,0) none 145 (71,7) general signs asthenia 54 (26,8) fever 58 (28,7) none 90 (44,5 place of residence 1st district 156 (77,2) 2nd district 24 (11,8) 3rd district 00 (00) neighboring towns (libreville, kango, cocobeach, andem, kougouleu, donguila) 22 (11) education level primary 19 (9,4) secondary 138 (68,3) university 37 (18,3) illiterate 8 (4,0) pa ge 39 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 36-44, 2025 occupation unemployed 139 (68,8) employed 63 (31,2) month of pregnancy 1 3 (1st trimester) 50 (24,7) 4 6 (2nd trimester) 105 (52) 7 9 (3rd trimester) 47 (23,3) insecticide-treated nets (itn) yes 97 (48,0) no 105 (52,0) insecticide spray yes 84 (41,6) no 118 (58,4) parasite density thick blood smears were used to assess parasite density in line with who recommendations (organization, 2020). plasmodium falciparum was the only plasmodial species observed in all examined biological samples. among the pregnant women infected with this parasite, 83% (n = 39) had a low parasite density (< 1,000 parasites/µl), 12.8% (n = 6) a moderate parasitaemia (1,000 9,999 parasites/µl) and 4.2% (n = 2) a high parasitaemia (≥ 10,000 parasites/µl). of the 39 patients with low parasitaemia, 42.6% were aged between 20 and 30, 23.4% were over 30 and 17% under 20. among women with moderate parasitaemia, 8.6% were aged between 20 and 30, and 4.2% were under 20. two cases (4.2%) of high parasitaemia were recorded, one in the age group 20 -30 and the other in the over 30 (table 2). table 2: parasite density by age among pregnant women in the commune of ntoum (2024) age ( years) low (< 1000 p/µl) moderate (1000 – 9 999 p/µl) high (≥ 10 000 p/µl) n % n % n % < 20 8 17 2 4,2 0 0 20 – 30 20 42,6 4 8,6 1 2,1 >30 11 23,4 0 0 1 2,1 total 39 83 6 12,8 2 4,2 malaria prevalence according to sociodemographic and obstetric characteristics, clinical symptoms, and preventive measures among pregnant women attending the ntoum departmental hospital all participants (n = 202) were tested for malaria. the results were positive in almost a quarter of the participants, giving a prevalence of malaria 23.3%. of the 47 pregnant females with malaria hematozoa in their circulating blood, 53.2% were aged between 20 and 30, and 25.5% were over 30. plasmodium infection was diagnosed predominantly (46.8%) in women in their second trimester of pregnancy, followed by those in their first and third trimesters (31.9% and 21.3%, respectively). clinically, cough was encountered in onefifth (21.3%) of them, and two-fifth (42.5%) complained of headaches. almost a sixth (15%) suffered from acute diarrhea, and over half (51.1%) had a fever. the largest number of participants (70.2%) lived in the first district of ntoum commune and had completed secondary school (87.2%)., malaria was detected in the majority (68.1%) of pregnant women with no income-generating activity. more than half of them did not use insecticide sprays or sleep under an insecticide-impregnated net (table 3). table 3: clinical symptoms, preventives measures, sociodemographic and obstetric characteristics variables (frequency (%) malaria + (%) odd ratio 95% ci p-value age (years) < 20 28 (13,8) 10 (21,3) 20 – 30 117 (58) 25 (53,2) 2.04 0.84 4.98 0.115 >30 57 (28,2) 12 (25,5) 2.08 0.76 5.67 0.150 month of pregnancy 1 3 (1st trimester) 50 (24,7) 15 (31,9) 4 6 (2nd trimester) 105 (52) 22 (46,8) 1.61 0.75 3.47 0.218 pa ge 40 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 36-44, 2025 7 9 (3rd trimester) 47 (23,3) 10 (21,3) 1.58 0.63 3.99 0.328 respiratory signs dyspnea 03 (1,5) 2 (4,3) suffocation 09 (4,5) 4 (8,5) 2.50 0.16 38.60 0.511 cough 38 (18,8) 10 (21,3) 5.60 0.45 68.68 0.178 choking 03 (1,5) 1 (2,1) 4.00 0.13 119.23 0.423 none 149 (73,7) 30 (63,8) 7.66 0.67 87.42 0.100 neurological signs dizziness 46 (22,7) 9 (19,1) headache 71 (35,1) 20 (42,5) 0.62 0.25 1.51 0.294 asthenia 1 (0,6) 00 (00) 0.76 0.03 20.17 0.869 none 84 (41,6) 18 (38,4) 0.89 0.36 2.18 0.802 digestive signs diarrhea 22 (10,8) 7 (15) vomiting 21 (10,4) 5 (10,6) 1.49 0.39 5.74 0.559 epigastralgia 12 (6,1) 5 (10,6) 0.65 0.15 2.8 0.566 vomiting 2 (1,0) 00 (00) 2.42 0.10 56.97 0.583 none 145 (71,7) 30 (63,8) 1.79 0.66 4.78 0.246 general signs asthenia 54 (26,8) 8 (17) fever 58 (28,7) 24 (51,1) 0.24 0.09 0.61 0.002 none 90 (44,5 15 (31,9) 0.86 0.34 2.21 0.769 place of residence 1st district 156 (77,2) 33 (70,2) 2nd district 24 (11,8) 9 (19,1) 0.44 0.18 1.11 0.083 3rd district 00 (00) 00 (00) 0.27 0.005 13.92 0.516 neighboring towns 22 (11) 5 (10,7) 0.91 0.31 2.65 0.866 education level primary 19 (9,4) 3 (6,4) secondary 138 (68,3) 41 (87,2) 0.44 0.12 1.60 0.215 university 37 (18,3) 1 (2,1) 6.75 0.65 69.97 0.109 illiterate 8 (4,0) 2 (4,3) 0.56 0.07 4.24 0.576 occupation unemployed 139 (68,8) 32 (68,1) employed 63 (31,2) 15 (31,9) 0.95 0.47 1.93 0.902 use of insecticide spray yes 84 (41,6) 19 (40,4) no 118 (58,4) 28 (59,6) 0.94 0.48 1.82 0.854 use of bed net yes 97 (48,0) 21 (44,7) no 105 (52,0) 26 (55,3) 0.84 0.43 1.61 0.601 factors associated with malaria infection this study revealed that pregnant women aged 20-30 and over 30 were two times more likely to test positive to malaria compared to those under 20. likewise, women in their second trimester were more susceptible to malaria infection (or = 1.61 [0.75 – 3.47]). women with university education were nearly seven (7) times less prone to malaria infection compared to the other groups (or = 6.75 [0.65 – 69.97]). employed women appeared to be less exposed to malaria infection than the unemployed ones (or = 0.95 [0.47 – 1.93]). neither the clinical manifestations, nor the obstetric or the preventive measures were significantly associated with malaria (p˃0.05). in contrast, women who reported pa ge 41 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 36-44, 2025 episodes of fever during the survey were less prone to malaria infection, and the difference was found statistically significant (or = 0.24 [0.09 0.61]; p =0.002). discussion a sixmonth cross-sectional study was conducted at the departmental hospital of ntoum to assess the prevalence and risk factors associated with malaria among pregnant women. among participants in this study, the prevalence of malaria infection was 23.3%. this is comparable to the prevalence of malaria reported in north-west ethiopia (almaw et al., 2022) and burkina faso (rouamba et al., 2021), but these results are higher than those found in studies carried out in kenya (okoyo et al., 2021) and nigeria (oyerogba et al., 2023). nevertheless, the prevalence among pregnant women in komo mondah is lower than that found in the san district of mali (diarra et al., 2022), or that reported in south-east nigeria (okoroiwu, 2023) and uganda (mangusho et al., 2023). these discrepancies in prevalence observed in high-transmission countries justify the view that malaria prevalence is a function of sample size and socio-environmental factors. in our study, the average age of pregnant women surveyed was 27.4 years, with extremes of 15 and 44 years. these values are similar to those found in many african health facilities. indeed, the average age of pregnant women attending antenatal clinics were 27.3 years, 26, and 24.6 years respectively in cameroon, burkina faso, and mali, (nguefack, 2018; yameogo,2023 ; delbaere et al., 2020). these similar results are due to the fact that a woman’s fertility is at its peak between the end of adolescence and the approach of her thirties. after the age of 30, fertility begins to decline, collapsing around the age of 38 due to a decrease in the number and quality of oocytes (delbaere et al., 2020). this study revealed that 21.3% of pregnant women under 20 suffered from malaria. these results are lower than those found in the peripheral maternity wards of parakou in benin and at the toamasina university hospital in madagascar, where malaria rates among pregnant women under 20 were 56.9% and 47.5% respectively (obossou et al., 2024 ; botolahy et al., 2011). among pregnant women aged between 20 and 30, malaria prevalence was 53.2% in our study. these results are slightly lower than those observed in benin (obossou et al., 2024), but higher than those observed at the commune ii reference health center in bamako, mali (santara, 2024). in the case of pregnant women over 30 years of age, 25.5% of our participants suffered from malaria, compared with 42.1% in the peripheral public maternity units of parakou, benin. (obossou et al., 2024). younger women and primiparous women are traditionally considered to be at greater risk of malaria, as they do not benefit from sufficient immunity with age (nankabirwa et al., 2014). in our study, malaria was more frequent in pregnant women in their second trimester (46.8%) than in those in their first or third trimester (31.9% and 21.3% respectively). our results are in line with those found in ghana, and north-eastern nigeria (dwumfour et al., 2023; ali, 2022). where the categories of pregnant women most affected were those in their second trimester, followed by those in their third trimester. those in their first trimester had the lowest malaria prevalence. nevertheless, our results contrast with those found in the democratic republic of congo (drc) and eastern sudan. indeed, their work emphasizes that it is during the third trimester of pregnancy that women are more likely to suffer from malaria (bihingoyi, 2013; idris nour et al., 2023). other studies carried out in southern ghana and southern malawi, on the other hand, showed that pregnant women were at greater risk of contracting malaria during the first trimester of pregnancy (ahadzie-soglie et al., 2022; verhoeff et al., 1999). these discrepancies could be explained by the fact that there is no significant association between gestational age and malaria, as highlighted by a study carried out in burkina faso (tahita et al., 2013). level of education also influenced the frequency of malaria in pregnant women. the highest incidence of malaria was found among pregnant women with secondary education (87.2%), followed respectively by primiparous (6.4%), illiterates (4.3%) and university graduates (2.1%). in general, the risk of contracting malaria decreases with the level of education, which is associated with better knowledge of malaria prevention measures and better access to health services. however, significant disparities may exist within study groups (salissou et al., 2016). most participants with malaria (70.2%) lived in the first district of the ntoum commune, followed by those living in the second district and neighboring towns; 19.2% and 10.7% respectively. in addition, to the tropical climate that prevails throughout gabon, the first district of the commune of ntoum is a region rich in bodies of water (inlets, mangroves, rivers and streams). the proximity habitations with watercourses and dense vegetation favors human contact with mosquitoes, and hence the occurrence of malaria (chaves et al., 2021; dufera et al., 2020). the study showed that the unemployment rate among participants was 68.8% (tonda, 2024).]. in our study, the prevalence of malaria among these unemployed people was equal to 68.1%. the low purchasing power of the poor means that they cannot afford medical consultations in gabonese hospitals, nor can they afford anti-mosquito bite tools (aerosol sprays, spiral fumigants) or antimalarial drugs. in most cases, the latter are forced to turn to cheaper traditional medicines, thus encouraging the transmission of malaria (diallo et al., 2007; kodio et al., 2023). in this study, clinical symptoms were dominated by fever (51.1%), followed by headache (42.5%). according to the results of a study carried out in malawi, the main clinical signs in favor of malaria were headaches (24.3%) and chills (16.9%). fever and muscle pain were less common, with 7.1% and 6.7%, respectively (namwera et al., 2025). in another study, carried out in north-west ethiopia, the clinical signs of malaria encountered in pregnant women were mainly fever (91.8%) and headache (81.4%) (almaw et al., 2024). the probability of malaria was significantly pa ge 42 https://journals.e-palli.com/home/index.php/ajlsi am. j. life sci. innov. 4(1) 36-44, 2025 higher in pregnant women presenting with fever and headaches than in asymptomatic pregnant women. a study conducted in burkina faso also found that fever, history of fever and headache were the most common malaria-related clinical signs in pregnant women (tahita et al., 2013). on the basis of these findings, the presence of fever and headaches in pregnant women in tropical zones points to malaria and emphasizes the need for early detection. to protect themselves from mosquito bites in the mining town of moanda (south-east gabon), 21.1% of people used insecticide sprays (nno mabiala et al., 2021). this frequency of chemical agent use is lower than that found in our study (41.6%), but higher in households in niger (63%) (mahaman toudou et al., 2021). the proliferation of insect pests on agricultural fields in niger has prompted farmers to use large volumes of synthetic insecticides on legumes and in irrigated perimeters, justifying the high use of insecticides in this country (abdou et al., 2024). this study revealed that more than half (59.6%) of malaria sufferers did not use chemical agents to protect themselves from mosquitoes, justifying that the risk of contracting malaria is lower when insecticides are used as an alternative to itns. however, the massive use of insecticides can, on the other hand, cause gene mutations in anopheles gambiae strains, making them resistant to many insecticides (ddt and pyrethroids), as some studies have shown (organization, 2023 ; akono et al., 2022). sleeping under an insecticide-treated mosquito net to prevent insect bites is a practice recommended by the who [1]. in this study, 48.0% of pregnant women declared sleeping under an itn. our results are similar to those found in nigeria (42.2%) (ogomaka, & obeagu, 2021); but, lower than those obtained in rwanda (57.9%) (kawuki et al., 2023). this discrepancy in results may be explained by the sample size and the study region, which differed from one study to another. nevertheless, this study reveals that more than half (55.3%) of pregnant women who did not sleep under an itn suffered from malaria. this finding is consistent with the fact that sleeping under an itn reduce the incidence of malaria (organization, 2023). study limitations our study has a number of limitations which must be taken into account when interpreting the results. indeed, the relatively small sample size, the absence of data on intermittent preventive treatment during pregnancy (iptp), and the lack of explanatory variables such as the reasons why participants did not sleep under an insecticide-treated net, or did not use aerosol sprays may reduce the statistical power to detect a true association in the population. conclusion this study shows that the rate of malaria during pregnancy is widespread among women consulting for antenatal care at ntoum hospital. the study also shows that regular use of malaria control tools (insecticide-treated nets, indoor residual spraying) and early antenatal care visits can significantly reduce the risk of malaria during pregnancy. to reduce the burden of malaria in the region, we recommend the distribution of free insecticidetreated mosquito nets to all pregnant women attending antenatal care at ntoum hospital. biological diagnosis and early treatment of malaria should be combined with the use of insecticide-treated nets. municipal authorities should implement environmental control measures to control the population of anopheles larvae. finally, the gabonese government should ensure the implementation of vector control interventions based on entomological surveillance, the correct and rational use of insecticides, and the monitoring and management of insecticide resistance. references abdou, l., rabo, y., kailou, m. h. s., assane, m. m., & mahamane, a. 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