166 © 2019 by the authors; licensee Asian Online Journal Publishing Group Agriculture and Food Sciences Research Vol. 6, No. 2, 166-171, 2019 ISSN(E) 2411-6653/ ISSN(P) 2518-0193 DOI: 10.20448/journal.512.2019.62.166.171 © 2019 by the authors; licensee Asian Online Journal Publishing Group Nutrient Content Assessment of Six Citrus Species Parts and their Feedstuff Significance Chinelo A. Ezeabara1 Okeke C.U.2 ( Corresponding Author) 1,2Department of Botany, Nnamdi Azikiwe University, P.M.B. 5025, Awka, Anambra State, Nigeria. Abstract Insufficient and high cost of good quality feed is the primary limitation to livestock operation. Hence, assessment of surplus atypical feed resources for their nutritive potential to support livestock productivity becomes topical. Dry matter bases of the roots, stems, stem barks, leaves and peels of six Citrus species that are commonly available in Southeastern Nigeria were investigated for nutrient composition. High value of crude protein ranging from 15 to 18 percent was found in the leaves of all the species. High carbohydrate values ranged from 70 to 81 percent, in the peels of C. grandis, C. reticulata, C. paradisii and C. sinensis. High percentage of fat was also present in the range of 12 to 13% in the peels of C. aurantifolia and C. sinensis. Ash contents were very high in the leaves of C. aurantifolia, C. grandis, C. limon and C. paradisii, ranging from 10 to 14%. Therefore, leaves and peels of these Citrus species could be regarded as low cost and locally available alternative high-quality animal feeds. Keywords: Animal feed, Animal protein, Animal nutrition, Citrus leaves, Citrus peels, Crude protein, feed quality, Nutritive value. Citation | Chinelo A. Ezeabara; Okeke C.U. (2019). Nutrient Content Assessment of Six Citrus Species Parts and their Feedstuff Significance. Agriculture and Food Sciences Research, 6(2): 166-171. History: Received: 28 March 2019 Revised: 8 May 2019 Accepted: 10 June 2019 Published: 22 July 2019 Licensed: This work is licensed under a Creative Commons Attribution 3.0 License Publisher: Asian Online Journal Publishing Group Contribution/Acknowledgement: Both authors contributed to the conception and design of the study. Funding: This study received no specific financial support. Competing Interests: The authors declare that they have no conflict of interests. Transparency: The authors confirm that the manuscript is an honest, accurate, and transparent account of the study was reported; that no vital features of the study have been omitted; and that any discrepancies from the study as planned have been explained. Ethical: This study follows all ethical practices during writing. Contents 1. Introduction .................................................................................................................................................................................... 167 2. Materials and Methods ................................................................................................................................................................. 167 3. Results .............................................................................................................................................................................................. 168 4. Discussion ........................................................................................................................................................................................ 169 5. Conclusion ....................................................................................................................................................................................... 169 References ............................................................................................................................................................................................ 170 http://crossmark.crossref.org/dialog/?doi=10.20448/journal.512.2019.62.166.171&domain=pdf&date_stamp=2017-01-14 http://crossmark.crossref.org/dialog/?doi=10.20448/journal.512.2019.62.166.171&domain=pdf&date_stamp=2017-01-14 http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ http://www.asianonlinejournals.com/index.php/AESR/article/view/866 Agriculture and Food Sciences Research, 2019, 6(2): 166-171 167 © 2019 by the authors; licensee Asian Online Journal Publishing Group Contribution of this paper to the literature This study suggested that Citrus aurantifolia (Christm.) Swingle, C. grandis Osbeck, C. limon (L.) Burm. f., C. paradisii Macf., C. reticulata Blanco and C. sinensis (L.) Osbeck leaves and peels are potential alternative high-quality animal feeds. 1. Introduction Citrus belongs to the family Rutaceae. Six species of Citrus are commonly available in southeastern Nigeria. They include: Citrus aurantifolia (Christm.) Swingle (Lime), C. grandis Osbeck (Shaddock/Pummelo), C. limon (L.) Burm. f. (Lemon), C. paradisii Macf. (Grapefruit), C. reticulata Blanco (Mandarin/Tangerine), and C. sinensis (L.) Osbeck (Sweet orange).The members of this genus are small trees or large shrubs, reaching 5 to 15 meters tall, with spiny shoots and alternately arranged evergreen leaves with an entire margin [1, 2]. The coriaceous evergreen leaves are gland dotted and unifoliate-compound with a joint between leaf blade and petiole [1, 3]. Citrus fruits are one of the world’s most crucial, tasty and juicy fruit of Rutaceae [4, 5].They have long been regarded as important sources of vitamin C; medicinal plant[6]and in the manufacture of household products. However, only the juices and the essential oils, which the antimicrobial property [7] and anti-aflatoxigenic activity [8] have been investigated. The use of Citrus pulp as a common animal feed had also been reported [9]. Thus, it becomes absolutely imperative to investigate other Citrus plants parts for nutritional composition with the view to support livestock productivity. Good nutrition is an integral part of successful livestock operation. Nutrients are chemical substances in food that nourish the body by providing energy, building materials, and factors to regulate needed chemical reactions in the body [10]. The groundwork of livestock production system is the accessibility of quality and quantity of animal feed, with feed costs taking up to 70% of the cost of production [11]. Moreover, improper animal feeding and nutrition, either unbalanced diet, under or over feeding of livestock affect the general well-being of the animals, including lowering their body immune system. Protein is one of the most expensive nutrients and an extremely important nutrient in all animal feed. Consumption of small protein and food energy could develop protein-energy malnutrition [12]. The deficiency eventually results in body wasting, primarily of lean tissue, and increased susceptibility to infections. Moreover, the deliberation for herdsmen to rear cattle in ranches in Nigeria is ongoing. Nigeria had over 14.73 million cattle consisting of 1.47 million milking cows and 13.26 million beef cattle in 2008 [13, 14]. The number must have undoubtedly increased by now. Plant materials are the chief diets of livestock. High cost of animal feeds is among the major factors limiting animal production in Nigeria [15, 16]. There is shortage of good quality feeds needed to sustain livestock growth especially during dry season, as a result of scarcity of forages. Although many livestock farmers formulate their own animal feed, they have challenges of access to low cost, good quality and easily available raw materials. Besides, feeding animals with crops could reduce the available food for humanity, considering the problem of food shortage as a result of human population explosion in developing countries. Hence, other alternative sources such as agro-industrial by-products and crop residues are being assessed for their nutritive quality to support livestock productivity. The objective of this study therefore, is to determine the nutritive value of various Citrus species parts that are predominant in southeastern, Nigeria. 2. Materials and Methods 2.1. Sources of Materials The roots, stems, stem barks, leaves and fruits of Citrus aurantifolia, C. grandis, C. limon, C. paradisii, C. reticulata and C. sinensis were collected in the months of November – December at optimum maturity, from Agricultural and Natural Resources Department Market Garden, Amawbia, Anambra State, Nigeria. The voucher specimens were deposited at Department of Botany Herbarium, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria. 2.2. Preparation of Plant Materials The rinds of healthy ripe fruits of the six Citrus species were peeled off with a knife. The roots, stems, stem barks and peels were sun dried for seven days whereas the leaves were air dried in the laboratory at room temperature for ten days. The dried samples (about 1.5kg) were then crushed with mortar and pestle before grinding into fine powder using a manual grinder (Corona, USA). 2.3. Proximate Determination The nutrient content of the six Citrus species parts were determined according to the methods described by Onwuka [17]. 2.3.1. Moisture Content Determination The dishes were washed thoroughly and dried in the oven. They were latter put inside the dessicator to cool. After which they were weighed. Sample was put into the weighed dish and weight taken. The sample was dried in the oven at 70 ᴼC for 2 hours and at 105 ᴼC for the next 4 hours. The sample was cooled in the dessicator and the dry weight of sample plus dish taken. The moisture content was calculated as follows: Percentage (%) moisture = W2− W3 × 100 W2− W1 1 Where:- W1 = Initial weight of empty crucible W2 = Weight of crucible + sample before drying W3 = Final weight of crucible + sample after drying 2.3.2. Ash Content Determination Agriculture and Food Sciences Research, 2019, 6(2): 166-171 168 © 2019 by the authors; licensee Asian Online Journal Publishing Group Five grams (5g) of finely ground dry sample was weighed into a tarred silica crucible. The sample was charred on a heater inside a fume cupboard, to dry off most of the smoke. The sample was transferred into a pre-heated muffle furnace at 550 ᴼC. It was left at this temperature for 2 hours. After which it was cooled in a dessicator and reweighed. Percentage (%) Ash = Weight of ash × 100 Weight of original sample 1 = W3 –W1 × 100 W2 –W1 1 Where:- W1 =Weight of empty crucible W2 =Weight of crucible +sample before drying W3 = Weight of crucible + ash 2.3.3. Crude Fibre Content Determination Water reflux was boiled for 30 minutes with 200 ml of a solution containing 1.25g of H2So4 per 100 ml of solution. The solution was filtered through two fold of cheese cloth on a fluted funnel. The residue was washed with boiling water until there was no longer acid. The residue was transferred to a beaker and boiled for 30 minute with 200 ml of a solution containing 1.25g of carbonate-free NaOH per 100ml.The final residue was filtered through a thin but closed pad of washed and ignited asbestos in a Gooch crucible. It was dried in an electric oven and weighed. It was finally incinerated, cooled and weighed. The loss in weight after incineration × 100 is the percentage of crude fibre. 2.3.4. Crude Protein Content Determination A measured weight (2g) of sample was weighed into a 250ml beaker. After which 75ml of hot water was added and brought to boil. It was stirred vigorously and added 25ml of 6% copper sulphate solution. It was again brought to boil, stirred vigorously and added 25ml of the 1.25% sodium hydroxide solution. The mixture was stirred vigorously, removed from the flame and allow to settle. It was filtered through a 15 cm, No. 4 Whatman paper. The precipitate was cleaned from the sides of the beaker with a ‘policeman’. The paper was washed free from sulphate with very hot water 6 times. It was allowed to drain well, and then transferred to a Kjeldahl flask containing about 10g anhydrous sodium hydroxide and a trace of selenium. Thirty milliliters (30ml) of conc. H2so4 was added, nitrogen content determined and hence the protein content of the sample. Percentage (%) protein = % N × F Where:- F = Conversion factor (6.25) Percentage (%) Nitrogen = VS − VB × Nacid × 0.028 × 100 W Where:- VS = Volume of acid required to titrate sample in milliliters VB = Volume of acid required to titrate blank in milliliters Nacid = normality of acid (0.1N) W = weight of sample in grams Therefore, Percentage (%) protein = % Nitrogen ×6.25. 2.3.5. Fat Content Determination The sample was mixed with a mixture of methanol and chloroform in such proportions to give a single phase miscible with water. Further, extra chloroform was added to give a separation of phases. The solvents were separated by centrifugation. The chloroform layer containing the dissolved fat was removed and fat residue left behind weighed. Percentage (%) Fat = Weight of fat × 100 Weight of sample 1 2.3.6. Carbohydrate Content Determination Carbohydrate content was determined by difference method: Percentage (%) Carbohydrate = 100 – (% Moisture + % Ash + % Crude fibre + % Crude protein + % Fat) 2.4. Statistical Analysis The data obtained were statistically analyzed using One-Way-ANOVA and the significant difference was established at (p>0.05). The results were expressed as mean + SD. 3. Results Considerable high levels of the basic nutrients were present in parts of the six Citrus species Table 1. High levels of carbohydrate were detected in the stems of C. aurantifolia (67.53+0.08%) and C. limon (50.13+0.04%) as well as in the peels of C. grandis (81.2+0.06%), C. reticulata (75.4+0.08%), C. paradisii (80.6+0.4%) and C. sinensis (70.4+0.05%). High percentages of fat and crude fibre occurred in the peels of C. aurantifolia (12.0+0.4, 8.30+0.4), C. limon (9.01+0.04, 9.37+0.02) and C. sinensis (13.89+0.02, 7.86+0.02) respectively. Fat content was also high in the leaf of C. reticulata (11.10+0.05%) and in the stem bark and root of C. paradisii (6.51+0.06, 6.28+0.4%) respectively. In addition, high levels of crude fibre were contained in the leaf of C. grandis (7.27+0.03%); peel of C. reticulata (9.75+0.03%) and leaf of C. paradisii (8.31+0.02%). Ash contents were very high in the leaves of C. aurantifolia (14.11+0.02%), C. limon (14.15+0.02%) and C. paradisii (13.01+0.01%).It was also high in the roots of C. Agriculture and Food Sciences Research, 2019, 6(2): 166-171 169 © 2019 by the authors; licensee Asian Online Journal Publishing Group grandis (12.72+0.04%) as well as in the stem bark of C. reticulata (15.18+0.02%) and C. sinensis (17.01+0.01%). Appreciable high values of ash were also detected in the leaves of C. grandis (10.18+0.02%) and root of C. paradisii (10.14+0.01%) Table1. 4. Discussion High carbohydrate values were ranged from 70 to 81 percent in the peels of C. grandis, C. reticulata, C. paradisii and C. sinensis. Carbohydrate and fat provide animal body with energy. Energy (mainly from carbohydrates) and protein are the primary macro nutrients to take into account, for beef cattle nutrition [9]. High percentage of fat was also present in the range of 12 to 13 in the peels of C. aurantifolia and C. sinensis. Fat is a solvent for fat-soluble vitamins and hormones. Reasonable levels of crude fibre were found in the leaves (except C. sinensis) and peels (with the exception of C. grandis), of all the Citrus species tested. Fibre promotes digestion, cleanses the digestive tract, prevents absorption of excess cholesterol [18] and aids in bowel movement by adding mass to the intestinal content [19]. Digestion of animal feed is therefore, determined by the crude fibre content [20]. Ash contents were very high in the leaves of C. aurantifolia, C. grandis, C. limon and C. paradisii, ranging from 10 to 14%. The ash values were higher than the ones found in leaves 10.85% of Ipomoea batatas (L.) Lam. (sweet potatoes) [15]. Ash of a food material contains mineral matter. Mineral salts are needed for regulation of body metabolism. Crude protein was found in high levels ranging from 15 to 18 percent in the leaves of all the Citrus species investigated. The percentage crude protein values were higher than the levels present in the Zea mays L. (corn) grain 9.5−11.2, Sorghum bicolor (L.) Moench (milo, sorghum) grain 10.3−11.0 and Glycine max L. Merr. (soybean) leaf 11.0−13.1 [21]. Moreover, the values in the peels of C. aurantifolia, C. limon, C. reticulata and C. sinensis that ranged from 6.4−10.7% were higher than the levels found in leaf 6.2−7.8%, husk 3.0−4.0%, cob 2.1−3.8% and stalk 3.4−4.9% of corn; stem 3.6−4.5% and pod 4.5−9.0% of soybean, and stalk 3.3−3.9% of milo. Beef is the major meat consumed in Nigeria and the meat quality is primarily dependent on the feed quality. Pastoralism, agro-pastoralism and ranching systems are three major beef cattle production systems in Nigeria [22] with the pastoralism and agro-pastoralism being the principal systems. Ranching system involves semi-intensive and intensive feeding systems, where the animals’ nutritional needs are supplemented with diets as additional feed. The nutrient requirements of animals differ because of a number of factors, including age, body condition, body size, pregnancy and lactation. These factors as well as forage quantity and forage quality are considered in determining the supplementation need of animals. For sustainable farming, easy availability of local feeds and supplements is essential. This can be achieved by sourcing for local plant species of high nutritional values. According to United States Department of Agriculture [23] the development of animal feeds entails mixing various readily obtainable and low cost feed materials into a ration that will provide nutrient requirements for animals. In addition, having the information on the chemical composition of the various available feed ingredients is a critical factor. The dietary crude protein used in diets formulated by some consultants ranged from 12.5 to 14.4% but most consultants use at least 13% crude protein [24] whilst at least 15 percent crude protein is required for creep feeds or forages for nursing calves [25]. This indicated that leaves of these Citrus species could be used as protein supplement to enrich animal feed, because protein is needed by animal body in large amounts. Sweet potato vine silage contained 136.1 g kg-1 DM crude protein and had been suggested for supplementing tropical cattle [26]. Soybean contained 44% [27] and 49.0-52.0% crude protein value[21]. Soybean meal is the most common protein source for all compound feeds for pigs, poultry and dairy cattle globally; however, it is seldom too costly [27].The finding therefore, suggested an inclusion of Citrus plants leaves and peels in plant food protein, indicating that they can potentially be used as easily available protein sources for livestock production. Providing adequate protein in beef cattle diets is essential for animal health and productivity as well as ranch profitability [25]. Besides, young, growing cattle, in particular, need relatively high levels of crude protein in their diets to support muscle growth. Moreover, when forage crude protein levels fall below 6 to 8%, the protein supplementation is highly needed[28]. This is as a result of low quality of the forage. Citrus parts could therefore, be used to solve the problem of shortage of good quality feeds needed to sustain livestock growth especially during the dry season, which in turn, will support livestock productivity. Insufficient good quality animal feeds result to serious effects on cattle such as lessened pregnancy rates; loss of body condition of the cow, and reduce milk production, hence lowers weaning weights[29]. In addition, other alternative feeds are mostly in short supply and costly. Cost of feeding is the most expensive part of livestock production [30]. Cost is also the primary factor for supplementing additional protein in finishing diets [24]. These indicated that the leaves and peels of these Citrus species could potentially be used as inexpensive and readily accessible feedstuffs for livestock production. Moreover, high level of crude protein in these Citrus species leaves indicated that they can serve as cheap sources of protein. 5. Conclusion The study revealed that the leaves and peels of these Citrus species contained high nutritive values. They are also environmentally friendly, readily available and relatively cheap. Hence, suggesting their potential use in development of animal feed for more sustainable feeding system. In addition, protein is the most expensive and critical nutrient needed by animal body in large amounts. Therefore, incorporation of these parts as protein supplement in animal feeds to enhance animal nutrition is recommended. Further research however, is necessary in order to assess the effect of the diet on animal nutrient digestibility and growth performance. Agriculture and Food Sciences Research, 2019, 6(2): 166-171 170 © 2019 by the authors; licensee Asian Online Journal Publishing Group Table-1. Proximate composition of leaf, peel, stem, stem bark and root of six Citrus species. Citrus Species Nutrients Plant Parts (%) Leaf Peel Stem Stem Bark Root Citrus aurantifolia Crude protein 16.69+0.07 6.38+0.4 6.27+0.04 7.81+0.01 9.38+0.02 Carbohydrate 41.76+0.04 53.89+0.4 67.53+0.08 56.86+0.12 55.17+0.01 Fat 9.39+0.01 12.0+0.4 2.88+0.03 6.17+0.03 5.71+0.01 Ash 14.11+0.02 5.82+0.04 6.47+0.01 11.27+0.03 13.61+0.01 Crude fibre 6.77+0.02 8.30+0.4 5.74+0.03 5.68+0.03 5.23+0.4 Moisture 12.86+0.04 11.29+0.06 11.13+0.01 12.07+0.02 10.87+0.02 C. grandis Crude protein 16.27+0.04 1.0+0.02 6.52+0.01 6.32+0.4 8.77+0.3 Carbohydrate 49.72+0.1 81.2+0.06 66.63+0.10 62.14+0.05 56.85+0.4 Fat 5.24+0.4 0.7+0.01 2.77+0.03 5.85+0.4 5.25+1.01 Ash 10.18+0.02 2.1+0.05 7.27+0.2 8.18+0.02 12.72+0.04 Crude fibre 7.27+0.03 4.5+0.4 5.28+0.01 4.81+0.01 5.85+0.4 Moisture 11.33+0.03 19.2+0.03 11.45+0.05 12.69+0.01 10.57+0.01 C. limon Crude protein 15.51+0.04 7.30+0.01 4.80+0.01 7.02+0.03 10.25+0.01 Carbohydrate 39.03+0.01 41.35+0.03 50.13+0.04 47.05+0.1 42.03+0.02 Fat 7.23+0.03 9.01+0.04 1.13+0.03 5.25+1.01 4.35+0.01 Ash 14.15+0.02 5.91+0.01 6.01+0.01 12.08+0.04 11.05+0.05 Crude fibre 6.80+0.01 9.37+0.02 5.86+0.01 6.30+0.01 7.52+0.02 Moisture 12.31+0.04 15.31+0.05 13.01+0.05 14.07+0.02 12.70+04 C. reticulata Crude protein 15.86+0.04 8.95+0.03 7.29+0.01 6.21+0.02 8.79+0.04 Carbohydrate 42.17+0.06 75.4+0.08 69.31+0.06 56.84+0.05 56.14+0.07 Fat 11.10+0.05 9.02+0.4 3.07+0.01 6.29+0.01 6.04+0.02 Ash 11.53+0.08 13.04+0.04 4.28+0.02 15.18+0.02 13.34+0.06 Crude fibre 6.81+0.3 9.75+0.03 4.91+0.01 4.74+0.02 5.30+04 Moisture 12.54+0.02 15.32+0.02 11.16+0.04 10.76+0.03 10.41+0.02 C. paradisii Crude protein 18.13+0.05 0.4+0.01 6.43+0.4 6.01+0.03 8.41+0.03 Carbohydrate 40.65+0.04 80.6+0.4 60.48+0.07 58.31+0.05 51.02+0.01 Fat 5.03+0.4 0.3+0.01 2.83+0.03 6.51+0.06 6.28+0.4 Ash 13.01+0.01 1.30+0.02 6.32+0.04 7.82+0.04 10.14+0.01 Crude fibre 8.31+0.02 2.3+0.05 5.01+0.01 4.98+0.02 4.71+0.01 Moisture 13.31+0.02 17.4+0.03 9.33+0.02 14.05+0.05 12.31+0.04 C. sinensis Crude protein 16.03+0.02 10.74+0.01 8.09+0.02 6.35+0.01 9.31+0.04 Carbohydrate 39.97+0.07 70.4+0.05 61.48+0.02 53.71+0.02 41.08+0.02 Fat 11.53+0.08 13.89+0.02 5.01+0.03 7.31+0.03 8.01+0.02 Ash 10.61+0.02 11.90+0.07 5.31+0.05 17.01+0.01 11.57+0.03 Crude fibre 4.93+0.02 7.86+0.02 5.05+0.01 5.31+0.07 4.27+0.01 Moisture 14.61+0.02 15.71+0.03 12.09+0.01 10.93+0.03 9.91+0.4 Note: Results are in Mean ± SE of triplicate determinations, (p>0.05). 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