Dietary Supplementation of S. platensis and S. cerevisiae in Laying Hens (H.I. Wahyuni et al.) 47 J I T A A Journal of the Indonesian Tropical Animal Agriculture Accredited by Ditjen Riset, Teknologi dan Pengabdian kepada Masyarakat No. 164/E/KPT/2021 J. Indonesian Trop. Anim. Agric. pISSN 2087-8273 eISSN 2460-6278 http://ejournal.undip.ac.id/index.php/jitaa 48(1):47-57, March 2023 DOI: 10.14710/jitaa.48.1. 47-57 Dietary supplementation of Spirulina platensis and Saccharomyces cerevisiae on egg quality, physiological condition and ammonia emission of hens at the late laying period H. I. Wahyuni*, T. Yudiarti, E. Widiastuti, T. A. Sartono, I. Agusetyaningsih, S. Sugiharto Department of Animal Science, Faculty of Animal and Agricultural Sciences, Universitas Diponegoro, Semarang, 50275, Indonesia *Corresponding E-mail: hihannyiw123@gmail.com Received November 11, 2022; Accepted February 27, 2023 ABSTRACT The study was aimed to evaluate the effect of Spirulina platensis and Saccharomyces cerevisiae or its combination on egg quality, physiological condition and ammonia excretion of hens at the late lay- ing period. At 81 weeks old, 144 Lohmann Brown layer chickens were divided into four treatment groups included CON (hens fed basal feed), SP (basal feed + 0.3% S. platensis), SC (basal feed + 0.2% S. cerevisiae), and SPSC (basal feed + 0.3% S. platensis + 0.2% S. cerevisiae). At the end of the study, eggs, intestinal mucosa, digesta, excreta and blood sample were collected. Results showed that albumin index was higher (P<0.05) in SPSC group than in CON, SP and SC. The yolk index and yolk colour were greater (P<0.05) in SP and SPSC groups than in CON and SC. The erythrocyte values were high- er (P<0.05) in SP group compared to CON, SC and SPSC groups. The ileum pH was higher (P<0.05) in SP than in CON, SC and SPSC. Lactic acid bacteria counts were lower (P<0.05) in the caecum of SC and SPSC than in CON and SP groups. The lower (P<0.05) counts of lactose-negative Enterobacte- riaceae were shown in SC and SPSC than in CON. The excreta pH was lower (P<0.05) in SC group compared to CON, SP and SPSC groups. There were better (P<0.05) protein digestibility coefficient and nitrogen retention in SPSC group than others. Faecal ammonia decreased (P<0.05) in SP, SC and SPSC groups. In conclusion, S. platensis improved egg yolk index and colour, increased erythrocyte counts and played an important role in maintaining the balance of bacteria in the intestine resulting in reduced ammonia excretion. Dietary inclusion of S. cerevisiae reduced ammonia excretion of laying hens during the late laying period. Keywords: Laying hens, Microalgae, Probiotic, Yeast INTRODUCTION Laying hens are one of the livestock com- modities that primarily produce eggs. The rearing period of laying hens is typically divided into several phases, included starter, grower, laying and finisher phase. Laying hens start producing eggs in the pullet phase and decrease after reach- ing the peak of production. Molnar (2017) stated, laying hens are kept in production until they are 48 J. Indonesian Trop. Anim. Agric. 48(1):47-57, March 2023 75-80 weeks old. Yet, farmers generally extend the egg production period till the age of 100 weeks, which is not only contributed to higher profitability but also more sustainable food pro- duction (Pottgüter, 2016). There were, however, a number of challenges, such as decreasing egg production and quality as well as the physiology of the hens in the later stages of commercial lay- ing hens. It takes a diverse strategy to solve these problems. Antibiotic growth promoters (AGP) were commonly used to maintain the productivity and health of laying hens during the late stage of egg production, but their use is now prohibited in the vast majority of countries worldwide included Indonesia (Wongsuvan et al., 2018; Afandi et al., 2020). Spirulina platensis has attracted the atten- tion of poultry nutritionists because of its high protein content and other active ingredients (Mullenix et al., 2021). The microalgae S. platensis contains 53.31 g/100 g crude protein, 9.25 g/100 g lipids, 23.38 g/100 g carbohydrate, 7.44 mg/g chlorophyll a, 6.41 mg/g chlorophyll b, and 1.69 mg/g carotenoid (Rahim et al., 2021). S. platensis also contains antioxidant, immuno- modulatory, anti-inflammatory, antiviral, and antimicrobial properties (Langers et al., 2012; Abdel-Daim et al., 2013; Shokri et al., 2014). In laying hens, dietary supplementation of S. platensis resulted in a significant improvement in egg physical characteristics (Omri et al., 2018; Zahroojian et al., 2013; Selim et al., 2018). Moreover, Mariey et al. (2012) showed that die- tary inclusion of S. platensis (at 0.1, 0.15, and 0.2% of diets) increased egg weight, egg mass, and laying rate. In other commodities, the inclu- sion of 1% S. platensis into pig and broiler feeds was capable of improving the blood parameters, nutrient digestibility and performance, antioxi- dant activity, while reducing ammonia emission (Sugiharto et al, 2018; Park et al., 2018; Evans et al., 2015; Furbeyre et al., 2017). Saccharomyces cerevisiae has been widely used as an additive and an alternative to AGP for poultry (Ogbuewu et al., 2018; Morales-Lopez and Brufau, 2013). S. cerevisiae contains vita- mins, amino acids, and enzymes like mannases, glucanases, amylases, lipases, and proteases (Sugiharto, 2022; Ahiwe et al., 2021). The cell wall of S. cerevisiae is primarily composed of α- mannan (31% dry mass), mannoprotein, which accounts for approximately 40% dry mass, β- glucan (approximately 60% dry mass), and chitin (approximately 2% dry mass) (Ahiwe et al., 2021). Study in laying hens by Pinar (2013) and Swain et al. (2011) found that dietary supple- mentation of 1% or 1.5 g/kg S. cerevisiae, re- spectively, increased the percentage of hen day production, improved feed conversion ratio (FCR), increased albumen and shell thickness, increased egg weight, and improved feed effi- ciencies. Another study by Yalcin et al. (2014) found that adding yeast cell wall at 1 or 2 g/kg diets improved egg nutritional quality by lower- ing egg yolk cholesterol levels in 26-weeks brown laying hens. It has been very common to combine sever- al active ingredients to achieve synergistic and complementary effects on poultry. In this study, S. platensis and S. cerevisiae were used together for laying hens. Indeed, S. platensis contains a hepatotoxin called microcystin, which is toxic to the liver in high concentrations (Sugiharto et al., 2018). Study by Valerio et al. (2014) revealed that S. cerevisiae can ameliorate the toxic effect of microcystin in S. platensis. In this regard, the use of S. platensis and S. cerevisiae together was expected to get a synergistic effect from the two additives without causing toxic effects from S. platensis. So far, the use of combination of S. platensis and S. cerevisiae for commercial laying hens has not been studied. This study therefore aimed to evaluate the effect of S. platensis and S. cerevisiae or its combination on egg quality, physiological condition and ammonia excretion of hens at the late laying period MATERIALS AND METHODS Animals and Experimental Diets One hundred forty four of 81 weeks old Isa Brown laying hens with an average body weight Dietary Supplementation of S. platensis and S. cerevisiae in Laying Hens (H.I. Wahyuni et al.) 49 of 1,915±131.92 g from the commercial farm were used in this study. This study used an bat- tery cage (20 × 40 cm 2 ). The temperature and humidity of the cage was between 22 to 30°C and 70 to 90%, respectively, while the light was provided intermittently every 2 hr from 7 PM until 5 AM. Four treatment groups with 6 repli- cations for each group were arranged, so there were 24 experimental units. Each unit containing 6 laying hens that were divided into 3 cages, each with 2 hens. The treatment groups included CON (hens fed basal feed), SP (basal feed + 0.3% S. platensis), SC (basal feed + 0.2% S. cerevisiae), and SPSC (basal feed + 0.3% S. platensis + 0.2% S. cerevisiae). S. platensis was purchased from PT. Algaepark Indonesia Man- diri (Tangerang, Banten, Indonesia). Based on manufacturer’s label, S. platensis contained 60- 64 g/100 g protein, 5.6-7.2 g/100 g water, 20-24 g/100 g carbohydrates, <0.05 omega-6, 110-145 mg/100 g magnesium, 4.2-5.0 mg/100 4.2-5.0 mg/100 g vitamin B3, 1.5-2.5 mcg/100 mg vita- min B12, 6.0-7.2 mg/100 g vitamin E, 250-295 mg/1000 g carotene, 720-840 mg/100 g chloro- phyll, 9150-10450 mg/100 g phycocyanobilin. S. cerevisiae contained 9.82 × 10 11 cfu/g. The yeast was purchased from Angel Yeast Co. Ltd., Hu- bei, China. The hens were provided with manual feeders and drinking water facilities. The nutrition con- tent of basal feed was 13% water, 16% protein, 3% fat, 7% crude fibre, 12% ash, 3% calcium, and 0.6% phosphor, with 2800 kcal/kg metabo- lizable energy. From the beginning to the end of the rearing period, drinking water was provided ad libitum, while the feed was offered twice a day (morning and afternoon). The treatment last- ed 35 days. Egg was taken from each experi- mental unit in the last 3 days of the study (33-35 days) to determine the egg quality. Following that, blood and excreta samples were collected at the last day of the experiment. Data Collection and Laboratory Analysis Table 1. Nutritional Compositions of Basal Feeds Items Content (%) Corn 55.0 Rice bran 18.0 Soybean meal 11.0 Corn gluten meal 5.00 Meat bone meal 3.00 Bone meal 2.00 Limestone 4.00 MCP 1.00 Premix** 1.00 Analysed nutritional composition* Metabolizable energy (kcal/kg) 2800 Crude protein 15.41 Crude fibre 7.00 Crude fat 3.00 Ash 12.00 Water content 13.00 Calcium 3.00 Phosphor 0.60 *Crude protein level was according to proximate analysis, while the other content corresponds to the feed label. **Premix containing (per 10 kg of diet): Vitamin A 12,000,000 IU; Vitamin D3 2,000,000 IU; Vitamin E 8,000 IU; Vitamin K3 2,000 mg; Vitamin B1 2,000 mg; Vitamin B2 5,000 mg; Vitamin B6 500 mg; Vitamin B12 12,000 µg; Vitamin C 25,000 mg; Calcium D-pantothenate 6,000 mg; Niacin 40,000 mg; Choline chloride 10,000 mg; Methionin 30,000 mg; Lysine 30,000 mg; Manganese 120,000 mg; Iron 20,000 mg; Iodine 200 mg; Zinc 100,000 mg; Cobalt 200 mg; Copper 4,000 mg; Santoquine (antioxidant) 10,000 mg. 50 J. Indonesian Trop. Anim. Agric. 48(1):47-57, March 2023 Egg Quality Observation on egg quality were carried out at the end of the study. One egg was collected for each experimental unit to be examined for albu- min index, yolk index, yolk colour, Haugh unit, shell thickness and egg weight. The height of albumen and yolk was measured using a standard micrometre, while yolk diameter was determined using calliper. Shells were washed using water and dried, then shell thickness was measured by micrometre. Haugh units was calculated by for- mula as described by Khaleel et al. (2019) as follows = 100*log(H+7.57)-(1.7*WE 0.37 ). Yolk index was determined by dividing the yolk height by yolk diameter. Complete Blood Counts Blood sample was collected from the wing vein of each hen for complete blood count deter- mination. The collected blood was placed in eth- ylenediaminetetraacetic acid (EDTA)-containing vacutainers prior to analysis. Routine haematolo- gy measurements were carried out with a fully automatic haematology analyser from PT. Prima Alkesindo Nusantara (PRIMA, Power 100- 400V~50/60Hz 150 VA). Intestinal pH Twenty-four laying hens were used to assess the intestinal pH from intestine. The digestive tract was immediately prepared after euthanasia. A pH meter (OHAUS ST300) was used to meas- ure the pH of the duodenum, jejunum, ileum, and caecum. Ileum and Caecum Bacterial Population The determination of intestinal bacterial population of the ileum and caecum was con- ducted according to Sugiharto et al. (2022). After euthanasia, the digesta of ileum and caecum were transferred into 15 mL containers and immedi- ately analysed for the number of lactic acid bac- teria (LAB), coliform and lactose-negative bacte- ria (LNE). Viable counts of LAB in the digesta sample were conducted by plating onto De Man Rogosa and Sharpe (MRS) agar and on Mac- Conkey agar plates for coliform and LNE. The incubation of the MRS agar medium was carried out anaerobically for 48 h at 37°C and 24 hours at 37°C under aerobic condition for MacConkey agar. After incubation, the bacterial colonies were counted and presented in log cfu/g. Water Content, Acidity, Temperature of Ex- creta and Protein Digestibility Sample of excreta were collected at the end of study. Total nitrogen (N) was determined by standard Kjehdahl method. The Kjehdahl proce- dure was adopted for the crude protein measure- ment (Jabbar et al., 2020). Measurement of am- monia level was carried out using ammonia de- tector (Smart Sensor AR8500, Intell Instrumens Plus) with accuracy of 1 ppm according to the manufacturer instruction. Excreta temperature was determined by inserting a thermometer (ThermoONE, Onemed) into hen excreta. Mois- ture content was measured by weighing the wet weight and dry weight of excreta. Nitrogen reten- tion was calculated according to Tillman (1998). Nitrogen retention was measured by calculating the amount of nitrogen consumed minus the ni- trogen excretion corrected for endogenous nitro- gen. The formula for calculating the nitrogen retention is as follow = N consumption – (N Ex- creta – N Endogenous) Statistical Analysis SPSS program version 16.0 (IBM SPSS Sta- tistics for Windows) was used to analyse data. The data obtained were statistically analysed us- ing one-way ANOVA with a 5% significance level. Duncan’s multiple range test was used to evaluate the variations among treatment groups. RESULTS Egg Quality Table 2 shows data on egg quality. The treatment of SP, SC, and SPSC had substantial effects (P<0.05) on albumin index, yolk index, and yolk colour. The albumin index was higher in the SPSC group than in the CON, SP, and SC Dietary Supplementation of S. platensis and S. cerevisiae in Laying Hens (H.I. Wahyuni et al.) 51 groups. There was no significant difference in albumin index between CON, SP, and SC groups. In contrast to the previous parameters, the SP and SPSC groups had differences (P<0.05) in yolk index and yolk colour when compared to the CON group, but the SC group was not significantly different when compared to the CON or SP and SPSC groups. The treatment had no effect (P>0.05) on Haugh Unit, shell thickness, or egg weight. Complete Blood Counts Data on complete blood counts of laying hens are listed in Table 3. The values of erythro- cytes were greater (P<0.05) in SP group than in CON, SC and SPSC. There was no influence (P>0.05) of treatments on the serum concentra- tions of haemoglobin, haematocrits, MCV, MCH, MCHC, leukocytes, heterophils, lympho- cytes and thrombocytes in laying hens. Small Intestine pH Data on the small intestine pH of laying hens are listed in Table 4. The pH level of ileum was higher (P<0.05) in SP than in CON, SC and SPSC groups. There was no significant change in pH in all groups. Intestinal Ecology of Laying Hens The bacterial populations in the intestine are presented in Table 4. There was no difference (P>0.05) in the numbers of LAB, coliform and LNE enumerated in ileum. However, the SP and SPSC groups showed a decrease (P<0.05) in LAB populations when compared to CON and SC groups. Likewise, the LNE population in the Table 2. Egg Quality of Laying Hens Items CON SP SC SPSC SEM P value Albumin index 0.083 b 0.095 ab 0.093 ab 0.108 a 0.032 0.046 Yolk index 0.308 b 0.331 a 0.330 ab 0.332 a 0.003 0.012 Yolk colour 11.77 b 12.27 a 11.77 ab 12.05 a 0.147 0.019 Haugh Unit 78.73 82.61 84.18 86.83 0.006 0.113 Shell thickness (mm) 0.52 0.49 0.48 0.48 0.007 0.195 Egg weight 65.00 64.14 64.18 64.43 0.234 0.563 a,b Means marked with superscript letters in the same row are significantly different (P<0.05) CON (hens fed by basal feed), SP (basal feed + S. platensis 0.3%), SC (basal feed + S. cerevisiae 0.2%), and SPSC (basal feed + S. platensis 0.3% + S. cerevisiae 0.2%), SEM: standard error of the mean Table 3. Complete Blood Counts of Layer Chickens Items CON SP SC SPSC SEM P value Haemoglobin (g/dL) 10.67 12.33 10.25 10.83 0.12 0.349 Erythrocytes (10 6 /µL) 3.00 b 3.66 a 2.77 b 3.01 b 0.43 0.038 Haematocrit (%) 38.67 44.67 36.08 38.67 1.52 0.238 MCV (fL) 128.81 122.18 130.45 128.36 1.63 0.312 MCH (pg) 35.52 35.25 37.00 35.88 1.52 0.238 MCHC (g/dL) 27.62 27.58 28.42 28.02 0.25 0.624 Leukocytes (10 3 /µL) 77.50 80.25 73.16 77.00 2.81 0.865 Heterophils (10 3 /µL) 3.00 2.08 2.75 1.83 0.20 0.112 Lymphocytes (10 3 /µL) 74.50 78.16 70.41 75.16 2.78 0.825 Thrombocytes (10 3 /µL) 9.16 11.50 10.66 9.00 0.58 0.375 a,b Means marked with superscript letters in the same row are significantly different (P<0.05) CON (hens fed by basal feed), SP (basal feed + S. platensis 0.3%), SC (basal feed + S. cerevisiae 0.2%), and SPSC (basal feed + S. platensis 0.3% + S. cerevisiae 0.2%), SEM: standard error of the mean, MCV: mean corpuscular volume, MCH: mean corpuscular haemoglobin, MCHC: mean corpuscular haemoglobin concentration 52 J. Indonesian Trop. Anim. Agric. 48(1):47-57, March 2023 SC and SPSC groups decreased (P<0.05) com- pared to CON. Laying hens treated with SP did not show a significant difference from the CON, SC and SPSC groups. Water Content, Acidity and Excreta Temper- ature Data of water content, acidity and excreta temperature of laying hens are listed in Table 6. At the end of study, the treatments applied had no significant effect (P>0.05) on water content (%) and excreta temperature. In the contrary, pH of excreta was significantly affected by the treat- ment. The hens in SC group have a greater ex- creta pH than others (CON, SP and SPSC). Protein Digestibility Research data on protein digestibility are shown in Table 7. It was shown that the treat- ment had an effect on the protein digestibility coefficient, nitrogen retention and excreta am- monia. The SC group had a lower protein digest- ibility coefficient (P<0.05) compared to SPSC group. Higher N retention (P<0.05) found in SPSC group than in SC. Treatment with SP, SC and SPSC lowered (P<0.05) the ammonia con- tent in laying hen excreta. However, the treat- ment had no significant effect on N excreta. DISCUSSION S. platensis and S. cerevisiae have received attention as a potential alternative to AGP that can maintain production performance, and also acts as health promoting agent in poultry. Alt- hough having no positive influence on the egg production, feed consumption, FCR and mortali- ty (unpublished data), data in the present study showed that the albumin index increased in the SPSC group. Furthermore, the yolk index and yolk colour in the SP and SPSC groups increased significantly when compared with the CON and SC groups. Indeed, these present findings were consistent with those of Dogan et al. (2016), who revealed that feeding 2% S. platensis increased quail yolk index. Different from our study, Selim Table 4. Small Intestine pH of Laying Hens Items CON SP SC SPSC SEM P value Duodenum 6.27 6.38 6.58 6.04 0.07 0.081 Jejunum 6.30 6.55 6.37 6.07 0.09 0.305 Ileum 6.58 b 7.34 a 6.90 b 6.60 b 0.08 0.001 Cecum 7.00 7.37 7.29 7.05 0.08 0.284 a,b Means marked with superscript letters in the same row are significantly different (P<0.05) CON (hens fed by basal feed), SP (basal feed + S. platensis 0.3%), SC (basal feed + S. cerevisiae 0.2%), and SPSC (basal feed + S. platensis 0.3% + S. cerevisiae 0.2%), SEM: standard error of the mean Table 5. Bacterial Populations in Ileum and Caecum of Laying Hens Items CON SP SC SPSC SEM P value Ileum (log CFU/g) LAB 11.51 11.51 11.44 11.36 0.074 0.900 Coliform 6.03 5.70 5.75 5.55 0.208 0.891 Lactose negative-enterobacteria 7.48 7.82 6.85 5.55 0.367 0.127 Caecum (log CFU/g) LAB 11.70 a 11.64 a 11.19 b 11.17 b 0.082 0.021 Coliform 6.96 7.33 6.34 6.48 0.287 0.629 Lactose negative-enterobacteria (LNE) 9.08 a 7.74 ab 6.90 b 6.80 b 0.333 0.045 a,b Means marked with superscript letters in the same row are significantly different (P<0.05) CON (hens fed by basal feed), SP (basal feed + S. platensis 0.3%), SC (basal feed + S. cerevisiae 0.2%), and SPSC (basal feed + S. platensis 0.3% + S. cerevisiae 0.2%), SEM: standard error of the mean Dietary Supplementation of S. platensis and S. cerevisiae in Laying Hens (H.I. Wahyuni et al.) 53 et al. (2018) found no significant effect of S. platensis on albumin index and yolk index of hens. The higher flavonoid content of S. platensis added to feed can explain the improvement in yolk colour in the SP and SPSC groups (Tufarelli et al., 2021). The latter authors further revealed that higher flavonoid levels in feed corresponded to higher pigment deposition in yolk, which de- termined colour intensity. Moreover, the effect of S. platensis on yolk colour may also be due to the biomass’s β-carotene content due to S. platensis treatment (Khan et al., 2021). It was apparent in this present study that, treatment with S. platensis increased erythrocyte counts in laying hens. Similar to this study, Sarker et al. (2022) reported the increased con- centration of erythrocytes in laying hen blood after supplementation with S. platensis. In this case, erythrocytes improved due to phycocya- nine, a bioactive compound in S. platensis that can stimulate erythropoietin hormone production (Sarker et al., 2022). This hormone stimulates stem cells in the bone marrow to produce more red blood cells. Similar to Sarker et al. (2022), Pankaj and Varma (2013) discovered that oral administration of S. platensis to rats increased red blood cell count. In the current study, feeding S. cerevisiae and combining it with S. platensis reduced the population of LAB and LNE in the caecum di- gesta of laying hens. With regard particularly to S. cerevisiae, such probiotic yeast has reportedly been shown to possess antimicrobial characteris- tics, hence lowering the number of dangerous bacteria, such as LNE, in the intestine of poultry (Sugiharto et al., 2022). Different from Erya et al. (2020) showing no effect of S. cerevisiae on the caecal LAB population, results in this pre- sent study showed reduced caecal LAB counts due to S. cerevisiae treatment. So far, the exact reason for such condition remains unknown. In term of S. platensis, flavonoid compound of S. platensis was actually expected to function as a bacterial clearing agent (Akbarbaglu et al., 2022). In accordance with this, S. platensis ad- ministration resulted in reduced caecal LNE con- tent in this study, although the values did not reach the significant levels. In line with our find- ings, Nuhu (2013) revealed that S. platensis was effective at inhibiting the growth of harmful bac- teria in the intestine as a result of antibacterial activity. The water content of laying hen excreta ranged from 79.9 to 84.7% in this study. The treatment had no discernible effect on the excre- ta water content in any of the treatment groups Table 6. Water Content, Acidity and Temperature of Laying Hens Excreta Items CON SP SC SPSC SEM P value Water content (%) 84.7 79.9 81.7 84.6 0.004 0.274 pH 6.7 b 6.5 b 6.8 a 6.7 b 0.029 <0.001 Temperature (°C) 33.8 34.2 34.5 34.3 0.133 0.271 a,b Means marked with superscript letters in the same row are significantly different (P<0.05) CON (hens fed by basal feed), SP (basal feed + S. platensis 0.3%), SC (basal feed + S. cerevisiae 0.2%), and SPSC (basal feed + S. platensis 0.3% + S. cerevisiae 0.2%), SEM: standard error of the mean Table 7. Protein Digestibility of Laying Hens Items CON SP SC SPSC SEM P value Protein digestibility coefficient (%) 57.76 ab 60.96 ab 48.60 b 70.40 a 2.73 0.031 Nitrogen retention 3.80 bc 4.07 ab 3.01 c 4.90 a 0.21 0.009 Nitrogen excreta 5.29 5.26 5.65 4.59 0.16 0.124 Faecal ammonia 14.2 a 7.0 b 6.8 b 6.7 b 0.818 <0.001 a,b,c Means marked with superscript letters in the same row are significantly different (P<0.05) CON (hens fed by basal feed), SP (basal feed + S. platensis 0.3%), SC (basal feed + S. cerevisiae 0.2%), and SPSC (basal feed + S. platensis 0.3% + S. cerevisiae 0.2%), SEM: standard error of the mean 54 J. Indonesian Trop. Anim. Agric. 48(1):47-57, March 2023 because the basal feed distributed to each group had the same nutritional composition. Indeed, high protein encourages high water content in excreta because of higher glomerulus uric acid concentration, which causes laying hens to drink more water. Due to the little percentage inclusion in this investigation, it seemed as S. platensis inclusion did not significantly increase the crude protein content of the basal diets. The excreta temperatures in the SP, SC, and SPSC groups did not differ significantly from one another. This suggests that the amount of metabolizable energy in each group was equal to the amount required to maintain a normal body temperature, indicat- ing that there were no appreciable differences in homeostasis between the treatment groups. In this study, S. platensis, S. cerevisiae, and both combinations were found to reduce ammo- nia levels in excreta. The dietary supplementa- tion of S. platensis and S. cerevisiae was thought to have resulted in optimal intestinal microbe function. The probiotic microbes could inhibit the activity of the urease enzyme (Rezaee et al., 2019), which can reduce the amount of uric acid in the digestive tract of laying hens. Acid pro- ducing bacteria combined with oligosaccharide- containing ingredients were more effective at reducing ammonia. The level of ammonia in ex- creta is typically determined by the amount of N in the excreta (Jeong and Kim, 2014). However, there was no significant effect of N excreta treat- ment in this study. The higher protein digestibil- ity coefficient in the SPSC group compared to the SC group suggests that both supplements had a synergistic effect. Protein digestibility appears to be related to the balance of essential and non- essential amino acids in S. platensis, which can aid in the digestion of crude protein in poultry (Park et al., 2018; Bleakley and Hayes, 2017; Evans et al., 2015). Maximum protein digestibility can increase N retention, as demonstrated by the findings of this study. Nitrogen retention is the amount of nitrogen that hens absorb and use. The results showed that higher N retention in SPSC groups corresponded to lower excreta ammonia in the SPSC group. This means that N from amino ac- ids was used more for production and maintain- ing health than nitrogen from the consumed ra- tion (Park et al., 2018). Overall, several parameters showed better results with single administration of S. platensis or S. cerevisiae, when compared to the combined administration of S. platensis and S. cerevisiae. This may be because the combination of S. platensis and S. cerevisiae does not always have a synergistic effect on poultry conditions (Sugiharto et al., 2022). The results of this study indicate that single administration of S. platensis or S. cerevisiae will be more efficient in improv- ing egg quality and health of laying hens in the period after peak production. In conclusion, S. platensis improved yolk index and colour, increased erythrocyte counts and played an important role in maintaining the balance of microbes in the digestive tracts result- ing in reduced ammonia excretion. 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