Effect of papaya (Carica papaya) seed as phytogenic feed additives on egg performance, egg quality and blood serum biochemical constituents of layer hens

Abstract Background Supplementing poultry diets with certain natural phytogenic additives has positive impacts on performance parameters like feed intake, egg production and quality of layer hens. Objectives The study aimed to examine how supplementing papaya seed as phytogenic feed additive (PFA) to the diets of layer chickens affected their egg production, quality and blood serum biochemical parameters. Methods One hundred twenty 28‐week‐old Bovans brown laying hens were randomly assigned to one of four treatments with varying levels of papaya seed supplementation at 0 g/kg (T1), 5 g/kg (T2), 10 g/kg (T3) and 15 g/kg (T4) in a complete randomized design. Result Layer chickens supplemented with 0.5% (T2), 1% (T3) and 1.5% (T4) papaya seed as a PFA had significantly (p < 0.05) improved dry matter intake, egg production%, egg mass and feed conversion. On the other hand, papaya seed supplementation decreased (p < 0.05) the bodyweight gain of laying hens. Papaya seed supplementation significantly (p < 0.05) increased egg weight, length, width, yolk height and yolk colour compared to the control group. Serum total protein on T4 and liver enzyme of alkaline phosphatase on T3 were significantly greater (p < 0.05) than in the control group. However, there were no significant differences (p > 0.05) in serum albumin, glucose, total cholesterol, triglyceride and aspartate aminotransferase levels among treatments laying hens. Conclusion Papaya seed can be a potential candidate as a PFA at 5–15 g/kg of the basal diet to improve egg‐laying performance and quality without deteriorating effect.

weight, quality and overall health.For example, researchers found that including fennel seed, black cumin seed and hot red pepper at a ratio of 0.5% of the basal diet significantly increased egg production, egg weight (EW), egg mass and feed conversion ratio (FCR) when compared to a non-supplemented group (Abou-Elkhair et al., 2018).Additionally, Abd El-Hack and Alagawany (2015) found that adding rosemary leaf at a rate of 3 g/kg led to an increase in egg production and egg mass.Moreover, thyme and oregano essential oils have improved feed efficiency, egg mass and egg production at supplementation levels around 1%-2%.Furthermore, garlic powder has been shown to improve EW and shell quality (Chowdhury et al., 2018).Ginger extract supplemented at 200-400 mg/kg resulted in better feed utilization and internal egg quality measures like Haugh units (Hus) and yolk weight in layers (Herkel' et al., 2018).
Papaya seed is one prospective natural supplement for poultry, as papaya is a common tropical fruit cultivated worldwide and the seeds are often discarded as an agricultural by-product.Papaya peels and seeds are generated in large amounts each year, especially in Ethiopia (7772.77tons), and the seeds make up roughly 14.3% of the overall fruit (Li et al., 2015;Saleh et al., 2019).In papaya seed, there are significant amounts of crude protein, essential amino acids, fatty acids, crude fibre and minerals like calcium, phosphorus, magnesium, iron and salt (Beski et al., 2015).In addition, there are vitamins C and E (Muazu & Aliyu-Paiko, 2020).Furthermore, papaya seeds contain beneficial bioactive like carotenoids, phenolic acids, benzyl isothiocyanates, carpaine, caricine, papaya oil and glucotropaeolin (Maisarah et al., 2014;Salla et al., 2016).
However, limited research has explored the direct impacts of supplemented papaya seeds on productivity metrics like egg yield and quality in laying hens.Earlier studies found 0.5%-2% papaya seed powder reduced parasite loads and increased feed intake in pullets but did not assess egg performance (Soedji et al., 2017).Additionally, papaya pomace supplementation did not influence feed intake in layers, though egg cholesterol content decreased (Rahmasari et al., 2022).
Therefore, this study aims to evaluate the effect of papaya seed as feed additives on egg production, quality and blood serum biochemical parameters of laying hens.The results will elucidate the potential for discarded papaya seeds to improve productivity in layers as an inexpensive, natural supplement.

Preparation of papaya seed additive
A ripened papaya (Carica papaya) seed was collected from local juice shops and all unnecessary materials (peels, pulps and dirt) were removed by hand-picking from the seeds.After removing coarse peels and pulps, the seeds were washed with tap water to remove small pulps and slimy substances.The washed seeds were soaked in a 3% hydrogen-peroxide (H 2 O 2 ) solution (1-L tap water with 3 mL of H 2 O 2 ratio) for 30 min to clean the fungus and any contaminants (Sanna et al., 2022).The soaked seeds were filtered after 30 min, air-dried for Dried papaya seeds were coarsely ground using a mortar and pestle and sieved through a 5 mm mesh screen for adding PFAs in the layer rations.

Experimental diets
The conventional mash diet for laying chickens was based on corn and soybean meal.The grounded meals were weighed to formulate a quintal of layers of mash, as indicated in Table 2.Only a basal diet was offered to the control group T1 (without papaya seed PFAs) until the end of the experimental period.The treatment groups were fed the same amount of basal diet with air-dried papaya) seed at 5 g/kg (T2), 10 g/kg (T3) and 15 g/kg (T4).Proximate analysis was carried out to estimate the experimental diets according to the standard procedure of Association of Official Analytical Chemists AOAC (2005).The experimental diets were formulated to meet the nutrient requirements for layer hens according to the recommendations of the commercial layer (Bovans brown lite layers) commercial management guide.
Table 1 presents the proximate chemical composition of the papaya seed.

Experimental design and management of layer chickens
Bovan brown layer chickens at 28-week-old with similar body weight were obtained from a private farm.Individual laying hens were weighed and randomly assigned in a complete randomized design into four dietary treatment groups (T1): basal diet + 0 g/kg papaya seed; (T2): basal diet + 5 g/kg papaya seed; (T3): basal diet + 10 g/kg papaya seed; (T4): basal diet + 15 g/kg papaya seed.Each treatment has four replicates consisting of 10 layers each.The experiment was conducted between 28 and 52 weeks of age (24 weeks experimental period).
The cage was designed at 1.8 × 1 × 0.5 m 3 for each replicate with feed and water trough.Clean water was provided ad libitum during the experimental period.The layers were vaccinated against common poultry diseases (fowl typhoid at 32 weeks of age).The lighting program was 16 h of continuous light per day.The layers were kept in optimal, standard bioclimatic and welfare conditions and managed according to the experimental animal protocol approved by the ethical committee and institutional review board (IRB) (reference number WSU/41/14/1023).E 40 mg, vitamin K3 3 mg, vitamin B1 2 mg, vitamin B2 6 mg, vitamin B6 5 mg, vitamin, B12 0.02 mg, niacin 45 mg, biotin 0.075 mg, folic acid 2 mg, pantothenic acid 12 mg, manganese 100 mg, zinc 600 mg, iron 30 mg, copper 10 mg, iodine 1 mg, selenium 0.2 mg and cobalt 0.1 mg.

DM intake and egg production performance
The dry matter intake (DMI) was determined by the difference between daily feed offered and daily feed refused at the dry matter base.The body weight gain of layers was calculated as the difference between the final and initial body weight to observe the body weight change of layers.The FCR was expressed as grams of feed consumed per grams of egg produced, and layers efficiently convert the feed into an egg (Abou-Elkhair et al., 2018).
The egg production performances were expressed as the percentage of each replicate's hen-day and hen-housed egg production %.The egg mass was computed as described by North (1984) by multiplying the average EW in grams with the hen-day egg production for each pen.

External and internal egg quality
For the measurement of external and internal egg quality, four eggs per replicate were randomly collected at 30, 40 and 50 weeks of age to assess external and internal egg quality traits' eggs were weighed each time using a sensitive balance with a precision of 0.01 g.Digital caliper measured egg length from the tip of the narrow (sharp) end to the broad end.A digital caliper measured the width of each egg at the centre, and the egg shape index was computed by dividing egg width by egg length × 100 (Doyon et al., 1986).The eggshell weight (g) was measured by electronic balance by removing the shell membrane.The shell thickness (mm) was calculated from the narrow (sharp end), equator and broad end (air cell) of the shell by using a digital caliper by removing the shell membrane.The average of three sites was considered eggshell thickness (Hosseinzadeh et al., 2010).
Each egg was carefully opened on a flat glass plate and kept for 3 min for internal egg quality analysis.A gauge micrometre measured individual egg yolk height (calibrated in mm).Yolk diameters were measured using a digital caliper.The colour of the yolk was determined by the Roche colour fan from 1 to 15 colour (Hoffman-La Roche Ltd.).
After separating the yolk from the albumen, the yolk weight was measured using an electronic balance.The yolk index was calculated as yolk height divided by the yolk diameter × 100 (Doyon et al., 1986).Albumen height (AH) was measured by gauge micrometre at two sides, the average was considered AH, and the weight of albumen was measured by sensitive balance.HUs were calculated from the records of AH and EW using the HU formula: HU = 100 × (log AH + 7.57 − 1.7EW 0.37 ) (Haugh, 1937).

Blood serum preparation and biochemical constituents
At the end of the experiment, blood samples were obtained from 24 layers.Two layers per replication were randomly selected, and 3 mL of blood per bird was drawn from the brachial vein using a 5 mL sterile disposable syringe and transferred into a sterilized tube.The blood serum was extracted from the tubes using a centrifuge and kept at −20 • C after being spun for 5 min (Kilinc & Karaoglu, 2020).Serum blood biochemical constituents were determined by the chemistry auto analyser COBAS 6000.Moreover, measurements of the liver's working enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were performed.Commercially available kits (RANDOX) were used to analyse the serum for total protein, albumin, glucose and total cholesterol in the enzymatic endpoint method triglycerides in the GPO-PAP method.

Statistical analysis
The collected data were subjected to a one-way analysis of vari-

Effects of papaya seed feed additive on egg production and FCR
The effect of dietary supplementation or additives of papaya seed on DMI, body weight gain, egg production, egg mass and FCR of the layer is presented in Table 3. Papaya seed additives significantly (p < 0.05) increased daily DMI, egg production, egg mass and FCR of the layer hen.The daily DMI was increased (p < 0.05) as the papaya seed additives increased.Papaya seed as a PFA at 5, 10 and 15 g/kg significantly (p < 0.05) improved egg production % by 3.27, 3.11 and 4.98, respectively.FCR was improved by 1.97%, 2.96% and 3.45% at the levels of the PFA increased from T2-T4 groups, respectively.The layer body weight change was significantly (p < 0.05) different among treatments.
Papaya seed slightly (p < 0.05) decreased the body weight gain of layer chickens compared to the control group.However, there was no difference (p > 0.05) in body weight among papaya seed PFAs groups.

Effects of papaya seed feed additive on egg quality
Table 4 shows the effect of papaya seed as a PFA on the quality of layers' eggs.EW increased by 2.1, 2.9 and 3.4 g in the T2, T3 and T4 groups, respectively (p < 0.05).In addition, the length, width, yolk height and colour of T2, T3 and T4 groups eggs have all been increased (p < 0.05).
The albumen weight was not significantly (p > 0.05) increased in the T2 and T4 groups.However, in treatments T3 and T4 group, the yolk weight, index, as well as albumen weight, were significantly (p < 0.05) increased.However, there is no significant (p > 0.05) difference in yolk index.The addition of papaya seed feed additive, on the other hand, had no effect (p > 0.05) on yolk diameter, HU, AH or shell weight of chicken eggs.

Effect of papaya seed feed additive on serum biochemical constitutes
The effect of papaya seed as a PFA on layers of blood serum biochemical constituents is presented in Table 5.The PFA of papaya seed at 15 g/kg (T4) significantly (p < 0.05) raised blood serum total protein.However, there was no significant (p > 0.05) difference in serum albumin, glucose, total cholesterol, triglyceride and AST liver enzyme compared to the control group.Papaya seed PFA significantly (p < 0.05) decreased ALT in T2, T3 and T4, respectively, compared to the control group.Papaya seed supplementation substantially (p < 0.05) decreased ALP in T2, whereas it increased in T3 group.

DISCUSSION
During the experimental period, supplementation of papaya seeds as feed additive increased DMI linearly.Among the supplemented groups, the highest daily DMI was observed on T4 group (15 g/kg of PFA of papaya seed).The current result agrees with that of Soedji et al. (2017), who supplemented papaya seed to ISA Brown male (layer-type) chicks at 0.5%, 1% and 2% of basal diet and observed an increased feed intake.
This finding disagrees with Tamiru et al. (2021), who reported that 20-32 weeks-age layer chickens fed papaya pomace (seeds, peels and pulps) at 2.5%, 5% and 7.5% inclusion levels showed no effect on feed DM intake.According to Salla et al. (2016), the active ingredients in papaya seeds, such as alkaloids, flavonoids, phenols and amino acids, can stimulate saliva and bile secretions, enhance the activities of digestive enzymes like proteolytic enzyme and increase the palatability of the basal diet.These factors could be possibly attributed to the fact that responsible for improving the DMI of papaya seed PFA groups.
In the current study, the decrease in body weight gain of layer chickens supplemented with papaya seed at 5, 10 and 15 g/kg, maybe layers utilized the feed they consumed to produce more eggs than the  et al. (2018), who reported that supplementation levels of 1%, 3% and 5%, Moringa oleifera seed to layer diet, lower the body weight gain of layer chickens.
The improvement observed in the present study may be an effect of the active compounds of papaya (benzyl isothiocyanate and papain), which had positive effects on the destruction or lowering of parasites, as a result, ample availability of nutrients for absorption and egg production.Additionally, papaya seed has high in nutritional value, like protein, amino acids and fatty acids, which may improve the egg production performance of the chickens.The current result agrees with Kaya et al. (2014), who reported that supplementing grape seed to 44-56-week-aged Lohman layer chickens at 0.5%, 1% and 1.5% increased egg production by 13.88%, 12.84% and 18.03%, respectively.
The current study observed the highest hen day and hen-housed egg production % was obtained from laying hens supplemented with 15 g/kg of PFA of papaya seed.This might be because the increasing level of papaya seed also increased the level of active compounds like the flavonoids in the diet and positively enhanced egg production by facilitating digestion and nutrient absorption.Novitasari et al. (2018) and Leke et al. (2018) revealed that the flavonoid components (myricetin and kaempferol) contained in natural plant feed additives had an estrogenic activity-inducing effect through regulating the hypothalamic gonadal axis of laying chicken.Previous studies employing papaya pomace, flaxseeds and fenugreek seeds for layers have confirmed this hypothesis (Saleh et al., 2019;Tamiru et al., 2021).
The highest egg mass was recorded in the PFA of papaya seed at 15 g/kg group hens compared with 5 and 10 g/kg group hens.The current result agrees with El-Saadany et al. (2022), who revealed that the PFAs of pumpkin seed products in 24-32 weeks age laying hens improved egg mass.Since, both pumpkin and papaya seeds contain high levels of protein, essential amino acids, unsaturated fatty acids and fibre, which can provide added nutritional value to poultry diets (Nakasone & Paull, 1998;Nkukwana et al., 2014).
Similarly observed in the current study may be due to the increased availabil-ity of dietary protein, which was achieved by supplementing papaya seed PFAs at 15 g/kg.However, papaya seed does not contain a hepatoprotein inhibitor (Eid et al., 2021), which can lead to an accumulation of protein in the blood.Therefore, the absence of a hepato-protein inhibitor in papaya seed may have also increased serum total protein levels.The total albumin, glucose, cholesterol and triglycerides were not changed due to the PFAs of papaya seed to layer chicken and the serum total albumin were within the standard reference range 1.17-2.74g/dL (Meluzzi et al., 1992); serum glucose 130-270 mg/dL and total cholesterol: 125-200 mg/dL (Reece et al., 2015).A similar result was reported by Gozde and Mevlut (2020), who noted that laying hens supplemented with 100, 200 and 300 mg/kg of grape seed oil and Hypericum perforatum to layer diet showed no change in total cholesterol and triglyceride.
Supplementation of papaya seed as a PFA decreased the liver enzyme ALT in T2 and T3.This might be due to the presence of antioxidants (Vitamin C, Vitamin E and β-carotene) and phenolic components in papaya seeds that may also sustain layer chicken liver function by lowering inflammatory liver enzymes.The decreases in AST and ALP activities support this interpretation, as their primary function is to channel amino acids into the citric acid cycle (Huang et al., 2006).The current finding disagrees with that of Kaya et al. (2014), who reported that supplementation of grape seed to 44-56-week-aged Lohman layer chickens at 0.5%, 1% and 1.5% did not affect serum ALT levels.According to Bolu et al. (2006), the ALT values become high when toxic substances cause liver damage.There is no particular change in AST concentration level among treatment of layer chickens.This may be because papaya seed supplementation does not negatively affect liver AST enzyme, and the finding at standard reference ranges for chicken is 70-220 U/L (Meluzzi et al., 1992).A similar result was reported by Gozde and Mevlut (2020), who noted that laying hens supplemented with H. perforatum to layer diet did not affect the liver enzyme of AST.

CONCLUSION
The dietary supplementation of papaya seed at 5, 10 and 15 g/kg to layer hens improved DMI, egg production, egg mass and FCR.Papaya seed supplementation positively affects some external and internal egg qualities.It increases serum total protein at a 15 g/kg supplementation level and maintains the liver function of layer chickens by reducing liver inflammation.
Proximate analysis of papaya seed diet.
TA B L E 1

TA B L E 2
Ingredients and nutrient composition of the diet.
Effect of papaya seed feed additive on the performance of layer chickens.Mean values within the same row having different letters (a, b and c) are significantly different at (p < 0.05); T1: basal diet + 0 g/kg papaya seed; T2: basal diet + 5 g/kg papaya seed; T3: basal diet + 10 g/kg papaya seed; T4: basal diet + 15 g/kg papaya seed.Abbreviations: BWC, body weight change; DMI, dry matter intake; FBW, final body weight; FCR, feed conversion ratio; HDEP, hen day egg production; HHEP, hen housed egg production; IBW, initial body weight; p-Value, probability.
ance using SAS statistical package version 9.4.Duncan Multiple Range test evaluated treatment differences between group means at a 5% significance level.TA B L E 3 The effect of papaya seed feed additive on egg quality of layers chickens.Mean values within the same row having different letters (a, b and c) are significantly different at (p < 0.05); T1: basal diet + 0 g/kg papaya seed; T2: basal diet + 5 g/kg papaya seed; T3: basal diet + 10 g/kg papaya seed; T4: basal diet + 15 g/kg papaya seed.Abbreviations: ALP, alkaline phosphatase; ALT, alanine transferase; AST, aspartate aminotransferase; g/dl, gram per decilitre; p-Value, probability; TCH, total cholesterol; TRIG, triglyceride; U/L, unit per litre.control group.This result aligned with previous finding of Mabusela TA B L E 4Note: Mean values within the same row having different letters (a, b and c) are significantly different at (p < 0.05); T1: basal diet + 0 g/kg papaya seed; T2: basal diet + 5 g/kg papaya seed; T3: basal diet + 10 g/kg papaya seed; T4: basal diet + 15 g/kg papaya seed.Abbreviation: p-Value: probability.TA B L E 5Effect of papaya seed feed additives on biochemical blood constitutes of layer chickens.