Introduction
⌅Laying hens can continue to produce eggs for several years; however, their productivity often declines significantly after the first year, with this decline varying widely among breeds (Albatshan et al. 1994Albatshan H.A., Scheideler S.E., Black B.L., Garlich J.D. & Anderson K.E. 1994. Duodenal Calcium uptake, Femur Ash, and Eggshell quality decline with age and increase following Molt. Poultry Science, 73: 1590-1596, ISSN: 1525-3171. https://doi.org/10.3382/ps.0731590.). To enhance the performance of aging hens, it is crucial to delay ovarian aging and manage the depletion of the primordial follicle reserve after the peak egg production period (Barzegar et al. 2020Barzegar, Y.A., Sharifi, D. & Mohammadi, S.A. 2020. Efficacy of dietary supplementation of nanoparticles-chromium, chromium-methionine and zinc-proteinate, on performance of Japanese quail under physiological stress. Italian Journal of Animal Science, 19: 1123-1134, ISSN: 1828-051X. https://doi.org/10.1080/1828051X.2020.1822763.).
Previously, many researchers have reported the antioxidant and anti-inflammatory effects of melatonin (Bantounou et al. 2022Bantounou, M., Josip, P. & Galley, H.F. 2022. Melatonin and related compounds: Antioxidant and anti-inflammatory actions. Antioxidants, 11: 532, ISSN: 2076-3921. https://doi.org/10.3390/antiox11030532. ). Melatonin (MT) has been shown to influence both the quantity and maturity of ovarian follicles, though research on its specific mechanisms in birds remains limited (Hao et al. 2020Hao, E.Y., Chen, H., Wang, D.H., Huang, C.X., Tong, Y.G., Chen, Y.F., Zhou, R.Y. & Huang, R.L. 2020. Melatonin regulates the ovarian function and enhances follicle growth in aging laying hens via activating the mammalian target of the rapamycin pathway. Poultry Science, 99(4): 2185-2195, ISSN: 1525-3171. https://doi.org/10.1016/j.psj.2019.11.040. ). Recent studies have highlighted melatonin's beneficial effects on reproduction in various animals. For instance, melatonin has been found to promote oocyte and early embryo development in mammals, including mice (Ganji et al. 2015Ganji, R., Nabiuni, M. & Faraji, R. 2015. Development of mouse preantral follicle after in vitro culture in a medium containing melatonin. Cell Journal (Yakhteh), 4: 546, ISSN: 2228-5814. https://doi.org/10.22074/cellj.2015.499. ). In laying hens, common issues during the later stages of production include reduced laying performance, decreased follicle counts in the ovaries (Zakaria et al. 1983Zakaria, A.H., Miyaki, T. & Imai, K. 1983. The effect of aging on the ovarian follicular growth in laying hens. Poultry Science, 62: 670-674, ISSN: 1525-3171. https://doi.org/10.3382/ps.0620670. and Ferlazzo et al. 2020Ferlazzo, N., Andolina, G., Cannata, A., Costanzo, M. G., Rizzo, V., Currò, M., Ientile, R. & Caccamo, D. 2020. Is melatonin the cornucopia of the 21st century. Antioxidants, 9: 1-29, ISSN: 2076-3921. https://doi.org/10.3390/antiox9111088. ), and higher egg breakage rates (Albatshan et al. 1994Albatshan H.A., Scheideler S.E., Black B.L., Garlich J.D. & Anderson K.E. 1994. Duodenal Calcium uptake, Femur Ash, and Eggshell quality decline with age and increase following Molt. Poultry Science, 73: 1590-1596, ISSN: 1525-3171. https://doi.org/10.3382/ps.0731590.).
Follicular development, maturation, and atresia play significant roles in various stages of follicular function, providing insight into the molecular mechanisms underlying egg production. FSH and LH work in tandem to ensure normal follicular growth (Raju et al. 2013Raju, G.A., Chavan, R., Deenadayal, M., Gunasheela, D., Gutgutia, R., Haripriya, G., Govindarajan, M., Patel, N.H. & Paktil, A.S. 2013. Luteinizing hormone and follicle-stimulating hormone synergy: A review of role in controlled ovarian hyper-stimulation. Journal of Human Reproductive Sciences, 6(4): 227-234, ISSN: 1998-4766. https://doi.org/10.4103/0974-1208.126285. ). FSH, produced by the anterior pituitary in response to gonadotropin-releasing hormone (GnRH) from the hypothalamus, plays a crucial role in reproductive physiology and fertility in both male and female animals (Ferlazzo et al. 2020Ferlazzo, N., Andolina, G., Cannata, A., Costanzo, M. G., Rizzo, V., Currò, M., Ientile, R. & Caccamo, D. 2020. Is melatonin the cornucopia of the 21st century. Antioxidants, 9: 1-29, ISSN: 2076-3921. https://doi.org/10.3390/antiox9111088. ). FSH enhances follicular development by promoting angiogenesis in the theca externa follicles, especially in hens with low egg production (Bi et al. 2021Bi, Y.L., Yang, S.Y., Wang, H.Y., Chang, G.B. & Chen, G.H. 2021. Follicle-stimulating hormone is expressed in ovarian follicles of chickens and promotes ovarian granulosa cell proliferation. Journal Integrated. Agriculture, 20(10): 2749-2757, ISSN: 1338-4376. https://doi.org/:10.1016/S2095-3119(21)63606-.). While FSH stimulates ovum development and maturation, LH is responsible for ovulation. Optimal levels of both hormones accelerate follicle growth, leading to increased egg production through the development and ovulation of more follicles (Prastiya et al. 2022Prastiya, R.A., Madyawati, S.P., Sari S.Y. & Nugroho, A.P. 2022. Effect of follicle-stimulating hormone and luteinizing hormone levels on egg-laying frequency in hens. Veterinary World, 15(12): 2890-2895, ISSN: 2231-0916. https://doi.org/10.14202/vetworld.2022.2890-2895. ). It also plays a role in regulating reproductive hormones by influencing FSH and LH synthesis and secretion, affecting the circadian rhythm, embryonic growth, and ovary development (Duo et al. 2014). Melatonin has been linked to improved immune responses and reduced oxidative stress in poultry. For example, a study by Liu et al. (2022)Liu, G., Li, J., Yang, L. & Zhang, X. 2022. Melatonin supplementation improves immune response and reduces oxidative stress in broilers. Journal of Animal Science, 100(3): 1124-1132, ISSN: 1525-3163. https://doi.org/10.1080/09064702.2023.2222733. found that melatonin supplementation enhanced the immune system and reduced markers of oxidative stress in broilers, contributing to better overall health and productivity. Despite these findings, there are limited research on the effects of melatonin on laying hens beyond their egg-laying peak, particularly in Nigeria.
Therefore, this experiment was designed to evaluate the effect of melatonin on hormonal profile, follicular development and production performance of spent layers of two prominent breeds of laying hen in Nigeria.
Materials and Methods
⌅Experimental site
⌅This experiment was approved by the University Ethical and Research Community with reference number EBSU/2022/2087. After the approval, it was then carried out at the Poultry Unit of the Teaching and Research Farm of Ebonyi State University, Abakaliki, Nigeria. The experiment was carried out during the dry season between October 2022 and March 2023.The mean temperature was 37.8 oC - 39 oC during the hot dry season.
Experimental animals and management
⌅The study involved Isa Brown and Nera Black hens, both 54 weeks old at the start of the 20-week experiment. The experiment was conducted in two phases, referred to as Experiment I and Experiment II, each focusing on a different breed of laying hens. A total of 117 laying hens from each breed were used, with the hens divided into three treatment groups for each experiment. Each treatment group consisted of 39 birds, which were further subdivided into three replicates of 13 hens each, housed on rice husk bedding.
The average live weight for Isa Brown birds was 1.65 kg, while Nera Black birds averaged 1.96 kg. These layers were sourced from Agrited, a breeding company based in Ibadan, Nigeria. Throughout the experiment, the birds were fed a layer’s mash containing 16 % crude protein, 3.5 % ether extract, 4.7 % crude fiber, and 11.2968 MJ/kg metabolizable energy. Below is the diet composition (table 1)
| Ingredients | % composition |
|---|---|
| Maize | 48.50 |
| Wheat offal | 10.30 |
| Palm kernel meal | 12.70 |
| Groundnut cake | 10.70 |
| Fish meal | 6.30 |
| Oyster shell | 6.0 |
| Bone meal | 4.0 |
| Salt | 0.5 |
| Premix | 0.5 |
| Lysine | 0.25 |
| Methionine | 0.25 |
| Total | 100 |
| Calculated | Values |
| Crude protein % | 16 |
| Crude fiber % | 4.70 |
| Ether extract % | 3.20 |
| Metabolizable (Mj/kg) | 11.30 |
*Premix to provide the following per kg of feed; Vit A-500 iu, Vit D3- 1200 mg, Vit.E-11 mg, Vit.K-2 mg, Riboflavin- 20 mg, Nicotinic acid- 10 mg, Pantothenic acid- 7 mg, Cobalamin- 0.08 mg, Choline chloride- 900 mg, Folic acid- 1.5 mg, Biotin-1.5 mg, Iron- 25 mg, Manganese-80 mg, Copper-2 mg, Zinc-50 mg, Cobalt-1.25 mg and Selenium-0.1 mg
Experimental materials
⌅The experimental material was melatonin supplement. The melatonin was dissolved in 10 % ethanol at the rate of 5 mg/1 mL or 10 mg/1 mL and administered orally through drinking at the rate of 2 mL per liter of water daily to the layers.
Hormonal analysis
⌅At the end of the experiment (72 weeks of age), blood samples (5 mL) (two samples in each replicate) were obtained from the brachial vein of each hen’s wing and stored in a vacutainer plain tube in a cool box (4 °C) filled with ice gel and ice cubes before moving them to the laboratory. An enzyme-linked immunosorbent assay (ELISA) was used to determine the Progesterone, FSH, and LH levels in the blood samples. In the ELISA, blood samples were centrifuged. The plasma luteinizing hormone (L.H.), follicle-stimulating hormone (FSH), and progesterone were quantified utilizing a homologous RIA (Krishnan et al. 1993) or determined by carrying out a quantitative test based on a solid phase enzyme-linked immuno-absorbent assay (ELISA) kit.
Reproductive morphological evaluations
⌅At the end of the experiment (72 weeks of age), 6 birds were randomly selected from each group (2 birds per replicate making it 18) and weighed and decapitated. The follicles were collected and promptly counted under the microscope for small white follicles, small yellow follicles and slightly larger follicles within 5mm to 2mm (SWF, SYF, F5, and F2).
Statistical analysis
⌅A 2×3 factorial arrangement in a completely randomized block design was used for the study. The differences between the treatment groups and the control group were analyzed with a Mini-Tab Version 12. Duncan’s new multiple-range tests after fisher test were used to identify which treatment conditions were significantly different from each other at a significance level of p<0.05 (Duncan 1955Bi, Y.L., Yang, S.Y., Wang, H.Y., Chang, G.B. & Chen, G.H. 2021. Follicle-stimulating hormone is expressed in ovarian follicles of chickens and promotes ovarian granulosa cell proliferation. Journal Integrated. Agriculture, 20(10): 2749-2757, ISSN: 1338-4376. https://doi.org/:10.1016/S2095-3119(21)63606-.)
Results
⌅Effect of Melatonin on the Hormonal Profile of Isa Brown
⌅Results from the table 2 showed that melatonin significantly (p<0.05) influenced the hormonal profile of the Isa Brown breed of laying birds. The control group T1 (2.96 pg/mL) had the lowest level followed by T2 (6.15 pg/mL) of progesterone while T3 (8.24 pg/mL) had the highest level. Follicle-stimulating hormone and Luteinizing hormone levels were equally significantly (p<0.05) affected by melatonin treatment. The highest average FSH levels were found in the hens under 5 mg of melatonin. Higher levels of FSH were seen in T2 which had 629.140 pg/mL followed by T3 (279.45 pg/mL) while control group T1 (198.78 pg/mL) had the least level. A similar trend was followed in the LH level where T2 (55.48 mIU/mL) had higher levels followed by T3 (39.64 mIU/mL) and T1 (30.68 mIU/mL), respectively
| Parameter | T1 (0 mg) | T2 (5 mg) | T3 (10 mg) | SEM | P - value |
|---|---|---|---|---|---|
| Progesterone, ng/mL | 2.96b | 6.15a | 8.24a | 0.41± 0.01 | 0.000** |
| FSH, pg/mL | 198.78b | 629.140a | 279.45b | 6.41± 0.07 | 0.053** |
| LH, mIU/mL | 30.68b | 55.48a | 39.64b | 0.32 ± 0.0 | 0.000** |
a-b Means in the same row with different superscripts differ significantly (p<0.05) according to Duncan's Multiple Range Test. FSH: Follicle Stimulating Hormone, LSH: Luteinizing Hormone
Results from table 3 indicate that melatonin significantly (p<0.05) improved the hen day egg production of layers after 50 weeks of age. Groups on 5 mg had 86.33 % followed by those on 10mg which had 61.66 % while the control group (0 mg) had the lowest HDEP. A similar trend was equally observed on the oviduct weight. The size of the follicles was equally influenced by melatonin administration. The LYF, SYF and SWF were all increased by melatonin administration. From F5-F1 there were numerical increases with increasing levels of melatonin, but it was not statistically significant.
| Parameter | T1 (0 mg) | T2 (5 mg) | T3 (10 mg) | SEM | p-value |
|---|---|---|---|---|---|
| HDEP (%) | 39.11b | 86.33a | 61.66a | 3.63 ± 0.07 | 0.005** |
| Oviwt (g) | 34.40b | 64.59a | 68.18a | 2.92 ± 0.02 | 0.000** |
| F5 (mm) | 3.00b | 6.00a | 5.00a | 0.86 ± 0.00 | 0.001** |
| F4 (mm) | 4.00 | 6.00 | 4.00 | 0.61 ± 0.00 | 0.012 |
| F3 (mm) | 4.00 | 5.00 | 5.00 | 0.50 ± 0.00 | 0.057 |
| F2 (mm) | 4.00 | 4.00 | 5.00 | 0.99 ± 0.10 | 0.001 |
| F1 (mm) | 4.00 | 4.00 | 5.00 | 0.48 ± 0.00 | 0.058 |
| LYF (mm) | 30.55b | 46.33a | 38.11a | 4.60 ± 0.10 | 0.000** |
| SYF (mm) | 18.55c | 40.66a | 30.12a | 0.59 ± 0.0 | 0.007** |
| SWF (mm) | 20.35a | 44.44a | 35.33b | 2.03 ± 0.03 | 0.000** |
a-b Means in the same row with different superscripts differ significantly (p<0.05) according to Duncan's Multiple Range Test. HDEP: Hen-Day-Egg Production, Oviwt: Oviduct weight.
F5: Fifth yellow follicle, F4: Fourth Yellow Follicle, F3: Third Yellow Follicle, F2: Second Yellow Follicle and F1: First Yellow Follicle, LYF: Large Yellow Follicle, SYF: Small Yellow Follicle, SWF: Small White Follicle
Effect of Melatonin on the Hormonal Profile of Nera Black
⌅Progesterone, Follicle-stimulating hormone, and Luteinizing hormone levels were significantly (p<0.05) affected by melatonin treatment (table 4). The highest average FSH levels were found in the hens under 5 mg of melatonin which was (T2429.68 pg/mL) followed by groups on 10 mg (T3198.46 pg/mL) and 0 mg (T152.74 pg/mL), respectively. A similar trend was equally recorded in LH where higher levels were recorded in T2 followed by T3 and T1, respectively. Progesterone levels were equally increased with increased levels of melatonin across the groups.
| Parameter | T1 (0 mg) | T2 (5 mg) | T3 (10 mg) | SEM | p- value |
|---|---|---|---|---|---|
| Progesterone ng/mL | 3.16c | 8.15b | 9.94a | 0.41± 0.01 | 0.012** |
| FSH pg/mL | 52.748c | 429.68a | 198.45b | 6.41 ± 0.05 | 0.009** |
| LH mIU/mL | 23.55b | 51.78a | 31.14b | 0.32 ± 0.00 | 0.000** |
a-b Means in the same row with different superscripts differ significantly (p<0.05) according to Duncan's Multiple Range Test. FSH: Follicle Stimulating Hormone, LSH: Luteinizing Hormone
Results from table 5 show that melatonin significantly (p<0.05) influenced some of the parameters. The HDEP was improved with melatonin administration. Treatment T2 (70.86 %) on 5 mg of melatonin had higher HDEP followed by T3 (58.60 %) while the control group had the least (31.10 %). The oviduct weight increased with increased level of melatonin where T3 (84.18 g) was followed by T2 (78.59 g) while T1 (54.40 g) recorded the lowest weight. Some of the follicles were equally influenced by the treatment while some had a numerical increase but were not statistically (p>0.05) significant.
| Parameters | 0 mg | 5 mg | 10 mg | SEM | p-value |
|---|---|---|---|---|---|
| HDEP (%) | 31.10c | 70.86a | 58.60b | 1.13 ± 0.00 | 0.007** |
| Oviwt (g) | 54.40b | 78.59a | 84.18a | 2.12 ± 0.02 | 0.000** |
| F5 (mm) | 2.00b | 4.00a | 3.00a | 0.61 ± 0.00 | 0.000** |
| F4 (mm) | 2.00 | 4.00 | 4.00 | 0.31 ± 0. 00 | 0.031 |
| F3 (mm) | 2.00 | 3.00 | 2.00 | 0.30 ± 0.00 | 0.001 |
| F2 (mm) | 3.00 | 4.00 | 4.00 | 0.81 ± 0.01 | 0.000 |
| F1 (mm) | 2.00 | 4.00 | 4.00 | 0.28 ± 0.1 | 0.120 |
| LYF (mm) | 18.55b | 28.30a | 27.11a | 4.60 ± 0.04 | 0.000** |
| SYF (mm) | 10.00 | 15.60 | 15.10 | 0.20 ± 0.00 | 0.201 |
| SWF (mm) | 10.35 | 14.44 | 13.33 | 1.43 ± 0.02 | 0.057 |
a-b Means in the same row with different superscripts differ significantly (p<0.05) according to Duncan's Multiple Range Test. HDEP = Hen-Day-Egg Production, Oviwt = Oviduct weight. F5: Fifth yellow follicle F4: Fourth Yellow Follicle. F3: Third Yellow Follicle. F2: Second Yellow Follicle and F1: First Yellow Follicle, LYF Large Yellow Follicle, SYF = Small Yellow Follicle, SWF Small White Follicle
Discussions
⌅Aging and environmental stress significantly impact an animal’s reproductive potential, suppressing its ability to reproduce effectively (Pandi-Perumal 2013Pandi-Perumal, S.R., BaHammam, A.S., Brown, G.M., Spence, D.W., Bharti, V.K., Kaur, C., Hardeland, R. & Cardinali, D.P. 2013. Melatonin antioxidative defense: Therapeutical implications for aging and neurodegenerative processes. Neurotoxicity Research, 23(3): 267-300, ISSN: 1476-3524. https://doi.org/10.1007/s12640-012-9337-. and Barzegar et al. 2020Barzegar, Y.A., Sharifi, D. & Mohammadi, S.A. 2020. Efficacy of dietary supplementation of nanoparticles-chromium, chromium-methionine and zinc-proteinate, on performance of Japanese quail under physiological stress. Italian Journal of Animal Science, 19: 1123-1134, ISSN: 1828-051X. https://doi.org/10.1080/1828051X.2020.1822763.). The increased FSH and LH levels in both breeds, could be attributed to melatonin antioxidative and nutraceutical properties in disease management (Kamfar et al. 2024Kamfar, W.W., Khraiwesh, H.M., Ibrahim, M.O., Qadhi, A.H., Azhar, W.F., Ghafouri, K.J., Alhussain, M.H., Aldairi, A.F., AlShahrani, A.M., Alghannam, A.F., Abdulal, R.H., Al-Slaihat, A.H., Qutob, M.S., Elrggal, M.E., Ghaith, M.M. & Azzeh, F.S. 2024. Comprehensive review of melatonin as a promsing nutritional and nutraceutical supplement. Heliyon, 10(4): e24266, ISSN: 2405-8440. https://doi.org/10.1016/j.heliyon.2024.e24266. ) The lowest FSH levels were observed in the 10 mg melatonin and control groups. Statistical analysis of the results confirmed that the 5 mg melatonin group exhibited significantly (p<0.05) higher FSH levels. This finding aligns with Ragil et al. (2022)Ragil, A.P., Sri, P.M., Sera, Y.S. & Aras, P.N. 2022. Effect of follicle-stimulating hormone and luteinizing hormone levels on egg-laying frequency in hens. Veterinary World, 15(12): 2890-2895, ISSN: 2231-0916. https://doi.org/10.14202/vetworld.2022.2890-2895., who reported that elevated FSH and LH levels in laying hens promote follicle development and ovulation.
The increased follicle development, maturation, and growth in both Isa Brown and Nera Black hens in this study led to a notable increase in hen-day egg production. This outcome is consistent with Jonak et al. (2017)Jonak, C.R., Lainez, N.M., Roybal, L.L., Williamson, A.D. & Coss, D. 2017. c-JUN dimerization protein 2 (JDP2) is a transcriptional repressor of follicle-stimulating hormone β (FSHβ) and is required for preventing premature reproductive senescence in female mice. Journal of Biological Chemistry, 292(7): 2646-2659, ISSN: 1083-351X. https://doi.org/10.1016/0300-9629(93)90275-9. , who observed that FSH enhances granulosa cell differentiation in pre-hierarchical follicles and supports steroid hormone synthesis in granular cells. Similarly, Ragil et al. (2022)Ragil, A.P., Sri, P.M., Sera, Y.S. & Aras, P.N. 2022. Effect of follicle-stimulating hormone and luteinizing hormone levels on egg-laying frequency in hens. Veterinary World, 15(12): 2890-2895, ISSN: 2231-0916. https://doi.org/10.14202/vetworld.2022.2890-2895. noted that higher FSH levels were associated with increased egg-laying frequency in Isa Brown hens. FSH plays a key role in follicular development by promoting angiogenesis in the theca externa follicles of hens with low egg production rates and aged hens (Tamura et al. 2017Tamura, H., Kawamoto, M., Sato, S., Tamura, I., Maekawa R., Taketani T., Aasada H., Takaki E., Nakai A. & Reiter, R.J. 2017. Long-term melatonin treatment delays ovarian aging. Journal of Pineal Research, 62: 12381, ISSN: 1600-079X. https://doi.org/10.1111/jpi.12381. and Bi et al. 2022).
The hormonal status of a hen significantly influences egg production and quality (Prastiya et al. 2022Prastiya, R.A., Madyawati, S.P., Sari S.Y. & Nugroho, A.P. 2022. Effect of follicle-stimulating hormone and luteinizing hormone levels on egg-laying frequency in hens. Veterinary World, 15(12): 2890-2895, ISSN: 2231-0916. https://doi.org/10.14202/vetworld.2022.2890-2895. ). The high FSH levels in Isa Brown hens likely contributed to their higher follicle counts compared to Nera Black hens, resulting in greater hen-day egg production in the Isa Brown group. Additionally, progesterone levels were elevated in the melatonin treatment groups compared to the control, indicating that higher melatonin doses increased progesterone levels, unlike FSH and LH. However, high doses of melatonin (e.g., 10 mg) significantly reduced FSH and LH levels in both breeds, as observed in treatment 3. This result supports Kang et al. (2023)Kang, B., Erying, H., Chen-xuan, H., Qiao-xian, Y., De-He, W., Lei S., Yi-fan, C., Hui, C. & Ren-Lu, H. 2023. Melatonin alleviates ovarian function damage and oxidative stress induced by dexamethasone in the laying hens through FOXO1 signaling pathway. Poultry Science, 102(8): 102745, ISSN: 1525-3171. https://doi.org/10.1016/j.psj.2023.102745. , who reported that higher doses of melatonin (e.g., 10 mg/kg) reduced FSH and LH levels, potentially due to feedback mechanisms or altered endocrine responses.
Luteinizing hormone (LH) levels fluctuated across treatment groups, with the highest concentrations observed in those administered 5 mg of melatonin. This suggests that melatonin enhances follicle development, particularly of the largest follicles (F1). This finding is consistent with the study by Marques et al. (2022)Marques, P., Skorupskaite, K. & George, J.T. 2022. Physiology of GNRH and gonadotropin secretion. In: Feingold, K.R., Anawalt, B., Boyce, A. editors. Endotext. MDText.com, Inc., South Dartmouth (MA) WWW.ENDOTEXT.ORG., which reported increased LH levels leading to a higher number of mature follicles in laying birds. The increase in progesterone synthesis observed in this study was linked to a reduction in estrogen levels, which occurs as larger follicles in the follicular hierarchy, especially F1, grow. This growth contributed to improved laying performance in the birds, despite the natural decline in production due to aging.
Hen-day egg production (HDEP) was significantly enhanced by melatonin administration. At 50 weeks (350 days), birds typically pass their egg production peak, and a decline in egg production often leads farmers to sell the layers as spent layers before recovering their production costs (Tamura et al. 2017Tamura, H., Kawamoto, M., Sato, S., Tamura, I., Maekawa R., Taketani T., Aasada H., Takaki E., Nakai A. & Reiter, R.J. 2017. Long-term melatonin treatment delays ovarian aging. Journal of Pineal Research, 62: 12381, ISSN: 1600-079X. https://doi.org/10.1111/jpi.12381. ). However, melatonin administration improved HDEP in both Isa Brown and Nera Black hens. Isa Brown achieved the highest HDEP at 86.33 %, followed by Nera Black at 70.86 %. These results align with Yaxiong et al. (2016)Yaxiong, Jia MinghuiYang, KuanfengZhu, LiangWang, Yukun Song, JingWang, WenxiangQin, ZhiyuanXu, YuChen & Guoshi Liu. 2016. Melatonin implantation improved the egg-laying rate and quality in hens past their peak egg-laying age. Scientific Reports, 6: 39799, ISSN: 2045- 2322. https://doi.org/10.1038/srep39799. , who found that melatonin improved egg production in layers after 400 days. The results of this experiment equally corroborate the work of Bocheva et al. (2024)Bocheva, G., Bakalov, D., Iliev, P., Tafradjiiska-Hadjiolova. 2024. The Vital Role of Melatonin and its Metabolites in the Neuroprotection and Retardation of Brain Aging. International Journal of Molecular Science, 25(10): 5122, ISSN: 1422-0067. https://doi.org/10.3390/ijms25105122. who noted that melatonin improves egg production in laying birds. The improved production could be attributed to protective effect of melatonin against stress through its direct free radical scavenging activity and indirect antioxidant activity via production of antioxidant enzymes (Yuanyuan et al. 2021Yuanyuan, T., Song, E., Zhenzhen, W., Na, J., Linling, Z., Wang, K., Sun, H., Yuting, Z., Qiujian, Z., Xiaojuan, L. & Zhu, M. 2021. Melatonin attenuates oxidative stress and inflammation of Müller cells in diabetic retinopathy via activating the Sirt1 pathway. Biomedicine & Pharmacotherapy, 137: 111274, ISSN: 1950-6007. https://doi:10.1016/j.biopha.2021.111274.)
In this experiment, the highest HDEP levels were recorded in treatment 2 (5 mg melatonin), followed by treatment 3, with treatment 3 showing the lowest performance. This led to the decreased expression of inflammatory mediators, such as cytokines, adhesion molecules and enzymes (Ferlazzo et al. 2020Ferlazzo, N., Andolina, G., Cannata, A., Costanzo, M. G., Rizzo, V., Currò, M., Ientile, R. & Caccamo, D. 2020. Is melatonin the cornucopia of the 21st century. Antioxidants, 9: 1-29, ISSN: 2076-3921. https://doi.org/10.3390/antiox9111088. ) leading to improved follicular growth. This indicates that a lower melatonin dosage of 5 mg was more effective in both breeds compared to a higher dosage.
The improved performance could be attributed to the increase in follicle development, from F5 to F1, as well as the small white follicles (SWF) and small yellow follicles (SYF) in both breeds. This is in line with Yaxiong et al. (2016)Yaxiong, Jia MinghuiYang, KuanfengZhu, LiangWang, Yukun Song, JingWang, WenxiangQin, ZhiyuanXu, YuChen & Guoshi Liu. 2016. Melatonin implantation improved the egg-laying rate and quality in hens past their peak egg-laying age. Scientific Reports, 6: 39799, ISSN: 2045- 2322. https://doi.org/10.1038/srep39799. , who reported that a higher melatonin dosage (20 mg implant) had a slightly inhibitory effect on the egg-laying rate. Additionally, increased melatonin administration led to an increase in oviduct weight, which was also reflected in higher egg weights and average body weight in the birds.
Before the experiment, egg production occurred once every three to four days, but within five days of melatonin administration, production increased to once every two days, and eventually to daily laying, especially in Isa Brown hens. This suggests that melatonin may serve as a growth promoter in animals.
Conclusions
⌅Enhancing hen-day egg production in aged birds beyond 364 days is a crucial goal that can significantly increase farmers' income in the poultry industry. In this experiment, we observed that melatonin positively influenced the hormonal profile of the birds, leading to a resurgence in their peak egg production due to the further maturation of ovarian follicles. Isa Brown birds exhibited superior performance with a higher number of follicles, resulting in greater hen-day egg production. However, Nera Black birds had a higher oviduct weight compared to Isa Brown, which contributed to increased egg weight and overall body weight.