Ovarian granulosa cells (GCs) are a critical approach to investigate the mechanism of gene regulation during folliculogenesis. The objective of this study was to investigate the role of MT2 in bovine GCs, and assess whether MT2 silencing affected GCs response to melatonin. We found that MT2 silencing significantly decreased the secretion of progesterone and estradiol, and increased the concentration of inhibin B and activin B. To further reveal the regulatory mechanism of MT2 silencing on steroids synthesis, it was found that the expression of CYP19A1 and CYP11A1 enzymes (steroid hormone synthesis) were down-regulated, while genes related to hormonal synthesis (StAR, RUNX2, INHA and INHBB) were up-regulated without affecting the expression of INHBA, suggesting that MT2 silencing may regulate hormone abundance. Furthermore, MT2 silencing significantly increased the expression of TGFBR3 and BMP6, and decreased the expression of LHR and DNMT1A without significant difference in the expression of FSHR and EGFR. In addition, MT2 silencing didn’t affect the effect of melatonin on increasing the expression of DNMT1A, EGFR, INHBA and LHR, and progesterone level, or decreasing INHA, TGFBR3 and StAR expression, and production of inhibin B. Moreover, MT2 silencing could disrupt the role of melatonin in decreasing the FSHR, INHBB and BMP6 expression, and activin B secretion. In conclusion, these results reveal that melatonin and MT2 are essential regulator of bovine GCs function by modulating reproduction-related genes expression, hormones secretion and other regulators of folliculogenesis.

Granulosa cells play an essential physiological role in mediating the follicle development and survival or apoptosis of granulosa cells dictate the follicle development or atresia. The aim of this study was to investigate the role of high dose (10⁻⁵ M) and low dose (10⁻⁹ M) melatonin in bovine granulosa cells, and assess whether MT1 and MT2 inhibiter affect granulosa cells response to melatonin. We found that the high dose (10⁻⁵ M) and low dose (10⁻⁹ M) both could act as an essential role in modulating granulosa cells apoptosis, cell cycle and antioxidant. The beneficial effect could be related to that melatonin promoted the expression of Bcl2, Bcl-xl, SOD1 and GPX4, and inhibited Bax, caspase-3 and p53 expression. Moreover P21 expression was decreased in granulosa cells treated with the high dose (10⁻⁵ M) melatonin and increased in that treated with the low dose (10⁻⁹ M) melatonin. To further reveal the role of MT1 and MT2 in mediating the effect of melatonin on granulosa cells apoptosis, cell cycle and antioxidant, we found that the luzindole and 4P-PDOT did not affect the effect of high dose (10⁻⁵ M) melatonin on regulating Bcl2, Bax, caspase-3, SOD1, GPX4 and p53 expression, while blocked its effect on modulating Bcl-xl and P21expression. However, luzindole and 4P-PDOT disturbed the effect of low dose (10⁻⁹ M) melatonin on regulating Bcl2, Bax, caspase-3, Bcl-xl, SOD1, GPX4, and p53 expression. In conclusion, these results reveal that the effect of low dose (10⁻⁹ M) melatonin on granulosa cells apoptosis are mediated by MT1 and MT2, and the high dose (10⁻⁵ M) melatonin affect the granulosa cells apoptosis by other pathway, besides MT1 and MT2. Moreover MT1 and MT2 may work in concert to modulate bovine granulosa cells function by regulating cellular progression and apoptosis.

Introduction: Melatonin is a neurohormone involving various biological processes, including restoration of cyclicity in animals with seasonal breeding patterns. The use of melatonin in different forms has gained broader acceptance in different species, particularly in summer anestrous buffaloes.Objectives: The objective of the current study was to evaluate the melatonin effect on the reproductive and productive performance of crossbred buffaloes during the low breeding season.Methods: Sixty-five cyclic and reproductively sound crossbred buffaloes were randomly allocated to three groups: the G1 (n = 20) served as the control group and received no single melatonin, G2 received melatonin (n = 22; 18 mg/50 kg, body weight) once prior to synchronization and G3 group was administered multiple melatonin injections (n = 23; 6 mg/50 kg body weight) for three consecutive days before the start of the synchronization protocol. The reproductive performance, milk yield traits, and serum immunoglobulin M (IgM) and melatonin levels were evaluated in treated and untreated crossbred buffaloes.Results: The results revealed that a single dose of melatonin administration has (p < 0.05) improved estrus response, ovulation occurrence and follicular growth in crossbred buffaloes compared to control groups. Higher pregnancy rates were observed in both melatonin-treated buffalo groups compared to the control. Following the administration of melatonin, serum IgM level increased in G2 and G3; however, an increment in melatonin level (p < 0.05) was detected in the G2 group only as compared to the control group subsequent day of melatonin administration. The milk compositions were not affected by melatonin administration except for milk urea nitrogen and somatic cell count (SCC). The melatonin administration (p < 0.05) decreased the somatic cell count in buffalo milk compared to untreated.Conclusion: In conclusion, single or multiple doses of melatonin before initiating the synchronization protocol improved the ovulation, ovulatory follicle diameter and pregnancy rates in crossbred buffaloes during the low breeding season. Moreover, the administration of melatonin enhanced the IgM values along milk traits in terms of milk protein, MUN and somatic cell count in treated buffaloes.

This study assessed the effects of dietary honeycomb supplementation on laying performance, intestinal inflammation, ovarian function, and yolk precursor synthesis in laying hens. A total of 320 Dawu Golden Phoenix laying hens (288-d-old) were randomly assigned into four treatment groups with eight replicates of 10 hens each. Hens were provided diets containing 0, 0.5, 1.0, or 2.0 g/kg honeycomb for 30 d. Dietary honeycomb significantly increased average egg weight, average daily feed intake, and laying rate, while decreasing the feed conversion ratio (p < 0.05). It enhanced serum antioxidant capacity, as reflected by higher GSH, SOD, and CAT activity (p < 0.05). Meanwhile, honeycomb modulated the expression of Nrf2/Keap1 pathway-related genes, accompanied by increased expression of downstream antioxidant-related genes. Additionally, honeycomb downregulated intestinal inflammatory- and apoptosis-related factors (p < 0.05), which effectively alleviated intestinal inflammation responses. In the liver, honeycomb showed favorable histological changes, including fewer lipid droplets and less vacuolar degeneration. Furthermore, it decreased serum ALT, AST, and TG levels and promoted yolk precursor synthesis by upregulating genes associated with lipid transport and vitellogenesis (p < 0.05). In the ovary, honeycomb optimized ovarian morphology and follicle development; elevated serum FSH, E2, and MLT levels; and enhanced the expression of steroidogenesis-related genes (p < 0.05), thereby promoting steroid hormone synthesis and reducing follicular atresia. In conclusion, dietary honeycomb supplementation could maintain intestinal health, modulate hepatic and ovarian metabolism functions, promote yolk precursor synthesis, improve follicle development, and consequently, enhance the laying performance of hens.

The present study investigated the role of dietary capsaicin (CPS) supplementation on milk yield (liters/head) and milk composition (total solids, lactose, albumin, protein, fat, milk urea nitrogen (MUN), somatic cell count (SCC) and somatic cell score (SCS), serum metabolites (lipoprotein esterase (LPL) and aspartate aminotransferase (AST)), and reproductive physiology (follicular development, estrus response, ovulation and pregnancy) following synchronization during the low breeding season. One hundred (n = 100) crossbred buffaloes were randomly assigned to four dietary groups consisting of CPS supplementation dosages (0, 2, 4 or 6 mg/kg of total mixed ration; TMR) as CPS-0 (n = 26), CPS-2 (n = 22), CPS-4 (n = 25) and CPS-6 (n = 27), respectively, in a 30-day feed trial. The results revealed that the CPS-4 group of buffaloes had a better estrus rate (72%) along with improved (p < 0.05) ovulatory follicle diameter (13.8 mm), ovulation rate (68%) and pregnancy rate (48%) compared to other treatment groups. Milk yield improved (p < 0.05) in CPS-4 supplemented buffaloes after day 20 of the trial, comparatively. There was a significant effect (p < 0.05) of milk sampling day (day 30) on total milk solids, lactose, milk protein and MUN levels, whereas lactose, MUN, SCC and SCS were influenced by supplementation dosage (CPS-4). Glucose levels were affected in buffaloes by sampling time (artificial insemination (AI) and 50-day post-AI) and CPS-dose (CPS-4 and CPS-6), respectively. LPL level changed in CPS-2 and CPS-4 groups at AI time and 50 days after AI. In addition, the AST level was different in CPS-4 at AI time and 50 days after AI. Therefore, our data suggest that a medium dose (~4 mg/kg of TMR) of CPS provided a better response in the form of milk yield, milk composition, serum metabolites and reproductive performance in crossbred buffaloes during the low breeding season.

Introduction: Following the application of different artificial insemination and synchronization protocols, the synchronized buffaloes had a higher incidence of early embryonic or fetal death, thus impairing reproductive performance. Therefore, there is a need to devise a synchronizing program that can improve conception, allow early pregnancy diagnosis, reduce early embryonic losses, and provide an early clue for pregnancy diagnosis and establishment. The present study aimed (1) to determine the effect of administration of different GnRH doses at day 20 of artificial insemination (AI) on the reproductive performance of buffaloes and (2) to observe the influence of synchronization protocol, parity, age, milk production, and body condition score (BCS) on early embryonic loss and progesterone (P4) levels in crossbred buffaloes.Methods: Crossbred buffaloes (n = 101) were synchronized using the GPGMH protocol. At day 20 of AI, the synchronized buffaloes were randomly divided into four groups and administrated GnRH doses (0, 100, 150, and 200 μg). The buffaloes were monitored for ovarian dynamics, P4 level, non-return rate, embryonic/fetal losses, and pregnancy rates. The previously synchronized buffaloes were also classified for synchronization protocol (with or without GnRH), parity (nulli- or multiparous), milk production (high or low), BCS (low, medium, or good) or age (>3 or < 3 years) groups for observing the embryonic loss and P4 level variations.Results: The results indicated no difference (P > 0.05) in CL size, P4 level, pregnancy rate and embryo/fetal losses across the treatment groups at different observation periods. There was a high (P < 0.05) incidence of early embryonic mortality in aged, multiparous, low BCS and low milk-producing buffaloes treated without GnRH.Conclusion: The data suggest that GnRH 200 μg at day 20 of AI improves embryo survival and pregnancy maintenance in crossbred buffaloes.