Administration of melatonin prior to modified synchronization protocol improves the productive and reproductive efficiency of Chinese crossbred buffaloes in low breeding season.

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.

Administration of GnRH at day 20 of AI and embryonic losses in synchronized crossbred buffalo.

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.

High and low dose of luzindole or 4-phenyl-2-propionamidotetralin (4-P-PDOT) reverse bovine granulosa cell response to melatonin.

Background: Communication between oocytes and granulosa cells ultimately dictate follicle development or atresia. Melatonin is also involved in follicle development. This study aimed to investigate the effects of melatonin and its receptor antagonists on hormone secretion, as well as gene expression related to hormone synthesis, TGF-ß superfamily, and follicle development in bovine granulosa cells, and assess the effects of melatonin in the presence of 4-P-PDOT and luzindole. Methods: Bovine ovaries were collected from a local abattoir and follicular fluid (follicle diameter 5-8 mm) was collected for granulosa cell isolation and culture. Granulosa cells and culture medium were collected 48 h after treatment with melatonin at high dose concentrations (10-5 M) and low dose concentrations (10-9 M) in the absence/presence of 4-P-PDOT and luzindole (10-5 M or 10-9 M). Furthermore, the expression level of genes related to hormonal synthesis (CYP11A1, CYP19A1, StAR, and RUNX2), TGF-ß superfamily (BMP6, INHA, INHBA, INHBB, and TGFBR3), and development (EGFR, DNMT1A, and FSHR) were detected in each experimental group by real-time quantitative PCR. In addition, the level of hormones in culture medium were detected using ELISA. Results: Both 10-5 M and 10-9 M melatonin doses promoted the secretion of inhibin A and progesterone without affecting the production of inhibin B and estradiol. In addition, both promoted the gene expression of INHA, StAR, RUNX2, TGFBR3, EGFR, and DNMT1A, and inhibited the expression of BMP6, INHBB, CYP11A1, CYP19A1, and FSHR. When combined with different doses of 4-P-PDOT and luzindole, they exhibited different effects on the secretion of inhibin B, estradiol, inhibin A, and progesterone, and the expression of CYP19A1, RUNX2, BMP6, INHBB, EGFR, and DNMT1A induced by melatonin. Conclusion: High and low dose melatonin receptor antagonists exhibited different effects in regulating hormone secretion and the expression of various genes in response to melatonin. Therefore, concentration effects must be considered when using luzindole or 4-P-PDOT.

Effects of Sodium Chromate Exposure on Gene Expression Profiles of Primary Rat Hepatocytes (In Vitro).

Chromium exposure has adverse impacts on human health and the environment, whereas chromate-induced hepatotoxicity's detailed mechanism is still unclear. Therefore, the purpose of the current study was to reveal the crucial signaling pathways and genes linked to sodium chromate-induced hepatotoxicity. GSE19662, a gene expression microarray, was obtained from Gene Expression Omnibus (GEO). Six primary rat hepatocyte (PRH) samples from GSE19662 include sodium chromate-treated (n = 3) and the control PRH samples (n = 3). A total of 2,525 differentially expressed genes (DEGs) were obtained, especially 962, and 1,563 genes were up- and downregulated in sodium chromate-treated PRHs compared to the control. Gene ontology (GO) enrichment analysis suggested that those DEGs were involved in multiple biological processes, including the response to toxic substances, the positive regulation of apoptotic process, lipid and cholesterol metabolic process, and others. Signaling pathway enrichment analysis indicated that the DEGs were mainly enriched in MAPK, PI3K-Akt, PPAR, AMPK, cellular senescence, hepatitis B, fatty acid biosynthesis, etc. Moreover, many genes, including CYP2E1, CYP1A2, CYP2C13, CDK1, NDC80, and CCNB1, might contribute to sodium chromate-induced hepatotoxicity. Taken together, this study enhances our knowledge of the potential molecular mechanisms of sodium chromate-induced hepatotoxicity.

Dietary Supplementation of Capsaicin Enhances Productive and Reproductive Efficiency of Chinese Crossbred Buffaloes in Low Breeding Season.

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.

Luzindole and 4P-PDOT block the effect of melatonin on bovine granulosa cell apoptosis and cell cycle depending on its concentration.

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.

The Mechanism of Melatonin and Its Receptor MT2 Involved in the Development of Bovine Granulosa Cells.

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.

RNAi-mediated knockdown of MTNR1B without disrupting the effects of melatonin on apoptosis and cell cycle in bovine granulose cells.

Melatonin is well known as a powerful free radical scavenger and exhibits the ability to prevent cell apoptosis. In the present study, we investigated the role of melatonin and its receptor MTNR1B in regulating the function of bovine granulosa cells (GCs) and hypothesized the involvement of MTNR1B in mediating the effect of melatonin on GCs. Our results showed that MTNR1B knockdown significantly promoted GCs apoptosis but did not affect the cell cycle. These results were further verified by increasing the expression of pro-apoptosis genes (BAX and CASP3), decreasing expression of the anti-apoptosis genes (BCL2 and BCL-XL) and anti-oxidant genes (SOD1 and GPX4) without affecting cell cycle factors (CCND1, CCNE1 and CDKN1A) and TP53. In addition, MTNR1B knockdown did not disrupt the effects of melatonin in suppressing the GCs apoptosis or blocking the cell cycle. Moreover, MTNR1B knockdown did not affect the role of melatonin in increasing BCL2, BCL-XL, and CDKN1A expression, or decreasing BAX, CASP3, TP53, CCND1 and CCNE1 expression. The expression of MTNR1A was upregulated after MTNR1B knockdown, and melatonin promoted MTNR1A expression with or without MTNR1B knockdown. However, despite melatonin supplementation, the expression of SOD1 and GPX4 was still suppressed after MTNR1B knockdown. In conclusion, these findings indicate that melatonin and MTNR1B are involved in BCL2 family and CASP3-dependent apoptotic pathways in bovine GCs. MTNR1A and MTNR1B may coordinate the work of medicating the appropriate melatonin responses to GCs.

The role of Melatonin receptor MTNR1A in the action of Melatonin on bovine granulosa cells.

Granulosa cells (GCs) play an important role in ovarian follicle growth, development, and follicular atresia. In the present study, we investigated the effects of Melatonin on bovine GCs, and asked if MTNR1A was involved in their response to this indole hormone. Our results indicated that Melatonin inhibited GC apoptosis by up-regulating the expression of BCL2, BCL-XL, GPX4, and SOD1, and down-regulating the expression of BAX, CASP3, and TP53. Moreover, Melatonin modulated bovine GC function by decreasing the expression of INHA, INHBB, FSHR, and TGFBR3, and the abundance of Inhibin ß and Activin B, while increasing the expression of LHR, INHBA, and secretion of progesterone by GCs. In contrast, knockdown of MTNR1A significantly increased the expression of BAX, CASP3, TP53, INHA, FSHR, and TGFBR3, as well as Inhibin ß abundance, while decreasing the expression of BCL2, GPX4, SOD1, and LHR, and production of progesterone and estradiol; no effect was observed on the expression of BCL-XL, INHBA, or INHBB. These results suggest that Melatonin and MTNR1A play an important role in modulating bovine GC function by regulating cellular progression, apoptosis, hormones secretion, and reproduction-related genes. Furthermore, altered expression of MTNR1A could affect how bovine GCs respond to Melatonin.

Overexpression of hypoxia-inducible factor prolyl hydoxylase-2 attenuates hypoxia-induced vascular endothelial growth factor expression in luteal cells.

Vascular endothelial growth factor (VEGF)-dependent angiogenesis has a crucial role in the corpus luteum formation and their functional maintenances in mammalian ovaries. A previous study by our group reported that activation of hypoxia­inducible factor (HIF)­1α signaling contributes to the regulation of VEGF expression in the luteal cells (LCs) in response to hypoxia and human chorionic gonadotropin. The present study was designed to test the hypothesis that HIF prolyl­hydroxylases (PHDs) are expressed in LCs and overexpression of PHD2 attenuates the expression of VEGF induced by hypoxia in LCs. PHD2-overexpressing plasmid was transfected into LC2 cells, and successful plasmid transfection and expression was confirmed by reverse transcription quantitative polymerase chain reaction and western blot analysis. In addition, the present study investigated changes of HIF­1α and VEGF expression after incubation under hypoxic conditions and PHD2 transfection. PHD2 expression was significantly higher expressed than the other two PHD isoforms, indicating its major role in LCs. Moreover, a significant increase of VEGF mRNA expression was identified after incubation under hypoxic conditions, which was, however, attenuated by PHD2 overexpression in LCs. Further analysis also indicated that this hypoxia­induced increase in the mRNA expression of VEGF was consistent with increases in the protein levels of HIF­1α, which is regulated by PHD-mediated degradation. In conclusion, the results of the present study indicated that PHD2 is the main PHD expressed in LCs and hypoxia­induced VEGF expression can be attenuated by PHD2 overexpression through HIF­1α­mediated mechanisms in LCs. This PHD2-mediated transcriptional activation may be one of the mechanisms regulating VEGF expression in LCs during mammalian corpus luteum development.

[Construction and identification of mammary expressional vector for cDNA of human lactoferrin].

The aim of this study was to construct a mammary gland-specific expressional vector pBC1-hLF-Neo for Human Lactoferrin (hLF) gene and then investigate its expression in the mammary gland epithelium cells. The constructed vector contained the 6.2 kb long 5' flank regulation region including promoter, other elements and the 7.1 kb long 3' flank regulation region including transcriptional ending signal of a goat's beta-casein gene. A cassette of Neo gene was also inserted into the vector which gave a total length of 26.736 kb identified by restriction fragment analysis and partial DNA sequencing. The results revealed that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, we transfected the lined vector DNA into the dairy goat's mammary gland epithelium cells and C127 cells of a mouse's mammary epithelium by Lipofectamine. After selection with G418 for 8-10 days, G418-risistant clones were obtained. PCR analysis demonstrated that hLF gene cassette had been integrated into the genomic DNA of G418-risistant clones. After proliferation culture, the two kinds of transgenic cells were cultured in serum-free DMEM-F12 medium with prolactin, insulin and hydrocortisone- a medium capable of inducing recombinant hLF expression. RT-PCR, Western blotting and anti-bacteria bioactivity experiments demonstrated that the constructed mammary gland specific vector pBC1-hLF-Neo possessed the desirable bioactivity to efficiently express and could secrete hLF in both mammary gland cells and have the effect of E. coli proliferation inhibition. Paramount to everything, this study laid a firm foundation for preparing the hLF gene transgenic goat fetal-derived fibroblast cells.