Melatonin is a unique multifunctional molecule that mediates reproductive functions in animals. In this study, we investigated the effects of melatonin on bovine parthenogenetic and androgenetic embryonic development, oocyte maturation, the reactive oxygen species (ROS) levels in parthenogenetic and androgenetic embryos and cumulus—oocyte complexes (COCs) hormone secretion with melatonin supplementation at four concentrations (0, 10, 20, and 30 pmol/mL), respectively. The results showed that melatonin significantly promoted the rates of bovine parthenogenetic and androgenetic embryonic cleavage and morula and blastocysts development (P < 0.05). The rate of cleavage was higher in the androgenetic embryo than that in the parthenogenetic embryo. Compared with the parthenogenetic embryos, the androgenetic embryos had a poor developmental competence from morula to blastocyst stage. Moreover, the levels of ROS were significantly lower in the parthenogenetic and androgenetic embryoes with melatonin-treated group than that of the control group (P < 0.05). Melatonin supplemented significantly increased the maturation rate of oocytein vitro(P < 0.05). More importantly, melatonin significantly promoted the secretion of progesterone and estradiol by COCs (P < 0.05). To reveal the regulatory mechanism of melatonin on steroids synthesis, we found that steroidogenic genes (CYP11A1, CYP19A1andStAR) were upregulated, suggesting that melatonin regulated estradiol and progesterone secretion through mediating the expression of steroidogenic genes (CYP11A1,CYP19A1andStAR). In addition, MT1 and MT2 were identified in bovine early parthenogenetic and androgenetic embryos using western blot. It could be concluded that melatonin had beneficial effects on bovine oocytein vitromaturation, COC hormone secretion, early development of subsequent parthenogenetic and androgenetic embryos. It is inferred that melatonin could be used to enhance the efficiency ofin vitrodeveloped embryos.

Estrus is crucial for cow fertility in modern dairy farms, but almost 50% of cows do not show the behavioral signs of estrus due to silent estrus and lack of suitable and high-accuracy methods to detect estrus. MiRNA and exosomes play essential roles in reproductive function and may be developed as novel biomarkers in estrus detection. Thus, we analyzed the miRNA expression patterns in milk exosomes during estrus and the effect of milk exosomes on hormone secretion in cultured bovine granulosa cells in vitro. We found that the number of exosomes and the exosome protein concentration in estrous cow milk were significantly lower than in non-estrous cow milk. Moreover, 133 differentially expressed exosomal miRNAs were identified in estrous cow milk vs. non-estrous cow milk. Functional enrichment analyses indicated that exosomal miRNAs were involved in reproduction and hormone-synthesis-related pathways, such as cholesterol metabolism, FoxO signaling pathway, Hippo signaling pathway, mTOR signaling pathway, steroid hormone biosynthesis, Wnt signaling pathway and GnRH signaling pathway. Consistent with the enrichment signaling pathways, exosomes derived from estrous and non-estrous cow milk both could promote the secretion of estradiol and progesterone in cultured bovine granulosa cells. Furthermore, genes related to hormonal synthesis (CYP19A1, CYP11A1, HSD3B1 and RUNX2) were up-regulated after exosome treatment, while exosomes inhibited the expression of StAR. Moreover, estrous and non-estrous cow-milk-derived exosomes both could increase the expression of bcl2 and decrease the expression of p53, and did not influence the expression of caspase-3. To our knowledge, this is the first study to investigate exosomal miRNA expression patterns during dairy cow estrus and the role of exosomes in hormone secretion by bovine granulosa cells. Our findings provide a theoretical basis for further investigating milk-derived exosomes and exosomal miRNA effects on ovary function and reproduction. Moreover, bovine milk exosomes may have effects on the ovaries of human consumers of pasteurized cow milk. These differential miRNAs might provide candidate biomarkers for the diagnosis of dairy cow estrus and will assist in developing new therapeutic targets for cow infertility.

Milk spectral data on 2118 cows from nine herds located in northern China were used to access the association of days open (DO). Meanwhile, the parity and calving season of dairy cows were also studied to characterize the difference in DO between groups of these two cow-level factors. The result of the linear mixed-effects model revealed that no significant differences were observed between the parity groups. However, a significant difference in DO exists between calving season groups. The interaction between parity and calving season presented that primiparous cows always exhibit lower DO among all calving season groups, and the variation in DO among parity groups was especially clearer in winter. Survival analysis revealed that the difference in DO between calving season groups might be caused by the different P/AI at the first TAI. In addition, the summer group had a higher chance of conception in the subsequent services than other groups, implying that the micro-environment featured by season played a critical role in P/AI. A weak linkage between DO and wavenumbers ranging in the mid-infrared region was detected. In summary, our study revealed that the calving season of dairy cows can be used to optimize the reproduction management. The potential application of mid-infrared spectroscopy in dairy cows needs to be further developed.