Cervical mucus not only provides energy for sperm but also forms a barrier to block sperm. This paper aims to study the microstructure of cervical mucus in dairy cows during the proestrus, estrus, and metestrus and its effect on sperm permeability. The experiment collected cervical mucus from 60 Holstein cows during these phases, then observed the different shapes of the mucus after crystallization, classified the mucus, and analyzed its proportions. Scanning electron microscopy was used to observe the ultrastructure of the cervical mucus and measure the micro-pore sizes, followed by sperm permeability tests using mucus from different estrous stages and counting the number of permeated sperm. The results indicate that cervical mucus from cows in different estrous phases includes four types (L, S, P, G), with each type constituting a different proportion. During the proestrus, the L type was significantly more prevalent than the other types (p < 0.05); during estrus, the S type was significantly more prevalent than the other types (p < 0.05); and during the metestrus, the p type was significantly more prevalent than the other types (p < 0.05). The micro-pore sizes of the same type of cervical mucus did not show significant differences across different estrous phases (p > 0.05). However, within the same estrous phase, there were significant differences in the micro-pore sizes among the four types (p < 0.05). The number of sperm that permeated the cervical mucus during estrus and metestrus was significantly higher than during the proestrus (p < 0.05). This study provides data support for the research on cervical mucus in dairy cows.

Semen quality plays a crucial role in bovine in vivo embryo production. This study aimed to compare the effects of 0 °C-refrigerated semen and liquid nitrogen-frozen semen on embryo quality in Simmental cattle. Semen collected from five bulls was equally divided into two groups: one diluted with a 0 °C refrigeration solution and stored at 0 °C, and the other diluted with a cryopreservation solution and stored in liquid nitrogen for 24 h. We evaluated sperm motility, progressive motility (assessed via a computer-assisted sperm analyzer), acrosome integrity, and plasma membrane integrity in both groups. Fifty superovulated Simmental cows were artificially inseminated with semen from both groups. Embryos were non-surgically flushed on day seven, followed by BrdU proliferation staining and TUNEL apoptosis staining. Proliferation and apoptosis levels were quantified using marker genes. Results showed that 0 °C-refrigerated semen exhibited significantly higher sperm motility, progressive motility, acrosome integrity, and plasma membrane integrity compared to liquid nitrogen-frozen semen (p < 0.05). While total embryo numbers showed no significant difference between groups (p ≥ 0.05), embryos from 0 °C-refrigerated semen contained significantly more proliferative cells (p < 0.05) and fewer apoptotic cells (p < 0.05) than those from frozen semen. These findings demonstrate that 0 °C-refrigerated semen outperforms liquid nitrogen-frozen semen in both sperm quality parameters and resultant embryo quality.

This study aimed to investigate the selective effect of the reproductive tract mucus in cows that have consistently produced offspring of a single sex on X/Y spermatozoa. We collected mucus from the reproductive tract of cows that had given calvings to offspring of the same sex, or alternated between sexes, for more than five consecutive calvings. We evaluated the pH of reproductive tract mucus. Subsequently, we conducted a spermatozoa penetration assay; the proportions of X and Y spermatozoa after penetration were then identified by dual TaqMan qPCR and flow cytometry. This was followed by in vitro fertilization and embryo sex determination experiments. Immediately afterwards, computer-aided spermatozoa analysis was employed to analyze the spermatozoa that had penetrated through different types of mucus in the reproductive tract. The analysis indicated that the reproductive tract mucus of cows consistently producing male or female calves exhibited selectivity towards X/Y spermatozoa. The differences in the pH values of the reproductive tract mucus among cows continuously producing male calves, those continuously producing female calves, and those alternately giving birth to male and female calves were not significant (p ≥ 0.05). The outcome of dual TaqMan qPCR for cows consistently producing male calves was Y: 79.29 ± 4.28% vs. X: 21.67 ± 4.53%; for cows consistently producing female calves, the equation was Y: 25.05 ± 4.88% vs. X: 75.34 ± 5.13%. The results of flow cytometry processing revealed the following proportions: for cows consistently producing male calves: Y: 83.33 ± 5.52% vs. X: 17.23 ± 4.74%; for cows consistently producing female calves: Y: 24.81 ± 4.13% vs. X: 76.64 ± 4.21%. The outcomes of embryo sex determination for cows consistently producing male calves were as follows: male embryos vs. female embryos (79.60 ± 2.87% vs. 21.07 ± 2.51%); for cows consistently producing female calves, the outcomes for male embryos vs. female embryos were 25.58 ± 3.96% vs. 75.63 ± 3.55%. Computer-aided analysis revealed that the concentration of spermatozoa penetrating the reproductive tract mucus in cows alternating between male and female calves (9.09 ± 0.72 million/mL) was significantly higher than that in cows consistently producing male calves (6.01 ± 1.19 million/mL) and cows consistently producing female calves (5.61 ± 0.60 million/mL). There were no significant differences in spermatozoa motility, the proportion of progressive motile spermatozoa, and curvilinear, straight-line, and average path velocities. Collectively, these findings indicate that the reproductive tract mucus of cows consistently producing offspring of a single sex exhibits selectivity towards either X or Y spermatozoa. This finding is of great significance for studying the impact of maternal factors on offspring sex.