Preservation of sperm significantly contributes to the advancement of assisted reproductive technologies, genetic conservation and improvement efforts, and precision breeding of livestock. This review distills knowledge from the existing information and emerging patterns in the field of buck sperm cryopreservation. The primary focus is on the challenges and opportunities associated with improving extender formulations and freezing techniques in order to enhance the vitality of sperm after thawing and to increase the potential for conception. This review assesses the efficacy and limitations of conventional extenders derived from egg yolk or soybean lecithin, and the adverse impacts of seminal plasma enzymes on sperm quality during the processes of chilling and cryopreservation. Significant progress has been made in the fields of molecular biology namely lipidomics, proteomics, metabolomics, DNA methylation providing valuable knowledge regarding the unique reactions of sperm to cryopreservation. The utilization of the “omics” technologies has shown intricate molecular transformation that occur in sperm during freezing and thawing. Moreover, detection of molecular biomarkers that indicate the quality of sperm and their ability to withstand freezing provides opportunities to choose the best sperm samples for cryopreservation. This, in turn, enhances the results of artificial insemination and genetic conservation endeavors. This review emphasizes the necessity for adopting a comprehensive approach that combines molecular and cellular knowledge with practical methods in the field of sperm cryopreservation to ensure production of goats as major food animals in the global scale.

This study aimed to determine the effect of microfluidic sperm sorting chip on embryo development and quality in the sperm treatment step in in vitro embryo production in cattle. Only A‐quality oocytes obtained from the ovaries of Holstein cattle were included in the study. These oocytes were first placed in in vitro maturation medium, and matured oocytes were randomly divided into two groups at the 24th hour of maturation. Oocytes in the first group with the Microfluidic Sperm Sorting Chip (MFSC, n = 154) were put into a fertilization medium with spermatozoa prepared with microfluidic sperm sorting device. Oocytes in the second group (Con, n = 169) were fertilized with spermatozoa prepared by the routine sperm treatment step of the commercial company. The rate of cleavage (85.71% vs. 76.33%, respectively) and of reaching the blastocyst (44.15% vs. 32.54%, respectively) in the MFSC group were higher than the control group. In addition, it was determined that the numbers of ICM (45.8 ± 2.04 vs. 39.2 ± 1.85, respectively), TE (122.13 ± 2.19 vs. 115.0 ± 2.61, respectively), TC (167.93 ± 2.89 vs. 154.2 ± 2.62, respectively) increased in the MFSC group compared to the control group. A statistically significant difference was found in the number of cells with apoptosis per embryo (5.14 ± 0.77 vs. 11.91 ± 0.79) and apoptotic index rates (3.06 ± 0.47 vs. 7.72 ± 0.55%) of the MFSC and Con groups. As a result, we concluded that using microfluidic sperm sorting chips during sperm treatment in bovine IVEP increases the rate of reaching the blastocyst, embryo development, and quality and reduces the possibility of apoptosis in developing blastocysts. For this reason, it is also thought that the use of microfluidic sperm sorting devices during sperm treatment in bovine IVEP may be a new alternative in this field.

The identification of molecular markers for fertility is critical for the sustainability of livestock production. We profiled small non-coding RNAs (sncRNAs) in sperm from rams with high fertility (HF) and low fertility (LF) phenotypes to uncover their roles in ram sperm fertility. Rams were categorized into high-fertility (HF, n = 31; 94.5 ± 2.8%) and low-fertility (LF, n = 25; 83.1 ± 5.73%) phenotypes based on pregnancy rates (average 89.4 ± 7.2%). From these, sperm samples of HF (n = 4; pregnancy rate 99.2 ± 1.6%) and LF (n = 4; pregnancy rate 73.6 ± 4.4%) rams underwent sncRNA sequencing. Small RNA sequencing produced 14,962,876 reads in LF rams and 17,401,094 reads in HF rams, showing distinct sncRNA biotypes, including miRNAs, tRNAs, snoRNAs, snRNAs, and rRNAs. Among these, miRNAs comprised 7.12% of reads in LF rams and 3.78% in HF rams, while rRNAs and repeats formed significant proportions in both groups. A total of 1673 known and 627 novel miRNAs were identified, with 227 differentially expressed miRNAs between the HF and LF groups. We showed that key miRNAs, such as oar-miR-200b and oar-miR-370-3p, were upregulated in HF sperm, while downregulated miRNAs in LF, such as oar-miR-26b and oar-let-7d, were associated with impaired sperm function and DNA fragmentation. A functional enrichment analysis of miRNA target genes highlighted pathways related to ribonucleoprotein complex biogenesis, RNA processing, and gene expression regulation. These findings establish the critical role of sperm sncRNAs as regulators of fertility and potential biomarkers in breeding soundness tests for the precision farming of livestock for global food security.

This study aimed to investigate the effect of putrescine supplementation to maturation medium during in vitro embryo production in cattle on maturation and embryo development/quality. Oocytes obtained from the ovaries of Holstein cattle were used in the study. Obtained cumulus-oocyte complexes were evaluated according to morphological structure, cytoplasmic features, and cumulus cell number, and only Category-I ones were used in the study. Before the in vitro maturation step, oocytes were randomly divided into two groups. In the first group (Putrescine group, n = 159), 0.5 mM putrescine was added to the maturation medium before in vitro maturation. No addition was applied to the maturation medium of the second group (Control group, n = 149). Cumulus expansion degrees of oocytes following maturation (Grade I: poor, Grade II: partial, and Grade III: complete) were determined. In addition, the meiosis of oocytes after maturation was evaluated by differential staining. Then the oocytes were left for fertilization with sperm and finally, possible zygotes were transferred to the culture medium. After determining the developmental stages and quality of the embryos after in vitro culture, only the embryos at the blastocyst stage were stained with the differential staining method to determine the cell numbers. When the cumulus expansion degrees of the groups were evaluated, the Grade III cumulus expansion rate in the putrescine group was higher than the control group (74.21% and 60.4%; respectively) and the Grade I expansion rate (11.95% and 26.17%; respectively) was found lower (p < .05). When the resumption of meiosis was evaluated according to the cumulus expansion degrees, it was determined that the rate of resumption of meiosis increased as the cumulus expansion increased. In addition, the cleavage rates of oocytes and reaching the blastocyst in the putrescine group were found to be higher than in the control group (p < .05). Moreover, inner cell mass, trophectoderm cells, and total cell counts were found to be higher in blastocysts obtained after the putrescine supplementation to the maturation medium compared to the control group (p < .05). As a result, it was determined that the putrescine supplementation to the maturation medium during in vitro embryo production in cattle increased the degree of cumulus expansion and the rate of resumption of meiosis. In addition, putrescine supplementation was thought to increase the rate of reaching the blastocyst of oocytes due to better cell development in embryos.