Cryopreservation of semen and artificial insemination have an important, positive impact on cattle production, and product quality. Through the use of cryopreserved semen and artificial insemination, sperm from the best breeding bulls can be used to inseminate thousands of cows around the world. Although cryopreservation of bull sperm has advanced beyond that of other species, there are still major gaps in the knowledge and technology bases. Post-thaw viability of sperm is still low and differs significantly among the breeding bulls. These weaknesses are important because they are preventing advances both in fundamental science of mammalian gametes and reproductive biotechnology. Various extenders have been developed and supplemented with chemicals to reduce cryodamage or oxidative stress with varying levels of success. More detailed insights on sperm morphology and function have been uncovered through application of advanced tools in modern molecular and cell biology. This article provides a concise review of progress in the cryopreservation of bull sperm, advances in extender development, and frontiers using diverse techniques of the study of sperm viability. This scientific resource is important in animal biotechnology because with the advances in discovery of sperm fertility markers, there is an urgent need to improve post-thaw viability and fertility of sperm through enhanced cryopreservation for precision agriculture to produce food animals to ensure food security on the global scale.

Sperm cryopreservation is an important technique for fertility management, but post-thaw viability of sperm differs among breeding bulls. With metabolites being the end products of various metabolic pathways, the contributions of seminal plasma metabolites to sperm cryopreservation are still unknown. These gaps in the knowledge base are concerning because they prevent advances in the fundamental science of cryobiology and improvement of bull fertility. The objective of this study was to test the hypothesis that seminal plasma amino acids are associated with freezability of bull sperm. To accomplish this objective, amino acid concentrations in seminal plasma from seven bulls of good freezability (GF) and six bulls of poor freezability (PF) were quantified using gas chromatography–mass spectrometry (GC–MS). Multivariate and univariate analyses were performed to identify potential freezability biomarkers. Pathways and networks analyses of identified amino acids were performed using bioinformatic tools. By analyzing and interpreting the results we demonstrated that glutamic acid was the most abundant amino acid in bull seminal plasma with average concentration of 3,366 ± 547.3 nM, which accounts for about 53% of total amino acids. The other most predominant amino acids were alanine, glycine, and aspartic acid with the mean concentrations of 1,053 ± 187.9, 429.8 ± 57.94, and 427 ± 101.3 nM. Pearson’s correlation analysis suggested that phenylalanine concentration was significantly associated with post-thaw viability (r = 0.57, P-value = 0.043). Significant correlations were also found among other amino acids. In addition, partial least squares-discriminant analysis (PLS-DA) bi-plot indicated a distinct separation between GF and PF groups. Phenylalanine had the highest VIP score and was more abundant in the GF groups than in the PF groups. Moreover, pathway and network analysis indicated that phenylalanine contributes to oxidoreductase and antioxidant reactions. Although univariate analysis did not yield significant differences in amino acid concentration between the two groups, these findings are significant that they indicate the potentially important roles of amino acids in seminal plasma, thereby building a foundation for the fundamental science of cryobiology and reproductive biotechnology.

Agarwal A, et al. World J Mens Health. 2023 Apr;41:e43. https://doi.org/10.5534/wjmh.220282

Agarwal A, et al. World J Mens Health. 2024 Jan;42(1):202-215. https://doi.org/10.5534/wjmh.230076

The objective of the current study was to determine the fatty acid composition of sperm from Holstein bulls with different freezability (Good and Poor;n= 12). Fatty acids were extracted from frozen sperm in 1:2 (v/v) chloroform–methanol solvent, fractionated into neutral and polar fractions, and composition determined by gas chromatography–mass spectrometry. Thirty-four fatty acids were quantified and their concentrations and percentages within each lipid fraction were calculated. Overall, saturated fatty acids (SFA) were predominant, accounting for 71 to 80% of fatty acids in neutral and polar lipid factions. There were marked differences in fatty acid composition between the lipid fractions (P< 0.001). The branched chain fatty acid (BCFA) concentration (15 to 18 µg) was almost twice as much as polyunsaturated fatty acids (PUFA) concentration found in the polar lipid fraction (8 to 9 µg;P< 0.001). Sperm with different freezability phenotypes only had a few differences in 22:0, 18:1 cis 9, and 14:0 13-methyl fatty acids (P≤ 0.011). These results are significant because they reveal key understandings of fatty acid composition of sperm membrane and lay a foundation for the manipulation of membrane integrity, fluidity, and stability to advance the assisted reproductive technologies.