Proteomic analysis of donkey sperm reveals changes in acrosome enzymes and redox regulation during cryopreservation.

Sperm cryoinjuries caused by cryopreservation restrict the application of donkey frozen semen in artificial insemination (AI). Identification of differentially represented proteins in fresh and frozen-thawed spermatozoa is of great significance to optimize the cryopreservation process and modify the component of cryopreservation extender. In this study, protein samples prepared from fresh (F) and frozen-thawed (FT) donkey spermatozoa were compared. 2682 proteins were quantitatively identified by tandem mass spectrometry (TMT) polypeptide labeling technique and LC-MS/MS method, of which 28 were more abundant in thawed samples and 147 in fresh spermatozoa. The differential abundant proteins (DAPs) were analyzed by bioinformatics. Most of the DAPs in intensive bioinformatic analysis were involved in the process of regulation of biological process and metabolism. Functional protein analysis showed that DAPs process mainly protein hydrolase activity and oxidoreductase activity. Cellular Component analysis showed that DAPs were related to vesicle transport and membrane system. This is the first analysis and study on differential proteomics of donkey sperm proteins before and after cryopreservation, which has a certain guiding significance for studying the mechanism of sperm damage caused by cryopreservation and improving the freezing and thawing procedure. SIGNIFICANCE: In recent years, the commercial value of donkey products has been discovered. Improving the breeding efficiency of donkeys can save the stock of donkeys which is decreasing rapidly, and allow people to continuously benefit from the nutritional value brought by donkey milk. Sperm cryopreservation technology has laid the foundation for encouraging the spread of artificial insemination in donkey reproduction, but the freezing and thawing process causes damage to sperm, which dramatically reducing the viability of frozen sperm and leading to low fertility. At present, the mechanism of damage to donkey sperm caused by cryopreservation is still unclear, and studying this mechanism can provide a direction for improving the quality of frozen semen. Protein is a potential key factor affecting sperm cryopreservation activity. Studying changes in the sperm proteome during cryopreservation can provide promising evidence for revealing sperm cryopreservation damage, which is of great significance for optimizing the cryopreservation process, improving the composition of cryopreservation extender, and seeking directions for improving the quality of frozen semen.

Expression profiles and polymorphic identification of the ACSL1 gene and their association with body size traits in Dezhou donkeys.

Finding out the genetic mechanism of growth and development traits and the development of related molecular markers can help improve the breeding of livestock. The long-chain acyl coenzyme A synthase 1 (ACSL1) gene plays a major role in lipid synthesis and fatty acid catabolism. However, there are few studies on the ACSL1 gene polymorphism of Dezhou donkeys. This study analyzed the expression level of the ACSL1 gene in different tissues of young and adult Dezhou donkeys, as well as association analysis of four gene polymorphic loci in 450 individuals. The results showed that expression levels of the ACSL1 gene are higher in heart, liver, spleen, lung, renal, gastric and muscle tissues of adult donkeys than in those of young donkeys. In the association analysis between genotype and body size traits, the wild genotype DD at the ACSL1-1 locus in female and male donkeys was greater than the mutant genotype II ( P < 0.05 ); genotype II of ACSL1-2 was significantly higher than that of DD in withers height, body length, rump width and body weight of male donkeys ( P < 0.05 ); and ACSL1-3 showed a tendency for the wild genotype II to be greater than the mutant genotype DD in female and male donkeys ( P < 0.05 ). In addition, among the five haplotype combinations constructed, Hap3Hap3 (II-II-DD-DD) and Hap6Hap6 (DD-II-II-II) haplotype combinations were superior to other haplotype combinations in growth traits, which also indicated that the results of haplotype combination association analysis and genotype association analysis tended to be the same. In conclusion, the results of this study indicate that the polymorphic loci of the ACSL1 gene can be used as candidate molecular markers for the growth and development of Dezhou donkeys, and provide a theoretical reference for the breeding of Dezhou donkeys.