Sperm cryopreservation is one of the sublime biotechnologies for assisted reproduction. In recent decades, there has been an increasing trend in the use of preserved semen. Post-thaw semen quality and values vary among animals of the same species. Similarly, there are species-specific variations in sperm morphology, i.e., sperm head, kinetic properties, plasma membrane integrity, and freezability. Similarly, the viability of sperm varies in the female reproductive tract, i.e., from a few hours (in cattle) to several days (in chicken). Various steps of sperm cryopreservation, i.e., male health examination, semen collection, dilution, semen centrifugation, pre- and post-thaw semen quality evaluation, lack standardized methodology, that result in differences in opinions. Assisted reproductive technologies (ART), including sperm preservation, are not applied to the same extent in commercial poultry species as in mammalian species for management and economic reasons. Sperm preservation requires a reduction in physiological metabolism by extending the viable duration of the gametes. Physiologically and morphologically, spermatozoa are unique in structure and function to deliver paternal DNA and activate oocytes after fertilization. Variations in semen and sperm composition account for better handling of semen, which can aid in improved fertility. This review aims to provide an update on sperm cryopreservation in farm animals.

The skeletal muscle fiber profile is closely related to livestock meat quality. However, the molecular mechanisms determining muscle fiber types in donkeys are not completely understood. In this study, we selected the psoas major muscle (PM; mainly composed of oxidative-type muscle fibers) and biceps femoris muscle (BF; mainly composed of glycolytic-type muscle fibers) and systematically compared their mRNA and microRNA transcriptomes via RNA-seq. We identified a total of 2881 differentially expressed genes (DEGs) and 21 known differentially expressed miRNAs (DEmiRs). Furthermore, functional enrichment analysis showed that the DEGs were mainly involved in energy metabolism and actin cytoskeleton regulation. The glycolysis/gluconeogenesis pathway (including up-regulated genes such as PKM, LDHA, PGK1 and ALDOA) was more highly enriched in BF, whereas the oxidative phosphorylation pathway and cardiac muscle contraction (including down-regulated genes such as LDHB, ATP2A2, myosin-7 (MYH7), TNNC1, TPM3 and TNNI1) was more enriched in PM. Additionally, we identified several candidate miRNA–mRNA pairs that might regulate muscle fiber types using the integrated miRNA–mRNA analysis. Combined with the results of protein–protein interaction (PPI) analysis, some interesting DEGs (including ACTN3, TNNT3, TPM2, TNNC2, PKM, TNNC1 and TNNI1) might be potential candidate target genes involved in the miRNA-mediated regulation of the myofibril composition. This study is the first to indicate that DEmiRs, especially eca-miR-193a-5p and eca-miR-370, and potential candidate target genes that are mainly involved in actin binding (e.g., ACTN3, TNNT3 and TNNC1) and the glycolysis/gluconeogenesis pathways (e.g., PKM) might coregulate the myofibril composition in donkeys. This study may provide useful information for improving meat quality traits in Dezhou donkeys.

The intramuscular fat (IMF) content is considered an important factor for assessing meat quality, and is highly related to meat flavor. However, in donkey meat, the influences of IMF content on lipid and volatile profiles remain unclear. Thus, we conducted lipidomic and volatilomic investigations on high- and low-IMF samples from donkey longissimus dorsi muscle. When the IMF level increased, the monounsaturated fatty acid (especially oleic acid) content significantly increased but the saturated fatty acid content decreased (p < 0.05). Twenty-nine of 876 lipids showed significant differences between the two groups. Volatile profiles from differential IMF content samples were also distinct. Five differential volatile odorants were identified in the two groups: 2-acetyl-2-thiazoline, octanal, 2-pentylfuran, pentanal, and 1-(2-pyridinyl) ethanone. Additionally, strong correlations were found between differential fatty acids and lipids with differential odorants. Thus, the difference in volatile odorants may result from the change in the fatty acid composition and lipid profiles induced by different IMF contents, highlighting the urgent need to increase IMF levels in donkey meat.

The growing recognition of the role of milk-derived exosomes in metabolic and immunological processes has brought attention to the potential utility of donkey milk. However, the efficacy and bioactive components of donkey milk are underexplored. This study aimed to elucidate the proteomic profiles of exosomes isolated from donkey colostrum and mature milk using advanced four-dimensional (4D) label-free quantitative proteomics. A comprehensive analysis identified and quantified a total of 2293 exosomal proteins from donkey milk, including 276 differentially expressed exosomal proteins (DEEPs). The results revealed marked proteomic differences between colostrum and mature milk exosomes, particularly in proteins associated with immune responses and metabolic pathways. Exosomal proteins derived from colostrum were found to be enriched in immune-modulatory factors and glycan-related pathways, which may contribute to the enhancement in neonatal immune system development. In contrast, exosomal proteins from mature milk were predominantly associated with metabolic processes and cellular senescence. Protein–protein interaction (PPI) analysis further suggested that specific exosomal proteins highly expressed in colostrum could serve as nutraceutical components with potential health benefits for humans. In conclusion, this study underscores the distinct proteomic features and potential physiological roles of exosomes from donkey colostrum versus mature milk.

The gut microbiota plays an important role in the digestion, absorption, and metabolism of nutrients, as well as in the immunity, health, and behavior of donkeys. While reference genomes and gut microbial gene catalogs have been helpful in understanding the composition of the donkey, there is still a significant gap in sequencing and understanding the functional aspects of donkey gut microbial genomes. In this study, we analyzed metagenomic sequencing data from 26 donkeys’ gut samples and successfully assembled 844 microbial metagenome-assembled genomes (MAGs). Surprisingly, 678 (80.33%) of these MAGs appear to belong to previously unidentified species. Our analysis further revealed a total of 292,980 predicted carbohydrate-active enzymes (CAZymes) and 257,893 polysaccharide utilization loci (PULs). Interestingly, these enzymes and loci displayed relatively low similarity matches in public databases. We found that the higher abundances of 36 MAGs in the cecum (such as Prevotella, Desulfovibrio, Alistipes, and Treponema_D) and 9 MAGs in the dorsal colon (such as Limimorpha, Saccharofermentans, and Lactobacillus) were associated with a diverse array of carbohydrate-degrading pathways. Network analysis identified Prevotella and Dysosmobacter as connectors, while Saccharofermentans and Akkermansia were shown as provincial hubs. This suggests their crucial roles in complex carbohydrate degradation and hindgut metabolism in donkeys. These findings underscore the complexity of hindgut metabolism in donkeys and expand our understanding of their gut microbiome. Overall, this study provides a comprehensive catalog of donkey gut microbial genes, revealing novel carbohydrate-degrading enzymes and offering new insights for future research on the donkey gut microbiome.