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 fibrolytic enzymes and the hindgut fungi in donkey cecum-colon ecosystem play an important role in dietary fiber digestion. A better understanding of the fibrolytic enzyme profiles and the fungal community along donkey caecum and colon is key for optimizing hindgut function. In the present study, the fibrolytic enzyme activities within donkey caecum and colon were firstly measured by spectrophotometry. Activities of carboxymethyl cellulase, avicelase, xylanase, and acetyl esterase were greater in donkey dorsal colon than in caecum, indicating that the colon microorganisms may be more efficient in producing fibrolytic enzymes compared to caecum microbes. The fungal community composition along donkey hindgut was determined by sequencing ITS region using Illumina MiSeq. Three fungal phyla were identified by sequence comparison: Ascomycota (66.8%–74.4%), Basidiomycota (21.6%–30.9%), and Neocallimastigomycota (0.9%–3.3%). The Aspergillus, Wallemia, Phanerochaete, Fusarium, and Penicillium were detected as the dominant genera, but their metabolic and functional significance in donkey cecum-colon ecosystem need further investigation. In terms of the anaerobic fungi Neocallimastigomycota, its abundance was greater in donkey colon than in caecum (p < 0.05), indicating that the donkey hindgut region was associated with differences in fungal community composition. Moreover, the relative abundance of enzymes related to plant cell wall degradation were predicted by PICRUSt, and they were also lower in caecum than in colon. The present study provided new information about fibrolytic enzyme profiles and fungal composition in donkey hindgut ecosystem.

As the initiation point of digestion, the oral microbiome is important in maintaining oral and systemic health. However, the composition of oral microbial communities and the influence of weaning on the oral microbiota of donkey foals remains poorly characterized. The present study used buccal swab samples to determine the changes in oral microbial communities occurring at the time of weaning. A total of 20 oral swab samples were collected from two groups: preweaning donkey foals (PreW group, n = 10) and postweaning donkey foals (PostW group, n = 10). The donkey oral microbiome was analyzed by 16S rRNA gene sequencing using Illumina MiSeq. This study is the first report of the donkey oral microbiome in association with weaning. Compared to the preweaning donkeys, the oral bacteria diversity in the postweaning donkeys was increased, with a higher Simpson index. Changes in the composition of the oral microbiota between the PreW and PostW groups were observed in the present study. At the phylum level, the relative abundance of Firmicutes and Myxococcota was significantly greater in the PostW than in the PreW group. At the genus level, the Gemella, unclassified_o__Lactobacillales, and Lactobacillus were increased in the postweaning donkeys. The donkeys’ oral microbial functions were predicted using PICRUSt, and the functions related to carbohydrate metabolic pathways were significantly enriched in the oral microbiome in the PostW donkeys. In summary, the current study provides a deeper insight into the oral microbiota changes during the weaning period, and the influence of weaning together with the documented changes in diversity and composition will help us to obtain a better understanding of their long-term health impact within the oral cavities of donkey foals. However, a major limitation of the present study was that the samples were obtained from different animals in the PreW and PostW groups, which may have resulted in variability among the different individuals. Further investigation is needed to monitor the shift in oral microbes in the same individuals during the weaning period.

Donkey hindgut is an enlarged fermentative chamber that harbors a highly complex and extremely abundant community of anaerobic bacteria. It can be divided into two different ecological sites: liquid (Lq) phase and adherent fraction (Ad) colonized by bacteria. However, the Ad bacteria have not previously been specifically collected or directly compared with the Lq bacteria. In the present study, the digesta collected from the caecum, ventral colon and dorsal colon of nine Dezhou donkeys was separated into Lq and Ad fractions. The bacterial community structure was comparatively determined using 16S rRNA gene sequences by Illumina MiSeq. The Ad bacteria had a higher bacterial diversity than Lq bacteria due to the higher Chao and ACE index (p < 0.05). The predominant bacteria at the phylum level were Firmicutes (55.4~74.3%) and Bacteroidota (13.7~32.2%) for both the Lq and Ad fraction. The relative abundance of Bacteroidota, Spirochaetota, Fibrobacterota and Patescibacteria in the Ad fraction was greater than Lq (p < 0.05), suggesting that bacteria associated with feed particles were mainly responsible for plant fiber degradation. At the genus level, the abundance of Lactobacillus in Lq was greater than that in the Ad fraction (p < 0.05), indicating that the bacteria in the Lq fraction were better at hydrolyzing readily fermentable carbohydrates. PICRUSt showed that the activities of enzymes related to fiber degradation in the Ad fraction were also greater than Lq. In addition, the hindgut region also had a significant effect on the bacterial community composition. The relative abundance of Rikenellaceae_RC9_gut_group, Clostridium_sensu_stricto_1, Christensenellaceae_R-7_group and norank_Bacteroidales_BS11_gut_group was increased (p < 0.05) along the donkey hindgut. In summary, the present study provides evidence that bacteria adherent to plant biomass were different to those in the liquid phase within the donkey caeco-colic digesta, and bacteria associated with feed particles may mainly be responsible for plant fiber degradation.

Grape seed proanthocyanidin (GSP) contains polyphenolic bioflavonoids ubiquitously found in the lignified portions of grape seeds from the winery and distillery industries, as an antioxidant. To explore its potential as a rumen modifier in methanogenesis inhibition, a 2 × 5 factorial experiment was conducted to determine the effect of GSP at 0, 15, 30, 60 and 120 mg/g of substrate on the rumen fermentation and methanogenesis of two representative total mixed rations (HY, a diet for high-yield (>2 kg/d) lactating cows, and LY, a diet for low-yield (<25 kg/d) lactating cows). By using the MIXED procedure, after a 48 h in vitro rumen incubation, increasing the GSP addition linearly decreased the in vitro dry matter digestion (IVDMD) and slowed down the rates of ration fermentation (RmaxS, g/h) and kinetic gas production (RmaxG, mL/h), with the decreases being more pronounced in the LY than HY group (p < 0.05). The GSP addition decreased hydrogen recovery (2Hrec) and altered the fermentation gas composition. The molar CH4 proportion was significantly reduced with both 60 and 120 mg GSP addition (p < 0.01). The total volatile fatty acid production was linearly decreased with the increasing GSP addition (p < 0.01). In addition, the GSP addition significantly decreased the ratio of methanogens to total bacteria (p < 0.05), and the reduction was notably greater in the HY than in the LY substrate (45.3% vs. 15.2% decrease), although the diversity of rumen methanogenic archaea was not affected in either the HY or the LY group. Bioinformatic analysis illustrated that the rumen archaeal community was predominated by a Methanobrevibacter genus (>72.5%), followed by Methanomassiliicoccus (>20.9%) and Methanosphaera (>1.0%). Methanobrevibacter could play an important role in methanogenesis in the presence of GSP, though it is usually considered to be the main hydrogenotrophic methanogen. In brief, the GSP addition presented high potential as a rumen modifier to mitigate methanogenesis by decreasing the ratio of methanogens to total bacteria. Methanobrevibacter could play an important role in methanogenesis in the presence of GSP. However, a relatively low administration level of GSP should be taken into consideration in order to obtain its inhibitory effect on CH4 emission, with a minimal negative effect on rumen digestion and fermentation.