Department of Animal Production and Health, Veterinary Public Health and Food Science and Technology (PASAPTA), Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, Alfara del Patriarca, Valencia, Spain; Unidad Asociada UCH-CEU -IVIA, Valencia, Spain.
Centro de Investigación y Tecnología Animal (CITA), Instituto Valenciano de Investigaciones Agrarias, Segorbe, Castellón, Spain; Unidad Asociada UCH-CEU -IVIA, Valencia, Spain.
Theriogenology. 2023 Oct 1;209:202-212. doi: 10.1016/j.theriogenology.2023.06.013. Epub 2023 Jun 17.
Ejaculates present their own microbiota, and a link between ejaculates' microbiota and sperm quality and fertility exists. With the development of artificial insemination in animal breeding, ejaculates must be manipulated by diluting them with extenders and storing them at temperatures below body temperature. The effects that these processes have on the original semen microbiota have never been studied. This study explores the effects of the protocol for preparing refrigerated goat buck semen doses and storing on seminal microbiota. Semen from six adult goat bucks of the Murciano-Granadina breed (24 ejaculates) was used, cooled to 4 °C in a skimmed milk-based extender, and stored at this temperature for 24 h. Samples were taken in different steps: in the raw ejaculates (ejaculates), after dilution with the refrigeration extender (diluted), immediately after reaching 4 °C (chilled 0 h) and the samples refrigerated at 4 °C and stored at this temperature for 24 h (chilled 24 h). Sperm quality (motility and integrity of plasma and acrosomal membrane, and mitochondrial functionality) was also evaluated. Bacterial 16S rRNA sequencing was used to study the seminal microbiota. Our results indicated that both refrigeration and storage at 4 °C negatively affected sperm quality parameters. Preparing semen doses and their subsequent conservation caused a significant change in the bacterial community structure. Raw ejaculates showed a lower Pielou's evenness index than the other samples (diluted, chilled 0 h and chilled 24 h). Ejaculates also had a lower Shannon's diversity index (3.44) than the diluted semen (4.17) and the semen chilled for 24 h (4.43). Regarding beta diversity, significant differences were detected between ejaculates and the other treatments. Differences were also found in unweighted UniFrac distances between the semen chilled for 0 h and that chilled for 24 h. At the genus level, marked effects of preparing doses and their subsequent conservation were also evident: 199 genera that were absent in ejaculates were found in the semen chilled and stored for 24 h; 177 genera that were present in ejaculates disappeared after 24-h refrigeration. In conclusion, the extender and protocol for preparing refrigerated goat buck semen doses considerably modify microbial ejaculate composition.
精液中存在自身的微生物群，并且精液微生物群与精子质量和生育力之间存在联系。随着动物繁殖中人工授精的发展，必须通过用稀释剂稀释精液并将其储存在低于体温的温度下来处理精液。这些过程对原始精液微生物群的影响从未被研究过。本研究探讨了制备冷藏山羊公鹿精液剂量和储存对精液微生物群的影响。使用了 6 只成年穆拉诺-格拉纳迪纳品种（Murciano-Granadina）山羊公鹿的精液（24 个样本），在基于脱脂乳的稀释剂中冷却至 4°C，并在该温度下储存 24 小时。在不同步骤中采集样本：在原始精液（精液）中、用冷藏稀释剂稀释后（稀释后）、达到 4°C 后立即（冷却 0 小时）以及在 4°C 下冷藏并在该温度下储存 24 小时（冷却 24 小时）后。还评估了精子质量（运动性和质膜及顶体膜的完整性以及线粒体功能）。使用细菌 16S rRNA 测序来研究精液微生物群。我们的结果表明，冷藏和 4°C 储存都对精子质量参数产生负面影响。制备精液剂量及其随后的保存会导致细菌群落结构发生显著变化。原始精液的皮耶罗均匀度指数（Pielou's evenness index）低于其他样本（稀释后、冷却 0 小时和冷却 24 小时）。精液的香农多样性指数（Shannon's diversity index）也低于稀释后的精液（4.17）和冷藏 24 小时的精液（4.43）。关于 beta 多样性，在精液和其他处理之间检测到显著差异。在冷却 0 小时和冷却 24 小时的精液之间的非加权 UniFrac 距离也存在差异。在属水平上，制备剂量及其随后的保存也产生了明显的效果：在冷藏和储存 24 小时的精液中发现了 199 个在精液中不存在的属；在 24 小时冷藏后，在精液中存在的 177 个属消失了。总之，用于制备冷藏山羊公鹿精液剂量的稀释剂和方案极大地改变了精液微生物群的组成。
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