Study on the correlation between L-citrulline supplementation and gastrointestinal flora structure and semen antifreeze performance in sheep

Cryopreservation of semen can give full play to the reproductive advantages of male animals. However, in actual production, due to the poor frost resistance of sheep semen and the low conception rate, the promotion of sheep frozen semen is greatly hindered. Therefore, it is urgent to improve the frost resistance of semen to improve the quality of frozen semen. At present, most studies on improving the quality of frozen semen are based on the improvement of semen dilutions, and few studies on improving the freezing resistance of ram semen by feeding functional amino acids. Therefore, 24 Turpan black rams were divided into high antifreeze group (HF) and a low antifreeze group (LF) Each of these groups was further randomly divided into control and experimental subgroups. The control subgroup was fed a basal diet, while the experimental subgroup received an additional 12 g/d of L-Cit supplementation based on the control group for a duration of 90 days. The results showed that Following L-Cit supplementation, the experimental group demonstrated significantly elevated sperm density and VSL (Velocity of straight line), T-AOC, GSH-Px, and NO levels in fresh semen compared to the control group (P < 0.01). After thawing, the experimental group exhibited significantly higher levels of T-AOC, GSH-Px, and NO compared to the control group (P < 0.01). Additionally, the HFT group, after thawing frozen semen, displayed significantly higher HK1 protein expression compared to the control group. The number of spermatogonia, spermatocytes, and sperm cells in the HFT group was significantly higher than that in the HFC group. Moreover, 16S rRNA sequence analysis showed that Candidatus_Saccharimonas, Staphylococcus, Weissella, succinivbrionaceae_UcG_002, and Quinella were significantly enriched in the rumen of the HFT group, while Ureaplasma was significantly enriched in the HFC group. In the duodenum, Clostridiales_bacterium_Firm_14, Butyrivibrio, and Prevotellaceae_NK3831_group were significantly enriched in the HFT group, whereas Desulfovibrio and Quinella were significantly enriched in the HFC group. Under the conditions employed in this study, L-Cit supplementation was found to enhance the intestinal flora composition in rams, thereby improving semen quality, enhancing the antifreeze performance of semen, and promoting the development of testicular spermatogenic cells.

精液冷冻保存可充分发挥雄性动物的繁殖优势。然而实际生产中，绵羊精液抗冻性能较差且受胎率偏低，极大制约了绵羊冷冻精液的推广应用。因此，提升精液抗冻性能以改善冷冻精液品质迫在眉睫。当前，多数改善冷冻精液品质的研究聚焦于精液稀释液的优化，而通过饲喂功能性氨基酸提升公羊精液抗冻性能的相关研究较为匮乏。为此，本研究选取24头吐鲁番黑公羊，分为高抗冻组（HF）与低抗冻组（LF），每组再随机分为对照组与试验组亚群；对照组亚群饲喂基础日粮，试验组亚群则在基础日粮基础上每日额外添加12 g/d L-瓜氨酸（L-Cit），试验周期为90天。结果显示，与对照组相比，试验组鲜精的精子密度、直线运动速度（VSL）、总抗氧化能力（T-AOC）、谷胱甘肽过氧化物酶（GSH-Px）活性以及一氧化氮（NO）水平均显著升高（P < 0.01）。解冻后，试验组的T-AOC、GSH-Px活性及NO水平仍显著高于对照组（P < 0.01）。此外，解冻后高抗冻试验组（HFT）的己糖激酶1（HK1）蛋白表达量显著高于对照组（HFC）；HFT组的精原细胞、精母细胞与精子细胞数量均显著高于HFC组。进一步的16S rRNA测序分析显示，HFT组瘤胃内的候选囊菌属（Candidatus_Saccharimonas）、葡萄球菌属（Staphylococcus）、魏斯氏菌属（Weissella）、琥珀弧菌科未培养菌属UcG_002（succinivbrionaceae_UcG_002）以及昆氏菌属（Quinella）显著富集，而HFC组瘤胃内的脲原体属（Ureaplasma）显著富集；在十二指肠中，HFT组的梭菌目细菌Firm_14（Clostridiales_bacterium_Firm_14）、丁酸弧菌属（Butyrivibrio）以及普雷沃氏菌科NK3831群（Prevotellaceae_NK3831_group）显著富集，而HFC组的脱硫弧菌属（Desulfovibrio）与昆氏菌属（Quinella）显著富集。本研究条件下，日粮添加L-Cit可改善公羊肠道菌群结构，进而提升精液品质、增强精液抗冻性能并促进睾丸生精细胞发育。