Data_Sheet_1_Temperature limits for storage of extended boar semen from the perspective of the sperm's energy status.zip

The optimum storage temperature for liquid-preserved boar semen has been empirically determined to be between 15 and 20°C. Lower temperatures provide an advantage to inhibit bacterial growth, but are regarded as critical due to the high sensitivity of boar spermatozoa to chilling injury. Higher storage temperatures are supposed to induce energy deficiency due to an insufficient depression of metabolic cell activity. However, experimental evidence for alterations of the sperm's energy status in relation to storage temperature and duration is missing. Therefore, we aimed to revisit the upper and lower storage temperature limits for liquid-preserved boar semen from the perspective of the sperm's energy metabolism. Ejaculates (n = 7 boars) were cooled down in Beltsville Thawing Solution (BTS) to 25, 17, 10, or 5°C and stored for up to 120 h. ATP and adenylate energy charge (EC) levels were assessed at storage temperature (24, 72, and 120 h storage) and after subsequent re-warming (38°C). Sperm quality and energy status remained at a stable level in samples stored at 25 and 17°C. Chilling to and storage at 10 or 5°C in BTS provoked cold shock in a subset of sperm as shown by a loss in viability and motility (P < 0.05), which was accompanied by a significant release of adenine nucleotides into the semen extender. Prolonged storage for 120 h resulted in significantly lower mean ATP concentrations in viable spermatozoa at 5 or 10°C compared to 17°C (P < 0.05). Cluster analysis revealed that the main sperm subpopulation, i.e., sperm with moderate speed and linearity, decreased from 50 to 30% (P < 0.05) in favor of slow-moving spermatozoa (5°C) or spermatozoa with a hyperactivation-like motility pattern (10°C). The results point to a sublethal imbalance in available ATP in a subset of the surviving sperm population, rather than a general decrease in available ATP in all spermatozoa. In conclusion, storing diluted boar semen at a stable temperature between 17 and 25°C is a safe procedure concerning the spermatozoa's energy status. Future concepts for hypothermic boar semen preservation below 17°C require measures which ameliorate the imbalanced energy status in viable spermatozoa.

经实验测定，液态保存的公猪精液的最佳储存温度为15~20℃。低温可有效抑制细菌生长，但公猪精子对低温损伤极为敏感，因此低温储存被视为关键风险因素；而较高的储存温度则会因细胞代谢活性未得到充分抑制，引发能量不足。然而，目前尚无实验证据表明精子能量状态会随储存温度与时长发生改变。为此，本研究从精子能量代谢的角度，重新探讨液态保存公猪精液的上下限储存温度。实验选取7头公猪的射精精液，以贝尔茨维尔解冻液（Beltsville Thawing Solution, BTS）稀释后，分别降温至25、17、10或5℃，并储存长达120小时。分别在储存24、72及120小时时，以及后续将样本复温至38℃后，检测ATP水平与腺苷酸能荷（adenylate energy charge, EC）。结果显示，储存于25℃与17℃的样本，其精子质量与能量状态均保持稳定。以10或5℃降温并储存的样本中，部分精子出现冷休克：表现为存活率与运动能力显著下降（P<0.05），同时精液稀释液中腺嘌呤核苷酸的释放量显著升高。当储存时长延长至120小时时，与17℃组相比，5℃与10℃组的有活力精子平均ATP浓度显著降低（P<0.05）。聚类分析结果显示，占主导的精子亚群（即运动速度中等且呈线性运动的精子）占比从50%降至30%（P<0.05），该亚群的减少伴随慢速运动精子（5℃组）或呈超激活样运动模式的精子（10℃组）占比上升。本研究结果表明，存活精子亚群中存在亚致死性的可利用ATP失衡，而非所有精子的可利用ATP普遍降低。综上，将稀释后的公猪精液稳定储存于17~25℃的温度区间内，在精子能量状态层面是安全的储存方式。未来针对17℃以下的公猪精液低温保存方案，需采取措施改善有活力精子的能量失衡问题。