Temperature Elevation during Semen Delivery Deteriorates Boar Sperm Quality by Promoting Apoptosis.
Temperature Elevation during Semen Delivery Deteriorates Boar Sperm Quality by Promoting Apoptosis.
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DOI：
10.3390/ani13203203
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发表时间：
2023-10-13
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猪精液质量是影响人工授精效果的关键因素。此外，精液交付实践可能会对公猪精子质量和功能产生负面影响。尽管如此，精液输送过程中温度变化的具体影响以及精子对温度变化的分子反应尚未完全清楚。在这种情况下，本研究的目的是探讨如何在精液运输过程中的温度变化影响公猪精子的质量和功能。我们的研究结果表明，使用密封良好的聚苯乙烯泡沫塑料输送24小时后，猪精液的温度从最初的17 °C升高到环境温度37 °C。精子的质量和功能受到了极大的损害，当精液温度回到17 °C时，这些都无法恢复。除此之外，猪精子经历了一个显着的早期凋亡的转化为晚期凋亡引起的温度升高。在此过程中，公猪精子通过上调热休克蛋白水平和激活关键的能量调节因子AMP激活蛋白激酶来调节自身的存活。这些发现有助于了解温度变化如何影响公猪精子的质量和功能，以及精子如何生存，为改善公猪精液的交付实践提供理论和技术信息。输精技术是保证高质量公猪精子人工授精效果的关键。本研究旨在评估一种常见的精液输送方法，聚苯乙烯泡沫塑料盒，在高温下对公猪精子质量和功能的影响，并调查精子对温度升高的潜在分子反应。本研究使用了10头杜洛克公猪（每头公猪3次射精）的3份混合精液样品。将每份混合精液样本分成两等份。将一份等分试样储存在恒定的17 °C下作为对照组。另一个被包装在密封良好的聚苯乙烯泡沫塑料盒中，并放置在37 °C的培养箱中24小时，以模拟炎热夏季的精液交付，随后转移到17 °C的冰箱中3天。连续监测精液温度。储存0 h时精液温度为17 °C，5 h时达到20 °C，14 h时达到30 °C，24 h时达到37 °C。对于每个时间点，通过CASA、流式细胞术和Western印迹法测定精子质量和功能、凋亡变化、磷酸化AMPK和热休克蛋白HSP 70和HSP 90的表达水平。结果表明，分娩过程中的温度升高显着恶化公猪精子的质量和功能后，14小时的交付。储存回到17 °C没有恢复精子活力。分娩过程中温度升高明显促进了精子早期凋亡向晚期凋亡的转化，Bax和Caspase 3的表达水平显著增加。在递送期间，温度升高至20 °C极大地诱导了磷酸化AMPK的水平，但此后降低。随着温度的升高，HSP70和HSP90的表达量显著增加。我们的研究结果表明，在精液输送过程中温度升高会促进精子凋亡，从而极大地损害精子质量和功能。HSP 70和HSP 90可能通过激活AMPK和抗凋亡过程参与猪精子对温度变化的抵抗。
Boar semen of high quality is a key factor that influences the outcome of artificial insemination. Furthermore, semen delivery practices could exert a negative effect on boar sperm quality and functionality. Nonetheless, the specific effect of temperature changes during semen delivery and molecular responses of sperm to temperature variations have not been fully understood. In this context, the present study aimed to investigate how temperature changes during semen transport influence boar sperm quality and functionality. Our results showed that the temperature of boar semen increased from the initial 17 °C to the ambient temperature of 37 °C after 24 h of delivery using a well-sealed Styrofoam. Sperm quality and functionality were greatly damaged, which could not be recovered when the semen temperature went back to 17 °C. Beyond this fact, boar sperm experienced a significant conversion of early apoptosis to late apoptosis caused by an elevated temperature. During this process, boar sperm adjusted themselves to survive by up-regulating the levels of heat shock proteins and activating the key energy regulator, AMP-activated protein kinase. The findings help to understand how temperature variations influence boar sperm quality and functionality and how the sperm struggles to survive, providing theoretical and technical information to improve the delivery practice of boar semen. Semen delivery practice is crucial to the efficiency of artificial insemination using high-quality boar sperm. The present study aimed to evaluate the effect of a common semen delivery method, a Styrofoam box, under elevated temperatures on boar sperm quality and functionality and to investigate the underlying molecular responses of sperm to the temperature rise. Three pooled semen samples from 10 Duroc boars (3 ejaculates per boar) were used in this study. Each pooled semen sample was divided into two aliquots. One aliquot was stored at a constant 17 °C as the control group. Another one was packaged in a well-sealed Styrofoam box and placed in an incubator at 37 °C for 24 h to simulate semen delivery on hot summer days and subsequently transferred to a refrigerator at 17 °C for 3 days. The semen temperature was continuously monitored. The semen temperature was 17 °C at 0 h of storage and reached 20 °C at 5 h, 30 °C at 14 h, and 37 °C at 24 h. For each time point, sperm quality and functionality, apoptotic changes, expression levels of phosphorylated AMPK, and heat shock proteins HSP70 and HSP90 were determined by CASA, flow cytometry, and Western blotting. The results showed that elevated temperature during delivery significantly deteriorated boar sperm quality and functionality after 14 h of delivery. Storage back to 17 °C did not recover sperm motility. An increased temperature during delivery apparently promoted the conversion of sperm early apoptosis to late apoptosis, showing a significant increase in the expression levels of Bax and Caspase 3. The levels of phosphorylated AMPK were greatly induced by the temperature rise to 20 °C during delivery but reduced thereafter. With the temperature elevation, expression levels of HSP70 and HSP90 were notably increased. Our results indicate that a temperature increase during semen delivery greatly damages sperm quality and functionality by promoting sperm apoptosis. HSP70 and HSP90 could participate in boar sperm resistance to temperature changes by being associated with AMPK activation and anti-apoptotic processes.
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