Temperature Elevation during Semen Delivery Deteriorates Boar Sperm Quality by Promoting Apoptosis

SIMPLE SUMMARY: 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. ABSTRACT: 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.