Abstract

Heat shock proteins (HSPs) are highly represented among oviductal epithelial cell (OEC) plasma membrane proteins (Sostaric et al. 2006 J. Proteome Res. 5, 3029–3027); their abundance increases in oviductal fluid in response to the presence of spermatozoa (Georgiou et al. 2005 Mol. Cell. Proteomics 4, 1785–1796), and they are capable of binding directly to spermatozoa (Boilard et al. 2004 Biol. Reprod. 71, 1879–1889). As a result of these observations, a role for HSPs in prolonging the functional life of spermatozoa in the oviduct prior to fertilization has been proposed. Poor fertility rates are often observed when fresh ram semen stored in conventional extenders is used for cervical AI (Salamon and Maxwell 2000 Anim. Reprod. Sci. 62, 77–111). This is due, in part, to extenders being unable to maintain the functional life of ram spermatozoa to the same extent as the oviduct. Therefore, in this study, we aimed to determine the effect of supplementing extenders with the HSPs, HSC70 and HSP70, on the maintenance of ram sperm function during storage. Eight freshly collected ram semen ejaculates were split and diluted in the extenders, INRA96 and RSD-1, to 25 × 10⁶ spermatozoa mL^–1, either alone (control) or supplemented with HSC70, HSP70, or α-tubulin (protein with a molecular weight similar to that of the two HSPs). The final concentration of protein in each case was 4 µg mL^–1, and the samples were stored at 17°C for 48 h. At 1 h, 6 h, 24 h, and 48 h, the viability and DNA integrity of the stored spermatozoa was determined using the live and dead stains, SYBR^®-14 and ethidium homodimer, and the Sperm-Ovis-Halomax kit, respectively. In each case, the mean percentage (%) of viable and DNA-fragmented (DF) spermatozoa was determined and log-transformed prior to analysis using factorial ANOVA. Statistical significance was defined as P < 0.05. In INRA96, but not RSD-1, the beneficial effect of protein supplementation on the % of viable spermatozoa observed during storage was significant (P < 0.001). Most noteworthy was INRA96 supplemented with HSC70, which maintained the % of viable spermatozoa observed during storage significantly (P < 0.001) better than INRA96 alone. Protein supplementation had no significant effect on the % of DF spermatozoa observed during storage. However, the % of DF spermatozoa observed increased significantly (P < 0.001) during storage, irrespective of the presence or absence of protein. On the basis of these findings, it is not implausible to suggest that supplementing the commercially available INRA96 extender with HSC70 would improve the fertility rates observed following cervical AI using stored ram semen. Furthermore, the increase in the incidence of DNA-fragmented spermatozoa observed during storage offers a potential explanation as to why poor fertility rates are often observed when ram semen stored in conventional extenders is used for cervical AI.