82 CRYOPRESERVATION OF BOAR EPIDIDYMAL SPERMATOZOA; ADDITION OF SEMINAL PLASMA TO THAWING SOLUTION IMPROVES REPRODUCTIVE PERFORMANCE BY ARTIFICIAL INSEMINATION

Epididymal spermatozoa are one of the available male germ cells for cryopreservation. It has been reported that frozen–thawed porcine epididymal spermatozoa have a high fertilization competence in vitro as compared with that in ejaculated one. However, there is little information about reproductive performance, such as conception rate or litter size, after artificial insemination (AI) using frozen–thawed epididymal spermatozoa. Recently, we demonstrated that the addition of seminal plasma to thawing solution improves membrane and acrosomal integrity, and enhanced both in vivo and in vitro fertilizing activity of frozen–thawed ejaculated spermatozoa. Moreover, the injection of seminal plasma to uterus with frozen–thawed spermatozoa significantly increased the number of implantation site (Okazaki et al. 2009 Theriogenology 71, 491–498). Thus, to apply those positive functions of seminal plasma to AI using frozen–thawed epididymal sperm, in this study, we added seminal plasma to thawing solution and then analysed the sperm functions including AI test using frozen–thawed epididymal spermatozoa. Epididymal spermatozoa collected by flushing caudal epididymis were frozen as described in our previous study (Okazaki et al. 2009). Frozen-spermatozoa were thawed in Modena solution with or without different percentages of seminal plasma. Protein tyrosine phosphorylation as a marker of capacitation was detected by western blotting. To examine the reproductive performance, the sows of natural oestrus were artificially inseminated two times (5 × 109 50 mL–1 per injection). When the frozen–thawed ejaculated or epididymal sperm was incubated up to 6 h, the motility of epididymal sperm was significantly higher than that of ejaculated sperm (19.6 v. 37.6%). However, the acrosomal membrane was damaged in epididymal sperm group at 3-h incubation period (15.2 v. 36.0%). The addition of seminal plasma [0, 10, 15, 20% (v/v)] in Modena solution protected the acrosomal injury (3 h; 35.2, 19.5, 15.6, 14.6%) and maintained high rate of motility (6 h; 38.8, 48.8, 62.5, 60.0%) in a dose-dependent manner. Furthermore, the addition of seminal plasma suppressed the expression of the 15 kDa phosphoprotein (early capacitation status), and the maximum effect was detected at 15% (v/v) seminal plasma. When the frozen–thawed epididymal spermatozoa with 15% (v/v) seminal plasma were artificially inseminated to swine (n = 15), the conception rate and the mean number of litter size were increased as compared with control (93 v. 43%, 10.0 v. 5.0). From these results, we concluded that the addition of seminal plasma to thawing solution was a beneficial method for artificial insemination using frozen–thawed epididymal spermatozoa in the pig. This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, and JST-Grant (No. 12-068 and No. 12-104).