Frozen-thawed epididymal spermatozoa have good fertilization capability in vitro; however, their artificial insemination conception rate is less than half of that of frozen-thawed ejaculated spermatozoa. Because the addition of seminal plasma to the thawing solution enhances the in vivo fertilizing ability of frozen-thawed ejaculated spermatozoa, we hypothesized that the reproductive performance of frozen-thawed epididymal spermatozoa could also be improved by the inclusion of seminal plasma. When frozenthawed epididymal spermatozoa were incubated for up to 6 hours, the motility of the sperm significantly decreased in a time-dependent manner. The acrosomal membrane was damaged in the majority of frozen-thawed epididymal spermatozoa. The addition of seminal plasma to the thawing solution significantly decreased the percentage of sperm with abnormal acrosomes and increased their total motility in a dose-dependent manner. Furthermore, the addition of seminal plasma reduced the abundance of a 15-kDa tyrosinephosphorylated protein in frozen-thawed sperm, and the maximum effect was observed at 15% (vol/vol) seminal plasma. When cryopreserved epididymal spermatozoa from 3 different boars were thawed with a 15% (vol/vol) seminal plasma-containing solution, the conception rate and mean litter size obtained by artificial insemination were significantly increased as compared with those in the control without seminal plasma. From these results, we concluded that the addition of seminal plasma to the thawing solution is a key step in obtaining an optimal number of piglets by artificial insemination using frozen-thawed boar epididymal spermatozoa.
Oxytocin (OXT) contained in boar semen is known to produce uterine contraction; therefore, we hypothesized that the co-injection of OXT with sperm would improve artificial insemination (AI) using liquid or frozen-thawed boar sperm. We initially examined whether OXT added to semen extender improved sperm transport to the oviduct. Although the addition of OXT did not affect the fresh or frozen-thawed sperm motility or acrosomal integrity, it significantly increased the number of sperm in the oviduct at 6 h after AI injection with OXT, as compared with the control (P < 0.05). Moreover, some sperm were observed in the sperm reservoir of the isthmus in the OXT treatment group, whereas few sperm were observed in the control. When OXT was added to the semen extender immediately prior to AI, the conception rates were significantly higher in both fresh semen and frozen-thawed semen than in the control group (P < 0.05: liquid, 87.5% vs. 70.5%; frozen-thawed, 89.8% vs. 75.0%). From these results, we concluded that the addition of OXT to the semen extender assisted in sperm transportation from the uterus to the oviduct, which resulted in improved reproductive performance.
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).
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