Cryopreservation of Boar Spermatozoa: the Curious Functions of Seminalplasma during Freezing or Thawing

There are various advantages, such as the preservation of genetic resources, an improvement of the reproduction performance in summer, long-distance transportation of semen and the possibility of progeny test, in the cryopreservation technique of boar semen. However, cryopreserved boar spermatozoa have not been routinely available to swine artificial insemination (AI) due to the lower conception and farrowing rates than those of fresh semen. To improve the reproductive performance after AI, many researchers have approached technical modification of temperature program during freezing, freezing extender, thawing solution and AI method (e.g. deep intra uterine insemination). In this study, we focused and analyzed on the role of the seminal plasma of ejaculated semen, and succeed to develop novel freezing and thawing method as following. In the freezing process from semen collection: 1. Ejaculated seminal plasma is immediately removed by centrifugation after semen collection because the fraction contains negative factors, such as bacteria or lipopolysaccharide (LPS). 2. The sperm pellet is resuspended in a semen extender that contains both common antibiotics and polymyxin B (an inactivator of LPS). 3. Hyperosmotic pressure (400 mOsm/ kg) and low concentration glycerol (2%) solution are used for freezing extender (conventional extender; 300 mOsm/ kg, 3% glycerol). Next step, in thawing process, the frozen straws picked out from the liquid nitrogen tank are melted in 38oC for 1 min, and the content injects into 10% (v/v) seminal plasma-containing thawing solution. When the frozen-thawed spermatozoa treated with seminal plasma were artificially inseminated to sows, the reproductive performance (conception rate; 80.2% and litter size; 10.1 piglets) was comparable to that of liquid semen (73.9%, 10.3 piglets). These results indicated that seminal plasma has positive roles on the AI using cryopreserved boar semen. However, because boar seminal plasma is contaminated with various kinds of bacteria and/or viruses, the development of thawing solution without animal derived materials is required for more sanitary AI. Cryo-capacitation just after thawing which arises from increasing of intercellular Ca2+ ([Ca2+]i) in sperm, decreases the motility and fertilization ability of frozen-thawed sperm. To suppress this induction, EGTA was added to diluent to chelate the transient increment of the ion. The addition of EGTA significantly suppressed both the increase [Ca2+]i level and the induction of cryo-capacitation. To yield a high pregnancy rate, not only improvement of sperm function mentioned above but remodeling of the uterine environment after AI is also a key step. We found that cortisol, which is a known immunosuppressive agent, existed in boar seminal plasma and regulated intrauterine environment to enhance implantation in sows. In this symposium, we will also introduce the reproductive performances obtained by AI using the novel diluent.