In vitro Fertilization and Development of Pig Oocytes Inseminated with Boar Sperm by Different Sperm Washing Media after Thawing of the Frozen Straws

Y J Yi,H K Kim,C S Park,C B Yang,H J Ko,S H Lee,D S Son

doi:10.5713/ajas.2004.164

Abstract

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing 500 µl mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to 24°C over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were resuspended in a lactose-egg yolk and N-acetyl-D- glucosamine (LEN) diluent to contain 1×10 9 sperm/ml and cooled to 5°C over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with 2×10 7 sperm concentration. Oocytes were coincubated for 6 h in 500 µl mTBM fertilization. At 6 h after IVF, oocytes were transferred into 500 µl NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 2 : 164-167)

Highlights

We found out that frozen-thawed boar sperm, even with a low motility, contributed to the penetration rates of oocytes
Polyspermic penetration has been the persistent problem in IVM-IVF systems (Funahashi and Day, 1997)
This study was the first to compare the in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws

Summary

Introduction

Potential explanations may be due to boar effects, to the purity of the sperm rich fraction, and to different semen treatment protocols for IVF. This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the same boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Since Mattioli et al (1989) succeeded in getting piglets from IVM/IVF oocytes in pigs, in vitro production of pig embryos have been developed gradually. Efficient production of pig embryos through IVM/IVF techniques has been hampered by the high incidence of polyspermy and a low incidence of male pronuclear formation. Deep frozen boar sperm had poorer motility, acrosomal morphology and viability than fresh sperm (Courtens and Paquignon, 1985; Weitze et al, 1986; Clarke and Johnson, 1987; Almlid and Johnson, 1988; Almlid et al, 1989; Hofmo and Almlid, 1991), and the accompanying poor farrowing rates (40-50%) and low litter size have made frozen boar semen impractical for the commercial swine producer (Johnson, 1985; Almlid et al, 1987; Hofmo and Almlid, 1991; Crabo and Dial, 1992)

Methods

Results

Conclusion