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

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 1×109 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 2×107 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.