V. G. Pursel

Two hours after semen collection, aliquots containing 6×109 spermatozoa were centrifuged for 10 min at 300 g. The seminal plasma was removed and the spermatozoa were resuspended to 5 ml with Beltsville F5 (BF5) extender. The semen was cooled gradually to 5 C over a 2-hr period, 5 ml of BF5 containing 2% glycerol was added and the semen was mixed and frozen immediately into pellets of 0.15 to 0.2 ml on Dry Ice. The pellets were transferred to liquid nitrogen for storage. For each insemination 10 ml of pellets were removed from liquid nitrogen, dumped into an empty styrofoam shipping container and held for 3 minutes. The pellets were then dumped into a 250-ml beaker containing 25 ml of Beltsville thawing solution (BTS) at 50 C and were swirled in the beaker until thawed. The thawed semen was poured into an insemination bottle, the beaker was rinsed with 15 ml BTS and the rinse solution was added to the insemination bottle.

Genetic engineering of livestock is expected to have a major effect on the agricultural industry. However, accurate assessment of the consequences of transgene expression is impossible without multigenerational studies. A systematic study of the beneficial and adverse consequences of long-term elevations in the plasma levels of bovine growth hormone (bGH) was conducted on two lines of transgenic pigs. Two successive generations of pigs expressing the bGH gene showed significant improvements in both daily weight gain and feed efficiency and exhibited changes in carcass composition that included a marked reduction in subcutaneous fat. However, long-term elevation of bGH was generally detrimental to health: the pigs had a high incidence of gastric ulcers, arthritis, cardiomegaly, dermatitis, and renal disease. The ability to produce pigs exhibiting only the beneficial, growth-promoting effects of growth hormone by a transgenic approach may require better control of transgene expression, a different genetic background, or a modified husbandry regimen.

The present study examined the kinetics of glutathione (GSH) concentration during maturation and after fertilization in pig oocytes and its relevance to the ability of pig oocytes to form a male pronucleus after in vitro fertilization. The GSH concentration was significantly higher in pig oocytes matured in Waymouth medium than in pig oocytes matured in either modified (m) TCM-199 or mTLP media. The addition of 0.04-0.57 mM cysteine (CySH) to mTLP significantly increased both the GSH concentrations in oocytes matured in vitro and the rate of male pronucleus formation as compared to those in oocytes cultured in mTLP alone. When pig oocytes were cultured 12, 24, or 36 h in mTLP plus 0.14 mM CySH, their GSH concentrations were significantly higher than in uncultured oocytes. After fertilization, the GSH concentration in pig oocytes declined significantly. GSH concentrations in oocytes matured in vivo did not differ from those in oocytes matured in mTLP plus 0.14 or 0.57 mM CySH. The results indicate that 1) the composition of maturation medium affects the GSH concentration in pig oocytes; 2) the addition of CySH to maturation medium permits GSH synthesis by the pig oocytes; 3) GSH levels in pig oocytes change during maturation and after fertilization; and 4) GSH synthesis during oocyte maturation is an important factor for promoting their ability to form a male pronucleus after fertilization.