In vitro culture of porcine embryos to the blastocyst stage after in vivo or in vitro fertilization

THERIOGENOLOGY

IN VITRO CULTURE OF PORCINE EMBRYOS TO THE BLASTOCYST STAGE AFTER IN VIVO OR IN VITRO FERTILIZATION

C. Renz Lorenzo Tortes and D. Rath Institut fiir Tierzucht und Tierverhalten (FAL), Mariensee, 3057 Neustadt 1, Germany Under in vitro conditions the development of porcine embryos is usually blocked at the four cell stage. The objective of this experiment was to overcome the "four cell block" of in vivo or in vitro produced embryos by means of a coculture system and various media. A.) Development of in vivoproduced embryos: Zygotes were recovered from 28 slaughtered prepuberal gilts, which were superovulated (PMSG-1500 i.U./hCG-500 i.U.) and mated twice (24h to 48h) after hCG injections. Zygotes (347) were either cocultured in vitro in the presence of oviduetal epithelial cells (monolayer) collected at different stages ( pre-estrus, estrus and post-estrus) of the cycle, or were cultured in modified Whitten's medium, modified KRB (Beckmann and Day, Therio. 35: 184, 1991) or medium NCSU 23 (Petters and Reed, Therio. 35: 253, 1991). At day 2 of the in vitro culture 315 embryos reached the 2 (n= 116) - 4 ( n= 199) cell stage. The table summerizes the results of further development until day 6. Co-culture Whitten's with epithelial cells medium*

stage of develop. at day 2 n

% expanded blastocysts at day 6

2cell 4cell 36

113

0a

0a

NCSU 23

2cell

4cell

31

37

28.8b 29.7b

KRB*

2cell 4cell 2cell 4cell 34

37

15

12

91.2c 75.7c 73.3c 33.3b * modified a:b,c; c:d p<0.01

B.) Development of in vitroproduced embryos: After in vitro fertilization of in vivo matured oocytes recovered from superovulated prepuberal gilts, 177 embryos were cultured either in TCM 199 or in NCSU 23 for two days until they reached the 2-4 ceil stage (39°C, 5% CO~, max. humidity) and then cultured in the presence of NCSU 23 medium for 5 dayL Only 26.3 % of the embryos previously cultured in TCM 199 and 59.7% ( X2 p < 0.01) of the embryos continuously cultured in medium NCSU 23 overcame the four cell block. At day 6/7 out of 65 embryos 10% developed to hatched blastocysts after culture in TCM 199 and 40.3% of the embryos hatched after culture in medium NCSU 23 ( p< 0.01). These results clearly demonstrate the ability of medium NCSU 23 to support the development of more advanced embryonic stages derived from both in vitro and in vivo fertilization. Coculture with oviduct epithelial cells did not support development sufficiently. Four-cell embryos seem to have less potency for further development under in vitro conditions than two-cell embryos. This difference is significant when the embryos are cultured in modified KRB. Embryos produced by in vitro fertilization have a reduced developmental capacity.

J A N U A R Y 1992 VOL. 37 NO. 1

283