113 RETINOIC ACID DOES NOT PROTECT AGAINST THE SELECTIVE DAMAGE TO THE BOVINE INNER CELL MASS INFLICTED BY VITRIFICATION

Abstract

Successful cryopreservation of in vitro-produced embryos is a major objective in reproductive biotechnology. It was reported that in vitro culture with high BSA concentrations improved bovine embryo survival after vitrification (Díez et al. 2005 Reprod. Dom. Anim. 40, 384). All-trans retinoic acid (ATRA) increases cell numbers in the inner cell mass (ICM) and the trophectoderm (TE) (Rodríguez et al. 2006 Hum. Reprod. 21, 2149–2157). This work analyzed the effect of ATRA on bovine embryo development, survival to vitrification, and cell allocation before and after cryopreservation. Bovine cumulus–oocyte complexes were matured and fertilized in vitro, and presumptive zygotes cultured in SOF + 20 g L^-1 BSA. At 139 h post-insemination (Day 6), a total of 917 morulae + early blastocysts were cultured for 24 h with: (1) 1.4 µM ATRA, (2) 0.7 µM ATRA, and (3) no ATRA (control). Embryos were subsequently cultured up to Day 9 in SOF + 20 g L^-1 BSA. Development was recorded and differential cell counting was performed on Day 8 and 9 hatched blastocysts. Simultaneously, Day 7 and 8 expanded blastocysts were vitrified (OPS; Vajta 2000 Anim. Reprod. Sci. 60–61, 357–364). After warming, blastocysts were cultured for 72 h in B2 + 5% FCS with Vero cells, and cell counts were performed in fully expanded or hatched blastocysts. Data (7 replicates for cell counts before and 4 after vitrification) were processed by GLM and Duncan's test, and were expressed as LSM ± SE (^x,y: P = 0.01; ^a,b: P < 0.05; ^α,β: P < 0.002). Developmental rates did not differ among groups. Blastocysts cultured in 0.7 µM ATRA survived vitrification at rates similar to those of controls, and only hatching rates 24 h post-warming were significantly lower than those of controls (4.0 ± 8.2^a vs. 31.2 ± 8.2^b). ATRA at 1.4 µM was detrimental to survival of Day 7 embryos, whereas differences were not detected in Day 8 blastocysts. In all groups, the vitrification procedure significantly reduced the cells of the ICM (1.4 µM ATRA: 28.3 ± 3.1^α vs. 8.6 ± 4.1^β; 0.7 µM ATRA: 27.7 ± 3.5^α vs. 2.2 ± 4.1^β; Control: 31.3 ± 3.1^α vs. 7.0 ± 5.1^β). Total cell counts were: 1.4 µM ATRA: 160.0 ± 9.8^a vs. 130.0 ± 12.2^b; 0.7 µM ATRA: 165.3 ± 8.8^a vs. 123.2 ± 11.7^b; Control: 161.2 ± 9.2^a vs. 131.0 ± 15.1^b. The ratios of ICM/TE cells were: 1.4 µM ATRA: 16.9 ± 2.7^x vs. 6.1 ± 3.2^y; 0.7 µM ATRA: 17.2 ± 2.3^x vs. 2.0 ± 3.0^y; Control: 20.6 ± 2.4^x vs. 4.3 ± 3.9^y. All values are before and after vitrification, respectively. When considered together, the differences in the cell counts before and after vitrification were highly significant (*P < 0.0001): 1.4 µM ATRA: 29.2 ± 1.9* vs. 5.9 ± 2.6; 0.7 µM ATRA: 162.5 ± 5.5* vs. 127.2 ± 7.6; Control: 18.3 ± 1.5* vs. 4.2 ± 2.0. Our results show that ATRA did not improve the embryo survival to vitrification. Although 1.4 µM ATRA was used to avoid a 'binding effect' related to an elevated protein level (Klaassen et al. 1999 Biochim. Biophys. Acta 1427, 265–275), the BSA concentrations used in culture could mask any ATRA effect. The vitrification procedure used in this study produced a selective damage within the ICM cells, which can explain the reduced survival rates obtained after warming.

This work was supported by Grant AGL2005-04479.