60 THE EFFECT OF l-ASCORBIC ACID DURING CULTURE, CRYOPRESERVATION, OR BOTH ON PORCINE EMBRYOS PRODUCED IN VITRO

Abstract

The benefits of adding l-ascorbic acid during the cryopreservation procedure have been reported before in mouse and bovine. In this study, the effects of l-ascorbic acid (AC) supplementation during culture, cryopreservation, or both procedures on the developmental ability and embryo quality of in vitro produced porcine blastocysts were examined. Embryo quality criteria consisted of total cell number, percentage of apoptosis, and cryotolerance. After in vitro fertilisation, presumptive zygotes were randomly assigned to 2 culture treatments in which the culture medium NCSU23 was supplemented with 100 µM AC (n = 1162) or nonsupplemented (n = 1163) for a 144-h period. On Day 6, blastocyst formation was assessed by stereomicroscopy, and a representative fraction of Grade I- and II-blastocysts of each culture treatment was evaluated using 4′,6-diamidino-2-phenylindole-TUNEL co-staining and considered as fresh-control. The remaining fraction of Grade I- and II-blastocysts was vitrified/warmed following the Cryotop^® method. To determine the effect of AC supplementation during cryopreservation procedures, each culture treatment was divided into 2 groups: (1) embryos exposed to 100 µM AC, and (2) nonexposed embryos (vitrified-control). Survival was determined according to reexpansion rates after 24 h of recovery in NCSU23 medium. After 24 h, reexpanded blastocysts were co-stained using the 4′,6-diamidino-2-phenylindole-TUNEL technique, and total number of cells and apoptosis indexes were determined. Experiment was replicated 9 times for each group. Data were analyzed by t-test for independent variables and a 2-way ANOVA. Results are expressed as means ± SE, and the significant level was set at 5% (Table 1). After culture, supplementing NCSU23 medium with AC showed no significant differences in blastocyst formation (fresh-control 11.6 ± 7.8 v. AC 11.6 ± 7.7), in number of cells (fresh-control 36.7 ± 15.8 v. AC 36.1 ± 15.9), or in apoptosis index (fresh-control 2.9 ± 5.7 v. AC 3.5 ± 4.7). On the other hand, only when both culture and vitrified media were supplemented with AC was there a significant increase of blastocyst survival. In contrast, no significant differences in embryo survival were observed when only 1 of these 2 media (culture or vitrification) was supplemented. Supplementing culture media or cryopreservation solutions with AC did not affect the total cell number or apoptosis index in vitrified blastocysts. In conclusion, the addition of 100 µM l-ascorbic acid to the culture and cryopreservation solutions improves the cryotolerance of in vitro-produced porcine blastocysts.

Table 1.  Survival of blastocysts (24 h), total cell number, and percentage of apoptosis after vitrification/warming