70 Effect of cryoprotectant concentration on the in vitro survival and cell proliferation of porcine blastocysts vitrified using the open pulled straw system

Abstract

The objective of the present project was to study the effect of the concentrations of ethylene glycol (EG) and dimethyl sulfoxide (DMSO) during vitrification on the survival and hatching rates of porcine blastocysts. Embryos were collected by laparotomy from weaned crossbred sows (n = 18), vitrified, and warmed (one-step dilution) as described by Cuello et al. (2004 Theriogenology 62, 1144-1152). Vitrification was performed in different concentrations of EG and DMSO (15%, 16%, and 17% v/v for each cryoprotectant) or in an EG-based medium (40% v/v) using superfine open pulled straws. Fresh and vitrified blastocysts were cultured for 24 h in TCM199 and assessed by stereomicroscopy during the culture. Blastocysts that reformed their blastocoelic cavities after warming, displaying a normal zona pellucida and excellent appearance, were considered viable. The in vitro survival rate was defined as the ratio of viable embryos divided by the total number of embryos cultured. The hatching rate is determined as the ratio of the number of embryos hatched in vitro to the total number embryos cultured. Some vitrified and fresh embryos classified as viable were processed for Hoechst 33342 staining and proliferation cell nuclear antigen (PCNA) inmunolocalization. The proliferation index was defined as the number of PCNA-positive nuclei divided by the total number of nuclei stained with Hoechst 33342. Data were analyzed by ANOVA using the MIXED procedure (SPSS version 11.5; SPSS, Inc., Chicago, IL, USA). Data were expressed as mean values � SEM. The survival rate was similar for fresh and vitrified blastocysts, except for blastocysts vitrified using 15% cryoprotectants, which displayed a lower (P < 0.05) survival rate (84.2 � 4.8%) than fresh blastocysts (94.6 � 5%) and blastocysts vitrified using 40% EG, 16%, or 17% EG-DMSO (88.8 � 4.9, 96.8 � 4.9, and 96.4 � 5%, respectively). Fresh blastocysts showed a higher (P < 0.05) hatching rate (80.7 � 4.5%) than their vitrified counterparts (range: 48.4 � 7.7-55.3 � 7.8%). Vitrified and fresh blastocysts showed similar cell proliferation indexes (range: 75.8 � 3.2-83.7 � 3). When only hatched blastocysts among groups were compared, the proliferation rate decreased (P < 0.05) after vitrification with 17% EG-DMSO. Among vitrification groups, there was no significant difference in the number of total cells. However, vitrified blastocysts had a lower (P < 0.05) total cell number (range: 116.6 � 6.7-124.8 � 6.6) than fresh blastocysts (195.5 � 11.4). In conclusion, under our experimental conditions, the concentration of EG-DMSO can be decreased until 16% in the vitrification medium with no reduction of the in vitro developmental ability of the blastocysts. In addition, a 40% EG-based medium can be used for vitrification with results similar to those achieved using a medium containing 16% EG-DMSO.