69 QUALITY OF IN VITRO PRODUCED AND CRYOPRESERVED PORCINE EMBRYOS ASSESSED BY CELL NUMBER, TUNEL-POSITIVE NUCLEI, AND CASPASE-3 ACTIVITY

Abstract

The aim of this experiment was to investigate the quality of in vitro cultured and cryopreserved porcine expanded blastocysts from Day 5, 6, and 7 of culture. The quality of the preimplantation embryos was determined by counting the number of cells, observing a TUNEL-positive reaction (TUNEL reagent; In Situ Cell Detection kit, Roche Diagnostics, Germany) and by caspase-3 labelling (PhiPhiLuxG₂D₂ Kit, Calbiochem, Germany). Embryos were collected from 32 superovulated donor gilts. All were crossbreds of Polish Landrace and Large White, age 6–8 months, weighing 90–100 kg. The experiment was done on 2–4 cell embryos produced in vivo and cultured in vitro for 7 days in NCSU-23 medium until expanding blastocyst stage. The embryos of this stage were obtained on Day 5, 6, and 7 of in vitro culture and divided into two groups: control-(1) 210 nonvitrified (NV) embryos and -(2) vitrified/thawed (VT) 169 embryos. The expanded blastocysts were vitrified the open pulled straw (OPS) method (Vajta 2000 Anim. Reprod. Sci. 60–61, 357–364). The results were analyzed by Student’s t-test, and all values were significant at P ≤ 0.05. The NV group of embryos showed significant differences in the number of cells (66.5 ± 24.0 v. 54.8 ± 15.9) and in TUNEL-positive nuclei (8.8 ± 12.5 v. 16.2 ± 14.9) between Day 5 and Day 7 of culture, respectively. Analysis of VT embryos also revealed significant differences in the number of cells (65.2 ± 17.4 v. 55.5 ± 14.3) and in TUNEL-positive nuclei (25.5 ± 16.4 v. 35.8 ± 19.3) between Day 5 and Day 7 of culture, respectively. Lower percentage of NT and VT blastocysts produced on Day 5 of culture revealed caspase-3 activity (51.3 v. 64.8%) compared with embryos on Day 7 (76.8 v. 89.3%), respectively. In conclusion, blastocysts cultured in vitro for 5 days consist of a high number of nuclei, have a low incidence of TUNEL-positive nuclei, and low caspase-3 activity compared with blastocysts cultured for 6 and 7 days in all analysed groups. Our results revealed that expanding blastocysts produced on Day 5 of in vitro culture had higher ability to survive vitrification/thawing procedure.