Morphokinetic changes and apoptotic cell death in vitrified and non-vitrified in vitro-produced ovine embryos

Karolina Fryc; Agnieszka Nowak; Barbara Kij-Mitka; Joanna Kochan; Pawel M Bartlewski; Maciej Murawski

doi:10.1016/j.repbio.2023.100750

Morphokinetic changes and apoptotic cell death in vitrified and non-vitrified in vitro-produced ovine embryos

Reprod Biol. 2023 Jun;23(2):100750. doi: 10.1016/j.repbio.2023.100750. Epub 2023 Feb 27.

Authors

Karolina Fryc¹, Agnieszka Nowak², Barbara Kij-Mitka², Joanna Kochan², Pawel M Bartlewski³, Maciej Murawski⁴

Affiliations

¹ Department of Animal Nutrition and Biotechnology, and Fisheries, University of Agriculture in Kraków, 24/28 Mickiewicza Ave., 30-059 Cracow, Poland. Electronic address: karolina.fryc@student.urk.edu.pl.
² Department of Animal Reproduction, Anatomy and Genomics, University of Agriculture in Kraków, 24/28 Mickiewicza Ave., 30-059 Cracow, Poland.
³ Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, 50 Stone Rd., Guelph, ON N1G 2W1, Canada.
⁴ Department of Animal Nutrition and Biotechnology, and Fisheries, University of Agriculture in Kraków, 24/28 Mickiewicza Ave., 30-059 Cracow, Poland.

PMID: 36857927
DOI: 10.1016/j.repbio.2023.100750

Abstract

This article addresses morphokinetic changes and the extent of apoptosis in vitrified and non-vitrified in vitro-derived ovine blastocysts. Cumulus-oocyte complexes were collected after ovarian scarification obtain after slaughter and in vitro maturation was performed in TCM 199 medium supplemented with Earle's Salt, 10 % of FBS, and 5 µg/mL of LH/FSH at 38 °C for 24 h. After maturation, the oocytes were co-incubated with thawed ram semen (IVF) for 19 h.Embryo development was monitored with the aid of the Primo Vision Time-Lapse (TL) system. Twenty-five out of thirty-one ovine blastocysts that were vitrified using the Cryotop system at the early blastulation stage of development subsequently re-expanded. Both the vitrified (n = 25) and non-vitrified (control group: n = 28) blastocysts were examined for detection of apoptosis (TUNEL assay) and total blastomere counts at the time they attained the expanded blastocyst stage. Blastocyst formation occurred earlier in non-vitrified than in vitrified ovine embryos (147:49 ± 20:23 compared with 156:46 ± 19:24; hours:minutes post-insemination; mean ± SD; P < 0.05). The average number of blastocyst collapses was greater (2.45 ± 1.64 compared with 1.45 ± 1.64), but the number of weak contractions was less for vitrified than non-vitrified ovine blastocysts (P < 0.05). The mean number of blastomeres was greater (131.8 ± 38.6 compared with 91.5 ± 18.3; P < 0.05) while the number of TUNEL-positive cells (4.4 ± 1.6 compared with 6.3 ± 2.3) and apoptotic index (3.4 ± 1.2 % compared with 6.9 ± 2.6 %) were less (P < 0.05) in non-vitrified compared with vitrified blastocysts. Vitrification of ovine embryos was associated with a delayed blastocyst formation, greater numbers of apoptotic cells, significant reduction in the number of blastomeres, and higher/lower incidence of blastocyst collapse/weak contractions.

Keywords: Blastocyst contractions; Embryo; Sheep; TUNEL; Time-lapse imaging; Vitrification.

MeSH terms

Animals
Apoptosis
Blastocyst
Cryopreservation* / veterinary
Male
Sheep
Sheep, Domestic
Vitrification*