Use of Antifreeze Protein from Tenebrio molitor (TmAFP) in Vitrification of In Vitro -Produced Bovine Embryos: An Ultrastructural Study.

The objective of this study was to evaluate the effects of different concentrations of antifreeze protein (AFP) extracted from the larva of the beetle, Tenebrio molitor ( Tm AFP), on vitrification of in vitro -produced bovine embryos. In vitro -produced blastocysts were divided into three experimental groups and vitrified using a cryotop. Tm AFP was added to the equilibrium solution (ES) and vitrification solution (VS) at a concentration of 0 ng/mL (control), 500 ng/mL (500 Tm AFP), or 1000 ng/mL (1000 Tm AFP). Vitrification was carried out by first placing the blastocysts in ES for 2 minutes (7.5% ethylene glycol [EG] and 7.5% dimethyl sulfoxide [DMSO]). The blastocysts were then transferred to VS (15% EG and 15% DMSO) and promptly deposited on a cryotop stem and submerged in liquid nitrogen. Warming was carried out in three steps with decreasing sucrose concentrations. After warming, the blast cells were cultured for 24 hours for subsequent survival analysis and ultrastructural evaluation. There was a significant difference in the survival rate and expansion in the 500 Tm AFP group compared with the other groups. The ultrastructural analysis revealed intracellular lesions in all vitrified embryos; however, the embryos of the 500 Tm AFP and 1000 Tm AFP groups showed fewer cytoplasmic lesions compared with the control group. Taken together, addition of Tm AFP can mitigate cellular changes that involve organelles and cellular components essential for proper functioning and improve the viability of warmed and vitrified in vitro -produced bovine embryos.