Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification.
The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition-CONTROL; after 60 h-PES Day 2.5 of embryo culture; and after 96 h-PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P < 0.05), increased apoptosis in fresh (2.5%: 19.1 ± 1.8 vs 10%: 28.4 ± 2.3, P < 0.05; mean ± SEM) and vitrified (2.5%: 42.8 ± 2.7 vs 10%: 69.2 ± 3.4, P < 0.05) blastocysts, and reduced blastocoele re-expansion after vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P < 0.05). The addition of PES in culture media, either from Days 2.5 or 4, reduced lipid accumulation (P < 0.05) and increased blastocoele re-expansion after vitrification (


METHODS
72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P < 0.05). However, just the use of PES from D4 reduced apoptosis in vitrified blastocysts (


METHODS
52.0 ± 3.0 vs PES Day 4: 39.2 ± 2.4, P < 0.05). Independent of FCS withdrawal or PES addition to culture media, the in vivo control group had lesser lipid accumulation, a lower apoptosis rate, and greater cryotolerance (P < 0.05). The increased lipid content was moderately correlated with apoptosis in vitrified blastocysts (r = 0.64, P = 0.01). In contrast, the increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts (r = 0.94, P < 0.0001). Therefore, using only 2.5% FCS and the addition of PES from Day 4, increased the survival of IVP embryos after vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification.
São Paulo State University, UNESP, School of Veterinary Medicine and Animal Science, FMVZ, Department of Animal Reproduction and Veterinary Radiology, Botucatu, SP, Brazil.
Mateus José
MJ
Sudano
Daniela Martins
DM
Paschoal
Tatiana da Silva
Tda S
Rascado
Luis Carlos Oña
LC
Magalhães
Letícia Ferrari
LF
Crocomo
João Ferreira
JF
de Lima-Neto
Fernanda da Cruz
Fda C
Landim-Alvarenga
D000465:Algorithms; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002417:Cattle / Q000196:embryology* / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian; D005260:Female; D005307:Fertilization in Vitro; D005615:Freezing / Q000009:adverse effects; D050356:Lipid Metabolism / Q000502:physiology; D008055:Lipids / Q000032:analysis*; D008297:Male; D058989:Vitrification
D008055:Lipids