73 Cytokine addition does not increase developmental competence of in vitro-produced bovine embryos
C. M. Helland A , M. Barcelo-Fimbres B and L. F. Campos-Chillon AA California Polytechnic State University, San Luis Obispo, CA, USA;
B Sierra Nevada Reproduction, Galt, CA, USA
Reproduction, Fertility and Development 32(2) 162-163 https://doi.org/10.1071/RDv32n2Ab73
Published: 2 December 2019
Abstract
Recently in porcine, the addition of 3 cytokines (FGF2, LIF, and IGF1) improved oocyte maturation, quadrupling the number of piglets born per oocyte collected (Yuan et al. 2017 Proc. Natl. Acad. Sci. USA 114, E5796-E5804). We hypothesised that in the bovine, addition of these cytokines to maturation (MCyt) and culture media (CCyt) would lower lipid content and increase mitochondrial activity, representing an improved developmental competence when compared with standard maturation (MCon) and culture (CCon) conditions. The experimental design was a 2 (MCon and MCyt) × 2 (CCon and CCyt) factorial in 8 replicates, testing the interactions of each maturation medium with each culture medium. In vitro-produced embryos produced aspirating cumulus-oocyte complexes (COCs) from 2 to 8 mm follicles of abattoir ovaries. The COCs (n = 2156) were matured for 23 h in MCon or MCyt media at 6% CO2 in air, fertilised with semen from one of two bulls, and cultured in CCon or CCyt media at 38.5°C, 6% CO2, 5% O2, and 89% N2. On Day 7.5 post-fertilisation, blastocyst rates were evaluated and embryos (n = 4/replicate/group) were stained with 1 µg mL−1 Nile Red for lipid quantification or 300 nM MitoTracker Red CMX-Rosamine for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes for Health) adjusted per cell number. Data were analysed by GLM using ANOVA and l.s.d. with SAS (SAS Institute Inc.). Results (Table 1) indicated similar cleavage and blastocyst rates between all groups (P > 0.05). The combination of MCon × CCon resulted in higher mitochondrial activity than any other combination (P < 0.05). The MCon × CCyt showed the highest lipid levels, whereas MCyt × CCyt showed the lowest lipid levels (P < 0.05). Results suggest that the combination of MCyt × CCyt media produces the lowest lipid levels, whereas the MCon × CCon media lead to the highest mitochondrial activity. The addition of cytokines to both maturation and culture media maintains competence and lowers lipid content; however, it also seems to lower mitochondrial activity. Cryopreservation studies and evaluation of pregnancy rates are ongoing.
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