125 EFFECT OF BOVINE OVIDUCTAL FLUID ON DEVELOPMENT AND QUALITY OF IN VITRO-PRODUCED BOVINE EMBRYOS

Abstract

Although fertilization and early embryonic development take place in the oviduct, the consequences of tubal fluid supplementation during in vitro embryo culture have not been explored. The aim of this study was to evaluate the effect of bovine oviducal fluid (bOF) supplementation during in vitro embryo culture of bovine embryos on their development and quality. The bOF was aspirated from oviducts of slaughtered heifers in the early luteal phase. In vitro-produced zygotes were cultured in SOF (C–; n = 927) or SOF + 5% FCS (C+; n = 872) or in SOF + bOF (n ~900/group) at different concentrations (0.62, 1.25, 2.5, 5, 10, or 25%) in 10 replicates. Blastocysts on Days 7/8 were used for quality evaluation through (a) differential cell count, (b) survival after vitrification/warming, and (c) gene expression (qRT-PCR). One-way ANOVA (development and quality) and t-test (cell count) were used for statistical analysis. The bOF concentrations over 5% were detrimental for blastocysts development (<7% at Day 7) and were discarded. Embryos cultured in absence of FCS exhibited a delay in the kinetics of blastocyst development; at Day 7, the groups cultured without FCS (bOF 0.62–2.5% and C–) had fewer blastocysts (range:12.0 ± 1.7 to 17.4 ± 1.5%) compared with C+ group (22.9 ± 1.2%). However, blastocyst yield at Day 9 was similar in 0.62 and 1.25 bOF groups (27.5 ± 1.7% and 27.5 ± 1.2%, respectively) compared with C+ (27.7 ± 1.0%) and significantly higher than 2.5 bOF (22.7 ± 1.5%) and C– (21.5 ± 1.4%; P < 0.05). In terms of blastocyst quality, 48 h after vitrification/warming, embryos from bOF 1.25%, 0.62%, and C– groups survived significantly higher than C+ (61.3 ± 2.1; 61.6 ± 4.1; 59.3 ± 3.2; and 30.3 ± 2.5, respectively; P < 0.05). This difference was even higher at 72 h (53.6 ± 1.7; 57.7 ± 3.8; 56.1 ± 2.9; and 25.9 ± 2.3%, respectively; P < 0.05). Total cell number of the embryos cultured in bOF 1.25 and 0.62% groups were significantly higher than C+ and C– (165.1 ± 4.7 and 156.2 ± 4.2 v. 143.1 ± 4.9 and 127.7 ± 4.9, respectively), which was associated with an increased TE cell number in 1.25 and 0.62% bOF groups (119.9 ± 3.7 and 127.0 ± 4.5, respectively). Culture with 2.5% bOF had no effect on either blastocyst yield or quality. Gene expression analysis was performed in 1.25% bOF, C–, and C+ groups. The result suggested a higher glucose (SCL2A1) and lipid (CYP51 and FADS1) metabolism in those groups cultured without serum. Gene DNMT3A and the imprinted gene IGF2R were also significantly up-regulated in bOF and C– compared with C+ (P < 0.05). Interestingly, AQP3, a gene positively correlated with survival after vitrification, was significantly up-regulated in bOF compared with C– and C+ (P < 0.05). In conclusion, in vitro culture with low concentrations of bOF has a positive effect in development and quality of bovine embryos cultured in absence of FCS.

Funded by the Spanish Ministry of Science and Innovation (AGL2012–37510).