Embryotrophic effect of bovine luteal cells co-culture: an alternative to improve IVF embryo production

MARURI, A.; CRUZANS, PR.; TELLO, MF.; LORENZO, MS.; LOMBARDO, DM.

PARIS

Conferencia; 18th International Conference on Animal Reproduction (ICAR); 2016

Local Organizing Committee ICAR

In vitro embryo production in cattle has been widely developed for several decades and the culture systems have been refined. However, the rates of development could be improved. The quality of embryos produced in vitro remains lower than those produced in vivo. Embryo-somatic cell co-culture is an alternative to improve the development and viability of mammalian embryos. However, little is known about the mechanisms underlying their beneficial effects. Somatic cells could remove deleterious components, secrete growth factors and modulate medium conditions. Despite various cell types have been used, very few studies have focused on the interaction between luteal cells and early embryos in co-culture systems. The aim of this study was to evaluate the effect of bovine luteal cells on early embryonic development. Two embryo quality markers were employed, the TUNEL assay for detecting late apoptosis and immunofluorescence detection of antigen Ki-67, a protein that is associated with cellular proliferation. Early corpora lutea were retrieved from slaughter ovaries and the cells were isolated to obtain a primary culture. The bovine luteal cells were purified by Percoll density gradient centrifugation and then were cryopreserved and stored until its use. Purity was determined by anti-3 beta-HSD immunocytochemistry. In vitro embryo production was carried out using cumulus-oocyte complexes (COC) recovered from antral ovarian follicles. After 22 h of in vitro maturation, COC were co-incubated with sperm for 5 h. Then, presumptive zygotes were denuded and randomly allocated to one of two culture conditions: without cells (C: control group; n = 141) or with bovine luteal cells (L: co-culture group; n = 133) using in both cases SOF medium. On day 2, anti-Ki-67 immunofluorescence (n = 119) was performed, cleavage was recorded and the embryos were transferred to new droplets of SOF medium. Embryo development was evaluated on day 8 and the embryos were classified according to the IETS standards. Late apoptosis was assessed by TUNEL assay in 7-Day blastocysts from each experimental group (n = 52). Data were statistically analyzed using Proportion test and non-parametric Kruskal-Wallis test. Differences with P < 0.05 were considered statistically significant. It was found that there were no differences in cleavage rate (L: 83% vs. C: 83%), whereas proliferation index (Ki-67-positive cells/total blastomeres) in the co-culture group was higher than the control (L: 0.45 vs. C: 0.13). On the other hand, the bovine luteal cells co-culture increased the blastocysts rate (L: 50% vs. C: 30%) and blastocysts-stage 6 rate (L: 37% vs. C: 24%) and decreased late apoptosis rate (L: 4.10% vs. C: 10.90%). In conclusion, bovine luteal cells exert an important trophic effect on bovine early embryos. Therefore, this co-culture system is able to be used successfully to optimize the in vitro embryo production in cattle.