Abstract
Assisted reproductive technologies in canine species are limited due to the low efficiency of in vitro maturation (IVM). Unlike other mammals, bitches ovulate oocytes in the germinal vesicle stage and complete metaphase II (MII) after 48–72 h in the oviductal environment and become fertilizable. For this reason, we compared two different IVM media, synthetic oviductal fluid (SOF) supplemented with 8% bovine serum albumin (BSA) or a mixture of 8% BSA–2.5% fetal bovine serum (FBS) and TCM-199 with 10% FBS. Additionally, we evaluated the effect of supplementation with insulin-transferrin-selenium (ITS) and low O2 tension in oocyte maturation, reactive oxygen species (ROS) levels, membrane integrity, and embryo development following parthenogenetic activation (PA). After 72 h of culture, SOF + BSA, SOF + BSA + FBS, and TCM-199 + FBS show 5, 7, and 4% of MII, respectively, without a statistical difference. However, SOF + BSA produced significantly higher degeneration rates compared to SOF + BSA + FBS (44 and 23%, respectively). Remarkably, supplementation with 1 μl/ml of ITS under high O2 tension demonstrated a beneficial effect by improving maturation rates up to 20% compared to the other groups. Low O2 tension increased maturation rates to 36.5%, although there were no statistical differences compared to high O2 tension in the presence of ITS. Lower ROS levels and higher integrity of the cytoplasmic membrane were found in the presence of ITS despite no differences in maturation rates under low O2 tension groups. Additionally, after PA, 1% development until the eight-cell stage was obtained after activation of in vitro-matured oocytes in the presence of ITS. Taken together, these results indicate that SOF supplemented with 8% BSA and 2.5% FBS is suitable for IVM of canine oocytes and ITS supplementation was beneficial for both high and low O2 tension. Furthermore, the addition of ITS in the cultured system lowers ROS levels and increases membrane integrity in domestic dog oocytes after IVM.
Highlights
The low efficiency of in vitro maturation (IVM) in canine remains a real challenge for reproductive biologists (Van Soom et al, 2014)
Experiment 1: Media and Protein Supplementation Effect on Canine in vitro Maturation We compared the ability of two different media to resume meiosis and reach metaphase II (MII) stage of immature oocytes recovered after ovariectomy of bitches with unknown stage of the estrous cycle
A total of 1,305 cumulus–oocyte complexes (COCs) were collected from bitches of unknown age, breed, and stage of the estrous cycle
Summary
The low efficiency of in vitro maturation (IVM) in canine remains a real challenge for reproductive biologists (Van Soom et al, 2014). Canine oocytes reach 10% of metaphase II (MII) after IVM, while most oocytes (30– 40%) degenerate (Farstad, 2000; Luvoni, 2000). Some authors such as No et al (2018) have used coculture with oviduct epithelial cells to improve maturation rates and have achieved 13% of MII using oocytes recovered from bitches at diestrus and 47% of MII with bitches at estrus. Previous authors have reported the use of SOF media for canine IVM with different supplementation and results (Hewitt and England, 1999; Bolamba et al, 2002; Saikhun et al, 2008; Lange-Consiglio et al, 2017)
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