174 Use of mesenchymal stem cell treatment to improve oocyte yield and in vitro embryo production in cattle

Abstract

Cumulative tissue damage and chronic inflammation associated with frequent ovum pickup (OPU) may lead to a progressive reduction in the number and quality of the oocytes recovered, particularly in donors with a high antral follicle count. The aim of this study was to evaluate the effect of an intraovarian treatment with mesenchymal stem cells (MSC) on oocyte yield, quality, and development potential during in vitro embryo production (IVEP) in cattle donors undergoing repeated OPU. Mesenchymal stem cells were previously isolated from adipose tissue, cultured in Dulbecco's modified Eagle medium until reaching 80% confluence, isolated with trypsin, and frozen in liquid N₂ until use. Characterisation of MSC was carried out according to the guidelines of the International Society for Cellular Therapy. Nelore (Bos indicus) cows (n = 5) were used in this study, with the ovaries as replicates. The cows underwent eight OPU sessions at 15-day intervals, and the oocytes recovered were graded and used for IVEP with the semen of a single sire and batch under similar in vitro culture conditions. To ensure a high inflammatory response, immediately after the fourth OPU session all ovaries received 30 additional punctures, performed with a 16-gauge Jelco needle. Six hours later, the left ovary of each cow was injected with 500 µL of Dulbecco's modified phosphate buffered saline (control ovary) and the right ovary received 500 µL of Dulbecco's modified phosphate buffered saline with 2.5 × 10⁶ allogenic MSC (treated ovary). Oocyte yield and embryo production before and after treatment were recorded for each ovary and donor. Grade I blastocysts produced from control and treated ovaries were used for gene expression evaluation. Data was analysed using the repeated-measures procedure of SAS (SAS Institute Inc.) to account for the effects of treatment, time, and interactions. There was no difference (P > 0.05) in any endpoint before treatment (sessions 1-4) between the right and left ovaries. Thus, differences between ovaries observed in OPU sessions 5-8 were assumed to be due to the treatment. After the injection of MSC, more total and viable oocytes were collected from the right ovaries compared with the left ovaries (15.3 ± 2.2 vs. 8.7 ± 1.2 (P < 0.02) and 13.6 ± 2.1 vs. 7.1 ± 1.0 (P < 0.01), respectively), resulting in more embryos produced in vitro (7.6 ± 1.2 vs. 3.6 ± 0.6, respectively; P < 0.01) as well as more initial and expanded blastocysts (1.4 ± 0.3 vs. 0.4 ± 0.1 and 4.4 ± 0.9 vs. 2.1 ± 0.4, respectively; P < 0.04). The proportion of viable oocytes recovered from the right ovary after treatment was greater than that from the left ovary (89.1% vs. 81.5%; P < 0.05). However, blastocyst rates did not differ between ovaries before or after treatment (50.4% vs. 55.5%: P > 0.05). In the blastocysts produced from treated ovaries, SLC2A3 was overexpressed (P < 0.04), whereas there was no difference for the expression of KRT8, PLAC8, SLC2A1, CASP3, PRDX3, or SOD2 (P > 0.05), suggesting potential differences in glucose uptake and metabolism. In conclusion, intraovarian treatment with MSC improved oocyte yield and quality and may be an alternative to increase IVEP from donors under intensive OPU schedules.

This research was supported by CNPq, CAPES, and Fazenda Grupo Esplanada.