250 EFFICIENCY COMPARISON BETWEEN FOLLICULAR ASPIRATION AND SCRAPING FOR EX VIVO RECOVERY OF EQUINE CUMULUS ENCLOSED OOCYTES

Abstract

Collection of cumulus enclosed oocytes from excised ovaries has become an ambitious task in recent years as a result of a decrease in slaughtering of horses because of ethical and political reasons. This increases the demand for highly efficient procedures to collect the maximum amount of oocytes out of these ovaries. The main objective of this study was to compare the efficiency of 2 oocyte recovery procedures. Oocyte aspiration is a quick and easy-to-perform technique with one potential limitation: the loss of the cumulus oophorus through suction forces. Recovery by follicular scraping is a more laborious procedure, but seems to be superior with regard to collection of cumulus-enclosed oocytes. This study was conducted to figure out the most efficient technique to collect high-quality equine cumulus-enclosed oocytes. The study was performed with ovaries of 32 slaughtered mares on 9 experimental days. All follicles of both ovaries from each mare were randomly allocated either to oocyte recovery by aspiration (15 mares) or by scraping (17 mares). Follicular scraping was performed with a bone curette after incision of the follicle with a scalpel blade, and follicular fluid was evacuated in watch glasses. After scraping, the follicles were flushed with PBS. Aspiration was performed using an 18-gauge needle with Tuohy bevel on a syringe. In a first step, follicular fluid was aspirated using scraping movements with the needle on the follicular wall. The empty follicular cavity was flushed once with the follicular fluid, the aspiration fluid was transferred to Falcon tubes and the aspirate settled for 10 min. Sediment of aspirated follicles was transferred to culture dishes, oocytes were searched under the stereomicroscope, and scraped follicles were searched directly in the watch glasses. Oocytes were classified in oocytes with compact cumulus oophorus, oocytes with expanded cumulus oophorus, oocytes with corona radiata only, denuded oocytes, and degenerated oocytes. Recovery rates were analysed for the 2 methods with special regard to oocyte classification. The overall recovery rates were similar for scraped (53.74%) and aspirated (52.70%) follicles. The 2 methods performed differently with respect to oocyte qualities. Significantly more of the oocytes recovered by scraping were collected with compact cumulus oophorus (72.16%) than in the aspiration group (33.77%; P = 0.004). Both methods collected similar amounts of oocytes with expanded cumulus oophorus (10.12% by scraping, 7.79% by aspiration). Using the aspiration more oocytes presented denuded (24.67 v. 6.33%, P = 0.08). The results of the presented study confirm that the scraping procedure is superior with regard to collection of cumulus-enclosed oocytes. These results prioritize follicle scraping for ex vivo collection of oocytes for research purposes because cumulus cells may support oocytal well-being during maturation and represent a unique biomarker source for the developmental competence of their corresponding oocytes.