Influence of oocyte retrieval methods and maturation media on in vitro development of porcine embryos

Abstract

Oocyte recovery, in vitro maturation (IVM) and in vitro fertilization (IVF) are reproductive technologies used to advance an oocyte to an embryo stage. The objectives of the study were to (1), compare oocyte retrieval methods (aspiration and slicing) from pig ovaries on oocyte quality and quantity; (2), compare IVM media (NCSU 37, TCM 199 and mpFF= porcine follicular fluid + FSH + LH) on oocytes polar body extrusion; (3), compare the effect of electrical stimulation (ES) and frozen-thawed semen on cleavage rates and (4) compare the effect of culture media, cNCSU 37 and cpFF, on zygote cleavage rates. Follicular fluid collected from the retrieval methods were assessed for the presence of oocytes using a microscope and categorized as Grades A, B or C. The IVM media were placed in a four-well multidish, thereafter Grades A and B oocytes were randomly allocated per treatment groups, matured for 44 hours and checked for polar body extrusion. Matured ootids were randomly allocated to either ES treatment or IVF treatment with frozen-thawed semen. The ES treatment used a medium consisting mainly of mannitol while BO-IVF was used for the frozen-thawed treatment. ES and IVF took place in conjuncture with in vitro culture (IVC), where both results were obtained following placement in IVC media. Zygotes were then checked and recorded for cleavage, single cell or lysed. The treatment means were compared using the Fisher’s protected t-test least significant difference. Results showed significant differences with Grades A and B accounting for 50.8% of total oocytes (193.8) for aspiration and 58.7% of 488.6 for slicing. Oocytes’ polar body extrusion was recorded as 25.3%, 84.2% and 73.8% for NCSU 37, TCM 199 and mpFF respectively. The percentage of cleaved zygotes for IVF with frozen-thawed semen was 48.1% and 56.8% for ES. The percentage of cleaved zygotes for cNCSU 37 was 66.6% and was 45.7% for cpFF. The slicing method proved to be better than aspiration for the retrieval of Grades A and B oocytes and the total number of oocytes retrieved. TCM 199 and mpFF media had a higher percentage of oocytes with polar body extrusion than NCSU 37. Cleavage rate of zygotes was higher when using ES and cNCSU 37.