317 EMBRYONIC DEVELOPMENT AFTER HOLDING BOVINE OOCYTES IN UNDILUTED FOLLICULAR FLUID FOR A 6-HOUR PREMATURATION PERIOD

Abstract

Follicular fluid has been implicated in follicular growth, initiation of steroidogenesis, regulation of granulosa cell function and oocyte maturation. Although individual components of the follicular fluid have been analyzed, it is still unclear how undiluted follicular fluid may affect subsequent in vitro oocyte maturation (IVM), in vitro fertilization (IVF) and embryo development. The objective of this study was to simulate recovery of bovine oocytes as if under field conditions to determine the effect of holding these oocytes in follicular fluid for a 6-h holding period before performing IVF. Ovaries were obtained from a local abattoir and transported to the laboratory at ∼22°C in a 0.9% saline and antibiotic solution. At the laboratory, ovaries were rinsed with an ethanol solution and randomly allotted to four treatment groups. Follicles ranging in size from 2 to 9 mm were aspirated using a 20-gauge needle attached to a sterile plastic syringe. Dominant follicles were not included. Oocytes harvested from the ovaries in Treatment 1 (Control) were placed directly into a standard laboratory maturation medium consisting of TCM-199 supplemented with LH, FSH, fetal bovine serum, estradiol and gentamicin for 22 h. Standard laboratory IVF was then performed with frozen-thawed semen from a fertile bull. In Treatment 2, 3 mL of pooled follicular fluid was dispensed into a 6-mL conical centrifuge tube and co-incubated with the harvested oocytes at room temperature (22°C) for 6 h. Oocytes recovered from the ovaries in Treatment 3 were placed into 3 mL of Ringer’s lactate solution for a 6-h holding period at 22°C. Oocytes obtained from the ovaries in Treatment 4 were placed into a mixture of 2 mL of Ringer’s lactate plus 1 mL of the same pooled follicular fluid and were held for 6 h at 22°C. After being held for a 6-h period, oocytes were recovered from each centrifuge tube and were placed into IVM medium for 22 h and then subjected to standard IVF. Embryo development in CR1aa culture medium was assessed at 72, 168 and 216 h post-insemination in each treatment group. In summary, no significant difference was detected between the standard IVF procedure and oocytes held in follicular fluid 6 h prior to IVF (Treatment 1 v. 2). Also, follicular fluid apparently had a positive effect on the oocytes over that of holding oocytes 6 h in Ringer’s lactate alone, as indicted by a significantly greater rate of blastocyst development (Treatment 3 v. 4). In conclusion, it should not be overlooked that bovine oocytes, aspirated under field conditions, could be held up to 6 h in 22°C undiluted bovine follicular fluid until they can be delivered to a full-service IVF laboratory.