317EMBRYONIC 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 9mm 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 22h. Standard laboratory IVF was then performed with frozen-thawed semen from a fertile bull. In Treatment 2, 3mL 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 6h. Oocytes recovered from the ovaries in Treatment 3 were placed into 3mL 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 2mL of Ringer’s lactate plus 1mL of the same pooled follicular fluid and were held for 6h 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 22h and then subjected to standard IVF. Embryo development in CR1aa culture medium was assessed at 72, 168 and 216h post-insemination in each treatment group. In summary, no significant difference was detected between the standard IVF procedure and oocytes held in follicular fluid 6h prior to IVF (Treatment 1 v. 2). Also, follicular fluid apparently had a positive effect on the oocytes over that of holding oocytes 6h 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 6h in 22°C undiluted bovine follicular fluid until they can be delivered to a full-service IVF laboratory.