335 A PROCEDURE COMBINING ISTAT^® ANALYSIS WITH OPU TO STUDY BOVINE FOLLICULAR ENVIRONMENTS

Abstract

Our previous work successfully analysed follicular fluid (45/61, 74% sampling success) from live cows using a portable clinical gas tension analyser (iSTAT Corp., Princeton, NJ). However, the oocyte recovery rate (7/24, 29%) from preovulatory follicles (POF) was disappointingly low, perhaps due to a need for the second puncture to aspirate the remaining follicular contents. This work describes a modified procedure that allowed a follicular fluid (FF) sample to be collected and stored in a gas-impermeable manner and the remaining follicular contents to be directly aspirated with or without follicular flushing from a single puncture. This modified dual port slip-luer hub, designed for use with a disposable needle ovum pick-up (OPU) system (PieMed, Netherlands), used a low negative pressure (30 mmHg), medium flow rate (25 mL/min) aspiration system with syringe flushing. FF samples for analysis were collected and stored in glass capillary tubes and the rest of the follicular contents/flushings were collected into tubes containing 10 mL collection medium and searched for oocytes. The hub's larger port, with a fitted Y connector, allows an initial flow of FF to be drawn into a capillary tube which has a polyvinylchloride (PVC) powder plugged end. When the PVC powder is permeated with FF it forms a seal effectively capping the tube. The remaining FF is diverted down the other leg of the Y connector using negative pressure. Flushing medium can be injected through the smaller port into the follicle while negative pressure is interrupted. A pilot trial examined gas tension, pH and ion concentrations from POF, dominant (D), and hormonally stimulated (S) follicles. Twenty synchronised Friesian cattle were scanned daily and follicles were tracked and mapped. The POF and D follicles were sampled on Day 20 or Day 10 respectively of the estrous cycle. The cows were then subjected to four weekly stimulations (used CIDR-B device inserted Day 2 and removed Day 5, 120 mg NIH-FSH-P1 administered twice daily on Day 5 and 6). Follicles were sampled and aspirated on Day 7. Successful FF analysis was achieved in 88% (110/125) of follicles attempted while oocyte recovery rates from POF, D, and S follicles were 56% (9/16), 54% (6/11) and 43% (36/83) respectively, showing it is possible to combine portable gas tension analysis with effective OPU. However, further work is needed to elucidate the correlation between the follicular environment and the oocyte developmental competence.

Table 1.
Follicular fluid sample analysis