205 DEVELOPMENT OF OOCYTES FROM COWS TREATED WITH RETINOL IS COMPROMISED PRIOR TO IMPLANTATION

Abstract

In the cow, sheep, and gilt, a retinoid support enhances the oocyte development (reviewed by Hidalgo C et al. 2003 Reproduction 125, 409–416). The shortest effective time interval for these animals injected with retinol (ROH) is four days, but the reasons for this have not been described. Furthermore, the development after transfer of embryos derived from oocytes of donors treated with ROH is not known. The objective of the present work was to elucidate the two above questions. We analyzed the ROH and retinyl palmitate (REP) contents in plasma and follicular fluid (FF). The estrus cycles of heifers (n = 12) were synchronized with progestagen and PGF2α. Blood samples were taken at progestagen removal (Day 0). Animals were injected with 1 × 10⁶ ROH units (n = 6) or corn oil (vehicle; n = 6) on Days 0, 1, and 4. Contents of follicles between 3 and 10 mm were aspirated on Day 4 by an ultrasound-guided procedure. All samples were analyzed for ROH and REP by HPLC. To produce embryos for transfer, follicles were aspirated from donors treated four times with 1 × 10⁶ ROH units/week or vehicle, starting four days before the first aspiration. Both groups of cumulus-oocyte complexes (COCs) were matured in vitro with or without 5 nM 9-cis-retinoic acid (RA). The presence of RA prolonged the exposure to retinoids (cows treated with ROH) or acted as a positive control (heifers with vehicle). Oocytes were fertilized and cultured in mSOF + 5% FCS. Embryo transfer (ET) to recipients was performed with fresh (one) or vitrified (two) good-morphology Day 7 embryos, and pregnancy monitored on Days 21, 35, and 60. Data analysis was by GLM (ROH and REP concentrations) or CATMOD (pregnancy monitoring), and Duncan's test (^a,bP < 0.05; ^v,x,y,zP < 0.02). Average FF volume recovered were 351 ± 127 μL (controls; range 100–700) and 393 ± 127 μL (ROH; range 80–950). Concentrations of REP were unaffected by timing, follicle or blood, and ROH treatment (data not shown). Concentrations of ROH (μg/dL) for vehicle and ROH-treated cows were (LSM ± SE) 42.0 ± 1.8^vx and 42.0 ± 2.4^vx (plasma-Day 0, before ROH injection), 37.3 ± 1.8^v and 47.64 ± 2.4^x (plasma-Day 1), 42.6 ± 1.9^vx and 45.5 ± 2.3^vx (plasma-Day 4), and 6.1 ± 3.0^y and 16.8 ± 2.6^z (FF-day 4), respectively. Cumulatively, embryos from donors receiving ROH did not exhibit pregnancy on Day 21 (0/15^x; confirmed on Day 35), which differed from vehicle donors on Day 21 (44%, 8/18^y) and Day 35 (33%, 6/18^y), and tended to differ (P = 0.07) on Day 60 (22%, 4/18). Pregnancy rates were independent of fresh or vitrified embryos within ETs. Injected ROH elevated blood concentrations of ROH, although values became normalized on Day 4. However, a coasting period of 4 days for ROH administration seems to be justified by increased intrafollicular levels of ROH. Development into blastocysts is disrupted before implantation, showing that ROH directly affects the oocyte during its intrafollicular growth.

This work was supported by MCYT-AGL-2002-0117.