Effect of GnRH antagonist-induced prolonged follicular phase on follicular atresia and oocyte developmental competence in vitro in superovulated heifers.
A GnRH antagonist (Antarelix) was used to suppress endogenous pulsatile secretion of LH and delay the preovulatory LH surge in superovulated heifers to study the effect of a prolonged follicular phase on both follicle and oocyte quality. Oestrous cycles were synchronized in 12 heifers with progestagen (norgestomet) implants for 10 days. On day 4 (day 0 = day of oestrus), heifers were stimulated with 24 mg pFSH for 4 days and luteolysis was induced at day 6 with PGF2 alpha (2 ml Estrumate). Animals in the control group (n = 4) were killed 24 h after the last FSH injection. At this time, heifers in group A36h (n = 4) and group A60h (n = 4) were treated with 1.6 mg of Antarelix every 12 h for 36 and 60 h, respectively, and then killed. After dissection of ovarian follicles, oocytes were collected for individual in vitro maturation, fertilization and culture; follicular fluid was collected for determination of steroid concentrations, and granulosa cells were smeared, fixed and stained for evaluation of pycnosis rates. Granulosa cell smears showed that 90% of follicles were healthy in the control group. In contrast, 36 and 58% of the follicles in group A36h showed signs of early or advanced atresia, respectively, while 90% of the follicles in group A60h showed signs of late atresia. Intrafollicular concentrations of oestradiol decreased (P < 0.0001) from healthy follicles (799.14 +/- 40.65 ng ml-1) to late atretic follicles (3.96 +/- 0.59 ng ml-1). Progesterone concentrations were higher (P < 0.0001) in healthy follicles compared with atretic follicles, irrespective of degree of atresia. Oestradiol:progesterone ratios decreased (P < 0.0001) from healthy (4.58 +/- 0.25) to late atretic follicles (0.07 +/- 0.009). The intrafollicular concentrations of oestradiol and progesterone were significantly higher (P < 0.0001) in the control than in the treated groups. The oestradiol:progesterone ratio was higher (P < 0.0001) in the control (4.55 +/- 0.25) than in the A36h (0.40 +/- 0.05) and A60h (0.07 +/- 0.009) groups. Unexpectedly, the cleavage rate of fertilized oocytes, blastocyst rate and number of cells per blastocyst were not significantly different among control (85%, 41% and 95 +/- 8), A36h (86%, 56% and 93 +/- 5) and A60h (88%, 58% and 79 +/- 4) groups. In addition, there were no significant differences in the blastocyst rates from oocytes derived from healthy (45%), early atretic (54%), advanced atretic (57%) and late atretic follicles (53%). In conclusion, the maintenance of the preovulatory follicles in superovulated heifers with a GnRH antagonist induced more atresia and a decrease in oestradiol and progesterone concentrations. However, the developmental potential in vitro to day 8 of the oocytes recovered from these atretic follicles was not affected.
Oussaid
B
B
INRA, Unité Physiologie de la Reproduction des Mammiféres Domestiques, Nouzilly, France.
Lonergan
P
P
Khatir
H
H
Guler
A
A
Monniaux
D
D
Touze
J L
JL
Beckers
J F
JF
Cognie
Y
Y
Mermillod
P
P
D000735:Androstenedione / Q000097:blood; D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002417:Cattle / Q000502:physiology*; D016009:Chi-Square Distribution; D005314:Embryonic and Fetal Development; D004958:Estradiol / Q000097:blood; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005496:Follicular Atresia / Q000187:drug effects*; D005498:Follicular Phase / Q000187:drug effects*; D007987:Gonadotropin-Releasing Hormone / Q000037:antagonists & inhibitors*; D007986:Luteinizing Hormone / Q000097:blood / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology*; D009866:Oogenesis / Q000187:drug effects*; D011374:Progesterone / Q000097:blood; D013480:Superovulation
D009842:Oligopeptides; D007987:Gonadotropin-Releasing Hormone; D000735:Androstenedione; D011374:Progesterone; D004958:Estradiol; D007986:Luteinizing Hormone; C084350:antarelix