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J. A. Southee, M. G. Hunter, and W. Haresign

Summary. Anoestrous Romney Marsh ewes with and without progesterone treatment (+P, −P) were treated with small-dose (250 ng) multiple injections of GnRH at 2-h intervals for 48 h. Animals were slaughtered on Days 4, 5, 7 and 11 after the end of GnRH treatment and luteal function was assessed by the measurement of daily plasma progesterone concentrations.

In all animals which ovulated (29/32, 91%) peripheral progesterone concentrations rose to 0·5–1·0 ng/ml within 3 days of the end of GnRH treatment. In 7/7 (100%) +P animals and 5/22 (23%) −P animals, progesterone concentrations continued to rise and were maintained at levels > 1·5 ng/ml until slaughter. In the remaining −P animals, plasma progesterone concentrations declined to reach basal levels by Day 5. Corpora lutea recovered from these animals showed signs of premature regression on Day 5 and were fully regressed by Day 7. Progesterone priming delayed the occurrence of the LH surge which occurred 39·1 ± 3·6 h after the end of GnRH treatment in the +P animals compared to 20·2 ± 1·74 h (P < 0·001) in the −P animals in which luteal function was abnormal and 22·4 ± 4·35 h in the −P animals in which luteal function was normal.

These results show that abnormal luteal function occurs in the majority of GnRH-treated ewes in the absence of progesterone pretreatment. It is characterized by a transient rise in plasma progesterone concentration to 0·5–1·0 ng/ml 3 days after GnRH treatment, which then declines to basal values between Days 4 and 5, coincident with the rapid premature regression of this abnormal corpus luteum which is complete by Day 7.

Keywords: GnRH; abnormal luteal function; sheep; anoestrus

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M. G. Hunter, J. A. Southee, and G. E. Lamming

Summary. Normal and abnormal corpora lutea were recovered from anoestrous Romney Marsh ewes on Days 3, 4, 5 and 6 after treatment with small-dose (250 ng) multiple injections of GnRH followed by a bolus injection (125 μg) with (+P) and without (−P) progesterone pretreatment and a study made of their characteristics in vitro. Plasma progesterone concentrations initially rose concurrently in all animals but abnormal luteal function occurred in 70% of the −P ewes and was defined on Day 5 when plasma progesterone concentrations declined relative to those in the +P ewes.

All corpora lutea recovered on Days 3 and 4 appeared macroscopically similar and there were no significant differences between the +P and −P groups in terms of luteal weight, progesterone content and binding of 125I-labelled hCG on these days. However, corpora lutea from the −P animals only exhibited a decline in progesterone production in vitro on Day 4 (P < 0·01), and morphological differences became apparent on Days 5 and 6 when the abnormal corpora lutea from the −P animals also decreased in weight (P < 0·01) and progesterone content (P < 0·001). Binding of 125I-labelled hCG increased on Day 5 in the normal corpora lutea only.

These results show that, although abnormal luteal function induced by GnRH treatment of anoestrous ewes could not be distinguished from normal corpora lutea before Day 5 by measurement of progesterone in peripheral plasma, a significant decline in progesterone production in vitro occurred on Day 4 in the abnormal corpora lutea. This was followed by significant decreases in weight and progesterone content and a failure to increase 125I-labelled hCG binding. Abnormal corpora lutea are therefore capable of some initial growth and progesterone production, before undergoing a rapid and premature regression from Day 4, which has similar characteristics to natural luteolysis.

Keywords: abnormal luteal function; in vitro; sheep; anoestrus

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J. A. Southee, M. G. Hunter, A. S. Law, and W. Haresign

Summary. Anoestrous Romney Marsh ewes were treated with small-dose (250 ng) multiple injections of GnRH. Ewes in Groups 1 and 3 were hysterectomized 2 weeks before treatment, while those in Groups 2 and 4 were intact controls. Groups 1 and 2 were primed with progesterone (+P) and treated with 2 h injections of GnRH (250 ng) for 36 h, while Groups 3 and 4 were not pretreated (−P) but were given 2 h injections of GnRH (250 ng) for 18 h. Both treatment regimens were terminated with a bolus injection of GnRH (125 μg), given to synchronize the timing of the LH surge and subsequent luteal progesterone production.

The plasma progesterone profiles of 5/5 animals in Group 2 (+P controls) and 2/5 animals in Group 4 (−P controls) were indicative of normal luteal function, while the remaining 3/5 animals in Group 4 produced plasma progesterone profiles typical of abnormal luteal function. However, in all the hysterectomized animals (Groups 1 and 3) peripheral plasma progesterone concentrations rose to reach a mean peak value of 1·3 ng/ml plasma on Day 8 which was maintained in all animals irrespective of progesterone pretreatment. The absence of a fall in progesterone concentrations precluded the identification of any animal in Group 4 showing abnormal luteal function. It was also noted that, after hysterectomy, although the corpus luteum was maintained, it was with reduced secretory capacity.

The prevention of the expected proportion (70%) of −P animals from displaying a decline in plasma progesterone concentration after hysterectomy provides firm evidence that the uterus is involved in the premature regression of the short-cycle corpus luteum.

Keywords: abnormal luteal function; hysterectomy; sheep; anoestrus

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M. G. Hunter, V. J. Ayad, C. L. Gilbert, J. A. Southee, and D. C. Wathes

Summary. Anoestrous Romney Marsh ewes with (+P) and without (−P) progesterone pretreatment were induced to ovulate by multiple low-dose injection of GnRH followed by a bolus injection of GnRH. Luteal function was assessed by twice daily measurement of plasma progesterone. Animals were slaughtered on Days 3 or 5 after the end of GnRH treatment and CL and endometrium were recovered. In all Day-5 ewes, blood samples were collected at 30-min intervals for 8 h on Days 3 and 5 for measurement of PGFM and oxytocin. At slaughter 92% of the Group +P ewes had ovulated compared with 54% of the Group −P ewes. The ovaries of some of the Group −P ewes only contained luteinized cysts either alone or in association with CL. In the ewes that ovulated, progesterone profiles were normal in all Group +P ewes, whereas Group −P ewes had 'normal' or 'abnormal' profiles in which plasma progesterone was declining prematurely. All of the CL from ewes with abnormal progesterone profiles were associated with follicular cysts, and were significantly smaller and with a lower progesterone content on Day 5. PGFM levels decreased (P < 0·05) between Days 3 and 5 in ewes in Groups +P and −P with 'normal' CL but increased (P < 0·01) in Group −P ewes with 'abnormal' CL. Oxytocin levels were lower in Group −P ewes with 'abnormal' CL on Day 5, than in 'normal' ewes in Groups −P (P < 0·01) or +P (P < 0·05). In 3/5 Day-5 ewes with 'abnormal' CL there was a clear association between a major peak of oxytocin and a rise in PGFM during the frequent sampling period on Day 3 or Day 5, and endometrial oxytocin binding sites were present at slaughter. This suggests that the premature regression of 'abnormal' CL occurs via the normal luteolytic mechanism. Although ewes in Groups +P and –P with 'normal' CL had similar progesterone profiles, plasma oxytocin was significantly higher (P < 0·05) in the Group −P ewes and oxytocin binding sites were present only in this group, suggesting that progesterone pretreatment can influence the production of both oxytocin and its receptor.

Keywords: abnormal corpus luteum; oxytocin; prostaglandin F-2α; ewe; luteolysis

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M. G. Hunter, J. A. Southee, B. J. McLeod, and W. Haresign

Summary. In two experiments carried out during seasonal anoestrus, Romney Marsh ewes were treated with small-dose (250 ng) multiple injections of GnRH at 2-h intervals with and without progesterone pretreatment. In Exp. 1, 8/8 progesterone-primed ewes ovulated and produced functionally normal corpora lutea compared with 2/9 non-primed ewes.

Follicles were recovered from similarly treated animals 18 or 28 h after the start of GnRH treatment (at least 14 h before the estimated time of the LH peak) and assessed in terms of diameter, granulosa cell number, oestradiol, testosterone and progesterone concentrations in the follicular fluid, oestradiol production in vitro and binding of 125I-labelled hCG to granulosa and theca. There were no significant differences in any of these measures in 'ovulatory' follicles recovered from the progesterone-pretreated compared to non-pretreated animals.

In Exp. 2, follicles were removed from similar treatment groups just before and 2 h after the start of the LH surge. Unlike 'ovulatory' follicles recovered from the nonpretreated ewes, those recovered from progesterone-pretreated ewes responded to the LH surge by significantly increasing oestradiol secretion (P < 0·01) and binding of 125I-labelled hCG (P < 0·05) to granulosa cells. Overall there was also more (P < 0·05) hCG binding to granulosa and theca cells from progesterone-pretreated animals. Non-ovulatory follicles recovered from progesterone-primed ewes had more (P < 0·05) binding of 125I-labelled hCG to theca and a higher testosterone concentration in follicular fluid (P < 0·05) than did those from non-primed ewes.

These results suggest that inadequate luteal function after repeated injections of GnRH may be due to a poor response to the LH surge indicative of a deficiency in the final maturational stages of the follicle.