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Peripheral plasma progesterone concentrations were determined throughout the reproductive cycle of ewes grazing oestrogenic (Yarloop clover) pastures to provide an index of corpus luteum function. In ewes failing to conceive, luteal function declined after Day 13 and plasma progesterone concentrations had fallen to low `oestrus' values by the 15th day after mating. In contrast, normal cyclic ewes grazing non-oestrogenic pastures maintained significant luteal function until the 15th day after mating. The mean periods between matings in both groups were, however, similar (17·5 and 17·1 days).

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B. A. Stone and R. F. Seamark

Summary. Between Days 9 and 15 after oestrus, concentrations of pregnenolone, pregnenolone sulphate, dehydroepiandrosterone (DHEA), DHEA sulphate, androstenedione, oestrone and oestrone sulphate in free uterine fluid collected from non-pregnant gilts were greater than respective values in plasma (P < 0·05). The total contents of pregnenolone, progesterone, DHEA, testosterone, oestrone and oestradiol in washings from pregnant uteri exceeded (P < 0·05) respective non-pregnancy levels during this same period.

Concentrations of pregnenolone, pregnenolone sulphate, DHEA, DHEA sulphate, androstenedione, oestrone, oestrone sulphate and oestradiol in free uterine fluid recovered from gravid uteri were also higher (P < 0·05) than respective plasma values. By contrast, the progesterone concentration in uterine fluid from pregnant animals was lower (P < 0·001) than the plasma value.

Concentrations of DHEA, DHEA sulphate, androstenedione and oestrone sulphate in plasma of pregnant gilts between Days 9 and 15 after mating exceeded (P < 0·05) the respective concentrations in unmated gilts between Days 9 and 15 after oestrus. Plasma levels of pregnenolone sulphate were lower (P < 0·05) in the pregnant animals.

We therefore suggest that the endometrium of the pig can concentrate steroid hormones in uterine fluid and that increases in steroid levels in this milieu between Days 9 and 15 after coitus reflect steroidogenesis by embryonic tissues and modification of enzyme activities within uterine tissues under the influence of progestagens. The pool of steroid sulphoconjugates present in uterine fluid between Days 9 and 15 post coitum could serve as an important precursor source for progestagen, androgen and oestrogen synthesis by tissues of pig embryos before implantation.

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Department of Obstetrics and Gynaecology, University of Adelaide, South Australia 5001

(Received 18th April 1975)

The key role of the fetus in the endocrine changes preceding parturition in the sheep is now well established. Liggins et al. (1974) have shown that the complex series of events which result in parturition are initiated by the fetal hypothalamus. As yet, however, the factors which activate the hypothalamus itself are not known. In the adult, the pineal gland has been shown by many workers to exert an important modifying influence on the function of the hypothalamopituitary system (see Reiter, 1973) and the possible involvement of the pineal at parturition must be considered.

Among the substances which mediate the endocrine functions of the pineal gland are the methoxyindoles melatonin and 5-methoxytryptophol. Present methods for measurement of these hormones in body tissues and fluids are, however, unsatisfactory and most assessments of pineal function are based

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Agricultural Research Council Unit of Reproductive Physiology and Biochemistry, 307 Huntingdon Road, Cambridge CB3 0JQ and Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide, South Australia 5000


Important changes occur in the follicle that is destined to ovulate and also in the surrounding ovarian tissue of sheep during the 72 hr preceding ovulation. It is our purpose to describe the morphological and functional changes that take place in large follicles during the preovulatory period and to relate these to the local ovarian environment and general endocrine status prevailing at this time. A consideration of changes in the structure, blood supply and function of the ovaries in vivo, is followed by a discussion of the functional capacity of the individual ovarian components when isolated and studied in vitro. Against this background, the nature and sequence of events that occur in the preovulatory follicle and the mechanisms which regulate them

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Large intact follicles and granulosa cells were obtained from sheep ovaries at various stages of the oestrous cycle and were cultured in vitro for 7 days. The daily output of steroid hormones into the culture medium was determined using a procedure capable of measuring a wide range of steroids including immunoreactive oestrogen, testosterone, androstenedione, progesterone, 20α-hydroxy-pregn-4-en-3-one, 17α-hydroxyprogesterone, 17α,20α-dihydroxy-pregn-4-en-3-one, pregnenolone, 17α-hydroxypregnenolone and certain hydrogenated metabolites.

Intact follicles explanted from sheep on Day 4 of the cycle produced oestrogen (40 ng/mg follicular tissue per 24 hr) during the first 3 days in culture followed by large amounts of testosterone (100 ng/mg per 24 hr) and finally 17α-hydroxylated progestin. Follicles explanted at midcycle produced constant amounts of oestrogen (30 ng/mg per 24 hr) throughout the culture period; no other steroids were produced in significant quantities. The highest levels of oestrogen (75 ng/mg per 24 hr) were produced by follicles which had been explanted from sheep on Day 14 of the cycle. Oestrogen production declined rapidly in follicles explanted just before oestrus (late Day 15) and was replaced by the production of testosterone and 17α-hydroxylated progestin. The production of oestrogen and testosterone was very low in follicles explanted at oestrus; these follicles produced a transient peak of 17α-hydroxypregnenolone (30 ng/mg per 24 hr) followed by large amounts of progesterone (300 ng/mg per 24 hr) and 20α-hydroxy-pregn-4-en-3-one (250 ng/mg per 24 hr).

Monolayer cultures of granulosa cells produced only progesterone, 20α-hydroxy-pregn-4-en-3-one and pregnenolone, thus implicating the cells of the theca interna as the principal source of oestrogen, androgen and 17α-hydroxylated progestin.

Our findings indicate that the biosynthesis of oestrogen in the cells of the theca interna involve a sequence of steps including pregnenolone → 17α-hydroxypregnenolone→17α-hydroxyprogesterone→ testosterone or androstenedione→ oestrogen. During the transformation of follicles from oestrogen to progesterone secretors, steroid synthetic capacity is transferred from the theca interna to the membrana granulosa. The accumulation first of testosterone and then of 17α-hydroxypregnenolone suggests that the aromatase and then the desmolase systems limit steroid production in the theca interna during the period of transformation.

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P. Holm, S. K. Walker and R. F. Seamark

The influence of various in vitro procedures on embryo survival and the production of normal offspring was investigated in sheep. Zygotes produced from in vitro matured (IVM) and fertilized (IVF) oocytes derived from slaughtered Merino ewes were allocated to three culture treatments for 6.5 days. Two groups were cultured in vitro in the absence or presence of oviduct epithelial cells while the third group was cultured in vivo in the oviducts of synchronized ewes. A fourth group of zygotes obtained from superovulated Merino ewes was also cultured in vivo and served as controls. After culture, IVM–IVF morulae and blastocysts, and control embryos were transferred to final recipient ewes. Pregnancy was diagnosed at day 50 of gestation by ultrasonography and pregnancies were allowed to go to term. The survival to term of IVM–IVF zygotes cultured in vitro was reduced compared with both in vivo cultured IVM–IVF zygotes and control zygotes 25–35% versus 51–60%, respectively, P < 0.05). Day 6.5 IVM–IVF morulae had a lower survival rate than did control morulae regardless of culture treatment (P < 0.05), while survival rates of day 6.5 IVM–IVF blastocysts cultured in vivo did not differ from those of control blastocysts (P > 0.1). Both the gestation period and birth weight of IVM–IVF lambs were increased when compared with controls, the former significantly in all groups (154.0–154.9 days versus 150.6 days; P < 0.01), while the latter increase was on the borderline of significance (4·5–4·8 kg versus 4.0 kg; 0.01 ≤ P ≤ 0.1, respectively) and dependent on the prolongation of the gestation period. It is concluded that in vitro maturation and fertilization compromise subsequent embryonic and fetal development in sheep irrespective of the subsequent in vivo or in vitro culture treatment. Subjecting IVM–IVF zygotes to in vivo culture for 6.5 days minimizes only some of these effects, thus leading to the aberrant production of some offspring.

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C. D. Matthews, R. F. Seamark and M. V. Guerin

Summary. Plasma melatonin was measured at the summer and winter solstices and the autumn and spring equinoxes in Romney Marsh sheep held under natural conditions in South Australia (35°S). The amount of melatonin detected was generally related to the extent of natural darkness, though the melatonin onset was particularly delayed after dusk in winter compared with other seasons. The duration of detectable melatonin was shorter in summer than at any other season.

After each initial 24 h sampling, the sheep were resampled for a further 24 h in acutely extended darkness to mark the phase and duration of suprachiasmatic nuclei activity which is believed to be the source of the melatonin signal. The onset of high plasma melatonin was earlier than the time of natural sunset in spring and summer, but not different from the time of natural sunset in autumn and winter. The offset of high plasma melatonin was later than the time of natural sunrise at all times of year and particularly so in summer. Under the extended dark conditions, the duration of detectable melatonin was longer than that under natural photoperiod at all seasons of the year and the duration of melatonin was again shorter in summer than winter.

If melatonin measurements under the conditions of extended darkness do reflect the phase and duration of suprachiasmatic nuclei function then the natural light of the photoperiod can, particularly during long photoperiod conditions, mask the expression of the pacemaker. The findings may have implications for the timing of the breeding season in Romney Marsh sheep.

Keywords: melatonin; suprachiasmatic nuclei; seasonal breeding; sheep

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S. K. Walker, D. H. Smith and R. F. Seamark

Summary. Ovulation rate, median time to first ovulation, median time of all ovulations and median time from first to last ovulation were studied by repeated laparoscopy in Merino ewes. Treatments with FSH or PMSG significantly affected ovulation rate (8·4 ± 0·81 and 7·3 ± 1·21 respectively, P < 0·05) and in median time of all ovulations (60 and 54h respectively after progestagen sponge removal, P < 0·05). Differences in the median time to first ovulation (60 and 48 h) and median time from first to last ovulation (6 and 6 h) for the respective treatments were not significant. The synchrony of ovulation after both treatments was adversely affected by (1) the occurrence of premature ovulations before the onset of superovulation, (2) variability in the time of commencement of superovulation, and (3) variability in the time from first to last ovulation.

Administration of GnRH synchronized the timing of ovulation with both gonadotrophin treatments. This synchrony was due to a reduction in the period during which superovulation began and in the interval from first to last ovulation. The median time of all ovulations was significantly less with FSH + GnRH than with PMSG + GnRH (45 and 48 h after progestagen sponge removal, respectively, P < 0·05). Administration of GnRH at 16, 20 or 24 h after progestagen sponge removal significantly affected all traits examined except ovulation rate. Administration at 20 and 24 h produced an equally good synchrony of ovulation which was better than that obtained at 16 h.

We suggest that the use of GnRH in embryo collection programmes appears justified and is likely to improve embryo yields due to improved rates of fertilization.

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Progesterone concentrations in the peripheral plasma of pregnant ewes grazing oestrogenic (yarloop clover) and non-oestrogenic (rye grass) pastures were compared. Whilst the overall pattern of plasma progesterone throughout pregnancy in the two groups was similar, the mean of values determined from the 90th day of gestation in ewes on yarloop was significantly lower than those on grass.

No significant differences were recorded in the length of pregnancy or birth weight of lambs (P>0·05) although some evidence of maternal dystocia was obtained for the ewes on yarloop.

In relation to parturition, mean plasma progesterone values in both groups began to fall 96 hr before birth, at first gradually then rapidly in the 16 hr preceding birth. Ewes on grass had a higher (P<0·01) plasma progesterone concentration 0 to 8 hr before birth but neither the level nor the pattern of progesterone disappearance appeared to be related to the lambing difficulties observed.

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D. O. Kleemann, S. K. Walker and R. F. Seamark

Two experiments were conducted to determine whether administration of progesterone during early pregnancy affects fetal growth in sheep and if any effect is specific to the days of treatment. In the first experiment, Merino ewes were randomly allocated to four treatment groups and inseminated at a synchronized oestrus. Three groups received progesterone on days 1–3, 3–6 or 1–6 of pregnancy while the fourth group was untreated. Concentrations of progesterone in peripheral plasma increased (P < 0.05) in all treatment groups. Fetal growth (to day 74) was greater in all treatment groups than in the control group (P< 0.001) and was greatest when treatments started on day 1. Pregnancy rate was not affected by progesterone treatment on days 3–6, but was reduced (P < 0.05) when treatment began on day 1. In the second experiment, embryos that had been exposed to either a normal (control) or a high concentration of progesterone on days 1–3 were randomly transferred, within groups, to recipient ewes that had or had not been treated with progesterone on days 1–3. In another group, embryos were exposed to a high concentration of progesterone on days 1–3 and the oviducts of the ewe were ligated. An increase in fetal mass was observed in the recipient group that had been treated with progesterone (P < 0.01) but was not observed in the initial group treated with progesterone. A greater fetal mass was also obtained when embryos that had been ligated in the oviducts of ewes treated with progesterone (P < 0.05) were transferred. This effect occurred irrespective of whether the final recipients received progesterone. These findings indicate that progesterone supplementation to ewes during the first 3 days of pregnancy enhances the growth of surviving fetuses. It is concluded that progesterone treatment or ligation of the oviducts during the first three days of pregnancy induce changes in embryo development that affect subsequent development.