Variation in the oxytocin content of caprine corpora lutea across the breeding season

Ovarian tissues were obtained from cyclic goats during the early, mid and late breeding season. Immunoreactive oxytocin was measured by RIA in tissue extracts after chromatography on octadecylsilica cartridges. Luteal oxytocin concentrations were significantly greater during the early breeding season than during the mid or late breeding season. Oxytocin is luteolytic in goats. High concentrations of luteal oxytocin may be related to the high incidence of short estrus at the onset of the breeding season.     

Effects of intrauterine infection on the function of the corpora lutea formed after first postpartum ovulations in dairy cows

This study was carried out to determine the relationship between postpartum intrauterine infections, endocrine patterns and the function of corpora lutea formed following the first postpartum ovulations in dairy cows. Blood samples were collected daily starting from the day of parturition until 30 d after parturition or until the second postpartum estrus, whichever occurred first. Sera were assayed for progesterone (P(4)), prostaglandin F(2alpha) metabolite (PGFM), and luteinizing hormone (LH) concentrations. Palpations per rectum and real-time ultrasound scanning of the reproductive tracts were carried out in all cows once every 4 d for 1 mo, starting from Day 4 after parturition. In addition, endometrial swabs were collected aseptically from each cow once every 4 d during the first month postpartum. The swabs were cultured for aerobic and anaerobic bacteria. Twelve cows (60%) exhibited short estrous cycles (SC; 6 to 14 d long) following first postpartum ovulations. The mean preovulatory LH surges and LH patterns during the first postpartum cycles were similar in both groups, leading us to believe that lack of luteotrophic stimulation was not a factor in the occurrence of SC. Bacterial isolations were frequent in SC cows. The occurrence of moderate to heavy bacterial growth patterns and the repeated isolations of the similar organisms during postpartum suggests the persistence of uterine infections in SC cows. Increases in PGFM concentrations prior to luteolysis in SC cows were associated with moderate to heavy infection. Thus, postpartum uterine infections do not appear to affect ovulations, but prostaglandin (PGF(2alpha)) released in response to uterine infection may contribute to early demise of the corpus luteum formed after the first postpartum ovulation.     

Progesterone synthesis and histology of postpartum bovine corpora lutea

In vitro progesterone (P(4)) synthesis by corpora lutea (CL) from the first, second or third ovulation after calving was compared and correlated with their histology and cytology. The CL were removed 7 to 12 days after ovulation, and luteal cells isolated by digestion with collagenase. The response of isolated cells to luteinizing hormone (LH) was determined. Hematoxylin-eosin stained tissues were used to study histology, and the distribution of cell types was estimated by stereological methods. Ovulation occurred within 25 days of calving and interovulatory intervals were short, 12.1 +/- 3.9 days and 12.6 +/- 4.8 days, respectively. The CL removed after first ovulation were smaller and contained fewer live cells than those obtained after subsequent ovulations. Stimulation by LH in vitro was independent of cycle number or day of cycle but was related to the histology of the tissue. The CL composed of large cells (> 24 mum) with vacuolated cytoplasm contained high amounts of P(4) but were not stimulated by LH. Conversely, CL composed of small and medium- sized cells (10 to 20 mum) and/or intact larger cells contained little P(4) but were stimulated by LH. These observations indicate that the response of postpartum CL to LH in vitro is dependent upon the structural integrity of the tissue at the time of removal. Furthermore, these observations suggest that the short life of CL during the postpartum period may not be due to the absence of luteotrophic support, but to the action of a luteolytic mechanism.     

Formation and function of GnRH-induced subnormal corpora lutea in cyclic ewes

Of 19 dioestrous ewes given 50 micrograms GnRH on Day 10 of the oestrous cycle, 15 (79%) formed corpora haemorrhagica within 2 days after injection of GnRH. After excision of the Day 10 spontaneous CL, the GnRH-induced CL were short lived when compared to spontaneous CL in saline-treated ewes (3.1 +/- 0.4 vs 17.3 +/- 0.3 days, respectively). Hysterectomy of ewes bearing the GnRH-induced CL prevented regression of the short-lived CL, thus extending functional lifespan greater than or equal to 38 days. Serum concentrations of progesterone produced by the GnRH-induced CL in hysterectomized ewes were less than those observed during a comparable interval (Days 7-14) in saline-treated, non-hysterectomized ewes (2.24 +/- 0.1 vs 3.67 +/- 0.15 ng/ml, respectively; P less than or equal to 0.001). When GnRH was given before (5 h before) or during (5 h after) PGF-2 alpha-induced regression of the Day 10 spontaneous CL, the GnRH-induced CL which formed were also short-lived. In contrast, when GnRH was given following (36 h after) PGF-2 alpha-induced regression of the Day 10 spontaneous CL, the CL which formed were not different in lifespan or production of progesterone from spontaneous CL. Efforts to enhance function of the GnRH-induced subnormal CL by treating ewes with the synthetic progestagen, norgestomet, to suppress follicular development after CL formation, were unsuccessful.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of corpora lutea numbers in Booroola--Texel ewes using transrectal ultrasound

Ovulation rates in Booroola--Texel ewes are currently determined by laparoscopic examination of the ovaries during the luteal phase allowing for carrier status of the ewe to be diagnosed. However, this is an invasive procedure and the aim of this work was to determine whether or not transrectal ultrasound would be an acceptable and accurate alternative to laparoscopy for this purpose in ewes. Transrectal ultrasound was performed using a 5.0 MHz transducer in 18 ewes restrained in the standing position. They were then euthanized and laparoscopy was performed, following which the reproductive tracts were recovered. An additional 22 ewes were subjected to scanning in dorsal recumbency, using a 7.5 MHz transducer, at various stages of the luteal phase before being euthanized and the reproductive tracts recovered. The ovarian structures identified using each method were then compared. The results demonstrated that laparoscopy is an accurate method of determining ovulation rates in ewes. Transrectal ultrasound examination appeared to be an acceptable and non-invasive procedure. The use of a 7.5 MHz transducer with the ewes in dorsal recumbency produced the best results and clearest images of the ovaries, which allowed for the determination of the number of corpora lutea (CL) and the presence of other structures. Accuracy improved with experience in the technique, the stage of the luteal phase at which the examination was performed, the number of CL present in the ovary and the age of the ewe. Further work is needed to confirm this finding due to the small numbers of animals in the present study. This ultrasound technique, however, has the potential to provide an alternative to laparoscopy for the determination of numbers of CL in ewes.     

Function and lifespan of corpora lutea in ewes treated with exogenous oxytocin

Oxytocin was administered to Dorset and Shropshire ewes in one experiment and to Dorset ewes in a further 4 experiments. In Exp. 1, concentrations of plasma progesterone and lengths of the oestrous cycle in ewes given oxytocin subcutaneously twice a day on Days 0-3, 2-5, 4-7, 6-9, 8-11, 10-13, 12-15 or 14-17 were similar to those of control ewes. In Exp. 2, intraluteal infusions of oxytocin from Day 2 to Day 9 after oestrus had no effect on concentration of progesterone, weight of CL collected on Day 9 or length of the oestrous cycle. In Exp. 3, intraluteal infusions of oxytocin on Days 10-15 after oestrus had no effect on weight of CL collected on Day 15. In Exp. 4, s.c. injections of oxytocin on Days 3-6 after oestrus had no effect on weight of CL collected on Day 9, concentrations of progesterone or length of the oestrous cycle. In Exp. 5, s.c. injections of oxytocin twice a day did not affect the maintenance and outcome of pregnancy in lactating and nonlactating ewes. Exogenous oxytocin, therefore, does not appear to affect luteal function at any stage of the ovine oestrous cycle although oxytocin has been reported by others to alter ovine CL function.     

Effect of immunization of ewes against prostaglandin F-2 alpha on the life-span of corpora lutea and oestrous behaviour during two breeding seasons

Two adjuvants, Freund's complete adjuvant (FCA) and GNE (proprietary product; Intervet Ltd, The Netherlands), were used to immunize cyclic Finnish Landrace ewes (4-6/treatment) against a prostaglandin F-2 alpha-human serum albumin (PGF-HSA) conjugate. Ewes were randomized to the following treatments: (a) control-untreated, (b) control-5 mg HSA in FCA (control-HSA), (c) 5 mg PGF-HSA in FCA (FCA-5 mg), (d) 15 mg PGF-HSA in FCA (FCA-15 mg), (e) 5 mg PGF-HSA in GNE (GNE-5 mg) and (f) 15 mg PGF-HSA in GNE (GNE-15 mg). Ewes were monitored for oestrus (twice daily) and ovarian activity (progesterone concentrations in blood samples taken twice weekly) for 2 consecutive breeding seasons. In the first breeding season, the mean number of oestrous periods detected was 6.0, 5.7, 0.0, 0.2, 1.8 and 0.5 in control, control-HSA, FCA-5 mg, FCA-15 mg, GNE-5 mg and GNE-15 mg-assigned ewes, respectively [pooled standard error of difference (s.e.d.) = 1.2]. A persistent CL formed, on average, 10.0, 10.0, 29.8 and 32.5 days after primary immunization (pooled s.e.d. = 14.6) in 6/6 FCA-5 mg, 6/6 FCA-15 mg, 5/6 GNE-5 mg and 4/4 GNE-15 mg-assigned ewes, respectively; these CL were maintained for, on average, 138.7, 139.0, 127.8 and 129.0 days, respectively (pooled s.e.d. = 15.9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian function in ewes at the onset of the breeding season

Transrectal ultrasonography of ovaries was performed each day, during the expected transition from anoestrus to the breeding season (mid-August to early October), in six Western white-faced cross-bred ewes, to record ovarian antral follicles > or = 3 mm in size and luteal structures. Jugular blood samples were collected daily for radioimmunoassay (RIA) of follicle-stimulating hormone (FSH), oestradiol and progesterone. The first ovulation of the breeding season was followed by the full-length oestrous cycle in all ewes studied. Prior to the ovulation, all ewes exhibited a distinct increase in circulating concentrations of progesterone, yet no corpora lutea (CL) were detected and luteinized unovulated follicles were detected in only three ewes. Secretion of FSH was not affected by the cessation of anoestrus and peaks of episodic FSH fluctuations were associated with the emergence of ovarian follicular waves (follicles growing from 3 to > or = 5 mm). During the 17 days prior to the first ovulation of the breeding season, there were no apparent changes in the pattern of emergence of follicular waves. Mean daily numbers of small antral follicles (not growing beyond 3 mm in diameter) declined (P < 0.05) after the first ovulation. The ovulation rate, maximal total and mean luteal volumes and maximal serum progesterone concentrations, but not mean diameters of ovulatory follicles, were ostensibly lower during the first oestrous cycle of the breeding season compared with the mid-breeding season of Western white-faced ewes. Oestradiol secretion by ovarian follicles appeared to be fully restored, compared with anoestrous ewes, but it was not synchronized with the growth of the largest antral follicles of waves until after the beginning of the first oestrous cycle. An increase in progesterone secretion preceding the first ovulation of the breeding season does not result, as previously suggested, from the ovulation of immature ovarian follicles and short-lived CL, but progesterone may be produced by luteinized unovulated follicles and/or interstitial tissue of unknown origin. This increase in serum concentrations of progesterone does not alter the pattern of follicular wave development, hence it seems to be important mainly for inducing oestrous behaviour, synchronizing it with the preovulatory surge of luteinizing hormone (LH), and preventing premature luteolysis during the ensuing luteal phase. Progesterone may also enhance ovarian follicular responsiveness to circulating gonadotropins through a local mechanism.     

A definition of the breeding season of Pool Dorset ewes

The seasonality of oestrus and ovulation of 62 Poll Dorset ewes was examined over a period of 16 months commencing October, 1981. Laparoscopic observations showed that the majority of these ewes were anovular during the South Australian spring and early summer (September to December). The number of ewes with corpora lutea (93.6%) and the mean ovulation rate (1.62) reached a peak in June. Oestrus was stimulated in anovular ewes in late spring by the introduction of vasectomised rams. The mean ovulation rate at the teased ovulation was 1.68, although teased ewes did not exhibit oestrus at this ovulation.     

Effects of season and lactation on luteinizing hormone secretion in postpartum ewes

Effects of season, postpartum interval and short-term weaning were investigated on luteinizing hormone (LH) secretion in ewes. Blood samples were collected at 10-min intervals for 4 h (basal period). Then gonadotropin-releasing hormone (GnRH) was administered and 10 more blood samples were collected over an additional 4 h period. The effects of day post partum (5, 20 or 40) and short-term weaning (weaned Day 37, tested Day 40 post partum) on basal and GnRH-induced LH secretion were tested. Mean basal concentrations of LH for ewes on Day 5, 20 or 40 post partum ranged from 1.6 to 4.6 ng/ml and did not differ. Mean concentrations of LH during the post-GnRH sampling interval were greater (P<0.01) for ewes bled on Day 20 or 40 post partum (12.3 and 11.8 ng/ml, respectively) than for ewes bled on Day 5 or for unbred control ewes (6.7 and 5.8 ng/ml, respectively). Weaning on Day 37 depressed GnRH-induced LH secretion on Day 40 post partum (8.18 ng/ml; P<0.05). Seasonal changes in LH secretion on Day 20 or 40 post partum in January, March or June lambing ewes were also tested. There was no difference in basal or GnRH-induced LH secretion between Day 20 or 40 post partum among groups in January or March.. In June, ewes had lower (P<0.01) basal and GnRH-induced LH secretion on Day 20 post partum than ewes did on Day 40 post partum. Across month of the year, on Day 20 post partum, ewes lambing in March released more LH in response to GnRH than ewes lambing in January (P=0.07) or June (P<0.05). Response to GnRH on Day 20 post partum was similar for ewes lambing in January or June (P>0.1). Ewes lambing in January released less (P<0.01) LH on Day 40 post partum than ewes lambing in March or June; however, no difference was detected between the latter two groups (P>0.1). Thus, seasonal modifications of the releasable pool of LH may mask or modify the effect of the postpartum interval upon this endocrine response.     

Ovarian function in ewes during the transition from breeding season to anoestrus

Transrectal ovarian ultrasonography was conducted in six Western white-faced ewes for 35 days from the last oestrus of the breeding season, to record the number and size of all ovarian follicles > or = 3 mm in diameter and luteal structures. Blood samples were collected once a day for estimation of serum concentrations of follicle-stimulating hormone (FSH), oestradiol and progesterone. Each ewe had five follicular waves (follicles growing from 3 to > or = 5 mm in diameter) over the scanning period. The duration of the growth phase of the largest ovarian follicles did not differ (P > 0.05) between waves, but follicular static and regressing phases decreased significantly (P < 0.05) after the decline in serum progesterone concentrations at the end of the last luteal phase of the breeding season. The intervals between the five follicular waves were: 9.2+/-0.4, 5.2+/-0.7, 8.3+/-0.8 and 5.8+/-0.7 days; the two shorter intervals differed (P < 0.05) from the two longer intervals. Using the cycle-detection program, rhythmic increases in serum FSH concentrations were detected in all ewes; the amplitude, duration and periodicity of FSH fluctuations did not vary (P > 0.05) throughout the period of study. The number of identified FSH peaks (7.8+/-0.5 peaks per ewe, per scanning period) was greater (P < 0.05) than the number of emerging follicular waves. Serum concentrations of oestradiol remained low (< or = 1 pg/ml) on most days, in five out of the six ewes studied, and sporadic elevations in oestradiol secretion above the non-detectable level were not associated with the emergence of follicular waves. The ovulation rate was lower than that seen during the middle portion of the breeding season (November-December) in white-faced ewes but the transitional ewes had larger corpora lutea (CL). Maximal serum concentrations of progesterone appeared to be lower and the plateau phase of progesterone secretion appeared to be shorter during the last luteal phase of the ovulatory season in comparison to the mid-breeding season of Western white-faced ewes. During the transition into anoestrus in ewes, the endogenous rhythm of FSH release is remarkably robust but the pattern of emergence of sequential follicular waves is dissociated from FSH and oestradiol secretion. Luteal progesterone secretion is suppressed because of fewer ovulations and diminished total luteal volume, but it may also result from diminished gonadotropic support. These season-related alterations in the normal pattern of ovine ovarian cycles appear to be due to reduction in ovarian responsiveness to gonadotropins and/or attenuation in secretion of luteinizing hormone (LH) occurring at the onset of the anovulatory season in ewes.     

Immunocytochemical localization of oxytocin in corpora lutea and luteinized cysts from anoestrous ewes stimulated with gonadotrophin-releasing hormone

Summary Anoestrous Romney Marsh ewes with or without progesterone pretreatment were injected with multiple low-doses of gonadotrophin-releasing hormone followed by a single, larger bolus. Blood samples were taken at twelve-hourly intervals for progesterone radioimmunoassay. Ewes were slaughtered on day 3 or 5 after the bolus injection, and the ovaries were collected for histology and immunocytochemical examination for oxytocin-immunocreactivity. The corpora lutea of all ewes killed on day 3 had similar weights and morphology. The ovaries of those ewes which were not pretreated with progesterone also contained some luteinized cysts. Ewes slaughtered on day 5 were separated into 2 groups according to plasma progesterone profiles, which were either rising (normal), or falling after a transitory rise (abnormal). Those ewes pretreated with progesterone all had a normal progesterone profile whereas, of 14 ewes not pretreated with progesterone, 6 were normal and 8 abnormal. Corpora lutea were significantly lighter in the abnormal group and the ovaries of most of these ewes also contained luteinized cysts. All corpora lutea and luteinised cysts showed staining for oxytocin-immunoreactivity although the staining intensity was variable. In corpora lutea from normal ewes oxytocin was restricted to large luteal cells. In addition tissues from abnormal ewes also contained many cells with an atypical elongated shape which stained for oxytocin-immunoreactivity. These results show that progesterone pretreatment is needed for both normal morphological and endocrine development of corpora lutea in anoestrous ewes stimulated with gonadotrophin-releasing hormone.     

Role of prostaglandin F-2 alpha and oxytocin in the regression of GnRH-induced abnormal corpora lutea in anoestrous ewes

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 less than 0.05) between Days 3 and 5 in ewes in Groups +P and -P with 'normal' CL but increased (P less than 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 less than 0.01) or +P (P less than 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 less than 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.     

Cytokeratin expression in bovine corpora lutea

Cytokeratin (CK)-positive cells were obtained from bovine corpora lutea. When cultured, these cells behave like CK-positive endothelial cells obtained from bovine large blood vessels. The origin of CK-positive cells has now been studied in 45 bovine corpora lutea of different estrous cycle stages. Additionally, 7 corpora lutea of pregnant cows were examined. The tissues were grouped into early stage (days 2 to 4), secretory stage (days 5 to 17) and late stage (days 18 to 21) according to gross morphology, wet weight and total progesterone content. One portion of a corpus luteum was used for immunohistochemistry, and another for Western blot analysis. Twenty-six of the 45 corpora lutea showed CK expression, as confirmed by immunostaining and Western blotting. Cytokeratin expression was found in all corporalutea from the early stage, in 14 of 26 corpora lutea from the secretory stage, and 3 of 10 from the late stage. Early stage corpora lutea displayed zonation such that a high number of CK-positive luteal cells occurred in the region of the previous granulosa layer and a very low number in the previous thecal layer. Secretory CK-positive corpora lutea showed uniformly distributed, predominantly large luteal cells. In secretory corpora lutea of group A, CK-positive cells and a distinct microvascular tree were seen, the latter visualized by factor VIII-related antigen immunolabelling of endothelial cells. Group B showed none or very few CK-positive cells. Corpora lutea of pregnant cows behaved like corpora lutea of group B. Roughly 1% of CK-positive cells closely associated with the capillary wall were sometimes reminiscent of endothelial cell sprouts.