A. Meikle

The effect of subluteal levels of exogenous progesterone on follicular dynamics and endocrine patterns during the early luteal phase of the ewe

Nineteen Corriedale ewes were treated with an im dose of a PGF2alpha during the luteal phase to synchronize estrus. After ovulation had been detected by using ultrasonography (Day 0); the ewes were randomly assigned to 2 different groups. In 11 ewes a CIDR, which had previously been used for 10 d, was inserted on the fourth day after ovulation. The ewes then received a dose of PGF2alpha on Day 5 to induce luteolysis. The CIDR remained in place until the end of the experiment (Day 9). Control ewes (n = 8) received no treatment. Blood samples were taken daily for estradiol, progesterone and FSH determinations. In the untreated ewes, 2 follicular waves were detected in all of the animals throughout the monitoring period, with a mean wave interval of 4.5 d. The total number of follicles which were > or =2 mm decreased from Day 0 to Day 4 (8.8+/-1.0 to 5.3+/-0.6; P< or =0.05) and then increased at Day 7 (7.5+/-0.9; P< or =0.05). The growth profiles of both the largest and the second largest follicles of Wave 1 showed significant divergence, while no divergence was observed in Wave 2. Serum estradiol concentrations decreased significantly from the day before to the day of ovulation and then increased again during the growing phase of the largest follicle of Wave 1. Concentrations of FSH were high on the day of emergence of both waves, but while a significant decline was observed after emergence in Wave 1, the levels remained high in Wave 2. In 8 of the 11 treated ewes, the largest follicle of Wave 1 was still present on the ninth day after ovulation (persistent follicle). In the other 3 ewes, the largest follicle of Wave 1 was already regressing on the day that the treatment was administered, and the largest follicle that was present on Day 9 originated from Wave 2 (nonpersistent follicle). In persistent follicle ewes, the largest follicle of Wave 1 prolonged its lifespan significantly, attaining the maximum diameter (Day 8.1+/-0.8) later than in untreated (Day 3.0+/-0.4) and nonpersisted follicle ewes (Day 2.0+/-0.6). The total number of follicles decreased in persistent follicle ewes between Day 0 and Day 4 (7.9+/-1.5 to 4.5+/-0.5, respectively; P< or =0.05) and remained low until the end of the experiment. Progesterone concentrations (nmol/L) between Days 6 and 9 were significantly different between untreated and persistent follicle ewes (12.8+/-1.0 vs. 9.4+/-1.0, P< or =0.02). The present study confirms that the largest follicle of Wave 1 is dominant in the ewe and that subluteal progesterone concentrations can prolong its lifespan and extend this dominance.

EFFECTS OF ESTRADIOL AND PROGESTERONE ON THE REPRODUCTIVE TRACT AND ON UTERINE SEX STEROID RECEPTORS IN FEMALE LAMBS

The effects of estradiol-17 beta (E(2)) and progesterone (P) on the reproductive tract and on uterine estrogen receptors and P receptors were studied in 2-mo-old female lambs (n = 11). On Days 0, 1 and 2, E(2) (1 ug/kg, Group E, n = 4), P (0.3 mg/kg, Group P, n = 4) or corn oil (control) vehicle (Group C, n = 3) were administered, and in Day 3 all lambs were slaughtered. Group E (n = 12) had E(2) serum concentrations (mean +/- SEM) of 43.8 +/- 2.2 pmol L , similar to that of the follicular phase; while P concentrations in Group P (n = 12) were similar (2.8 +/- 0.18 nmol L ) to those of the luteal phase of the ewe estrous cycle. The E(2) treatment increased the reproductive tract weight, while P treatment increased only the uterine weight. Both E(2) and P receptors from upper and middle uterine zones (including the myometrium, endometrium and caruncles) were determined by binding assays with tritiated hormones, dextran-charcoal separation and inverse Scatchard analysis. Both the E(2) and P treatments decreased E(2) and P receptor concentrations in upper and middle zones, although the upper zone had higher receptor concentrations than the middle zone (P < 0.01). E(2) receptor concentrations in the upper zone (mean +/- SEM, fmol mg prot) were 1236 +/- 34, 667 +/- 80 and 444 +/- 103 for Groups C, P and E, respectively. The P receptor concentrations were 2434 +/- 135, 1273 +/- 102 and 1536 +/- 213 for the same groups. The high uterine P receptor concentrations allowed P action without prior estrogen priming of female lambs. The present results suggest that E(2) and P might down-regulate their own and each others receptors during development. The biological responses induced by E(2) and P, as measured by the reproductive tract weight, demonstrated that at an early stage of development uterine receptors are physiologically active.

Accuracy of evaluation of ovarian structures by transrectal ultrasonography in ewes

The objective of this study was to assess the accuracy of estimating the size and number of corpora lutea (CL) and ovarian follicles by ultrasonography (US) of the ewe in standing position. US observations were compared with those made at subsequent postmortem examination of the ovaries. Corriedale ewes (n=50) of unknown reproductive history and at random stages of the oestrus cycle were used for the study. Transrectal US was performed using a 7.5 MHz transducer with the ewe in standing position. The ewes were slaughtered 12 h after the US examination and the ovaries collected, dissected and the number and size of the CL and follicles evaluated. CL were classified as functional or non-functional on the basis of their colour and follicles were classified by size (2, 3, 4 and > or =5 mm). Accuracy of US was assessed by calculating its positive predictive value and sensitivity. The data were evaluated by Pearson correlation and linear regression analysis. The predictive value and sensitivity of US was 100% for the presence and 96% for the absence of CL. In four out of five ewes with double ovulations, the presence of both CL was correctly diagnosed by US. For functional CL, the sensitivity of US was 100%. The regression coefficient for CL diameter was significant (P< or =0.001; r2=0.4; n=35). The size of 85 of 117 follicles was accurately determined. The correlation between numbers of follicles counted by US and postmortem increased with size of follicle from r=0.44 (P< or =0.01) for 2 mm to r=0.85 (P< or =0.001) for >or =5 mm diameter follicles. The regression between the diameter of follicles determined by the two methods was significant (P< or =0.001; r2=0.8; n=117). The predictive value of US for the number of follicles was high (98-100%) for all follicle sizes except for 3 mm diameter follicles (predictive value 71%). Similarly, the sensitivity was high for all sizes of follicles (90-95%) except for those of 2 mm diameter (62%). It was concluded that ultrasound scanning provides a highly accurate method for determining the number of CL and follicles > or =4 mm diameter but that its predictive value and sensitivity are lower for smaller diameter follicles. The regression equation for diameter measured by US on that evaluated postmortem explained more of the variation for follicles than for CL.

Circulating gonadotrophins and follicular dynamics in anestrous ewes after treatment with estradiol-17β

Plasma FSH, LH, estradiol (E2) and progesterone (P4) profiles and patterns of follicular growth and regression by ultrasonography were determined after E2 treatment (1 microg/kg) in anestrous ewes. Fifteen ewes were treated with one (group I, n=7) or two (group II, n=4) i.m. injections of E2 with a 24h interval, or two oil injections with a 24h interval (group C, n=4). Blood samples for E2, P4, FSH and LH determinations were collected daily 4 days before the initiation of the treatment (day 0), when bleeding increased to every 2h starting 2h before treatment until 56h after the first injection and from then on every 6h until day 8, and twice per day till the end of the experiment (day 9). During the experimental period (days -4 to 9), transrectal ultrasonic examinations were carried out daily using a 7.5 MHz linear array probe. Number and size of follicles > or =3mm in diameter were recorded. No estrous was detected before, during or after treatment. LH and FSH surges were observed 10-18h after the first E2 injection. The second E2 injection stimulated another release of LH but no surges. E2 inhibited FSH levels before the surge and the second E2 injection induced a longer inhibition. No ovulation was detected by ultrasonography during the experimental period and P4 levels remained low (<0.7 nmol/l) before, during and after the treatment in all ewes. There was an effect of E2 treatment on the diameter of the largest follicle, a decrease could be observed 3 days after the first injection in both ewes of groups I and II. The E2-treated groups had a higher frequency of ewes showing wave emergence on day 3 (day 1.5+/-1,2.4+/-0.4 and 2.5+/-0.5 for control, groups I and II). LH and FSH surges were observed after E2 treatment, but were not able to provoke ovulation neither luteinization. In contrast, the treatment was associated with the regression of the largest follicle and with emergence of a new follicular wave on day 3.

Priming effect of exogenous oestradiol on luteinizing hormone secretion in prepubertal lambs

The effect of repeated administration of oestradiol-17beta on luteinizing hormone (LH) secretion was studied in 3-month old female lambs (n = 20). Animals were randomly divided in five groups and treated or not treated (group I) with 1, 2 or 3 i.m. injections of oestradiol in corn oil vehicle (1 microg/kg). Animals were slaughtered 12 (group II) and 24 h (group III) after the first injection, 24 h after the second injection (group IV) and 24 h after the third injection (group V). Animals in groups IV and V were catheterized and blood samples were collected every 4 h starting before treatment until time of sacrifice. In the rest of the groups blood samples were taken at the time of slaughter. The number of follicles > 1 mm in diameter on the ovarian surface were recorded and classified according to size. Maximum levels of oestradiol ranged from 103 to 250 pmol/l and returned to baseline within 12 to 16 h after each injection. LH secretion showed a consistent pattern in all lambs, with increases between 8 and 16 h after each oestradiol injection. The highest amplitude and longest duration (8-12 h) of LH secretion was recorded after the second oestradiol injection. Preliminary data indicated that FSH secretion resembled that of LH. There was an increase in the number of follicles with a diameter of more than 2 mm. Plasma concentrations of progesterone and cortisol were low and did not differ within groups or between treatments. The findings confirm that the pituitary LH release system in ewe lambs is sensitive to the stimulatory effects of oestradiol long before puberty. Results indicate that priming with oestradiol increases pituitary LH release to subsequent challenges of oestradiol, but long time exposure to oestradiol may have a negative effect on LH secretion. Although none of the oestradiol-treated lambs ovulated, the increase in the number of large follicles with repeated injections of oestradiol suggests that small follicles were gonadotrophin-responsive and stimulated by the gonadotrophin release.

Estrogen and progesterone receptor content in the pituitary gland and uterus of progesterone-primed and gonadotropin releasing hormone-treated anestrous ewes

The objective of this work was to investigate the effect of progesterone (P) and gonadotropin-releasing hormone (GnRH) treatment on estrogen receptor (ER) and P receptor (PR) concentrations in the pituitary gland and uterus of anestrous ewes. Ewes were either not treated (group C, n = 4); were treated with 0.33 g P-controlled internal drug release (P-CIDR) for 10 days (group P, n = 4), with GnRH, 6.7 ng i.v. injections every 2 h for 18 h followed by a 4 microg bolus administration of Receptal at 20 h (group GnRH, n = 4), or with a combination of the P and GnRH treatment (group P + GnRH, n = 3). Ewes were humanely killed either at the beginning of the experiment (group C), when the CIDR was removed (group P), or 24 h after the GnRH bolus treatment (groups GnRH and P + GnRH). Progesterone treatment increased serum P concentrations, indicating that the treatment was effective. All GnRH treated ewes had similar luteinizing hormone (LH) surges, which lasted 8 h. At slaughter, estradiol (E2) concentrations in the GnRH group were higher than in groups C, P, and P + GnRH. Treatment with GnRH increased more than 10-fold the content of ER and PR in the pituitary gland without altering steroid receptor concentrations in the uterus. When GnRH was combined with P the uterine receptor contents were higher than with P treatment alone. The treatment with P decreased ER and PR content in the uterus, but had no effect on the pituitary gland. The results show that regulation by P and GnRH of ER and PR content in anestrous ewes is tissue-specific.

Restoration of estrogen and progesterone uterine receptors during the ovine postpartum period

Estrogen and progesterone uterine cytosolic receptors (E2R and P4R) were investigated in postpartum ewes that lambed during the breeding season. Reproductive organs of 10 multiparous Corriedale ewes were studied after ovarihysterectomies at Day 1 (n = 2), 5 (n = 4), 17 (n = 2) or 30 (n = 2) post partum. E2R and P4R were determined by binding assays with tritiated hormones, dextrancharcoal separation and inverse Scatchard analysis. Follicles present at ovarian surface at time of ovarihysterectomy were recorded and progesterone profiles were determined to evaluate ovarian activity restoration. High-affinity binding sites for both receptors were found in all uterine tissue samples; Kd values (Mean ± SEM) were 0.42 ± 0.04 nM (n = 10) for E2R and 0.76 ± 0.10 nM (n = 10) for P4R. Binding affinity did not differ in the different postpartum days. Both E2R and P4R. levels in early post partum (Days 1 to 5) were significantly lower than late postpartum (Days 17 to 30). Mean values (± SD) expressed as fmolmg protein for E2R were 173 ± 54 (n = 6) and 322 ± 77 (n = 4; P < 0.05), and for P4R they were 253 ± 84 (n = 6) and 604 ± 356 (n = 4; P < 0.05). There was an inverse relationship between uterine weights and E2R contents, (r = −0.64, n = 10; P < 0.05) and P4R contents, (r = −0.75, n = 9; P < 0.05). While only 1 ovarihysterectomized ewe (16) at Days 1 to 5 had a follicle larger than 4 mm (presumptive estrogen-active follicle) all the ewes (4/4) ovarihysterectomized at Days 17 to 30 presented follicles of this size (P < 0.05). This data suggests that the postpartum restoration of uterine receptor levels was probably due to the recovery of the ovarian activity and the subsequent up-regulation exerted by estrogen of follicular origin. The P4R values were positively correlated with E2R concentrations, (r = 0.78; P < 0.05) and support this hypothesis. In conclusion, we have shown that the uterine tissue recovers its steroid receptor contents as uterine involution and reassumption of ovarian activity takes places.

A polymorphism in the insulin-like growth factor 1 gene is associated with postpartum resumption of ovarian cyclicity in Holstein-Friesian cows under grazing conditions

BackgroundInsulin-like growth factor 1 (IGF-1) gene is considered as a promising candidate for the identification of polymorphisms affecting cattle performance. The objectives of the current study were to determine the association of the single nucleotide polymorphism (SNP) IGF-1/Sna BI with fertility, milk production and body condition traits in Holstein-Friesian dairy cows under grazing conditions.MethodsSeventy multiparous cows from a commercial herd were genotyped for the SNP IGF-1/Sna BI. Fertility measures evaluated were: interval to commencement of luteal activity (CLA), calving to first service (CFS) and calving to conception (CC) intervals. Milk production and body condition score were also evaluated. The study period extended from 3 wk before calving to the fourth month of lactation.Results and discussionFrequencies of the SNP IGF-1/Sna BI alleles A and B were 0.59 and 0.41, respectively. Genotype frequencies were 0.31, 0.54 and 0.14 for AA, AB and BB, respectively. Cows with the AA genotype presented an early CLA and were more likely to resume ovarian cyclicity in the early postpartum than AB and BB ones. No effect of the SNP IGF-1/Sna BI genotype was evidenced on body condition change over the experimental period, suggesting that energy balance is not responsible for the outcome of postpartum ovarian resumption in this study. Traditional fertility measures were not affected by the SNP IGF-1/Sna BI.ConclusionTo our knowledge this is the first report describing an association of the SNP IGF-1/Sna BI with an endocrine fertility measure like CLA in cattle. Results herein remark the important role of the IGF-1 gene in the fertility of dairy cows on early lactation and make the SNP IGF-1/Sna BI an interesting candidate marker for genetic improvement of fertility in dairy cattle.

Estrogen and progesterone receptors in the ovine cervix during the postpartum period

Cervical estrogen (E) and progesterone (P) receptors were characterized and quantified during the postpartum period in Corriedale ewes lambing in the late breeding season. Cervices and uteri were collected after ovariohysterectomy at 1 d (n = 2), 5 d (n = 4), 17 d (n = 2) or 30 d (n = 2) post partum. The estrogen and progesterone receptors were measured using binding assays with tritiated hormones, dextran charcoal separation and inverse Scatchard analysis. Similar kinetic parameters in cytosolic binding sites for both hormones were found in all cervical and uterine samples, indicating that the binding protein in both tissues is of the same nature. Receptor concentrations (fmol/mg cytosolic protein) in the cervix of early (1 to 5 d, n = 6) and late (17 to 30 d, n = 4) postpartum ewes were 348 +/- 66 vs 994 +/- 145 (P < 0.05) for E and 618 +/- 126 vs 1170 +/- 201 (P < 0.05) for P, respectively. These data suggest an increased synthesis of receptors, probably due to the presence of ovarian estrogen-active follicles. Cervical E and P receptor concentrations were similar or higher than those in the uterus (1.40 +/- 0.15, n = 10 and 1.51 +/- 0.19, n = 10; for E and P respectively), and these receptor ratios did not differ between the early and late postpartum period. The high ratio between cervical/uterine receptors suggests that the ovine cervix may be a very sensitive to steroid action. In conclusion, it was shown that restoration of steroid receptors during the postpartum period in the ovine cervix is similar to receptor dynamics in the uterus, and is probably associated with the recovery of ovarian cyclicity.

Cortisol secretion after adrenocorticotrophin (ACTH) and dexamethasone tests in healthy female and male dogs.

BackgroundFor the conclusive diagnosis of Cushings Syndrome, a stimulating ACTH test or a low suppressive Dexamethasone test is used. Reports in other species than the dog indicate that plasma cortisol concentration after ACTH administration is affected by gender. We investigated the effect of gender on the cortisol response to ACTH and Dexamethasone tests in dogs.MethodsSeven healthy adult Cocker Spaniels (4 females and 3 males) were assigned to a two by two factorial design: 4 dogs (2 females and 2 males) received IV Dexamethasone 0.01 mg/kg, while the other 3 dogs received an IV saline solution (control group). Two weeks later the treatments were reversed. After one month, ACTH was given IV (250 μg/animal) to 4 dogs (2 female and 2 males) while the rest was treated with saline solution (control group). Cortisol concentrations were determined by a direct solid-phase radioimmunoassay and cholesterol and triglycerides by commercial kits.Results and DiscussionNo effect of treatment was observed in metabolite concentrations, but females presented higher cholesterol concentrations. ACTH-treated dogs showed an increase in cortisol levels in the first hour after sampling until 3 hours post injection. Cortisol concentrations in Dexamethasone-treated dogs decreased one hour post injection and remained low for 3 hours, thereafter cortisol concentrations increased. The increase in cortisol levels from one to two hours post ACTH injection was significantly higher in females than males. In Dexamethasone-treated males cortisol levels decreased one hour post injection up to 3 hours; in females the decrease was more pronounced and prolonged, up to 5 hours post injection.ConclusionWe have demonstrated that cortisol response to ACTH and Dexamethasone treatment in dogs differs according to sex.