Kisspeptin,c‐Fos and CRFR Type 2 Co‐expression in the Hypothalamus after Insulin‐Induced Hypoglycaemia

Normal reproductive function is dependent upon availability of glucose and insulin‐induced hypoglycaemia is a metabolic stressor known to disrupt the ovine oestrous cycle. We have recently shown that IIH has the ability to delay the LH surge of intact ewes. In the present study, we examined brain tissue to determine: (i) which hypothalamic regions are activated with respect to IIH and (ii) the effect of IIH on kisspeptin cell activation and CRFR type 2 immunoreactivity, all of which may be involved in disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and at 28 h after progesterone withdrawal (PW), animals received saline (n = 6) or insulin (4 IU/kg; n = 5) and were subsequently killed at 31 h after PW (i.e., 3 h after insulin administration). Peripheral hormone concentrations were evaluated, and hypothalamic sections were immunostained for either kisspeptin and c‐Fos (a marker of neuronal activation) or CRFR type 2. Within 3 h of treatment, cortisol concentrations had increased whereas plasma oestradiol concentrations decreased in peripheral plasma (p < 0.05 for both). In the arcuate nucleus (ARC), insulin‐treated ewes had an increased expression of c‐Fos. Furthermore, the percentage of kisspeptin cells co‐expressing c‐Fos increased in the ARC (from 11 to 51%; p < 0.05), but there was no change in the medial pre‐optic area (mPOA; 14 vs 19%). CRFR type 2 expression in the lower part of the ARC and the median eminence was not altered by insulin treatment. Thus, disruption of the LH surge after IIH in the follicular phase is not associated with decreased kisspeptin cell activation or an increase in CRFR type 2 in the ARC but may involve other cell types located in the ARC nucleus which are activated in response to IIH.     

Integration Between Different Hypothalamic Nuclei Involved in Stress and GnRH Secretion in the Ewe

This study investigated possible integrated links in the neuroanatomical pathways through which the activity of neurones in the paraventricular nucleus and arcuate nucleus may modulate suppression of gonadotrophin‐releasing hormone (GnRH) secretion during stressful situations. Double‐label immunofluorescence and laser scanning confocal microscopy were used to examine the hypothalamic sections from the follicular phase ewes. Noradrenergic terminals were in close contact with 65.7 ± 6.1% corticotrophin‐releasing hormone (CRH) and 84.6 ± 3.2% arginine vasopressin (AVP) cell bodies in the paraventricular nucleus but not with β‐endorphin cell bodies in the arcuate nucleus. Furthermore, γ‐amino butyric acid (GABA) terminals were close to 80.9 ± 3.5% CRH but no AVP cell bodies in the paraventricular nucleus, as well as 60.8 ± 4.1%β‐endorphin cell bodies in the arcuate nucleus. Although CRH, AVP and β‐endorphin cell terminals were identified in the medial pre‐optic area, no direct contacts with GnRH cell bodies were observed. Within the median eminence, abundant CRH but not AVP terminals were close to GnRH cell terminals in the external zone; whereas, β‐endorphin cells and terminals were in the internal zone. In conclusion, neuroanatomical evidence is provided for the ewe supporting the hypothesis that brainstem noradrenergic and hypothalamic GABA neurones are important in modulating the activity of CRH and AVP neurones in the paraventricular nucleus, as well as β‐endorphin neurones in the arcuate nucleus. These paraventricular and arcuate neurones may also involve interneurones to influence GnRH cell bodies in medial pre‐optic area, whereas the median eminence may provide a major site for direct modulation of GnRH release by CRH terminals.     

Post‐glucose load changes of plasma key metabolite and insulin concentrations during pregnancy and lactation in ewes with different susceptibility to pregnancy toxaemia

Insulin resistance during late gestation may act as a predisposing factor of ovine pregnancy toxaemia (OPT). To evaluate the insulin action on energy metabolism in ewes with different susceptibilities to OPT, intravenous glucose tolerance tests (1 mmol glucose/kg body weight) were performed in 5.6 ± 0.7 year old, slightly underfed German Blackheaded Mutton ewes [high‐risk (HR) ewes] and 2.5 year old, overnourished Finnish Landrace ewes [low‐risk (LR) ewes] during mid and late pregnancy, during early lactation and during the dry period. Plasma samples were analysed for glucose, insulin, non‐esterified fatty acids (NEFA) and β‐hydroxybutyrate (β‐HB). The glucose elimination rate and the glucose‐stimulated first‐phase insulin secretion were significantly (p < 0.05) lower in the HR, in relation to the LR group combining the data of all gestational stages. The basal rate of lipolysis was significantly increased in the HR ewes during late pregnancy, but the NEFA clearance after the glucose load was similar in both groups during all reproductive stages. Plasma β‐HB concentrations decreased only in the LR ewes after the glucose load during late pregnancy. Results indicate an insulin resistance in the HR ewes regarding the glucose utilization and the ketone body formation during late pregnancy. The insulin resistance in the HR ewes may represent one predisposing factor responsible for the susceptibility to OPT. Further scientific work is necessary to elucidate whether this insulin resistance was due to breed, age or nutritional state.     

Insulin Sensitivity during Late Gestation in Ewes Affected by Pregnancy Toxemia and in Ewes with High and Low Susceptibility to this Disorder

Background

Insulin resistance during late gestation might act as 1 etiologic factor causing pregnancy toxemia in ewes.

Objective

Evaluation of pancreatic insulin secretion and peripheral insulin sensitivity in ewes with differing susceptibility to pregnancy toxemia and in ketotic ewes.

Animals

Pregnant ewes suffering from (PT, n = 5) and ewes with high (HR, n = 7) and low risk (LR, n = 5) of being affected by pregnancy toxemia.

Methods

In a case‐control study, the pancreatic insulin release and the peripheral insulin sensitivity were assessed by means of the intravenous glucose tolerance test with subsequent measurement of the plasma concentrations of glucose, insulin, nonesterified fatty acids (NEFA), and β‐hydroxybutyrate (β‐HB). The ewes were tested during late pregnancy within 5 and 15 days antepartum.

Results

The insulin secretion after glucose administration was significantly lower in the HR and PT than in the LR ewes. The baseline rate of lipolysis was significantly increased in the HR ewes, but the NEFA clearance was similar in both risk groups, albeit delayed in the PT ewes. The baseline β‐HB concentration was significantly higher in the PT than in the HR and LR ewes. In the HR and in the PT ewes, the plasma β‐HB concentrations did not decrease after glucose administration.

Conclusion and Clinical Importance

There is reduced pancreatic first‐phase insulin response and impaired insulin‐dependent inhibition of the ketone body formation during late pregnancy in the HR and PT ewes. This insulin resistance might represent 1 causative factor in the pathogenesis of ovine pregnancy toxemia.     

Maternal obesity in ewes results in reduced fetal pancreatic β-cell numbers in late gestation and decreased circulating insulin concentration at term

About 30% of U.S. women of reproductive age are obese, a condition linked to offspring obesity and diabetes. This study utilized an ovine model of maternal obesity in which ewes are overfed to induce obesity at conception and throughout gestation. At mid-gestation, fetuses from these obese ewes are macrosomic, hyperglycemic, and hyperinsulinemic, and they exhibited markedly increased pancreatic weight and β-cell numbers compared with fetuses of ewes fed to requirements. This study was conducted to establish fetal pancreatic phenotype and function in late gestation and at term in this ovine model. Multiparous ewes were fed a control (C, 100% National Research Council [NRC] recommendations) or obesogenic (OB, 150% NRC) diet from 60 days before conception to necropsy at day 135 of gestation or to lambing. No differences were observed in fetal size or weight on day 135 or in lamb birth weights between C and OB ewes. In contrast to our previously published results at mid-gestation, pancreatic weights (P < 0.01) and β-cell numbers (P < 0.05) of OB fetuses were markedly lower than those from C fetuses, whereas the β-cell apoptotic rate was increased (P < 0.05) in day 135 OB versus C fetuses. At birth, blood insulin concentration was lower (P < 0.05) and glucose level was higher (P < 0.05) in newborn lambs from OB versus C ewes. These data demonstrate differential impacts of maternal obesity on fetal pancreatic growth and β-cell numbers during early and late gestation. During the first half of gestation there was a marked increase in pancreatic growth, β-cell proliferation, and insulin secretion, followed by a reduction in pancreatic growth and β-cell numbers in late gestation, resulting in reduced circulating insulin at term. It is speculated that the failure of the pancreas to return to a normal cellular composition and function postnatally could result in glucose/insulin dysregulation, leading to obesity, glucose intolerance, and diabetes in postnatal life.     

Changes in Ovarian Follistatin Levels During the Oestrous Cycle in Sheep may Serve as an Intraovarian Regulator

The expression and concentration of follistatin and activin change during oestrous cycle suggesting their involvement in the regulation of follicular development. The aim of this study was to determine the level, source and potential role of follistatin in the sheep ovary. Follistatin in ovarian venous blood, measured by radioimmunoassay, remained at its low level from follicular phase (day ?1 and 0) to mid‐luteal phase (days 11–13) phase but were significantly elevated during the late luteal phase (days 14 and 15) when corpora lutea underwent regression. Western blot analyses of follicular fluid at day 15 of the cycle showed two strong bands at 42 and 45 kDa and weakly stained bands at 39 and 31 kDa. At day 0, these bands became weaker and the 39 kDa band became undetectable. However, there were no differences in follistatin concentrations between ovaries with and without functional corpus luteum (CL) during the whole luteal phase. In addition, although the ovaries of Booroola ewes normally contain more corpora lutea than those of normal merino ewes, follistatin concentrations in both jugular and ovarian venous blood were similar in Booroola and normal merino ewes. It is concluded that the secretion of follistatin from the ovary is not related to the formation of CL or high ovulation rate of Booroola ewes. The elevation in follistatin concentration in follicular fluid and ovarian blood during late luteal phase may indicate a dual role of follistatin in the luteolysis of existing CL and development of new follicle cohort.     

Pancreatic insulin release and peripheral insulin sensitivity in German black headed mutton and Finish Landrace ewes: evaluation of the role of insulin resistance in the susceptibility to ovine pregnancy toxemia

German black headed mutton (GBM) ewes are recognized as being highly susceptible to ovine pregnancy toxemia (OPT). The present trial was performed to evaluate whether a breed-dependent gestational diabetes mellitus-like insulin resistance during late pregnancy might be responsible for the high incidence of OPT in the GBM breed. Modified frequently sampled intravenous glucose tolerance tests (300 mg glucose and 0.03 IU insulin per kg of BW) were performed during mid and late pregnancy, the periparturient, and the dry period in polytocous 3.5-yr-old GBM and Finnish Landrace (FL) ewes fed according to their requirements. The corresponding blood samples were analyzed for plasma concentrations of glucose, insulin, nonesterified fatty acids (NEFAs) and β-hydroxybutyrate (β-HB). In addition, the baseline plasma cortisol concentrations were determined during late pregnancy. The BW gain during pregnancy and the rearing success did not differ between the GBM and FL ewes. In both breeds, late pregnancy was associated with decreased basal plasma glucose concentrations and enhanced glucose disposal, as well as elevated baseline β-HB values. Only in the GBM ewes did the plasma NEFA concentrations increase significantly during advancing pregnancy. Moreover, significantly higher baseline plasma NEFA concentrations as well as lower (P < 0.05) basal plasma glucose values were recorded during late pregnancy in the GBM than in the FL ewes. The first-phase insulin secretion, the peripheral insulin sensitivity, and the baseline plasma cortisol values did not differ between both breeds during late pregnancy. It is concluded that increased lipolysis during late pregnancy is a characteristic of the GBM breed. Moreover, elevated plasma NEFA concentrations may contribute to impaired pancreatic insulin response and peripheral insulin resistance in GBM ewes and thus promote OPT.     

Immunolocalization of c-Fos,ELOVL5 and oestradiol in the ewe vulva in relation to oestrus behaviour after treatment with lipopolysaccharide

Sudden activation of the stress axis by a lipopolysaccharide endotoxin (LPS) significantly reduces ewes' sexual attractivity to rams by delaying all signs of oestrous behaviour. To understand mechanisms involved in attracting male interest, we examined c-Fos (nuclear activation), ELOVL5 (enzyme involved in pheromone synthesis) and oestradiol receptors (ER) using immunohistochemistry on ewe vulval tissue at 0, 31 and 40 hr in the ovarian follicular phase with or without exposure to LPS at 28 hr (5 groups of 4 ewes per group). While there was intense staining for immunoreactive (IR)-c-Fos and IR-ELOVL5 in the vulval epithelium and sebaceous glands, there were no differences in intensity between groups of ewes. The absence of IR-ER staining in vulval epithelium and sebaceous/sweat glands was unexpected. Differences in ram behaviour towards ewes in the ovarian follicular phase and after LPS treatment do not appear to involve quantitative changes in vulval c-Fos, ELOVL5 or ER, but subtle qualitative differences in individual-specific compounds (attraction pheromones) remain an option.     

Regulation of adipose tissue metabolism during pregnancy and lactation in the ewe: the role of insulin

This study was conducted to examine the effect of insulin on lipid metabolism of adipocytes during pregnancy and lactation in ewes. During the first 3 mo of pregnancy, metabolism of adipocytes from omental adipose tissue was characterized by a high rate of de novo lipogenesis (90 to 125 nmol of acetate incorporated into lipids.2 h-1.10(6) cells-1) and a 38% reduction in response to beta-lipolytic stimulus (isoproterenol 10(-6) M). Simultaneously, there was a rise in the number of high-affinity insulin receptors (Kd = .2 nM), and insulin binding characteristics showed a decrease in the negative cooperativity phenomenon. Moreover, lipogenesis stimulated by insulin (1 mU/ml) increased in comparison with observations in nonpregnant ewes. The last third of pregnancy and early lactation were characterized by a marked fall in lipogenesis and a simultaneous increase in isoproterenol-stimulated lipolysis. During lactation, the number of total insulin receptors was decreased by 62% and insulin stimulation of lipogenesis became inefficient. Results suggest that insulin plays a direct role in adipose tissue metabolism during pregnancy.     

Evaluation of a bovine visual pregnancy test for the detection of pregnancy‐associated glycoproteins in sheep

As sheep produce similar pregnancy‐associated glycoproteins (PAGs) to cattle, a commercially available bovine visual pregnancy test based on the detection of PAGs (visual‐PAG‐test) was evaluated in sheep. The test was performed with whole blood (WhB), plasma (P) and serum (S) of 163 pregnant and 153 non‐pregnant ewes. Additionally, 11 pregnant ewes were tested weekly from day 14 to 49 of gestation and monthly from day 60 to day 149. Ten ewes were sampled weekly from the date of lambing until day 63 post‐partum (p.p.). The sensitivity in mid‐pregnancy (n = 163) was 98.16% (WhB), 99.39% (P) and 99.39% (S) compared to transabdominal ultrasonography as the gold standard while the specificity (n = 153) was 94.12% (WhB), 76.47% (P) and 93.46% (S), respectively. During early pregnancy, all 11 ewes were correctly identified as pregnant on day 42 (100%); however, the test sensitivity decreased to 54.6% (WhB) and 63.6% (S and P, respectively) at day 49. The ewes were again consistently identified as pregnant on day 63 (P) or on day 119 (S, WhB). The test was consistently negative from day 42 p.p. onwards in eight out of ten ewes. Two ewes remained consistently positive until the last sample on day 63 p.p. In conclusion, the test could be used to accurately select pregnant ewes at day 42 with a drop in sensitivity at day 49. The sensitivity of the visual‐PAG‐test was good in mid to late pregnancy, and early detection of pregnancy was possible in individual animals. In some ewes, the PAGs were however detectable for more than 63 days p.p.—the previous breeding history should therefore always be taken into account for correct interpretation of the test results.     

Follicular Dynamics,Interval to Ovulation and Fertility After AI in Short‐Term Progesterone and PGF2α Oestrous Synchronization Protocol in Sheep

The study was aimed to assess the influence that short‐term progesterone treatments have on follicular dynamics, oestrus and ovulation in sheep. The treatment was tested thereafter in a field trial to assess its fertility after AI with fresh semen. In a first experiment, 12 ewes without CL were grouped to receive a new (n = 6) or used CIDR (n = 6) for 7 days and blood samples were obtained to follow plasma progesterone profiles. In a second experiment, 39 cycling ewes were synchronized by a 7‐day P4+PGF2α protocol using a new (n = 20) or a 7‐day used CIDR (n = 19). Half of both groups received 400 IU eCG and half remained untreated as controls. Ultrasound ovarian examination and oestrous detection were used to compare follicular dynamics, oestrus and ovulation in both groups. In a third experiment, 288 ewes in 3 farms were synchronized by the short‐term P4+PGF2α+eCG protocol and ewes were AI with fresh semen 24 h after oestrous detection. Lambing performance was used to test the fertility of the treatment. In Experiment 1, ewes with new inserts presented higher P4 concentration than ewes with used inserts throughout the sampling period (p < 0.05) and exhibited a P4 peak at days 1‐2 of the treatment that was not observed in ewes with used inserts. In Experiment 2, ewes treated with new and used inserts show similar ovarian and behavioral traits (p > 0.10). However, ewes treated with eCG show shorter interval to oestrus (p = 0.004) and tend to have larger mature CL (p = 0.06). In Experiment 3, oestrous presentation and lambing performance after AI with fresh semen was considered normal compared to published results. Results suggest that the oestrous synchronization protocol based on P4+PGF2α allows little control of follicular dynamics without compromising fertility after AI with fresh semen provided that eCG is added at the end of the treatment.     

Effect of ammonia‐generating diet on ovine serum and follicular fluid ammonia and urea levels,serum oestrogen and progesterone concentrations and granulosa cell functions

This study was undertaken to elucidate the effect of ammonia‐generating diet on serum and follicular fluid ammonia and urea levels, serum oestrogen and progesterone concentrations and granulosa cell growth and secretion parameters in ewes (Ovis aries). Ewes were fed with 14% CP diet (control) or ammonia‐generating diet or ammonia‐generating diet plus soluble sugar. The serum and follicular fluid ammonia and urea level, serum oestrogen and progesterone levels and granulosa cell (obtained from ovaries of slaughtered ewes) growth parameters and secretory activities were estimated. Ammonia‐generating diet (high‐protein diet) increased the serum ammonia and urea concentration. Supplementation of soluble sugar significantly reduced the ammonia concentration in serum with comparable levels as in control group; however, the urea level in the same group was higher than that observed in control group. Supplementation of soluble sugar significantly reduced the follicular fluid ammonia concentration; however, the level was significantly higher compared to control group. Supplementation of soluble sugar brought down the follicular fluid urea level comparable to that observed in control group. Oestrogen and progesterone levels remained unchanged in ewes fed with different types of diet. Oestrogen and progesterone secretion were significantly lowered from granulosa cells recovered from ewes fed with high ammonia‐generating diet. Low metabolic activity and high incidence of apoptosis were observed in granulosa cells obtained from ovaries of ewes fed with ammonia‐generating diet.     

Induction of ovarian cystic follicles in sheep

Cystic follicles are a significant cause of infertility in women, dairy cattle and sheep. Sheep were used as a model to identify factors that may elicit formation of cystic follicles. Insulin resistance and elevated LH activity were tested in overweight ewes because of associations among these factors and the formation of cystic follicles. Sheep were synchronized using a progesterone-releasing pessary and insulin resistance was induced during the synchronization period through administration of bovine somatotropin. Following removal of pessaries follicular growth was stimulated by treatment with eCG or eCG and hCG (PG-600). Follicular growth was monitored via daily transrectal ultrasonography and blood samples were collected for hormonal analyses. Six of 18 ewes had a subnormal or absent preovulatory gonadotropin surge and developed cystic follicles. Neither insulin resistance nor elevated LH activity were associated with formation of cystic follicles. Ewes that developed cystic follicles were heavier (93 +/- 4 kg) than ewes that ovulated (81 +/- 3 kg; P = 0.02). Furthermore, following pessary removal and initiation of daily ultrasonography, ewes that developed cystic follicles lost body weight (-3 +/- 1%), while ovulatory ewes continued to gain body weight (1 +/- 1%; P = 0.005). It is speculated that in heavy ewes metabolic factors associated with acute body weight loss inhibit the positive feedback of estradiol and thereby suppress the preovulatory gonadotropin surge leading to formation of cystic follicles.     

Metabolic responses to mid-pregnancy shearing that are associated with a selective increase in the birth weight of twin lambs

Previous studies have shown that shearing pregnant ewes at mid- or late-pregnancy is associated with an increase in lamb birth weight. The present study was designed to investigate metabolic responses that may underlie this response. Single- and twin-bearing ewes were either unshorn or shorn at mid-pregnancy (Day 69 of pregnancy; P69), and insulin, glucose and epinephrine challenges were conducted on P109–111 and P132–134. Shearing increased the birth weight of twin lambs by over 1 kg (P < 0.001) without having any effect on singleton birth weight. This response was associated with a 10–20% reduction in the insulin response to a glucose challenge (P < 0.05) without a change in glucose clearance following either glucose or insulin challenges. The lipolytic response to epinephrine challenge increased as pregnancy progressed, but was not associated with the increased birth weight of twin lambs born to shorn ewes. By late pregnancy, a 25% reduction in maternal IGF-I concentration and a two- to threefold increase in maternal IGFBP-1 concentration (P < 0.05) associated with shearing were observed. The increase in lamb birth weight associated with mid-pregnancy shearing may have been associated with an increase in the non-insulin dependent uptake of glucose by the placental-fetal unit.     

Sex differences in oestrogen receptor levels in adrenal glands of sheep during the breeding season

The concentrations of the oestrogen receptor (ER), and the mRNA levels of ER, progesterone receptor (PR) and insulin-like growth factor I (IGF-I) were characterised in adrenal glands and uterine tissue of adult Corriedale sheep during the breeding season. The sheep were of different sex and gonadal status. Ewes had higher levels of cytosolic ER in the adrenals than the rams (mean±S.E.M.: 7.3±2.0 fmol/mg protein and 2.5±1.0 fmol/mg protein, respectively; P=0.0091) and gonadectomy increased ER (mean±S.E.M.: 2.9±1.2 fmol/mg protein and 8.6±2.3 fmol/mg protein, intact and gonadectomised sheep, respectively; P=0.0071). No differences could be observed in mRNA levels for ER and IGF-I in the adrenal glands of all of the sheep. PR mRNA levels were reduced in ovariectomised ewes and enhanced in castrated rams (sex×gonadal status: P=0.009). PR mRNA levels tended to be higher in ewes in the follicular phase than in ovariectomised ewes and intact rams (P<0.1). All of the animals had positive nuclear staining for ER in the adrenal cortex, but no differences were observed between the groups. In this study, we demonstrated the existence of ER in the adrenal gland of sheep and found varying sensitivity to oestrogens as the ER levels differed among sex and gonadal status. These findings indicate that oestrogens most likely affect steroidogenesis directly at the adrenal cortex and suggest that oestrogens are partly responsible for the sex differences in cortisol secretion in sheep.     

Ultrasonographic Characteristics of Ovulatory Follicles and Associated Endocrine Changes in Cyclic Ewes Treated with Medroxyprogesterone Acetate (MAP)-releasing Intravaginal Sponges and Equine Chorionic Gonadotropin (eCG)

The aim of this study was to assess the ultrasonographic characteristics of ovulatory follicles in cyclic Western White Face ewes (December) that had received intravaginal sponges containing medroxyprogesterone acetate (MAP; 60 mg) for 12 days, with or without an injection of 500 IU of equine chorionic gonadotropin (eCG) at sponge removal. We hypothesized that quantitative echotextural attributes of the follicles in ewes treated only with MAP would differ from those in MAP/eCG-treated ewes, reflecting the increased antral follicular growth and secretory function under eCG influence. Digital images of ovulatory follicles obtained at 0 and 24 h after MAP sponge removal and at 24 h before ovulation in the eCG-treated (five ewes, 13 follicles) and control (six ewes, 9 follicles) animals, were subjected to computerized analyses. The mean diameter of ovulatory follicles increased (p < 0.001) 24 h after eCG treatment. The mean pixel intensity and heterogeneity of the follicular antrum (p < 0.001), as well as mean pixel intensity of the follicular wall and perifollicular ovarian stroma (p < 0.05), were greater in eCG-treated animals compared with control ewes 24 h after sponge removal and at 24 h before ovulation. Mean serum concentrations of oestradiol-17beta tended to increase (p = 0.06) 24 h after eCG treatment and the eCG-treated ewes exceeded (p < 0.05) control animals in progesterone concentrations from days 9-15 after ovulation. Our results support the hypothesis that large antral follicles in eCG-treated ewes exhibit distinctive echotextural characteristics. Follicular image attributes in eCG-treated ewes appear to be indicative of the changes in follicular morphology and secretory activity caused by the administration of the exogenous gonadotropin, which has both FSH- and LH-like activities.     

Characterization of Endocrine Events During the Periestrous Period in Sheep After Estrous Synchronization with Controlled Internal Drug Release (CIDR) Device

The Controlled Internal Drug Releasing (CIDR) device is an intravaginal pessary containing progesterone (P₄) designed for synchronizing estrus in ruminants. To date, there has been little information available on the timing, duration, and quality of the follicular phase after CIDR removal and how those characteristics compare with natural periovulatory endocrine events. The present communication relates the results of methods we used to characterize the endocrine events that followed CIDR synchronization. Breeding-season ewes were given an injection (10 mg) of Lutalyse (PGF_2α), and then studied during three consecutive estrous cycles, beginning in the luteal phase after the estrus induced by PGF_2α. Cycle 1 estrus was synchronized with 1 CIDR (Type G) inserted for 8 d beginning 10 d after PGF_2α. Cycles 2 and 3 were synchronized with two CIDRs for 8 d beginning 10 d after previous CIDR removal. Cycle 1 estrous behavior and serum gonadotropins showed a follicular phase (the interval from CIDR withdrawal to gonadotropin surge [surge] peak) of 38.2 ± 1.5 hr. Two CIDRs lengthened the interval to 46.2 ± 1.5 hr (P < 0.0001). At CIDR removal, circulating P₄ concentrations were higher in ewes treated with two CIDRs (5.1 ± 0.3 and 6.4 ± 0.4 ng/mL in Cycles 2 and 3 vs. 2.7 ± 0.3 ng/mL in Cycle 1), whereas estradiol concentrations were higher in the 1 CIDR cycle (3.3 ± 0.5 pg/mL in Cycle 1 vs. 0.5 ± 0.1, and 0.7 ± 0.2 pg/mL in Cycles 2 and 3), suggesting that the lower levels of P₄ achieved with one CIDR was not sufficient to arrest follicular development. There were no differences in any other endocrine variable. Both one and two CIDR synchronization concentrated surges within a 24-hr period in 92% of the ewes in Cycles 1 and 2. Cycle 3 ewes were euthanized at estimated luteal, early follicular, late follicular, LH surge, and secondary FSH rise timepoints. Endocrine data and ovaries showed that 88% of the ewes synchronized with two CIDRs were in the predicted stage of the estrous cycle. These data demonstrate that the CIDR device applied during the luteal phase effectively synchronizes estrus and results in a CIDR removal-to-surge interval of similar length to a natural follicular phase.     

Synchrony of Ovulation and Follicular Dynamics in Merino Ewes Treated with GnRH in the Breeding and Non-breeding Seasons

The effect of gonadotropin releasing hormone (GnRH) treatment on the time of ovulation and the occurrence of follicular dominance during the non-breeding and breeding seasons (experiment 1), and on fertility after artificial insemination (AI) in the non-breeding season (experiment 2), was examined in Merino ewes. Oestrus was synchronized in 40 nulliparous ewes (experiment 1; n = 20, in the non-breeding and breeding seasons) and in 79 multiparous ewes (experiment 2) using intravaginal sponges and pregnant mare serum gonadotropin. Thirty six hours after sponge removal (SR), half the ewes were injected (i.m.) with 40 microg of synthetic GnRH and the remainder used as controls. GnRH improved the synchrony of ovulation compared with the controls in the breeding (SD = 2.8 vs 5.7 days, p = 0.04) but not the non-breeding season (SD = 3.8 vs 4.4 days, p = 0.69), with ewes ovulating from 42 to 54 h (mean 50.4 +/- 4.08 h) and 42-60 h (mean 54.4 +/- 5.47 h) after SR for GnRH and control, respectively. For both treated and control ewes, ovulation occurred earlier in the non-breeding than the breeding season (50.1 vs 54.6 h; p = 0.002). GnRH had no effect on follicular dominance, as assessed by divergence (D: the time the ovulatory follicle exceeded the average size of the other non-ovulating follicles) or on the interval from D to ovulation (IDO). However, follicular dynamics differed between seasons. The mean follicle diameter increased at a faster rate up to 36 h after SR in the non-breeding compared with the breeding season and then rapidly declined, compared with a later peak (42 h after SR) in mean follicular size during the breeding season. IDO was shorter in the non-breeding than in the breeding season (26.7 +/- 4.30 h vs 39.6 +/- 4.53 h; p = 0.05). In experiment 2, ewes (n = 38 GnRH-treated, n = 40 controls) were inseminated in the uterus by laparoscopy 42 h or 48 h after SR with frozen-thawed sperm. The fertility of ewes treated with GnRH (nine of 39, 23%) was not different to the controls (eight of 38, 21%; p = 0.01). In conclusion the application of GnRH improved synchronization of ovulation but did not improve fertility rates after AI.