Neuronal Responses in the Brainstem and Hypothalamic Nuclei Following Insulin Treatment During the Late Follicular Phase in the Ewe

The aim of this study was to determine the neuronal responses following insulin administration during the late follicular phase. Intact ewes were given either saline or insulin (5 IU/kg, i.v.) at 35 h after progesterone withdrawal and killed 3 h later. There was a marked increase in the number of Fos‐positive noradrenergic neurones in the caudal brainstem of insulin‐treated ewes. In the hypothalamic paraventricular nucleus, insulin treatment increased the presence of Fos‐positive corticotrophin‐releasing hormone neurones (from 2% to 98%) and Fos‐positive arginine vasopressin parvocellular neurones (from 2% to 46%). Interestingly, after insulin treatment, despite a general increase in Fos‐positive neurones in the arcuate nucleus (ARC), there was a marked reduction (from 47% to 1%) in Fos‐positive β‐endorphin neurones. Similarly, colocalized Fos and oestradiol receptor (ER) α‐positive neurones decreased in the ARC after insulin (from 7% to 3%). Conversely, in the ventromedial nucleus, ERα‐positive neurones with Fos increased (from 7% to 22%) alongside a general increase in Fos‐positive neurones. Overall, a complex system of neurones in brainstem and hypothalamus is activated following insulin administration during the late follicular phase.     

Cortisol disrupts the ability of estradiol-17β to induce the LH surge in ovariectomized ewes

Stress disrupts the preovulatory luteinizing hormone (LH) surge in females, but the mechanisms are unknown. We tested the hypothesis that cortisol compromises the ability of estrogen to induce a preovulatory-like LH surge in ovariectomized ewes in both the breeding and nonbreeding season. Luteinizing hormone surges were induced in ovariectomized ewes by treatment with progesterone followed by a surge-inducing estradiol-17β (E2) stimulus using a crossover design. The experiment was replicated in the breeding and nonbreeding seasons. Cortisol reduced the incidence of LH surges irrespective of season. Cortisol increased the latency from E2 stimulus to the onset of the surge in the breeding season only and suppressed the LH surge amplitude during both seasons (P < 0.01). We conclude that cortisol can interfere with the LH surge in several ways: delay, blunt, and in extreme cases prevent the E2-induced LH surge. Furthermore, the effect of cortisol to delay the E2-induced LH surge is more pronounced in the breeding season. These results show that cortisol disrupts the positive feedback effect of E2 to trigger an LH surge and suggest the involvement of multiple mechanisms.     

Insights into the mechanism by which kisspeptin stimulates a preovulatory LH surge and ovulation in seasonally acyclic ewes: Potential role of estradiol

We have previously demonstrated that a constant intravenous infusion of kisspeptin (Kp) for 48 h in anestrous ewes induces a preovulatory luteinizing hormone (LH) surge followed by ovulation in approximately 75% of animals. The mechanisms underlying this effect are unknown. In this study, we investigated whether Kp-induced preovulatory LH surges in anestrous ewes were the result of the general activation of the whole gonadotropic axis or of the direct activation of central GnRH neurons required for the GnRH/LH surge. In the first experiment, a constant iv infusion of ovine kisspeptin 10 (Kp; 15.2 nmol/h) was given to 11 seasonally acyclic ewes over 43 h. Blood samples were taken every 10 min for 15 h, starting 5 h before the infusion, and then hourly until the end of the infusion. We found that the infusion of Kp induced a well-synchronized LH surge (around 22 h after the start of the Kp infusion) in 82% of the animals. In all ewes with an LH surge, there was an immediate but transient increase in the plasma concentrations of LH, follicle-stimulating hormone (FSH), and growth hormone (GH) at the start of the Kp infusion. Mean (± SEM) concentrations for the 5-h periods preceding and following the start of the Kp infusion were, respectively, 0.33 ± 0.09 vs 2.83 ± 0.49 ng/mL (P = 0.004) for LH, 0.43 ± 0.05 vs 0.55 ± 0.03 ng/mL (P = 0.015) for FSH, and 9.34 ± 1.01 vs 11.51 ± 0.92 ng/mL (P = 0.004) for GH. In the first experiment, surges of LH were observed only in ewes that also had a sustained rise in plasma concentrations of estradiol (E₂) in response to Kp. Therefore, a second experiment was undertaken to determine the minimum duration of Kp infusion necessary to induce such a pronounced and prolonged increase in plasma E₂ concentration. Kisspeptin (15.2 nmol/h) was infused for 6, 12, or 24 h in seasonally acyclic ewes (N = 8), and blood samples were collected hourly for 28 h (beginning 5 h before the start of infusion), then every 2 h for the following 22 h. Kisspeptin infused for 24 h induced LH surges in 75% of animals, and this percentage decreased with the duration of the infusion (12 h = 50%; 6 h = 12.5%). The plasma concentration of E₂ was greater in ewes with an LH surge compared to those without LH surges; mean (± SEM) concentrations for the 5-h period following the Kp infusion were, respectively, 2.23 ± 0.16 vs 1.27 ± 0.13 pg/mL (P < 0.001). Collectively, our results strongly suggest that the systemic delivery of Kp induced LH surges by activating E₂-positive feedback on gonadotropin secretion in acyclic ewes.     

Ovarian and Hormonal Responses to Follicular Phase Administration of Investigational Metastin/Kisspeptin Analog,TAK‐683, in Goats

This study evaluated the effects of follicular phase administration of TAK‐683, an investigational metastin/kisspeptin analog, on follicular growth, ovulation, luteal function and reproductive hormones in goats. After confirmation of ovulation by transrectal ultrasonography (Day 0), PGF_2α (2 mg/head of dinoprost) was administered intramuscularly on Day 10 to induce luteal regression. At 12 h after PGF_2α administration, intravenous administration of vehicle or 35 nmol (50 μg)/head of TAK‐683 was performed in control (n = 4) and treatment (n = 4) groups, respectively. Blood samples were collected at 6‐h intervals for 96 h and then daily until the detection of subsequent ovulation (second ovulation). After the second ovulation, ultrasound examinations and blood sampling were performed every other day or daily until the subsequent ovulation (third ovulation). Mean concentrations of LH and FSH in the treatment group were significantly higher 6 h after TAK‐683 treatment than those in the control group (12.0 ± 10.7 vs 1.0 ± 0.7 ng/ml for LH, 47.5 ± 28.2 vs 15.1 ± 3.4 ng/ml for FSH, p < 0.05), whereas mean concentrations of oestradiol in the treatment group decreased immediately after treatment (p < 0.05) as compared with the control group. Ovulation tended to be delayed (n = 2) or occurred early (n = 1) in the treatment group as compared with the control group. For the second ovulation, ovulatory follicles in the treatment group were significantly smaller in maximal diameter than in the control group (3.8 ± 0.5 vs 5.4 ± 0.2 mm, p < 0.05, n = 3). Administration of TAK‐683 in the follicular phase stimulates gonadotropin secretion and may have resulted in ovulation of premature follicles in goats.     

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.     

Effect of short‐term supplementation with rumen‐protected fat during the late luteal phase on reproduction and metabolism of ewes

This study was designed to study the effect of short‐term supplementation with rumen‐protected fat during the late luteal phase on reproduction and metabolism of sheep during breeding season. Seventy‐six ewes (Rahmani, Barki and Awassi × Barki) were allocated to two groups considering genotype: the control ewes (C‐group) received a maintenance diet, and the fat‐supplemented ewes (F‐group) received the maintenance diet plus 50 g/head/day of rumen‐ protected fat (Megalac) for 9 days during which oestrus was synchronized. The latter had been accomplished using double intramuscular injection of prostaglandin F_2α (PGF_2α) 11 days apart. Ovarian activity, serum concentration of cholesterol, glucose, insulin and reproductive performance variables were recorded. Data were analysed considering treatment (group) and genotype. Supplementation had positive effects on the overall mean serum concentrations of cholesterol (p < 0.05), glucose (p < 0.05) on day 6 of nutritional treatment and insulin (p = 0.07) on day 8. Fat supplementation did not affect the total number of follicles, follicle populations and ovulation rate. However, fat‐supplemented Rahmani ewes tended to have higher ovulation rate compared with other breeds (treatment × breed interaction, p = 0.06). Treatment also did not affect the mean concentration of serum estradiol or progesterone. Supplemented ewes had higher conception (p = 0.06) and lambing rates (p < 0.05) compared with control. In conclusion, short‐term supplementation with rumen‐protected fat as a source of energy around breeding time improved metabolism, conception and lambing rates of ewes without effects on steroidogenic capacity and ovarian activity being apparent.     

Effects of feed restriction on reproductive and metabolic hormones in ewes

The goal of this study was to determine the effects of short-term feed withdrawal on reproductive and metabolic hormones during the luteal phase of the estrous cycle in mature ewes. Mature ewes observed in estrus were assigned randomly to control and fasted groups (n = 10 per group Trials 1 and 2). For Trials 1 and 2, control ewes had ad libitum access to feed, whereas fasted ewes were not fed from d 7 through 11 of their estrous cycle; on d 12, all ewes were treated with 10 mg of PGF2alpha, and fasted ewes were gvien ad libitum access to feed. For Trial 1, blood samples were collected daily through fasting and at 2-h intervals following PGF2alpha for 72 h. Serum concentrations of insulin (P < or = 0.002) and IGF-I (P < or = 0.01), but not GH (P > or = 0.60), were decreased during fasting compared with fed ewes. Serum concentrations of 29 (P = 0.02) and 34 kDa (P = 0.04) IGFBP were greater in fasted ewes at 96 h after initiation of fasting than in control ewes. Two control and four fasted ewes in Trial 1 did not exhibit a preovulatory surge release of LH by 72 h. Therefore, Trial 2 was conducted so that the timing of the LH surge could be predicted following the collection of blood samples at 2-h intervals for 112 h and then at 6-h intervals until 178 h following PGF2alpha administration and realimentation. The magnitude of the preovulatory LH surge in Trial 2 was decreased (P = 0.009) and delayed (P = 0.04), and serum concentrations of estradiol were diminished (P < or = 0.03) 12 h before the LH surge in fasted ewes. Ovulation rates were not influenced (P > or = 0.32) by fasting in Trials 1 and 2. Serum concentrations of progesterone in both Trials 1 and 2 were, however, greater (P < 0.001) in fasted than in control ewes. A third trial with ovariectomized ewes was conducted to determine whether the increased serum concentrations of progesterone observed in fasted ewes during Trials 1 and 2 were ovarian-derived. Ovariectomized ewes were implanted with progesterone-containing intravaginal implants and allotted to control (n = 5) or fasted (n = 5) treatment groups and fed as described for Trials 1 and 2. Similar to intact ewes, serum concentrations of progesterone were approximately twofold greater (P < 0.001) in fasted than in control implanted ovariectomized ewes. In summary, feed withdrawal for 5 d during the luteal phase of the estrous cycle increased serum concentrations of progesterone and evoked endocrine changes that could perturb the subsequent estrous cycle.     

GnRH and prostaglandin‐based synchronization protocols as alternatives to progestogen‐based treatments in sheep

The study investigated, for cycling sheep, synchronizing protocols simultaneously to the standard “P” protocol using progestogens priming with intravaginal devices and gonadotropin. In November 2014, 90 adult Menz ewes were assigned to either the “P” protocol, “PGF” treatment where oestrus and ovulation were synchronized using two injections of prostaglandin 11 days apart or a “GnRH” treatment where the ewes had their oestrus and ovulation synchronized with GnRH (day 0)–prostaglandin (day 6)–GnRH (day 9) sequence. The ewes were naturally mated at the induced oestrus and the following 36 days. Plasma progesterone revealed that 92% of the ewes were ovulating before synchronization and all, except one, ovulated in response to the applied treatments. All “P” ewes exhibited oestrus during the 96‐hr period after the end of the treatments in comparison with only 79.3% and 73.3% for “PGF” and “GnRH” ewes, respectively (p < .05). Onset and duration of oestrus were affected by the hormonal treatment (p < .05); “GnRH” ewes showed oestrus earliest and had the shortest oestrous duration. Lambing rate from mating at the induced oestrus was lower for “P” than for “PGF” ewes (55.6% and 79.3%, respectively; p < .05). The same trait was also lower for “P” than for “PGF” and “GnRH” ewes (70.4%, 89.7% and 86.7%, respectively; p < .05) following the 36‐day mating period. Prostaglandin and GnRH analogue‐based protocols are promising alternatives for both controlled natural mating and fixed insemination of Menz sheep after the rainy season when most animals are spontaneously cycling.     

Follicle Diameter and Systemic Hormone Interrelationships during Induction of Follicle Collapse with Intrafollicular Prostaglandin E2 and F2α in Mares

The objectives were to determine: (i) whether intrafollicular administration of PGE2 and PGF2α to mares would hasten follicle collapse and (ii) the differences in reproductive hormone characteristics in mares with spontaneous and prostaglandin‐induced follicle collapses. Six mares were followed for two oestrous cycles each: when the mares reached a follicle diameter of 30–35 mm and showed mild‐to‐moderate endometrial oedema, mares were administered a single 0.5 ml dose containing 500 μg PGE2 and 125 μg PGF2α (treatment cycle) or a placebo (0.5 ml of water for injection; control cycle) into the preovulatory follicle (Hour 0). Blood samples were collected, and serial ultrasound examinations were performed until follicle collapse. Treated mares showed follicle collapse significantly earlier (20.0 ± 5.9 h) than the control mares (72.0 ± 10.7 h). The LH, progesterone, total oestrogens and oestradiol concentrations did not differ between groups; however, the progesterone concentration increased more between 48 and 72 h after follicle injection in the treatment compared to the control cycles (P < 0.05). In conclusion, intrafollicular treatment with PGE2 and PGF2α hastened follicle collapse in mares without the simultaneous use of an inductor of ovulation; despite the early induction of follicle collapse, the profiles of LH and oestradiol were not altered. This study provides information on the role of prostaglandins (PGs) in the process of follicle wall rupture and collapse and suggests that this may happen even before the beginning of the sharp rise in circulating LH at the final stage of the ovulatory surge.     

Concentrations of ovarian and pituitary hormones following prostaglandin F2 alpha-induced luteal regression in ewes varies with day of the estrous cycle at treatment

Prostaglandin F2 alpha (PGF2 alpha) was injected on d 5, 8 or 11 postestrus in ewes to determine how stage of the estrous cycle would affect PGF2 alpha-induced changes in concentrations of ovarian and pituitary hormones and intervals to the onset of estrus and the preovulatory surge of luteinizing hormone (LH). Initial concentrations of progesterone and average values during the 12 h after PGF2 alpha were related positively to the day of cycle on which PGF2 alpha was administered. Patterns of decline in progesterone after injection of PGF2 alpha were similar among the 3 d. Concentrations of LH in plasma increased in a similar manner from 0 to 12 h in all ewes. After 12 h LH continued to increase, plateaued or declined in ewes treated on d 5, 8 or 11, respectively. Initial concentrations of follicle stimulating hormone (FSH) in plasma were related positively to day of treatment. After treatment with PGF2 alpha, FSH increased within 2 h on d 5 but declined by that time on d 8 or 11. Concentrations of estradiol following treatment did not vary with day. The onset of estrus and the preovulatory surge of LH occurred at 36 and 35, 40 and 45, and 48 and greater than 48 h in ewes treated on d 5, 8 or 11, respectively. It is concluded that: 1) the initial increase in LH is dependent on a decrease in plasma progesterone and 2) differences in patterns of secretion of gonadotropins before the preovulatory surge of LH might be caused by differences in progesterone or progesterone:-estradiol ratio when luteal regression is induced on different days of the estrous cycle.     

Effects of realimentation after nutrient restriction during mid‐ to late gestation on pancreatic digestive enzymes,serum insulin and glucose levels,and insulin‐containing cell cluster morphology

This study examined effects of stage of gestation and nutrient restriction with subsequent realimentation on maternal and foetal bovine pancreatic function. Dietary treatments were assigned on day 30 of pregnancy and included: control (CON; 100% requirements; n = 18) and restricted (R; 60% requirements; n = 30). On day 85, cows were slaughtered (CON, n = 6; R, n = 6), remained on control (CC; n = 12) and restricted (RR; n = 12), or realimented to control (RC; n = 11). On day 140, cows were slaughtered (CC, n = 6; RR, n = 6; RC, n = 5), remained on control (CCC, n = 6; RCC, n = 5) or realimented to control (RRC, n = 6). On day 254, the remaining cows were slaughtered and serum samples were collected from the maternal jugular vein and umbilical cord to determine insulin and glucose concentrations. Pancreases from cows and foetuses were removed, weighed, and subsampled for enzyme and histological analysis. As gestation progressed, maternal pancreatic α‐amylase activity decreased and serum insulin concentrations increased (p ≤ 0.03). Foetal pancreatic trypsin activity increased (p < 0.001) with advancing gestation. Foetal pancreases subjected to realimentation (CCC vs. RCC and RRC) had increased protein and α‐amylase activity at day 254 (p ≤ 0.02), while trypsin (U/g protein; p = 0.02) demonstrated the opposite effect. No treatment effects were observed for maternal or foetal pancreatic insulin‐containing cell clusters. Foetal serum insulin and glucose levels were reduced with advancing gestation (p ≤ 0.03). The largest maternal insulin‐containing cell cluster was not influenced by advancing gestation, while foetal clusters grew throughout (p = 0.01). These effects indicate that maternal digestive enzymes are influenced by nutrient restriction and there is a potential for programming of increased foetal digestive enzyme production resulting from previous maternal nutrient restriction.     

Differential Effects of Continuous Exposure to the Investigational Metastin/Kisspeptin Analog TAK-683 on Pulsatile and Surge Mode Secretion of Luteinizing Hormone in Ovariectomized Goats

The aim of the present study was to determine if the estradiol-induced luteinizing hormone (LH) surge is influenced by the constant exposure to TAK-683, an investigational metastin/kisspeptin analog, that had been established to depress the pulsatile gonadotropin-releasing hormone (GnRH) and LH secretion in goats. Ovariectomized goats subcutaneously received TAK-683 (TAK-683 group, n=6) or vehicle (control group, n=6) constantly via subcutaneous implantation of an osmotic pump. Five days after the start of the treatment, estradiol was infused intravenously in both groups to evaluate the effects on the LH surge. Blood samples were collected at 6-min intervals for 4 h prior to the initiation of either the TAK-683 treatment or the estradiol infusion, to determine the profiles of pulsatile LH secretion. They were also collected at 2-h intervals from –4 h to 32 h after the start of estradiol infusion for analysis of LH surges. The frequency and mean concentrations of LH pulses in the TAK-683 group were remarkably suppressed 5 days after the start of TAK-683 treatment compared with those of the control group (P<0.05). On the other hand, a clear LH surge was observed in all animals of both groups. There were no significant differences in the LH concentrations for surge peak and the peak time of the LH surge between the TAK-683 and control groups. These findings suggest that the effects of continuous exposure to kisspeptin or its analog on the mechanism(s) that regulates the pulsatile and surge mode secretion of GnRH/LH are different in goats.     

Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats

Metastin/kisspeptin, the KiSS-1 gene product, has been identified as an endogenous ligand of GPR54 that reportedly regulates GnRH/LH surges and estrous cyclicity in female rats. The aim of the present study was to determine if metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges. We demonstrated that preoptic area (POA) infusion of the anti-rat metastin/kisspeptin monoclonal antibody blocked the estrogen-induced LH surge, indicating that endogenous metastin/kisspeptin released around the POA mediates the estrogen positive feedback effect on GnRH/LH release. Metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) may be responsible for mediating the feedback effect because the percentage of c-Fos-expressing KiSS-1 mRNA-positive cells to total KiSS-1 mRNA-positive cells was significantly higher in the afternoon than in the morning in the anteroventral periventricular nucleus (AVPV) of high estradiol (E(2))-treated females. The percentage of c-Fos-expressing metastin/kisspeptin neurons was not different between the afternoon and morning in the arcuate nucleus (ARC). Most of the KiSS-1 mRNA expressing cells contain ERalpha immunoreactivity in the AVPV and ARC. In addition, AVPV KiSS-1 mRNA expressions were highest in the proestrous afternoon and lowest in the diestrus 1 in females and were increased by estrogen treatment in ovariectomized animals. On the other hand, the ARC KiSS-1 mRNA expressions were highest at diestrus 2 and lowest at proestrous afternoon and were increased by ovariectomy and decreased by high estrogen treatment. Males lacking the surge mode of GnRH/LH release showed no obvious cluster of metastin/kisspeptin-immunoreactive neurons in the AVPV when compared with high E(2)-treated females, which showed a much greater density of these neurons. Taken together, the present study demonstrates that the AVPV metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges in female rats.     

Characterization of the LH peak after short and long fixed‐time artificial insemination protocols in sheep raised in the tropics

The aim of this study was to evaluate the peak in luteinizing hormone (LH) and the pregnancy rate of sheep (Texel × Santa Inês) in the tropics using short‐ (6 days) and long‐term (12 days) progesterone protocols followed by artificial insemination (AI) both in and out of the breeding season. Experiment 1 was conducted within (IN) the breeding season (autumn, n = 36), and experiment 2 was conducted outside (OUT) of the breeding season (spring, n = 43). In each experiment, the sheep were divided into two groups (6 or 12 days) according to the duration of treatment with a single‐use progesterone release vaginal device (CIDR^®, Pfizer, São Paulo, SP, Brazil), and blood samples were collected from 10 animals per group every 4 hr to measure the LH and progesterone concentrations. In the spring, the characteristics of the LH peak did not differ between groups; but in the autumn, there were differences between groups at the beginning (G‐6 IN: 36.44 ± 5.46 hr; G‐12 IN: 26.57 ± 4.99 hr) and end of the LH peak (G‐6 IN: 46.22 ± 7.51 hr; G‐12 IN: 34.86 ± 8.86 hr). The results showed alterations in the LH peak during the breeding season only in the sheep undergoing the short‐term protocol.     

α-Adrenergic control of gonadotropin secretion in the ewe

The effect of the centrally acting α-adrenoceptor agonist, clonidine, on plasma LH and FSH was studied in oestradiol-primed and unprimed ewes and in oestrous ewes. In unprimed anoestrous ewes, clonidine stimulated LH and FSH release after a lag period of 18 h, and noradrenaline intracarotid injection or i.v. infusions immediately stimulated LH release. In oestradiol-infused anoestrous ewes, clonidine produced either a delay or inhibition of the gonadotrophin surge and noradrenaline i.v. infusion advanced the LH surge. In oestrous ewes treated with clonidine, there was marked delay in the LH surge, but the magnitude of the LH and FSH surges were unaffected. Intravenous administration of α-adrenoceptor blockers, phentolamine and phenoxybenzamine, blocked the oestradiol-induced gondotrophin surge in anoestrous ewes. The effect of phenoxybenzamine on gonadotrophin surge was dose dependent in oestrous ewes. Small doses (4 mg/kg i.v.) of phenoxybenzamine delayed the synchronous LH and FSH surges. There was complete blockade of the LH surge and partial blockade of FSH surges in ewes given phenoxybenzamine (8 mg/kg i.v.) before the expected synchronous gonadotrophin surges. After this experiment, the initial rise of plasma progesterone concentrations did not occur until day 6 of oestrous cycle. Administration of phenoxybenzamine before the expected second FSH surge had no effect on the second FSH surge. Gonadotrophin release induced by gonadotrophin-releasing hormone was attenuated by phenoxybenzamine, but not by clonidine. The results suggest that the LH surge is under α-adrenergic control and the first FSH surge is under partial α-adrenergic control, but the second FSH surge is not under α-adrenergic control. The results also suggest oestradiol modulation of α-adrenergic receptor action.     

Quantitative expression of mRNA encoding BMP/SMAD signalling genes in the ovaries of Booroola carrier and non‐carrier GMM sheep

The GMM sheep is a carrier of Booroola fecundity (FecB) gene, which produces the twins and triplets in one lambing. The homozygous carrier GMM (FecB^BB), non‐carrier GMM and Malpura (FecB⁺⁺) ewes were synchronized by progesterone sponges, and the plasma progesterone concentration was measured by RIA. The results showed that the progesterone concentration did not differ significantly (p > .05) in homozygous carrier GMM (5.74 ± 1.2 ng/ml), non‐carrier GMM (5.42 ± 1.4 ng/ml) and non‐carrier Malpura ewes (5.67 ± 1.5 ng/ml). Further, quantitative expression of BMP factors/receptors and SMAD signalling genes were analysed in the ovaries of sheep by qRT‐PCR. The study showed that the expression of BMP2 was slightly higher (p > .05) in carrier GMM than that of non‐carrier GMM, but it was almost similar to Malpura ewes. Expression of BMP4 and BMP7 was significantly higher (p < .001; p < .05) in carrier GMM than that of non‐carrier GMM and Malpura ewes. Although BMP6 expression was higher (p > .05) in carrier GMM than that of non‐carrier GMM, but lower (p > .05) than the Malpura ewes. Expression of BMP15 (p < .05), GDF5 (p < .01) and GDF9 (p < .05) was significantly higher in carrier GMM than non‐carrier GMM ewes. Surprisingly, BMPR1B expression was significantly higher (p < .001) in non‐carrier GMM and Malpura than the carrier GMM ewes, while TGFβRI did not differ significantly (p > .05) among both GMM genotypes. On the other hand, expression of BMPR1A (p > .05) and BMPRII (p < .05) was higher in carrier GMM than the non‐carrier GMM, but significantly lower (p < .001) than the Malpura ewes. It was interesting to note that the expression of SMAD1 (p > .05), SMAD2 (p < .001), SMAD3 (p < .05), SMAD4 (p < .001), SMAD5 (p < .001) and SMAD8 (p < .001) was lower in the carrier GMM than that of non‐carrier GMM ewes. It is concluded that the FecB mutation alters the expression of BMPR1B and SMAD signalling genes in the ovaries of homozygous carrier GMM ewes.     

Characterization of ovine follicles destined to form subfunctional corpora lutea

A study was done to test whether ovulatory follicles destined to form subfunctional corpora lutea differed from normal ovulatory follicles in steroidogenic function. Twenty-five ewes were treated with prostaglandin F2 alpha on d 11 of the estrous cycle, then unilaterally ovariectomized before (n = 13) or after (n = 12) the surge of luteinizing hormone (LH) at the induced estrus to collect "control" follicles, which would have produced normal corpora lutea. In 15 ewes, the second ovary was removed 63 to 84 h later to collect "treated" follicles before (n = 7) or after (n = 8) the second expected surge of LH. Five ewes (control) were allowed to ovulate from the remaining ovary at first estrus and another five (treated) at the second estrus (3 to 4 d later). Treated ewes had lower serum progesterone than control ewes during the ensuing cycle (P less than .05). Treated follicles contained less estradiol in the theca (4.4 +/- .6 vs 10.0 +/- 2.5 ng; P less than .05), less androstenedione (.1 +/- .1 vs 1.0 +/- .2 ng) and estradiol (.5 +/- .1 vs 2.9 +/- 2.2 ng) in the granulosa (P less than .05) and less progesterone in the follicular fluid (.8 +/- .4 vs 3.3 +/- .8 ng; P less than .05) than control follicles, when removed before the surge of LH. Follicles removed after the surge of LH did not differ. In conclusion, ovulatory follicles with low steroidogenic function became corpora lutea that secreted lower-than-normal quantities of progesterone.