Characterization and distribution of gonadotrophs in the pars distalis and pars tuberalis of the equine pituitary gland during the estrous cycle and seasonal anestrus

Little is known about the neuroendocrine control of fertility in the horse. In this species, unusual features characterize the normal estrous cycle such as a prolonged preovulatory LH surge during the follicular phase and a distinctive FSH surge during the midluteal phase. This study investigated the distribution and hormonal identity of gonadotrophs in the pars distalis (PD) and pars tuberalis (PT) of the equine pituitary gland as possible morphological bases for the referred unusual endocrine characteristics. In addition, the proportion of gonadotrophs in relation to other pituitary cell types during both the estrous cycle and anestrus were investigated. Pituitary glands were collected from sexually active (n = 5) and seasonally anestrous (n = 5) mares in November, and single or double immunofluorescent staining was carried out on 6-microm sections using monoclonal antibodies to the LHbeta or FSHbeta subunits and a polyclonal antibody to ovine LHbeta. Gonadotrophs were densely distributed around the pars intermedia in the PD and in the caudal ventral region of the PT. In addition to isolated cells, clusters of gonadotrophs were found surrounding the capillaries. No significant differences were detected in the number of gonadotrophs between sexually active and anestrous mares in either the PD or PT. In the PD, gonadotrophs represented 22.7 +/- 5. 8% and 19.1 +/- 2.1% of the total cell density in sexually active and anestrous animals, respectively (P: > 0.05). However, in the PT, gonadotrophs accounted for a higher proportion of the total cell population in sexually active (6 +/- 0.1%) than in anestrous (1.2 +/- 0.05%) mares (P: < 0.02). Double immunofluorescence revealed that the majority of gonadotrophs were bihormonal (i.e., positive for LH and FSH); however, in the sexually active mare, a larger proportion of gonadotrophs (22.5 +/- 3.6%) were monohormonal for either LH or FSH, when compared to anestrous animals (9.7 +/- 1.2%; P: < 0.02). Based on these findings we conclude that: 1) although the relative distribution of gonadotrophs is similar to those reported for other species, a significantly larger proportion of gonadotroph cells is present in the equine pituitary gland; 2) gonadotroph density does not appear to differ between sexually active and anestrous mares in the PD; 3) a larger proportion of gonadotrophs is apparent in the PT of sexually active animals; and 4) although a large incidence of bihormonal gonadotrophs is present in the horse, specific LH or FSH cells differentiate predominantly during the sexually active phase.     

Induction of ovulation in seasonally anestrous mares under ambient lights using recombinant equine FSH (reFSH)

Traditionally, mares are put under artificial lights to advance the first ovulation of the year. The aim of the present study was to determine the efficacy of recombinant equine FSH (reFSH) in stimulating follicular development and advancing the first ovulation of the year in seasonally anestrous mares compared with anestrous mares given a placebo. Both groups of mares were housed under ambient light conditions. Sixty deep anestrous mares of light horse breeds (follicular diameters ≤20 mm in diameter and progesterone <1 ng/mL) were maintained under a natural photoperiod at three different sites: University of California, Davis, Colorado State University, and University of Kentucky Gluck Centre. Twenty mares at each site were randomly allocated to receive either 0.65 mg of reFSH (group A: treatment; n = 10) or a placebo (group B: control; n = 10) twice daily by im beginning on January 31. Treatment continued until one or more preovulatory follicles developed or up to a maximum of 15 days. Randomized treatments were blinded. Follicular development was closely monitored by transrectal ultrasonography. When the largest follicle reached ≥35 mm in diameter, reFSH treatment was discontinued and an injection of 2500 international units of hCG was administered iv 36 hours later to induce ovulation. Jugular blood samples were collected daily from all mares at University of California, Davis, and processed for LH, FSH, progesterone, estradiol-17β, and immunoreactive-inhibin by RIA. All 30 mares receiving reFSH (group A) developed follicles ≥35 mm within 7.4 ± 1.6 days of treatment. Twenty-three of the 30 reFSH-treated mares (group A) ovulated within 72 hours after hCG administration. In contrast, mares in group B (placebo, control) did not exhibit significant follicular development and none ovulated within the 15-day observation period. Mares in group A had significantly higher plasma levels of FSH, estradiol-17β, and immunoreactive-inhibin during treatment but did not exhibit a preovulatory LH surge. Mares administered reFSH returned to anestrus and spontaneously ovulated at a similar calendar date as control mares. These data indicate that reFSH was effective in stimulating the development of ovarian follicles and advancing the first ovulation of the year in seasonally anestrous mares under ambient lights but was not successful in inducing continued cyclicity.     

Fluctuations in the plasma levels of the follicle-stimulating hormone during estrous cycle, anestrus, gestation and lactation in the ewe: evidence for an endogenous rhythm of FSH release

Two experiments were carried out to analyse FSH secretion in the ewe. The first was a long-term study during which four ewes under controlled photoperiods were checked for plasma concentrations of FSH twice daily for a period of 16 months. They were successively anestrous, cycling, gestating and lactating. The results suggested that an endogenous secretion rhythm of FSH persisted throughout each of the physiological states of the ewes. The periodic cycles of FSH production lasted about 5 days during anestrus and gestation but extended to about 6 days during estrus. One of the three waves of secretion we noted during one cycle was represented by the two periovulatory surges, the first coincident with the LH peak, the second occuring 30-40 h later. Plasma levels of FSH were similar during estrous cycles and anestrus, whereas the FSH secretion decreased gradually throughoug gestation. During lactation, large differences were observed among animals before the recovery of cyclic ovarian activity. The second experiment consisted of frequent blood sampling (every ten minutes) of eight ewes for 6 hours during anestrus. FSH was secreted differently compared to LH. No pulsatile production of FSH was demonstrated and no increase in FSH levels was seen at the time of the episodic LH surge.     

Progesterone concentration, follicular development and induction of cyclicity in dairy cows receiving intravaginal progesterone inserts

Objectives were to evaluate progesterone concentrations after cows had initiated estrous cycles following calving and induction of estrous cycles in postpartum anovular high-producing Holstein dairy cows treated with controlled internal drug releasing (CIDR). In experiment 1 (EXP1), 62 cows that had initiated estrous cycles received a new CIDR (NCIDR) containing 1.38 g of progesterone or a 7-d used autoclaved CIDR (UCIDR) 48h after luteolysis for 7 d. Ovaries were examined by ultrasonography, and plasma analyzed for concentrations of progesterone. In experiment 2 (EXP2), 515 cows diagnosed as anestrus were randomly assigned to untreated control, NCIDR or UCIDR for 6d. Plasma was analyzed for concentration of progesterone 12 d after CIDR removal to determine ovulation. In EXP1, milk yield and body condition did not influence progesterone concentrations. Concentration of progesterone tended to increase faster (P=0.10) in cows receiving UCIDR than NCIDR, but both treatments reached a plateau at 90min. Cows receiving the NCIDR had greater (P=0.04) concentrations of progesterone during the 7-d treatment, but they were mostly subluteal (<1.0 ng/mL) after d 2. After removal, concentrations of progesterone were greater for NCIDR than UCIDR for the first 45 min, and were similar thereafter. Multiparous cows had lesser (P=0.004) concentrations than primiparous cows throughout the study. The pattern of ovarian follicular development was not affected by treatment. In EXP2, induction of onset of estrous cycles increased (P<0.01) with progesterone treatments, but was similar between NCIDR and UCIDR. Proportion of cows experiencing shorter than typical length estrous cycles after first AI tended to be greater (P=0.09) for control cows than those receiving the CIDR, and for cows remaining anestrous than those in which onset of estrous cycles was induced. Pregnancy per AI and pregnancy loss were similar among treatments. Cows that resumed estrous cyclicity prior to first AI had greater (P=0.01) pregnancy per AI. Treatment of high-producing Holstein cows that had previously initiated onset of estrous cycles with CIDR resulted in subluteal concentrations of progesterone, but in anestrous high-producing cows increased induction of estrous cycles with no effect on fertility at first insemination.     

Influence of reproductive stage at PRID insertion on synchronization of estrus and ovulation in mares

In the present study, we investigated the effects of reproductive status, size of follicles and plasma progesterone concentrations of mares at PRID insertion on the efficacy of the treatment, estrous cycle patterns, plasma concentrations of progesterone and LH. The progesterone-releasing device (PRID) was administered intravaginally to 28 Haflinger mares for 11 days at different reproductive stages: anestrus (n=6), estrus (n=11) and diestrus (n=11). Plasma concentrations of progesterone at insertion (Day 1) of PRID differed among treatment groups (anestrus: 0.2-0.6 ng mL(-1), estrus: 0.2-0.5 and diestrus: 1.6-10.8 ng mL(-1); P<0.001). Total secretion of progesterone (area under curve (AUC)) during treatment period revealed highest values in diestrus (38.2+/-3.1 ng mL(-1)h(-1)) followed by estrus (25.1+/-2.7) and anestrus (21.0+/-0.4 ng mL(-1)h(-1); P<0.05). Progesterone area under curve (AUC) was positively correlated with initial progesterone concentrations (R=0.5; P<0.05), but it did not correlate with the interval from PRID removal to ovulation. Plasma concentrations of LH during treatment period, were significantly lower in anestrous mares (184.6+/-28.6 ng mL(-1)h(-1)) when compared to estrous and diestrous mares (349.7+/-53.3 and 370.5+/-40.3 ng mL(-1)h(-1); P<0.05). Follicular size at PRID insertion had no effects on the intervals from PRID removal to subsequent estrus and ovulation. Follicle diameters at removal of PRID were significantly correlated with the interval from coil removal to estrus (R=-0.55, P<0.05) and ovulation (R=-0.72, P<0.0004) in cyclic mares. In anestrus 0 of 6 (0%) mares, in estrus 5 of 11 (45.5%) and in diestrus 6 of 11 (54.5%) mares ovulated within a defined interval of 1 day before to 1 day after mean interval from PRID removal to ovulation. In cyclic mares, response to treatment was significantly higher when compared to anestrous mares: almost all mares responded with estrus and ovulation independent from the stage of the estrous cycle at the start of treatment. However, accuracy of synchronization was still unsatisfactory. In cyclic mares, the plasma progesterone concentrations at insertion of PRID seem to be more important for the efficacy of the treatment than the assignment to estrous cycle stages.     

Ex vivo influence of carbetocin on equine myometrial muscles and comparison with oxytocin

To determine the intercyclic effect of oxytocin and carbetocin on equine myometrial tissue, the effect of the drugs was evaluated through pharmacokinetic and pharmacodynamic studies. The complete pharmacokinetic profile for oxytocin was unknown and had to be established. To do so, 25 IU of oxytocin were administered intravenously to six cycling mares and blood samples were collected before and 2, 4, 8, and 15 min after administration. The half-life of oxytocin was determined to be 5.89 min, the clearance rate 11.67 L/min, mean residence time (MRT) 7.78 min. The effective plasma concentration was estimated to be 0.25 ng/mL. This was similar to the concentration achieved for the organ bath study where the concentration that produced 50% of the maximum effect (EC₅₀) was calculated at 0.45 ng/mL. To determine the intercyclic effect of oxytocin and carbetocin uterine myometrial samples were collected from slaughtered mares in estrus, diestrus, and anestrus. The samples were mounted in organ baths and exposed to four ascending, cumulative doses of oxytocin and carbetocin. Area under the curve and amplitude, maximum response (E_max), and concentration that produced 50% of the maximum effect were studied for each agonist and statistically evaluated. The effect of oxytocin on equine myometrial tissue was higher during diestrus, and surprisingly anestrus, than during estrus, whereas the effect of carbetocin was the same independent of the stage of estrous cycle. A significant difference was found for estrous and anestrous samples when oxytocin was used but not when carbetocin was used.     

Induction of reproductive function in anestrous mares using a dopamine antagonist

We investigated the role of dopamine in the regulation of seasonal reproductive activity in mares. Nine seasonal anestrous mares, maintained under a natural photoperiod, were treated daily with a dopamine D2 antagonist, [-]-sulpiride (200 mg/mare, im), beginning February 5 (day of year = 36) until the first ovulation of the year or for a maximum of 58. Nine untreated anestrous mares were maintained under the same conditions. The ovaries were examined by ultrasonography twice a week, and blood was collected three times a week for progesterone, LH, FSH and prolactin determinations. Mean day of first ovulation was significantly advanced for [-]-sulpiride-treated mares than control mares (mean day of year +/- SEM = 77.3 +/- 7.9 and 110.0 +/- 6.8, respectively; P < 0.01). Eight mares ovulated during [-]-sulpiride treatment while one mare failed to ovulate. Ovulation occurred 91 d after the start of treatment or on Day 127. All mares continued to have normal estrous cycles after the first ovulation. First cycle length and luteal progesterone concentrations did not differ between [-]-sulpiride-treated and control mares. Plasma prolactin concentrations were significantly increased at 2 and 9 h after [-]-sulpiride administration (P < 0.05), and had returned to basal levels by 24 h. At the time of the LH surge associated with the first ovulation, mean LH and FSH secretion was significantly higher in [-]-sulpiride-treated mares than in control mares (P < 0.05). These results suggest that dopamine plays a role in the control of reproductive seasonality in mares and exerts a tonic inhibition on reproductive activity during the anovulatory season.     

Reproduction in high body condition mares with high versus low leptin concentrations

Previous results from our laboratory indicated that a majority of mares with high body condition scores (BCS) displayed estrous cycles or had considerable follicular activity during the winter. Among these high BCS mares, about 35% of them exhibited a persistent hyperleptinemia and hyperinsulinemia. The current experiment was designed to compare the reproductive characteristics of high BCS mares with hyperleptinemia to those with normal (low) plasma concentrations of leptin during the winter and the first estrous cycle (or the first full cycle encountered for those already cycling). Light horse mares with high BCS (6-8.5) were assigned to groups based on leptin concentrations (8/group): low (<5 ng/mL) and high (>10 ng/mL). Beginning 7 January, mares were assessed every 3d for follicular activity and then daily once a follicle >25 mm was detected. Mares were subsequently monitored through their first and second ovulations. Leptin concentrations remained higher (P<0.001) in mares in the high leptin group over the duration of the experiment. Also, high leptin mares had greater (P<0.0001) insulin response to glucose infusion and a faster (P<0.05) rate of glucose clearance. One mare with high leptin and three mares with low leptin had progesterone concentrations indicative of the presence of a corpus luteum at the onset of the experiment. Plasma concentrations of LH, FSH, and progesterone did not differ between groups (P>0.1) during the first estrous cycle occurring after 7 January. Date of first ovulation after 7 January and interovulatory interval were similar (P>0.1) for the two groups, as were estimates of follicular numbers on the ovaries (small, medium, and large; P>0.1). It is concluded that the perturbations in leptin and insulin secretion observed in some high BCS mares are not associated with alterations in ovarian activity or the estrous cycle during winter and into the period of vernal transition.     

The effect of the manipulation of follicle-stimulating hormone (FSH)-peak characteristics on follicular wave dynamics in sheep: does an ovarian-independent endogenous rhythm in FSH secretion exist?

We designed three experiments to investigate the relationship between FSH peaks and ovarian follicular waves and to examine whether an endogenous rhythm of FSH peaks exists in sheep. In experiment 1, anestrous ewes were treated with ovine FSH (oFSH) or vehicle (6 ewes per group) at the expected time of an endogenous FSH peak, to double the FSH-peak amplitude in treated ewes. In experiment 2, anestrous ewes were treated with either oFSH or vehicle (6 ewes per group) at the expected time of two consecutive interpeak nadirs, such that the treated ewes had 5 FSH peaks in the time frame of 3 FSH peaks in control ewes. In experiment 3, to measure FSH concentrations, daily blood samples were collected from 5 cyclic ewes for a control period during the estrous cycle and then for three 17-day periods after ovariectomy. Daily blood samples were collected from another group of 8 ovariectomized ewes that were treated with estradiol-releasing implants and intravaginal progestogen sponges. Doubling the FSH-peak amplitude did not alter the characteristics of the following follicular wave. Increasing the frequency of FSH peaks stimulated the emergence of additional follicular waves, but did not alter the rhythmic occurrence of FSH peaks and follicular wave emergence. Endogenous follicular waves in oFSH-treated ewes emerged and grew in the presence of the growing largest follicle of the induced follicular waves. Finally, based on the observation of serum FSH concentrations in ovariectomized ewes, it appears that there exists an endogenous rhythm for peaks in daily serum FSH concentrations, which is, at least in part, independent of regulation by ovarian follicular growth patterns.     

Ultrasound and endocrine evaluation of the ovarian response to a single dose of 500 IU of eCG following a 12-day treatment with progestogen-releasing intravaginal sponges in the breeding and nonbreeding seasons in ewes

A standard dose of 500 IU of eCG is commonly given to progestogen pre-treated anestrous ewes for induction of estrus. Twelve seasonally anestrous and 12 cyclic Western White Face ewes were treated for 12 days with intravaginal sponges impregnated with medroxyprogesterone acetate (MAP). In trials in both the breeding and nonbreeding seasons, six randomly selected ewes were given 500 IU of eCG at sponge removal to determine the effects of low dose of eCG on ovarian antral follicular dynamics and ovulation. Ultrasound scanning and blood sampling were done daily. Treatment with eCG did not have marked effects on antral follicular growth. All ewes ovulated, except for five of six control anestrous ewes. Luteal structures and progesterone secretion were confirmed in all but the control anestrous ewes. In the breeding season, peak progesterone concentrations were greater (P<0.05) in eCG-treated compared to control ewes. Daily serum estradiol concentrations were greater in the periovulatory period in eCG-treated compared to control ewes (treatment-by-day interaction; P<0.05), particularly in anestrus. Progestogen-treated ewes ovulated follicles from several follicular waves, in contrast to ovulations of follicles from the final wave of the cycle in untreated, cyclic ewes. Anestrous ewes exhibited more frequent follicular waves and FSH peaks compared to cyclic ewes after a progestogen/eCG treatment. In conclusion, 500 IU of eCG given after 12 days of progestogen treatment had limited effects on the dynamics of ovarian follicular waves. However, eCG treatment increased serum concentrations of estradiol during the periovulatory period, particularly in anestrous ewes; this probably resulted in the synchronous estrus and ovulation in anestrous ewes.     

Shifts in gonadotropin and steroid levels that precede anestrus in female golden hamsters exposed to a short photoperiod

Female golden hamsters exposed to short photoperiods become anestrous and exhibit daily surges of gonadotropins and progesterone. Since little is known about the transition between the cycling and anovulatory states, the following experiments were done to determine whether there are hormonal changes that precede cessation of estrous cyclicity. Females killed on the morning of estrus, up to the tenth estrous cycle in short days, showed no hormonal or ovarian morphologic evidence of changes in reproductive function. When assessed on the afternoon of estrus, however, serum levels of luteinizing hormone and progesterone increased significantly before vaginal and ovarian cyclicity ceased. Females sampled in both the morning and afternoon at increasing durations since their last vaginal estrus revealed that maximal daily surges of both gonadotropins and progesterone were not consistently manifested until the vaginal cycle had been absent for 2 weeks. By then, estrogen levels and uterine weights were low and ovaries showed hypertrophied interstitia and arrested follicular growth. We have demonstrated that there are hormonal changes in females before the loss of the vaginal cycle and onset of major daily hormonal surges. Our results suggest that alterations in feedback relationships between steroid hormones and gonadotropins may precede photoperiod-induced anestrus.     

Influence of day length and endocrine status on luteinizing hormone secretion in intact and ovariectomized adult ferrets

The purpose of this study was to examine the pituitary-ovarian relationship of both estrous and anestrous female ferrets. The endocrine status of the animals was induced by manipulating photoperiod: females in estrus were housed in long days (16L:8D); females in anestrus were housed in short days (8L:16D). For studies of intact animals in both photoperiods, plasma luteinizing hormone (LH) levels were quantified in blood samples collected from adult ferrets at 5-min intervals over a 24-h period. Similar groups of females (estrous and anestrous) were ovariectomized (while remaining in their assigned photoperiods) and blood samples were collected at 5-min intervals for 4-h periods on Days 1, 2, 4, 10, 17, and 35 after ovariectomy. Intact, estrous females exhibited continuously low or undetectable levels of LH with no evidence of episodic secretion. Ovariectomy of these estrous animals resulted in rapid onset (within 24 h) of episodic LH secretion, with pulses occurring in excess of 1 pulse/h. No substantial further change in frequency or amplitude of pulses occurred in these females from 1 to 35 days postovariectomy. In contrast, intact anestrous ferrets exhibited clear episodic LH secretion at a frequency of about 0.4 pulses/h. Removal of ovaries from these females caused no change in LH secretion for 24-48 h, after which LH pulses gradually increased in frequency. By 18 days after ovariectomy, LH patterns were indistinguishable among ovariectomized females in long and short days. These studies suggest a major site of ovarian negative feedback on LH secretion during anestrus is the hypothalamus, whereas the site of the ovarian feedback in estrous females is not yet evident.     

Effect of daily clenbuterol and exogenous melatonin treatment on body fat,serum leptin and the expression of seasonal anestrus in the mare

A small percentage of mature mares continue to exhibit estrous cyclicity during the non-breeding season which is of interest because of the importance of timing of the breeding season to the equine breeding industry. Previously, it was demonstrated that the continuation of estrous cycles was more likely to occur in mature than young mares. Additionally, an apparent association exists between elevated body fat and increased circulating concentrations of leptin, and the occurrence of estrous cycles during the non-breeding season. Two experiments were conducted to test the hypothesis that pharmacological manipulation of body fat and leptin by administration of the beta(2)-adrenergic receptor agonist clenbuterol, with and without continuous treatment with melatonin, would increase the proportion of mares entering anestrus or advance its timing. In Experiment 1, eight mature mares were administered clenbuterol (3.2 microg/kg) daily from October through January and six mares were untreated. In Experiment 2, eight mares were administered clenbuterol daily from June through December, eight mares were treated with continuous release melatonin implants, and seven mares were treated with both clenbuterol and melatonin. Melatonin treatment was included to confirm previous results that constant treatment with melatonin did not advance the timing of anestrus. In both studies, leptin concentrations (P<0.01, both experiments) and body fat percentage (P<0.01, Experiment 1; P<0.05, Experiment 2) declined in response to daily clenbuterol treatment. Unexpectedly, however, this decrease was attenuated in mares also treated with melatonin (P<0.05). Although treatment with clenbuterol was associated with a decline in body fat and circulating concentrations of leptin, the timing or proportion of mares exhibiting anestrus was not modified (P>0.05). The results demonstrate the manipulation of body condition via pharmacological intervention does not modify the mechanisms controlling seasonal anestrus in the mare.     

Studies on the effect of manual massage of the ovaries on the reproductive activity of the mare

The aim of this study was to determine the influence of hand massage of mares' ovaries on breeding activity and hormonal changes in the winter anestrous period and during the luteal phase of the oestrus cycle. The experiment was conducted on 5 experimental and 5 control mares. In winter, (January) the experimental mares underwent 30-sec daily massage of both ovaries, for 30 d, and in summer (August) from the 6th day of the cycle to the occurrence of estrus. The sexual behavior of all mares was determined each day by individual teasing by a vigorous stallion, and the ovaries were checked by palpation per rectum and with an USG. Every second day blood samples were drawn from each mare to determine progesterone and estradiol in the plasma. Ovarian massage during deep winter anestrus had no significant effect on acceleration of the mares' active breeding season. Nevertheless, a higher concentration of estradiol was observed in the experimental group. These differences occurring on the 11th, 17th and 20th days were found to be significant (P ≤ 0.05). It was shown that during the summer period, in the luteal phase of the cycle, ovarian massage shortened the length of the estrous cycle, and ovulation was brought on somewhat earlier.     

The postpartum buffalo. II. Acyclicity and anestrus

Prolonged postpartum acyclicity (absence of ovarian cyclic activity) and anestrum (absence of overt estrous signs) are major sources of economic loss to buffalo breeders. Studies on the epidemiology of these two problems are highly recommended to achieve successful control. Review of the available literature on controlled studies in dairy buffaloes revealed that first ovulation as detected by rectal palpation and progesterone analysis occurred between 28-71 and 24-55 days, respectively, after calving. Postpartum estrus in the same studies occurred between 44 and 87 days. Reports concerned with data compiled from breeding records of research stations, breeding farms and small holders where estrus is a subjective measure, gave much longer periods. Also data from Egypt, India and Pakistan indicate that only 34-49% of buffaloes showed estrus during the first 90 days after calving and 31-42% remained anestrus for more than 150 days. In swamp buffaloes both postpartum ovulation and estrus are more delayed than in dairy buffaloes. The role of suckling, nutrition, body condition score at calving, milk yield, parity, season of calving and other minor factors were discussed. First postpartum ovulation is frequently followed by one or more short estrous cycles (<18 days). Long anovulatory and anestrous periods due to prolonged inter-luteal phase were reported to occur after short cycles. Also long anestrous periods due to cessation of cyclic activity (true anestrus) for 3 or more weeks and prolonged luteal activity for 28 days or more were described to occur in about 25 and 8-11% of the buffaloes, respectively, after the first or second ovulation. These cycle irregularities certainly impose difficulties on estrus detection programs in postpartum buffaloes. Four main forms of anestrus i.e. true anestrus (inactive ovaries and small and medium sized anovulatory follicles), subestrus, prolonged luteal activity and ovarian cysts in addition to pregnancy are reviewed in this article. Differentiation between true anestrus and subestrus is particularly important in buffaloes because of their weak estrous signs. However, the accuracy of a single rectal palpation of the ovaries is limited with an overestimation of the frequency of true anestrus due to misdiagnosis of the corpus luteum. The possible causes are discussed.     

Short- and long-term effects of unilateral ovariectomy in sheep: causative mechanisms

The mechanisms of ovulatory compensation following unilateral ovariectomy (ULO) are still not understood. In the present study, we investigated the short- and long-term effects of ULO in sheep using transrectal ovarian ultrasonography and hormone estimations made during the estrous cycle in which surgery was done, the estrous cycle 2 mo after surgery, and the 17-day period during the subsequent anestrus. The ULOs were done when a follicle in the first follicular wave of the cycle reached a diameter > or =5 mm, leaving at least one corpus luteum and one ovulatory-sized follicle in the remaining ovary. Ovulation rate per ewe was 50% higher in the ULO ewes compared with the control ewes at the end of the cycle during which surgery was performed, but it did not differ between groups at the end of the cycle, 2 mo later. This compensation of ovulation rate in ULO ewes was due to ovulation of follicles from the penultimate follicular wave in addition to those from the final wave of the cycle. Ovulation from multiple follicular waves appeared to be due to a prolongation of the static phase of the largest follicle of the penultimate wave of the cycle. Interestingly, the length of the static phase of waves was prolonged in ULO ewes compared with control ewes in every instance where the length of the static phase could be determined. Changes in follicular dynamics due to ULO were not associated with alterations in FSH and LH secretion. In conclusion, ovulatory compensation in ULO sheep involves ovulation from multiple follicular waves due to the lengthened static phase of ovulatory-sized follicles. These altered antral follicular dynamics do not appear to be FSH or LH dependent. Further studies are required to examine the potential role of the nervous system in the enhancement of the life span of the ovulatory-sized follicles leading to ovulatory compensation by the unpaired ovary in ULO sheep.     

Treatment with recombinant equine follicle stimulating hormone (reFSH) followed by recombinant equine luteinizing hormone (reLH) increases embryo recovery in superovulated mares

The dynamics of ovarian follicular development depend on a timely interaction of gonadotropins and gonadal feedback in the mare. The development and efficacy of genetically cloned recombinant equine gonadotropins (reFSH and reLH) increase follicular activity and induce ovulation, respectively, but an optimum embryo recovery regimen in superovulated mares has not been established. The objective of this study was to determine if treatment with reFSH followed by reLH would increase the embryo per ovulation ratio and the number of embryos recovered after superovulation in mares. Sixteen estrous cycling mares of light horse breeds (4-12 years) were randomly assigned to one of two groups: Group 1; reFSH (0.65mg)/PBS (n=8) and Group 2; reFSH (0.65mg)/reLH (1.5mg) (n=8). On the day of a 22-25mm follicle post-ovulation mares were injected IV twice daily with reFSH for 3 days (PGF(2α) given IM on the second day of treatment) and once per day thereafter until a follicle or cohort of follicles reached 29mm after which either PBS or reLH was added and both groups injected IV twice daily until the presence of a 32mm follicles, when reFSH was discontinued. Thereafter, mares were injected three times daily IV with only PBS or reLH until a majority of follicles reached 35-38mm when treatment was discontinued. Mares were given hCG IV (2500IU) to induce ovulation and bred. Embryo recovery was performed on day 8 day post-treatment ovulation. Daily jugular blood samples were collected from the time of first ovulation until 8 days post-treatment ovulation. Blood samples were analyzed for LH, FSH, estradiol, progesterone and inhibin by validated RIA. Duration of treatment to a ≥35mm follicle(s) and number of ovulatory size follicles were similar between reFSH/reLH and reFSH/PBS treated mares. The number of ovulations was greater (P<0.01) in the reFSH/reLH group, while the number of anovulatory follicles was less (P<0.05) compared to the reFSH/PBS group. Number of total embryos recovered were greater in reFSH/reLH mares than in the reFSH/PBS mares (P≤0.01). The embryo per ovulation ratio tended to be greater (P=0.07) in the reFSH/reLH mares. Circulating concentrations of estradiol, inhibin, LH and progesterone were not statistically different between groups. Plasma concentrations of FSH were less (P<0.01) in the reFSH/reLH treated mares on days 0, 1, 4, 6, 7 and 8 post-treatment ovulation. In summary, reFSH with the addition of reLH, which is critical for final follicular and oocyte maturation, was effective in increasing the number of ovulations and embryos recovered, as well as reduce the number of anovulatory follicles, making this a more viable option than treatment with reFSH alone. Further evaluation is needed to determine the dose and regimen of reFSH/reLH to significantly increase the embryo per ovulation ratio.     

Effects of canine serum collected from dogs at different estrous cycle stages on in vitro nuclear maturation of canine oocytes

Canine oocytes are ovulated at prophase of the first meiotic division and undergo maturation in the distal part of the oviduct for at least 48-72 h. Because of these differences from other domestic mammals, the efficiency of in vitro maturation (IVM) of canine oocyte is very low. The present study was conducted to evaluate the effects of canine serum on IVM of canine oocytes recovered from ovaries in various reproductive states (follicular, luteal or anestrous stages). Oocytes were recovered by mincing ovaries from bitches presented for ovariohysterectomy at various stages of the estrous cycle. Heat-inactivated canine serum was prepared with blood taken from dogs at the anestrous, estrous or diestrous stage of the estrous cycle as determined by progesterone concentration and vaginal cytology. Oocytes were cultured for 72 h in tissue culture medium (TCM)-199 supplemented with 10% canine anestrous, estrous or diestrous serum or fetal bovine serum (FBS) (experiment 1), or supplemented with 0 (control), 5%, 10% or 20% canine estrous serum (experiment 2). In experiment 1, IVM of oocytes collected at the follicular stage of the estrous cycle to metaphase II (MII) stage was higher (p < 0.05) with canine estrous serum (14.2%) than with canine anestrous (5.2%) or diestrous serum (6.3%), FBS (2.2%) or in the control (2.2%). In experiment 2, oocytes collected at the follicular stage of the estrous cycle cultured in TCM-199 with 10% canine estrous serum showed a higher maturation rate to MII stage (13.5%, p < 0.05) compared with those cultured with 5% (1.3% MII) or 20% canine estrous serum (5.1% MII) or the control (2.7% MII). In conclusion, our results demonstrate that supplementing culture medium with 10% canine estrous serum improves IVM of canine follicular stage oocytes.     

Effects of repeated transvaginal ultrasound–guided aspirations performed in anestrous and cyclic mares on P4 and E2 plasma levels and luteal function

The aim of the present study was to verify how repeated ovum pick-up (OPU), performed in anestrous and cyclic mares, affect ovarian activity, measured by progesterone (P4) and 17ß-estradiol (E2) plasma levels. Ovum pick-up of all visible follicles was performed every 9 to 12 days, and four sessions were carried out during anestrous (A) and breeding season (BS). The number of aspirated follicles per mare at each session was not significantly different between the two periods (BS: 6.1 ± 2.4; A: 7.5 ± 4.4; P > 0.05), but the mean follicular diameter was significantly higher during BS (16.0 ± 7.1 vs. 10.2 ± 5.1 mm; P < 0.05); during A the number of aspirated follicles less than 15 mm in diameter resulted significantly higher than that registered in BS (5.1 ± 2.7 vs. 3.0 ± 1.8; P < 0.05). The total mean value of P4 was higher in BS than in A (6.3 ± 4.4 vs. 0.3 ± 1.8 ng/mL; P < 0.05), whereas the total mean level of E2 was not different between the two periods (3.8 ± 3.4 vs. 2.5 ± 2.7 pg/mL; P > 0.05). Estradiol plasma values resulted positively correlated, in A and BS, with diameter of follicles detected on the ovaries (R = 0.345 and R = 0.331, respectively), whereas a negative correlation was observed between P4 and follicular diameter in BS (R = −0.162). Both E2 and P4 presented a high individual variability during BS; in particular, in three of seven mares, P4 trend was compatible with a normal estrous cycle, and the interval between two consecutive peaks was 21 days. In two of seven mares, with CL at first OPU, P4 concentrations remained more than 3 ng/mL throughout the entire treatment period. Finally, in two of seven animals, P4 levels initially showed a similar pattern to that of a normal estrous cycle, then, after the second aspiration, they remained consistently higher than 3 ng/mL. When the procedure was carried out in cyclic animals, the influence of this technique on ovarian activity seemed to be related to individual variability although, according to progesterone values, structures observed on the ovaries after aspirations presented luteal function. Furthermore, the resumption of normal ovarian activity, after repeated OPU sessions, occurred in a period not much longer than the duration of a normal estrous cycle (25.4 ± 5.2 days). Data recorded during nonbreeding period showed that repeated OPU in anestrous mares do not affect ovarian activity and do not anticipate the resumption of ovarian cyclicity. However, based on the number of aspirated follicles in anestrous and cyclic mares, both types of subjects could be considered as oocyte donors.