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Successful reproductive management of domestic mammals depends primarily upon timely identification of oestrous cycle stages. There is a need to develop an alternative non-invasive, welfare-friendly, accurate and reliable method to identify reproductive cycle stages. This is of particular interest for horse breeders, because horses are high-value farm animals that require careful management and individual monitoring. Saliva sampling is non-invasive, painless and welfare-friendly. Thus, we performed a metabolomic analysis of equine saliva during different reproductive stages to identify changes in the salivary metabolome during anoestrus, the oestrous cycle and early gestation. We compared the saliva and plasma metabolomes to investigate the relationship between the two fluids according to the physiological stage. We collected saliva and plasma samples from six mares during seasonal anoestrus, during the follicular phase 3 days, 2 days and 1 day before ovulation and the day when ovulation was detected, during the luteal phase 6 days after ovulation, and during early gestation 18 days after ovulation and insemination. Metabolome analysis was performed by proton-nuclear magnetic resonance spectroscopy. We identified 58 and 51 metabolites in saliva and plasma, respectively. The levels of four metabolites or groups of metabolites in saliva and five metabolites or groups of metabolites in plasma showed significant modifications during the 4 days until ovulation, ie 3 days prior to and on the day of ovulation. The levels of 11 metabolites or groups of metabolites in saliva and 17 metabolites or groups of metabolites in plasma were significantly different between the seasonal anoestrus and the ovarian cyclicity period. The physiological mechanisms involved in the onset of ovarian cyclicity and in ovulation induced modifications of the metabolome both in plasma and saliva. The metabolites whose salivary levels changed during the reproductive cycle could be potential salivary biomarkers to detect the reproductive stage in a welfare friendly production system. In particular, we propose creatine and alanine as candidate salivary biomarkers of ovulation and of the onset of ovarian cyclicity, respectively. However, extensive validation of their reliability is required. Our study contributes to extend to domestic mammals the use of saliva as a non-invasive alternative diagnostic fluid for reproduction in a welfare-friendly production system., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Kisspeptins are neuropeptides encoded by the Kiss1 gene that was discovered as a metastasis suppressor gene in melanoma and breast cancer. Kisspeptin has pivotal functions for gonadotropin-releasing hormone secretion and plays integrated roles in the hypothalamic-pituitary-gonadal axis. However, little is known about the peripheral expression of kisspeptin in ruminants, especially in the female reproductive tract. Here, the objectives of the current study were to investigate the spatial localization of kisspeptin and mRNA expression of Kiss1 and its receptor (Kiss1r) in the fallopian tubes (FT) and uterus of goats at varied reproductive activity (cyclic versus true anoestrous goats, n=6, each). Specimens of the uterus and FT were collected and fixed using paraformaldehyde to investigate the localizations of kisspeptin in the selected tissues by immunohistochemistry. Another set of samples was snape-frozen to identify the expressions of mRNAs encoding Kiss1 and Kiss1r using real-time PCR. Results revealed immunolocalizations of kisspeptin in the uterus and the FT. The staining of kisspeptin was found mainly in the mucosal epithelium of the uterus the FT, and the endometrial glands. Very intense staining of kisspeptin was found in the uterine and FT specimens in the true anoestrous goats compared to that in cyclic ones. The expression of mRNA encoding Kiss1 gene was significantly higher in the uterine specimen of cyclic goats (1.00±0.09) compared to that in the true anoestrous goats (0.62±0.08) (P ˂0.05), while the expression of mRNA encoding Kiss1r was significantly (P ˂0.001) higher in the uterine tissues of true anoestrous goats (1.78±0.17) compared to that in cyclic ones (1.00±0.11). In conclusion, immunohistochemical localization of kisspeptin and the expression of mRNA encoding Kiss1/Kiss1r revealed spatial changes in the uterus and FT of goats according to the reproductive potential of goats (cyclic versus true anoestrous goats). However, the definitive local role of kisspeptin in the uterus and FT need further investigation., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest for this paper., (Copyright © 2024. Published by Elsevier Inc.)

Most sheep are seasonal estrus, and they breed in autumn when the days get shorter. Seasonal estrus is an important factor that affects the productivity and fertility of sheep. The key point to solve this problem is to explore the regulation mechanism of estrus in sheep. Therefore, in this study, transcriptomic sequencing technology was used to identify differentially expressed mRNAs in the hypothalamus, pituitary and ovary of Small Tail Han sheep (year-round estrus) and tan sheep (seasonal estrus) among luteal, proestrus and estrus stages. There were 256,923,304,156 mRNAs being identified in the hypothalamus, pituitary and ovary, respectively. Functional analysis showed that the photosensor, leucine and isoleucine biosynthesis pathways were enriched significantly. It is speculated that photoperiod may initiate estrus by stimulating the corresponding pathways in hypothalamus. ODC1 , PRLH , CRYBB2 , SMAD5 , OPN1SW , TPH1 are believed to be key genes involved in the estrogen process. In conclusion, this study expanded the database of indigenous sheep breeds, and also provided new candidate genes for future genetic and molecular studies on the seasonal estrus trait in sheep.

Background: As the milk production of dairy cows increases, the reproductive capacity gradually declines. Ovarian quiescence has become one of the concerns of the dairy industry., Objective: To explore the different plasma metabolite levels between estrus and anestrus in energy balanced (EB) dairy cows., Methods: Ten estrous and 10 anestrus EB Holstein cows in early lactation were selected for the study. 1 H nuclear magnetic resonance technology was used to detect plasma metabolites and screen different plasma metabolites between anestrous and estrous cows at 60-90 days postpartum using multivariate statistical analysis., Results: Within an elective waiting period of 60-90 days postpartum mean plasma concentration of total estrogens was significantly higher in estrus cows as compared to anestrus cows (71.2 ± 26.0 and 42.4 ± 16.7 pg/mL, respectively). Seven plasma metabolites (isoleucine, leucine, valine, alanine, arginine, choline and phosphatecholine) demonstrated significant decreases in estrous dairy cows relative to anestrous subjects. The main pathway was leucine, isoleucine and valine biosynthesis., Conclusion: Anestrus in dairy cows is accompanied by alterations in amino acid, glucose and lipid metabolism based on 1 H NMR analysis.

This study aimed to explain how prolonged inhibition of central dopaminergic activity affects the cellular processes governing gonadotrophin-releasing hormone (GnRH) and LH secretion in anoestrous sheep. For this purpose, the study included two experimental approaches: first, we investigated the effect of infusion of sulpiride, a dopaminergic D2 receptor antagonist (D2R), on GnRH and GnRH receptor (GnRHR) biosynthesis in the hypothalamus and on GnRHR in the anterior pituitary using an immunoassay. This analysis was supplemented by analysis of plasma LH levels by radioimmunoassay. Second, we used real-time polymerase chain reaction to analyse the influence of sulpiride on the levels of kisspeptin (Kiss1) mRNA in the preoptic area and ventromedial hypothalamus including arcuate nucleus (VMH/ARC), and RFamide-related peptide-3 (RFRP-3) mRNA in the paraventricular nucleus (PVN) and dorsomedial hypothalamic nucleus. Sulpiride significantly increased plasma LH concentration and the levels of GnRH and GnRHR in the hypothalamic-pituitary unit. The abolition of dopaminergic activity resulted in a significant increase in transcript level of Kiss1 in VMH/ARC and a decrease of RFRP-3 in PVN. The study demonstrates that dopaminergic neurotransmission through D2R is involved in the regulatory pathways of GnRH and GnRHR biosynthesis in the hypothalamic-pituitary unit of anoestrous sheep, conceivably via mechanisms in which Kiss1 and RFRP-3 participate.

Exposure to a ram during spring stimulates luteinizing hormone (LH) secretion and can induce ovulation in sexually quiescent ewes ("ram effect"). Kisspeptin (Kiss) present in the arcuate nucleus (ARC) and the preoptic area (POA) is a potent stimulators of LH secretion. Our aim was to investigate whether Kiss neurons mediate the increase in LH secretion during the ram effect. With double immunofluorescent detection, we identified Kiss neurons (Kiss IR) activated (Fos IR) by exposure to a ram for 2 hours (M2) or 12 hours (M12) or to ewes for 2 hours (C). The density of cells Kiss + Fos IR and the proportion of Kiss IR cells that were also Fos IR cells were higher in M2 and M12 than in C in ARC (P < 0.002) and POA (P < 0.02). In ARC, these parameters were also higher in M12 than in M2 (P < 0.02 and P < 0.05). Kiss antagonist (P234 10-6M) administered by retrodialysis in POA for 3 hours at the time of introduction of the ram reduced the amplitude of the male-induced increase in LH concentration compared with solvent (P < 0.02). In ARC, P234 had a more limited effect (P < 0.038 1 hour after P234) but pulse frequency increased less than after solvent (P = 0.07). In contrast, Kiss antagonist (P271 10-4M) infused in ARC but not POA 6 to 18 hours after introduction of the ram prevented the LH surge in the ewe (0/6 vs 4/5 and 4/6 in C). These results suggest that both populations of Kiss neurons are involved in the ram-induced pulsatile LH secretion and in the LH surge., (Copyright © 2017 Endocrine Society.)

Two modes of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion are necessary for female fertility: surge and episodic secretion. However, the neural systems that regulate these GnRH secretion patterns are still under investigation. The neuropeptide somatostatin (SST) inhibits episodic LH secretion in humans and sheep, and several lines of evidence suggest SST may regulate secretion during the LH surge. In this study, we examined whether SST alters the LH surge in ewes by administering a SST receptor (SSTR) 2 agonist (octreotide) or antagonist [CYN154806 (CYN)] into the third ventricle during an estrogen-induced LH surge and whether endogenous SST alters episodic LH secretion. Neither octreotide nor CYN altered the amplitude or timing of the LH surge. Administration of CYN to intact ewes during the breeding season or anestrus increased LH secretion and increased c-Fos in a subset GnRH and kisspeptin cells during anestrus. To determine if these stimulatory effects are steroid dependent or independent, we administered CYN to ovariectomized ewes. This SSTR2 antagonist increased LH pulse frequency in ovariectomized ewes during anestrus but not during the breeding season. This study provides evidence that endogenous SST contributes to the control of LH secretion. The results demonstrate that SST, acting through SSTR2, inhibits episodic LH secretion, likely acting in the mediobasal hypothalamus, but action at this receptor does not alter surge secretion. Additionally, these data provide evidence that SST contributes to the steroid-independent suppression of LH pulse frequency during anestrus., (Copyright © 2017 Endocrine Society.)

The aim of this study was to determine the mRNA LHR and LHR protein expression pattern in the canine ovarian follicles at different stage of development throughout the estrous cycle. Dog ovaries were obtained from 1-6y bitches at proestrus/estrus, anestrus and diestrus stages following ovariohysterectomy. Follicular cells were mechanically recovered from follicles distributed into four types (preantral, small antral, medium antral and large antral). Total RNA extraction was performed and the evaluation of gene expression levels was achieved by relative quantification q-PCR analysis. Intrafollicular amounts of LHR were assessed by western blot method. All results were evaluated by ANOVA. The expression levels of mRNA LHR in follicular cells were observed in every stage of development, however this gene expression varied over the estrous cycle. LHR transcripts increased (P < 0.05) from preantral to antral stage. There were not differences in LHR gene expression among follicles at preantral stages; however, at antral stages the lowest (P < 0.05) LHR mRNA expression was found at anestrus and the highest (P < 0.05) at proestrus/estrus. The LHR protein was also detected in dog follicles in all reproductive phases with patterns varying with stage of follicular development over the reproductive cycle. The antibody against human LHR revealed two bands at ∼90 and ∼67 kDa, probably representing the matured protein and its precursor respectively. Both bands LHR appeared already at preantral follicles increasing (P < 0.05) with growth. A high proportion of LHR was presented as immature forms in all follicles stages during different phases of the estrous cycle. In conclusion, the gene and protein of LHR are differentially expressed in dog follicles over the estrous cycle, increasing with growth and the precursor protein is the most predominant LHR form present in canine follicles., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study was performed to explain how the molecular processes governing the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamic-pituitary unit are reflected by luteinizing hormone (LH) secretion in sheep during anoestrous period and during luteal and follicular phases of the oestrous cycle. Using an enzyme-linked immunosorbent assay (ELISA), we analyzed the levels of GnRH and GnRHR in preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk-median eminence (SME), and GnRHR in the anterior pituitary gland (AP). Radioimmunoassay has also been used to define changes in plasma LH concentrations. The study provides evidence that the levels of GnRH in the whole hypothalamus of anoestrous ewes were lower than that in sheep during the follicular phase of the oestrous cycle (POA: p < 0.001, AH: p < 0.001, VM: p < 0.01, SME: p < 0.001) and not always than in luteal phase animals (POA: p < 0.05, SME: p < 0.05). It has also been demonstrated that the GnRHR amount in the hypothalamus-anterior pituitary unit, as well as LH level, in the blood in anoestrous ewes were significantly lower than those detected in animals of both cyclic groups. Our data suggest that decrease in LH secretion during the long photoperiod in sheep may be due to low translational activity of genes encoding both GnRH and GnRHR.

The aim of the study was to localize oxytocin receptors (OTR) and measure mRNA expression of OTR in the canine uterus with and without the influence of progesterone. Uterine samples were taken from nine anoestrous and eight dioestrous bitches during ovariohysterectomy. Histological changes were evaluated in haematoxylin and eosin (HE)-stained samples. Purified polyclonal antibody for OTR was used in immunohistochemistry to localize receptors in uterine layers. Relative mRNA concentration of OTR was evaluated with real-time PCR from full-thickness uterine samples taken from the middle horn and the body. Myometrial smooth muscle cells, endometrial luminal epithelium (LE) and deep and superficial glandular epithelium were positively stained for oxytocin receptors in non-pregnant animals. No significant difference in staining intensity was detected between uterine middle horn and body. However, the staining intensity of LE was significantly higher in dioestrous than in anoestrous uteri (p < .05). Leucocytes and endothelium of blood vessels were also positively stained for OTR. Real-time PCR showed no significant differences in OTR mRNA expression between the middle horn and the body of the uterus, or between anoestrous and dioestrous uterus. No correlation was noted between OTR mRNA expression and blood progesterone concentration. In conclusion, despite the apparent inactivity, the uterus of the non-pregnant bitch expresses OTR. The distribution or relative expression of OTR does not differ between uterine horn and body in dioestrus or anoestrus except in LE. LE may have more oxytocin-dependent activity during dioestrus than anoestrus., (© 2016 Blackwell Verlag GmbH.)

This study investigated the distribution of lipid droplets (LD) in immature canine oocytes in relation to their size and the reproductive stage. Oocytes were collected from the ovaries of bitches at different estrous stages, divided according to their size (110-120 µm; >120 µm), and stained with Nile Red to detect lipid droplet distribution. At the follicular phase most of the oocytes displayed a diffuse pattern of LD distribution, whereas at anestrus and luteal phase oocytes showed LD mainly in a peripheral/ perinuclear LD distribution. A significantly higher intensity of LD has been recorded in the oocytes > 120 µm compared to those of smaller size (110 - 120 µm) at all stages of the estrous cycle. At follicular phase, oocytes > 120 µm displayed LD intensity similar to that of oocytes > 120 µm at luteal phase and higher compared to the oocytes of the other groups., (© 2015 Japanese Society of Animal Science.)

High ambient temperature during summer in tropical and subtropical countries predisposes water buffaloes (Bubalus bubalis) to develop oxidative stress having antigonadotropic and antisteroidogenic actions. Melatonin is a regulator of seasonal reproduction in photoperiodic species and highly effective antioxidant and free radical scavenger. Therefore, a study was designed to evaluate the effect of sustained-release melatonin on biomarkers of oxidative stress i.e., the serum malondialdehyde (MDA) and nitric oxide (NO), and the total antioxidant capacity (TAC). For the study, postpartum buffaloes diagnosed as summer anestrus (absence of overt signs of estrus, concurrent rectal examination, and RIA for serum progesterone) were grouped as treated (single subcutaneous injection of melatonin at 18 mg/50 kg body weight dissolved in sterilized corn oil as vehicle, n = 20) and untreated (subcutaneous sterilized corn oil, n = 8). Blood sampling for estimation of serum TAC and MDA (mmol/L) and NO (μmol/L) was carried out at 4 days of interval from 8 days before treatment till 28 days after treatment or for the ensuing entire cycle length. Results showed serum TAC concentration was higher in the treatment group with a significant (P < 0.05) increasing trend, whereas MDA and NO revealed a significant (P < 0.05) decline. Serum MDA and NO were higher in control compared with those of treatment group. Moreover, buffaloes in the treatment group showed 90% estrus induction with 18.06 ± 1.57 days mean interval from treatment to the onset of estrus. These results report that melatonin has a protective effect by elevating antioxidant status and reducing oxidative stress resulting in the induction of cyclicity in summer-stressed anestrous buffaloes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Sheep are seasonal breeders, but exposure of anoestrus ewes to rams results in a rapid increase in luteinizing hormone (LH) secretion, eventually leading to surge in LH. Although LH secretion is known to be under the control of many neurotransmitters, noradrenaline (NA) is of particular importance for the LH surge in induced ovulators, although little is known about its role in LH secretion induced by males in spontaneous ovulators. To address this question, anoestrus ewes fitted with guide-tubes in the medial preoptic area (MPOA) or the ventromedial hypothalamus were subjected to microdialysis and blood sampling every 15 min for an hour before and 2 h after exposure to rams, and the concentrations of LH, monoamine and amino acid transmitters were measured. In ewes implanted in the posterior MPOA that responded to the ram by an increase in LH pulses, NA concentrations changed after exposure to the ram (P<0.018) and were higher at 15 (P<0.054) and 45 min (P<0.03) after male introduction than before. By contrast, no change in NA could be detected in ewes implanted in the same region, but not responding to the ram, or in those showing increased LH pulsatility, but implanted in the anterior MPOA or in the ventromedial hypothalamus. No changes were observed in other neurotransmitters or when the ewes were exposed to male odour alone. These results suggest that NA release in the posterior MPOA is selectively involved in the triggering of LH secretion by rams in anoestrus ewes.

The first ovulation induced by male effect in sheep during seasonal anoestrus usually results in the development of a short cycle that can be avoided by progesterone priming before ram introduction. In elucidating the involvement of the hypothalamic-pituitary-gonadal axis in the occurrence of short cycles, the effects of progesterone and the time of anoestrus on the development of male-induced preovulatory follicles were investigated in anoestrous ewes using morphological, endocrine and molecular approaches. Ewes were primed with progesterone for 2 (CIDR2) or 12 days (CIDR12) and untreated ewes used as controls during early (April) and late (June) anoestrus. The duration of follicular growth and the lifespan of the male-induced preovulatory follicles were prolonged by ∼1.6 days in CIDR12 ewes compared with the controls. These changes were accompanied by a delay in the preovulatory LH and FSH surges and ovulation. Intra-follicular oestradiol concentration and mRNA levels of LHCGR and STAR in the granulosa and theca cells of the preovulatory follicles were higher in CIDR12 ewes than the control ewes. The expression of mRNA levels of CYP11A1 and CYP17A1 also increased in theca cells of CIDR12 ewes. CIDR2 ewes gave intermediate results. Moreover, ewes ovulated earlier in June than in April, without changes in the duration of follicular growth, but these effects were unrelated to the lifespan of corpus luteum. Our results give the first evidence supporting the positive effect of progesterone priming on the completion of growth and maturation of preovulatory follicles induced by male effect in seasonal anoestrous ewes, thereby preventing short cycles., (© 2014 Society for Reproduction and Fertility.)

Negative energy balance is a known risk factor for decreased fertility in dairy cows. This study evaluated the accuracy of plasma concentrations of nonesterified fatty acids (NEFA), β-hydroxybutyrate (BHBA), and insulin-like growth factor 1 (IGF-1)-factors related to negative energy balance-in predicting decreased fertility. One plasma sample per cow was collected from 480 cows in 12 herds during the period from d 4 to 21 in milk and analyzed for NEFA, BHBA, and IGF-1. For each cow, data on breed, parity, calving date, gynecological examinations, and insemination dates were obtained. Milk samples from 241 cows in 7 of the participating herds were analyzed for progesterone concentration to define the first day of luteal activity. The diagnostic sensitivity (Se) and specificity (Sp) at different cut-off concentrations of NEFA, BHBA, or IGF-1 were calculated and related to individual cow fertility status, measured as anestrus (ANEST), delayed first artificial insemination (DFAI), or delayed conception. Positive and negative predictive values (PV+; PV-) were calculated considering different levels of (within-herd) prevalence. Strata (i.e., subgroup)-specific Se and Sp and associations between test results and fertility parameters were investigated using logistic regression. The NEFA and BHBA tests for ANEST and DFAI had the highest combined Se and Sp and were thus evaluated further. Cut-off values with Sp around 80% were used in this step to provide a reasonable number of test-positive cows, representing a practical situation. This corresponded to a cut-off value for the NEFA test of 400 µEq/L (Se 0.27-0.45) and for the BHBA test of 1.8mM (Se 0.15-0.30) across all cows included in the study. The estimated Sp was generally higher than the original 80%, but the corresponding Se was further decreased when the test was used in heifers compared with older cows. The true prevalence of ANEST in the study population was 27%, which gave a PV+ of 0.36 to 0.45 and a PV- of 0.76 to 0.79. With 35% true prevalence of DFAI, PV+ was 0.29 to 0.38 and PV- was 0.64 to 0.66. Thus, overall test performance was low when metabolic indicators measured as single values in early lactation were used to predict fertility in dairy cows, but accuracy was influenced by cow-level factors such as parity. The prevalence of the target condition (in this case, decreased fertility) also influences test usefulness and should be considered when planning test systems and interpreting test results., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

We investigated whether live vocalizations emitted by bucks interacting with anestrous females stimulate secretion of LH, estrous behavior and ovulation in anestrous goats. In experiment 1, bucks rendered sexually active by exposure to long days followed by natural photoperiod were exposed in a light-proof-building to five anestrous females. Buck vocalizations were reproduced through a microphone-amplifier-loudspeaker system to an open pen where one group of goats (n=6) was exposed for 10 days to these live vocalizations. Another group of females (n=6) was isolated from males and vocalizations. The proportion of goats displaying estrous behavior was significantly higher in females exposed to buck vocalizations than in females isolated from males. The proportion of goats that ovulated did not differ between the 2 groups (exposed to males versus isolated). In experiment 2, female goats that either had previous contact with males (n=7), or no previous contact with males (n=7) were exposed to live buck vocalizations, reproduced as described in experiment 1, for 5 days. The number and amplitude of LH pulses did not differ between groups before exposition to buck vocalizations. Five days of exposure to male vocalizations significantly increased LH pulsatility only in females that had previous contact with males, while LH pulse amplitude was not modified. We concluded that live buck vocalizations can stimulate estrous behavior and LH secretion in goats if they have had previous contact with bucks., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Little is known about the role of activin B during folliculogenesis. This study investigated the expression levels of activin/inhibin subunits (βA, βB, and α), steroid enzyme, and gonadotrophin receptors in theca (TC) and granulosa cells (GC) by QPCR and activin A and B and inhibin A protein levels in follicular fluid (FF) of developing sheep follicles during estrus and anestrus. The effect of activin B on androgen production from primary TC cultures in vitro was also assessed. During folliculogenesis, in anestrus and estrus, FF activin B concentrations and thecal and GC activin βB mRNA levels decreased as follicle diameter increased from 1-3 to >6  mm regardless of estrogenic status. Estrogenic preovulatory follicles had reduced concentrations of FF activins B and A, and TC and GCs expressed higher levels of activin βA mRNA at 3-4  mm, and TCs more inhibin α mRNA at >4  mm stages of development compared with nonestrogenic follicles. Activin B decreased androstenedione production from primary TCs in vitro, an effect blocked by inhibin A. Thus, sheep follicles 1-3  mm in diameter contained high FF levels of activin B, which decreased as the follicle size increased, and, like activin A, suppressed thecal androgen production in vitro, an effect blocked by inhibin. Furthermore, the theca of large estrogenic follicles expressed high levels of inhibin α and activin βA mRNA suggesting local thecal derived inhibin A production. This would inhibit the negative effects of thecal activins B and A ensuring maximum androgen production for enhanced estradiol production by the preovulatory follicle(s).

Human genetic studies have suggested that kisspeptin and neurokinin B (NKB) play pivotal roles in the control of gonadotropin-releasing hormone (GnRH) secretion. However, the role of NKB in this context is less clear compared with that of kisspeptin. In the present study, we investigated the ratio of colocalization of kisspeptin and NKB in neurons in the arcuate nucleus (ARC), the effects of intracerebroventricular infusion of NKB on luteinizing hormone (LH) secretion and whether the treatment activates ARC kisspeptin/NKB neurons in seasonally anestrous ewes. Double-labeling immunohistochemistry revealed that the majority of kisspeptin neurons coexpressed NKB in the ARC. Infusion of NKB for 2 h into the lateral ventricle elicited a discharge of LH, which resulted in significant increases in LH concentrations between 20 and 50 min after the start of infusion compared with a saline-infused control. Animals were sacrificed immediately after the end of infusion, and Fos expression in ARC kisspeptin neurons was immunohistochemically examined. The NKB treatment activated kisspeptin neurons throughout the ARC, and approximately 70% of kisspeptin neurons expressed Fos immunoreactivity at the caudal portion of the nucleus. The present study demonstrated that a central infusion of NKB elicited a discharge of LH, which was associated with the activation of a large population of ARC kisspeptin/NKB neurons in seasonally anestrous ewes. The results suggest that NKB plays a stimulatory role in the control of pulsatile GnRH secretion and that the population of ARC kisspeptin/NKB neurons is one of sites of the NKB action in sheep.

The objective of this study was to determine if pulsatile LH secretion was needed for ovarian follicular wave emergence and growth in the anestrous ewe. In Experiment 1, ewes were either large or small (10 x 0.47 or 5 x 0.47 cm, respectively; n = 5/group) sc implants releasing estradiol-17 beta for 10 d (Day 0 = day of implant insertion), to suppress pulsed LH secretion, but not FSH secretion. Five sham-operated control ewes received no implants. In Experiment 2, 12 ewes received large estradiol-releasing implants for 12 d (Day 0 = day of implant insertion); six were given GnRH (200 ng IV) every 4 h for the last 6 d that the implants were in place (to reinitiate pulsed LH secretion) whereas six Control ewes were given saline. Ovarian ultrasonography and blood sampling were done daily; blood samples were also taken every 12 min for 6 h on Days 5 and 9, and on Days 6 and 12 of the treatment period in Experiments 1 and 2, respectively. Treatment with estradiol blocked pulsatile LH secretion (P < 0.001). In Experiment 1, implant treatment halted follicular wave emergence between Days 2 and 10. In Experiment 2, follicular waves were suppressed during treatment with estradiol, but resumed following GnRH treatment. In both experiments, the range of peaks in serum FSH concentrations that preceded and triggered follicular wave emergence was almost the same as control ewes and those given estradiol implants alone or with GnRH; mean concentrations did not differ (P < 0.05). We concluded that some level of pulsatile LH secretion was required for the emergence of follicular waves that were triggered by peaks in serum FSH concentrations in the anestrous ewe., ((c) 2010 Elsevier Inc. All rights reserved.)

An immune/inflammatory challenge can affect reproduction at the level of the hypothalamus, pituitary gland, or gonads. Nonetheless, the major impact is thought to occur within the brain or the pituitary gland. The present study was designed to examine the effect of intravenous (i.v.) lipopolysaccharide (LPS) injection on the expression of gonadotropin-releasing hormone (GnRH) and the gonadotropin-releasing hormone receptor (GnRHR) genes in the hypothalamic structures where GnRH neurons are located as well as in the anterior pituitary gland (AP) of anestrous ewes. We also determined the effect of LPS on luteinizing hormone (LH) release. It was found that i.v. LPS injection significantly decreased GnRH and GnRHR mRNAs levels in the preoptic area (40%, p

In ewes, immunization against GnRH blocks LH pulses but mean serum FSH concentrations are only partly reduced; the fate of the FSH peaks that precede ovarian follicular waves has not been studied. In this study, we used immunization against GnRH to examine the need for pulsed GnRH secretion in the genesis of FSH peaks in the anestrous ewe. Six anestrous ewes were given a GnRH immunogen on Day 0 and a booster injection on Day 28. Control ewes (n=6) received adjuvant only. Transrectal ultrasonography was performed daily for 2 days prior to and 10 days following both the primary (Days -2 to 10) and booster (Days 26-38) injections and for a 13-day period beginning 26 days after booster injection (Days 54-66). Blood samples were collected daily. Intensive bleeding (every 12min for 7h) was performed on Days 9, 37, and 65 of the experimental period to characterize the pulsatile pattern of LH secretion. GnRH antibody titers were increased and LH pulses were abolished immediately after booster immunization (P<0.05). The number of FSH peaks, FSH peak concentration and amplitude and basal FSH concentrations were only decreased in immunized ewes in the period of observations starting 26 days after booster immunization (P<0.05); however, some peaks were still seen. The number of follicular waves was decreased in the period around booster immunization and no follicular waves were seen during the period starting 26 days after booster immunization in immunized ewes (P<0.05). In summary, in anestrous ewes, when pulsed LH secretion was abolished by immunization against GnRH, the peaks in serum concentrations of FSH that trigger ovarian follicular waves continued for a period of time. We concluded that although blocking the effects of GnRH gradually causes a diminution of FSH secretion, there is no acute requirement for GnRH in the regulation of FSH peaks. The existence of FSH peaks in the absence of follicular waves, and pulsed LH secretion, suggests that some endogenous rhythm may drive the occurrence of FSH peaks, independent of both changes in negative feedback by secretory products from ovarian antral follicles and GnRH., (Copyright 2010 Elsevier B.V. All rights reserved.)

The presence of the mu-opioid receptor (MOR) was investigated in the mare oviduct during oestrus and anoestrus, by means of immunoblotting and immunohistochemistry. Immunoblotting analysis showed that the MOR protein is expressed as 65, 50 and 30 kDa forms in the infundibulum and ampulla both in oestrus and anoestrus, while the 30 kDa form is absent in the isthmus. Moreover, different levels of expression were observed along the ampulla in the two periods examined. Immunohistochemistry revealed MOR in the mucosal epithelium, stromal cells, myocytes and blood vessels. Ciliated cells expressed MOR in the apical cytoplasm and, except for the isthmus of oestrous mares, also in the nucleus. Non-ciliated cells showed MOR only in the isthmus segment during oestrus. Stromal cells showed different immunoreactivity along the oviduct segments and during the oestrous and anoestrous phases. The myosalpinx displayed immunostained myocytes in the intrinsic musculature of the ampulla and in the extrinsic and intrinsic musculature of the isthmus without significant differences between anoestrus and oestrus. Blood vessels expressed MOR in endothelial cells and smooth muscle cells in the isthmus myosalpinx of oestrous mares only. In conclusion, these findings show diverse MOR expression in the three segments constituting the oviduct, as well as changes in MOR expression linked to the mare's physiological condition., (Copyright 2009 Elsevier B.V. All rights reserved.)

The objective was to develop and test radioimmunoassays (RIAs) to measure fecal progestogens (P) and estrogens (E) to monitor ovarian activity in the bottlenose dolphin (Tursiops truncatus). Fecal samples were collected at least once a week for 20 mo from three peripubertal female bottlenose dolphins. Blood samples were collected at least once a month to compare serum and fecal steroid concentrations. Moreover, random fecal samples from three pregnant females, one lactating female, and one sexually mature female receiving oral altrenogest treatment were also collected. Fecal samples were collected behaviorally with a probe to avoid water contamination and extracted with petroleum ether (for P analysis) or diethyl ether (for E analysis). When possible, vaginal cytology and ovarian ultrasonography were used to monitor the estrous cycle. The RIA for fecal P had good reproducibility and negligible matrix effect. In addition, when fecal samples (N=25) were extracted with ethanol, the results with the two methods of extraction were highly correlated (r=0.923). Therefore, extraction of fecal samples with petroleum ether represented a valid alternative to other, more time-consuming methods of determining fecal P concentrations. In the absence of luteal activity, fecal P concentrations were consistently < 10 pmol/g feces, although they never decreased below 10 pmol/g during pregnancy. Thus, the threshold to confirm the presence of an active corpus luteum was provisionally set at 10 pmol/g. Around the onset of puberty, luteal phases appeared shorter and irregular in the bottlenose dolphin, as in other mammalian species. Additional HPLC-MS studies should be performed to identify predominant P metabolites to be used as fecal indicators of luteal activity in this species.

Background: Kisspeptins are recognized as important stimulators of gonadotropin-releasing hormone (GnRH) and gonadotropin secretion. In sheep, the nonbreeding (anestrous) period is characterized by reduced Kiss1 mRNA expression in the hypothalamus. The aim of the present study was to determine whether the response to kisspeptin treatment varies between the breeding and nonbreeding seasons., Methods: Corriedale ewes were treated intravenously with murine kisspeptin-10 (submaximal dose: 20 microg or maximal dose: 250 microg) or GnRH (250 ng) during either the anestrous period or during the breeding season at the luteal, mid-follicular, or late-follicular phase of the estrous cycle., Results: Kisspeptin treatment increased the concentration of luteinizing hormone (LH) in all animals. We found the LH response to kisspeptin (20 microg) was greater in ewes during the anestrous period, being 4-fold that seen during the luteal phase of the estrous cycle. Within the breeding season, kisspeptin (20 microg) was most effective during the late-follicular phase of the estrous cycle. Kisspeptin treatment (both 20 and 250 microg) had no effect on plasma levels of growth hormone, prolactin or cortisol. The variable effect of kisspeptin (20 microg) on LH secretion appeared to mirror that of GnRH treatment indicating that the effect may be mediated by a change in the pituitary response to GnRH., Conclusion: We conclude that the gonadotropic response to kisspeptin depends upon the reproductive status of the female.

The relative proportion of the circulating luteinizing hormone isoforms in goats during follicular phase (pre-ovulatory peak; F) and anestrus (A) was investigated. Estrus was synchronized in six goats with a prostaglandin analogue. After estrus was detected, blood samples were taken at 1 h intervals for 24 h. Four anestrous goats received 100 microg i.v. of GnRH and blood samples were collected every 15 min for 5 h. Samples with the greatest LH concentration in follicular phase and after GnRH administration (anestrus) were analyzed by chromatofocusing and eluted with a pH gradient from 10.5 to 3.5. For quantification purposes eluted LH was grouped into basic (pH> or =7.5), neutral (pH 7.4-6.5) and acidic isoforms (pH< or =6.4) as well as by pH unit. In both physiological conditions (PC), basic and acidic isoforms were greater than the neutral. With this grouping criteria, there was an interaction between PC and pH group, with the proportion of neutral isoforms being greater (p<0.05) in A (12.0+/-0.8%) as compared with F (5+/-2%). Analysis by pH unit showed a very basic group of eluted isoforms (pH> or =10), which amounted to a percentage of 6.0+/-0.4% of the total observed during A, and 3+/-1% during F (p<0.05). Predominant isoforms in A eluted in the pH range 9.99-9.0 (42+/-3%) as compared to 7+/-3% (p<0.01) in that pH range in F. In contrast, the predominant isoforms in F eluted in the pH range 8.99-8.0, representing 55+/-8%, while in A the proportion was 11+/-2% (p<0.01). Isoforms eluted at the pH range 7.9-7 represented a significantly greater proportion during A (5.0+/-0.6%) as compared with F (3+/-1%). This is the first report on goat LH circulating isoforms. During A the LH isoforms secreted by the pituitary are more basic than during F.

Seasonal anoestrus in the ewe results from enhanced oestrogen negative feedback. Recent data have implicated the ventromedial preoptic area (vmPOA) as an important site of oestrogen action. This study addressed whether NO acts within the vmPOA to inhibit LH during seasonal anoestrus. In Experiment 1, microimplants containing Nomega-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor), S-methyl thiocitrulline (SMTC, neural NOS (nNOS) inhibitor) or empty implants (control) were administered during mid-anoestrus to the vmPOA. l-NAME, but not SMTC, significantly increased LH pulse frequency. For Experiment 2, ewes in late anoestrus were administered 7-nitroindazole (7NI; nNOS inhibitor), l-NAME, SMTC, or empty implants. 7NI, but not l-NAME or SMTC, increased LH pulse frequency. In Experiment 3, the effects of microimplants and microinjections of l-NAME were compared in mid-anoestrus. Microinjections of l-NAME (300 nl at 10 microg/microl) increased LH pulse frequency, but microimplants did not. In late anoestrus, similar microinjections were ineffective. Taken together, the results of Experiments 1-3 suggested that NO inhibition may be stronger during the middle than at the end of seasonal anoestrus. To test this hypothesis, ewes in Experiment 4 received microinjection of l-NAME or vehicle thrice during the non-breeding season; none of the treatments increased LH pulse frequency. These results indicate that NO plays a role in the vmPOA in suppressing LH secretion during seasonal anoestrus because NOS inhibitors were consistently stimulatory when LH pulse frequency was low. However, the inconsistent and modest effects of these inhibitors suggest that NO actions in this area cannot completely account for the effects of inhibitory photoperiod.

The objective of this work was to study the effect of small doses of naloxone (Nx) on the pulsatile secretion of prolactin (Pr). For this purpose 12 crossbreed ewes were selected and allocated to three groups of four. Group 1 was treated with two injections (at 7 and 19 h) of 40 microg of GnRH. Group 2 was treated with two i.m. injections (at 7 and 19 h) of 0.5mg of naloxone. And the control group 3 was sham treated with injections of 3 ml saline. Blood samples were taken at 20 min intervals during six consecutive hours after injections. When ewes were treated at 7h no significant changes were observed in concentrations of prolactin following treatment with GnRH. Values fluctuated between 200 and 210 ng/ml. In group 2 treated with naloxone there was no change in plasma Pr concentrations during the first 100 min of sampling, however 60 min after Nx treatment Pr decreased significantly (p<0.01) and thereafter Pr plasma levels were consistently less (p<0.001) than control and GnRH treated ewes for the duration of the experiment. The response of the three groups after the second injection (19 h): After the injection of GnRH plasma Pr levels followed much the same pattern observed after the initial treatment, Pr concentrations were similar to those of control ewes. Ewes treated with a second small dose of naloxone (0.5mg i.m.) however, showed a decrease in plasma Pr 60 min after the administration of the endogenous opioid antagonist. Thereafter Nx treated ewes had lesser (p<0.001) plasma Pr levels until the termination of the experiment. It was concluded that Nx an opioid antagonist administered in small intermittent doses can alter Pr plasma concentrations in the ewe, showing that endogenous opioids are important modulators of endocrine function and that the administration of small intermittent doses of opioid antagonists produce significant endocrine changes in ewes.

In the rats with regular estrous cycles effects of letrozole, a steroid aromatase inhibitor, on the enzyme activity in ovarian tissues and reproductive system condition were studied. Letrozole was administered per os daily in a dose of 1 mg/kg b.w. over 10 days. As the result of treatment, 20.5-fold decrease in aromatase activity was obtained. All animals have developed persistent anestrus with vaginal smear patterns being similar to diestrus. Uterine weights were two-fold less than that of controls, and ovarian weights increased by one third. The ovaries contained multiple cystoidal follicles. Results of the study demonstrate the key role of ovarian steroid aromatase in letrozole-induced disorders of reproductive cycles. Letrozole might be recommended for experimental modeling of reproductive cycle disorders in female rats.

The corpus luteum (CL) is a temporary endocrine gland, whose life span depends on the interaction of luteotrophic and luteolytic factors. Since development and maintenance of CL is based on angiogenesis, angiogenic growth factors may play a role in CL-function of the bitch, as described for other species. The aim of this study was to detect the presence of the vascular endothelial growth factor (VEGF) system in the bitch CL throughout diestrus and early anestrus. For that purpose, blood samples from 24 bitches were collected and analyzed for progesterone to determine ovulation time and the animals were subjected to ovariosalpingohysterectomy 10, 20, 30, 40, 50, 60 or 70 days after ovulation. The corpora lutea were fixed in formalin and embedded in Paraplast resin. Five micrometers sections were submitted to standard immunohistochemistry protocol using three primary antibodies (SC-315, SC-316 and VG76e) for detection of kinase domain region (KDR), fms-like tyrosine kinase 1 (Flt-1) and VEGF, respectively. The VEGF system expression could be detected in all diestrus stages in endothelial as well as luteal cells (responsible for blood vessel formation and progesterone production, respectively), indicating time dependent changes: immunostaining tended to increase from Day 10 to 50 and to decrease until Day 70 post-ovulation. In the CL of the bitch, structure related cells, like pericytes and stroma cells, expressed it in determined time points of diestrus with little intensity variation. We concluded that VEGF might have a modulatory effect in the CL of the dog acting as paracrine and autocrine factor through its receptors, Flt-1 and KDR.

GABA has been shown to play an important role in the control of gonadotropin-releasing hormone (GnRH) and luteinizing hormone secretion in many mammals. In sheep, seasonal differences in the ability of GABA-B receptor antagonists to alter pulsatile luteinizing hormone secretion have led to the hypothesis that this receptor subtype mediates the increased inhibitory effects of estradiol on GnRH and luteinizing hormone pulse frequency seen during the non-breeding season (anestrus). The aim of the present study was to use multiple-label immunocytochemistry to determine if ovine GnRH neurons contain the GABA-B receptor subunits R1 and/or R2, and to determine whether there are seasonal differences in the colocalization of these subunits in GnRH neurons. A majority of GnRH cells in the preoptic area, anterior hypothalamic area, and medial basal hypothalamus of both breeding season and anestrous ewes contained either GABA-B R1 or R2 subunits; a subset of GnRH neurons in breeding season (42%) and anestrous ewes (60%) contained both subunits. In contrast to colocalization within cell bodies, GnRH fibers in the median eminence did not colocalize GABA-B receptor subunits. Although the percentage of GnRH neurons expressing GABA-B receptor subunits tended to be higher in anestrus than in the breeding season, there were no significant seasonal differences in R1 and R2 subunit colocalization in GnRH cell bodies. Thus, while GABA may act directly on GnRH cell bodies via GABA-B receptors in the sheep, any role that GABA-B receptors may play in seasonal reproductive changes is likely mediated by other neurons afferent to GnRH cells.

The paper reviews data concerning the involvement of GABA(A) and GABA(B) receptors in the control of GnRH secretion in anestrous ewes. Generally, GABA influences the GnRH release through GABA(A) and GABA(B) receptors located on perikaria of the GnRH neurons in the preoptic area (MPOA) or through the influence on beta-endorphinergic and catecholaminergic systems activity in MPOA and in ventromedial-infundibular region of the hypothalamus (VEN/NI). Stimulation of GABA(A) receptors in VEN/NI and MPOA attenuates GnRH release, while activation of GABA(B) receptors in MPOA decreases GnRH secretion, and in VEN/NI increases concentration of GnRH. The different neural mechanisms could be involved in this process: direct ligand action on the GABA(A) and GABA(B) receptors located on GnRH cells and axon terminals or indirect effect through the changes in the beta-endorphinergic and catecholaminergic systems activity in these structures of the brain.

This study describes progesterone receptor (PR) location within uterine cells and associated morphological changes to the uterine luminal and glandular epithelium in seasonally anoestrous brushtail possums treated with oestradiol and/or progesterone. Twenty-four adult female possums (n = 6/group) were treated with oestradiol, progesterone, oestradiol followed by progesterone or with the oil vehicle alone for 6-day periods. Uterine tissue was recovered, weighed and processed for light and transmission electron microscopy and for immunohistochemistry for PRs. Stereological techniques were used to quantify epithelial cell and constituent volumes for both luminal and glandular tissues. Plasma concentrations of oestradiol and progesterone were determined by radioimmunoassay. Mean uterine wet weights were significantly heavier (P < 0.001) following oestradiol/progesterone treatment and maximum gland dilation, cellular and stromal growth, maximum cell height, and cell and constituent volumes were recorded after this treatment regimen. Cell nuclei and debris were commonly observed in gland lumina, and nuclear PRs were found predominantly in stromal cells following oestradiol-only treatment. Sequential treatment with oestradiol and then progesterone caused a decline in the number of positively stained cells. Epithelial cells contained extensive secretory organelles and degenerating cells were common within the glands. Oestradiol treatment induced cell and cell constituent growth and promoted PR formation in anoestrous possum uterine tissue. Subsequent exposure to progesterone stimulated uterine tissues to reach maximum wet weights and led to the cellular maturation necessary to remodel the uterus in preparation for pregnancy.

The present review summarises knowledge of the reproductive response of anoestrous ewes to the introduction of rams - in other words, the ram effect. The ovarian and endocrine response, the factors that determine whether ewes will respond or not (associated with both the stimulus and the receptivity of the ewes) and some aspects of practical management are discussed. Information on the use of the ram effect to stimulate post-partum, prepubertal and cyclic ewes is also given. New insights are provided on ovarian response patterns, including recently collected information on luteal responses. The existence of delayed ovulation (5-7 days after the introduction of the rams) followed by luteal phases of normal or short length, luteal cysts and luteinised follicles is reported after scanning the ovaries with ultrasound. Endocrine parameters for depth of anoestrus, such as LH pulsatility and FSH concentrations, and how the concentrations of these hormones should be considered are discussed. Particular attention is paid to the observation of spontaneous, higher LH pulsatility and higher FSH concentrations in anoestrous ewes that respond to rams with luteal phases than in those that fail to respond. The use of progestogen priming and single progestogen administration and the possible advantages for synchronisation of oestrus are also discussed. Other factors that should be considered before the ram effect is applied, such as the strength of the stimulus and some practical considerations, are also included.

Neuropeptide Y (NPY) is a putative mediator of many physiological processes in the central nervous system including reproductive functions. In this study we examined the effect of NPY on the GnRH/LH secretory activity in early anoestrous ewes. Crossbreed ewes one week (group 1w, n=7) and six weeks (group 6w, n=7) after the last oestrous cycle were infused with Ringer solution (control) or 50 microg of NPY to the third ventricle for 5 minutes. Blood samples were collected during the days of infusion over six hours at ten minute intervals and then the ewes were slaughtered and their brains fixed in situ. Immunoreactive (ir) GnRH in the median eminence and LH cells in the adenohypophysis were localised by immunohistochemistry technique. Data were generated using computer analysis to determine the optical density for immuno-staining and the proportion of ir LH cells. Plasma LH was determined by radioimmunoassay. It was found that investigated anoestral groups differed only in the optical densities of ir GnRH and ir LH, which were lower in group 6w compared to group 1w (p<0.001). LH concentrations and parameters of pulsatile LH secretion were similar in both anoestral groups. Acute NPY infusions induced in group 1w decreased both in ir GnRH and ir LH optical densities and in area fraction for ir LH cells (p<0.001) whilst it increased in plasma LH concentration (p<0.05) compared to vehicle infused animals. There was no changes in the secretory activity of the GnRH/LH axis following NPY infusion in group 6w. In conclusion, we suggest that NPY may be involved in the central regulation of reproductive function in ewes, however, the sensitivity of the GnRH/LH axis to NPY modulation declines throughout the early anoestrous period.

Pharmacokinetics for one growth hormone secretogogue (NNC 26-0722), but not for another (NN703), differ between dogs in estrus or anestrus. We examined if the differences could be mimicked by administering estradiol during anestrus and if there was a relationship with rates of small intestine absorption. Pharmacokinetics for oral doses of NN703 (1.0-1.6 mg kg(-1)) did not differ among dogs in estrus, anestrus, or anestrus and given estradiol for 1 week (days 1, 3, and 6; 40 micro g kg(-1)), whereas plasma concentrations of NNC 26-0722 increased from undetectable in untreated, anestrus dogs to several hundred nanograms per milliliter in dogs given estradiol, with maximal concentrations measured 5 min after oral dosage. Estradiol treatment increased small intestinal absorption of NNC 26-0722 by 100% (P<0.05), but did not increase absorption of NN703, and caused a 64% increase in carrier-mediated glucose transport at 50 mmol l(-1) (P<0.05) due to increased densities of transporters. These findings indicate estrus and estradiol enhance absorptive functions of the dog proximal small intestine and can affect pharmacokinetics for some orally administered drugs.

Follicular waves and associated circulating hormone concentrations were studied during the anovulatory season in pony mares (n=8). Follicles were monitored by ultrasonography and a blood sample was taken daily from 29 January until ovulation (mean, 28 April). A mid-anovulatory period (largest follicle, 16.0+/-0.5 mm in diameter) and transitional period (largest follicle, 22.4+/-0.5 mm) were distinctive in each mare. The two periods were delineated by an increase in the diameter of the largest follicle to >/=21.0 mm. Follicular waves, identified by significant increases in the mean diameter of the second to sixth largest follicles, were detected during both the mid-anovulatory and transitional periods. The mean number of follicles >/=15.0 mm in diameter and the diameter of the second to sixth largest follicles increased in association with statistically identified FSH surges. The pattern of the FSH concentration changes during surges did not change during the mid-anovulatory and transitional periods. During the declining portion of the FSH surge, follicle growth continued and circulating total inhibin increased, indicating suppression of FSH by inhibin from the growing follicles. Circulating oestradiol or LH did not change relative to wave emergence. Results indicated that follicular waves occurred during the second-half of the anovulatory season, even during the period of lowest follicular activity. On a temporal basis, follicular wave emergence was stimulated by surges in circulating FSH. However, the increase in follicle growth to >/=21.0 mm in diameter for the wave at the beginning of the transitional period and for the subsequent waves was not attributable to a change in the characteristics of the associated FSH surges.

There is increasing evidence that estrogen has beneficial effects on cognition, both in humans and in rodents, and may delay Alzheimer's disease onset in postmenopausal women. Several rodent studies have utilised the ovariectomy model to show estrogen regulation of the p75 neurotrophin receptor, TrkA, and markers of acetylcholine synthesis in the cholinergic basal forebrain. We studied estrogenic effects in aged (16-17-month-old), noncycling rats. Estrogen treatment for 10 days drastically reduced p75(NTR) immunoreactivity in the rostral parts of the basal forebrain. The number of p75(NTR)-immunoreactive neurons was decreased, and those neurons remaining positive for p75(NTR) showed reduced p75(NTR) staining intensity. In vehicle-treated rats, almost all choline acetyltransferase-immunoreactive neurons were p75(NTR) positive (and vice versa), but, in estrogen treated rats, large numbers of choline acetyltransferase-immunoreactive cells were negative for p75(NTR). Similar levels of p75(NTR) down-regulation in the rostral basal forebrain were found when estrogen treatment was extended to 6 weeks. There was no reduction in the number of p75(NTR)-immunoreactive neurons in the caudal basal forebrain after 10 days of treatment. After 6 weeks of treatment, however, there was evidence of p75(NTR) down-regulation in the caudal basal forebrain. There was no evidence of hypertrophy or atrophy of cholinergic neurons even after 6 weeks of estrogen treatment. Considering the evidence for the role of p75(NTR) in regulating survival, growth and nerve growth factor responsiveness of cholinergic basal forebrain neurons, the results indicate an important aspect of estrogen's effects on the nervous system., (Copyright 2002 Wiley-Liss, Inc.)

The aim of this immunohistochemical study was to describe the cellular distribution of the estrogen receptor-alpha (ERalpha), progesterone receptor (PR) and androgen receptor (AR) in canine uterine tubes. Samples of uterine tubes were taken from dogs in different stages of the estrous cycle, and dogs that were pregnant or had just delivered. Nuclear staining for sex steroid hormone receptors was observed in the surface epithelium, stromal cells and smooth muscle cells of the muscular layer. Only slight differences in staining pattern were observed between the ampulla and fimbriae. The staining for ERalpha and PR showed changes throughout the estrous cycle. Some of these changes were related to changing concentrations of sex steroid hormones. High staining scores for ERalpha and PR were found during proestrus and low scores during early metestrus. The staining for AR showed only minor cyclic changes. However, during proestrus and estrus, cytoplasmic staining for AR was observed in differentiated secretory epithelial cells, when nuclear staining in these cells was nearly absent. For the three hormone receptors, stromal cells generally stained with a higher intensity than epithelial cells. It is likely that many steroid hormone actions on the epithelium are mediated through stromal cells. During pregnancy, rather high staining scores were found for ERalpha and AR in the uterine tube. This is in contrast to observations in the canine pregnant uterus.

To examine the role of the GABA(A) receptor mediating systems in the control of gonadotropin-releasing hormone (GnRH) release from the ventromedial-infundibular region (VEN/IN) of anestrous ewes, the extracellular concentrations of GnRH, beta-endorphin, noradrenaline (NE), dopamine (DA), 4-hydroxy-3-methoxy-phenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC) were quantified during local stimulation or blockade of GABA(A) receptors with muscimol or bicuculline respectively. In most animals stimulation of GABA(A) receptors significantly attenuates GnRH release with concomitant increase of beta-endorphin and DA release, and MHPG and DOPAC levels. Blockade of the GABA(A) receptors generally did not affect GnRH and NE release but inhibited in most animals beta-endorphin release and decreased dopaminergic activity. These results suggest, that GABA may suppress GnRH release directly by GABA(A) receptor mechanism on the axon terminal of GnRH neurons or indirectly by GABA(A) receptor processes activating beta-endorphin-ergic and dopaminergic neurons in the VEN/NI. On the basis of these results in could not be distinguish between these two events. The decrease in extracellular beta-endorphin and dopamine concentration without evident changes in the GnRH level during GABA(A) receptor blockade may suggest that other neuronal systems are involved in this effect.

Ovaries of bitches are relatively inactive during anoestrus despite apparently adequate circulating FSH concentrations. Alternative FSH receptor (FSH-R) transcripts in bitches might hinder the follicular response to gonadotrophins, which may account for anoestrus. The expression of the full length FSH-R and novel isoforms in bitches was examined using in situ hybridization and RT-PCR analysis. Various PCR primers to the FSH-R were used and its expression was assessed in ovarian tissue at different stages of the oestrous cycle. RT-PCR amplification of the extracellular domain (exon 1-10) was generally successful, indicating that cFSH-R expression (> 90%) occurs throughout the oestrous cycle. Two FSH-R isoforms were sequenced, but there were no clear differences in the pattern of expression between anoestrus and other stages of the oestrous cycle, except that isoform expression was less frequent (30% occurrence) in prepubertal bitches. Data from in situ hybridization showed clear expression of the FSH-R in secondary and antral follicles, and corpora lutea. It was concluded that there is no evidence of a change in the expression of the FSH-R specific to anoestrus.

The influence of lactation and weaning on the number, distribution, and structure of mammotroph cells (PRL cells) in ewes was studied using immunocytochemical procedures for light and electron microscopy, as well as morphometric and stereological techniques. The adenohypophyseal gland of 12 ewes of the Segureña breed at different stages of milk production and weaning were analysed, while the same organ of 3 ewes in anestrus served as control. During lactation, PRL cells increased in number and decreased in size. Ultrastructurally, these cells showed a well-developed rough endoplasmic reticulum (RER) and Golgi complex (GC), and large secretory granules. These findings point to increased synthesis and secretion activities during lactation, and a decrease after weaning. One day after weaning 'storage' cells increased in number, and crinophagy became evident 7 days after weaning, when the first 'inactive' cells were seen (10% of total cells). In anestrus these last cells represented 40% of the total, and showed scarce granules, signs of inactivity, lysosomes and lipid droplets.

The effect of central, short-term melatonin administration on daily GnRH and LH secretion was studied in ewes during seasonal anestrus. Melatonin, in a total dose of 32 micrograms and the vehicle were perfused for 4 hours into the mediobasal hypothalamus/median eminence (MBH/ME). The mean GnRH concentration during perfusion with melatonin decreased significantly (P < 0.05), as compared to the concentration during the preceding perfusion with the vehicle only. This change resulted from high variations in GnRH concentration noted during the initial phase of perfusion rather than from an action of melatonin. Melatonin perfused into the MBH/ME did not significantly affect LH secretion. A higher dose of melatonin and vehicle were also infused intracerebroventricularly (icv.) in either intact (300 micrograms for 3 hours) or ovariectomized (OVX) ewes (400 micrograms for 4 hours, 100 micrograms/100 microliters/h). In the intact animals, melatonin did not significantly affect LH secretion. Interestingly, melatonin significantly decreased (P < 0.05) the number of LH peaks in OVX ewes. These results demonstrate that melatonin delivered for a few hours directly into the central nervous system did not affect either daily hypothalamic GnRH release or pituitary LH secretion in intact ewes during seasonal anestrus, but did modify pulsatile LH secretion in ewes deprived of the negative feedback of estradiol.

The objectives of this study were to compare the timing and mechanisms controlling the onset of anestrus in young and mature mares treated either continuously with melatonin and in those that remained untreated. Changes in body weight, subcutaneous body fat measured to provide an estimate of total body fat, and circulating concentrations of leptin were compared throughout the 1-yr experimental period. The results demonstrate that in young mares the timing of anestrus occurs significantly earlier in the year than in mature mares and that mature mares are more likely to exhibit continuous reproductive activity during the nonbreeding season. The propensity of mature mares to exhibit this phenomenon is not modified by continuous treatment with melatonin but is associated with higher mean circulating concentrations of leptin, body weight, and estimated percent of body fat. In both young and mature mares, body weight, percent of body fat, and circulating concentrations of leptin are higher during summer than winter months. We conclude that, in the mare, the reproductive response to a decrease in photoperiod or a presumptive inhibitory melatonin signal is modified by energy availability, which may be signaled to the hypothalamus-pituitary axis via a change in the circulating concentration of leptin. An additional observation confirmed that the prolactin axis is responsive to continuous treatment with melatonin but that a suppression of prolactin secretion is limited to the spring months.

There are few published reports of an alternative, less invasive method than blood sampling to obtain reproductive hormone concentrations from captive dolphins. The aims of this study were to: (1) validate milk as an effective alternative to blood plasma for determining progesterone concentrations; and (2) utilize milk samples collected frequently to obtain progesterone concentration profiles and determine reproductive status. During the course of this study 16 plasma/milk sample pairs were collected from four adult bottlenose dolphins to correlate plasma and milk concentrations of progesterone. Milk samples were also collected approximately weekly for 4-5 months during three independent lactational periods. Additionally, milk samples were collected daily for approximately 1 year during three other independent lactational periods. A highly significant correlation was found between progesterone concentrations in plasma and milk (r(2) = 0.91, P < 0. 01). Progesterone contained in milk whey, fat, and solids were 3.95 +/- 1.3, 8.5 +/- 1.1, and 52.0 +/- 0.6%, respectively. Progesterone profiles from milk samples collected from two dolphins during 1995 indicated pregnancies (with progesterone concentrations between 8 and 46.5 ng/ml) which resulted in parturition. High progesterone concentrations in a third dolphin that did not give birth indicated a possible pseudopregnancy or fetal resorption. A possible ovulation not resulting in pregnancy was evident in one female in 1998, follicular activity in another female in 1998, and a year-long anestrous period in the third animal studied in 1998. It is confirmed that dolphins can become pregnant while lactating and that the approximate time of conception is identifiable in milk profiles, illustrating the potential application of this method in pregnancy detection and reproductive monitoring., (Copyright 2000 Academic Press.)

Purpose: This study was designed to evaluate the effects of pregnancy on the kinetics of lamotrigine (LTG)., Methods: Five pregnant dogs were given a daily dose of LTG (100 mg) for a period of 1 week. Two months after parturition, the same subjects were given the LTG dose (100 mg) over the same period. On both occasions, plasma LTG concentrations were determined by a sensitive, high-performance liquid chromatographic (HPLC) method, over a 30-h period after the last dose., Results: The mean maximum plasma concentration (Cmax), volume of distribution (Vd/F), and oral body clearance (Cl/F) for LTG (+/- SD) during pregnancy were 7.63+/-2.46 microg/ml 1.74+/-0.29 L/kg, and 0.19+/-0.04 L/h/kg, respectively. After pregnancy, the same variables were 6.12+/-2.24 microg/ml, 2.36+/-1.10 L/kg, and 0.30+/-0.13 L/h/kg, respectively. None of these pharmacokinetic parameters was found to be significantly different between the two groups., Conclusions: The apparent lack of change in the relevant pharmacokinetic parameters of LTG during pregnancy may indicate that pregnancy has little or no effect on glucuronidation; the principal pathway for the drug's elimination.

To clarify the effect of beta-endorphin released under stress condition on LHRH secretion, the concentration of LHRH and beta-endorphin-like-immunoreactivity (beta-END-LI) were analysed in perfusates from the nucleus infundibularis-median eminence of anestrous ewes subjected to footshocks stimulation. The dynamics of LHRH and beta-END-LI release during time-course of footshocks stimuli was altered. A brief facilitatory influence of stress on LHRH and beta-END-LI release was observed at the beginning of the stimulation on the first and third day; however the peaks of beta-END-LI were delayed about half an hour in relation to LHRH. Than on the first day of the stimulation LHRH returned to the control value but on the third day it declined below the control levels. Prolonged stress had stimulatory effect on beta-END-LI secretion during first and third day. The presented results indicate that: 1) short stress activates LHRH and beta-END-LI release, 2) prolonged stress has stimulatory effect on beta-END-LI release but leads to suppression of LHRH release. It is suggested that stress-induced beta-endorphin release may be one of the factors responsible for suppression of LHRH activity.

The importance of androgens for the female reproductive system has been investigated for decades and a number of androgen sensitive processes has now been identified in female reproductive organs. For carnivore species no data were available so far about uterine androgen sensitivity and its regulation. The present study therefore aimed to investigate whether androgen receptors (AR) are present in the dog uterus, whether they are regulated throughout the ovarian cycle and whether pyometra affects their expression rate. Uterine tissue samples were collected from 28 bitches of different ages and various breeds. The samples were grouped according to the stage of estrous cycle (metestrus ME or anestrus AE) and the pathological status of the uterus (i.e. suffering from pyometra or not). Androgen receptor mRNA (AR mRNA) was quantified from 500 ng of total RNA isolated from the tissue samples using an internally standardized reverse transcription polymerase chain reaction (RT-PCR) described previously. The amount of total RNA extractable per g tissue was elevated during pyometra. The successful amplification of the expected 172 bp fragment from canine uterine RNA together with the confirmation of the identity of this fragment by sequence analysis, demonstrates that AR is expressed in this particular tissue. Comparing the expression rates in uteri from bitches during ME or AE being healthy (H) or suffering from pyometra (P), the only significant (p < 0.01) difference was found between H and P uteri during ME with 3.5-fold lower expression rates in P. Although the same seems true for AE bitches, a significant difference could not be demonstrated due to the low number (n = 2) of diseased animals in the AE group. There was no evident effect of the stage of ovarian cycle on uterine AR mRNA levels.

In order to clarify the effect of exogenous corticotropin-releasing factor (CRF) on catecholaminergic and serotoninergic system activity in the mediobasal hypothalamus-median eminence (MBH-ME) of ewes the changes in extracellular levels of noradrenaline (NA) and serotonin (5HT), and main metabolites of monoamines, 4-hydroxy-3-methoxyphenylglycol (MHPG), 3,4-dihydroxy-phenylacetic acid (DOPAC), homovanilic acid (HVA), and 5-hydroxy-indolo-3-acetic acid (5-HIAA) were quantified in the perfusates collected from MBH-ME. NA, 5-HT and monoamine metabolites in the perfusates were analyzed using high performance liquid chromatography with electrochemical detection. CRF induced a rise in extracellular concentration of NA and 5-HT only in the estrous ewes prior to a preovulatory LH surge. CRF treatment caused a heterogenous effect on extra-cellular concentrations of 5-HT in ewes during the preovulatory LH surge. Except for DOPAC and HVA in some estrous ewes during the preovulatory LH surge, CRF caused an increase in monoamine metabolites levels in the MBH-ME in anestrous and estrous animals. These results indicate that CRF facilities NA release in the MBH-ME during the presurge LH period in ewes, and that CRF increases metabolic activities of the monoaminergic systems in this structure in the anestrous and estrous ewes, except dopaminergic system in the ewes during the preovulatory LH surge. It is suggested that: 1) the responses of monoaminergic systems activity in the MBH-ME to CRF in large degree is dependent upon physiological state of ewes and 2) in some endocrinological phases CRF may affect LHRH and other hypothalamic hormone secretion indirectly by altering monoaminergic system activity in the MBH-ME.

It is well established that the thyroid gland is essential for termination of seasonal reproductive activity in a variety of birds and mammals. In the present study, we examined when during the breeding season the thyroid exerts this effect in female sheep. Previous results suggest that the presence of thyroid hormones during the first 4-6 wk (20-25%) of the breeding season is not sufficient for the neuroendocrine changes that lead to anestrus. We therefore hypothesized that thyroid hormone action is exerted at some point during the latter 75-80% of the breeding season. To test this hypothesis, ewes thyroidectomized early in the breeding season received replacement of thyroxine at various times to create gaps during the mid- to late breeding season when thyroid hormones were absent. We then examined the effect, if any, of this absence on development of seasonal neuroendocrine anestrus. Each ewe was ovariectomized and treated with a constant-release Silastic capsule containing estradiol. Serum concentrations of LH were used as an index of seasonal changes in reproductive neuroendocrine activity. We found that when thyroid hormones were removed for a 60-day period in mid- to late breeding season (from mid-Oct. to late Dec., which is approximately 40% of the entire breeding season), anestrus still developed at the normal time. We conclude, therefore, that thyroid hormones need not be present for much of the breeding season (mid-Sept. through late Dec.) for anestrus to develop in the ewe. Rather, we postulate that thyroid hormones need to be present for only a brief period of time near the end of the breeding season for the neuroendocrine changes that lead to anestrus.

The localization and characterization of bradykinin B2 receptors in sheep uterus was carried out using a radiolabelled B2 receptor ligand, 3,4-hydroxyphenypropionyl-D-Arg0-[Hyp3,Thi5,D-++ +Tic7,D-Oic8] bradykinin (HPP-HOE140). Competition of the radioligand, [125I]HPP-HOE140, from membrane preparations of anoestrus sheep uterus by bradykinin agonists and antagonists revealed the presence of high- and low-affinity binding sites with ligand specificity typical of the bradykinin B2 receptor. Using in vitro autoradiography on tissue sections, intense binding was visible over the superficial epithelial layer of the endometrium and inner third of the myometrium of anoestrus sheep uterus. Bradykinin B2 receptors in the myometrium were down regulated in pregnant sheep uterus. We demonstrate that binding studies using [125I]HPP-HOE140 offer high sensitivity and specificity for characterization, quantitation and localization of the bradykinin B2 receptors in tissues and offers new information on uterine bradykinin B2 receptors.