Expression dynamics of adipokines during induced ovulation in the bovine follicles and early corpus luteum.

The study aimed to assess the local gene expression of adipokine members, namely vaspin, adiponectin, visfatin, resistin and their associated receptors - heat shock 70 protein 5 (HSPA5), adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2) - in bovine follicles during the preovulatory period and early corpus luteum development. Follicles were collected before gonadotropin-releasing hormone (GnRH) treatment (0 h) and at 4, 10, 20, 25 and 60 h after GnRH application through transvaginal ovariectomy (n = 5 samples/group). Relative mRNA expression levels were quantified using real-time reverse transcription polymerase chain reaction (RT-qPCR). Vaspin exhibited high mRNA levels immediately 4 h after GnRH application, followed by a significant decrease. Adiponectin mRNA levels were elevated at 25 h after GnRH treatment. AdipoR2 exhibited late-stage upregulation, displaying increased expression at 20, 25 and 60 h following GnRH application. Visfatin showed upregulation at 20 h post-GnRH application. In conclusion, the observed changes in adipokine family members within preovulatory follicles, following experimentally induced ovulation, may constitute crucial components of the local mechanisms regulating final follicle growth and development.

Induced-ovulation in female dromedary camel involves kisspeptin neuron activation by ß nerve growth factor†.

The dromedary camel (Camelus dromedarius) is a short-day desert breeder in which female ovulation is induced by mating. Current data indicate that male-induced ovulation is triggered by its seminal plasma nerve growth factor beta (ß-NGF), but the exact mechanisms involved in the induction of ovulation are still unknown. In this study, we report that an intramuscular injection of ß-NGF in sexually active short-day-adapted female camels induces an ovulation attested by a surge of circulating LH (2-6 h after treatment) followed by an oocyte release with its cumulus oophorus (confirmed by ultrasonography 72 h after treatment) and a large and progressive increase in circulating progesterone (significant from the 2nd to the 10th days after ß-NGF injection). In addition, this ß-NGF treatment induces a broad nuclear c-FOS activation in cells located in various hypothalamic areas, notably the preoptic area, the arcuate nucleus, the dorso- and ventromedial hypothalamus, the paraventricular nucleus, and the supraoptic nucleus. A double immunostaining with neuropeptides known to be involved in the central control of reproduction indicates that ~28% kisspeptin neurons and 43% GnRH neurons in the proptic area, and ~10% RFRP-3 neurons in the dorso- and ventromedial hypothalamus are activated following ß-NGF injection. In conclusion, our study demonstrates that systemic ß-NGF induces ovulation in the female dromedary camel and indicates that this effect involves the central activation of hypothalamic neurons, notably the kisspeptin neurons.

An experimental test of the ovulatory homolog model of female orgasm.

The ovulatory homolog model of female orgasm posits that the neuro-endocrine mechanisms underlying female orgasm evolved from and are homologous to the mechanisms mediating copulation-induced ovulation in some mammals. This model predicts that pharmacological agents that affect human orgasm, such as fluoxetine, should also affect ovulation in animals with copulation-induced ovulation, such as rabbits. We tested this prediction by treating rabbits with daily doses of fluoxetine for 2 wk and found that fluoxetine treatment reduces the number of ovulations postcopulation by 30%. In a second experiment we tested whether this result was mediated by an effect on the brain or via peripheral serotonin functions. We treated animals with fluoxetine and induced ovulation with a single injection of human chorionic gonadotropin. In this experiment ovulation rate was nominally reduced by only 8%, which is statistically not significant. We conclude that the effect of fluoxetine on copulation-induced ovulation rate supports the ovulatory homolog model of female orgasm, suggesting that female orgasm has very deep evolutionary roots among the early eutherian mammals.

Combined treatment of prednisone and hydroxychloroquine may improve outcomes of frozen embryo transfer in antinuclear antibody-positive patients undergoing IVF/ICSI treatment.

BACKGROUND: The influence of anti-nuclear antibody (ANA) on induced ovulation was controversial, and the effect of prednisone plus hydroxychloroquine (HCQ) treatment on frozen embryo transfer outcomes of in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) for ANA-positive women was unclear. METHODS: Fifty ANA-positive women and one-hundred ANA-negative women matched for age and anti-Mullerian hormone (AMH) were included from a Reproductive Medical Central of a University Hospital. Sixty-one oocytes pick-up (OPU) cycles in ANA+ group and one-hundred OPU cycles in ANA- group were compared; 30 frozen embryo transfer cycles without treatment and 66 with prednisone plus HCQ treatment among ANA-positive women were compared. RESULTS: There was no statistical difference in number of retrieved oocytes (13.66 ± 7.71 vs 13.72 ± 7.23, p = .445), available embryos (5.23 ± 3.37 vs 5.47 ± 3.26, p = .347), high-quality embryos (3.64 ± 3.25 vs 3.70 ± 3.52, p = .832), and proportion of high-quality embryos (26.5% vs. 26.7%, p = .940). Biochemical pregnancy rate (33.3% vs. 68.2%, p < .05), clinical pregnancy rate (20.0% vs. 50.1%, p < .05), and implantation rate (5.6% vs. 31.8%, p < .05) were lower, and pregnancy loss rate (83.3% vs. 23.1%, p < .05) was higher in patients with treatment than no treatment. CONCLUSION: The influence of ANA on number of retrieved oocytes, available embryos, high-quality embryos, and proration of high-quality embryos was not found. The treatment of prednisone plus HCQ may improve implantation rate, biochemical pregnancy rate, and clinical pregnancy rate, and reduce pregnancy loss rate in frozen embryo transfer outcomes for ANA-positive women.

Physiological impacts of housing maned wolves (Chrysocyon brachyurus) with female relatives or unrelated males.

The maned wolf is a threatened canid species native to South America. Previous studies have suggested the species exhibits induced ovulation. In captive breeding facilities, reproductive success is low while rates of neonatal mortality are high. Females that are not recommended for breeding are frequently housed together. However there has never been a systematic study of the reproductive consequences of co-housing females. This study was conducted for three purposes, to: (1) corroborate the presence of induced ovulation, (2) determine whether elevated cortisol is implicated in neonatal pup mortality, and (3) evaluate the endocrine correlates of group housed females. Using fecal hormone monitoring for estrogen, progesterone, and cortisol, 43 cycles from 33 female maned wolves were studied from 2002 to 2015. Females were categorized by their reproductive status: pregnant and successfully raised pups (PR; n = 11), pregnant with neonatal pup demise within 3 days (PL; n = 7), housed with a male but no signs of breeding or pregnancy (PP; n = 10), housed singly (S; n = 8), or housed with related females (F; n = 7). Estrogen and progestagen remained at baseline for all females not housed with a male (S, F), while elevations consistent with ovulation were seen in females housed with a male (PP, PL, PR). Compared to PR females, PL individuals showed similar cortisol levels throughout the cycle and slightly lower progesterone levels during gestation. As for the effect of co-housing related females, F females showed estrogen and progesterone levels lower even than S females while cortisol levels were elevated compared to all other groups. These findings support the previous evidence of induced ovulation in the maned wolf. Although elevated cortisol does not seem to be implicated in pup loss, a non-significant trend towards lower progesterone during gestation could be implicated. Future studies should assess depressed progesterone levels as a correlate to neonatal pup mortality. Female maned wolves housed with related females experience suppressed reproductive hormones and elevated adrenal hormones. Therefore, a more systematic study of hormonal and behavioral correlates to co-housing with related females is warranted.

Comparison of clomiphene citrate and letrozole for ovulation induction in women with polycystic ovary syndrome: a prospective randomized trial.

To compare the therapeutic efficacy of clomiphene citrate (CC) and letrozole (LE) on ovulation, pregnancy, and live birth in women with polycystic ovary syndrome (PCOS); and to ensure if LE can replace CC as the first-line therapy for ovulation induction in these women. This is a prospectively, randomized, controlled trial in the tertiary hospital. Two-hundred and sixty-eight anovulatory PCOS patients were treated by CC or CC plus metformin and LE or LE plus metformin for three continuous cycles or conception; their ovulation rates, pregnancy rates, and live birth rates were calculated and compared. No significant difference was noted among the four groups regarding to the baseline data of clinical manifestations, serum sex hormone levels, and serum insulin levels. A total of 240 patients completed the therapies. The ovulation rate was significantly higher in the group LE than the group CC; however, no significant difference was noted between the groups LE and CC, CC, and CC + MET, or LE and LE + MET in the pregnancy rate, abortion rate, and live birth rate. No birth defect was found in the total of 63 newborns. CC regimen was still recommended to be the first-line therapy of ovulation induction for PCOS.

Spawning performance of Clarias gariepinus (Burchell, 1822) induced with ethanol preserved and fresh catfish pituitary extract.

This study was conducted to determine the efficacy of preserved and fresh pituitary extract of Clarias gariepinus (Burchell, 1822) to induced spawning in the same species. Growth performance of fry was also monitored for 15 days to determine the possible effect of natural hormone treatment on the fry. Pituitary glands (PG) were obtained from 12 male broodstocks, six extracted PG were preserved in 96% ethanol 24 h before injection, while the other were extracted and used in their fresh state. The PG solutions (1 g ml-1 of saline water) were injected at 1 ml kg-1 of the female broodstock. Ovaprim® (a synthetic hormone) was used as the control and administered at a rate of 0.5 ml kg -1 of fish. After a uniform latency period of 9 h 30 min, fish were striped, fertilized, incubated and the performance evaluated. Results obtained revealed better hatching performance using Ovaprim® synthetic hormone (64.52%), however, preserved PG gave better hatchability (59.74%) than fresh PG extract (51.39%). After 15 days of feeding ad libitum with shell-free Artemia cysts, the growth of fry spawn with preserved PG was comparable with that of the control, while least performance was observed using the fresh PG. It was concluded that preservation of PG in ethanol 24 h before injection had a positive effect on breeding performance and could be exploited in the commercial production of C. gariepinus fingerlings.

Sex steroids as pheromones in mammals: the exceptional role of estradiol.

This article is part of a Special Issue (Chemosignals and Reproduction). Whether from endogenous or exogenous sources, 17ß-estradiol (E2) has very powerful influences over mammalian female reproductive physiology and behavior. Given its highly lipophilic nature and low molecular mass, E2 readily enters excretions and can be absorbed from exogenous sources via nasal, cutaneous, and other modes of exposure. Indeed, systemic injection of tritiated estradiol ((3)H-E2) into a male mouse or bat has been shown to produce significant levels of radioactivity in the reproductive tissues and brain of cohabiting female conspecifics. Bioactive E2 and other steroids are naturally found in male mouse urine and other excretions, and males actively direct their urine at proximate females. Very low doses of E2 can mimic the Bruce effect (disruption of peri-implantation pregnancy by novel males), the Vandenbergh effect (early reproductive maturation induced by novel males), and male-induced estrus and ovulation. Males' capacities to induce the Bruce and Vandenbergh effects can both be diminished by manipulations that reduce their urinary E2. Uterine dynamics during the Bruce and Vandenbergh effects are consistent with the actions of E2. Collectively, these data demonstrate a critical role of male-sourced E2 in these major mammalian pheromonal effects.

Morphologic, Steroidogenic, and Transcriptomic Assessment of the Corpus Luteum in Holstein Cows after Spontaneous or Hormone-Induced Ovulation.

There is evidence that replacing the gonadotropin-releasing hormone (GnRH) with porcine luteinizing hormone (pLH) to synchronize ovulation prior to artificial insemination (AI) increased pregnancy per AI in dairy cows without affecting blood progesterone (P4) concentrations. Whether morphologic, steroidogenic, and transcriptomic differences exist among corpora lutea (CL) formed after ovulation induced by GnRH and pLH is unclear. Our main objective, therefore, was to compare CL characteristics between GnRH- and pLH-induced CL. In 24 non-lactating Holstein cows, ovulations were spontaneous (Spont-Ov) or induced with 100 µg GnRH, 25 mg pLH, or 1 mg estradiol benzoate (EB), with CL excised 12 d after ovulation. In pLH- versus GnRH-treated cows, the duration of elevated LH (above baseline) was prolonged (10 versus 6 h, respectively, p < 0.01), but CL dimensions, pixel intensity of CL images, proportions of steroidogenic and non-steroidogenic luteal cells, and mean plasma LH did not significantly differ. Post-ovulation mean plasma P4 (ng/mL) did not differ among Spont-Ov (3.0) pLH (3.1) or GnRH (3.0) cows but were lower in EB cows (2.0). In vitro P4 concentration was greater in luteal explants of pLH-treated cows than in all other groups (combined means, 16.0 vs. 12.3 µg/mL, p < 0.02). Relative abundance of mRNA for oxytocin receptor (OXTR) was 2-fold higher (p < 0.01) in CL of pLH vs. GnRH cows and highest in Spont-Ov CL. In summary, pLH-treated cows had a longer LH peak, and greatest luteal tissue concentrations and in vitro production of P4. We inferred that increased P4 concentrations at the ovarian-uterine level in pLH-treated cows could have promoted embryo development and increased pregnancy per AI.

Spawning Performance and Sex Steroid Levels in Female Pikeperch Sander lucioperca Treated with Poly(lactic-co-glycolic acid) Microparticles.

Pikeperch Sander lucioperca is a piscivorous species considered a promising candidate for the diversification of intensive aquaculture. This study aimed to determine the effect of a sustained-release delivery system incorporating mammalian gonadotropin-releasing hormone agonist (mGnRHa) into poly(lactic-co-glycolic acid) (PLGA) microparticles on the sex steroid levels and aspects of artificial reproduction of pikeperch. Fish were divided into four groups and injected with 20 µg mGnRHa/kg, 5-day release microparticles encapsulated with 5 µg GnRHa/kg BW (PLGA 5), 20 µg GnRHa/kg (PLGA 20), or 1 mL/kg 0.9% NaCl (control). Cumulative percentage ovulation was 100% in the PLGA 5 group, significantly higher than in other tested groups. No differences among groups were observed in latency or fecundity. The level of 11-ketotestosterone (11-KT) peaked at 40 h post-injection, and was sustained during ovulation, in all treated groups. The 17ß-estradiol (E2) concentration increased in the mGnRHa-only group immediately after hormone injection, while both PLGA groups showed a reduction in E2 after injection, continuing to decrease until ovulation. A low dose of mGnRHa in PLGA microparticles significantly improves induction of ovulation and results in acceptable reproductive performance, which may positively affect pikeperch production under controlled conditions.

Comparison of Perinatal Outcomes of Letrozole-Induced Ovulation and Hormone Replacement Therapy Protocols in Patients With Abnormal Ovulation Undergoing Frozen-Thawed Embryo Transfer: A Propensity Score Matching Analysis.

Background: With the increasing use of frozen embryo transfer (FET), the best endometrial preparation protocol is continuously being discussed. The hormone replacement therapy (HRT) cycle and letrozole-induced ovulation (L-OI) cycle are available protocols for patients with abnormal ovulation. Previous comparisons of the two protocols have focused on pregnancy outcomes, with less attention to perinatal outcomes, and population heterogeneity was large; thus, convincing conclusions about which protocol is more appropriate could not be drawn. Methods: We performed a retrospective cohort study using propensity score matching (PSM) analysis for a population of patients undergoing FET cycles in the reproductive center of the Third Affiliated Hospital of Zhengzhou University from January 2016 to September 2020. The main outcome measures were clinical pregnancy rate, live birth rate, very preterm delivery (VPTD), preterm delivery (PTD), low birth weight (LBW), macrosomia, small for gestational age (SGA), large for gestational age (LGA), hypertensive disorders of pregnancy (HDP), gestational diabetes mellitus (GDM), premature rupture of membranes (PROM), placenta previa, and congenital abnormality. Results: A total of 8010 women were enrolled. Due to the large heterogeneity among the patients, we conducted 1:1 PSM, and 1461 women matched in each group. Compared with the HRT group, the L-OI group had a smaller proportion of thin endometrium (27.38% vs. 41.07%) and thicker endometrium on the day of embryo transfer (9.63 ± 1.82 vs. 8.91 ± 1.38). There were no significant differences in clinical pregnancy rate, early abortion rate or live birth rate between the groups. There was no significant difference in perinatal outcomes of singleton live birth, including VPTD, PTD, postterm delivery, LBW, macrosomia, SGA, LGA, GDM, HDP, placenta previa, and congenital malformation. Conclusion: For women with abnormal ovulation, the pregnancy and perinatal outcomes of HRT and L-OI protocols are reassuring. It seems that both protocols are safe and effective for endometrial preparation in frozen-thawed embryo transfer in the clinic.

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle.

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography-high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.

Incorporation of a fixed-time artificial insemination protocol results in improved reproductive management and genetics of the beef herd. However, a subset of animals exposed to such protocols will not display estrus prior to insemination. Behavioral estrus is indicative of the preovulatory follicle's physiological maturity and is essential for both the production of an oocyte with optimal developmental competence and preparation of the maternal environment for pregnancy establishment. Animals that do not display estrus prior to insemination and are induced to ovulate a physiologically less advanced follicle have reduced oocyte developmental competence that leads to reduced embryo cleavage rates, embryo quality, and pregnancy rates. This study investigated the impacts of reduced follicle maturity at the initiation of ovulation on the energy production capacity of the oocyte as well as follicular fluid metabolic composition. Results from this study demonstrated that follicle maturity, indicated by increased serum estradiol concentration at the initiation of ovulation, resulted in increased ATP within the oocyte as well as an increased level of metabolites involved in glucose metabolism in the follicular fluid. Increased energy production ability in the oocytes from more mature follicles could contribute to the increased cleavage rates and embryo quality seen in previous studies.

Screening and Identification of Differential Ovarian Proteins before and after Induced Ovulation via Seminal Plasma in Bactrian Camels.

Camelidae are induced ovulators whose ovulation is tightly regulated by multiple factors. Understanding the biological mechanisms underlying follicular development, hormone secretion, and ovulation requires investigating the potential molecular pathways involved. However, little is known about these pathways in Bactrian camels. To screen and identify candidate biomarkers after inducing ovulation, this study performed comprehensive proteomic and molecular biological analyses of the ovaries from two camel groups (n = 6). We identified 5075 expressed ovarian proteins, of which 404 were differentially expressed (264 upregulated, 140 downregulated) (p < 0.05 or p < 0.01), in samples from plasma-induced versus control camels. Gene ontology annotation identified the potential functions of the differentially expressed proteins (DEPs). These results validated the differential expression for a subset of these proteins using Western blot (p < 0.05) and immunofluorescence staining. Three DEPs (FST, NR5A1, and PRL) were involved in neurochemical signal transduction, as well as endocrine and reproductive hormone regulatory processes. The Kyoto Encyclopedia of Genes and Genomes analysis indicated the involvement of several pathways, including the calcium, cAMP, gonadotropin-releasing hormone, MAPK, and neuroactive ligand-receptor signaling pathways, suggesting that induced ovulation depends on the hypothalamic-pituitary-ovarian axis. Identifying these candidate biomarkers enables a better understanding of Bactrian camel reproduction. Ovarian proteomic profiling and the measurement of selected proteins using more targeted methods is a promising approach for studying induced-ovulation mechanisms.

Differential Transcript Profiles in Cumulus-Oocyte Complexes Originating from Pre-Ovulatory Follicles of Varied Physiological Maturity in Beef Cows.

Small dominant follicle diameter at induced ovulation, but not at spontaneous ovulation, decreased pregnancy rate, fertilization rate, and day seven embryo quality in beef cows. We hypothesized that the physiological status of the follicle at GnRH-induced ovulation has a direct effect on the transcriptome of the Cumulus-Oocyte complex, thereby affecting oocyte competence and subsequent embryo development. The objective of this study was to determine if the transcriptome of oocytes and associated cumulus cells (CC) differed among small (≤11.7 mm) and large follicles (≥12.7 mm) exposed to a GnRH-induced gonadotropin surge and follicles (11.7-14.0 mm) exposed to an endogenous gonadotropin surge (spontaneous follicles). RNA sequencing data, from pools of four oocytes or their corresponding CC, revealed 69, 94, and 83 differentially expressed gene transcripts (DEG) among oocyte pools from small versus large, small versus spontaneous, and large versus spontaneous follicle classifications, respectively. An additional 128, 98, and 80 DEG were identified among small versus large, small versus spontaneous, and large versus spontaneous follicle CC pools, respectively. The biological pathway "oxidative phosphorylation" was significantly enriched with DEG from small versus spontaneous follicle oocyte pools (FDR < 0.01); whereas the glycolytic pathway was significantly enriched with DEG from CC pools obtained from large versus small follicles (FDR < 0.01). These findings collectively suggest that altered carbohydrate metabolism within the Cumulus-Oocyte complex likely contributes to the decreased competency of oocytes from small pre-ovulatory follicles exposed to an exogenous GnRH-induced gonadotropin surge.

Correlation between Pre-Ovulatory Follicle Diameter and Follicular Fluid Metabolome Profiles in Lactating Beef Cows.

Induced ovulation of small pre-ovulatory follicles reduced pregnancy rates, embryo survival, day seven embryo quality, and successful embryo cleavage in beef cows undergoing fixed-time artificial insemination. RNA-sequencing of oocytes and associated cumulus cells collected from pre-ovulatory follicles 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the pre-ovulatory gonadotropin surge suggested reduced capacity for glucose metabolism in cumulus cells of follicles ≤11.7 mm. We hypothesized that the follicular fluid metabolome influences metabolic capacity of the cumulus-oocyte complex and contributes to reduced embryo cleavage and quality grade observed following induced ovulation of small follicles. Therefore, we performed a study to determine the correlation between pre-ovulatory follicle diameter and follicular fluid metabolome profiles in lactating beef cows (Angus, n = 130). We synchronized the development of a pre-ovulatory follicle and collected the follicular contents approximately 20 h after GnRH administration. We then performed ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) metabolomic studies on 43 follicular fluid samples and identified 38 metabolites within pre-ovulatory follicles of increasing size. We detected 18 metabolites with a significant, positive correlation to follicle diameter. Individual and pathway enrichment analysis of significantly correlated metabolites suggest that altered glucose and amino acid metabolism likely contribute to reduced developmental competence of oocytes when small pre-ovulatory follicles undergo induced ovulation.

Can jaguar (Panthera onca) ovulate without copulation?

Threatened of extinction in Brazil, the jaguar is the largest predator in Latin America, playing an important role in the ecosystem where it is inserted. Despite of some important studies in this species, its reproductive physiology needs to be better understood for the development of more effective reproductive biotechnologies. One well studied biological aspect is the occurrence of ovulation following stimulation of the vaginal floor during copulation. This mechanical stimulation is responsible for the induction of ovulation in domestic and wild cats. Thus, the objective of the present study was to evaluate whether ovulation in captive jaguars can be induced by the above-mentioned mechanical stimulation but also by other forms of sensory stimulation. Nine jaguar (Panthera onca) females and six males were divided into three groups: Group 1 (four females), in which the females were close to the males, but with no possibility of copulation; Group 2 (three females), in which the females were in the same enclosure as the males, thereby being able to copulate; and Group 3 (two females), in which the females were completely isolated from any male of the species. Follicular growth was stimulated by administration of 800 IU of eCG, conducted three or five days before LOPU. In order to have certainty about the occurrence of ovulation, the ovaries were assessed by laparoscopy allowing the visualization of ovarian structures including follicles, recent ovulations, corpora hemorrhagica (CH) and corpora lutea (CL). Multiple CH were observed in the 2 females housed with males, as well as the two females kept in proximity of males, i.e. only visual contact (Group 1). None of the females in complete isolation from males showed CH or ovulation spots (Group 3). In summary, the seven females in contact or near proximity with males had multiple ovulations, regardless of copulation or not. Based on this evidence, we have established that jaguars are capable of two forms of induced ovulation: 1) the "mechanical" coitus-induced form that is traditional and thoroughly described for felines; 2) a "sensorial" form in which the nearby presence of a male can induce ovulation through the visual, olfactory and/or auditive senses. Further research is required for establishing the mechanisms and chemical mediators of sensorial stimulation. In addition, and consistent with expected results, we confirmed that females that received the same stimulation are incapable of ovulating when not exposed to any form of stimulation by males.

Distribution of GnRH and Kisspeptin Immunoreactivity in the Female Llama Hypothalamus.

Llamas are induced non-reflex ovulators, which ovulate in response to the hormonal stimulus of the male protein beta-nerve growth factor (ß-NGF) that is present in the seminal plasma; this response is dependent on the preovulatory gonadotrophin-releasing hormone (GnRH) release from the hypothalamus. GnRH neurones are vital for reproduction, as these provide the input that controls the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland. However, in spontaneous ovulators, the activity of GnRH cells is regulated by kisspeptin neurones that relay the oestrogen signal arising from the periphery. Here, we investigated the organisation of GnRH and kisspeptin systems in the hypothalamus of receptive adult female llamas. We found that GnRH cells exhibiting different shapes were distributed throughout the ventral forebrain and some of these were located in proximity to blood vessels; sections of the mediobasal hypothalamus (MBH) displayed the highest number of cells. GnRH fibres were observed in both the organum vasculosum laminae terminalis (OVLT) and median eminence (ME). We also detected abundant kisspeptin fibres in the MBH and ME; kisspeptin cells were found in the arcuate nucleus (ARC), but not in rostral areas of the hypothalamus. Quantitative analysis of GnRH and kisspeptin fibres in the ME revealed a higher innervation density of kisspeptin than of GnRH fibres. The physiological significance of the anatomical findings reported here for the ovulatory mechanism in llamas is still to be determined.

Geographical Origin, Delayed Implantation, and Induced Ovulation Explain Reproductive Seasonality in the Carnivora.

Patterns of reproductive seasonality in the Carnivora are difficult to study comparatively, due to limited numbers of species for which information is available. Long-term databases of captive populations could overcome this difficulty. We apply a categorical description and a quantitative high-resolution measure (birth peak breadth, the number of days in which 80% of all births occur) based on daily observations in captivity to characterize the degree of reproductive seasonality in the Carnivora for 114 species with on average 1357 births per species. We find that the majority of species retained the birth seasonality displayed in the wild. Latitude of natural origin, delayed implantation, and induced ovulation were the main factors influencing reproductive seasonality. Most species were short-day breeders, but there was no evidence of an absolute photoperiodic signal for the timing of mating or conception. The length of the gestation period (corrected for body mass) generally decreased with birth seasonality but increased in species with delayed implantation. Birth seasons become shorter with increasing latitude of geographical origin, likely because the length of the favorable season declines with increasing latitude, exerting a strong selective pressure on fitting both the reproductive cycle and the interval offspring needs for growth following the termination of parental care into the short time window of optimal environmental conditions. Species with induced ovulation exhibit a less seasonal reproductive pattern, potentially because mates do not have to meet during a short time window of a fixed ovulation. Seasonal species of Carnivora shorten their gestation period so reproduction can occur during the short time window of optimal environmental conditions. Alternatively, other Carnivora species lengthen their gestation periods in order to bridge long winters. Interestingly, this occurs not by decelerating intrauterine growth but by delaying implantation.

Urinary profiles of progestin and androgen metabolites in female polar bears during parturient and non-parturient cycles.

Due to the environmental and anthropogenic impacts that continue to threaten the reproductive success of polar bears, a more detailed understanding of their reproductive cycle is needed. Captive populations of polar bears provide an excellent opportunity to learn more about the reproductive physiology of the species. Progestin (P4) and androgen (T) metabolites in urine, and their ratio (P4:T), were examined during 11 reproductive cycles of captive female polar bears (n = 4) to characterize the steroid hormone profile during pregnancy and determine possible variations related to reproductive failure. The concentration of hormone metabolites in urine were determined through enzyme immunoassay. Reproductive cycles were classified as pregnant (n = 3), anovulatory (n = 4) and ovulatory-non-parturient (n = 4) based on the changes in urinary hormone metabolite values and cub production. In the absence of a lactational suppression of estrus, elevated androgen concentrations suggested resumption of follicular development within 3 weeks of parturition. Breeding behaviours were most often observed when androgen values were at their highest or in decline. Ovulation was identified by a return to basal androgen concentration and elevation of progestins within 1-4 weeks after breeding. As a result, urinary concentrations of progestins were greater than androgens (P4:T ratio ≥ 1.0) during ovulatory cycles whereas the P4:T ratio was <1.0 when females were anovulatory. Progestins and the P4:T ratio of parturient cycles were greatest beginning in June/July (17-20 weeks after breeding) and reached a peak at 24-37 weeks (mid-October/mid-November, 4-9 weeks before birth of cubs). Non-invasive monitoring of hormone metabolites in urine provided a rapid determination of endocrine function for improved husbandry and reproductive management of polar bears in captivity. Further research is warranted to understand the reproductive endocrinology of polar bears and its impact on conservation and management of this species in captivity and the wild.

LH release and ovulatory response after intramuscular, intravenous, and intrauterine administration of ß-nerve growth factor of seminal plasma origin in female llamas.

The objective of the study was to compare the pituitary and ovarian responses after intramuscular, intravenous, or intrauterine administration of ß-nerve growth factor (ß-NGF) of seminal plasma origin (SP-NGF) in llamas. In experiment 1, mature female llamas with a growing follicle of 7 mm or greater were assigned randomly to four groups (n = 7/group) and given 2 mg of purified SP-NGF in a volume of 2 mL by (1) intramuscular administration, (2) intravenous administration, and (3) intrauterine infusion, or (4) intrauterine infusion of 2 mL of PBS (negative control). Because ovulations were not detected after intrauterine infusion in experiment 1, a second experiment was done to determine if a higher dose of SP-NGF given by intrauterine infusion, similar to a natural dose during copulation, will elicit an ovulatory response. In experiment 2, llamas with a growing follicle of 7 mm or greater were assigned randomly to three groups (n = 6/per group) given an intrauterine infusion of (1) 4 mL of raw seminal plasma, (2) 4 mL of PBS containing 20 mg of purified llama SP-NGF, or 3) 4 mL of PBS (negative control). In both experiments, the ovaries were examined daily by transrectal ultrasonography using a B-mode scanner and power Doppler mode to detect ovulation and to monitor CL growth, regression, and vascularization. Blood samples were collected to determine plasma LH and progesterone concentrations. In experiment 1, only llamas treated by intramuscular or intravenous administration of SP-NGF ovulated (7 of 7 and 6 of 7, respectively). Plasma LH concentration did not differ between the intramuscular and intravenous SP-NGF-treated groups, nor did CL diameter, CL vascularization, or plasma progesterone concentration profiles. In experiment 2, the ovulation rate was 100% for llamas treated by intrauterine infusion of raw seminal plasma or llama SP-NFG, whereas no ovulations were detected in females treated with PBS. Plasma LH concentrations did not differ between groups that ovulated, nor did CL diameter, CL vascularization, or plasma progesterone concentration profiles. We conclude that ß-NGF from llama seminal plasma origin elicits a preovulatory LH surge, followed by ovulation and the development of a functional CL, regardless of the route of administration. However, the dose required to elicit pituitary and ovarian responses is higher when administered by intrauterine infusion than by intramuscular or intravenous routes.