Bacterial infection and imbalance of bacterial community in the genitourinary system of giant panda could affect the reproductive health. In severe cases, it can also lead to abortion. In this study, 13 of vaginal secretions in the estrue (E) group and seven of vaginal secretions in the non-estrue (NE) group were used to study the composition and diversity of vaginal bacterial communities between estrus and non-estrus by 16S rRNA gene sequencing analysis. The results showed that the vaginal microbiome in giant pandas shared the same top five abundant species between estrus and non-estrus at the phylum level. However, the vaginal microbiome changed significantly during estrus at the genus level. In top 10 genera, the abundance of Escherichia, Streptococcus, and Bacteroides in the E group was significantly higher than that in the NE group (p<0.05); Azomonas, Porphyromonas, Prevotella, Campylobacter, and Peptoniphilus in the NE group was significantly higher than that in the E group (p<0.05). The richness and diversity of vaginal microbiome in giant panda on estrus were significantly lower than those on non-estrus (p<0.05). It is noteworthy that the abundance of Streptococcus, Escherichia, and Bacteroides of vagina in giant pandas maintained low abundance in the daily. Whereas, they increased significantly during estrus period, which may play an important role in female giant pandas during estrus period. It was hypothesized that hormones may be responsible for the changes in the vaginal microbiome of giant pandas between estrus and no-estrus stages.

The co-evolution of gut microbes and the host plays a vital role in the survival and reproduction of the host. The dhole (Cuon alpinus) has been listed as endangered species by the International Union for Conservation of Nature; therefore, conservation and effective breeding of dholes are essential. Effective estrus can promote reproduction. However, little is known about the relative contribution of estrus in shaping the structure and the functions of fecal microbiota. Here, we investigated the potential association between estrus and the fecal microbiota in dholes using shotgun metagenomic sequencing. We found that the estrus stages in dholes vary significantly in terms of gut bacterial composition and microbiome metabolism and function. Compared with that of non-estrus, adult dholes, the microbiome of estrus adult dholes had a significantly higher abundance of Bacillus faecalis and Veillonella, which play a key role in the synthesis of sex hormones and nucleic acids, energy production, and reproductive cell division. The insulin and energy metabolism-related pathways are significantly enhanced in the gut microbes and the related gluconeogenic enzymes are significantly enriched during estrus. These findings suggest that the structure and metagenome of the fecal microbiome during estrus have a significant effect in promoting estrus in dholes, thus providing a new perspective for dhole conservation.

Mammalian behavior is typically monitored by observation. However, direct observation requires a substantial amount of effort and time, if the number of mammals to be observed is sufficiently large or if the observation is conducted for a prolonged period. In this study, machine learning methods as hidden Markov models (HMMs), random forests, support vector machines (SVMs), and neural networks, were applied to detect and estimate whether a goat is in estrus based on the goat's behavior; thus, the adequacy of the method was verified. Goat's tracking data was obtained using a video tracking system and used to estimate whether they, which are in "estrus" or "non-estrus", were in either states: "approaching the male", or "standing near the male". Totally, the PC of random forest seems to be the highest. However, The percentage concordance (PC) value besides the goats whose data were used for training data sets is relatively low. It is suggested that random forest tend to over-fit to training data. Besides random forest, the PC of HMMs and SVMs is high. However, considering the calculation time and HMM's advantage in that it is a time series model, HMM is better method. The PC of neural network is totally low, however, if the more goat's data were acquired, neural network would be an adequate method for estimation.

Among female rats, some individuals show estrus cycle-dependent irritability/aggressive behaviors, and these individual rats may be used as a model for premenstrual dysphoric disorder (PMDD). We wanted to investigate if these behaviors are related to the estrus cycle phase containing moderately increased levels of positive GABA-A receptor-modulating steroids (steroid-PAM), especially allopregnanolone (ALLO), and if the adverse behavior can be antagonized. The electrophysiology studies in this paper show that isoallopregnanolone (ISO) is a GABA-A-modulating steroid antagonist (GAMSA), meaning that ISO can antagonize the agonistic effects of positive GABA-A receptor-modulating steroids in both α1β2γ2L and α4β3δ GABA-A receptor subtypes. In this study, we also investigated whether ISO could antagonize the estrus cycle-dependent aggressive behaviors in female Wistar rats using a resident-intruder test. Our results confirmed previous reports of estrus cycle-dependent behaviors in that 42% of the tested rats showed higher levels of irritability/aggression at diestrus compared to those at estrus. Furthermore, we found that, during the treatment with ISO, the aggressive behavior at diestrus was alleviated to a level comparable to that of estrus. We noticed an 89% reduction in the increase in aggressive behavior at diestrus compared to that at estrus. Vehicle treatment in the same animals showed a minimal effect on the diestrus-related aggressive behavior. In conclusion, we showed that ISO can antagonize Steroid-PAM both in α1β2γ2L and α4β3δ GABA-A receptor subtypes and inhibit estrus cycle-dependent aggressive behavior.

Female mammals experience cyclical changes in sexual receptivity known as the estrus cycle. Little is known about how estrus affects the cortex, although alterations in sensation, cognition and the cyclical occurrence of epilepsy suggest brain-wide processing changes. We performed in vivo juxtacellular and whole-cell recordings in somatosensory cortex of female rats and found that the estrus cycle potently altered cortical inhibition. Fast-spiking interneurons were strongly activated with social facial touch and varied their ongoing activity with the estrus cycle and estradiol in ovariectomized females, while regular-spiking excitatory neurons did not change. In situ hybridization for estrogen receptor β (Esr2) showed co-localization with parvalbumin-positive (PV+) interneurons in deep cortical layers, mirroring the laminar distribution of our physiological findings. The fraction of neurons positive for estrogen receptor β (Esr2) and PV co-localization (Esr2+PV+) in cortical layer V was increased in proestrus. In vivo and in vitro experiments confirmed that estrogen acts locally to increase fast-spiking interneuron excitability through an estrogen-receptor-β-dependent mechanism.

To improve reproductive performance in cattle, the accurate detection of estrus and optimization of insemination relative to ovulation are necessary. However, poor heat detection by farm staff leads to a decreased conception rate, thus inflicting economic damage to the beef and dairy industries. This study aimed to develop monoclonal antibodies (mAb) that can specifically bind to the bovine lactoferrin (bLF) protein, which we have previously demonstrated to be overexpressed in bovine cervical mucus during estrus. Female rats were intraperitoneally immunized with bLF protein as the antigen. Anti-bLF mAbs were then purified by affinity chromatography, and their binding affinity for the bLF antigen was examined using ELISA. We found a high binding affinity between mAbs and bLF. Finally, we developed a rapid bovine heat detection kit using the anti-bLF mAbs that we generated and tested on cervical mucus from 12 cows (estrous synchronization, n = 2; natural cycling, n = 10). We found that the kits accurately detected estrus. Overall, our fabricated heat detection kit based on rat anti-bLF mAbs could pave the way for the development of potent tools for heat detection devices for dairy cattle, thereby preventing economic loss.

Depression and anxiety are diagnosed almost twice as often in women, and the symptomology differs in men and women and is sensitive to sex hormones. The basolateral amygdala (BLA) contributes to emotion-related behaviors that differ between males and females and across the reproductive cycle. This hints at sex- or estrus-dependent features of BLA function, about which very little is known. The purpose of this study was to test whether there are sex differences or estrous cyclicity in rat BLA physiology and to determine their mechanistic correlates. We found substantial sex differences in the activity of neurons in lateral nuclei (LAT) and basal nuclei (BA) of the BLA that were associated with greater excitatory synaptic input in females. We also found strong differences in the activity of LAT and BA neurons across the estrous cycle. These differences were associated with a shift in the inhibition-excitation balance such that LAT had relatively greater inhibition during proestrus which paralleled more rapid cued fear extinction. In contrast, BA had relatively greater inhibition during diestrus that paralleled more rapid contextual fear extinction. These results are the first to demonstrate sex differences in BLA neuronal activity and the impact of estrous cyclicity on these measures. The shift between LAT and BA predominance across the estrous cycle provides a simple construct for understanding the effects of the estrous cycle on BLA-dependent behaviors. These results provide a novel framework to understand the cyclicity of emotional memory and highlight the importance of considering ovarian cycle when studying the BLA of females.SIGNIFICANCE STATEMENT There are differences in emotional responses and many psychiatric symptoms between males and females. This may point to sex differences in limbic brain regions. Here we demonstrate sex differences in neuronal activity in one key limbic region, the basolateral amygdala (BLA), whose activity fluctuates across the estrous cycle due to a shift in the balance of inhibition and excitation across two BLA regions, the lateral and basal nuclei. By uncovering this push-pull shift between lateral and basal nuclei, these results help to explain disparate findings about the effects of biological sex and estrous cyclicity on emotion and provide a framework for understanding fluctuations in emotional memory and psychiatric symptoms.

The giant panda (Ailuropoda melanoleuca) reproduction is of worldwide attention, and the vaginal microbiome is one of the most important factors affecting the reproductive rate of giant pandas. The aim of this study is to investigate the diversity of vaginal mycobiota structure, and potential pathogenic fungi in female giant pandas during estrus and non-estrus.

Female mice homozygous for an engineered Gnrhr E90K mutation have reduced gonadotropin-releasing hormone signaling, leading to infertility. Their ovaries have numerous antral follicles but no corpora lutea, indicating a block to ovulation. These mutants have high levels of circulating estradiol and low progesterone, indicating a state of persistent estrus. This mouse model provided a unique opportunity to examine the lack of cyclic levels of ovarian hormones on uterine gland biology. Although uterine gland development appeared similar to controls during prepubertal development, it was compromised during adolescence in the mutants. By age 20 weeks, uterine gland development was comparable to controls, but pathologies, including cribriform glandular structures, were observed. Induction of ovulations by periodic human chorionic gonadotropin treatment did not rescue postpubertal uterine gland development. Interestingly, progesterone receptor knockout mice, which lack progesterone signaling, also have defects in postpubertal uterine gland development. However, progesterone treatment did not rescue postpubertal uterine gland development. These studies indicate that chronically elevated levels of estradiol with low progesterone and therefore an absence of cyclic ovarian hormone secretion disrupts postpubertal uterine gland development and homeostasis.

Women are more susceptible to functional bowel disorders than men and the severity of their symptoms such as diarrhea, constipation, abdominal pain and bloating changes over the menstrual cycle, suggesting a role for sex hormones in gastrointestinal function. Nitric oxide (NO) is a major inhibitory neurotransmitter in the gut and blockade of nitric oxide synthase (NOS; responsible for NO synthesis) increases colonic motility in male mice ex vivo. We assessed the effects of NOS inhibition on colonic motility in female mice using video imaging analysis of colonic motor complexes (CMCs). To understand interactions between NO and estrogen in the gut, we also quantified neuronal NOS and estrogen receptor alpha (ERα)-expressing myenteric neurons in estrus and proestrus female mice using immunofluorescence. Mice in estrus had fewer CMCs under control conditions (6 ± 1 per 15 min, n = 22) compared to proestrus (8 ± 1 per 15 min, n = 22, One-way ANOVA, p = 0.041). During proestrus, the NOS antagonist N-nitro-L-arginine (NOLA) increased CMC numbers compared to controls (189 ± 46%). In contrast, NOLA had no significant effect on CMC numbers during estrus. During estrus, we observed more NOS-expressing myenteric neurons (48 ± 2%) than during proestrus (39 ± 1%, n = 3, p = 0.035). Increased nuclear expression of ERα was observed in estrus which coincided with an altered motility response to NOLA in contrast with proestrus when ERα was largely cytoplasmic. In conclusion, we confirm a cyclic and sexually dimorphic effect of NOS activity in female mouse colon, which could be due to genomic effects of estrogens via ERα.

The breeding of yaks is highly seasonal, there are many crucial proteins involved in the reproduction control program, especially in follicular development. In order to isolate differential proteins between mature and immature follicular fluid (FF) of yak, the FF from yak follicles with different sizes were sampled respectively, and two-dimensional gel electrophoresis (2-DE) of the proteins was carried out. After silver staining, the Image Master 2D platinum software was used for protein analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was performed for differential protein identification. The expression level of transferrin and enolase superfamily member 1 (ENOSF1) was determined by Western blotting for verification analysis. The results showed that 2-DE obtained an electrophoresis map of proteins from mature and immature yak FF with high resolution and repeatability. A comparison of protein profiles identified 12 differently expressed proteins, out of which 10 of them were upregulated while 2 were downregulated. Western blotting showed that the expression of transferrin and ENOSF1 was enhanced with follicular development. Both the obtained protein profiles and the differently expressed proteins identified in this study provided experimental data related to follicular development during yak breeding seasons. This study also laid the foundation for understanding the microenvironment during oocyte development.

The induction of pseudopregnancy by the exogenous administration of estradiol dipropionate (EDP) was investigated in cyclic Microminipigs (MMpigs) and the effects of exogenous administration of prostaglandin (PG) F2α on estrus exhibition were assessed in pseudopregnant MMpigs. In experiment 1, ovariectomized MMpigs were given a single intramuscular injection of 0.5, 1.5, or 2.5 mg of EDP. The estradiol-17β level at each of these doses was significantly higher 1 to 3 days after EDP administration than on the day of the injection. In experiment 2, animals were given 1.5 mg of EDP once at 9 to 12 days after the end of estrus (D0) and then no (1.5 mg × 1 group), one (D0 and D4; 1.5 mg × 2 group), or two (D0, D4 and D7; 1.5 mg × 3 group) additional treatments. The pseudopregnancy rate was significantly higher in the 1.5 mg × 3 than in the 1.5 mg × 1 group. In experiment 3, PGF2α was administered twice between 26 and 28 days after EDP treatment to five pseudopregnant gilts with a 24-h interval between the two injections. Estrus after PGF2α treatment and LH surge were observed in 100% and 80% pseudopregnant MMpigs, respectively. The interval from the day of the first PGF2α treatment to the onset of estrus was 6.5 ± 0.2 days. These results indicate that multiple EDP treatments are required for induction of pseudopregnancy in MMpigs and estrus exhibition can be controlled in MMpigs by treatment with EDP and PGF2α.

Sexually selected infanticide (SSI) is often presumed to be rare among seasonal breeders, because it would require a near immediate return to estrus after the loss of an entire litter during the mating season. We evaluated changes in reproductive strategies and the reproductive fate of females that experienced litter loss during the mating season in a seasonal breeder with strong evidence for SSI, the brown bear. First, we used a long-term demographic dataset (1986-2011) to document that a large majority of females (>91%) that lose their entire litter during the mating season in fact do enter estrus, mate, and give birth during the subsequent birthing season. Second, we used high-resolution movement data (2005-2011) to evaluate how females changed reproductive strategies after losing their entire litter during the mating season. We hypothesized that females would shift from the sedentary lifestyle typical for females with cubs-of-the-year to a roam-to-mate behavior typical for receptive females in no more than a few (∼3) days after litter loss. We found that females with cubs-of-the-year moved at about 1/3 of the rate and in a less bimodal diurnal pattern than receptive females during the mating season. The probability of litter loss was positively related with movement rate, suggesting that being elusive and sedentary is a strategy to enhance cub survival rather than a relic of cub mobility itself. The movement patterns of receptive females and females after litter loss were indistinguishable within 1-2 days after the litter loss, and we illustrate that SSI can significantly reduce the female interbirth interval (50-85%). Our results suggest that SSI can also be advantageous for males in seasonally breeding mammals. We propose that infanticide as a male reproductive strategy is more prevalent among mammals with reproductive seasonality than observed or reported.

No abstract available

In mammals, gonadal function is controlled by the activity of hypothalamic gonadotropin-releasing hormone neurons, which control the secretion of adenohypophyseal and gonadal hormones. However, there are a number of unanswered questions in relation to gonadal function. It is currently unknown how erotogenic stimulation of the genitals influences the subpopulation of hypothalamic medial preoptic area neurons, antidromically identified as projecting to the median eminence at different periods of the estrous cycle. Additionally, the distinctiveness of hypothalamic medial preoptic area neurons, with respect to methods of feedback control by exogenous hormones, is also unknown. In this study, spontaneous discharges from individual neurons encountered within the medial preoptic area, gono-like neurons, were recorded extracellularly using glass microelectrodes. To confirm the cellular and histochemical properties of the recording units, antidromic stimulation was performed using a side-by-side bipolar stimulating electrode placed into the median eminence, alongside microiontophoretic injections of the conventional tracer, horseradish peroxidase. In addition, further immunohistochemical analyses were performed. Results showed that elevated gono-neuron activity was accompanied by increased background activity and greater responses to erotogenic stimuli during estrus. Application of clitoral traction stimulation resulted in increased activation of the gono-like neurons. This neuronal activity was noticeably inhibited by β-estradiol administration. Immunohistochemical analyses revealed the presence of gonadotropin-releasing hormone-reactive protein in hypothalamic cells in which electrophysiological recordings were taken. Thus, medial preoptic area neurons represent the subset of hypothalamic gonadotropin-releasing hormone neurons described from brain slices in vitro, and might serve as a useful physiological model to form the basis of future in vivo studies.

Recent evidence suggests that adolescent and young adult females may be particularly responsive to nicotine use interventions that include exercise or environmental enrichment. This possibility was addressed in the current study by comparing the efficacy of exercise versus non-exercise environmental enrichment (saccharin) during abstinence at reducing subsequent nicotine-seeking/relapse vulnerability in an adolescent-onset rat model. The efficacy of each intervention was examined as a function of estrous cycle phase given findings indicating that hormonal status influences relapse vulnerability and treatment outcome in females. Once adolescent female rats acquired nicotine self-administration, they were given 23-h/day access to nicotine (0.01mg/kg/infusion) for 10days. Following the last self-administration session, rats began a 10-day forced abstinence period with 2-h/day access to an unlocked wheel (exercise, n=15), a bottle containing a saccharin-sweetened solution (0.25%; saccharin, n=19), or without access to a wheel or saccharin (control, n=20). Nicotine-seeking, as assessed under an extinction/cued-induced reinstatement procedure, was examined on day 11 of abstinence. Levels of nicotine-seeking were highest in females tested during estrus as compared to females tested during non-estrus phases. Exercise or saccharin during abstinence reduced nicotine-seeking in females tested during estrus, but neither affected the low levels of nicotine-seeking observed in females tested during non-estrus phases, presumably due to a floor effect. These results demonstrate that exercise or saccharin during abstinence decrease nicotine-seeking, and suggest that either would be effective as an early intervention for nicotine use and addiction in females.

Superovulation is a widely used reproductive technique in livestock production, but the mechanism of sheep's superovulation is not yet clear. Here, a method of superovulation and estrus synchronisation was used to treat female Duolang sheep. After treatment, there were significant differences in serum FSH and LH levels and the number of dominant follicles between the two groups of sheep. We identified a total of 5021 differentially expressed genes (11, 13 and 15 days after treatment) and performed RT-qPCR analysis to identify several mRNA expression levels. GO and KEGG enrichment analysis revealed that differentially expressed genes were involved in the regulation of signalling pathways of follicular development, cell cycle, material synthesis, energy metabolism, such as COL3A1, RPS8, ACTA2, RPL7 RPS6 and TNFAIP6 may play a key role in regulating the development of follicles. Our results show a comprehensive expression profile after superovulation and estrus synchronisation treatment. We provide the basis for further research on breeding techniques to improve the ovulation rate and birth rate of livestock.

The hormones estrogen and progesterone alter physiological functions, including the estrus cycle and relevant neurological and synaptic activity. Here, we determined the extent to which estrus cycle stage interacts with an inflammatory stimulus, lipopolysaccharide (LPS), to alter synaptic mitochondrial respiration in female rats. LPS elevated synaptic mitochondrial respiration of rats in estrus, but not diestrus. Likewise, estrogen concentration correlated with multiple respiratory metrics in LPS treated females in estrus. These data suggest estrogen likely modulates synaptic mitochondrial respiration in a high progesterone environment.

We have determined uterine glycogen content, metabolizing enzyme expression and activity in the mink, a species that exhibits obligatory embryonic diapause, resulting in delayed implantation. Gross uterine glycogen concentrations were highest in estrus, decreased 50% by diapause and 90% in pregnancy (P ≤ 0.05). Endometrial glycogen deposits, which localized primarily to glandular and luminal epithelia, decreased 99% between estrus and diapause (P ≤ 0.05) and were nearly undetectable in pregnancy. Glycogen synthase and phosphorylase proteins were most abundant in the glandular epithelia. Glycogen phosphorylase activity (total) in uterine homogenates was higher during estrus and diapause, than pregnancy. While glycogen phosphorylase protein was detected during estrus and diapause, glycogen synthase was almost undetectable after estrus, which probably contributed to a higher glycogenolysis/glycogenesis ratio during diapause. Uterine glucose-6-phosphatase 3 gene expression was greater during diapause, when compared to estrus (P ≤ 0.05) and supports the hypothesis that glucose-6-phosphate resulting from phosphorylase activity was dephosphorylated in preparation for export into the uterine lumen. The relatively high amount of hexokinase-1 protein detected in the luminal epithelia during estrus and diapause may have contributed to glucose trapping after endometrial glycogen reserves were depleted. Collectively, our findings suggest to us that endometrial glycogen reserves may be an important source of energy, supporting uterine and conceptus metabolism up to the diapausing blastocyst stage. As a result, the size of uterine glycogen reserves accumulated prior to mating may in part, determine the number of embryos that survive to the blastocyst stage, and ultimately litter size.

To screen differentially expressed proteins in the blood dairy cows with inactive ovaries caused by a negative energy balance and to determine the roles of the identified proteins in the development of inactive ovaries.Holstein cows at 14 to 21 days postpartum in an intensive dairy farm were examined for their energy balance (EB) status by blood β-hydroxybutyrate (BHBA) and assigned to the inactive ovary (IO) group (n = 50) and the normal oestrus control (CON) group (n = 50) at 60 to 90 days postpartum by means of the oestrus manifestation, rectal examination and B-ultrasound examination. Fourteen differentially expressed proteins from 61 proteins in the plasma of dairy cows with IOs were identified by iTRAQ/LC-MS/MS and GO, KEGG, and PATHWAY analysis. Eleven expressed proteins were upregulated, and 3 expressed proteins were downregulated. Among the 10 differentially expressed proteins verified by Western blot or ELISA, the relative expression levels of ALDOB, IGFBP2, ITIH3 and LDHB in mixed samples and single samples were consistent with the proteomic protein results. PKM2, GPX3, ALDOB, RBP4 and AHSG were significantly different between the two groups (P < 0.05); APOA4 and SPAM1 were not significantly different (P > 0.05) but were still downregulated in the ovarian resting group. This study confirmed that 14 plasma differential proteins in the inactive ovaries of postpartum dairy cows were associated with follicular development, and these findings provide a foundation for further research on the mechanism and prevention of inactive ovaries in dairy cows.