Advancing 3Rs: The Mouse Estrus Detector (MED) as a Low-Stress, Painless, and Efficient Tool for Estrus Determination in Mice.

Determining the estrous cycle stages in mice is essential for optimizing breeding strategies, synchronizing experimental timelines, and facilitating studies in behavior, drug testing, and genetics. It is critical for reducing the production of genetically unmodified offspring in the generation and investigation of genetically modified animal models. An accurate detection of the estrus cycle is particularly relevant in the context of the 3Rs-Replacement, Reduction, and Refinement. The estrous cycle, encompassing the reproductive phases of mice, is key to refining experimental designs and addressing ethical issues related to the use of animals in research. This study presents results from two independent laboratories on the efficacy of the Mouse Estrus Detector (MED) from ELMI Ltd. (Latvia) for the accurate determination of the estrus phase. The female mice of five strains/stocks (CD1, FVB/N, C57Bl6/J, B6D2F1, and Swiss) were used. The results showed that the MEDProTM is a low-traumatic, simple, rapid, and painless method of estrus detection that supports the principles of the 3Rs. The use of the MEDProTM for estrus detection in mice caused minimal stress, enhanced mating efficiency, facilitated an increase in the number of embryos for in vitro fertilization, and allowed the production of the desired number of foster animals.

Effects of prenatal cocaine exposure on estrous cycle, and behavior and expression of estrogen receptor alpha and oxytocin during estrus and diestrus in mice offspring.

Increasing evidence indicates that prenatal cocaine exposure may result in many developmental and long-lasting neurological and behavioral effects. The behaviors of female animals are strongly associated with the estrous cycle. Estrogen receptors and oxytocin are important neuroendocrine factors that regulate social behavior and are of special relevance to females. However, whether prenatal cocaine exposure induces estrous cycle changes in offspring and whether neurobehavioral changes in estrus and diestrus offspring differ remains unclear. On gestational day 12, mice were administered cocaine once daily for seven consecutive days, then the estrous cycle was examined in adult female offspring, as well as locomotion, anxiety level, and social behaviors, and the expression of estrogen receptor alpha-immunoreactive and oxytocin-immunoreactive neurons were compared between estrus and diestrus offspring. Prenatal cocaine exposure resulted in the shortening of proestrus and estrus in the offspring. During estrus and diestrus, prenatally cocaine-exposed offspring showed increased anxiety levels and changed partial social behaviors; their motility showed no significant differences in estrus, but declined in diestrus. Prenatal cocaine exposure reduced estrogen receptor alpha-immunoreactive expression in the medial preoptic area, ventromedial hypothalamic nucleus, and arcuate nucleus and oxytocin-immunoreactive expression in the paraventricular nucleus in estrus and diestrus offspring. These results suggest that prenatal cocaine exposure induces changes in the offspring's estrous cycle and expression of estrogen receptor alpha and oxytocin in a brain region-specific manner and that prenatal cocaine exposure and the estrous cycle interactively change motility and partial social behavior. Estrogen receptor alpha and oxytocin signaling are likely to play important concerted roles in mediating the effects of prenatal cocaine exposure on the offspring.

Augmenting interpretation of vaginoscopy observations in cycling bitches with deep learning model.

Successful identification of estrum or other stages in a cycling bitch often requires a combination of methods, including assessment of its behavior, exfoliative vaginal cytology, vaginoscopy, and hormonal assays. Vaginoscopy is a handy and inexpensive tool for the assessment of the breeding period. The present study introduces an innovative method for identifying the stages in the estrous cycle of female canines. With a dataset of 210 vaginoscopic images covering four reproductive stages, this approach extracts deep features using the inception v3 and Residual Networks (ResNet) 152 models. Binary gray wolf optimization (BGWO) is applied for feature optimization, and classification is performed with the extreme gradient boosting (XGBoost) algorithm. Both models are compared with the support vector machine (SVM) with the Gaussian and linear kernel, k-nearest neighbor (KNN), and convolutional neural network (CNN), based on performance metrics such as accuracy, specificity, F1 score, sensitivity, precision, matthew correlation coefficient (MCC), and runtime. The outcomes demonstrate the superiority of the deep model of ResNet 152 with XGBoost classifier, achieving an average model accuracy of 90.37%. The method gave a specific accuracy of 90.91%, 96.38%, 88.37%, and 88.24% in predicting the proestrus, estrus, diestrus, and anestrus stages, respectively. When performing deep feature analysis using inception v3 with the same classifiers, the model achieved an accuracy of 89.41%, which is comparable to the results obtained with the ResNet model. The proposed model offers a reliable system for identifying the optimal mating period, providing breeders and veterinarians with an efficient tool to enhance the success of their breeding programs.

Participation of preovulatory follicles in the activation of primordial follicles in mouse ovaries.

The mechanisms behind the selection and initial recruitment of primordial follicles (PmFs) from the non-growing PmF pool during each estrous cycle in females remain largely unknown. This study demonstrates that PmFs closest to the ovulatory follicle are preferentially activated in mouse ovaries under physiological conditions. PmFs located within 40 µm of the ovulatory follicles were more likely to be activated compared to those situated further away during the peri-ovulation period. Repeated superovulation treatments accelerated the depletion of the PmF reserve, whereas continuous suppression of ovulation delayed PmF reserve consumption. Spatial transcriptome sequencing of peri-ovulatory follicles revealed that ovulation primarily induces the degradation and remodeling of the extracellular matrix (ECM). This ECM degradation reduces mechanical stress around PmFs, thereby triggering their activation. Specifically, Cathepsin L (CTSL), a cysteine proteinase and lysosomal enzyme involved in ECM degradation, initiates the activation of PmFs adjacent to ovulatory follicles in a distance-dependent manner. These findings highlight the link between ovulation and selective PmF activation, and underscore the role of CTSL in this process under physiological conditions.

Exploring the Nexus of Steroidal Hormone Receptor, Uterine VEGF Expression and NADPH-d Interaction in Buffalo Uterus During Oestrous Cycle With Seasonal Variation.

The reproductive efficiency in buffalo is highly influenced by seasonal variability. Angiogenesis in the reproductive cycle is important for optimal physiological functioning of uterus. Estrogen receptor-α (ERα), vascular endothelial growth factor (VEGF) and reduced nicotinamide adenine dinucleotide phosphatase diaphorase (NADPH-d) are vital indicators for the uterine angiogenic process. This study was conducted to see the effect of season on the expression of different uterine angiogenic factors. Season wise (winter and summer) and phase wise (follicular and luteal), immune staining intensity of buffalo uterus was measured by calculating the optical density value (OD) for ERα and VEGF. Percentage of immuno-positive cell count for ERα was done. Histoenzymic NADPH-d expression was analysed. Expression of all these factors increased during follicular phase of oestrous cycle in order to support the angiogenesis; however, the expression was significantly lower (p ≤ 0.05) in term of OD value as well as percentage count of immuno-positive cells during summer season indicating lower angiogenic activity that subsequently affected reproduction in buffalo.

Saliva and plasma metabolome changes during anoestrus, the oestrous cycle and early gestation in the mare: A pilot study.

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.

Smooth muscle electromyography for detecting major alterations in the estrus cycle in rats.

Determining the female animal cycle is crucial in preclinical studies and animal husbandry. Changes in hormone levels during the cycle affect physiological responses, including altered contractility of the visceral smooth muscle. The study aimed to identify estrus and anestrus using smooth muscle electromyographic (SMEMG) measurements, in vivo fluorescent imaging (IVIS) and in vitro organ contractility of the uterus and cecum. The study involved sexually mature female Sprague-Dawley rats, aged 10-12 weeks. The rats received a daily injection of cetrorelix acetate solution for 7 days, while another group served as the control. The animals were subjected to gastrointestinal and myometrial SMEMG. The change in αvß3 integrin activity was measured with IVIS in the abdominal cavity. Contractility studies were performed in isolated organ baths using dissected uterus and cecum samples. Plasma samples were collected for hormone level measurements. A 3-fold increase in spontaneous contraction activity was detected in SMEMG measurements, while a significant decrease in αvß3 integrin was measured in the IVIS imaging procedure. Cetrorelix reduced the level of LH and the progesterone / estradiol ratio, increased the spontaneous activity of the cecum rings, and enhanced KCl-evoked contractions in the uterus. We found a significant change in the rate of SMEMG signals, indicating simultaneous increases in the contraction of the cecum and the non-pregnant uterus, as evidenced by isolated organ bath results. Fluorescence imaging showed high levels of uterine αvß3 integrin during the proestrus-estrus phase, but inhibiting the sexual cycle reduced fluorescence activity. Based on the results, the SMEMG and IVIS imaging methods are suitable for detecting estrus phase alterations in rats.

Plasma lipidomics in early APP/PS1 female mouse model and its relationship with brain: Is it affected by the estrous cycle?

BACKGROUND: Alzheimer's disease (AD) is the most prevalent dementia, showing higher incidence in women. Besides, lipids play an essential role in brain, and they could be dysregulated in neurodegeneration. Specifically, impaired plasma lipid levels could predict early AD diagnosis. This work aims to identify the main plasma lipids altered in early AD female mouse model and evaluate their relationship with brain lipidome. Also, the possible involvement of the estrous cycle in lipid metabolism has been evaluated. METHODS: Plasma samples of wild-type (n = 10) and APP/PS1 (n = 10) female mice of 5 months of age were collected, processed, and analysed using a lipidomic mass spectrometry-based method. A statistical analysis involving univariate and multivariate approaches was performed to identify significant lipid differences related to AD between groups. Also, cytology tests were conducted to confirm estrous cycle phases. RESULTS: Three hundred thirty lipids were detected in plasma, 18 of them showed significant differences between groups; specifically, some triacylglycerols, cholesteryl esters, lysophosphatidylcholines, phosphatidylcholines, and ether-linked phosphatidylcholines, increased in early AD; while other phosphatidylcholines, phosphatidylethanolamines, ceramides, and ether-linked phosphatidylethanolamines decreased in early AD. A multivariate approach was developed from some lipid variables, showing high diagnostic indexes (70% sensitivity, 90% specificity, 80% accuracy). From brain and plasma lipidome, some significant correlations were observed, mainly in the glycerophospholipid family. Also, some differences were found in both plasma and brain lipids, according to the estrous cycle phase. CONCLUSIONS: Therefore, lipid alterations can be identified in plasma at early AD stages in mice females, with a relationship with brain lipid metabolism for most of the lipid subfamilies, suggesting some lipids as potential AD biomarkers. In addition, the estrous cycle monitoring could be relevant in female studies.

The Influence of Sex Steroid Hormone Fluctuations on Capsaicin-Induced Pain and TRPV1 Expression.

Sexual dimorphism among mammals includes variations in the pain threshold. These differences are influenced by hormonal fluctuations in females during the estrous and menstrual cycles of rodents and humans, respectively. These physiological conditions display various phases, including proestrus and diestrus in rodents and follicular and luteal phases in humans, distinctly characterized by varying estrogen levels. In this study, we evaluated the capsaicin responses in male and female mice at different estrous cycle phases, using two murine acute pain models. Our findings indicate that the capsaicin-induced pain threshold was lower in the proestrus phase than in the other three phases in both pain assays. We also found that male mice exhibited a higher pain threshold than females in the proestrus phase, although it was similar to females in the other cycle phases. We also assessed the mRNA and protein levels of TRPV1 in the dorsal root and trigeminal ganglia of mice. Our results showed higher TRPV1 protein levels during proestrus compared to diestrus and male mice. Unexpectedly, we observed that the diestrus phase was associated with higher TRPV1 mRNA levels than those in both proestrus and male mice. These results underscore the hormonal influence on TRPV1 expression regulation and highlight the role of sex steroids in capsaicin-induced pain.

Anti-Müllerian hormone as a promising novel biomarker for litter size in Romanov sheep.

Anti-Müllerian hormone (AMH) is a hormone produced by growing preantral and antral follicles of the ovary. AMH is accepted as an important biomarker for fertility and superovulation parameters in livestock species. This study aimed to evaluate changes in serum AMH level in the oestrous cycle, repeatability of AMH, the effect of age on serum AMH level and the effects of AMH on litter size in Romanov sheep. In the study, a total of 36 Romanov sheep were used as animal material. First blood samples (0th day) were collected from 36 ewes to evaluate AMH and progesterone levels. Second blood samples were collected randomly from 20 ewes 9 days after first sampling to compare AMH levels at two different periods of the oestrous cycle in Romanov ewes. The ewes were categorized into three groups as low, medium and high AMH based on their first AMH levels. Results indicated that serum AMH level did not change during the oestrous and dioestrous phases of the oestrous cycle and two random time points of the oestrous cycle (p > .05). Pearson correlation analysis revealed that there is a high (r = .95) and significant (p < .001) correlation between AMH levels at the 0th (AMH-1) and 9th (AMH-2) days. The effect of AMH level on litter size was found to be significant. Litter size was significantly higher in the high AMH group than in the low AMH group (p < .05). In addition, the age of ewes did not affect serum AMH levels (p > .05). ROC analysis indicates that AMH cut-off value >320 pg/mL with 70% sensitivity and 100% specificity can be used for litter size in Romanov ewes. In conclusion, AMH is highly repeatable and its serum AMH level did not change during the oestrous cycle in Romanov sheep. In addition, AMH affects litter size and can be reliably used as a marker for litter size in Romanov sheep.

Corticotropin releasing factor alters the functional diversity of accumbal cholinergic interneurons.

Cholinergic interneurons (ChIs) provide the main source of acetylcholine in the striatum and have emerged as a critical modulator of behavioral flexibility, motivation, and associative learning. In the dorsal striatum (DS), ChIs display heterogeneous firing patterns. Here, we investigated the spontaneous firing patterns of ChIs in the nucleus accumbens (NAc) shell, a region of the ventral striatum. We identified four distinct ChI firing signatures: regular single-spiking, irregular single-spiking, rhythmic bursting, and a mixed-mode pattern composed of bursting activity and regular single spiking. ChIs from females had lower firing rates compared with males and had both a higher proportion of mixed-mode firing patterns and a lower proportion of regular single-spiking neurons compared with males. We further observed that across the estrous cycle, the diestrus phase was characterized by higher proportions of irregular ChI firing patterns compared with other phases. Using pooled data from males and females, we examined how the stress-associated neuropeptide corticotropin releasing factor (CRF) impacts these firing patterns. ChI firing patterns showed differential sensitivity to CRF. This translated into differential ChI sensitivity to CRF across the estrous cycle. Furthermore, CRF shifted the proportion of ChI firing patterns toward more regular spiking activity over bursting patterns. Finally, we found that repeated stressor exposure altered ChI firing patterns and sensitivity to CRF in the NAc core, but not the NAc shell. These findings highlight the heterogeneous nature of ChI firing patterns, which may have implications for accumbal-dependent motivated behaviors.NEW & NOTEWORTHY Cholinergic interneurons (ChIs) within the dorsal and ventral striatum can exert a major influence on network output and motivated behaviors. However, the firing patterns and neuromodulation of ChIs within the ventral striatum, specifically the nucleus accumbens (NAc) shell, are understudied. Here, we report that NAc shell ChIs have heterogeneous ChI firing patterns that are labile and can be modulated by the stress-linked neuropeptide corticotropin releasing factor (CRF) and by the estrous cycle.

Unveiling the role of protein kinase A (PKA) activity in bovine oviductal epithelial cells: implications on apoptotic signaling pathways during the estrous cycle.

The complex interactome crucial for successful pregnancy is constituted by the intricate network of endocrine and paracrine signaling pathways, involving gametes, embryos, and the female reproductive tract. Specifically, the oviduct exhibits distinct responses to gametes and early embryos during particular phases of the estrus cycle, a process tightly regulated by reproductive hormones. Moreover, these hormones play a pivotal role in orchestrating cyclical changes within oviductal epithelial cells. To unravel the molecular mechanisms underlying these dynamic changes, our study aimed to investigate the involvement of protein kinase A (PKA) in oviductal epithelial cells throughout the estrus cycle and in advanced pregnancy, extending our studies to oviductal epithelial cell in primary culture. By a combination of 2D-gel electrophoresis, Western blotting, and mass spectrometry, we identified 17 proteins exhibiting differential phosphorylation status mediated by PKA. Among these proteins, we successfully validated the phosphorylation status of heat shock 70 kDa protein (HSP70), aconitase 2 (ACO2), and lamin B1 (LMNB1). Our findings unequivocally demonstrate the dynamic regulation of PKA throughout the estrus cycle in oviductal epithelial cells. Also, analysis by bioinformatics tools suggest its pivotal role in mediating cyclical changes possibly through modulation of apoptotic pathways. This research sheds light on the intricate molecular mechanisms underlying reproductive processes, with implications for understanding fertility and reproductive health.

Impact of diet-induced maternal obesity on the reproductive capacity of F1 female offspring and the early development of the second generation.

The aim of this study was to examine the impact of maternal obesity on the reproductive capacity of the female offspring (F1) and on the early development of the second generation (F2). To this end, rats were fed either standard (SD) or cafeteria (CD) diet. CD rats and their offspring were divided into 2 groups: rats with 18% and ≥25% overweight (CD18 and CD25, respectively) and offspring from CD18 and CD25 rats (OCD18 and OCD25, respectively). Both OCD groups achieved greater weight gain than controls, without changes in the serum levels of glucose, cholesterol or triglycerides. However, they showed increased gonadal cholesterol concentration and fat content compared to controls. Female OCD groups showed a slight prolongation of the estrous cycle and different pattern of changes in the weight gain during pregnancy. The OCD25 group displayed an increased fertility index and preimplantation losses, and changes in some fetal measurements. Some OCD25 dams gave birth to a larger litter of pups and displayed a lower viability index and lactation rate than controls. OCD25 dams also showed an increase in estradiol and a decrease in testosterone and anti-Müllerian hormone. OCD25 rats showed increased mRNA levels of steroidogenenic enzymes. The offspring from OCD25 females (F2OCD25 offspring) showed early vaginal opening and higher ovulation rate in females, and lower ano-genital distances in males, compared to controls. In conclusion, these results reflect that maternal obesity impacts on the reproductive health of successive generations, probably as a result of epigenetic changes in different systems, including germ cells.

Neuroactive steroids fluctuate with regional specificity in the central and peripheral nervous system across the rat estrous cycle.

Neuroactive steroids (i.e., sex steroid hormones and neurosteroids) are important physiological regulators of nervous function and potential neuroprotective agents for neurodegenerative and psychiatric disorders. Sex is an important component of such effects. However, even if fluctuations in sex steroid hormone level during the menstrual cycle are associated with neuropathological events in some women, the neuroactive steroid pattern in the brain across the ovarian cycle has been poorly explored. Therefore, we assessed the levels of pregnenolone, progesterone, and its metabolites (i.e., dihydroprogesterone, allopregnanolone and isoallopregnanolone), dehydroepiandrosterone, testosterone and its metabolites (i.e., dihydrotestosterone, 3α-diol and 17ß-estradiol) across the rat ovarian cycle to determine whether their plasma fluctuations are similar to those occurring in the central (i.e., hippocampus and cerebral cortex) and peripheral (i.e., sciatic nerve) nervous system. Data obtained indicate that the plasma pattern of these molecules generally does not fully reflect the events occurring in the nervous system. In addition, for some neuroactive steroid levels, the pattern is not identical between the two brain regions and between the brain and peripheral nerves. Indeed, with the exception of progesterone, all other neuroactive steroids assessed here showed peculiar regional differences in their pattern of fluctuation in the nervous system during the estrous cycle. These observations may have important diagnostic and therapeutic consequences for neuropathological events influenced by the menstrual cycle.

The modulatory role of serotonin-1A receptors of the basolateral amygdala and dorsal periaqueductal gray on the impact of hormonal variation on the conditioned fear response.

Despite significant advances in the study of fear and fear memory formation, little is known about fear learning and expression in females. This omission has been proven surprising, as normal and pathological behaviors are highly influenced by ovarian hormones, particularly estradiol and progesterone. In the current study, we investigated the joint influence of serotonin (5-HT) neurotransmission and estrous cycle phases (low or high levels of estradiol and progesterone) on the expression of conditioned fear in a group of female rats that were previously divided according to their response to stressful stimuli into low or high anxiety-like subjects. The baseline amplitude of the unconditioned acoustic startle responses was high in high-anxiety female rats, with no effect on the estrous cycle observed. Data collected during the proestrus-estrus phase revealed that low-anxiety rats had startle amplitudes similar to those of high-anxiety rats. It is supposed that high-anxiety female rats benefit from increased estradiol and progesterone levels to achieve comparable potentiated startle amplitudes. In contrast, female rats experienced a significant decrease in hormone levels during the Diestrus phase. This decrease is believed to play a role in preventing them from displaying a heightened startle response when faced with strongly aversive stimuli. Data collected after 5-HT and 8-OH-DPAT were administered into the basolateral nuclei and dorsal periaqueductal gray suggest that 5-HT neurotransmission works with progesterone and estrogen to reduce startle potentiation, most likely by activating the serotonin-1A receptor subtype.

Differing planes of nutrition alter serum and histotroph amino acid composition in nonpregnant, multiparous beef cows on day 3 of the estrous cycle.

Endometrial-derived uterine histotroph is a critical component of nutrient supply to a growing conceptus throughout gestation; however, the effect of nutritional plane on histotroph nutrient composition remains unknown in multiparous cows. We hypothesized that differing planes of nutrition would alter histotroph and serum nutrient composition in beef cattle. Thus, we evaluated serum and histotroph amino acid and glucose composition, and serum non-esterified fatty acids (NEFA) and blood urea nitrogen (BUN) in cows individually fed to maintain body weight (BW; 0 kd/d, n = 9; CON) compared with those losing moderate BW (-0.7 kg/d, n = 9; NEG). After 49 d of differing nutritional planes, cows were subjected to the 7-d CoSynch + controlled internal drug release device estrus synchronization protocol and then slaughtered on day 62. Blood serum (days 0 and 62) and uterine histotroph [day 62; from uterine horns ipsilateral and contralateral to the corpus luteum (CL)] were collected and analyzed for concentrations of amino acids, glucose, and NEFA. Performance characteristics, body composition via ultrasound (days 0 and 62), and carcass characteristics were collected. Body condition score, change in BW, average daily gain, dry matter intake, and gain:feed were decreased (P ≤ 0.05) in NEG vs. CON cows. There were no differences in body composition or carcass characteristics, except an increase (P ≤ 0.05) in dressing percentage in NEG cows due to differences in gut fill, consistent with study design. Serum NEFA increased (P ≤ 0.05) in the NEG group, but there were no differences between NEG vs. CON in glucose or BUN. Serum histidine increased (P ≤ 0.05) and alanine, isoleucine, and tryptophan decreased (P ≤ 0.05) in NEG vs. CON cows. Compared with that of the uterine horn ipsilateral to the CL, histotroph from the uterine horn contralateral to the CL had increased (P ≤ 0.05) isoleucine, asparagine, and proline concentrations in NEG cows, and decreased (P ≤ 0.05) tryptophan as a proportion of essential and total amino acids. There were no differences in glucose concentrations of histotroph contralateral or ipsilateral to the CL. Cow nutritional plane does alter serum and histotroph amino acid composition, although the presence of an embryo may be necessary to fully elucidate these changes. Differences in serum and histotroph tryptophan should be given consideration in future studies due to its importance as an essential amino acid in protein synthesis and bioactive affects.

Amino acids are important in protein synthesis and bioactive affects. Maternal diet could impact histotroph amino acid composition which serves as a nutrient supply to the conceptus throughout pregnancy and is especially critical during early pregnancy, before the placenta is fully functional. Cows were subjected to their diets for 62 d, resulting in decreased body condition, average daily gain, dry matter intake, G:F, and a greater change in body weight (BW) among moderate loss cows. These data demonstrate our model for moderate BW loss was successful. Moderate BW loss cows exhibited alterations in serum and histotroph amino acid composition in the uterine horn contralateral to the corpus luteum (CL). However, in the present study, histotroph amino acid alterations were in the uterine horn contralateral to the CL, which would be opposite of the developing conceptus. Nevertheless, because the 2 uterine horns communicate via the common uterine body, the pre-implantation conceptus should have access to the histotroph from the contralateral uterine horn. Thus, future studies are needed to fully elucidate effects of nutritional plane on histotroph nutrient composition, and its potential impact on pregnancy.

C/EBPß deletion in macrophages impairs mammary gland alveolar budding during the estrous cycle.

Macrophages have important roles in mammary gland development and tissue homeostasis, but the specific mechanisms that regulate macrophage function need further elucidation. We have identified C/EBPß as an important transcription factor expressed by multiple macrophage populations in the normal mammary gland. Mammary glands from mice with C/EBPß-deficient macrophages (Cebpb ΔM) show a significant decrease in alveolar budding during the diestrus stage of the reproductive cycle, whereas branching morphogenesis remains unchanged. Defects in alveolar budding were found to be the result of both systemic hormones and local macrophage-directed signals. RNA sequencing shows significant changes in PR-responsive genes and alterations in the Wnt landscape of mammary epithelial cells of Cebpb ΔM mice, which regulate stem cell expansion during diestrus. Cebpb ΔM macrophages demonstrate a shift from a pro-inflammatory to a tissue-reparative phenotype, and exhibit increased phagocytic capacity as compared to WT. Finally, Cebpb ΔM macrophages down-regulate Notch2 and Notch3, which normally promote stem cell expansion during alveolar budding. These results suggest that C/EBPß is an important macrophage factor that facilitates macrophage-epithelial crosstalk during a key stage of mammary gland tissue homeostasis.

The impact of estrous cycle on anxiety-like behaviour during unlearned fear tests in female rats and mice: A systematic review and meta-analysis.

Anxiety fluctuates across the human menstrual cycle, with symptoms worsening during phases of declining or low ovarian hormones. Similar findings have been observed across the rodent estrous cycle, however, the magnitude and robustness of these effects have not been meta-analytically quantified. We conducted a systematic review and meta-analysis of estrous cycle effects on anxiety-like behaviour (124 articles; k = 259 effect sizes). In both rats and mice, anxiety-like behaviour was higher during metestrus/diestrus (lower ovarian hormones) than proestrus (higher ovarian hormones) (g = 0.44 in rats, g = 0.43 in mice). There was large heterogeneity in the data, which was partially accounted for by strain, experimental task, and reproductive status. Nonetheless, the effect of estrous cycle on anxiety-like behaviour was highly robust, with the fail-safe N test revealing the effect would remain significant even if 21,388 additional studies yielded null results. These results suggest that estrous cycle should be accounted for in studies of anxiety in females. Doing so will facilitate knowledge about menstrual-cycle regulation of anxiety disorders in humans.

Female rodents in behavioral neuroscience: Narrative review on the methodological pitfalls.

Since the NIH 'Sex as biological variable' policy, the percentage of studies including female subjects have increased largely. Nonetheless, many researchers fail to adequate their protocols to include females. In this narrative review, we aim to discuss the methodological pitfalls of the inclusion of female rodents in behavioral neuroscience. We address three points to consider in studies: the manipulations conducted only in female animals (such as estrous cycle monitoring, ovariectomy, and hormone replacement), the consideration of males as the standard, and biases related to interpretation and publication of the results. In addition, we suggest guidelines and perspectives for the inclusion of females in preclinical research.

Human CYP2D6 varies across the estrous cycle in brains of transgenic mice altering drug response.

Cytochrome P450 (CYP) 2Ds are drug metabolizing enzymes found in brain and liver which metabolize numerous centrally acting drugs. Inhibition and induction of CYP2D-mediated metabolism in rodent brain alters brain drug and metabolite concentrations and resulting drug response. In female rats, brain CYP2D metabolism varies across the estrous cycle and with exogenous estrogen, changing brain drug concentrations and response. In this study harmine-induced hypothermia was lower in humanized CYP2D6 transgenic female mice during estrus compared to diestrus. Pretreatment into the cerebral ventricles with propranolol, a selective irreversible inhibitor of human CYP2D6 in brain, increased hypothermia in estrus but not in diestrus. In vivo enzyme activity was higher in brains of transgenic mice in estrus compared to diestrus and was lower after pretreatment with inhibitor in estrus, but not in diestrus. Hepatic activity and plasma harmine concentrations were unaffected by either estrous phase or inhibition of brain CYP2D6. In wild-type female mice, harmine-induced hypothermia was unaffected by either estrous phase or inhibitor pretreatment. Male mice were used as positive controls, where pretreatment with inhibitor increased harmine-induced hypothermia in transgenic but not wild-type, mice. This study provides evidence for female hormone cycle-based regulation of drug metabolism by human CYP2D6 in brain and resulting drug response. This suggests that brain CYP2D6 metabolism may vary, for example, during the menstrual cycle, pregnancy, or menopause, or while taking oral contraceptives or hormone therapy. This variation could contribute to individual differences in response to centrally acting CYP2D6-substrate drugs by altering local brain drug and/or metabolite concentrations.