Preovulatory Follicles Research Articles

Meiotic and developmental competence of growing pig oocytes derived from small antral follicles is enhanced in culture medium containing FGF2, LIF, and IGF1 (FLI medium)

BackgroundOocytes of large animal species isolated from small ovarian follicles (< 2 mm) are less competent to support early embryonic development after in vitro maturation and fertilization than their counterparts isolated from medium-sized and preovulatory follicles. This study aimed to assess the effect of a new maturation medium containing FGF2, LIF, and IGF1 (FLI medium) on the meiotic and developmental competence of pig cumulus-oocytes complexes (COCs) derived from the small and medium-sized follicles.MethodsThe growing oocytes were isolated from 1 to 2 (small follicle; SF) and the fully-grown ones from 3 to 6 (large follicle; LF) mm follicles and matured in a control M199 medium with gonadotropins and EGF and the FLI medium enriched by the triplet of growth factors. The matured oocytes were parthenogenetically activated and cultured to the blastocyst stage. Chromatin configuration before and during the culture and MAP kinase activity were assessed in the oocytes. Finally, the expression of cumulus cell genes previously identified as markers of oocyte quality was assessed.ResultsThe maturation and blastocyst rates of oocytes gained from LF were significantly higher than that from SF in the control medium. In contrast, similar proportions of oocytes from LF and SF completed meiosis and developed to blastocysts when cultured in FLI. Most of the oocytes freshly isolated from SF possessed germinal vesicles with fine filaments of chromatin (GV0) or chromatin surrounding the nucleolus (GVI; 30%); the oocytes from LF were mainly in GVI (or GVII) exhibiting a few small lumps of chromatin beneath the nuclear membrane. When cultured in the FLI medium for 16 h, an acceleration of the course of maturation in oocytes both from SF and LF compared to the control medium was observed and a remarkable synchrony in the course of chromatin remodeling was noticed in oocytes from SF and LF.ConclusionsThis work demonstrates that the enrichment of culture medium by FGF2, LIF, and IGF1 can enhance the meiotic and developmental competence of not only fully-grown, but also growing pig oocytes and significantly thus expanding the number of oocytes available for various assisted reproductive technology applications.

Follicular Dynamics and Pregnancy Rates during Foal Heat in Colombian Paso Fino Mares Bred under Permanent Grazing.

No studies have evaluated the peripartum follicular dynamics resulting in foal heat under tropical environments. We aimed to assess retrospectively the peripartum follicular dynamics in Colombian Paso Fino mares that were inseminated at the foal heat, becoming pregnant or not. Records including follicular dynamics of pregnant mares prepartum and from foaling until foal heat ovulation were assessed in Colombian Paso Fino mares (CPF, n = 24) bred under permanent grazing in a tropical herd in Colombia. The number of ovarian follicles >10 mm before foaling and the largest follicle (F1) growth rate (mm/day) from foaling until the F1 reached the largest diameter (pre-ovulatory size) at the foal heat were assessed. Mares were inseminated at foal heat with 20 mL of semen (at least 500 million live spermatozoa) with >75% motility and 80% viability from a stallion of proven fertility. Ovulation was confirmed the day after follicles had reached the largest diameter. Quantitative data from follicular growth, the day at ovulation, from mares that became pregnant (PM) or not (NPM) at 16 days post-insemination were compared by one-way ANOVA, repeated measures ANOVA (follicle growth rate data) or Chi-square test (edema and cytology scores data). Epidemiological data, gestation length, and the number of follicles on third prepartum days did not significantly differ between PM and NPM (p > 0.05). Seventy-one percent of mares (17/24) got pregnant. Ovulatory follicles grew faster in the NPM group (n = 7), which ovulated between the seventh and ninth postpartum days, compared to PM (n = 17), which ovulated between the 11th and 13th postpartum days. Pre-ovulatory follicle diameter in PM (48.57 ± 0.8 mm) was significantly larger than in NPM (42.99 ± 1.0 mm) (p < 0.05). In addition, the PM edema score (2.93 ± 0.32 mm) on ovulation day was significantly lower (p < 0.05) than NPM (4.47 ± 0.05 mm). First postpartum ovulation occurred at 12.6 ± 0.3 and 8.5 ± 0.4 days (p < 0.05) in PM and NPM, respectively. Colombian Paso Fino mares bred under permanent grazing under tropical rainforest conditions with no foaling or postpartum complications showed a 71% gestation rate when inseminated at foal heat when ovulation occurs between the second and third postpartum week.

Effects of Nesting Material Provision and High-Dose Vitamin C Supplementation during the Peripartum Period on Prepartum Nest-Building Behavior, Farrowing Process, Oxidative Stress Status, Cortisol Levels, and Preovulatory Follicle Development in Hyperprolific Sows.

Hyperprolific sows often experience increased oxidative stress during late gestation and lactation periods, which can adversely affect the farrowing process and overall lactation performance. This study examines the influence of providing a coconut coir mat (CCM; 1 × 1 m) as nesting material, supplementing high-dose vit-C (HVC; 20% vit-C, 10 g/kg feed) as an antioxidant, or both on maternal behavior, the farrowing process, oxidative status, cortisol levels, and preovulatory follicle developments in sows with large litters. In total, 35 sows (Landrace × Yorkshire; litter size 15.43 ± 0.27) were allocated to the following four treatment groups: control (n = 9, basal diet), vit-C (n = 8, basal diet + HVC), mat (n = 10, basal diet + CCM), and mat + vit-C (n = 8, basal diet + HVC + CCM). A post-hoc analysis showed that compared with sows that were not provided CCM, mat and mat + vit-C groups demonstrated increased durations of nest-building behavior during the period from 24 h to 12 h before parturition (p < 0.05 for both), reduced farrowing durations, and decreased intervals from birth to first udder contact (p < 0.01 for both). The mat group exhibited lower advanced oxidation protein product (AOPP) levels during late gestation and lactation periods than the control group (p < 0.05). Sows with HVC supplementation showed longer farrowing durations than those without HVC supplementation (p < 0.0001). The vit-C group had higher salivary cortisol levels on day 1 after farrowing than the other treatment groups (p < 0.05). Furthermore, the follicle diameters on day 3 after weaning in the vit-C group tended to be smaller than those in the control group (p = 0.077). HVC supplementation prolonged farrowing and increased the physiological stress on postpartum, and no advantageous effects on maternal behavior and developmental progression of preovulatory follicles were observed. Hence, alternative solutions beyond nutritional approaches are required to address increased oxidative stress in hyperprolific sows and secure their welfare and reproductive performance. The present results substantiated the positive impact of providing CCM as nesting material for sows with large litters on nest-building behavior and the farrowing process, which could mitigate the deleterious consequences induced by peripartum physiological and oxidative stress.

Altered expression of angiogenic factors in dominant preovulatory follicles of dairy cattle treated with ACTH

Studies in cows have reported that ovulation, steroidogenesis and angiogenesis are affected by stress and consequently fertility decreases. The purpose of this study was to evaluate the effects of ACTH administration during the preovulatory period on the expression of growth factors (CD-31, PDGF-A, PDGF-B, VEGFA-164, VEGFA-164b, VEGF-R1 and VEGF-R2) associated with the angiogenic process by immunohistochemistry in cows (n = 14). Results evidenced the expression of these growth factors in theca and granulosa cells from antral, atretic and dominant preovulatory follicles of ACTH-treated cows, suggesting that, under stress conditions, their expression continues to be required. VEGFA-164, VEGF-R1 and VEGF-R2 expression was greater in theca cells of dominant preovulatory follicles of the ACTH-treated group than in those of the control group. CD-31 protein expression was lower in the dominant preovulatory follicles of the ACTH-treated group than in those of the control group. PDGF-A and PDGF-B expression did not differ between groups, either in granulosa or in theca cells. These results suggest that VEGFA-164, its receptors and CD-31 are actors in the normal cycle of the ovaries and could have greater pathophysiological importance in the altered angiogenic process and other events that occur during anovulation and stress conditions. This dysregulation reinforces the importance of the angiogenic process in the pathophysiology of cystic ovarian disease in cows. This is the first report on the expression and localization of components of the VEGF and PDGF systems and CD-31 in cells from dominant preovulatory follicles after ACTH administration.

Selenium affects genes associated with immunity and apoptosis in in vitro follicles of ewes

Selenium plays an important role in bodily functions. It activates immune cells such as neutrophils and immunity through T cells. However, its role in ovarian follicular gene expression has not been reported. The effects of selenomethionine (SeMet) on gene expression in in vitro ewe follicles and their relationship with immunity and apoptosis were studied. Preovulatory follicles were randomly cultured, and SeMet was added to the culture, followed by incubation for 24 h. Total RNA was extracted from follicles and placed on a microarray chip. Gene expression was analyzed using GenArise and classified using the DAVID v6.8 bioinformatics program based on the Gene Ontology and Kyoto Encyclopedia for Genes and Genomes (KEGG). Gene pathways were designed using Cytoscape v3.7.2. There were 2,538 differentially expressed genes (DEGs): 1,228 upregulated and 1,310 downregulated. The upregulated genes were classified into 32, 18, and 12 nodes related to biological processes, cellular components, and molecular functions, respectively. KEGG assigned these genes to 41 metabolic and signaling pathways. 94 genes were involved in eight pathways associated with immune processes. The expression levels of CD8, NFAT2, and CD48 were quantified using RT-qPCR. Selenium activated gene expression in the in vitro preovulatory follicles of ewes, some of which were related to the immune system. The relationship among CD8, NFAT2, and CD48 genes suggests a possible immune pathway related to follicular apoptosis regulation.

Short Estrous Cycle Post-ovulation in Peri-pubertal Sahiwal and Jersey Crossbred Heifers

This investigation focuses on analysing the follicular dynamics exhibited during peri-pubertal period in Sahiwal and Jersey crossbred heifers. The aim of the study was to assess first ovulation in the heifers following puberty (N=20; 10 for each breed). Heifers were subjected to alternate day trans-rectal ultrasonography (TRUS) when the diameter of dominant follicle surpassed 9 mm diameter. Transrectal ultrasonography (TRUS) was performed to observe and assess first ovulation in heifers. The size of the first pre-ovulatory follicle in Sahiwal and Jersey Crossbred heifer in peri-pubertal stage was 10.52±0.13 and 10.15±0.51 mm, respectively and the corpus luteum attained its maximum size of 13.89 and 11.26 mm, was significantly different (p&lt;0.01) at day 8.6±0.4 and 8.2±0.48 in Sahiwal and Jersey crossbred heifers, respectively. The regression of corpus luteum (CL) adjudged by the Colour Doppler initiated on 10.6±0.4 and 11.0±0.63 day for Sahiwal and Jersey crossbred heifers, respectively. Some other follicular dynamics parameters i.e. day of wave onset, number of follicles recruited, inter-wave duration along with maximum diameter of dominant follicle, day of maximum diameter, growth rate, length of growth and static phase, duration of static phase and onset of atresia was recorded, however, no significant difference (p&gt;0.05) was recorded between breeds. On an interesting note, the next estrous cycle for heifers did not lead to ovulation despite having follicular size more than10 mm in both the breeds.&#x0D; Conclusion: In peroration, peri-pubertal Sahiwal and Jersey crossbred heifers had notably precocious luteal regression after first ovulation, without subsequent ovulation in next estrous cycle.

Nitrophenols disrupt the expression and activity of biotransformation enzymes (CYP3A and COMT) in chicken ovarian follicles in vivo and in vitro.

Nitrophenols are environmental pollutants and xenobiotics, the main sources of which are diesel exhaust fumes and pesticides. The biotransformation processes that take place in the liver are defence mechanisms against xenobiotics, such as nitrophenols. Our previous study showed that the chicken ovary is an additional xenobiotic detoxification place and that nitrophenols disrupt steroidogenesis in chicken ovarian follicles. Therefore, the present study aimed to determine the in vivo and in vitro effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on the expression and activity of phase I (CYP3A) and phase II (COMT) biotransformation enzymes in chicken ovary. In an in vivo study, hens were treated with a vehicle or 10mg PNP or PNMC/kg b.wt. per day for 6 days. In an in vitro study, prehierarchical white and yellowish follicles, as well as the granulosa and theca layers of the three largest preovulatory follicles (F3, F2 and F1), were isolated and then incubated in a control medium or medium supplemented with PNP (10-6M) or PNMC (10-6M) for 24 or 48 h. Both in vivo and in vitro studies showed that nitrophenols exert tissue- and compound-dependent (PNP or PNMC) effects on CYP3A and COMT gene (real-time PCR) protein (Western blot) expression and their activity (colorimetric methods). The inhibitory effect of nitrophenols in vivo on the activity of biotransformation enzymes suggest that the ovary has the capacity to metabolise PNP and PNMC.

P-39 Fictitious hyperestrogenism in perimenopause: Difficulties in diagnosis by laboratory pitfalls

Abstract Introduction Laboratory errors related to interfering substances can cause significant morbidity related to unnecessary investigations and interventions and patient anxiety. Estradiol (E2) is quantified mainly with competitive immunoassays. The prevalence of analytical interference can be up to 6% and is due to the impairment of the interaction between test antibody and analyte, causing falsely elevated levels. Nevertheless, interference due to heterophile antibody is extremely rare. Clinical Case A 51-year-old woman, normal weight, nulliparous, irrelevant previous medical history, menarche at 12 years old and regular menstrual cycles, was referred to the Endocrinology Department due to elevated E2 level. She was followed 4 years before in another institution with no formal diagnosis complaining of severe vasomotor symptoms, vaginal dryness and menometrorrhagias. She was on combined oral contraceptives for 10 years as a method of contraception, which she stopped two years ago. The first two E2 random values were 2362 and 3121 pmol/L, respectively when she had 47 and 49 years old. During our evaluation in January 2021, at the age of 51 years old, in the 4th day of her menstrual cycle, the laboratory evaluation revealed: FSH 9.56 mUI/mL (2.5-10.2), LH 5.43 mUI/mL (1.9-12.5), Prolactin 18.12 ng/mL (2.8-29.2), E2 3530.51 pmol/L (71.6-529.2), 17-Hydroxiprogesterone 0.52 ng/mL (0.32-1.47), DHEA-SO4 75.9 ug/dL (35-430), 4-Androstenedione 1 ng/mL (0.3-3.3) and SHBG 137 nmol/L (18-144). Transvaginal pelvic ultrasonography performed 15 days after menstrual period revealed 2 subserous leiomyomas, a 12 mm endometrial line and a right ovary with a pre-ovulatory follicle. CT scan of adrenal glands was unremarkable and a sellar MRI revealed a 4mm area of hypocaptation in the left region of the adenohypophysis. During this period the patient was treated with escitalopram 10 mg od, with vasomotor symptoms improvement. Two years later, after a 6-month period of amenorrhea, she repeated the analytical evaluation that revealed raised gonadotropins levels (FSH 102.12 mUI/mL, LH 25.37 mUI/mL), low progesterone 0.91 mUI/mL (&amp;lt; 4.45) but maintained a largely elevated estradiol level (1128.01 pmol/L) with undetectable free Estriol (&amp;lt; 0.10 ng/mL). This pattern motivated laboratorial review of blood samples by mass spectrometry and not by chemiluminescence as before. The value was substantially lower (447 pmol/L) hypothesizing the presence of heterophilic antibodies. Conclusion Although false E2 elevation due to analytical interference is rare, it must be suspected when results are discordant with the clinical picture and there is no evidence of neoplastic disease. Here, we report a case of falsely elevated E2 that led to prolonged investigation and major impact on a female's quality of life during the challenging period of perimenopause.

Evaluation of the expression of growth hormone and its receptor during the resumption of postpartum ovarian follicle development in dairy cows

Growth hormone is a key endocrine factor for metabolic adaptations to lactation and optimal reproductive function of the dairy cow. This study aimed to analyze the expression of GH and its receptor (GHR) in ovarian follicles, along with metabolic biomarkers, during the resumption of the postpartum follicular development, and to analyze the immunolocalization and protein expression of GH and GHR in preovulatory follicles. Thirty-six dairy cows were grouped according to the postpartum days (PPD) until the establishment of the first dominant follicle in: cows that established their first dominant follicle at fewer postpartum days (FPPD group; n = 15) and cows that established their first dominant follicle at more postpartum days (MPPD group; n = 22). For a second analysis, the same cows were regrouped according to the calving season (S), into cows calving in autumn (n = 20) and cows calving in winter (n = 17). During the PP, blood and follicular aspirates were obtained at two timepoints (T): when the first dominant follicle was established (T1, day 9 ± 2), and when the preovulatory follicle was established (T2, day 45 ± 2). Also, six dairy cows were ovariectomized in proestrus and ovarian histological sections were obtained. Growth hormone mRNA was detected in granulose cells from ovarian follicle sampled during PP. A PPD × T interaction was observed for GHR mRNA, where it was greater in the FPPD cows than in the MPPD cows at T1. Metabolic biomarkers and reproductive hormones showed differences or interaction between PPD, T, S, depending on the case. Also, GH and GHR were immunolocalized in granulosa and theca interna cells of preovulatory follicles. These results confirm the expression of GH and GHR in the mature ovarian follicles of dairy cows and show a possible association between greater GHR expression and an earlier resumption of postpartum follicular development.

Foal sex in Thoroughbred horses: related factors.

Reproductive biotechniques in the equine species have advanced in the last decade and horse breeders have started to question the possibilities of interfering in the determination of foal sex. The aim of the present study was to verify whether the variables mares and stallion's age, side of the ovary containing the preovulatory follicle, preovulatory follicle diameter, time between breeding and ovulation, and ovulation inducing hormones influence the sex of the foal. A total of 259 reproductive cycles of 160 mares and 22 Thoroughbred stallions were used. Statistical analysis was performed using R software, including Pearson's chi-square test and logistic regression. Of the total foals born, 136 were males (52.51%) and 123 were females (47.49%). In mares that ovulated with -24h after ovulation induction, 104 foals (54.74%) were males and 86 (45.26%) were females, while in mares that ovulated with +24h, 32 foals (46.38%) were males and 37 (53.62%) were females. Stallions up to 15 years old had 44.14% (n=49) females and those over 15 years had 49.66% (n=73) females. The simple logistic regression model showed that mares and stallions under 15 years old, mares with ovulation time less than 24 hours and treated with Deslorelin had a higher probability of having male foals, but the Pearson's chi-square test showed that foals gender were not influenced by the variables studied.

Serum anti-Müllerian hormone around the time of ovulation simulated by exogenous hormones in clouded leopards (Neofelis nebulosa).

Anti-Müllerian hormone (AMH) is produced by granulosa cells of the antral follicles. It serves as a promising biomarker for ovarian reserve and responsiveness to ovarian stimulation in humans and domestic animals. This study aimed to validate the AMH Gen II enzyme-linked immunosorbent assay (ELISA) and correlate ovarian structures with serum AMH concentrations after stimulation treatment in clouded leopards (Neofelis nebulosa). Serum samples were collected from 12 women (age 6.21 ± 3.56 years), and serum AMH concentrations were analysed using AMH Gen II ELISA. The animals were divided into two groups based on ovarian structures [preovulatory follicles (>2 mm) and/or corpora hemorrhagica] along with the presence of uterine tonicity visualized laparoscopically around the time of ovulation. Animals that exhibited these reproductive features were identified as the responder group (n = 9, aged 7.59 ± 2.96 years), whereas those lacking the corresponding features were assigned to the nonresponder group (n = 3, aged 2.06 ± 0.53 years). The intra-assay coefficient of variation (CV) and interassay CV was 3.56% and 7.75%, respectively. The linearity of AMH dilution was confirmed (r2 = .998), and the percentage of recovery ranged from 93% to 115%. The results demonstrated that overall serum AMH concentrations around the time of ovulation were negatively correlated with age (rs = -.692, p = .013). However, serum AMH concentrations were not correlated with the average number of ovarian structures (rs = -.535, p = .074). Thus, AMH Gen II ELISA was validated in clouded leopards. Around the time of ovulation, serum AMH decreased with advancing age and ovarian responsiveness cannot be evaluated using serum AMH.

Unique hyaluronan structure of expanded oocyte-cumulus extracellular matrix in ovarian follicles.

In preovulatory follicles, after the endogenous gonadotropin surge, the oocyte-cumulus complexes (OCCs) produce hyaluronan (HA) in a process called "cumulus expansion". During this process, the heavy chains (HCs) of the serum-derived inter-alpha-trypsin inhibitor (IαI) family bind covalently to synthesized HA and form a unique structure of the expanded cumulus HA-rich extracellular matrix. Understanding the biochemical mechanism of the covalent linkage between HA and the HCs of the IαI family is one of the most significant discoveries in reproductive biology, since it explains basis of the cumulus expansion process running in parallel with the oocyte maturation, both essential for ovulation. Two recent studies have supported the above-mentioned findings: in the first, seven components of the extracellular matrix were detected by proteomic, evolutionary, and experimental analyses, and in the second, the essential role of serum in the process of cumulus expansion in vitro was confirmed. We have previously demonstrated the formation of unique structure of the covalent linkage of HA to HCs of IαI in the expanded gonadotropin-stimulated OCC, as well as interactions with several proteins produced by the cumulus cells: tumor necrosis factor-alpha-induced protein 6, pentraxin 3, and versican. Importantly, deletion of these genes in the mice produces female infertility due to defects in the oocyte-cumulus structure.

Molecular crosstalk between insulin-like growth factors and follicle-stimulating hormone in the regulation of granulosa cell function.

The last phase of folliculogenesis is driven by follicle-stimulating hormone (FSH) and locally produced insulin-like growth factors (IGFs), both essential for forming preovulatory follicles. This review discusses the molecular crosstalk of the FSH and IGF signaling pathways in regulating follicular granulosa cells (GCs) during the antral-to-preovulatory phase. IGFs were considered co-gonadotropins since they amplify FSH actions in GCs. However, this view is not compatible with data showing that FSH requires IGFs to stimulate GCs, that FSH renders GCs sensitive to IGFs, and that FSH signaling interacts with factors downstream of AKT to stimulate GCs. New evidence suggests that FSH and IGF signaling pathways intersect at several levels to regulate gene expression and GC function. FSH and locally produced IGFs form a positive feedback loop essential for preovulatory follicle formation in all species. Understanding the mechanisms by which FSH and IGFs interact to control GC function will help design new interventions to optimize follicle maturation, perfect treatment of ovulatory defects, improve invitro fertilization, and develop new contraceptive approaches.

Analysis of cell-cell interaction between mural granulosa cells and cumulus granulosa cells during ovulation using single-cell RNA sequencing data of mouse ovary.

We investigated the interactions between mural granulosa cells (MGCs) and cumulus granulosa cells (CGCs) during ovulation after the LH surge. We performed clustering, pseudotime, and interactome analyses utilizing reported single-cell RNA sequencing data of mouse ovary at 6 h after eCG-hCG injection. Clustering analysis classified granulosa cells into two distinct populations, MGCs and CGCs. Pseudotime analysis divided granulosa cells into before and after the LH surge, and further divided them into two branches, the ovulatory MGCs and the ovulatory CGCs. Interactome analysis was performed to identify the interactions between MGCs and CGCs. Twenty-six interactions were acting from CGCs toward MGCs, involving ovulation and steroidogenesis. Thirty-six interactions were acting from MGCs toward CGCs, involving hyaluronan synthesis. There were 25 bidirectional interactions, involving the EGFR pathway. In addition, we found three novel interactions: Ephrins-Ephs pathway and Wnt-Lrp6 pathway from CGCs to MGCs, associated with steroidogenesis and lipid transport, respectively, and TGF-β-TGFBR1 pathway from MGCs to CGCs, associated with hyaluronan synthesis. MGCs and CGCs interact with each other in the preovulatory follicle after the LH surge, and their interactions have roles in corpus luteum formation, oocyte maturation, and follicle rupture.

Evaluation of the morphology and development of preantral ovarian follicles in mice submitted to a chronic diet of dietary supplementation with Pereskia aculeata Miller leaves.

This study aimed to investigate the effect of including mouse feed with different concentrations (5, 10, or 20%) of Pereskia aculeata Miller (PAM) leaves on the morphology and development of preantral ovarian follicles and ovarian stromal cell density. The oral toxicity was performed using repeated dose toxicity assays subdivided into experiments of 30 days and 90 days of treatment. After the experiments, the ovaries of each animal were collected and submitted to classical histology. At 30 and 90 days, there was an equivalent percentage of normal, primordial, and developing follicles (P > 0.05) between PAM treatments compared to the control. Regarding the different stages of follicular development, after 90 days, there was a higher percentage (P < 0.05) of developing follicles only in the control group compared to day 30. The PAM 5% treatment was the only one that affected the cell density in the stroma after 90 days of treatment. Thus, we observed that supplementing the diet with P. aculeata did not pose any risk concerning animal consumption; specifically, there were no toxic reproductive effects observed from adding Pereskia aculeata Miller to the mouse diet.

In vitro culture of mechanically isolated murine primary follicles in the presence of human platelet lysate PLTMax.

To develop a system for the culture of murine preantral ovarian follicles using Human Serum Albumin (HSA) and Human Platelet Lysate (PLTMax). Mechanically isolated preantral follicles (N=146) were obtained from Swiss mice and cultured in DMEM:F12 medium for ten days in a 96-well plate with conical bottom. The medium was supplemented with penicillin, streptomycin, and equine chorionic gonadotropin. Additional proteins were tested in 4 test groups: G1: human serum albumin (HSA), G2: human platelet lysate (PLTM), and G3 and G4: HSA + PLTMax at lower and higher concentrations, respectively. Cellular vitality and oocyte morphology were evaluated on day 11 of culture. The highest follicular growth (3.4 fold) was achieved in HSA (G1), while a significantly lower (1.8 fold) growth was achieved in the presence of PLTM (G2, G4) and even further reduced (1.2 fold) when HSA and PLTM were combined (G3). Cellular vitality was close to 70-80% among the four groups, and the highest number of intact oocytes were found in G1. PLTM did not improve follicular development and oocyte maturation compared to HSA but preserved cell vitality.

Access to an outdoor open pack promotes estrus activity in dairy cows.

Dairy cows have a partial preference to access an outdoor deep-bedded pack, but the effects of continuous access to an outdoor area on estrous behaviors has not been studied. Our objective was to investigate if access to an outdoor open deep-bedded pack improves the expression of estrus behaviors. We enrolled 60 lactating Holstein cows directly after calving and followed them each for 12 weeks. Cows were housed in a single freestall pen holding 36 cows at a time, with a dynamic group composition. Half of the cows were randomly assigned to the OUTDOOR treatment; these cows had access to an outdoor open pack via an automated selection gate. INDOOR cows were housed together with OUTDOOR cows but were not allowed outdoor access. All cows were fitted with an automated activity monitor (AAM) 21 ± 3 d before expected calving date. Estrous behaviors (i.e., standing to be mounted and mounting behaviors) were continuously monitored using video, and the intensity of mounting (i.e., the number of standing to be mounted and other mounting behaviors per hour) was calculated per estrus event per cow. Temperature and humidity were monitored by data loggers indoors, and these data were used to calculate the Temperature Humidity Index (THI). Following an alert from the AAM, cows were checked to detect the presence of a dominant preovulatory follicle and an absence of a mature corpus luteum (CL) by rectal ultrasonography following milking, as well as 7 d thereafter to confirm ovulation by the presence of a new CL. A total of 94 estrus events were used in the final analysis. INDOOR cows tended to have a lower mounting intensity with increasing indoor THI, but no such effect was noted for OUTDOOR cows. The total number of cows in estrus positively influenced mounting intensity. OUTDOOR cows had a longer duration of estrus, as measured by the automated monitors, compared with INDOOR cows (12.4 ± 0.7 h versus 9.9 ± 0.8 h). Estrus event number and the total number of cows in estrus were both positively associated with estrus duration. These results indicate that access to an outdoor pack tended to increase mounting behavior for freestall housed dairy cows, especially during periods of elevated THI. We conclude that providing dairy cows access to an outdoor area can help with estrus detection and may thus help improve the reproductive programs on dairy farms.

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.

Uterus histomorphometry of alpacas (Vicugna pacos) in induced luteal phase, with GnRH or Copulation

Background: Alpacas have reproductive traits such as induced ovulation and a higher gestation rate in the left uterine horn. Additionally, low fertility and high embryonic mortality are significant challenges in alpaca breeding. To address these issues, it is essential to study the histological changes occurring in the uterine wall during day 8 of the luteal phase. Aim: This research aims to describe these changes in alpacas with induced ovulation using GnRH or copulation. Methods: The study was conducted on 8 sexually mature fertile alpacas with a preovulatory follicle of ≥ 7 mm. Ovulation was induced with GnRH (G1, n=3) and natural mating’ (G2, n=5), confirmed by ultrasound showing the disappearance of the follicle and formation of the corpus luteum. On day 8 of the luteal phase, samples of the uterine wall from the right and left uterine horns were obtained through a midline laparotomy. A uterine lavage with PBS was also performed, and no embryo-ova was found. The tissues were fixed, processed, and stained with Hematoxylin and Eosin. Measurements of the myometrium and endometrium, the area of the superficial and deep endometrial glands, the height of the gland cells, and the height of the endometrial mucosal cells were performed using microscopy and software. The Measurements were taken in microns (um), from 12 different photographs per animal. Results: In G2 (copulation), greater thickness of the endometrium and myometrium, as well as a larger area of the superficial glands and cell height, were observed (p &lt; 0.005). Additionally, the area of the superficial glands in the left horn was greater compared to the right. Conclusion: The thickness of the myometrium and endometrium, and the area of the superficial glands of the left uterine horn in alpacas during the luteal phase (day 8), induced by copulation (G2), show better development. This research provides new insights into changes in the histomorphometry of the alpacas uterus during this stage and is the first report in this species.

Influence of Ovarian Follicle Sizes and Estrous Signs on Pregnancy Following Progesterone-Based Fixed Time Artificial Insemination in Water Buffaloes

The objectives of the present study were to elucidate the importance of follicle sizes and estrous signs during Controlled Internal Drug Release-Synch-human Chorionic Gonadotropin (CIDR-Synch-hCG) protocol for Fixed Time Artificial Insemination (FTAI) and to evaluate their association with pregnancy in water buffaloes. Data from riverine buffaloes (n = 207) under the CIDR-Synch-hCG protocol were analyzed. Buffaloes were administered with Gonadotropin-Releasing Hormone (GnRH) with insertion of CIDR on Day 0. Prostaglandin (PGF2α) was given on Day 7 with the removal of CIDR. hCG was given on Day 9, and AI was performed on Day 10. Follicle measurements by ultrasonography were done on Days 0, 7, and 10, and follicle sizes on those days were categorized into I, II, and III. Estrus signs were taken on the day of AI. The pregnancy diagnosis was done on Day 30-35 post-AI. The average size of follicles in Category III is significantly higher than those of Categories I and II, regardless of the Days of the protocol. Pregnancy is significantly (P&lt;0.001) associated with Pre-Ovulatory Follicle (POF) size and uterine tonicity on the Day of AI but not with follicle sizes on Days 0 and 7, nor with mucus discharge discharge (P&gt;0.05). The overall pregnancy rate is 44.44% while performing AI with POF size ≥12.0 mm increased the probability of pregnancy rate to 56.25%. In conclusion, the present study demonstrated a follicle size-based CIDR-Synch-hCG protocol providing new fertility indicators to improve FTAI efficiency in buffaloes with huge application in other livestock species.