Effects of Nerve Growth Factor-β From Bull Seminal Plasma on Steroidogenesis and Angiogenic Markers of the Bovine Pre-ovulatory Follicle Wall Cell Culture

Nerve growth factor-β (NGF) is critical for ovulation in the mammalian ovary and is luteotrophic when administered systemically to camelids and cattle. This study aimed to assess the direct effects of purified bovine NGF on steroidogenesis and angiogenic markers in the bovine pre-ovulatory follicle. Holstein heifers (n = 2) were synchronized with a standard protocol, and heifers with a preovulatory follicle (≥ 12 mm) had the ovary containing the dominant follicle removed via colpotomy. Pre-ovulatory follicles were dissected into 24 pieces containing theca and granulosa cells that were randomly allocated into culture media supplemented with either purified bovine NGF (100 ng/mL) or untreated (control) for 72 h. The supernatant media was harvested for quantification of progesterone, testosterone, and estradiol concentrations, whereas explants were subjected to mRNA analyses to assess expression of steroidogenic and angiogenic markers. Treatment of follicle wall pieces with NGF upregulated gene expression of steroidogenic enzyme HDS17B (P = 0.04) and increased testosterone production (P < 0.01). However, NGF treatment did not alter production of progesterone (P = 0.81) or estradiol (P = 0.14). Consistently, gene expression of steroidogenic enzymes responsible for producing these hormones (STAR, CYP11A1, HSD3B, CYP17A1, CYP19A1) were unaffected by NGF treatment (P ≥ 0.31). Treatment with NGF downregulated gene expression of the angiogenic enzyme FGF2 (P = 0.02) but did not alter PGES (P = 0.63), VEGFA (P = 0.44), and ESR1 (P = 0.77). Collectively, these results demonstrate that NGF from seminal plasma may interact directly on the theca and granulosa cells of the bovine pre-ovulatory follicle to stimulate testosterone production, which may be secondary to theca cell proliferation. Additionally, decreased FGF2 expression in NGF-treated follicle wall cells suggests hastened onset of follicle wall cellular remodeling that occurs during early luteal development.

Effects of Heat Stress on Follicular Physiology in Dairy Cows

Follicular organization starts during mid-to-late fetal life with the formation of primordial follicles. The bilateral interplay between the oocyte and adjoining somatic cells during follicular growth and ovulation may be sensitive to heat stress (HS). Mechanisms giving rise to pre-ovulatory temperature gradients across reproductive tissues are mostly regulated by the pre-ovulatory follicle, and because the cooling of the gonads and genital tract depends on a counter-current transfer system of heat, HS may be considered a major factor impairing ovulation, fertilization and early embryo development. There is evidence of a long-lasting influence of HS on oogenesis and final follicular maturation. Follicular stages that are susceptible to HS have not been precisely determined. Therefore, the aim of this review was to describe the influence of HS during the staged follicular development in dairy cattle, from the activation of primordial follicles to ovulation. Some clinical prospects are also considered.

Comparison of the effects of Presynch-Ovsynch, Presynch- Ovsynch+CIDR, and G6G protocols on the reproductive performance of lactating Holstein cows in the postpartum period

This study was performed on an industrial dairy farm near Tehran province, Iran, on 666 lactating Holstein cows from September 2019 to June 2020 in order to eliminate the harmful effects of heat stress on the reproductive performance of the cows. The hypothesis of the recent study was that by increasing the serum concentration of progesterone (P4) in the luteal phase before insemination in the form of Presynch-Ovsynch+CIDR (POC) and G6G protocols, the pregnancy rate of cows would be increased compared to the Presynch-Ovsynch (PO) protocol. The aim of the present study was to evaluate the effects of Presynch-Ovsynch (PGF2α-14d- PGF2α-12d-Ovsynch (OVS: GnRH(GnRH1)- 7d-PGF2α(PG)-2d-GnRH)-18h-Fixed-time artificial insemination (FTAI), n=212), Presynch-Ovsynch+CIDR (similar to the PO protocol, plus the use of a CIDR for 7 days, from the start of the OVS+FTAI protocol to the time of PG injection, n=230), and G6G (PGF2α- 2d-GnRH-6d-OVS+FTAI, n=224) protocols on the reproductive performance and pregnancy rate of lactating Holstein cows. The average body condition score (BCS) for the cows was about 2.5 at the time of the onset of the OVS+FTAI program. The average daily milk production for cows at the time of the start of the OVS+FTAI program was 38.3 kg/day. On average, the studied cows were inseminated 86 days postpartum. Implementation of the G6G protocol in comparison to the PO and POC protocols increased the serum concentration of P4 at GnRH1 (P=0.04). The cows that received the G6G protocol had a greater number of corpuses luteum (CL) on their ovaries at PG in comparison to the cows in the PO and POC protocols (P=0.03 and P=0.05, respectively). For all treatment protocols, the pregnancy rate of cows with >2 CLs on their ovaries at PG was significantly higher than for cows with ≤2 CLs on their ovaries at this time. The overall pregnancy rate for all cows studied was 42%. Separately, pregnancy rates for cows in PO, POC, and G6G protocols were calculated at 36.7%, 41.7%, and 47.3% respectively. The highest and the lowest pregnancy rates were obtained in the G6G and PO protocols, respectively (P=0.03). It can be concluded that the G6G protocol increased the pregnancy rate of cows in comparison to the PO but not to the POC protocol. This result can be attributed to the increase in serum concentration of P4 at GnRH1 and the greater number of CLs at PG following implementation of the G6G compared to the PO protocol. Although CIDR administration in the POC protocol led to an increase in the pregnancy rate of cows compared to the PO protocol, possibly by increasing the synchrony rate and the quality of the ovulatory follicle, this increase was not statistically significant (P=0.09).

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

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

EFFECT OF THE ADMINISTRATION OF SERUM AND RECOMBINANT EQUINE CHORIONIC GONADOTROPIN ON OVARIAN DYNAMICS AND CONCEPTION RATE IN ANESTRUS PRIMIPAROUS BRANGUS COWS

The objective was to determine the size of the dominant ovulatory follicle, the ovulation time and the conception rate of cows in anestrus treated with serum equine chorionic gonadotropin (eCG), recombinant chorionic gonadotropin and untreated controls. 57 anestrus primiparous Brangus cows were used with 70 ± 26 days of calving and a body condition of 2.50 ± 0.15. They were synchronized with a FTAI protocol based on estrogen and progesterone devices (DI). Upon removal of DI, 3 groups were made according to the type of eCG applied: serum eCG received 400 IU im of equine Chorionic Gonadotropin, recombinant eCG received 140 IU of recombinant Equine Chorionic Gonadotrophin and Control did without treatment. Follicular size and time of ovulation (normal or abnormal) was determined at DI removal, during FTAI and 7 days later. An ANOVA was performed to determine the effect of the treatment on ovarian dynamics and a chi-square test and correspondence analysis to establish associations. In normal ovulation cows at the FTAI, 94.4% of the serum eCG, 55.5% of the recombinant eCG and 71.4% of the Control cows presented a dominant ovulatory follicle (DOF), which in those treated with serum eCG, it was 1.4 mm greater in relation to the Control (P = 0.0073). The ovulation rate, in normal and abnormal ovulation cows, was 94.4% for serum eCG, and for recombinant eCG and 66.6% for Controls. A significant association was detected between ovulation time and the treated groups (P = 0.0042). Normal ovulation was 38.9% higher in cows with serum eCG in relation to those treated with recombinant eCG and 50.8% in comparison with Control. A significant relationship was observed between the conception rate and the groups treated with the different Gonadotropins (P = 0.0574), being 66.7% in serum eCG, 50% in recombinant eCG and 28.6% in the Control. The use of eCG, either serum or recombinant, in anestrus primiparous cows, stimulates the development of DOF at FTAI, increases ovulation rate and improves conception in relation to untreated cows.

The Proteome of Equine Oviductal Fluid Varies Before and After Ovulation: A Comparative Study

Equine fertilization cannot be performed in the laboratory as equine spermatozoa do not cross the oocyte's zona pellucida in vitro. Hence, a more profound study of equine oviductal fluid (OF) composition at the pre-ovulatory and post-ovulatory stages could help in understanding what components are required to achieve fertilization in horses. Our work aimed to elucidate the proteomic composition of equine OF at both stages. To do this, OF was obtained postmortem from oviducts of slaughtered mares ipsilateral to a pre-ovulatory follicle (n = 4) or a recent ovulation (n = 4); the samples were kept at −80°C until analysis. After protein extraction and isobaric tags for relative and absolute quantification (iTRAQ) labeling, the samples were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The analysis of the spectra resulted in the identification of a total of 1,173 proteins present in pre-ovulatory and post-ovulatory samples; among these, 691 were unique for Equus caballus. Proteins from post-ovulatory oviductal fluid were compared with the proteins from pre-ovulatory oviductal fluid and were categorized as upregulated (positive log fold change) or downregulated (negative log fold change). Fifteen proteins were found to be downregulated in the post-ovulatory fluid and 156 were upregulated in the post-ovulatory OF compared to the pre-ovulatory fluid; among the upregulated proteins, 87 were included in the metabolism of proteins pathway. The identified proteins were related to sperm–oviduct interaction, fertilization, and metabolism, among others. Our data reveal consistent differences in the proteome of equine OF prior to and after ovulation, helping to increase our understanding in the factors that promote fertilization and early embryo development in horses.

Immunological aspects of follicle ovulation and corpus luteum formation in cattle

Ovulation has been described as an inflammatory event, characterized by an influx of leukocytes into the ovulatory follicle and changes in the expression of immune factors in both the theca and granulosa tissue layers. Since information on this process is limited in cattle, our objective was to elucidate the contribution of the immune system to dominant follicle luteinization, ovulation and corpus luteum formation in cattle. Beef heifers (n=50) were oestrous synchronized, slaughtered and ovarian follicular or luteal tissue collected during a 96h window around ovulation. Follicular fluid cytokine concentration, temporal immune cell infiltration and inflammatory status were determined by Luminex multiplex analysis, immunohistochemistry and quantitative real time PCR-analysis, respectively, in pre- and peri-ovulatory follicular tissues. The concentrations of CXCL10 and VEGF-A were highest in pre-ovulatory follicular fluid samples. The pre and peri -ovulatory follicles play host to a broad repertoire of immune cells, including T-cells, granulocytes and monocytes. Dendritic cells were the most abundant cells in ovulatory follicular and luteal -tissue at all times. The mRNA expression of candidate genes associated with inflammation was highest in pre- and peri-ovulatory tissue, whereas tissue growth and modelling factors were highest in the post-ovulatory follicular and early luteal tissue. In conclusion, ovulation in cattle is characterized by the presence of neutrophils, macrophages and dendritic cells in the ovulatory follicle, reflected in compartmentalized cytokine and growth factor expression. These findings indicate a tightly regulated sterile inflammatory response to the LH surge in the ovulatory follicle which is rapidly resolved during early corpus luteum formation.

Hormonal Levels and Follicular Dynamics in Relation to the Oestrous Cycle in Barb and Arabian Mares, Algeria

This current study is an effort to understand the hormonal and follicular growth in the Barb and Arabian mares during the oestrous cycle; as mares are unique creatures. A total of 53 mares with 97 oestrous cycles were studied. The mares with a mean age of 10.38 ± 4.55 were examined by ultrasonography every day during their breeding season (2017). Two blood samples from each mare (n = 24) were obtained for progesterone (P4), oestrogen (oestradiol-17 beta) and follicle-stimulating hormone (FSH) determinations. The data revealed that the duration of the oestrous cycle was between 19 to 22 days. The pre-ovulatory follicle grew (3.02 ± 1.04) millimetre per day. The rate of cycles exploited in the mare (Arabian versus Barb) for conception was significantly different (P < 0.001). The maximal diameter of the follicle was 50.00 millimetre. The serum progesterone levels (P < 0.01) in mares were significantly higher in the luteal phase than those recorded during the time of oestrous. However, the levels of oestradiol and for FSH did not significantly change during the oestrous cycle in the mares. Determining the association between the size of the follicle and the hormone profiles were the most reliable criterion in the prediction of ovulation.