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Metabolic stress conditions caused by negative energy balance (NEB) have been associated with reduced fertility in cows. β-hydroxybutyrate (BHBA) is the main circulating ketone body, which accumulates within follicular fluid. The aim of this study was to evaluate the effects of BHBA on follicle growth and on ovulatory mechanisms in cattle. At 72 h after intrafollicular injection, there was a decrease in follicular diameter in BHBA group compared to control (P = 0.02). Furthermore, follicle growth rate was reduced post-treatment with BHBA in comparison to the control group (P 0.03). The BHBA intrafollicular injection in follicles ≥ 12 mm, however, did not affect E2 and P4 concentrations in the follicular fluid. In addition, the relative abundance of genes involved in the ovulatory cascade (ADAM 17, AREG, EREG, PTGS2), steroidogenesis (CYP19A1, 3BHSD, STAR), cellular stress (SOD1, CAT, GPX1, HSPA5, XBP1s, XBP1u, ATF4, ATF6), monocarboxylic acid transporters (SLC16A1, SLC16A7) and apoptosis (XIAP) was similar between groups. In conclusion, the results of this study indicate that the increase in intrafollicular concentrations of BHBA affects follicular growth, but it does not compromise the ovulatory cascade and cellular homeostasis in bovine granulosa cells.

There is growing evidence that greater than homeostatic blood concentrations of nonesterified fatty acids (NEFAs) and β-hydroxybutyrate (BHBA) have negative consequences on dairy cow’s fertility, but effects on cell homeostasis in the reproductive system is not completely understood. In this study, lipids accumulation, reactive oxygen species (ROS) concentrations, abundance of gene transcripts, and immunofluorescence signal of H3K4me3 and H3K9me3 were evaluated in endometrial epithelial cells of cattle cultured with NEFAs (Oleic (OA), Stearic (SA) and Palmitic (PA) acids), BHBA, NEFAs + BHBA or each of the three NEFAs alone. The cellular lipids were in greater concentrations as a result of NEFAs + BHBA, NEFAs, SA or OA supplementation, but not by BHBA or PA. The ROS concentrations were greater when there were treatments with NEFAs + BHBA, NEFAs or BHBA. The relative mRNA abundance for genes involved in the regulation of apoptosis (XIAP), glucose transport (GLUT3), and DNA methylation (DNMT1) were greater when there were NEFAs + BHBA, but not NEFAs, BHBA, OA, SA or PA treatments. The immunofluorescence signal for H3K9me3 was greater when there were NEFAs + BHBA, NEFAs or PA, but not by BHBA, OA or SA treatments. These findings indicate that NEFAs and BHBA have an additive effect on endometrial cells of cattle by altering epigenetic markers and the expression of genes controlling important cellular pathways. Furthermore, there was cellular lipid accumulation and increased H3K9me3 in cultured bovine endometrial cells that was mainly induced by OA and PA treatments, respectively.

Although it is well documented that leptin signals the body nutritional status to the brain, mechanisms of leptin regulation at the ovary are not well understood. This study was conducted to determine whether there was leptin and the receptor for leptin (LEPR) in cattle ovarian follicles and to investigate potential actions of leptin on follicular growth in vivo and on regulation of granulosa cell functions in vitro. There was leptin and LEPR in granulosa and theca cells of dominant and subordinate follicles, with greater immunostaining for leptin in granulosa cells of subordinate follicles. There was a lesser relative abundance of leptin receptor gene-related protein (LEPROT) and of the adiponectin receptors 1 (ADIPOR1) and 2 (ADIPOR2) mRNA transcripts in granulosa cells of subordinate than dominant follicles (P0.05). Intrafollicular injection of either 100 or 1000 ng/mL leptin did not affect the diameter and the growth of dominant follicles (P0.05). Supplementation of in vitro culture medium with different leptin concentations did not affect (P0.05) the relative abundance of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), signal transducer and activator of transcription 3 (STAT3) and X-linked inhibitor of apoptosis protein (XIAP) mRNA transcripts in granulosa cells. These findings indicate that leptin and LEPR are present in the follicular cells of cattle ovaries, but leptin apparently does not have essential functions in steroidogenesis and growth of dominant follicles.

Dairy cows frequently undergo a state of negative energy balance (NEB) after parturition and some have impaired ovarian functions that result in delayed resumption of estrous cyclicity and development of follicles without ovulation occurring. During the postpartum period, cows undergo body-fat store losses, hormonal changes, fat mobilization and increases in nonesterified fatty acid (NEFAs) concentrations in blood and follicular fluid. The effect of NEFAs on follicular development and function of follicular cells, however, is not fully understood. The aim of this study, therefore, was to study the effect of an intrafollicular injection of a mixture of oleic, stearic and palmitic NEFAs on dominant follicle development and function of granulosa cells in cows that were not in a NEB state. Follicular size was less at 24 and 48 h after administration of NEFAs compared to that of control follicles injected with vehicle only. At 24 h after intrafollicular injection, the relative mRNA transcript abundance for proteins involved in steroidogenesis (CYP19A1, 3BHSD, STAR, FSHR), metabolism (GLUT1, GLUT3, INSR, IRS1, IRS2, SLC27A1, PPARG), and cell proliferation and apoptosis (CCND2; XIAP) in granulosa cells, as well as estradiol concentrations in follicular fluid were similar in control and NEFA-treated follicles. In conclusion, the results of this study indicate increased intrafollicular concentrations of NEFAs in cows that are not in a NEB state has a detrimental effect on follicle development. We propose intrafollicular injection is a useful approach to further investigate the local effects of NEFAs on the function of follicular cells.

The aim of the present study was to evaluate the expression of mRNA encoding natriuretic peptides (NPs) and their receptors in the cumulus-oocyte complex in cattle, a monovular mammalian species, and also to investigate the role of NPs in oocyte meiotic resumption in vitro. mRNA was observed for the NP precursor type-A (NPPA), type-C (NPPC), NP receptor-1 (NPR-1), receptor-2 (NPR-2) and receptor-3 (NPR-3) in bovine cumulus cells, and NPR-2 mRNA was observed in oocytes. These results are different from those obtained in mouse and pig models. The effects of NPPA, NP precursor type-B (NPPB) and NPPC on the resumption of arrested meiosis maintained by forskolin were studied at three different doses (10, 100 and 1000nM) with a 12h culture system. The germinal vesicle breakdown rates were greater (P≤0.05) in oocytes that were cultured in the presence of one or a combination of NPs (from 44% to 73%) than the negative control (from 24% to 27%). Additionally, it was demonstrated that the concentration of cyclic guanosine 3',5'-monophosphate (cGMP) is increased by NPPA and NPPC in oocytes and cumulus cells after 3h of in vitro maturation. However, in both groups, the concentration of cyclic adenosine 3',5'-monophosphate (cAMP) in the oocyte did not increase between 3 and 6h of culture, even when forskolin was used. In summary, we observed the presence of mRNA for NPs and their receptors in the bovine cumulus-oocyte complex and demonstrated that, in vitro, NPPA, NPPB and NPPC stimulate oocyte meiotic resumption in a monovular species.

Bone morphogenetic proteins are known to be involved in determining ovulation rate in mammals. The mechanisms through which these proteins determine follicle fate are incompletely understood. In the present study, we used cattle as a model to evaluate the regulation of BMP15 and GDF9 receptors in granulosa cells during dominant follicle (DF) selection. Before follicular deviation (day 2 of the follicular wave), BMPR2 mRNA abundance tended to be higher in the second largest follicles (F2; P0.1) compared to the future dominant follicle (F1). At the expected time of follicular deviation (day 3), BMPR2 and BMPR1B mRNA levels were higher in subordinate follicles (SFs; P0.05) compared to dominant follicles (DFs). After deviation (on day 4), BMPR1B mRNA and protein were significantly more abundant in atretic SFs (as assessed by cleaved caspase 3) than in DFs. The fact that BMPR1B is more expressed in atretic follicles was further confirmed by using intrafollicular treatment with two agents known to induce atresia, namely an estradiol receptor antagonist (fulvestrant) and FGF10. In conclusion, the fact that BMPR-1B and -2 are more expressed in the second largest follicles before and at the expected time of follicular deviation is indicative of their inhibitory role in follicle differentiation and steroidogenesis. BMPR1B also seems to have a pivotal role during follicle regression since it is upregulated in advanced atretic follicles.

The aims of the present study were to determine the role of protein kinase C (PKC) on meiotic resumption and its effects on pronuclear formation and cleavage in the bovine. Oocytes were matured in the presence of 0, 1, 10 and 100 nM of phorbol 12-myristate 13-acetate (PMA), to evaluate the percentage of germinal vesicle breakdown. To study pronuclear formation and cleavage, oocytes were randomly distributed in four groups and matured in modified TCM-199 with LH and FSH (negative control); 10% of estrous cow serum (positive control); 100 nM of PMA (treatment); 100 nM of 4alpha-PDD (phorbol ester control). Oocytes were also matured in positive control medium, fertilized and transferred to KSOM with increasing concentrations of a PKC inhibitor. The protein profile and the presence of PKC at the end of maturation period were determined by SDS-PAGE followed by Silver Stain and Western blot, respectively. PMA stimulated meiotic resumption in a concentration-dependent manner. PKC stimulation during oocyte maturation caused an increase in pronuclear formation and did not cause parthenogenetic activation. Inhibitor of PKC (MyrPKC) inhibited cleavage in a dose-dependent and irreversible manner. A protein band around 74 kDa was not detected in PMA-treated oocytes and PKC was not detected by Western blot at the end of the maturation period. In conclusion, meiotic resumption was accelerated and the rate of oocytes with two pronuclei was increased when PKC was activated during oocyte maturation. Moreover, cleavage was inhibited in the presence of PMA.

Semen of four dairy bulls was inactivated by centrifugation twice through a 7% Ficoll solution. After the second centrifugation, the sperm fractions were resuspended in 14G buffer and stored for 0, 3, 6 and 9 days at room temperature. At the end of each storage period, inactivated semen was reactivated by centrifugation and the sperm plugs were resuspended to a final concentration of 400 × 106 sperm ml−1 in tris-based diluent supplemented with 5 mM caffeine or theophylline. Sperm motility and acrosome status were assessed during subsequent incubation. Sperm penetration into cervical mucus was also assessed. The fertilizing ability of the sperm was tested by using the zona-free hamster oocyte technique. The results showed that bovine semen inactivated by centrifugation through a 7% Ficoll solution could be stored beyond 9 days at room temperature. Satisfactory proportions of motile sperm and spermatozoa with intact acrosomes were recovered after re-activation. The inclusion of either caffeine or theophylline at a 5 mM level had a beneficial effect on the percentage of motile sperm (P

The aim of the present study was to characterize ovarian follicular development and steroid concentrations during postpartum and the estrous cycle of Brangus Ibagé cows (3/8 Nelore + 5/8 Aberdeen Angus) with different levels of fertility. Cows were classified as having high or low fertility according to the calving interval (CI). The average CI of the herd from which cows used in this study were selected was 404.6+/-5.44 and 711.2+/-20.89 days for the high and low fertility groups, respectively. Four cows of high fertility and five cows of low fertility had calves removed between 70 and 100 days after parturition. Ovarian activity was monitored daily by ultrasound for 16 days after calf removal. Days to emergency of the first follicular wave after calf removal, number of follicles with diameter9 mm, growth rate of largest follicle, maximum diameter of largest follicle, length (days) and number of follicular waves were recorded. During this period, blood was collected daily for measurements of serum progesterone (P(4)) and estradiol (E(2)) concentrations. In another experiment, ovarian activity and P(4) and E(2) concentrations were examined during estrous cycle in five cows of high fertility and four cows of low fertility. Ovarian activity and steroid concentrations were assessed from the day prior to estrus to the 15th day of the estrous cycle (estrus = day 0). In postpartum cows of high fertility, the total number of follicles5mm and the maximum diameter of the largest follicle were higher than in cows of low fertility (P0.05). Concentrations of P(4) and E(2) did not differ between groups in the postpartum cows. However, E(2) increased 5 days after calf removal (around 90 days of postpartum) in the high fertility group, followed by an increase in P(4) with average values indicating ovulation around 100 days postpartum. In cycling cows, the profile of follicular development was similar between cows of high and low fertility. There was no difference between groups for number of follicles5mm, but the day effect was significant (P0.01). Plasma concentrations of P(4) and E(2) were similar in both groups. These data suggest that cows, from a population raised in the same environment have different fertility as a consequence of individual physiological characteristics.

The present work investigated the effect of the interval of serial sections of ovarian tissue on the number of isolated preantral follicles in the goat. Goat ovaries were cut in the tissue chopper at eight different intervals. The quality of isolated follicles were evaluated by histology and transmission electron microscopy. Best results were obtained when the ovaries were cut in the tissue chopper at intervals of 75.0 microm (9664 preantral follicles per ovary). Histochemical and ultrastructural analysis showed that the follicular morphology was preserved after mechanical isolation as demonstrated by the normality of oocytes and granulosa cells as well as by preservation of basement membrane. The percentages of isolated primordial, primary and secondary follicles were 96.3%, 2.5%, and 1.2% and their average diameters were 21.5, 34.7 and 65.3 microm, respectively. It was concluded that the interval of serial sections of ovarian tissue in the tissue chopper affects the number of isolated preantral follicles, and that the follicles remained intact after mechanical isolation in goats.