Motility, oxidative status and morphology of frozen-thawed bovine semen are not impacted by fatty acid exposure in vitro.

While sperm migrate within the reproductive tract of cows experiencing negative energy balance (NEB), they come into contact with elevated concentrations of non-esterified fatty acids (NEFA). For this reason, this study aimed to investigate the effects of three different NEFA - palmitic acid (PA), stearic acid (SA), and oleic acid (OA) - on bovine sperm motility, kinetic parameters, oxidative status, and morphology. Frozen thawed semen samples from Bos taurus bulls were incubated with varying concentrations of each fatty acid, and the sperm's characteristics were analysed at different time points. Computer-Assisted Sperm Analysis (CASA) was employed to assess sperm motility and kinetic parameters. Concurrently, the production of the reactive oxygen species (ROS) and total antioxidant capacity were measured to determine the oxidative status. Additionally, sperm morphology was evaluated. In Experiment 1, different concentrations of PA did not show significant effects on total motility, progressive motility, or any kinetic parameters analysed. Similarly, PA did not have a significant impact on the oxidative status or sperm morphology. In Experiment 2, SA at various concentrations did not lead to significant changes in total motility, progressive motility, or any kinetic parameters evaluated. Furthermore, SA did not affect oxidative status or sperm morphology. In Experiment 3, the concentrations of OA used did not result in significant changes in total motility, progressive motility, or any kinetic parameters studied. Likewise, OA did not induce any alterations in oxidative status or sperm morphology. Overall, the results from all three experiments indicate that PA, SA and OA, at the in vitro conditions and tested concentrations, do not exert detrimental effects on bovine sperm function and morphology. These results provide insights that contribute to our understanding of how fatty acids can impact the reduction of fertility rates in cows facing NEB. This, in turn, lays the foundation for additional critical investigations in this area. Further studies are necessary to validate these findings in vivo.

Vitamin E reduces the reactive oxygen species production in dominant follicle during the negative energy balance in cattle.

In the postpartum period, there is an increase in non-esterified fatty acids (NEFA) in both serum and follicular fluid (FF) of cattle. The increase in fatty acid concentration results in increased production of reactive oxygen species (ROS) that can compromise bovine fertility. The objectives of this study were to characterize the lipid profile found in the FF of cows experiencing induced negative energy balance (NEB) and to evaluate the effect of α-tocopherol in the prevention of oxidative stress in the serum and FF of cows. Twenty-nine beef cows were divided into groups: (1) control; (2) Fasting for 24 days; and (3) Fasting + VitE. Between D0 and D4 blood samples were taken to assess concentrations of NEFA, ROS production, total antioxidant capacity (FRAP), lipid peroxidation, and α-tocopherol (vitamin E). On D4, follicular aspiration was performed for analysis of FF from the dominant follicle. Our results demonstrate that fasting was effective in causing increased fat mobilization in animals. The increase in serum concentration of C18:1c9 was reflected in the FF of fasting cows. Serum α-tocopherol concentration was higher in the control and Fasting + VitE groups compared to the Fasting group. In FF, there was an increase of α-tocopherol in the Fasting + VitE group in comparison to Fasting cows. There was an increase in ROS production in the serum of fasting cows. ROS production in FF was higher in the Fasting compared to the Fasting + VitE group. Vitamin E has beneficial effects in reducing ROS production in the dominant follicle of cows in NEB.

Analysis of nuclear maturation, DNA damage and repair gene expression of bovine oocyte and cumulus cells submitted to ionizing radiation.

Radiotherapy causes destruction of tumor cells, but also threatens the integrity and survival of surrounding normal cells. Then, woman submitted to irradiation for cancer treatment may present permanent ovary damage, resulting in impaired fertility. The objective of this study was to investigate the effects of therapeutic doses of ionizing radiation (IR), used for ovarian cancer treatment in humans, on bovine cumulus-oocyte complexes (COCs) as experimental model. Bovine ovaries were exposed to 0.9 Gy, 1.8 Gy, 3.6 Gy or 18.6 Gy IR, and then COCs were collected and used to evaluate: (a) oocyte nuclear maturation; (b) presence of phosphorylated H2A.X (γH2AX), as an indicator of DNA double-strand breaks (DSBs); and (c) expression of genes involved in DNA repair (TP53BP1, RAD52, ATM, XRCC6 and XRCC5) and apoptosis (BAX). The radiation doses tested in this study had no detrimental effects on nuclear maturation and did not increase γH2AX in the oocytes. However, IR treatment altered the mRNA abundance of RAD52 (RAD52 homolog, DNA repair protein) and BAX (BCL2-associated X protein). We conclude that although IR doses had no apparent effect on oocyte nuclear maturation and DNA damage, molecular pathways involved in DNA repair and apoptosis were affected by IR exposure in cumulus cells.

FSH Regulates YAP-TEAD Transcriptional Activity in Bovine Granulosa Cells to Allow the Future Dominant Follicle to Exert Its Augmented Estrogenic Capacity.

The molecular mechanisms that drive the granulosa cells' (GC) differentiation into a more estrogenic phenotype during follicular divergence and establishment of follicle dominance have not been completely elucidated. The main Hippo signaling effector, YAP, has, however, emerged as a potential key player to explain such complex processes. Studies using rat and bovine GC demonstrate that, in conditions where the expression of the classic YAP-TEAD target gene tissue growth factor (CTGF) is augmented, CYP19A1 expression and activity and, consequently, estradiol (E2) secretion are reduced. These findings led us to hypothesize that, during ovarian follicular divergence in cattle, FSH downregulates YAP-TEAD-dependent transcriptional activity in GC to allow the future dominant follicle to exert its augmented estrogenic capacity. To address this, we performed a series of experiments employing distinct bovine models. Our in vitro and ex vivo experiments indicated that indeed FSH downregulates, in a concentration-dependent manner, mRNA levels not only for CTGF but also for the other classic YAP-TEAD transcriptional target genes ANKRD1 and CYR61 by a mechanism that involves increased YAP phosphorylation. To better elucidate the functional importance of such FSH-induced YAP activity regulation, we then cultured GC in the presence of verteporfin (VP) or peptide 17 (P17), two pharmacological inhibitors known to interfere with YAP binding to TEADs. The results showed that both VP and P17 increased CYP19A1 basal mRNA levels in a concentration-dependent manner. Most interestingly, by using GC samples obtained in vivo from dominant vs. subordinate follicles, we found that mRNA levels for CTGF, CYR61, and ANKRD1 are higher in subordinate follicles following the follicular divergence. Taken together, our novel results demonstrate that YAP transcriptional activity is regulated in bovine granulosa cells to allow the increased estrogenic capacity of the selected dominant follicle.

Increased ß-hydroxybutyrate (BHBA) concentration affect follicular growth in cattle.

Metabolic stress conditions caused by negative energy balance (NEB) have been associated with reduced fertility in cows. ß-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.

The Hippo pathway effectors YAP and TAZ interact with EGF-like signaling to regulate expansion-related events in bovine cumulus cells in vitro.

PURPOSE: To determine if the inhibition of the interaction between the Hippo effector YAP or its transcriptional co-activator TAZ with the TEAD family of transcription factors is critical for the cumulus expansion-related events induced by the EGF network in cumulus-oocyte complexes (COCs). METHODS: We performed a series of experiments using immature bovine COCs subjected to an IVM protocol for up 24 h in which cumulus expansion was stimulated with EGF recombinant protein or FSH. RESULTS: The main results indicated that EGFR activity stimulation in bovine cumulus cells (CC) increases mRNA levels encoding the classic YAP/TAZ-TEAD target gene CTGF. To determine if important genes for cumulus expansion are transcriptional targets of YAP/TAZ-TEAD interaction in CC, COCs were then subjected to IVM in the presence of FSH with or without distinct concentrations of Verteporfin (VP; a small molecule inhibitor that interferes with YAP/TAZ binding to TEADs). COCs were then collected at 6, 12, 18, and 24 h for total RNA extraction and RT-qPCR analyses. This experiment indicated that VP inhibits in a time- and concentration-dependent manner distinct cumulus expansion and oocyte maturation-related genes, by regulating EGFR and CTGF expression in CC. CONCLUSIONS: Taken together, the results presented herein represent considerable insight into the functional relevance of a completely novel signaling pathway underlying cumulus expansion and oocyte maturation in monovulatory species. YAP/TAZ or CTGF may represent potential targets to improve the efficiency of IVM systems, not only for monovulatory species of agricultural importance as the cow, but for human embryo production.

Local regulation of antral follicle development and ovulation in monovulatory species.

The identification of mutations in the genes encoding bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) associated with phenotypes of sterility or increased ovulation rate in sheep aroused interest in the study of the role of local factors in preantral and antral folliculogenesis in different species. An additive mutation in the BMP15 receptor, BMPR1b, which determines an increase in the ovulatory rate, has been introduced in several sheep breeds to increase the number of lambs born. Although these mutations indicate extremely relevant functions of these factors, the literature data on the regulation of the expression and function of these proteins and their receptors are very controversial, possibly due to differences in experimental models. The present review discusses the published data and preliminary results obtained by our group on the participation of local factors in the selection of the dominant follicle, ovulation, and follicular atresia in cattle, focusing on transforming growth factors beta and their receptors. The study of the expression pattern and the functionality of proteins produced by follicular cells and their receptors will allow increasing the knowledge about this local system, known to be involved in ovarian physiopathology and with the potential to promote contraception or increase the ovulation rate in mammals.

High concentrations of ß-hydroxybutyrate alter the kinetics of bovine spermatozoa.

Postpartum cows, mainly with metabolic diseases, such as ketosis, usually experience an increased number of services per conception. During ketosis, high concentrations of ß-hydroxybutyrate (BHBA) in follicular, uterine and oviductal fluid have been considered to cause subfertility in cows. However, the effect of sperm exposure to an environment with high BHBA concentration is not known. This study investigated the influence of high levels of BHBA on kinetics, oxidative status and morphology of bovine spermatozoa. To assess the effect of BHBA after sperm selection, bovine spermatozoa were incubated (180 min) with different BHBA concentrations: 0 (Control), 0.8, 2.4 or 5 mM. Sperm kinetics was evaluated after 30, 60, 120 and 180 min, and oxidative status and morphology were analysed at 180 min. Oxidative status was evaluated through the production of reactive oxidative species (ROS), total antioxidant capacity and lipid peroxidation. High concentrations of BHBA decreased the curvilinear velocity, straight line velocity, mean path velocity, linearity, straightness and hyperactivity of spermatozoa. However, there was no effect of BHBA on oxidative and antioxidant capacity as well as on sperm morphology. In conclusion, exposure of bovine spermatozoa to high levels of BHBA impairs sperm kinetics without altering oxidative and antioxidant mechanisms.

Cushioned centrifugation during sperm selection increases the fertilization and cleavage rates of cattle embryos produced in vitro.

This study was conducted to evaluate the effect of utilization of an iodixanol-based solution as a cushioning method during the sperm selection utilizing discontinuous Percoll gradient centrifugation in in vitro production (IVP) of cattle embryos. In Experiment I, all aliquots of thawed semen were subjected to sperm selection using the same discontinuous Percoll® gradients, except for the following four conditions: presence of cushioning solution (Cushion Fluid, Minitube) during the first centrifugation process (C1), presence of cushioning solution during the second centrifugation process (C2), inclusion of cushioning solution in both centrifugation steps (C1-2), and no addiction of cushioning solution (C; control group). Recovery rates, sperm kinetics, and reactive oxygen species (ROS) production were evaluated. In Experiment II, sperm cells were processed using sperm selection conditions C and C1, and fertilization rates and embryonic development kinetics were compared between experimental groups. With use of condition C1, there was improvement in fertilization and cleavage rates when compared to use of condition C (56.4% compared with 45.5% and 80.0% compared 64.7%, respectively). In conclusion, results indicate the use of a cushioning solution during sperm selection positively affects the developmental potential of embryos.

Intrafollicular injection of nonesterified fatty acids impaired dominant follicle growth in cattle.

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.

Decreasing the dose of equine chorionic gonadotropin does not affect ovarian or pregnancy responses of purebred taurine and crossbred beef heifers.

In this study there was evaluation of effects of different doses of equine chorionic gonadotropin (eCG: 200, 300, or 400 IU) administrated at progesterone (P4) plus estradiol-based timed AI (TAI). A total of 1080 heifers were included in the study. There was insertion of the intravaginal P4-device plus administration of 2 mg of estradiol benzoate IM. On D7, 12.5 mg of dinoprost tromethamine IM was administered and on D9, the P4 insert was removed and 0.5 mg of estradiol cypionate IM was administered. Heifers were categorized according to Reproductive Tract Status (RTS; 1-5) and were assigned to one of three treatments: 200 IU (n = 387), 300 IU (n = 357), or 400 IU (n = 336) of eCG. Estrous occurrence was evaluated at TAI 48 h later (D11). A subset of heifers (n = 213) had the largest follicle (LF) evaluated on D9 and on D11, and the formation of a new CL evaluated on D18.There was no effect of eCG treatment on LF on D11 (P = 0.79), occurrence of estrus (P = 0.92), and pregnancy at 30 days after AI (P/AI; 52.2%, 49.8%, and 51.5% for 200 IU, 300 IU, and 400 IU, respectively; P = 0.46). Regardless of the treatment, there was a greater P/AI when heifers had a functional CL, at initiation of the estrous synchronization treatment regimen. It, therefore, is efficacious to reduce the dose of eCG to 300 or 200 IU in purebred taurine and crossbred beef heifers without negative effects on ovarian, estrous or pregnancy responses.

Mercury at environmental relevant levels affects spermatozoa function and fertility capacity in bovine sperm.

Over the last several years human sperm quality was found to be significantly reduced and the role environmental contaminants play in this phenomenon remain to be determined. Mercury (Hg) is one of the most widespread contaminants; however the correlation between metal exposure and adverse consequences on human and animals fertility are not completely established. The aim of this study was to determine the effects of direct exposure to inorganic Hg on male gametes using spermatozoa (bovine sperm) which characteristically resemble human sperm. Sperm were divided and incubated for 0.5, 1 or 2 h at low levels of Hg: i) Control: without exposure; ii) Hg8 nM: mercury chloride (HgCl2) at 8 nM and iii) Hg8 µM: HgCl2 at 8 µM. Sperm kinetics, morphology, sperm membrane integrity, and in vitro fertilization were assessed. In addition the levels of reactive oxygen species (ROS), lipid peroxidation and total antioxidant capacity were measured. Hg exposure for 2 h impaired sperm morphology and membrane integrity as well as kinetic parameters including curvilinear velocity and straight-line velocity, which are needed for fertilization as evidenced by the reduced fertilization rate in 8 µM Hg-treated gametes. Hg enhanced oxidative stress in male sperm as reflected by elevated levels of ROS and lipid peroxidation and decreased antioxidant capacity. Data demonstrated that low levels of Hg when incubated with spermatozoa are sufficient to increase oxidative stress, adversely affect sperm quality parameters, subsequently impairing sperm fertility capacity.

Effect of γ-oryzanol on testicular degeneration induced by scrotal insulation in rams.

The present study assessed the effects of daily supplementation with 33 mg/metabolic weight (MW) of γ-oryzanol on testicular degeneration induced by scrotal insulation in rams. Eight animals were divided into two groups: Control (subjected to scrotal insulation without treatment) and Gamma (subjected to scrotal insulation and γ-oryzanol treatment). The rams were subjected to scrotal insulation by covering the scrotum with a thermal bag for 72 h. Animals in the Gamma group received 33 mg/MW oral γ-oryzanol once-daily, beginning 7 days before insulation and continuing during insulation and for 20 days afterward, for a total treatment period of 30 days. Samples of semen and blood were collected during the experiment to perform biochemical evaluations of oxidative stress, seminal kinetics and morphology, and plasma testosterone concentrations. Ultrasound examinations of the testicular parenchyma and clinical evaluations of its consistency and the scrotal perimeter were also performed at weekly intervals. Testicular tissue was collected for biochemical analyses of oxidative stress parameters at the end of the experiment by orchiectomy. The results showed that testicular degeneration was induced by scrotal insulation, as was demonstrated by the reduced scrotal perimeter and increased in testicular flaccidity immediately after insulation. Moreover, a delayed increase in the number of hyperechoic points in the parenchyma and a delayed reduction in sperm motility were observed at 10 weeks after insulation by ultrasonography. Treatment with γ-oryzanol reduced levels of reactive oxygen species (ROS) levels in the testes, and increased the total antioxidant potential (assessed based on the ferric reducing ability (FRAP)) in week 10 and levels of lipid peroxidation (TBARS). It also increased the number of intact spermatozoa in week 3, but increased the total number of sperm defects from week 5 onwards. Although γ-oryzanol protected the semen and testes by reducing the levels of the parameters of oxidative stress evaluated herein, the other parameters studied were not improved by the treatment. In addition, supplementation with γ-oryzanol led to more morphological abnormalities in the sperm. This study presented new information on the oral administration of γ-oryzanol to rams with testicular degeneration, and described potential therapies for this pathology, which currently has no established treatment and has important impacts on reproductive health.

Superstimulation with eCG prior to ovum pick-up improves follicular development and fertilization rate of cattle oocytes.

This study aimed to evaluate the effect of equine chorionic gonadotrophin (eCG) stimulation prior to ovum pick-up (OPU) on follicular development, number and quality of recovered oocytes, fertilization rate, and early embryo development in vitro. There were four OPU sessions (cross over) conducted on 16 Braford cows to evaluate the effect of various eCG doses. The timing of the wave of ovarian follicular development was synchronized, and three days after, the respective eCG dose was administrated (0, 200, 400, or 800 IU). The OPU was performed on Day 6, and viable oocytes were used for IVM and IVF according to the respective treatment. After IVF treatment, the fertilization and cleavage rates, time of cleavage, and the cell number at 48 h were evaluated. There was no difference in the number of follicles, oocyte quantity, and morphological quality of oocytes among treatments (P > 0.05). The oocyte recovery rate was similar among the eCG-treated groups, but was less than in the control group (P < 0.01). The eCG800 group, however, had a greater recovery rate of follicles >6 mm in diameter (P < 0.01). In addition, the eCG800 group had a greater rate of normal fertilization (P < 0.01) and lesser rate of polyspermy (P < 0.02). The cleavage rate of the eCG800 group was greater than the other treatment groups but similar to that of the control. In conclusion, the use of eCG800 increased the proportion of follicles > 6 mm, with improved rate of normal fertilization and reduced occurrence of polyspermy, without affecting early embryonic development in vitro.

Reduction in Percoll volume increases recovery rate of sex-sorted semen of bulls without affecting sperm quality and early embryonic development.

The aim of the present study was to evaluate the effects of Percoll volume on recovery rate, sperm quality, and embryo development kinetics in in vitro production of cattle embryos. Straws of conventional and sex-sorted semen were allocated to three different volumes of Percoll: 300 µL of each Percoll gradient (90%, 60%, and 30%), Control; 100 µL of each Percoll gradient, P100; and 200 µL of each Percoll gradient, P200. Sperm quality, fertilization rate, and embryo morpho-kinetic development using time lapse cinematography up to 48 h post-insemination were evaluated. For conventionally processed semen, sperm motility, vigor, and recovery rate were greater in the P100 and P200 treatment groups compared to the Control (P < 0.05), whereas reactive oxygen species (ROS) concentrations, lipid peroxidation, and superoxide dismutase enzyme activity were not influenced by treatments. For sex-sorted semen, treatment with P100 increased sperm curvilinear velocity, average path velocity, and amplitude of lateral head displacement (P < 0.05). Recovery rate was greater in the P100 group than Control and P200 groups (P < 0.05), formation of ROS was less in the P100 than Control and P200 groups, and superoxide dismutase enzyme activity was less in the P100 than Control group. Fertilization and cleavage rates, time of first cleavage, and cell number were similar between the P100 and Control groups (P > 0.05). The inclusion of Percoll volumes of 100 µL resulted in an increased sperm recovery rate without damage to sperm quality or affecting early embryonic development.