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Although prostaglandins are important in the ovulation process, a precise role for prostaglandin F2α (PGF) has not been elucidated. This study aimed to evaluate the regulation of PGF receptor mRNA (PTGFR) in granulosa cells and the local effect of PGF on ovulation and luteinization. In Experiment 1, using samples collected in vivo before (Day 2), during (Day 3) and after (Day 4) follicular deviation, expression of PTGFR in bovine granulosa cells was more abundant in the dominant follicle after deviation than in subordinates (P 0.05). However, the expression of PTGFR was not regulated (P = 0.1) in preovulatory follicles at different time-points (0, 3, 6, 12 and 24 h) after ovulation induction with GnRH. In Experiment 2, to assess the role of systemic PGF treatment on luteinization and vascularization of preovulatory follicles, flunixin meglumine (FM), a nonsteroidal anti-inflammatory drug, was used to inhibit endogenous prostaglandin synthesis. Cows with preovulatory follicles were induced to ovulate with GnRH (0 h) and allocated to three groups: Control, with no further treatment; FM, treated with 2.2 mg/kg FM im 17 h after GnRH treatment; and FM + PGF, treated with FM 17 h after GnRH, followed by 25 mg dinoprost tromethamine (PGF) 23 h after GnRH treatment. FM injection was able to reduce the concentration of PGF in the follicular fluid (FF) (P 0.001). However, contrary to our hypothesis, color Doppler ultrasound evaluations revealed decreased vascular flow in FM + PGF group (P 0.05), and no effect of the treatments on intrafollicular P4 and E2 concentrations 24 h after GnRH. The prostaglandin metabolite (PGFM) concentrations in the FF were greater in cows receiving systemic PGF (P 0.001), which prompted us to further check its role on ovulation. Therefore, in Experiment 3, in a final attempt to demonstrate the local effect of PGF on ovulation, cows with preovulatory follicles received an intrafollicular injection (IFI) of PBS (Control) or 100 ng/mL purified PGF (PGF group). PGF treatment did not affect the time of ovulation after IFI (66 ± 6.4 and 63 ± 8.5 h for control and PGF, respectively; P 0.05), further suggesting that it has no direct effect in the ovulatory process. Based on our findings, we concluded that FM decreased PGF synthesis within the follicle, whereas PGF treatment decreased follicular vascularization. In addition, the in vivo model of intrafollicular injection evidenced that PGF alone is not able to locally induce ovulation.

This study evaluated reproductive indicators of gilts treated with altrenogest or an intravaginal device (IVD) containing medroxyprogesterone acetate (MPA) for estrous cycle synchronization, starting the protocol on different days of the estrous cycle or replacing the IVD in the middle of treatment. In Experiment 1, 126 gilts were assigned, according to the day of treatment onset (Day 5 or 10 of the estrous cycle), to the following treatments: Control-5 (no hormone); Control-10 (no hormone); IVD-5 (IVD with MPA); IVD-10 (IVD with MPA); ALT-5 (altrenogest); or ALT-10 (altrenogest). The first day of the previous estrus was considered as Day 0 of the estrous cycle, and progestogen groups were treated for 14 d. In Experiment 2, 63 gilts were assigned to Control, ALT, or IVD groups. Progestogen treatment started on Day 10 of the estrous cycle, and the IVD was replaced after 7 d of treatment. In both experiments, no gilts expressed estrus during progestogen administration. In Experiment 1, the interval hormonal withdrawal-to-estrus (IHE) tended to be shorter when treatment started on Day 10 than on Day 5 (3.6 vs. 4.1 d, respectively; P = 0.09). The percentage of gilts expressing estrus after hormone withdrawal was lower for IVD-gilts (76.3%) compared to ALT (100%) and Control-gilts (92.9%; P ≤ 0.07). The percentage of persistent follicles (PFOL) was greater in IVD-10 (60.0%) and ALT-10 (33.3%) than CONT-10 (0.0%; P ≤ 0.06). The adjusted farrowing rate (AFR) was lower in IVD (65.5%) and ALT (80.5%) compared with CONT (97.4%; P ≤ 0.08). In Experiment 2, the IHE was longer for ALT than IVD (4.9 vs. 3.9 d, respectively; P 0.01). No difference among groups was observed in the percentage of gilts expressing estrus (overall 86.4%), but the occurrence of PFOL was higher in IVD (61.5%) compared to ALT (5.3%), and Control groups (10.5%; P 0.01). The AFR was lower in IVD (53.8%) than in ALT (88.2%) and Control (94.7%; P ≤ 0.05). The total number of piglets born was not affected by hormonal treatments in either experiment. Estrous expression was delayed in gilts treated with altrenogest or IVD-MPA. However, the reproductive performance of IVD-gilts was compromised, which was not circumvented by IVD replacement in the middle of treatment. Therefore, further studies are necessary to understand MPA pharmacodynamics and investigate alternative devices for a steady release of progestogens in gilts.

The peroxisome proliferator-activated receptor gamma (PPARG, also called NR1C3) is a nuclear receptor of the peroxisome proliferator-activated receptor family (PPAR). PPARs are involved in the regulation of apoptosis, cell cycle, estradiol and progesterone synthesis, and metabolism. However, the role of PPARs and their regulation during follicular development and ovulation in monovular species remain poorly understood. In this study, a well-established intrafollicular injection model was used to investigate if the PPARG participates in the regulation of dominant follicle development and ovulation in cattle. Findings from this study revealed that the relative mRNA abundance of PPARG was similar between dominant and subordinate follicles around follicle deviation, decreased after the LH surge, and increased before ovulation. In addition, a quadratic correlation was found between PPARG mRNA levels in granulosa cells and progesterone concentration in the follicular fluid. Intrafollicular injection of 50 μM Troglitazone (TGZ; a PPARG agonist) inhibited follicular growth and decreased CYP19A1 mRNA abundance in granulosa cells. These findings indicate that PPARG is involved in the regulation of steroidogenesis, follicle growth and ovulation in cattle.

Oncostatin M (OSM) and its receptor (OSMR) are members of the interleukin-6 family cytokines. Although OSM and OSMR expression was detected in human ovaries, their function and regulation during follicle development, ovulation and luteolysis have not been studied in any species. The aim of the present study was to investigate the levels of OSM and OSMR mRNA in bovine ovaries and the effect of OSM treatment on cultured granulosa cells. OSM mRNA was not detected in granulosa cells obtained from follicles around the time of follicular deviation and from pre-ovulatory follicles, whereas OSMR transcript levels were greater in granulosa cells of atretic subordinate follicles (P 0.001). Abundance of OSMR mRNA increased in granulosa cells of preovulatory follicles, collected at 12 and 24 h after the ovulatory stimulus with gonadotropins (P 0.001). In the luteal tissue, OSM mRNA abundance levels were higher at 24-48 h after PGF-induced luteolysis (P 0.01) compared to 0 h, whereas OSMR mRNA was transiently increased at 2 h after PGF treatment (P 0.05). In cultured granulosa cells, 10 ng/mL OSM in the presence of FSH increased BAX/BCL2 mRNA ratio (P 0.05) compared to the control. Moreover, 100 ng/mL OSM in the presence of FSH increased OSMR (P 0.05) and decreased XIAP mRNA (P 0.05) levels, compared to the control group. These findings provide the first evidence that OSMR is regulated during follicle atresia, ovulation and luteolysis, and that OSM from other cells may mediate granulosa and luteal cell function, regulating the expression of genes involved in cell's viability.

During folliculogenesis, the luteinizing hormone (LH) surge triggers dynamic events in granulosa cells that culminate with ovulation. The aim of this study was to evaluate if the epidermal growth factor receptor (EGFR) is required for ovulation in cattle, and if it regulates the expression of the natriuretic peptide (NP) system in granulosa cells after gonadotropin-releasing hormone (GnRH)/LH stimulation. It was observed that GnRH induces amphiregulin (AREG) and epiregulin (EREG) mRNA at 3 and 6 h after in vivo treatment, but the expression of these genes was not regulated by atrial (ANP) and C-type (CNP) NPs in granulosa cells cultured in vitro. The abundance of mRNA encoding the NP receptors (NPR1, 2 and 3) was not altered by LH supplementation and/or EGFR inhibition (AG1478; AG) in granulosa cells after 6 h of in vitro culture. However, in the same conditions, mRNA encoding the natriuretic peptide precursor C (NPPC) was upregulated by LH, whereas AG (0.5 and 5 μM) inhibited the LH effect. In order to confirm those results, 5 μM AG or saline were intrafollicularly injected in preovulatory follicles and cows were simultaneously treated with GnRH intramuscularly. Granulosa cells harvested at 6 h after GnRH injection revealed higher NPR3 and lower NPPC mRNA levels in AG-treated, compared to control cows. However, intrafollicular injection of AG did not inhibit GnRH-induced ovulation. In granulosa cells cultured in vitro, ANP associated with LH increased prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA abundance. In conclusion, we inferred that LH modulated NPPC and NPR3 mRNA abundance through EGFR in bovine granulosa cells, but ovulation in cattle did not seem to depend on EGFR activation.

Supplementation of diets with omega-3 polyunsaturated fatty acids (PUFA) may benefit sow reproductive performance, but the efficiency of algae as a source of PUFA is still unknown. This study evaluated serum levels of metabolic markers and reproductive performance of sows supplemented with the heterotrophic microalgae Schizochytium sp. Sows (n = 596) were supplemented from the 85th day of gestation, during lactation and until the weaning-estrous interval (WEI). Microalgae were included at five levels: 0 (control); 3.5; 7.0; 14.0; and 28.0 g/d. Blood samples were collected at the 85th day of gestation, at the 10th d of lactation and at the last day prior to weaning (WEI). Serum levels of cholesterol, triglycerides, insulin-like growth factor type-1 (IGF-1) and non-esterified fatty acids (NEFA) were evaluated. At the parity concurrent with the supplementation, no effects were observed on stillborn rates (P > 0.05), sows fed 28.0 g/d farrowed heavier piglets than sows fed lower levels (P 0.05). The highest tested concentration (28.0 g/d) of Schizochytium sp. was associated with reduced serum levels of triglycerides during gestation and with increased piglet birth weight, but the supplementation generally did not benefit sow reproductive performance.

The techniques currently available for embryo collection in sheep include surgical (laparotomy; LP) and transcervical (TC) methods Although both methods are well established, there is no data about the levels of inflammatory markers in ewes after using such procedures. The objective of this study was to compare the systemic reaction to inflammation in ewes submitted to superovulation and embryo collection through LP or TC methods Blood samples were collected before each procedure (at D0) and at 3, 6 and 9 days after procedures to determine the levels of total protein, haptoglobin, fibrinogen and paraoxonase 1. The levels of inflammatory markers did not differ (P > 0.05) between methods Serum levels of haptoglobin and total protein and paraoxonase 1 activity increased after D0 (P

The aim of this study was to evaluate effects of intra-follicular (i.f.) treatment with lipopolysaccharide (LPS) on follicular and luteal development in cows. There were 18 non-lactating cows assigned to two groups to address this aim: control group (n = 9), which received an i.f. injection of saline; and LPS group (n = 9), which received an i.f. injection of 1 μg of LPS per mL of follicular fluid. Cows were treated with an intravaginal P4 releasing device (IVD) and estradiol benzoate on D0. On D4 and D5 cows were treated with cloprostenol sodium and on D7 the IVD was removed. At 12 h after IVD removal, cows were administered the i.f. injection of LPS or saline. After administration of these treatments, follicular development was evaluated every 12 h until ovulation. The LPS treatment increased blood flow in pre-ovulatory follicles (P = 0.05). Follicle growth was reduced by LPS injection (P0.02) resulting a longer period to the time of ovulation for cows in the LPS than control group (P = 0.03). The percentage of cows having ovulations was less for the LPS than control group (P = 0.03). The diameter of the CL, CL blood flow and P4 concentrations 5 and 12 days after ovulation did not differ between groups (P0.05). In conclusion, intra-follicular treatment with LPS resulted in a decreased rate of follicle growth, delayed timing of ovulations and a lesser number of cows having ovulations.

In Experiment I, during the non-breeding season, after intravaginal devices containing progesterone (P4) were withdrawn (n = 28), estrous rates were greater with treatment with 400 IU eCG (P 0.05). In Experiment II (non-breeding season), ewes of one group were treated with 300 IU eCG (n = 8) and of two groups were treated with 10 mg FSH. In one FSH group, 250 μg estradiol benzoate (EB) were administered after 24 h (n = 9); in the other, 4 μg GnRH were administered after 36 h (n = 10). Serum P4 concentrations were greater in eCG-treated ewes (P 0.05). In Experiment III (breeding season), the treatments were: 300 IU eCG; 250 μg estradiol cypionate; 250 μg EB; or control (n = 22). Follicular growth was greater for eCG-treated ewes within 0−24 h and for control ewes within 48−72 h (P = 0.001). Although estrous and ovulation rates did not differ (P > 0.05), all eCG-treated ewes had ovulations. During the non-breeding season, FSH treatment promoted follicular growth but did not induce ovulations. For FTAI regimens, eCG was more effective than FSH plus GnRH and estradiol esters in inducing estrus and ovulation.

Although combining of eCG and hCG administrations is known to enhance LH-like actions, there have been few studies where there was comparison of the effects of treatment of anestrous ewes with eCG and hCG and eCG alone. In Experiment 1, 18 ewes in seasonal anestrus were administered an intravaginal device (IVD) containing medroxyprogesterone acetate for 12 days, and at the time of IVD removal (D0), were allocated into the following groups (n = 6/group): no further treatment (control); 400 IU eCG (eCG); or 400 IU eCG and 200 IU hCG (eCG + hCG). There was greater ovarian follicular growth in the groups treated with gonadotropins, compared to the control, and there were greater progesterone concentrations in the eCG + hCG group on D9 (P 0.05). In Experiment 2, 66 ewe lambs were assigned to the same treatment groups described for Experiment 1, and subsequently there was natural mating with rams. There was a greater rate of behavioral estrous manifestation in the eCG (88.5 %; 23/26) and eCG+hCG (85.2 %; 23/27), than control (30.8 %; 4/13; P 0.05) group. Pregnancy rate was also greater in the eCG (34.6 %; 9/26) and eCG+hCG (18.6 %; 5/27) than control (0 %; 0/13; P 0.05) group, whereas conception rate, considering only ewe lambs that were mated, was only greater in the eCG group. Although there were greater progesterone concentrations 9 days after treatment in the eCG+hCG group, there was no difference in follicular growth in anestrous ewes, nor was there an effect on estrous behavior manifestation and pregnancy rates in ewe lambs, compared to treatment with only eCG.

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 intravulvosubmucosal (IVSM) route has been used to reduce the dose of prostaglandin F 2α (PGF 2α ), a luteolytic agent used in estrus synchronization programs. To validate the effectiveness of PGF 2α administered via IVSM, the estrus rate was monitored in 1937 beef cows for 5 days (25.6% of estrus). The cows that did not show signs of estrus by day 5 received 5 mg of dinoprost IVSM (1/5 of the standard dose; n =1440). By day 10, 68.2% of the cows were in estrus. In a second trial, using the same synchronization protocol, the proportion of heifers in estrus after receiving 5 mg dinoprost at day 5 via IVSM (47.4%; n =97) or via an intramuscular injection (IM; 54.7%; n =95) did not differ ( P >0.05). The occurrence of luteolysis (serum progesterone concentrations below 1 ng/mL) was dependent on the period of the estrous cycle in which the cows were treated with 5 mg of dinoprost via IM or IVSM. Luteolysis was observed in 5 out of 10 cows treated at day 10 but was not observed in any cows treated at day 5 of the estrous cycle. Luteolysis occurred in all animals treated with 25 mg dinoprost independent of the estrous cycle period (day 5 or 10). After IVSM injection, the PGF 2α concentration did not differ in the uterine and jugular veins. This was further confirmed by measuring the concentration of 13,14-dihydro-15-keto-PGF 2α (PGFM) after the injection of 5 mg of dinoprost via the IM or IVSM route. Dinoprost IM and IVSM injections resulted in a similar PGFM serum pattern over time, indicating the same rate of absorption into the systemic circulation. Taking the results together, we concluded that PGF 2α IVSM injections reached the systemic circulation before reaching the ovary, and the effectiveness of low doses of PGF 2α was dependent on the period of the estrous cycle and not on the route of administration.

Made available in DSpace on 2013-09-27T14:54:28Z (GMT). No. of bitstreams: 1 WOS000304231600006.pdf: 4200149 bytes, checksum: da68c3a014ed77979dfeca212097e21a (MD5) Previous issue date: 2012-06-01 Made available in DSpace on 2013-09-30T18:37:40Z (GMT). No. of bitstreams: 1 WOS000304231600006.pdf: 4200149 bytes, checksum: da68c3a014ed77979dfeca212097e21a (MD5) Previous issue date: 2012-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:50:02Z No. of bitstreams: 1 WOS000304231600006.pdf: 4200149 bytes, checksum: da68c3a014ed77979dfeca212097e21a (MD5) Made available in DSpace on 2014-05-20T13:50:02Z (GMT). No. of bitstreams: 1 WOS000304231600006.pdf: 4200149 bytes, checksum: da68c3a014ed77979dfeca212097e21a (MD5) Previous issue date: 2012-06-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Oocyte meiotic resumption is triggered by the ovulatory gonadotropin surge; in cattle, angiotensin II (AngII) and prostaglandins (PG) are key mediators of this gonadotropin-induced event. Here, we tested the hypothesis that progesterone (P-4) is also involved in oocyte meiotic resumption induced by the gonadotropin surge. In Experiment I, P-4 induced nuclear maturation in a dose-dependent manner using a coculture of follicular hemisections and cumulus-oocyte complexes. In the second experiment, using an in vivo model, an injection of mifepristone (MIFE; P-4 receptor antagonist) at the antrum of preovulatory follicles prevented GnRH-induced oocyte meiotic resumption in vivo. In Experiment III (coculture system similar to that of Experiment I), MIFE prevented stimulatory effects of AngII on resumption of meiosis, but saralasin (AngII receptor antagonist) did not inhibit P-4 actions. In Experiments IV and V, fibroblast growth Factor 10 (FGF10; known to suppress steroidogenesis in granulosa cells), blocked AngII-but not P-4-induced oocyte meiotic resumption. Therefore, we inferred that AngII is upstream to P-4 in a cascade to induce meiotic resumption. Previously, we had reported that AngII acted throughout the PGs pathway to modulate nuclear progression. In Experiment V, indomethacin inhibited resumption of meiosis induced by P-4, providing further support to the AngII-P-4, sequential effect on meiotic resumption. In conclusion, we inferred that AngII, P-4, and PGs are sequential steps in the same pathway that culminates with bovine oocyte maturation. (C) 2012 Elsevier B.V. All rights reserved. Universidade Federal de Santa Maria (UFSM), Lab Biotechnol & Anim Reprod BioRep, BR-97119900 Santa Maria, RS, Brazil State Univ São Paulo UNESP, Dept Physiol, Botucatu, SP, Brazil State Univ São Paulo UNESP, Dept Physiol, Botucatu, SP, Brazil