Bone morphogenetic protein 15 intrafollicular injection inhibits ovulation in cattle.

Oocyte-derived bone morphogenetic protein 15 (BMP15) is one of the main local regulators of ovarian physiology, but its role in the regulation of preovulatory follicles and ovulation is not well established. Therefore, this study was conceived to determine the effect of intrafollicular injection (IFI) of BMP15 on final follicular growth, ovulation and luteinization in cattle. Initially, it was observed that relative mRNA abundance of the BMP15 receptor BMPR1B in granulosa cells was regulated by GnRH treatment, and it was negatively correlated (R2 = 0.5; P < 0.001) to progesterone concentration in follicular fluid (FF) from preovulatory follicles. The IFI of recombinant human BMP15 tended to inhibit the growth of dominant follicles, as evidenced by an average increase of only 7.7% in the follicular diameter (from 8.8 mm to 9.1 mm) at 36 h post injection compared to 36.4% increase (from 8.9 mm to 14 mm) in the control group. Injection of BMP15 into preovulatory follicles (12-14 mm), simultaneously to im GnRH treatment, inhibited ovulation compared to control group, but did not prevent luteinization and progesterone production. Most of preovulatory follicles injected with BMP15 became luteinized cysts. Collectively, these findings indicate a suppressive role of BMP15 on later follicular development and ovulation in cattle, but not on luteogenesis and progesterone secretion.

Regulation and function of leptin during ovarian follicular development in cows.

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 (P < 0.05). Intrafollicular injection of either 100 or 1000 ng/mL leptin did not affect the diameter and the growth of dominant follicles (P> 0.05). Supplementation of in vitro culture medium with different leptin concentations did not affect (P > 0.05) 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.

Heat stress on oocyte or zygote compromises embryo development, impairs interferon tau production and increases reactive oxygen species and oxidative stress in bovine embryos produced in vitro.

Interferon tau (IFNT) is the cytokine responsible for the maternal recognition of pregnancy in ruminants and plays a role modulating embryo-maternal communication in the oviduct inducing a local response from immune cells. We aimed to investigate IFNT production, reactive oxygen species, and oxidative stress under the influence of heat stress (HS) during different stages of bovine in vitro embryo production. HS was established when the temperature was gradually raised from 38.5°C to 40.5°C in laboratory incubator, sustained for 6 hr, and decreased back to 38.5°C. To address the HS effects on IFNT production, reactive oxygen species, and oxidative stress, ovaries from a slaughterhouse were used according to treatments: control group (38.5°C); oocytes matured under HS; oocytes fertilized under HS; zygotes cultured in the first day under HS; and cells submitted to HS at oocyte maturation, fertilization, and the first day of zygote culture. The HS negatively affected cleavage and blastocyst rates, in all HS groups. On Day 7, all HS-treated embryos showed decrease IFNT gene and protein expressions, whereas reactive oxygen species were increased in comparison to the control. In conclusion, the compromised early embryo development due to higher temperatures during in vitro oocyte maturation, fertilization, and/or zygote stage have diminished IFNT expression and increased reactive oxygen species in bovine.

Partial inhibition of nitric oxide synthase activity stimulates the nuclear maturation progression of bovine cumulus-oocyte complex in vitro in the presence of hemisections of the follicular walls.

This study aimed to assess the effects of the inhibition of nitric oxide synthase (NOS) on events that modulate bovine in vitro oocyte maturation. Cumulus-oocyte complexes (COCs) were cultured with hemisections (HSs) of the follicular walls in a maturation medium supplemented with different concentrations (0.1-10.0 mM) of Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME). Controls consisted of COCs cultured in the presence (+HSs) or absence of HSs (-HSs) with no additional l-NAME supplementation. The following parameters were assessed: oocyte nuclear maturation stage; cumulus cell (CC) membrane integrity; nitrate/nitrite, progesterone, and estradiol concentrations in the culture medium at 22 h of cultivation; and the concentrations of cGMP and cAMP in COCs during the first hour of maturation. The addition of 1.0 mM l-NAME increased the percentage of oocytes that reached metaphase II (MII) and the percentage of intact CCs (P < 0.05). All l-NAME concentrations reduced the nitrate/nitrite concentrations (P < 0.05), but none affected steroid concentrations compared with control +HSs (P > 0.05). The addition of 1.0 mM l-NAME reduced cGMP concentrations at 3 h and increased cAMP concentrations in the first hour of culture (P < 0.05). Our findings suggest that the NOS/NO/cGMP pathway participates in meiosis progression (MI to MII) of the bovine oocytes matured in vitro in the presence of hemisections of the follicular walls. Lastly, the mechanisms that lead to the progression of meiosis after NOS inhibition do not involve changes in steroid production.

Activation of PPARG inhibits dominant follicle development in cattle.

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 and its receptors mRNA regulation in bovine granulosa and luteal cells.

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.

Luteinizing hormone upregulates NPPC and downregulates NPR3 mRNA abundance in bovine granulosa cells through activation of the EGF receptor.

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.

Parthenogenetic bovine embryos secrete type I interferon capable of stimulating ISG15 in luteal cell culture.

Interferon tau (IFNT) is the pregnancy recognition signal in ruminants and is secreted by trophoblast cells. Paracrine action in the endometrium is well established by inhibiting luteolytic pulses of prostaglandin F2 alpha. Recently, endocrine action was documented in the corpus luteum, blood cell and liver. It was hypothesized that conditioned medium (CM) obtained from days 7, 9 and 12 parthenogenetic embryos alters luteal cell gene expression. The aim was to establish a bovine mixed luteal cell culture to evaluate cellular response associated to interferon stimulated genes, steroidogenesis and apoptosis. Conditioned medium was obtained from Days 7, 9 and 12 parthenogenetic (PA) embryos culture. Moreover, antiviral assay was performed on CM from Days 7, 9 and 12 to verify Type I interferon activity. Luteal cell culture was validated by steroidogenic and apoptotic genes (CYP11A1 , HSD3B1, BAX, BCL2, AKT and XIAP mRNA expression), and concentration of progesterone as endpoint. Luteal cell culture was treated with interferon alpha (IFNA) and CM from parthenogenetic embryos. Antiviral assay revealed Type I interferon activity on CM from embryos increasing on Days 9 and 12. ISG15 mRNA was greater in the mixed luteal cells culture treated with 1, 10 and 100ng/ml of interferon alpha (IFNA) and also on Days 7, 9 and 12 CM treatments. Concentration of progesterone was not altered in luteal cell culture regardless of treatments. Steroidogenic and apoptotic genes were similar among groups in luteal cell culture treated with different doses of IFNA or CM from PA embryos. In conclusion, parthenogenetic embryo-derived CM has antiviral activity, luteal cell culture respond to Type I interferon by expressing IGS15. These data indicate this model can be used for IFNT endocrine signaling studies.

Prostaglandin F2α-induced luteolysis involves activation of Signal transducer and activator of transcription 3 and inhibition of AKT signaling in cattle.

Prostaglandin F2α (PGF) induces the precipitous loss of steroidogenic capabilities and cellular death in the corpus luteum of many species, yet the molecular mechanisms underlying this event are not completely understood. Signal transducer and activator of transcription 3 (STAT3) was activated in granulosa cells during follicle atresia, whereas AKT is immediately down-regulated in the corpus luteum after PGF treatment in cattle; however, their involvement in both functional and morphological luteolysis in monovular species still need to be determined. Blood samples and corpus lutea were collected from cows before (0) and 2, 12, 24, and 48 hr after PGF treatment on Day 10 of the estrous cycle (4-5 cows per time point). Serum progesterone concentrations decreased by threefold (p < 0.05) within 2 hr, confirming functional luteolysis. The mRNA abundance of the pro-apoptotic gene BAX increased 12-48 hr post-PGF treatment (p < 0.05), while morphological luteolysis was observed 24 and 48 hr after PGF treatment, based on the loss of plasma membrane integrity, reduction of cytoplasmic volume, and pyknotic nuclei. Phosphorylated STAT3 increased, peaking at 12 hr, and remained elevated until 48 hr after PGF treatment. SOCS3 transcript abundance also increased (p < 0.05) starting at 2 hr post-PGF treatment. In contrast, AKT phosphorylation decreased by 12 hr after treatment. Thus, activation of STAT3 and inactivation of AKT signaling are involved in structural regression of the corpus luteum.

Chemical castration in cattle with intratesticular injection of sodium chloride: Effects on stress and inflammatory markers.

Intratesticular injection (ITI) of sodium chloride (NaCl) is efficient for chemical castration of young calves, but its effects on calves welfare are unknown. Two experiments were conducted to evaluate the effects of ITI of 20% NaCl on stress and inflammatory markers in calves less than 20 days old and to assess the efficiency of ITI of 30% NaCl in 5 months old calves. In Experiment 1, control calves were only restrained and compared to calves submitted to castration through surgery (SC) and ITI with 20% NaCl (n = 9/group). No differences were observed for the eye corner temperature measured by thermography from 60 s before to 60 s after the procedures (P > 0.05). In the SC group, acute serum cortisol levels increased at 30 and 60 min after the procedure, but increased levels in the ITI group occurred only at 30 min (P < 0.05). Chronic discomfort markers were measured at 0, 24, 48, 72 and 96 h after the procedures (D0, D1, D2, D3 and D4, respectively). The serum levels of the paraoxonase 1 (PON1) enzyme and cortisol did not differ among groups (P > 0.05). Scrotal temperature was higher at D1 in the SC group than for the other groups, but lowest at D4 compared to the control (both P < 0.05). In Experiment 2, histological sections of testes were compared after ITI with either 30% NaCl or 30% calcium chloride (CaCl2), to intact calves (control). After 60 days, intact seminiferous tubules and mediastinum were observed after ITI with 30% NaCl, whereas coagulative necrosis, inflammatory infiltration and calcification occurred after ITI with 30% CaCl2. Efficient chemical castration through ITI of 20% NaCl in young calves was followed by slight stress and inflammatory responses compared to surgical castration. However, ITI of 30% NaCl was ineffective for chemical castration of 5 months old calves.

Altered expression of BRG1 and histone demethylases, and aberrant H3K4 methylation in less developmentally competent embryos at the time of embryonic genome activation.

Epigenetics is a fundamental regulator underlying many biological functions, such as development and cell differentiation. Epigenetic modifications affect key chromatin regulation, including transcription and DNA repair, which are critical for normal embryo development. In this study, we profiled the expression of epigenetic modifiers and patterns of epigenetic changes in porcine embryos around the period of embryonic genome activation (EGA). We observed that Brahma-related gene 1 (BRG1) and Lysine demethylase 1A (KDM1A), which can alter the methylation status of lysine 4 in histone 3 (H3K4), localize to the nucleus at Day 3-4 of development. We then compared the abundance of epigenetic modifiers between early- and late-cleaving embryos, which were classified based on the time to the first cell cleavage, to investigate if their nuclear localization contributes to developmental competence. The mRNA abundance of BRG1, KDM1A, as well as other lysine demethylases (KDM1B, KDM5A, KDM5B, and KDM5C), were significantly higher in late- compared to early-cleaving embryos near the EGA period, although these difference disappeared at the blastocyst stage. The abundance of H3K4 mono- (H3K4me) and di-methylation (H3K4me2) during the EGA period was reduced in late-cleaving and less developmentally competent embryos. By contrast, BRG1, KDM1A, and H3K4me2 abundance was greater in embryos with more than eight cells at Day 3-4 of development compared to those with fewer than four cells. These findings suggest that altered epigenetic modifications of H3K4 around the EGA period may affect the developmental capacity of porcine embryos to reach the blastocyst stage. Mol. Reprod. Dev. 84: 19-29, 2017. © 2016 Wiley Periodicals, Inc.

Resveratrol prevents oxidative damage and loss of sperm motility induced by long-term treatment with valproic acid in Wistar rats.

Valproic acid (VPA) is a drug widely use for the treatment of epilepsy in both children and adults. Evidence suggests that long-term use of VPA may lead to an impairment in the male reproductive function. Oxidative stress is considered to play a major role in VPA associated toxicity. In the present work, we demonstrated that the natural antioxidant compound resveratrol (RSV) can be use to prevent VPA oxidative damage. Wistar rats treated with VPA (400mgkg(-1)) by gavage for 28days showed decrease in sperm motility accompanied by increase in oxidative damage to lipids and proteins. Additionally, VPA administration leaded to depletion of reduced glutathione and decrease in total antioxidant potential in testes and epididymides of Wistar rats. The co-administration of RSV (10mgkg(-1)) efficiently prevented VPA pro-oxidant effects. In summary, RSV was shown to protect the reproductive system from the damage induced by VPA. Altogether, our data strongly suggests that RSV administration might be a valuable strategy to minimize reproductive impairment in patients requiring long-term VPA treatment.

Protective effect of vitamin E on sperm motility and oxidative stress in valproic acid treated rats.

Long-term administration of valproic acid (VPA) is known to promote reproductive impairment mediated by increase in testicular oxidative stress. Vitamin E (VitE) is a lipophilic antioxidant known to be essential for mammalian spermatogenesis. However, the capacity of this vitamin to abrogate the VPA-mediated oxidative stress has not yet been assessed. In the current study, we evaluated the protective effect of VitE on functional abnormalities related to VPA-induced oxidative stress in the male reproductive system. VPA (400 mg kg(-1)) was administered by gavage and VitE (50 mg kg(-1)) intraperitoneally to male Wistar rats for 28 days. Analysis of spermatozoa from the cauda epididymides was performed. The testes and epididymides were collected for measurement of oxidative stress biomarkers. Treatment with VPA induced a decrease in sperm motility accompanied by an increase in oxidative damage to lipids and proteins, depletion of reduced glutathione and a decrease in total reactive antioxidant potential on testes and epididymides. Co-administration of VitE restored the antioxidant potential and prevented oxidative damage on testes and epididymides, restoring sperm motility. Thus, VitE protects the reproductive system from the VPA-induced damage, suggesting that it may be a useful compound to minimize the reproductive impairment in patients requiring long-term treatment with VPA.

Exposure of Somatic Cells to Cytoplasm Extracts of Porcine Oocytes Induces Stem Cell-Like Colony Formation and Alters Expression of Pluripotency and Chromatin-Modifying Genes.

Cell permeabilization followed by exposure to cytoplasmic extracts of oocytes has been proposed as an alternative to transduction of transcription factors for inducing pluripotency in cultured somatic cells. The main goal in this study was to investigate the effect of treating porcine fibroblast cells with cytoplasmic extracts of GV-stage oocyte (OEx) followed by inhibition of histone deacetylases with Scriptaid (Scrip) on the formation of stem cell-like colonies and expression of genes encoding pluripotency and chromatin-modifying enzymes. Stem cell-like colonies start developing ∼2 weeks after treatment in cells exposed to OEx or OEx + Scrip. The number of cell colonies at the first day of appearance and 48 hours later was also similar between OEx and OEx + Scrip treatments. Transcripts for Nanog, Rex1, and c-Myc genes were detected in most cell samples that were analyzed on different days after OEx treatment. However, Sox2 transcripts were not detected and only a small proportion of samples had detectable levels of Oct4 mRNA after OEx treatment. A similar pattern of transcripts for pluripotency genes was observed in cells treated with OEx alone or OEx + Scrip. Transcript levels for Dnmt1 and Ezh2 were reduced at Day 3 after treatment in cells exposed to OEx. These findings revealed that: (a) exposure to OEx can induce a partial reprogramming of fibroblast cells toward pluripotency, characterized by colony formation and activation of pluripotency genes; and (b) inhibition of histone deacetylases does not improve the reprogramming effect of OEx treatment.

Effect of Cell Cycle Interactions and Inhibition of Histone Deacetylases on Development of Porcine Embryos Produced by Nuclear Transfer.

The aim of this study was to evaluate if the positive effects of inhibiting histone deacetylase enzymes on cell reprogramming and development of somatic cell nuclear transfer (SCNT) embryos is affected by the cell cycle stage of nuclear donor cells and host oocytes at the time of embryo reconstruction. SCNT embryos were produced with metaphase II (MII) or telophase II (TII) cytoplasts and nuclear donor cells that were either at the G1-0 or G2/M stages. Embryos reconstructed with the different cell cycle combinations were treated or not with the histone deacetylase inhibitor (HDACi) Scriptaid for 15 h and then cultured in vitro for 7 days. Embryos reconstructed with MII-G1-0 and TII-G2/M developed to the blastocyst stage with a higher frequency compared to the other groups, confirming the importance of cell cycle interactions on cell reprogramming and SCNT embryo development. Treatment with HDACi improved development of SCNT embryos produced with MII but not TII cytoplasts, independently of the cell cycle stage of nuclear donor cells. These findings provide evidence that the positive effect of HDACi treatment on development of SCNT embryos depends upon cell cycle interactions between the host cytoplast and the nuclear donor cells.

Gonadotoxic effects of busulfan in two strains of mice.

Busulfan is a chemotherapy drug that has side effects on spermatogonial stem cells (SSC). The effects of bulsufan treatment on male germ cells and fertility vary significantly between individuals. In this study, we have used molecular, cellular and histopathology approaches to investigate the effects of a single intraperitoneal dose of busulfan (40mgkg(-1)) in two mice strains, Balb/C and Swiss, at two different periods after treatment, 30 and 90 days. Testicular degeneration was observed in both Balb/C and Swiss mice after busulfan injection. Interestingly, testicular functions and fertility recovered spontaneously post busulfan treatment in Swiss mice, but not in Balb/C mice. Abnormal fertility induced by busulfan in Balb/C mice was associated with altered seminiferous tubules, sperm morphology and transcript levels of Nanos2, Nanos3, Gdnf and Plzf genes. These findings revealed that SSC of Balb/C mice are more sensitive to the toxic effects of busulfan then those of Swiss mice.

Effect of diets enriched with rutin on blood parameters, oxidative biomarkers and pituitary hormone expression in silver catfish (Rhamdia quelen).

The effects of adding rutin to the diet (0, 0.15 or 0.30%) of silver catfish for 21 days on blood parameters, oxidative stress biomarkers and pituitary hormones expression were investigated. Fish that received the diet containing 0.15% rutin exhibited reduced plasma cortisol levels. The levels of lipid peroxidation were lowered in the all tissues of animals receiving the diet containing rutin. Rutin increased the activity of the superoxide dismutase (SOD), catalase (CAT), nonprotein thiols (NPSH), ascorbic acid content (AA) and total reactive antioxidant potential (TRAP) in the brain; glutathione S-transferase (GST) activity and TRAP in the gills; SOD, CAT and GST activity, NPSH, AA levels and TRAP in the liver; CAT and GST activity and TRAP levels in the kidneys; and glutathione peroxidase activity, NPSH, AA levels and TRAP in the muscle. There were no changes regarding the expression of growth hormone, prolactin and somatolactin in fish fed with the diet containing rutin when compared with the control. The supplementation of rutin to the diet of fish is beneficial because it increases the antioxidant responses of tissues.

Rhamdia quelen (Quoy & Gaimard, 1824), submitted to a stressful condition: effect of dietary addition of the essential oil of Lippia alba on metabolism, osmoregulation and endocrinology

The aim of this study was to evaluate the effect of the essential oil of Lippia alba (EOLA) as a feed additive on ionoregulatory and metabolic parameters and pituitary hormones expression in silver catfish, Rhamdia quelen , submitted to a stressful condition (stocking density of 10.6 kg m-3 and limited space). Fish were fed with different concentrations of EOLA (0.0 - control, 0.25 and 0.50 mL kg food-1) for 20 days. Metabolic parameters were not affected by the diet, with the exception of alanine aminotransferase, which was higher in the liver of fish fed 0.50 mL EOLA kg food-1. Plasma ions and activity of H+-ATPase did not change, but fish fed 0.25 mL EOLA kg food-1 presented higher Na+/K+-ATPase activity. Somatolactin expression in the pituitary was higher in the fish fed 0.25 mL EOLA kg food -1, but the expression of growth hormone and prolactin did not change. Therefore, dietary EOLA does not exert a protective effect in R. quelen submitted to a stressful situation because it did not alter most measured parameters. The use of 0.25 mL EOLA kg food-1 seems to be more suitable than 0.50 mL EOLA kg food-1 since the latter may be related to liver damage.

O objetivo deste estudo foi avaliar o efeito do óleo essencial de Lippiaalba (OELA) como aditivo em rações na ionoregulação, parâmetros metabólicos e expressão de hormônios hipofisários em jundiás, Rhamdiaquelen, submetidos a uma situação estressante (densidade de estocagem de 10,6 kg m-3 e espaço limitado). Os peixes foram alimentados com diferentes concentrações de OELA (0,0 - controle, 0,25 e 0,50 mL kg de ração-1) durante 20 dias. Parâmetros metabólicos não foram afetados pela dieta, com a exceção da alanina aminotransferase, que foi mais elevada no fígado dos peixes alimentados com 0,50 mL de OELA kg de ração-1. Íons plasmáticos e a atividade da H+-ATPase não apresentaram nenhuma alteração, mas os peixes alimentados com 0,25 mL OELA kg de ração-1 apresentaram maior atividade da Na+/K+-ATPase. A expressão da somatolactina na hipófise de peixes alimentados com 0,25 mL OELA kg de ração-1 aumentou, porém a expressão do hormônio de crescimento e da prolactina não mudou. Portanto, a adição do OELA na ração não tem um efeito protetor em jundiás submetidos a uma situação estressante, pois não influiu na maioria dos parâmetros medidos. O uso de 0,25 mL OELA kg de ração-1 parece ser mais adequado que 0,50 mL OELA kg de ração-1, uma vez que este nível de inclusão pode estar relacionado a danos hepáticos.

Natriuretic peptides stimulate oocyte meiotic resumption in bovine.

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.

Functional status of STAT3 and MAPK3/1 signaling pathways in granulosa cells during bovine follicular deviation.

Follicle development is coordinated by gonadotropins, steroids, and growth factors, which activate multiple signaling pathways. Phosphorylated-MAPK (pMAPK) level was indicated as an early marker of follicle dominance, whereas phosphorylated STAT3 (pSTAT3) was increased in granulosa cells of hypophysectomized rats. We hypothesized that MAPK3/1 and STAT3 pathways are regulated in granulosa cells during follicle deviation in cattle. Cyclic beef cows were synchronized and ovariectomized to recover the two largest follicles. Follicular diameter did not differ on Day 2 but was significantly greater in dominant follicles (DFs) than that in subordinate follicles (SFs) on Days 3 and 4 of the follicular wave. The elevated abundance of CYP19A1 mRNA expression in granulosa cells of DFs and cleaved caspase 3 in Day-4 SFs further validated our in vivo model. Before deviation, pMAPK3/1 levels were significantly higher in granulosa cells of the future DF. STAT3 mRNA and total protein (tSTAT3) were higher in granulosa cells of SFs collected on Day 4. Furthermore, levels of pSTAT3 were dramatically increased in granulosa cells of Day-4 SFs. In conclusion, pMAPK3/1 was increased in the future DF, but such differential abundance between the DF and SF was not evident after deviation. The higher abundance of pSTAT3 in granulosa cells of SFs after deviation suggests that this pathway may be involved in granulosa cell death and follicular atresia.