2011 (SBTE) 25th Annual Meeting Proceedings - International ...

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Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A055 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION EFFICIENCY OF DIFFERENT TREATMENT MENTS OF OVARIAN CYST STS S IN DAIRY COWS Tochimar chimara a Apar parecida Miy iyauchi 1 , Car arlos Antônio de Car arvalho alho Fer ernandes 1 , Mar arina Resende Pimen imenta Por ortinar tinari 1 , Mar arilu Mar artins Gioso 1 , Bruno Fer ernandes Ludgero Alves 2 & Ana Cristina Silva de Figueiredo 1 1 UNIFENAS, ALFENAS, MG, BRAZIL. 2 BIOTRAN, ALFENAS, MG, BRAZIL. The incidence of the ovarian follicular cyst is close related to reproductive failure in dairy cattle. Effective treatment of this disease promotes the regression of this structure, the development of the corpus luteum and hastens the return of the normal ovarian cyclic activity. The benedict effect of the association between prostaglandin analogous and GnRH on treatment of the luteinized cystic structures has been reported. The study was designed to evaluate the Fertirelin acetate associated or not with cloprostenol on ovarian cyst treatments in dairy cattle. Hundredthirty-six Holstein cows with follicular cyst provided from four different were included for this purpose. Follicular cysts were detected by linear array 7.0 MHz probe (Esaote-Falco) by transrectal examination of the ovarian. Circular anechoic structures larger than 20 mm of the diameter without detecting luteal tissue were considered as follicular cysts. The animals were randomized in one of the five groups according to the drug injection: 2 mL of saline (Group 1; n = 16); 0.1mg of the Fertirelin acetate - Fertigen ® Schering Plough-Brazil (Group 2; n = 31); 0.1mg of the Fertirelin acetate and 0.530 mg of the Sodium Cloprostenol 10 days later - Ciosin ® Schering Plough-Brazil (Group 3; n = 28); 0.1mg of the Fertirelin acetate and 0.530 mg of the Sodium Cloprostenol at the same time (Group 4; n = 29); and 0.1mg of the Fertirelin acetate and two doses of the Sodium Cloprostenol, the first at the same time of the Fertirelin and the second 10 days later (Group 5; n = 32). All treated cows were reexamined by transrectal ultrasonography between 20 and 30 days after treatment. The therapy was considered efficient when the cystic structure was not present and the luteal tissue was detected at the second ultrasound examination. The percentage of recover after treatment was 18.8c, 54.8b, 53.5b, 79.3a, 81.2%a for groups 1 to 5, respectively. The interval from treatment to first artificial insemination (AI) was 61.2±17.9a, 44.5±16.4b, 30.9±12.6c, 26.2±14.2c, 18.3±10.2d days for groups 1 to 5, respectively. There was no effect of the farm and the number of AI per conception was similar among treatments. The interpretation of these results is that the association between Fertirelin and cloprostenol in two different protocols was efficient to promote luteolysis and to hasten the return to the reproductive activity after treatment. [Supported by Fapemig]. Keywords: bovine, ovarian cysts, reproductive efficience. A056 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION STABILIT ABILITY OF REFERENCE GENES AND LEVELS OF MRNA FOR THE IGF SYSTEM STEM IN BOVINE OVARIAN FOLLICLES GROWN IN VITRO AND IN VIVO Gisvani Lopes de Vasconcelos, Anderson Weiny Barbalho Silva, José Jackson do Nascimento Costa, Maria Juliane Passos, Rodrigo Otávio Decaria de Salles Rossi, Emanuela de Lima Rebouças & José Roberto Viana Silva UNIVERSIDADE FEDERAL DO CEARÁ - UFC, SOBRAL, CE, BRAZIL. It is important to know the profile of gene expression, as well as suitable reference genes for normalization of data from real-time PCR (qRT-PCR) as internal controls. However, the expression of mRNA for the IGF system in secondary and early antral follicles in the bovine is not yet known. The aim of this study was to investigate the stability of seven reference genes, and the relative expression of IGF-I and II, the receptors of IGF-I and II (IGFR-I and-II) and IGF proteins (IGFBPs 1- 6) in bovine follicles before and after culture in vitro. In order to evaluate the stability of reference genes and the expression levels of IGF system, follicles with ~ 0.2 (n = 30), ~ 0.5 (n = 30) and ~ 1.0 mm (n = 30) were micro-dissected and then subjected to total RNA extraction and cDNA synthesis. GeNorm software was used to evaluate the stability of Glyceraldehyde-3 phosphatedehydrogenase (GAPDH), β-tubulin, β-actin, Fosfogliceroquinase, 18S rRNA, Ubiquitin(UBQ) and ribosomal protein-19 (RPL-19). To compare the levels of mRNA of IGF system before and after in vitro culture, secondary follicles of ~ 0.2 mm (n = 24) were isolated and cultured for 12 days to reach ~ 0.5 mm. These follicles were subjected to total RNA extraction and cDNA synthesis. The deltadelta CT method was used to normalize the data of mRNA expression. The nonparametric Kruskal-Wallis test was used to analyze mRNA levels for components of the IGF system (P < 0.05). The results demonstrated that GAPDH and UBQ were the most stable reference genes in preantral and antral bovine follicles. The levels of mRNA for IGFR-I and IGFR-II in follicles of 0.5 mm were significantly higher than in follicles of 0.2 and 1.0 mm. A reduction in the levels of IGFBP-1 was observed during growth of the follicles. The levels of IGFBP-2 in follicles of 0.5 mm were significantly higher than in follicles of 1.0 mm. In contrast, 1.0 mm follicles showed higher levels of IGFBP-3 and IGFBP-4 than follicles of 0.2 and 0.5 mm. After in vitro culture, we observed significant reductions in levels of IGF-I, IGFR-I, IGFBP-3, -5 and -6 compared with noncultured follicles with ~ 0.2 mm. Comparing the 0.5 mm follicles grown in vitro with in vivo, the expression of IGFBP-1, -2, -3 e -6 were significantly lower in follicles cultured. Thus, we conclude that the UBQ and GAPDH are the most stable reference genes in bovine follicles with 0.2, 0.5 and 1.0 mm, while the IGF-I and II, their receptors (IGFR-I and IGFR-II) and IGFBPs 1-6 show variable expression in follicular categories analyzed. Keywords: igf, bovine, mrna. s364
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A057 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION EXPRESSION OF MRNA ENCODING FGFR1B AND FGFR2B IN BOVINE FETAL OVARIES DURING GESTATION TION Rubia Bueno Da Silva 1 , Ester Siqueira Caixeta 2 , Christopher Price 3 & José Buratini Junior 4 1,2,4 UNESP, BOTUCATU, SP, BRAZIL. 3 UNIVERSITÉ DE MONTREAL, MONTREAL, CANADÁ. Recent findings suggest the involvement of fibroblast growth factor 10 (FGF-10) in regulation of preantral follicle development. During gestational ovarian development, expression of RNAm FGF-10 is progressively upregulated with a significant increase at 120 days of gestation, coincident with the increase in primary follicles number (Castilho, data not published). FGF-10 signaling requires activation of FGFs receptors 1B or 2B (FGFR1B, FGFR2B). In the present study, we assessed mRNA expression patterns of FGFR1B and FGFR2B in bovine fetal ovaries during gestation. Bovine fetal ovaries were obtained in a local slaughterhouse, fetal age was estimated by the crown-rump length and samples were grouped according with days of gestation as follows: 60 (n = 7), 75 (n = 7), 90 (n = 7), 120 (n = 7), 150 (n = 7) and 210 (n = 7). Expression of mRNA encoding FGFR1B and FGFR2B was determined by semi-quantitative real time RT-PCR using bovine specific primers, and cyclophilin A as the endogenous control. Reverse transcription was performed with SuperScriptIII (Invitrogen, São Paulo, Brazil) and PCR with Power SYBR green master mix (Applied Biosystems, Foster City, USA) in an ABI Prism ® 7500. Relative expression values were determined by the Pfaffl equation, analyzed by ANOVA and groups were compared by orthogonal contrast test (JMP software). Expression of mRNA encoding FGFR1B and FGFR2B was detected throughout gestation. mRNA abundance of FGFR1B increased from 90 to 120 days of gestation, when we previously observed an increase in FGF-10 expression and in the number of primary follicles. In contrast, FGFR2B mRNA levels decreased from 60 to 90 days of gestation, around the time primordial follicle activation begins. In conclusion, both receptors of FGF-10 were shown to be developmentally regulated during gestation. The temporal association between gene expression patterns and fetal follicular dynamics suggests that FGFR1B and FGFR2B signalling may be involved in the control of follicle activation. Keywords: fgfr1b, fgfr2b, bovine. A058 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION LEUKEMIA INHIBITOR ORY FACT CTOR STIMULATES TES THE IN VITRO DEVEL VELOPMENT OF SHEEP PREANTRAL AL FOLLICLES AND THE PRODUCTION OF EMBRYOS Valesca Barreto Luz 1 , Ticiana Franco Pereira da Silva 1 , Valdevane Rocha Araújo 1 , Ana Beatriz Graça Duarte 1 , Juliana Jales de Hollanda Celestino 2 , Deborah de Melo Magalhães Padilha 1 , Roberta Nogueira Chaves 1 , Ivina Rocha Brito 1 , Anderson Pinto Almeida 1 , Cristiano Feltrin 3 , Marcelo Bertolini 3 , Gerlane Modesto da Silva 1 & José Ricardo de Figueiredo 1 1 UECE, FORTALEZA, CE, BRAZIL. 2 UNILAB, REDENÇÃO, CE, BRAZIL. 3 UNIFOR, FORTALEZA, CE, BRAZIL. The aim of this study was to investigate the effects of leukemia inhibitory factor (LIF), with or without FSH on the development of sheep preantral follicles (PAF) and the influence embryo production. Ovaries of sheeps were dissected, and follicles (> 200 µm) were selected and cultured individually in drops of 100 µL of base medium, that consisted of α-MEM supplemented with 3 mg/mL of BSA, ITS (insulin 10 µg/mL, transferrin 5.5 µg/mL and selenium 5 ng/mL), 2 mM of glutamine, 2 mM of hypoxantine, 50 µg/mL of ascorbic acid and LIF (50 ng/ mL) in the presence or absence of sequential rFSH (day 0-6: 100 ng/mL, days 6-12: 500 ng/mL and days 12-18: 1000 ng/mL). In vitro culture (IVC) was performed at 39°C and 5% CO2 for 18 days, and all medium was replenished every six days. At the end of the IVC period, all healthy follicles were carefully opened and oocytes (>110 µm) with a homogeneous cytoplasm and surrounded by at least one compact layer of cumulus cells were selected for in vitro maturation (IVM). The selected cumulus-oocyte complexes were washed three times in maturation medium: TCM199B (TCM199 + sodium bicarbonate) supplemented with BSA (1 mg/mL), pyruvate (1 mM/mL), rFSH (0.5 µg/mL), LH (5 µg/mL), 17β-estradiol (1 ug/mL), EGF (10 ng/mL), IGF-1 (50 ng/mL) and cysteamine (100 µM/mL). After washing, the oocytes were transferred to 50 µL drops of maturation medium under mineral oil and then incubated for 40 h at 39ºC in 5% CO2. Mature oocytes from treatment with the highest rate of MII were activated by exposure to 5 µM ionomycin in TCM-HEPES supplemented with 0.4% BSA for 5 min followed by 3.5 h incubation in 2 mM 6-DMAP in SOFaa + 0.4% BSA at 39°C. Then, activated embryos were cultured in SOFaa + 10% SFB for 5 days. Cleavage and embryo rates were determined on days 2 and 5, respectively (Day 0: embryo activation). The results were compared by Chi-square test and were expressed as percentages. There were no significant differences between treatments regarding oocytes for IVM (= 110 µm) and meiosis resumption. In the group of follicles cultured with LIF in the absence of FSH showed a rate of MII of 29.63%, however in the presence of FSH this rate was of 16.66%. After parthenogenesis, which was only performed in treatment without FSH, were obtained 7 embryos (8 cels) from 12 mature oocytes. We concluded that the LIF associated or not to FSH stimulates the in vitro development of sheep PAF. Furthermore, embryos can be obtained from PAF cultured in the presence of LIF. Keywords: leukemia inhibitory factor, follicule, embryo. N s365
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