Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos

Objective: To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. Design: Comparison between oocyte groups and between early embryo stages. Setting: Laboratories of embryo manipulation and molecular biology from Departamento de Genetica (FMRP) and Departamento de Ciencias Basicas (FZEA) - University of Sao Paulo. Sample(s): Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. Main Outcome Measure(s): Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). Result(s): Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good-and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. Conclusion(s): Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development. (Fertil Steril (R) 2010; 93: 2507-12. (C) 2010 by American Society for Reproductive Medicine.)

Human-animal interaction, stress, and embryo production in Bos indicus embryo donors under tropical conditions

This study investigated the effect of human-animal interaction (HAI) and the stress response on the quality of embryo production in superovulated Nelore (Bos indicus) cattle, under tropical conditions. Thirty-two females underwent a superovulation protocol for 5 days. Cortisol concentrations were determined in blood plasma collected on days 0, 4, and 5. Artificial insemination was performed on days 4 and 5, and nonsurgical embryo flushing on day 11. Embryo production and viability were determined. Human stimulation, animal behaviors, accidents, and handling time were recorded to assess HAI. Cattle age was negatively correlated with accidents, frequency of aversive behaviors, and negative stimuli by stockperson during transit through corral compartments to receive superovulation treatments. The factor analysis revealed two distinct groups. The first group was called stressed and had higher cortisol concentration than the nonstressed group, 16.0 +/- 2.1 and 12.5 +/- 1.0 ng/mL, respectively. Comparisons between these groups showed that the frequency of voice emissions by the stockperson and the number of accidents were higher in the stressed group, and also, the mean handling time was longer in the stressed group than for the nonstressed. As a result, viability rate of the embryos was 19% lower in the stressed group (P < 0.05). This indicates that intensive negative HAI is likely related to stress, which affects embryo production in a superovulation program.

Evaluation of the contribution of the placentomal fusion during gestation in cattle

The placenta of mammals is a structure formed by the juxtaposition of the fetal membranes and the maternal tissues. The main function of the placenta is to regulate the physiological interchange between the fetus and the mother as well as to operate as an important endocrine organ during the gestation. The placentomal fusions were characterized throughout gestation of cattle using macroscopic, histological and flow cytometry analyses. Analyzing the cell cycle phases with a flow cytometry, a balance between the G2M phase and apoptosis was observed, suggesting that the placentomal fusions do not interfere in the placentary maturation process, which is a pre-requirement for the fetal-maternal disconnection and the release of fetal membrane. (C) 2011 Elsevier B.V. All rights reserved.

Heat tolerance and the effects of shade on the behavior of Simmental bulls on pasture

The objectives were to assess the degree of thermolysis capacity as a characteristic of heat tolerance of the Simmental beef cattle and evaluate the effects of shade and shade type (artificial: AS, trees: TS, or no shade: NS) on daily behavior patterns during summer. Black globe temperature (BGT) was different under the two types of shade (P < 0.05) and was lower under the TS (P < 0.01) and under AS (P > 0.01) than average BGT in the sun. Animals when in AS used more intensely the shade (P = 0.002) mostly lying down under it (10.00-14.00 hours), while time standing was similar (P = 0.107) between TS and NS. Bulls without shade (NS) spent significantly more time at the water trough and most part of the day standing idle (72.4%, 10.1 h/14 h). TS bulls spent more time grazing/standing (P < 0.001). The Simmental bulls that were in TS and AS spent more time ruminating than bulls that stay without shade (NS). The availability of shade changes grazing, rumination and idling behavior of cattle in response to environmental conditions. Shade provided by trees can be more efficient than artificial shading as cattle spent more time grazing when tree shade was available. Thermolysis capacity can be used to select heat-tolerant animals.

Assessment of DGAT1 and LEP gene polymorphisms in three Nelore (Bos indicus) lines selected for growth and their relationship with growth and carcass traits

The aim of this study was to analyze LEP and DGAT1 gene polymorphisms in 3 Nelore lines selected for growth and to evaluate their effects on growth and carcass traits. Traits analyzed were birth, weaning, and yearling weight, rump height, LM area, backfat thickness, and rump fat thickness obtained by ultrasound. Two SNP in the LEP gene [LEP 1620(A/G) and LEP 305(T/C)] and the K232A mutation in the DGAT1 gene were analyzed. The sample consisted of 357 Nelore heifers from 2 lines selected for yearling weight and a control line, established in 1980, at the Estacao Experimental de Zootecnia de Sertaozinho (Sertaozinho, Brazil). Three genotypes were obtained for each marker. Differences in allele frequencies among the 3 lines were only observed for the DGAT1 K232A polymorphism, with the frequency of the A allele being greater in the control line than in the selected lines. The DGAT1 K232A mutation was associated only with rump height, whereas LEP 1620(A/G) was associated with weaning weight and LEP 305(T/C) with birth weight and backfat thickness. However, more studies, with larger data sets, are necessary before these makers can be used for marker-assisted selection.

Prematuration of bovine oocytes with butyrolactone I reversibly arrests meiosis without increasing meiotic abnormalities after in vitro maturation

Objectives: Asynchrony between nuclear and cytoplasmic maturation, and possibly damage to the oocyte meiotic spindle, limits the application of in vitro maturation (IVM) in assisted reproduction. Several studies have suggested that Prematuration with meiosis blockers may improve oocyte quality after IVM, favoring early embryogenesis. Thus, we investigated the effect of Prematuration with the nuclear maturation inhibitor butyrolactone I (BLI) on the meiotic spindle and chromosomal configuration of bovine oocytes. Study design: Immature oocytes obtained from cows slaughtered in a slaughterhouse (n = 840) were divided into the following groups: (1) control (n = 325), submitted only to IVM in TCM199 for 24 h; (2) BLI 18 h (n = 208) submitted to meiotic blockage with 100 mu M BLI for 24 h (Prematuration) and then induction of IVM in TCM199 for 18 h; and (3) BLI 24 h (n = 307), pre-matured with 100 mu m BLI for 24 h followed by 24 h of IVM in TCM199. The oocytes were then fixed, stained by immunofluorescence for morphological visualization of both microtubules and chromatin, and evaluated. Results: Meiotic arrest occurred in 90.2% of the oocytes cultured with BLI. Maturation rates were similar for all groups (80.3%, 73.6% and 82.7% for the control, BLI 18 h and BLI 24 h groups, respectively). We observed 81.3% normal oocytes in metaphase II in the control group, and 80.0% and 81.2% in the BLI 18 h and BLI 24 h groups, respectively. The incidence of meiotic anomalies did not differ between groups (18.7%, 20.0% and 18.8% for the control, BLI 18 h and BLI 24 h, respectively). Conclusion: Prematuration with butyrolactone I reversibly arrests meiosis without damaging the meiotic spindle or the chromosome distribution of bovine oocytes after in vitro maturation. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

Aflatoxins and cyclopiazonic acid in feed and milk from dairy farms in Sao Paulo, Brazil

The occurrence of aflatoxins (AF) B(1), B(2), G(1), G(2) and cyclopiazonic acid (CPA) in feeds, and AFM(1) and CPA in milk was determined in dairy farms located in the northeastern region of Sao Paulo state, Brazil, between October 2005 and February 2006. AF and CPA determinations were performed by HPLC. AFB(1) was found in 42% of feed at levels or 1.0-26.4 mu g kg(-1) (mean: 7.1 +/- 7.2 mu g kg(-1)). The concentrations of AFM(1) in raw milk varied between 0.010 and 0.645 mu g l(-1) (mean: 0.104 +/- 0.138 mu g l(-1)). Only one sample was above the tolerance limit adopted in Brazil (0.50 mu g l(-1)) for AFM(1) in milk. Regarding CPA in feed, six (12%) samples showed concentrations of 12.5-1533 mu g kg(-1) (mean: 57.6 +/- 48.7 mu g kg(-1)). CPA was detected in only three milk samples (6%) at levels of 6.4, 8.8 and 9.1 mu g l(-1). Concentrations of aflatoxins and CPA in feed and milk were relatively low, although the high frequency of both mycotoxins indicates the necessity to continuously monitor dairy farms to prevent contamination of feed ingredients.

Seroprevalence of orthopoxvirus in an Amazonian rural village, Acre, Brazil

Vaccinia virus strains from the family Poxviridae have been frequently isolated in Brazil and associated with outbreaks of exanthematic disease affecting cows and humans. An ELISA IgG was applied to evaluate the seroprevalence of orthopoxviruses in a community located in a rural settlement in the Amazon region, where no orthopoxvirus outbreaks have yet been reported. An overall seroprevalence of 27.89% was found, and it was 23.38% in the non-vaccinated population (smallpox vaccination). These results strongly suggest that orthopoxviruses circulate in this population, and it is the first finding of seropositivity for orthopoxviruses in a population without any previously reported outbreaks.

Intraspecific sequence variation in 16S rRNA gene of Ureaplasma diversum isolates

Ureaplasma diversum infection in bulls may result in seminal vesiculitis, balanoposthitis and alterations in spermatozoids. In cows, it can cause placentitis, fetal alveolitis, abortion and the birth of weak calves. U. diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. These microorganisms were submitted to Polymerase Chain Reaction for 16S gene sequence determination using Tact High Fidelity and the products were purified and bi-directionally sequenced. Using the sequence obtained, a fragment containing four hypervariable regions was selected and nucleotide polymorphisms were identified based on their position within the 16S rRNA gene. Forty-four single nucleotide polymorphisms (SNP) were detected. The genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains. (c) 2011 Elsevier B.V. All rights reserved.

Genetic Heteroscedasticity for Domestic Animal Traits

Animal traits differ not only in mean, but also in variation around the mean. For instance, one sire’s daughter group may be very homogeneous, while another sire’s daughters are much more heterogeneous in performance. The difference in residual variance can partially be explained by genetic differences. Models for such genetic heterogeneity of environmental variance include genetic effects for the mean and residual variance, and a correlation between the genetic effects for the mean and residual variance to measure how the residual variance might vary with the mean. The aim of this thesis was to develop a method based on double hierarchical generalized linear models for estimating genetic heteroscedasticity, and to apply it on four traits in two domestic animal species; teat count and litter size in pigs, and milk production and somatic cell count in dairy cows. The method developed is fast and has been implemented in software that is widely used in animal breeding, which makes it convenient to use. It is based on an approximation of double hierarchical generalized linear models by normal distributions. When having repeated observations on individuals or genetic groups, the estimates were found to be unbiased. For the traits studied, the estimated heritability values for the mean and the residual variance, and the genetic coefficients of variation, were found in the usual ranges reported. The genetic correlation between mean and residual variance was estimated for the pig traits only, and was found to be favorable for litter size, but unfavorable for teat count.

Utilização do grão tostado de soja (Glicine max (L) Merril) na alimentação de vacas em lactação

Soybeans were roasted in an experimental roaster during 2 or 3 minutes either at 380 or 490 ºC air temperature and kept or not for 30 minutes under steeping. The heat treatment effects on soybeans were evaluated in a first trial through two different procedures: a) after 16 hours of an “in situ” ruminal incubation period, all heat treated soybeans showed an increased (P<0,05) rumen undegraded protein (RUP) content in comparison to raw soybeans; b) a pepsin/pancreatin “in vitro” procedure showed that roasting at 380 ºC, during 2 minutes, with steeping, or at 380 ºC , during 3 minutes without steeping, did not affect the “in vitro” enzymatic intestinal digestibility of soybeans (P>0,05). In a second trial, soybeans roasted at 380 ºC, during 2 minutes and kept under steeping were included in isonitrogenous and isocaloric lactating dairy cows rations and evaluated in comparison to raw soybeans, with two other protein sources as references. There were no differences (P>0,05) between the roasted and raw soybeans diets related either to dry matter or crude protein intakes, milk yield and composition and plasma urea nitrogen (PUN) and milk urea nitrogen (MUN) concentrations, but roasted soybeans yielded significantly (P<0,05) more milk per kg of dry matter or crude protein intake than raw soybeans.

Padronização da técnica de imunoistoquímica para identificação de Listéria monocytogenes em placentas humanas e tecido nervoso central de ruminantes.

Este projeto foi desenvolvido no Laboratório de Patologia Experimental do Hospital de Clínicas de Porto Alegre (HCPA), com a aprovação da Comitê de Ética em Pesquisa do HCPA e com apoio financeiro parcial do Fundo de Incentivo à Pesquisa e Eventos do HCPA (FIPE). O experimento 1, chamado de projeto piloto, teve como objetivo implementar a técnica de IHQ para identificar a Listeria monocytogenes (L.m.), utilizando anticorpo policlonal antilisteria monocytogenes (Biodesig ). Vários testes foram realizados para acertar a diluição (1:1000) que foi diferente da preconizada pelo fabricante. Os blocos de parafina, de dez placentas provenientes de parto prematuro ou aborto foram utilizados para os cortes histológicos e a preparação das lâminas para a coloração Hematoxilina e Eosina (HE) e imunoistoquímica (IHQ). As lâminas foram identificadas por números para resguardar a identidade das pacientes. O resultado do HE mostrou alterações inflamatórias em oito placentas e L. m. foi identificada pelo IHQ em cinco dessas placentas. O objetivo do 2º experimento foi identificar a L. m. em tecido nervoso cerebral de ruminantes, utilizando a técnica implementada no projeto piloto. O material utilizado neste trabalho foi cedido pelo Setor de Patologia Veterinária do Departamento de Patologia da Universidade Federal de Santa Maria. Os casos estudados (2 ovinos, 1 caprino e 2 bovinos) tinham suspeitas clínicas diversas e as necropsias dos animais evidenciaram aspectos sugestivos da doença. Os cinco casos foram confirmados pelo IHQ, comprovando a importância da utilização desta técnica para o diagnóstico da listeriose no SNC de ruminantes. O 3º experimento objetivou identificar a L. m. em placentas encaminhadas ao Serviço de Patologia do HCPA no ano 2000. Da mesma forma que no experimento 1, as lâminas foram identificadas por números. Após o levantamento realizado nos registros dos exames anatomopatológicos (AP) deste setor, observou-se que 714 AP eram de placentas provenientes de aborto, parto prematuro e nascimento a termo examinados naquele período. Foram sorteados 254 AP para análise através de HE, revelando que 148 desses AP apresentavam alterações inflamatórias (corioamnionite, vilite e deciduite). Os blocos destas placentas foram utilizados para fazer as lâminas e realizar IHQ. A consulta aos prontuários dos casos com alterações inflamatórias permitiu observar que um deles tinha a confirmação bacteriológica de L. m. na placenta, tornando-se este o controle positivo. O controle negativo foi selecionado entre aqueles sorteados que não apresentavam alterações inflamatórias. A presença de L. m. foi identificada em 33,78% das placentas analisadas pela técnica IHQ. Corioamnionite e vilite foram as alterações inflamatórias que mostraram diferença estatística significativa nas placentas positivas. L. m. estava presente nas placentas de 1º, 2º e 3º trimestres gestacionais. A idade das gestantes, casos de aborto e/ou parto prematuro não mostraram diferença estatística significativa com a presença ou ausência de L. m. nas placentas. Abortos habituais ocorreram em pacientes com ou sem L. m. no tecido placentário. Conclusão: a técnica de imunohistoquímica pode ser utilizada para confirmar o diagnóstico histopatológico de listeriose em placentas e tecido nervoso central de ruminantes.

Manejo reprodutivo pós-parto de vacas primíparas e desenvolvimento de terneiros

O trabalho teve por objetivo avaliar os efeitos de cargas animais de 240 e 320 kg PV/ha em campo nativo (T1 e T2, respectivamente), de 400 kg PV/ha em pastagem melhorada com azevém (Lolium multiflorum L.) por 80 dias pós-parto e, após, carga animal igual a T2 em campo nativo (T3), e do desmame dos terneiros aos 100 dias (DP) ou aos 180 dias (DC) de idade sobre o desempenho reprodutivo de vacas de corte primíparas Hereford e Braford, e sobre o desenvolvimento de seus terneiros. A taxa de prenhez (TP) não foi influenciada (P>0,05) pelos tratamentos (T1 = 93,8%; T2= 90,6%; T3= 100%), nem pela idade de desmame (DP= 97,8%; DC= 91,3%). Houve efeito significativo dos tratamentos (P<0,05) sobre a TP aos 21 dias (T1= 15,6%; T2= 0,0%; T3= 17,9%) e aos 42 dias (T1= 46,9%; T2= 37,5%; T3= 71,4%) após iniciada a estação de acasalamento. O intervalo entre partos (IEP) e o intervalo parto-concepção (IPC) foram influenciados (P<0,01) pelos tratamentos. O IEP e o IPC para T1, T2 e T3 foram 390,9; 399,0 e 386,8 dias e 105,9; 114,0 e 101,8 dias, respectivamente Terneiros filhos de vacas Braford no T2 foram mais pesados ao desmame (P<0,05) do que terneiros filhos de vacas da mesma raça no T1 e filhos de vacas Hereford no T2. Os terneiros do DP tiveram pesos ajustados aos 180 dias menores (P<0,01) do que os do DC (174,6 kg vs. 197,2 kg, respectivamente). Não houve efeito (P>0,05) dos tratamentos, nem da idade de desmame sobre o peso dos terneiros aos 365 dias de idade. Os novilhos do DP tiveram pesos de abate e de carcaça, aos 14 meses, inferiores (P<0,01) aos novilhos do DC. Entretanto, o ganho de peso e o rendimento de carcaça dos novilhos do DP foram superiores (P<0,01) aos novilhos do DC. Não houve efeito (P>0,05) da idade de desmame sobre a espessura de gordura subcutânea.

Aspectos epidemiológicos da paratuberculose bovina no Rio Grande do Sul

Descrevem-se os achados clínicos e patológicos da paratuberculose em uma criação intensiva de bovinos de leite no municí pio de Capela de Santana, RS. Os sinais clínicos foram observados em oito de um total de 345 bovinos, consistindo em diarréia crônica refratária aos tratamentos, emagrecimento progressivo e queda na produção de leite. As principais lesões macroscópicas, observadas nos oito animais necropsiados, incluíam intestino delgado com acentuado espessamento da parede e superfície mucosa de aspecto reticulado, semelhante às circunvoluções cerebrais, lesão essa perceptível, através da serosa. A luz intestinal estava preenchida com conteúdo fluido e de aspecto leitoso. Os vasos linfáticos do mesentério mostravam-se mais evidentes sendo que alguns tinham aspecto varicoso. Os linfonodos mesentéricos estavam aumentados de volume e, ao corte, fluía grande quantidade de líquido leitoso. Focos de mineralização foram observados na íntima das artérias, nas válvulas cardíacas e na serosa do rúmen. As principais lesões macroscópicas incluíam enterite, linfadenite e linfangite granulomatosa caracterizada por infiltrado inflamatório com macrófagos, células gigantes de Langhans que continham grande quantidade de bacilos álcool-ácido-resistentes. As lesões vasculares consistiam em degeneração e mineralização das túnicas í ntimas e média das artérias de grande calibre associada a proliferação de colágeno. Havia calcificação da serosa do rúmen atrofia hepatocelular difusa e hepatite granulomatosa multifocal. O M. avium subsp. paratuberculosis (Map) foi isolado em amostras de intestino e linfonodos de 8 vacas Holandesas (3,5%) com doença de Johne, dentre 229 amostras cultivadas provenientes de um rebanho leiteiro. Amostras inoculadas em HEYM com micobactina produziram colônias identificadas como Map, segundo as caracterí sticas fenotí picas próprias como: crescimento lento, coloração álcool-ácido-resistente (A.A.R.) e dependência a micobactina. O laboratório de Referência da OIE confirmou a amostra isolada. O M. avium subsp. paratuberculosis (Map) foi isolado em amostras de intestino e linfonodos de 8 vacas Holandesas (3,5%) com doença de Johne, dentre 229 amostras cultivadas provenientes de um rebanho leiteiro Amostras inoculadas em HEYM com micobactina produziram colônias identificadas como Map, segundo as caracterí sticas fenotí picas próprias como: crescimento lento, coloração álcool-ácido-resistente (A.A.R.) e dependência a micobactina. O laboratório de Referência da OIE confirmou a amostra isolada. Não houve isolamento do agente em 221 amostras intestinais quando processadas, após 2 anos de sua colheita. O teste de IDGA aplicado como “screening”, detectou 26 vacas (11,4%) positivas, dentre 228 animais testados e sacrificados em matadouro. O teste de ELISA adsorvido, utilizando o antí geno PPA-3 detectou 125 (39,8%) amostras positivas. O ELISA não adsorvido detectou mais 32 (10,1%) reagentes positivos, dentre os 314 bovinos testados. A prevalência da infecção causada pelo Map em 36 rebanhos leiteiros procedentes de 25 municí pios do Rio Grande do Sul foi estimada em 44,6% das 1316 amostras testadas. A infecção foi identificada em 35 (97,2%) dos 36 rebanhos testados e presentes em todos os municí pios incluí dos. A ocorrência da doença de Johne foi enfatizada, tanto a forma clí nica quanto a infecção subclí nica no Rio Grande do Sul, sugerindo a adoção de medidas de controle sejam aplicadas na proteção dos rebanhos leiteiros nacionais.

Efeito de polimorfismos no gene LEP na expressão da leptina em adipócitos de bovinos de corte

Os caracteres produtivos são normalmente influenciados por muitos fatores, sendo difícil determinar todos os locos envolvidos em um fenótipo específico. Por isso, a seleção animal tem se baseado principalmente em uma estimativa direta ou indireta do fenótipo. A leptina é um importante regulador do metabolismo energético, da adiposidade e da reprodução. E por desempenhar diferentes funções, pode ser considerado um bom gene candidato para o estudo de associações entre marcadores moleculares e a eficiência reprodutiva ou ganho de peso. Em várias espécies, têm sido descritos diversos polimorfismos no gene da leptina, influenciando o ganho de peso, a reprodução, e outras características produtivas. Em bovinos, o STR IDVGA51 e o SNP LEPSau3A1, foram descritos por afetarem a performance reprodutiva, os alelos IDVGA51*181 e LEPSau3A1*2 estando associados a um aumento no intervalo entre partos de 79 e 81 dias, respectivamente, e os STRs BMS1074 e BM1500 afetam o ganho de peso, em vacas, no pós-parto: os alelos BMS1074*151 e BM1500*135 reduzindo e aumentando, respectivamente, o ganho de peso diário. Para confirmar o efeito ou não destes alelos na expressão do gene da leptina, este trabalho comparou os níveis de mRNA de leptina em animais portadores e não portadores dos alelos IDVGA51*181, LEPSau3A1*2, BMS1074*151 e BM1500*135, com os objetivos de: 1. Verificar as distribuições genotípicas e alélicas dos marcadores IDVGA51, BMS1074, BM1500 e LEPSau3A1, em uma amostra de 137 bovinos da raça Brangus-Ibagé, provenientes da Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA-Pecuária Sul, Bagé-RS). 2. Identificar, no rebanho, animais portadores e não portadores dos alelos IDVGA51*181, LEPSau3A1*2, BMS1074*151 ou BM1500*135, previamente descritos como associados à eficiência reprodutiva e ganho de peso. 3. Avaliar os animais, criados em campo nativo, com pastagem natural, quanto à sua condição corporal. 4. Realizar procedimento cirúrgico, para obtenção de amostras de tecido adiposo subcutâneo e omental. 5. Dosar os níveis de mRNA de leptina nos adipócitos destes dois tecidos, através do método quantitativo em tempo real (Real-Time RT-PCR), comparando a expressão do gene LEP entre indivíduos portadores e não portadores dos alelos de predisposição a ganho de peso e desempenho reprodutivo.