Efecto de adicionar ácido ascórbico al medio de vitrificación de folículos preantrales bovinos

The aim of this study was to evaluate the effect of adding ascorbic acid (AA) to the vitrification medium of bovine preantral follicles (PFs) subjected previously to cooling at 4 °C for 4 h or 24 h. Ovaries were collected from Nelore heifers at 14 months of age. In the laboratory, ovarian fragments were removed from the cortical region and distributed to fragments as fresh control (C0h), and fragments to cooling at 4 ºC for 4 and 24 hours in TCM-199 plus HEPES and antibiotics. Of the cooled fragments, two were fixed as controls for each cooling time (C4h, C24h), and the remaining fragments were distributed in four vitrification treatments, using the TCM-199 medium associated with ethylene glycol and dimethyl sulfoxide (V), sucrose (VSUC) or ascorbic acid (VAA), and the treatment with ethylene glycol, dimethyl sulfoxide, sucrose and AA (VSUC+AA). After 72 h, fragments were warmed and fixed for histological analysis and mechanical follicular isolation. No difference (p>0.05) between C0h and C4h for morphologically normal PFs was detected (99.3 and 96.0%, respectively). Vitrification reduced the morphological integrity and follicular viability in all treatments compared to C0h; nevertheless, VAA treatment maintained the follicular viability like C24h (p>0.05). It is concluded that bovine PFs were conserved efficiently at 4 °C during 4 h, and the addition of ascorbic acid to the vitrification medium improved survival rates and kept the morphological integrity of the follicles.El objetivo del estudio fue evaluar el efecto de la adición de ácido ascórbico (AA) al medio de vitrificación de folículos preantrales (FPs) bovinos previamente enfriados a 4 °C durante 4 o 24 h. Se recolectaron ovarios de novillas Nelore a los 14 meses de edad. En el laboratorio se extrajeron fragmentos de ovario de la región cortical y se distribuyeron a fragmentos para control fresco (C0h), y fragmentos para refrigeración a 4 ºC por 4 y 24 horas en TCM-199 más HEPES y antibióticos. De los fragmentos enfriados, dos se fijaron como controles para cada tiempo de enfriamiento (C4h, C24h), y los restantes se distribuyeron en cuatro tratamientos de vitrificación, utilizando el medio TCM-199 asociado con etilenglicol y dimetilsulfóxido (V), sacarosa (VSUC) o ácido ascórbico (VAA), y el tratamiento con etilenglicol, dimetilsulfóxido, sacarosa y AA (VSUC+AA). Después de 72 h, los fragmentos se calentaron y fijaron para el análisis histológico y el aislamiento folicular mecánico. No se detectó diferencia (p>0.05) entre C0h y C4h para FPs morfológicamente normales (99.3 y 96.0%, respectivamente). La vitrificación redujo la integridad morfológica y la viabilidad folicular en todos los tratamientos en comparación con C0h; sin embargo, el tratamiento VAA mantuvo la viabilidad folicular similar a C24h (p>0.05). Se concluye que los FAs bovinos se conservaron eficientemente a 4 °C durante 4 h, y la adición de ácido ascórbico al medio de vitrificación mejoró las tasas de supervivencia y mantuvo la integridad morfológica folicular

Methods for evaluating the efficiency of the genetic multiplication by bovine embryo production

O objetivo deste trabalho foi avaliar estratégias de incremento da eficiência da produção in vitro de embriões bovinos (PIVE). Dois artigos foram desenvolvidos, o primeiro objetivou avaliar se o volume ovariano, a presença e o diâmetro do corpo lúteo (CL) têm efeito sobre o número e a qualidade de oócitos bovinos recuperados. O segundo artigo teve como objetivo comparar diferentes métodos estatísticos para predizer a probabilidade de gestação em um programa comercial de PIVE. Artigo 1: Foram obtidos em abatedouro 110 ovários. Os complexos cumulus-oócitos foram aspirados e avaliados em microscópio estereoscópico. Os oócitos foram contados e classificados de acordo com sua qualidade (grau I, II, III e IV). O volume do ovário foi correlacionado com o número de oócitos de boa qualidade (r = 0.33; P < 0.05). Os ovários com CL mostraram maior número de oócitos de boa qualidade do que ovários sem CL (P < 0.05). Além disso, a presença do CL e o seu diâmetro influenciaram positivamente a probabilidade de recuperação de oócitos de boa qualidade (P < 0.05). Em conclusão, o volume ovariano não é um bom parâmetro para predizer características ovarianas importantes; além disso, a análise do CL, a sua presença e seu diâmetro, pode ser uma boa ferramenta para melhorar a eficiência em programas de PIVE. Artigo 2: Redes neurais artificiais (RNA) e árvores de decisão têm provado ser bem sucedidas em diferentes áreas de estudo, tais como medicina, genética e produção animal. No entanto, a utilização destas metodologias na área de reprodução bovina ainda é muito escassa. No presente estudo, um conjunto de dados reais foi usado, composto por 9.697 transferências de embriões produzidos in vitro, 6.788 observações foram utilizadas no modelo de treinamento e 2.909 foram utilizadas para validação. O conjunto de dados foi analisado por meio de regressão logística, rede neural feed-forward, multilayer percepetron com uma e com duas camadas ocultas, e uma árvore de decisão baseada no algoritmo ID3. Todas as cinco análises foram comparadas pela curva ROC, capacidade preditiva, teste de Kolmogorov-Smirnov e correlação ponto bisserial. Em conclusão, todas as análises foram muito semelhantes. No entanto, a análise pela rede neural feed-forward classificou corretamente mais de 70% das gestações positivas. Apesar de ter classificado incorretamente quase 60% das não gestações, este resultado se torna interessante porque não detectar uma gestação positiva é mais prejudicial a um programa comercial que não detectar uma gestação negativa. Ainda assim, são necessários estudos com conjuntos de dados maiores, incluindo não somente variáveis categóricas, mas também variáveis quantitativas para garantir resultados mais precisos.The objective of the present study was to evaluate some strategies for increasing the efficiency of bovine in vitro embryo production (IVP). Two articles were developed, first one aimed to evaluate whether ovarian volume, presence and diameter of the CL have effect on the number and quality of bovine recovered oocytes. The second article aimed to compare different statistical methods to predict pregnancy in a commercial program of IVP. Article 1: one hundred and ten ovaries were obtained from slaughterhouse. Cumulus oocytes complex were aspirated and evaluated under stereomicroscope. Oocytes were counted and classified according to their quality (Grade I, II, III and IV). Ovarian volume was weakly correlated to the number of good quality oocytes (P < 0.05). Ovaries with CL show greater number of good quality oocytes than ovaries without CL (P < 0.05). Besides, presence of CL and its diameter positively influenced the probability of recovering good quality oocytes (P < 0.05). In conclusion, ovarian volume is not a good parameter itself to predict important ovarian characteristics; moreover, analysis of CL, its presence and diameter, may be a good tool to improve efficiency on IVP programs. Article 2: Artificial neural networks (ANN) and decision trees have proved to be very successful in different fields of study such as medicine, genetics and animal production. However, the use of these methodologies in bovine reproduction is still very scarce. In this study, a real dataset was used, consisting of 9,697 embryo transfers, 6,788 observations were used in training model and 2,909 were used for validation. Dataset was analyzed by logistic regression, feed-forward neural network, multilayer perceptron with one and two hidden layers, and a based on ID3 algorithm decision tree. All analyses were compared by ROC curve, predictive capacity, Kolmogorov-Smirnov test, and point biserial correlation. In conclusion, all analyzes were very similar. Nevertheless, analysis by the feed-forward neural network correctly classified more than 70 % of positive pregnancies. Despite having incorrectly classified almost 60 % of non-pregnancies, this result becomes interesting because not detect a positive pregnancy is more harmful to a commercial program than not detect a negative pregnancy. Still, studies with larger dataset and more explanatory variables are required, including not only categorical variables, but also quantitative variables to ensure more accurate results.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Vitamin E on cryopreservation of goat semen.

O objetivo deste estudo foi verificar se a vitamina E afeta a integridade estrutural da membrana plasmática dos espermatozóides caprinos, bem como verificar o potencial uso desta vitamina em meios diluentes de criopreservação de sêmen caprino. Foram utilizados 2 machos adultos da raça Parda alpina. Para as coletas de sêmen foi utilizada vagina artificial onde se obteve 8 ejaculados por animal. Após a coleta, fez-se a avaliação física do sêmen, morfológica dos espermatozóides e da integridade funcional da membrana espermática pelo teste hiposmótico. Em seguida, o sêmen foi diluído com os seguintes tratamentos: BIOXCELL® (Controle), BIOXCELL® + Equex, BIOXCELL® + Vitamina E 25&#956;M, BIOXCELL® + Vitamina E 50&#956;M e BIOXCELL® + Vitamina E 100&#956;M. Após as diluições finais, foram avaliados a motilidade progressiva e vigor espermático e realizado teste hiposmótico de cada tratamento. O sêmen foi envasado em palhetas de 0,25mL, as palhetas foram resfriadas a 5 oC, durante 1 hora em refil plástico contendo álcool etílico. O pré-congelamento foi realizado em vapor de nitrogênio líquido durante 15 minutos. Após esse período, as palhetas foram imersas no nitrogênio para o congelamento final do sêmen. As partidas foram descongeladas em banho-maria a 37 oC por 30 segundos, acondicionadas em tubos plásticos e homogeneizadas para a análise imediata de motilidade e vigor espermático, teste hiposmótico e teste de termorresistência. No sêmen fresco, as características físicas e morfológicas mantiveram-se dentro de parâmetros normais. Não houve correlação da motilidade progressiva com outras variáveis. O volume apresentou correlação negativa com o vigor espermático, defeitos menores e defeitos totais (r = -0,55, r = -0,52 e r = - 0,53, respectivamente) e correlação positiva com o teste hiposmótico (r = 0,44). Houve correlação média negativa (r = -0,48) entre a concentração de espermatozóides e os defeitos maiores. Os valores de defeitos menores e defeitos totais apresentaram correlação forte e positiva (r = 0,98). A motilidade progressiva, o vigor espermático e o teste hiposmótico do sêmen diluído não diferiram entre os tratamentos (P > 0,05). As médias gerais de motilidade e vigor logo após o descongelamento e ao longo do TTR não diferiram (P > 0,05) entre os tratamentos. A integridade funcional da membrana espermática, avaliada pelo teste hiposmótico, não diferiu (P > 0,05) entre os tratamentos. Foi observada correlação positiva entre motilidade e vigor dos espermatozóides em todos os tratamentos no sêmen diluído (pré-resfriado) e em todos os tempos do TTR. Não houve correlação entre o vigor e o teste hiposmótico dos espermatozóides em nenhum dos tratamentos no sêmen diluído e nos tempos 0H e 2H do TTR, havendo correlação média positiva no tratamento Equex no tempo 1H (r = 0,51) e nos tratamentos Controle, Equex e Vit. E 50&#956;M no tempo 3H (r = 0,44, 0,69, 0,57, respectivamente). Houve correlação entre a motilidade e o teste hiposmótico do sêmen diluído no Tramento Vit. E 50&#956;M (r = 0,68). As amostras de sêmen dos tratamentos Equex e Vit. E 100&#956;M descongeladas mostraram correlações entre motilidade e teste hiposmótico. Conclui-se que: Nenhum dos parâmetros avaliados neste estudo foi afetado pela Vitamina E.The objectives of this study were to examine whether vitamin E has an effect on the structural integrity of goat sperm plasma membrane, as well to investigate the potential use of this vitamin in extenders medium for cryopreservation of goat semen. Two adult Parda Alpina breed males were used, totalizing 8 semen samples for each one. After collection, the physical characteristics of the semen, the sperm morphology and the functional integrity of sperm membrane by hypoosmotic swelling test were evaluated. Then the semen was diluted as follows: BIOXCELL® (Control), BIOXCELL® + Equex, BIOXCELL® + Vitamin E (25&#956;M), BIOXCELL® + Vitamin E (50&#956;M) and BIOXCELL® + Vitamin E (100&#956;M). After the final dilutions, the progressive motility, sperm vigor and hypoosmotic swelling test were performed for each treatment. The semen was packaged in 0.25 ml straws, the straws were cooled to 5 oC for 1 hour in plastic container containing ethyl alcohol. The pre-freezing was done in liquid nitrogen vapor for 15 minutes. After this period, the straws were immersed in liquid nitrogen. The samples were thawed in a water bath at 37 oC for 30 seconds, packaged in plastic tubes and homogenized for immediate analysis of sperm motility and vigor, hypoosmotic swelling test and thermoresistance test. In fresh semen, the physical and morphological characteristics remained within normal parameters. No correlation of motility with other variables were detected. The average volume correlated negatively with sperm vigor, minor defects and total defects (r = -0.55, r = -0.52 and r = -0.53, respectively) and positive correlation with the hypoosmotic swelling test (r = 0, 44). A negative correlation (r = -0.48) was found between sperm concentration and major defects. The minor defects and total defects values were markedly positive correlated (r = 0.98). The treatments did not differ (P > 0.05) for progressive motility, spermatic vigor and hypoosmotic swelling test of semen. The motility and vigor after thawing and during the TTR did not differ (P > 0.05) among treatments. No significant difference (P > 0.05) among treatments after the completion of the hypoosmotic swelling test was detected. Correlation was observed between motility and vigor in all treatments in the diluted semen (pre-cooled) and at all times of the TTR. No correlation between the vigor and the hypoosmotic swelling test in any of the treatments on 0H and 2H of TTR times was found, however a correlation at 1H time Equex treatment (r = 0.51) and the Control, Equex and Vit. E 50&#956;M treatment at time 3H (r = 0.44, 0.69, 0.57, respectively) were found. The thawed semen samples from Equex treatment and Vit. E 100&#956;M treatment showed correlation between motility and hypoosmotic swelling test. It is concluded that: The vitamin E did not affect any of parameters under study.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Análise de associação genômica ampla associa o gene Lin-28 à idade ao primeiro parto na raça Nelore

peer reviewe

Testosterone serum profile, semen characteristics and testicular biometry of Mangalarga Marchador stallions in a tropical environment

This study was conducted to characterize the daily profile of testosterone secretion and its mean concentrations in the four seasons as well as to evaluate the semen characteristics and testicular biometry of Mangalarga Marchador stallions throughout the year in a tropical region. Three stallions were submitted to semen collections and evaluation of testicular biometry every 14 days along a year. Blood samples were collected once at the middle of each season, in a 20-min interval during 24 hr in order to evaluate the testosterone secretion profiles among seasons. Testosterone concentrations along the day were higher at the beginning of the afternoon (from 12:00 to 15:00 hr), but a circadian secretion was not clearly observed. Mean testosterone concentrations did not differ among seasons (p > .05), but a pattern of secretion along the day showed variations with higher concentrations in the afternoon during the winter. Ejaculate volume was higher during summer; however, sperm motility decreased in summer and spring. Total sperm in ejaculate, sperm morphology and testicular biometry kept constant along the year showing no differences among the seasons. The results demonstrated that in a tropical region, reproductive aspects of stallions did not show a clearly defined seasonal variation, and months of autumn and winter were not unsuitable for reproduction of the males

Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus α-tocopherol

Abstract: The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles

Recombinant bovine somatotropin in the synchronization of ovulation in crossbred dairy cows (Bos taurus indicus × Bos taurus taurus)

Aim: The aim of this study was to evaluate the effects of the administration of recombinant bovine somatotropin (rbST) at the moment of implementation of the timed artificial insemination protocol, on follicular dynamics and pregnancy rate in crossbred cows. Materials and Methods: A total of 346 cows were used in two experiments with a factorial 2×2 design. The cycling cows (Tcycling) and the anestrous cows (Tanestrous) were considered as factor 1 and the administration of rbST (TrbST) or not (Tcontrol) as factor 2. The experimental protocol: (1) Tcontrol – day 0 (D0), insertion of a progesterone-release intravaginal device (PRID) plus 2 mg of estradiol benzoate (EB); D8, PRID removal, plus 0.150 mg of prostaglandin F2α, and 400 IU of equine chorionic gonadotropin; D9, 1 mg of EB; and with artificial insemination at day 10; (2) TrbST – similar to Tcontrol plus 500 mg of rbST on D0. In experiment I, ultrasound examinations were performed in all treatments. In experiment II, the cows' pregnancy rate was evaluated. Data were analyzed with 5% probability. Results: There was no effect of the protocols on cows cyclicity or follicular growth rate (p>0.05). There was no interaction of the effects, administration of rbST, and the cyclicity of cows on the pregnancy rate. The total pregnancy rate observed was 49.0%. The pregnancy rate in cows receiving rbST was lower for anestrous compared with cycling cows (p<0.05). Conclusion: The administration of rbST did not alter the patterns of follicular dynamics nor the ovulation rate. However, cows in anestrous that received rbST had lower pregnancy rates than cycling cows