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Estradiol benzoate (EB), estradiol cypionate (EC), and gonadotrophin releasing hormone (GnRH) were evaluated as ovulation inducers for TAI of crossbred lactating cows. In experiment I (n = 45), the follicular dynamics was evaluated, while in experiment II (n = 171) the reproductive efficiency was evaluated. All females were submitted to a synchronization protocol: insertion on day 0 (D0) of intravaginal progesterone -release intravaginal device (PRID; 1g) plus administration of EB 2mg; D8, PRID removal, administration of 0.150mg PGF2α and 400 IU of eCG, and cows were allocated into three treatments: 1) TEB9 - IM administration of 1mg EB on D9 and AI performed 52 hours after PRID removal; 2) TEC - IM administration of 1mg EC on D8 and AI performed 48 hours after withdrawal of PRID; and 3) TGnRH - administration of 100pg GnRH on D10 and AI performed 52 hours after removal of PRID. The follicular growth rate was lower in the EC group (p 0.05). The use of EB, EC, and GnRH as ovulation inducers in the TAI protocol did not show marked differences in the evaluated patterns of follicular dynamics and pregnancy. RESUMO Benzoato de estradiol (EB), cipionato de estradiol (EC) e hormônio liberador de gonadotrofinas (GnRH) foram avaliados como indutores da ovulação na IATF em vacas mestiças em lactação. No experimento I (n=45) a dinâmica folicular foi avaliada, enquanto no experimento II (n=171) a eficiência reprodutiva foi avaliada. Todas as fêmeas foram submetidas a um protocolo de sincronização: inserção no dia 0 (D0) de dispositivo intravaginal de liberação de progesterona (PRID; 1g) mais administração de EB 2mg; D8, remoção de PRID, administração de 0,150mg de PGF2α e 400 UI de eCG, e as vacas foram alocadas em três tratamentos: 1) TEB9 - administração IM de 1mg EB em D9 e AI realizada 52 horas após a remoção PRID; 2) TEC - administração IM de 1mg EC em D8 e AI realizada 48 horas após a remoção de PRID; e 3) TGnRH - administração de 100μg GnRH em D10 e AI realizada 52 horas após a remoção de PRID. A taxa de crescimento folicular foi menor no grupo EC (p 0,05). O uso de EB, EC e GnRH como indutores de ovulação em protocolos de IATF não mostrou diferenças marcantes nos padrões avaliados de dinâmica folicular e prenhez.

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

Experiments were conducted to investigate whether supplementation of cryopreservation medium with ascorbate, dithiothreitol (DTT) or an inhibitor of caspase-3 (z-DEVD-fmk) could improve post-thaw survival of bovine embryos produced in vitro (IVP). For all experiments, embryos were harvested on day 7 after insemination and subjected to controlled-rate freezing in medium containing 1.5 M ethylene glycol and treatments as described below. In experiments 1-3, embryos were cryopreserved in freezing medium with ascorbate (0, 0.1, 0.3 or 0.5 mM), DTT (0, 50, 100 or 200 μM) and z-DEVD-fmk (0, 50, 100 or 200 μM), respectively. Post-thaw survival was assessed at 24, 48 and 72 h. For experiments 4-5, embryos were cryopreserved in freezing medium with or without 0.1 mM ascorbate. At 24 h post-thaw, embryo total cell number, DNA fragmentation and levels of reactive oxygen species (ROS) were evaluated. Embryos subjected to freezing and thawing in medium supplemented with 0.1 mM ascorbate had greater (p .05) re-expansion rates at 24, 48 and 72 h and hatching rate at 72 h as compared to embryos not treated with ascorbate. Post-thaw cryosurvival was not affected by the addition of either DTT or z-DEVD-fmk to medium used for cryopreservation. Embryos cryopreserved in medium supplemented with 0.1 mM ascorbate had reduced (p .001) levels of intracellular ROS and fewer (p .001) cells with DNA fragmentation. In conclusion, post-thaw survival of bovine IVP embryos is enhanced by supplementation of freezing medium with ascorbate.

We aimed to characterize the expression of angiogenesis-related genes in corpus luteum (CL) and cumulus cells (CC) during estrous cycle in gilts of different genetic groups, as well as to study the relation between gene expression and phenotypic data. Forty five gilts were used as follows: L1, Commercial Line 1 (Large White x Landrace x Duroc) (n = 15); L2, Commercial Line 2 (Large White x Landrace x Pietrain) (n = 15); and Piau, Piau breed gilts (n = 15). Estrus observation started from 120 days of age. After the second observed estrus females were slaughtered (n = 3) on days 3, 5, 10, 14 and 18 of estrous cycle (first day of estrous cycle as Day 0). CL sampling was performed on days 3, 5, 10 and 14 and collection of CC and follicular fluid on days 14 and 18. Follicular fluid was used for analysis of estradiol levels and CC and CL samples for analysis of angiogenesis-related genes expression, ANGPT-1/-2 and TEK in CC and MMP-2, VEGFA, VEGFR-1/-2, ANGPT-1/-2 and TEK in CL. Piau gilts showed lower ovulation rate than both L1 and L2 gilts (P 0.05), lower number of large antral follicles (6 mm) at 18 days than L2 gilts (P 0.05), and smaller diameter of the largest follicles at 14 days than L1 gilts (P 0.05). Piau and L2 gilts showed higher estradiol levels in follicular fluid on day 18. Expression of ANGPT-1 and -2 genes in CC did not differ among genetic groups neither among days of the estrous cycle, but TEK gene expression was higher in L1 than L2 gilts on day 18. Expression of VEGFA, VEGFR-2 and MMP-2 genes in CL did not differ among genetic groups and days of cycle, but VEGFR-1 expression was higher in Piau than L2 gilts on days 10 and 14, and it was higher in L1 than L2 gilts on day 14. The ANGPT-1/-2 and TEK genes expression in CL were significantly higher in Piau than L1 gilts on day 10. The ANGPT-2/ANGPT-1 gene expression ratio in CL was higher in L1 than Piau and L2 gilts at 14 days, suggesting a shorter luteal phase for L1 gilts. Results indicated differences among genetic groups for the pattern of the angiogenesis-related genes expression in CL along estrous cycles, which may be reflected in phenotypic traits such as ovulation rate, estradiol levels in follicular fluid and number and diameter of antral follicles.

This study aimed to describe longitudinal testicular volume (TV) data of Nellore bulls by using nonlinear mixed models. Dataset consisted of 2,294 TV measurements from 505 bulls with ages ranging from 563 to 4,307 days. Nine nonlinear models were evaluated: Brody, Gompertz, Hill, Logistic I and II, Meloun, Michaelis-Menten, Mitscherlich and von Bertalanffy. Goodness of fit was evaluated by Akaike's information criterion (AIC), Bayesian information criterion (BIC), adjusted R2, percentage of convergence, error sum of squares (ESS), mean absolute deviation (MAD), and average prediction error (APE). These criteria were used to select the best model, then the absolute growth rate (AGR) for TV was estimated by the first derivate of the adjusted model related to time ( ∂ Y / ∂ t ). The values of adjusted R2, ESS, MAD and APE were similar among models. Percentage of convergence was higher for the Logistic I (76.8%), Logistic II (75.5%) and Mitscherlich (78.6%) models, but Logistic I and II showed the lowest values of AIC and BIC, indicating a better fit, so the Logistic I model was chosen for subsequent analyses. The TV growth occurred at a high rate until the inflection point, which was estimated at approximately 22 months of age; it stabilized and reached a plateau at approximately 2,500 days of age. This may suggest that TV is more related to sexual maturity than precocity. Additionally, the Logistic I model was used to estimate the growth curve and the AGR of the testicular length and width. As a result, testicular length increased at a higher rate than testicular width until approximately 1,600 days of age, indicating that the testes become longer with increasing age.

The growth hormone (GH) and growth insulin-like factor-1 (IGF-1) act directly upon the regulation and growth in the different phases of preantral follicles. Thus, it is necessary to define their sequentiality until the in vitro preovulatory development. Therefore, the study aimed to assess the effects of a sequential medium containing GH and/or IGF-1 in the long-duration in vitro culture of preantral ovarian follicles. Ovarian fragments were cultivated: first half (days 1-7), second half (days 7-14) or during 14 culture days. Treatments were identified as: αMEM+; GH → IGF-1; IGF-1 → GH and GH + IGF-1. The culture was designed in 24-well plates, in an incubator at 37°C and 5% CO2 . The parameters of normality, viability, follicles (primordial/in developing) and follicle diameter were evaluated. In addition, the ultrastructure was confirmed with electron transmission microscopy. The results showed that the culture treated with GH → IGF-1 kept the follicular normality and the viability until the 14th day of culture and increased both in the follicular development until 7th day and in the follicular diameter until 14th day, when compared to the control. The treatments IGF-1 → GH and GH + IGF-1 were not effective in the developing and follicular diameter after 7 days of culture, and also reduced the percentage of viability. It is concluded that the bovine preantral follicles cultured in the sequential medium treated with GH → IGF-1 improved the follicular development until the first half of the culture and kept these parameters with normality, viability and ultrastructure until the second half of the in vitro culture.

We aimed to evaluate apoptosis and myogenesis related genes expression in embryos and fetuses from two divergent genetic groups of pigs: Piau breed and a commercial line. Thirty females (15 Piau and 15 commercial line) were selected at 120 days of age. Estrous cycle was observed and on the third estrus females were considered sexually mature. Gilts were inseminated with semen from males of the respective breed. Three females from each breed were slaughtered at five different gestational ages: 15, 30, 45, 60 and 90 days. Whole embryos (15 and 30 d) and samples of longissimus dorsi muscle from fetuses (45, 60 and 90 d) were collected for RNA extraction. Expression of apoptosis and myogenesis related genes (BAX, BCL2, FGF4, IHH, HHIP, SHH, SOX2, WNT1 and WNT4) were evaluated by quantitative real time PCR. There was significant effect of interaction between breeds and gestational ages for all genes evaluated (P

This study aimed to describe the testicular growth curve in Mangalarga Marchador stallions fitting nonlinear models to data of total scrotal width (TSW) and testicular volume (TV). A cross-sectional study was conducted with 120 stallions, aging from 1.75 to 21.93 years. The parameters of nonlinear models (Brody, Gompertz, Hill, Logistic I and II, Meloun I and II, Michaelis-Menten, Mitscherlich, and von-Bertalanffy) were estimated by Gauss-Newton iterative process. Goodness of fit was evaluated by the corrected Akaike (AICc) and Bayesian (BIC) information criteria, adjusted R2, error sum of square, mean absolute deviation and average prediction error. Based on the goodness of fit, the Logistic I model fitted better for TSW while Logistic II fitted better for TV growth curve. The inflection point (IP) of TSW growth was estimated in 5.23 cm at 0.76 years; the IP of TV growth was estimated in 139.36 cm3 at 2.57 years. The TV growth curve reached a plateau later than TSW growth curve, mainly because testicular height maintains growth for a longer period. In addition, there were moderate and positive correlations between whiter height and TSW and TV (r = 0.51 and r = 0.53, respectively). Findings suggest that TV is more associated with sexual maturity than sexual precocity. In addition, the moderate and positive correlations between whiter height and TSW and TV indicate that testicular traits can be included in selection programs of Mangalarga Marchador stallions with the same relevance as the morphometric traits.

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.

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. Resumo: Os objetivos deste estudo foram avaliar a vitrificação de folículos pré-antrais bovinos com dimetilsulfóxido (D) e sacarose (S) adicionando α-tocoferol 5mmol/L (T5) ou 10mmol/L (T10) e, avaliar o aquecimento com meio essencial mínimo (m) com ou sem sacarose (s). Ovários de fêmeas bovinas foram coletados de abatedouro, para o experimento I (n= 66) e II (n= 51). No laboratório fragmentos ovarianos foram distribuídos aleatoriamente para o controle fresco e 8 tratamentos de vitrificação (Controle e Dm; Dms, a DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Os fragmentos ovarianos foram colocados na solução de vitrificação (5 min) e imersos em nitrogênio líquido (-196°C). Após uma semana os fragmentos foram aquecidos e analisados. No experimento I, folículos pré-antrais foram observados morfologicamente para avaliação histológica (normal, degenerados e estádio de desenvolvimento). No experimento II, folículos pré-antrais foram mecanicamente isolados do tecido ovariano e examinados com o azul de trypan, observando mortos e vivos corados e não corados respetivamente. Os tratamentos a DSm, DSms e DST10m foram eficazes na preservação da morfologia in situ. No entanto, a viabilidade de folículos pré-antrais isolados após a vitrificação manteve-se elevada apenas no tratamento DST10m. Assim, DST10m preservou as taxas de sobrevivência e integridade morfológica durante a vitrificação de folículos pré-antrais bovinos.

BACKGROUND Bulls are of great importance in the productive chain and for this reason they should have a good semen quality. There is no doubt that sperm morphology is very important to bull fertility, although little is known about how exactly the abnormal morphologies may affect sperm functions. OBJECTIVES To detail the morphological description of the aplastic midpiece defect (AMD), as well as to understand its consequences for male fertility based on membrane and acrosome status, mitochondrial membrane potential and DNA integrity parameters. MATERIALS AND METHODS The bulls were divided into two groups: control, consisting of satisfactory potential breeders (n = 3); and AMD, consisting of unsatisfactory potential breeders with a high percentage of AMD (n = 3). Bulls were evaluated by the breeding soundness evaluation; five ejaculates were collected from each animal and analyzed by flow cytometry. RESULTS Spermatozoa from AMD group exhibited lower sperm motility and vigor (p

This study aimed to evaluate the correlation between testicular biometry and semen variables, as well as, to relate testicular variables to the probability of selecting Nellore bulls with desirable sperm morphology when conducting breeding soundness evaluations (BSE). A total of 2055 BSEs from 506 bulls comprised the dataset. Biometric variables evaluated were: scrotal circumference, testicular volume, width, length, ratio and eccentricity; and semen variables were sperm motility, major sperm defects, minor sperm defects and normal sperm. Data of testicular biometry were correlated with data for semen variables using the Pearson’s correlation assessment. Effects of testicular variables in selecting for sperm morphology of bulls in the BSE were evaluated by logistic regression. Scrotal circumference, testicular volume, length and width were positively correlated to sperm motility (0.18 to 0.19) and normal sperm (0.24 to 0.27) and negatively correlated with values for major defects (−0.24 to −0.27), but for testicular ratio and eccentricity there were coefficients near zero for all semen traits. Testicular ratio and eccentricity were not suitable for predicting the probability of selecting a bull based on semen variables using the BSE, but scrotal circumference, testicular volume, length and width were highly significant (P

This study aimed to assess characteristics of bovine cryopreserved sperm and evaluate its relation to field fertility in fixed-time artificial insemination (FTAI). Semen samples of 16 bulls were used to inseminate 811 Nellore cows, and four of these bulls were also used to inseminate 101 Nellore heifers. Samples of the same ejaculate used for FTAI from each bull were analysed in the laboratory after thawing. Sperm motility and vigour were subjectively assessed by light microscope, and integrity of the plasma and acrosome membranes, and H2 O2 production were evaluated by flow cytometer. Relation among sperm characteristics and pregnancy rate of cows and heifers were evaluated by univariate and multivariate logistic regression. Subjective sperm motility and vigour did not affect the probability of pregnancy in cows or heifers. In univariate analysis for pregnancy in cows, sperm traits related to acrosome injury positively affected probability of pregnancy mainly when associated with plasma membrane integrity; H2 O2 production seems to be less important than plasma membrane integrity in affecting probability of pregnancy. In multivariate analysis, sperm traits related to injured acrosome positively affected probability of cow and heifer pregnancies while intact acrosome was negatively related to cow pregnancy. Intact plasma membrane and high H2 O2 production were positively related to cow pregnancy but negatively related to heifer pregnancy. Results suggest that a capacitation-like status of the acrosome may benefit probability of pregnancy in cows.

Cow fertility traits are key factors that influence beef production profitability, and is particularly important in tropical environments where achieving high reproductive rates is challenging. Genomic selection (GS) has the potential to improve genetic gain rates for reproduction, if genomic estimated breeding values (GEBV) for these traits are sufficiently accurate. Several Bayesian models have already been proposed for GS, but the benchmarks used to compare them are still scarce, mainly for age at first calving (AFC) in Nellore cattle. A total of 714 AFC records of Nellore cows and 70 K SNPs were used to compare five models, Bayes A (BA), Bayes B (BB), Bayes Cπ (BCπ), Bayesian LASSO (BL) and Bayesian Ridge Regression (BRR). These models were compared by cross validation, randomly partitioning the whole population into 7 subsets (7-fold) and replicated 15 times. The prediction accuracy were 0.24 (0.11), 0.23 (0.11), 0.33 (0.13), 0.24 (0.11) and 0.38 (0.13), for BA, BB, BCπ, BL and BRR, respectively. Thus, BRR resulted in 14%, 15%, 5% and 14% additional prediction accuracy compared to BA, BB, BCπ and BL, respectively. Pearson and Spearman correlations between GEBVs obtained from BRR and BB models were, 0.97 and 0.94, respectively. It suggested that little difference in terms of animal selection would result from these methods. A more parsimonious model, such as BRR, can be successfully used in breeding programs to generate GEBVs which further enable consistent selection decisions. Although moderate accuracies of GEBV for AFC can be achieved, we found low efficiency of GS for AFC in the present population due to the small sample size and low heritability, reinforcing that GS efficiency is highly dependent upon these factors.

Nonlinear mixed models were used to describe longitudinal scrotal circumference (SC) measurements of Nellore bulls. Models comparisons were based on Akaike's information criterion, Bayesian information criterion, error sum of squares, adjusted R2 and percentage of convergence. Sequentially, the best model was used to compare the SC growth curve in bulls divergently classified according to SC at 18-21 months of age. For this, bulls were classified into five groups: SC < 28cm; 28cm ≤ SC < 30cm, 30cm ≤ SC < 32cm, 32cm ≤ SC < 34cm and SC ≥ 34cm. Michaelis-Menten model showed the best fit according to the mentioned criteria. In this model, β1 is the asymptotic SC value and β2 represents the time to half-final growth and may be related to sexual precocity. Parameters of the individual estimated growth curves were used to create a new dataset to evaluate the effect of the classification, farms, and year of birth on β1 and β2 parameters. Bulls of the largest SC group presented a larger predicted SC along all analyzed periods; nevertheless, smaller SC group showed predicted SC similar to intermediate SC groups (28cm ≤ SC < 32cm), around 1200 days of age. In this context, bulls classified as improper for reproduction at 18-21 months old can reach a similar condition to those considered as good condition. In terms of classification at 18-21 months, asymptotic SC was similar among groups, farms and years; however, β2 differed among groups indicating that differences in growth curves are related to sexual precocity. In summary, it seems that selection based on SC at too early ages may lead to discard bulls with suitable reproductive potential.

In view of the importance of the genetic material of local breeds in the swine industry and the lack of information about reproductive performance of Piau females, two experiments were conducted to evaluate puberty and sexual maturity as well as the morphometry of embryos/fetuses, placenta and uterus during the first 90 days of gestation in Piau breed and Commercial line gilts. In experiment I, 37 Piau and 25 commercial line gilts were used. From the 120 days of age, detection of estrus was performed using mature boars from the first to third estrus of each gilt. Data regarding to age, body weight and estrus duration were recorded. After third estrus, females were slaughtered and ovaries were collected to determine ovulation rate. In experiment II, 36 Piau and 18 commercial line gilts were distributed into three groups according to the mating: Commercial, commercial line females x commercial line male; cross-mated, Piau females x commercial line male; and Piau, Piau females x Piau male. Gilts were slaughtered at 7, 15, 30, 45, 60 and 90 days of pregnancy. Piau females reached puberty and sexual maturity at the same age as commercial line females, but with lower weight; moreover, Piau group showed negative correlations of birth weight with puberty (−0.27) and sexual maturity (−0.29). Commercial gilts presented higher ovulation rate, weight and length of uterus, and length and thoracic circumference of fetuses. Nevertheless, number of fetuses was similar in all groups at 90 days of gestation suggesting that Piau females present higher survival rates of the conceptuses. The results showed differences between the genetic groups related to fetal and placental development, gestational losses, number of ovulations and uterine development. In addition, an intermediate status of fetal weight was observed in Piau/Commercial line crossbred conceptuses; thus, the selection of Piau females on reproductive traits to be mated with commercial line males would be an alternative to contribute to improvement of intramuscular fat content.

We performed a genome-wide mapping for the age at first calving (AFC) with the goal of annotating candidate genes that regulate fertility in Nellore cattle. Phenotypic data from 762 cows and 777k SNP genotypes from 2,992 bulls and cows were used. Single nucleotide polymorphism (SNP) effects based on the single-step GBLUP methodology were blocked into adjacent windows of 1 Megabase (Mb) to explain the genetic variance. SNP windows explaining more than 0.40% of the AFC genetic variance were identified on chromosomes 2, 8, 9, 14, 16 and 17. From these windows, we identified 123 coding protein genes that were used to build gene networks. From the association study and derived gene networks, putative candidate genes (e.g., PAPPA, PREP, FER1L6, TPR, NMNAT1, ACAD10, PCMTD1, CRH, OPKR1, NPBWR1 and NCOA2) and transcription factors (TF) (STAT1, STAT3, RELA, E2F1 and EGR1) were strongly associated with female fertility (e.g., negative regulation of luteinizing hormone secretion, folliculogenesis and establishment of uterine receptivity). Evidence suggests that AFC inheritance is complex and controlled by multiple loci across the genome. As several windows explaining higher proportion of the genetic variance were identified on chromosome 14, further studies investigating the interaction across haplotypes to better understand the molecular architecture behind AFC in Nellore cattle should be undertaken.

In order to evaluate whether ovarian volume, presence and diameter of the corpus luteum (CL) have effects on the number and quality of bovine recovered oocytes, 110 ovaries were obtained from the slaughterhouse. Cumulus oocytes complex were aspirated and evaluated under stereomicroscope. Oocytes were counted and classified according to their quality (Grades I, II, III and IV). Ovarian volume was weakly correlated to the number of good quality oocytes (P < 0.05). Ovaries with CL showed greater numbers of good quality oocytes than ovaries without CL (P < 0.05). Further, 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 in vitro embryo production programs.

The quality of oocytes is more critical for the success of bovine in vitro embryo production (IVP) than the conditions of in vitro culture of these structures. Despite knowledge about bovine follicular development, especially gonadotropin-dependent phase, be considerably wide, knowledge about effects of the estrous cycle phase, follicular or luteal, on recovery Mini -review Article

The objective of this study was to evaluate different concentrations of growth hormone (GH) on the development of bovine preantral follicles cultured included in the ovarian tissue (in situ) on the rates of morphologically normal, viable, primordial and developing follicles, as well as the oocyte and follicle diameter and ultrastructural analysis. Ovarian fragments collected from cows with no cross-breeds defined were cultured in situ for 1 and 7 days in minimal essential medium (α-MEM+) supplemented with different concentrations of recombinant human GH (0, 10, 25, 50 ng/ml). The ovarian fragments non-cultured (control) and cultured were processed for classic histology, mechanical isolation and electron transmission microscopy (MET). The parameters underwent anova (Tukey's and Dunnett's tests) and chi-square test (χ(2) ). After 7 days of culture, the treatment with 50 ng/ml GH showed no differences with fresh control (p > 0.05) and had greater effectiveness than in the 0, 10 and 25 ng/ml GH concentrations of the morphologically normal follicles. Regarding the primordial follicles, a reduction was observed in the 50 ng/ml GH concentration concomitant with the significant increase in developing follicles, differing from both the fresh control and the other GH concentrations tested. In addition, 50 ng/ml GH showed a larger follicle and oocyte diameter when compared to the other treatments cultured. Similar structures were ultrastructurally observed in the control group, 50 ng/ml GH. Follicles cultured in 10 ng/ml GH showed nuclear invagination, vacuoles and lesioned basal membrane. Hence, it is concluded that 50 ng/ml GH is the most effective concentration for the development of preantral follicles cultured in situ.

This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF-1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α-MEM(+) , supplemented with different concentrations of human recombinant IGF-1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO2 in 24-well plates with total replacement of the medium every 2 days. Non-cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF-1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF-1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p

The aim of this study was to determine the effectiveness of minimum essential medium alpha modification (α-MEM), tissue culture medium 199 (TCM-199), and McCoy's medium (McCoy's) on IVC of preantral follicles included in the bovine ovarian cortex (in situ). Bovine ovarian fragments were cultured in α-MEM, TCM-199, or McCoy supplemented ((+)) with glutamine, insulin, transferrin, selenium, ascorbic acid, BSA, penicillin, streptomycin, and HEPES buffer in 24-well plates, at 37 °C and 5% CO2 for 1 or 7 days. The morphology of follicles, normal, primordial and development (primary and secondary), as well as viability and morphometric variables of follicles and oocytes were assessed. The morphology and morphometry of preantral follicles were analyzed by ANOVA followed by the Tukey and Dunnett tests, and viability variables were determined by the chi-square test. The results showed that TCM-199(+) reduced significantly (P0.05) the percentage of morphologically normal and viable follicles after 7 days of culture compared to the control. Similar results were observed in McCoy(+), in which the percentage of viable follicles after 7 days of culture was significantly lower (P0.05) than in the control. However, it was similar (P0.05) between α-MEM(+) and TCM-199(+). Moreover, follicular diameters in McCoy(+) and TCM-199(+) were significantly smaller (P0.05) than in control and α-MEM(+) after 7 days of culture. In addition, the ultrastructure of preantral follicles was similar between the control and α-MEM(+) after 7 days of culture. In conclusion, α-MEM(+) showed to be the most effective medium to preserve morphology, morphometry and ultrastructure of bovine preantral follicles, ensuring their viability and growth after in situ culture.

A criopreservação do sêmen é de grande importância para diversas biotecnologias da reprodução, como Inseminação Artificial (IA), Produção de Embriões In Vitro (PIV) e Injeção Intracitoplasmática de Espermatozoides (ICSI). Avaliou-se a estabilidade e persistência da motilidade e vigor dos espermatozoides, assim como alterações da membrana plasmática, após a adição de Ringer com Lactato, citrato de sódio 2,92% ou solução TRIS ao sêmen caprino descongelado. O sêmen foi coletado de dois bodes da raça Parda Alpina, realizando-se os procedimentos padrões de análise e criopreservação seminal. Após a descongelação do sêmen, foram adicionados os diluentes Ringer Lactato, citrato de sódio 2,92% ou solução TRIS, realizando- se os Testes de Termorresistência (TTR), Supravital e Morfológico. No TTR, somente o grupo a que foi adicionada a Solução TRIS obteve motilidade e vigor por maior período (90 minutos; P< 0,05). Não foi encontrada influência da adição das soluções na análise morfológica, assim como no teste Supravital, o qual apresentou valores próximos aos encontrados para motilidade (P> 0,05). Concluiu-se que a adição das soluções não permite uma grande persistência da motilidade e vigor dos espermatozoides descongelados, porém a solução TRIS poderia ser utilizada para expansão de doses seminais utilizadas em biotecnologias reprodutivas in vitro. Cryopreservation of semen is of great importance for various reproductive biotechnologies such as artificial insemination (IA), In Vitro Embryo Production (PIV) and Intracytoplasmic Sperm Injection (ICSI). The objective of this work was to evaluate the stability and persistence of motility and strength of sperm, as well as changes in the plasma membrane after the addition of Ringer Lactate, sodium citrate 2.92% or TRIS solution in thawed goat semen. Semen was collected from two Alpine Brown goats, and standard procedures for cryopreservation and seminal analysis were performed. After thawing the semen, the extenders Ringer Lactate, sodium citrate 2.92% and TRIS solution were added and Thermoresistance (TTR), Supravital and Morphology Tests were carried out. In TTR, only the group received TRIS solution presented motility and strength for a longer period (90 minutes; P< 0,05). No influence of the addition of the solutions was found in the morphological analysis, as well as in the Supravital Test, which showed values close to those found for motility (P> 0.05). We concluded that the addition of the solutions does not allow a large persistence of motility and strength of thawed sperm, but TRIS solution could be used for expansion of seminal doses used in vitro reproductive biotechnology.

The aim of this study was to evaluate the effects of vitamin E associated with rapid thawing on cryopreserved goat semen. Two bucks were used and eight ejaculates per animal were collected using artificial vagina. Semen was diluted with the following treatments: BIOXCELL (control), BIOXCELL + Equex (sodium lauryl sulphate) and BIOXCELL + vitamin E 100 μM. Semen was packaged into 0.25 mL straws and cooled at 5°C for 1 hour. Freezing was performed in liquid nitrogen vapor (−155°C) during 15 minutes. Then, the straws were immersed in liquid nitrogen (−196°C). Straws were thawed at 38°C/60 seconds or at 60°C/7 seconds with immediate sperm analysis. Hypoosmotic swelling test was performed adding a 20 μL aliquot of thawed semen to 1 mL of hypoosmotic solution (100 mOsm·Kg−1) followed by incubation during 60 minutes in water bath (38°C). Vitamin E did not affect any studied parameters (P>0.05). Nevertheless, defrosting rate of 60°C/7 seconds improved sperm membrane functional integrity (P<0.05). Current knowledge about goat semen cryopreservation is not sufficient to ensure high post-thawing recovery rates; thus, this study brings important data about using antioxidants and different thawing rates on cryopreservation process.

Thirty adult male wistar rats were used in five groups. Two groups received oral treatment with aqueous solution of TORDON 2,4-D® 64/240 BR in different concentrations; group O1: 2 ml.L-1 (n = 6); group O2: 4 ml.L-1 (n = 6). Two groups received topical treatment in the dorsal cervical area, the following concentrations were used, group T1: 16.7 ml.L-1 (n = 6), and group T2: 400 ml.L-1 (n = 6). A control group was used without the addition of the herbicide (n = 6). At the end of the experiment, the animals were euthanized; the testes were collected and fixed for subsequent histological analysis in light microscope. The T1 group showed increase in albuginea weight (P < 0.05). The animals of the O2 group showed significant reduction (P < 0.05) of the height of seminiferous epithelium. The animals of the O1 and T2 groups did not present harmful effects on the spermatogenic morphometry.

In order to evaluate whether ovarian volume, presence and diameter of the corpus luteum (CL) have effects on the number and quality of bovine recovered oocytes, 110 ovaries were obtained from the slaughterhouse. Cumulus oocytes complex were aspirated and evaluated under stereomicroscope. Oocytes were counted and classified according to their quality (Grades I, II, III and IV). Ovarian volume was weakly correlated to the number of good quality oocytes (P0.05). Ovaries with CL showed greater numbers of good quality oocytes than ovaries without CL (P0.05). Further, presence of CL and its diameter positively influenced the probability of recovering good quality oocytes (P0.05). 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 in vitro embryo production programs.

Cryotolerance of bovine embryos produced in vitro (PIV) can be improved by l-carnitine. The objective of the present study was to determine whether the optimal concentration of l-carnitine is dependent on serum. Bovine embryos were produced in vitro with abattoir-derived oocytes. After fertilization (Day 0), oocytes (n = 2768) were randomly assigned in a 2 × 4 factorial design to culture in SOF-BE1 medium supplemented with or without 5% fetal bovine serum and l-carnitine at concentrations of 0.0, 0.75, 1.5, and 3.03 mM at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. The proportion of oocytes that cleaved was assessed on Day 3, and the proportion of oocytes that developed to the blastocyst and advanced blastocyst (expanded, hatching, and hatched) stages was determined on Day 7. Blastocysts and expanded blastocysts (n = 466) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. After thaw, embryos were cultured for 72 h in SOF-BE1 supplemented with 10% (v/v) fetal bovine serum and 50 mM dithiothreitol at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 9 times, and data were analysed by logistic regression. There was no interaction between serum and l-carnitine, at any of the concentrations tested, on embryo development or cryotolerance. Cleavage rates were not affected by serum or l-carnitine. Addition of serum during culture increased (P

The aim of the present experiments was to determine whether treatment with the antioxidants ascorbic acid and dithiothreitol, or an inhibitor of caspase-3, Z-DEVD-FMK, during cryopreservation could improve the cryotolerance of bovine embryos produced in vitro. For all experiments, bovine embryos were produced in vitro using abattoir-derived ovaries. At Day 7 after insemination, blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing following equilibration for 8 to 10 min in freezing medium [Hepes-TALP (Parrish et al. 1986) plus 1.5 M ethylene glycol and 0.5 M sucrose] with treatments as described below. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. For Experiment 1, embryos (n = 578) were equilibrated in freezing medium containing 0, 0.1, 0.3, or 0.5 mM ascorbic acid. For Experiment 2, embryos (n = 243) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM dithiothreitol. For Experiment 3, embryos (n = 227) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM Z-DEVD-FMK. Embryos frozen in freezing medium containing ascorbic acid had increased (P