Seminal quality comparison of first and second cryopreserved ejaculates of Alpine kid goats by flow cytometry.

Discarding the first ejaculate is recommended as an alternative for improving seminal quality after long sexual resting, especially when semen should be used for cryopreservation. However, when the males are not in sexual resting the necessity to discarding the first ejaculate is still unknown. Therefore, this study aimed to compare by flow cytometry the quality of the first and second ejaculates. Ten kids and uniform goats between 5 and 6 months of age were used in a completely randomized design. Semen collection was carried out every 4 days, until a total of five ejaculates per animal in each treatment was completed. The fresh and frozen semen collected were processed and analyzed using macroscopic and microscopic parameters, resistance test, hypo-osmotic medium test, and flow cytometry (FC). The FC parameters were production of reactive oxygen species, plasma and acrosomal membrane integrity, and lipid peroxidation of the plasma membrane. The ejaculates did not differ for the resistance test, the reactivity in the hypo-osmotic medium and for the macroscopic and microscopic seminal parameters, except for sperm volume and concentration. The first ejaculate had a higher percentage of minor and total defects. None of the FC parameters analyzed differed between the first and second ejaculates. The first and second ejaculates demonstrated similar seminal qualities, so for Alpine kid goats without a sexual resting period, discarding the first ejaculate it is not recommended.

Effect of addition of ascorbate, dithiothreitol or a caspase-3 inhibitor to cryopreservation medium on post-thaw survival of bovine embryos produced in vitro.

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.

Can maintenance intake reduce production costs without compromising the reproductive performance in bucks?

Excessive intake of nutrients can result in decreases in the reproductive efficiency of goat bucks and increase production costs in goat e nterprises. In this study, the aim was to compare the reproductive features of young bucks fed diets for maintenance of growth or were fed ad libitum. Ten bucks were used (n = 5/group), with a completely randomized design. Blood and semen were collected from each animal every 2 weeks for nine collections. Semen was frozen and later analyzed using flow cytometry to compare reactive oxygen species, plasma membrane integrity, acrosome integrity, and membrane lipid peroxidation. Body condition score, sexual behavior, testicular biometry and histomorphometry of the testis, as well as oxidative enzymes and carbonylated proteins, were also evaluated. Animals fed ad libitum had a greater body condition score (3.32 + 0.08 × 2.88 + 0.05), sperm volume (0.68 + 0.04 × 0.52 + 0.03 mL), and testis circumference (25.2 + 0.21 × 24.5 + 0.24 cm) and volume (121 + 5.83 × 107 + 4.38 cm³, left testicle; 116 + 5.26 × 100 + 3.75 cm³, right testicle). Ejaculates of the bucks fed diets to maintain growth rate had a larger number of minor and total sperm morphology defects. There were no differences in testosterone concentrations and sexual behavior. In conclusion, there can be feeding for growth rate maintenance instead of ad libitum feeding, for the reduction in production costs of young bucks without compromising semen quality and reproductive functions.

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.

Powdered coconut water (ACP 406®) as an alternative base culture medium for in vitro culture of goat preantral follicles enclosed in ovarian tissue.

This study evaluated a powdered coconut water solution (ACP 406®) as a base culture medium on the in vitro survival and development of in situ goat preantral follicles. The ovarian fragments were either immediately fixed in Carnoy solution (non-cultured control) or individually cultured for 2 or 6 days. The following culture media (all containing 100 µg/mL penicillin and 100 µg/mL streptomycin) were evaluated: α-MEM (α-MEM alone, without additional supplementation); α-MEM+ (supplemented α-MEM); ACP (ACP®406 alone); or ACP+ (supplemented ACP®406). Additional supplementation includes: 1.25 mg/mL bovine serum albumin, 10 µg/mL insulin, 5.5 µg/mL transferrin, 5 ng/mL selenium, 2 mM glutamine, and 2 mM hypoxanthine. The endpoints (i) follicular morphology; (ii) development; (iii) estradiol production; and (iv) reactive oxygen species (ROS) were recorded. Data were analyzed using chi-square, Turkey, t-test or One-Way ANOVA. Differences were considered significant when P < 0.05. At day 2 of culture, a greater (P < 0.05) percentage of morphologically normal follicles was observed between ACP+ and ACP treatments. Moreover, at day 2 of culture, no hormonal difference (P < 0.05) was observed between ACP+ and both α-MEM treatments. At day 6 of culture when ACP and α-MEM treatments were compared the percentage of healthy follicles were similar (P > 0.05) among treatments. Overall, all treatments had lower primordial follicles (P < 0.05) accompany by greater developing follicles (P < 0.05) percentages than non-cultured control treatment, indicating primordial follicle activation. However, at day 6 of culture, the percentage of primordial follicle development were similar (P > 0.05) among the treatments. Likewise, no differences (P > 0.05) were observed for ROS production and follicular and oocyte diameters among treatments. Therefore, ACP+ has the equivalent efficiency to MEM+ in maintaining the survival and development of goat preantral follicles, representing an alternative plant-based low-cost culture medium for in vitro culture.

The base medium affects ultrastructure and survival of bovine preantral follicles cultured in vitro.

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 (P < 0.05) 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 (P < 0.05) than in the control. However, it was similar (P > 0.05) between α-MEM(+) and TCM-199(+). Moreover, follicular diameters in McCoy(+) and TCM-199(+) were significantly smaller (P < 0.05) 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.

Influence of corpus luteum and ovarian volume on the number and quality of bovine oocytes.

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.

Association of vitamin E with rapid thawing on goat semen.

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.

Developmental competence and expression of the MATER and ZAR1 genes in immature bovine oocytes selected by brilliant cresyl blue.

The objective of this work was to evaluate the selection of immature bovine oocytes by brilliant cresyl blue dye (BCB) and expression of transcripts MATER and ZAR1. Cumulus-oocyte complexes (COCs) from slaughterhouse ovaries were exposed to BCB diluted in mDPBS and incubated for 60 min at 38.5 degrees C in humidified air. After exposure those COCs were distributed in two groups, according to their cytoplasm colour: BCB+ (coloured cytoplasm) or BCB- (colourless cytoplasm). The control group was submitted to in vitro maturation (IVM) immediately after morphological selection and holding control group COCs were exposed to mDPBS without BCB but in the same incubation conditions of BCB+ and BCB- group. The COCs of all groups were submitted to IVM, in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rate (72 h post-insemination) was similar between control (65.3%) and BCB+ (64.4%) groups, but greater than (p < 0.05) holding control (49.8%) and BCB- (51.3%) groups. Blastocyst rate (192 h post-insemination) was not different between BCB+ (18.5%) and control (16.3%) groups, but greater (p < 0.05) than BCB- (8.4%) group. No difference was found for blastocyst rate between holding control group (14.2%), control and BCB+ groups. The relative expression of MATER and ZAR1 genes was evaluated by real-time PCR in immature oocytes collected from the control, holding control, BCB+ and BCB- groups. Despite the relative expression of MATER in holding control, BCB+ and BCB- were down regulated in comparison to control group there was no statistical difference (p > 0.05) in the relative expression of MATER and ZAR1 transcripts among groups. The results indicate that the BCB dye detects immature oocyte populations with different developmental competence, although no improvement in in vitro embryo production using oocytes exposed or not to BCB was observed. Development competence of immature oocytes exposed to BCB does not seem to be associated with variations in the expression of MATER and ZAR1 transcripts.

Estrus behavior in Guzera (Bos taurus indicus) breed cows / Comportamento estral de vacas da raça Guzerá (Bos taurus indicus)

The estrus behavior was evaluated during an induced and natural subsequent estrus in twelve Guzera breed cows, in the winter and summer seasons in two consecutive years. The proportion of cows that responded to estrus synchronization was 84.8 percent. The effect of the interaction season x year of experiment on estrous cycle length was observed. Pro-estrus was longer in the summer (57.69±4.72h) and in the natural estrus (74.23±4.41h) than in the winter (38.95±4.02h) and in the induced estrus (22.40±4.36h). Estrus length was similar in the winter (11.48±0.70h) and in the summer seasons (13.40±0.82h) as well as among cows with induced (12.47±0.75h) and natural estrus (12.41±0.76h). The number of mounts accepted during estrus and the number of mounts accepted/ hour in estrus were similar in winter (29.17±2.86 and 2.59±0.22, respectively) and summer (31.45±3.36 and 2.42±0.26, respectively) as well as between induced (30.23±3.10 and 2.54±0.24, respectively) and natural estrus (30.40±3.14 and 2.47±0.24, respectively). The length and intensity of estrus were not influenced by synchronization or season of the year. Longer period of pro-estrus in summer may favor the identification of sexually active animals in this season.

A manifestação do comportamento estral foi avaliada durante um estro induzido e o estro subseqüente, em 12 vacas da raça Guzerá, no inverno e no verão em dois anos consecutivos. A proporção de vacas que respondeu à sincronização de estros foi de 84,8 por cento. Observou-se efeito de interação entre estação e ano sobre a duração do ciclo estral. O proestro foi maior no verão (57,69±4,72h) e no estro natural (74,23±4,41h), que no inverno (38,95±4,02h) e no estro induzido (22,40±4,36h). A duração do estro foi similar no inverno (11,48±0,70h) e no verão (13,40±0,82h), assim como entre as vacas com estro induzido (12,47±0,75h) e natural (12,41±0,76h). O número de montas recebidas durante o estro e o número de montas recebidas/hora em estro foram similares no inverno (29,17±2,86 e 2,59±0,22, respectivamente) e no verão (31,45±3,36 e 2,42±0,26, respectivamente), assim como entre o estro induzido (30,23±3,10 e 2,54±0,24, respectivamente) e o natural (30,40±3,14 e 2,47±0,24, respectivamente). A duração e a intensidade do estro não foram influenciadas pela sincronização ou pela estação do ano. O maior período de proestro no verão pode favorecer a identificação dos animais sexualmente ativos.

Efeito do manejo diferenciado de amamentaçäo sobre retorno da atividade ovariana e níveis sangüíneos de colesterol, glicose, uréia e progesterona, em vacas Nelore em anestro / The effect of different types of suckling management on ovarian activity reestablishment and on plasma levels of cholesterol, glucose, urea and progesterone in anestrous Nelore cows

O objetivo do presente experimento foi verificar o efeito de diferentes manejos de amamentaçäo sobre parâmetros reprodutivos e sua relaçäo com os níveis plasmáticos de colesterol, glicose, uréia e progesterona. Foram utilizadas 40 vacas pós-parto da raça Nelore distribuídas ao acaso em quatro tratamentos:T1 = shang mais uma mamada diária; T2 = shang e bezerro ao pé;T3 = uma mamada diária;e T4 = bezerro ao pé. O regime de amamentaçäo diferenciada durou 28 dias; neste período, coletaram-se amostras de sangue para análises. A interrupçäo da mamada por 67 horas(shang) foi feita no início do período experimental. O nível plasmático médio de colesterol e glicose foi inferior (P<0,05) nas vacas de T4 e T2, respectivamente. Em funçäo dos períodos avaliados dentro dos tratamentos, os níveis plasmáticos de uréia e glicose apresentaram variaçöes. Com relaçäo aos parâmetros ligados à reproduçäo, as concentraçöes plasmáticas médias de progesterona de 1,38; 2,03; e 1,39ng/mL, observados nos tratamentos T1, T2 e T3, respectivamente, foram maiores (P<0,05) que 0,503ng/mL do T4, apresentando comportamento linear crescente para os tratamentos T1, T2 e T3, mantendo-se baixa no tratamento T4. A manifestaçäo de estro sofreu influência do manejo (P<0,05), ao contrário do retorno da atividade ovariana e da gestaçäo

Efeito da alteração do peso corporal sobre o desempenho reprodutivo de vacas leiteiras submetidas a diferentes níveis nutricionais no pré e no pós-parto / Effect of Bodyweight Changes on Reproductive Perfomance in Dairy Cattle Cows under Two Levels of Nutrition during Pre-and Post-partum

A alteração do peso corporal foi avaliada em 40 vacas de três graus de sangue, distribuídas em quatro tratamentos (T1, T2, T3 e T4), com variação no nível de suplementação concentrada nos períodos pré (N = normal e A = alta) e pós-parto (N1 = normal e A1 + alta). Diferença significativa (P < 0,05) foi observada entre os graus de sangue em relação à alteração do peso corporal das fêmeas nos dias 30 e 60 do período pós-parto. Observou-se que animais com maior taxa de ganho de peso apresentaram menor intervalo, em dias, do parto à concepção: T2 = 83; T1 = 1 09,3; T4 = 114, T3 = 156,3.

The bodyweight changes were observed in 40 breeding dairy cows distributed in 4 treatments (T1, T2, T3, and T4) with variation in the leveis of concentrate supplementation in pre-partum: N = nor-mal and H = high and post-partum: N1 = normal and H i = high, where, T1 = HN1, T2 = NH1, T3 = HH1 and T4 = NN1 ). Significative difference (P < 0,05) was observed between treatments in relation to changes of bodyweight between breeding dairy cows in the days 30 and 60 postpartum. lt was observed that animais with major gain weight showed shorter interval between parturition and conception (T2 = 83.0, T1 = 1 09.3, T4 = 114.00 and T3 = 156.3).