Lincoln da Silva Amorim

The hypoosmotic swelling test in fresh rabbit spermatozoa

The hypoosmotic swelling test (HOST) has proved to be a good tool for evaluating the membrane integrity of spermatozoa of various domestic animals including cattle, horses, and swine. However, the best approach for using this technique in rabbit semen has not been tested. The present study aimed to establish the best hypoosmotic solution (HS) for testing membrane integrity in fresh rabbit semen. Sucrose solutions with the following osmolarities were used: 50, 60, 75, 100, 125 and 150mOsm/L. Semen samples (n=30) were collected from five mature White New Zealand rabbits (six collections per rabbit) at 72h intervals. After macroscopic evaluation, 10microL of semen was immediately added to 2mL of each solution and incubated for 1h at 37 degrees C. Sequentially, 20microL of semen diluted in HS were evaluated with oil immersion using a phase-contrast microscope. A total of 200 spermatozoa were counted in at least five different fields, and sperm tails were classified as non-coiled, coiled, and strongly coiled. The respective percentages of spermatozoa with coiled tails (coiled plus strongly coiled) in the six solutions listed above were 54.8, 65.2, 54.3, 53.9, 38.9 and 29.4%. Percentage of strongly coiled spermatozoa was: 40.2, 51.0, 43.2, 41.5, 32.7 and 26.9 for the six solutions, respectively. According to total and strong coiling 60mOsm/L was superior to others treatments (P<0.05). Results suggest that the 60mOsm/L solution would be most desirable for use in HOST in fresh rabbit spermatozoa.

Interrelationships among morphology, echotexture, and function of the bovine corpus luteum during the estrous cycle.

It has been suggested that ultrasound image attributes are a potential indicator of physiological and functional status of the corpus luteum (CL) in several species, including cattle. The aims of this study were to evaluate CL morphological, functional and echotextural characteristics, and also to investigate the hypothesis that those attributes are correlated and change similarly throughout an estrous cycle. Ovaries of crossbred (Bos taurus taurus x Bos taurus indicus) heifers were evaluated using ultrasonography daily throughout an interestrus interval using a B-mode, real-time ultrasound machine equipped with a 5 MHz linear-array rectal transducer, during a natural estrous cycle (Experiment 1; n=12) or during a shortened cycle, with luteolysis induction 10d after estrus (Experiment 2; n=6). Blood samples were collected for assay of plasma progesterone concentrations. Corpora lutea areas were measured and daily images of each CL were videotaped and digitized for computer-assisted analysis using custom-developed software. In Experiment 1, area of luteal tissue increased until a maximum value 10d after estrus (P<0.001), followed by a plateau phase, and then a decline beginning 14 d after estrus. Luteal tissue area was highly correlated to plasma progesterone concentrations (r=0.86; P<0.001). When luteolysis was induced in Experiment 2, loss of CL function (decrease in plasma progesterone concentrations to metestrous values) preceded tissue regression by 48 h (24h compared with 72 h; P<0.001). The mean pixel value of ultrasound images did not change in Experiment 1 (P>0.70), but a day effect on this attribute was observed in Experiment 2 (P=0.052). In contrast, mean pixel value was correlated to plasma progesterone concentrations in Experiment 1 (r=-0.63; P<0.05), but not in Experiment 2 (r=-0.28; P>0.10). In regard to CL heterogeneity, defined as the standard deviation of the mean pixel value of the luteal tissue, a time effect was observed following both natural (Experiment 1; P<0.009) and luteolysis-induced (Experiment 2; P<0.05) estrous cycles (P<0.05). Moreover, this variable was correlated with plasma progesterone concentrations (r=-0.71 and -0.58 in Experiments 1 and 2, respectively; P<0.01), indicating that CL images were more heterogeneous during metestrus and after luteolysis (functional regression). In summary, morphological and echotextural attributes were correlated with CL function and underwent similar changes during the estrous cycle. Luteal tissue heterogeneity, assessed by ultrasonography, is considered a potential indicator of CL functional status, because it is correlated to circulating progesterone concentrations.

Dinâmica folicular em cabras da raça Toggenburg em lactação tratadas ou não com somatotropina bovina recombinante

The effect of the r-bST injection was evaluated on the goats estrous cycles. Twenty-four Toggenburg does were used in two treatments: T1 (n=12) treated with r-bST; and T2 (n=12) treated with saline solution (control). After the first r-bST injection, the estrous cycles were checked between two estrus. The estrous cycle length and interovulatory period of the goats did not differ between treatments (P>0.05). Estrous cycles with two, three and four waves of follicular growth were observed. The number of waves during the estrous cycle were not affected by the r-bST treatment (P>0.05). The number of 3mm follicles was different between T1 and T2 (P 0.05) between treatments. The r-bST did not affect the ovarian response during the estrous cycle. The r-bST not affected the follicular dynamic of Toggenburg lactating does, but increased the number of emerging follicles ≥3mm during the estrous cycle.

Condição corporal e desempenho produtivo de cabras Alpinas no início de lactação

The objective of this study was to evaluate the influence of body condition at birth on Alpine goat productive parameters in early lactation. Sixty-eight females were assigned to three groups according to the body condition score (BCS) - low (1.00 - 2.75); intermediate (2.75 - 3.50) and high (3.50 - 5.00) and evaluated from kidding to the eighth week of lactation. The feed intake and milk production were recorded on a daily basis and the body weight, body condition score and milk composition at weekly intervals. Animals with a high body condition score presented lower dry matter intake, neutral detergent fiber and crude protein compared to those with low and intermediate body condition scores. The average time for dry matter intake recovery of the animals with the three body condition scores was 16 days (2.3 weeks) postpartum, and stabilization by the end of the experiment. In the sixth week of lactation, animals with low body condition score had the same body weight of animals with intermediate body condition score. There was no effect of the body condition at birth on the body condition score changes and milk production during the experiment. The milk fat content of animals with high body condition score was lower in the first two weeks postpartum compared to animals with low and intermediate body condition score. The high body condition at birth influences negatively the ad libtum intake of dairy goats in the early lactation, but when it is used feed of high quality with high energy levels, the influence of the body condition at birth is nil to variations in weight, BCS and milk production.

Body condition and metabolic profile of Alpine goats at the onset of lactation

Objetivou-se avaliar a influencia da condicao corporal ao parto sobre alguns metabolitos sanguineos em cabras no inicio da lactacao. As cabras (68 no total) foram distribuidas em tres grupos de acordo com o escore da condicao corporal (ECC) ao parto: entre 1,00 e 2,75 (animais magros); entre 2,75 e 3,50 (condicao corporal intermediaria); e entre 3,50 e 5,00 (animais gordos). O experimento foi realizado nos primeiros 60 dias de lactacao e as coletas de sangue, apos o parto e semanalmente ate a oitava semana de lactacao. Foram feitas analises para determinacao das concentracoes de acidos graxos nao-esterificados (AGNE), beta-hidroxibutirato (BHBA), glicose, colesterol total e lipoproteina de alta densidade (HDL). As concentracoes plasmaticas dos metabolitos nao diferiram entre os grupos. As concentracoes de AGNE, nos animais dos tres grupos, ao parto e nas oito primeiras semanas de lactacao, foram de 658,46 e 232,90 mg/dL, respectivamente, e os valores de BHBA ao parto, de 10,31 ± 3,0 mg/dL para os animais magros; 6,88 ± 0,9 mg/dL para os intermediarios; e 4,21 ± 0,8 mg/dL para os animais gordos. Os valores de glicose ao parto foram de 112,54 ± 15,4 mg/dL para os animais magros; 90,93 ± 13,5 mg/dL para os animais intermediarios; e 132,47 ± 26,7 mg/dL para os animais gordos. Os valores medios de colesterol total e de HDL nas oito primeiras semanas pos-parto foram de 109,01 ± 2,5 e 54,9 ± 1,39 mg/dL para os animais magros; 83,0 ± 21,7 e 56,0 ± 1,30 mg/dL para os intermediarios; e 84,3 23,9 e 54,5 ± 1,93 mg/dL para os gordos. As alteracoes no metabolismo energetico de cabras leiteiras de media producao diminuem, independentemente da condicao corporal ao parto, quando fornecida dieta de alta qualidade e com altos niveis de energia.

Follicular dynamics and gene expression in granulosa cells, corpora lutea and oocytes from gilts of breeds with low and high ovulation rates.

Follicular dynamics and the expression of candidate genes using real-time polymerase chain reaction (PCR) were compared during the oestrous cycle of pig breeds with high (commercial line; n=24) and low (local Brazilian Piau; n=21) ovulation rates and prolificacy. Gilts were killed on Days 0, 4, 10 and 18 of the oestrous cycle and visible ovarian follicles were classified by follicular diameter. Recovered cumulus-oocyte complexes were classified as normal or atretic and frozen in liquid nitrogen until RNA extraction. Low ovulation rates and/or prolificacy in Piau gilts was associated with a different pattern of follicle development, with lower numbers of small follicles on Day 18, fewer large follicles on Days 0 and 18 (P≤0.05) and a higher proportion of atretic follicles on Days 0 and 18 (P≤0.05). Compared with commercial line gilts, less-prolific Piau gilts exhibited higher expression of apoptotic genes during luteolysis (CASP3 and FASL; P≤0.05), decreased expression of TGFBR2 and BAX mRNA in the corpus luteum (P≤0.05), higher expression of apoptotic genes (FAS, BCL2 and CASP8; P≤0.05) in granulosa cells and a greater abundance (P≤0.05) of genes controlling oocyte-secreted factors (GDF9, BMP15 and BMP6), suggesting underlying mechanisms controlling differences in follicular development, ovulation rate and inherent prolificacy in this pig breed.

Criopreservação de sêmen de suínos da raça Piau submetido a três protocolos de congelamento

The objective of this study was to verify the effect of three protocols of cryopreservation on the sperm viability post-thawing of swine from the Piau breed (Sus scrofa), by means of evaluation assays in vitro and in vivo. Twenty-two ejaculates of 5 adult males of the Piau breed trough the gloved-hand method. In order to verify the sperm viability of the raw semen and post-thawing, the following traits were evaluated: sperm motility and vigor, hypoosmotic test, supra-vital color test and percentage of normal acrosomes. To freezing, the ejaculates were fractioned and submitted in: method 1, method of freezing recommended by Furst et al. (2005), modified according to media extender; method 2, recommended by Furst et al. (2005), modified according to the cooling curve; and method 3, recommended by Ohata et al. (2001). To test the in vivo fertility of the frozen/thawed semen, 23 intra-uterine (post-cervical) inseminations were performed in 14 females of the Piau breed, with semen from method 3. The mean values obtained with the use of methods 1 and 2 and post-thawing were inferior to those obtained with method 3. Four adult sows got pregnant (28.6% fertility rate). The freezing method recommended by Ohata et al. (2001), with balance period of 90 minutes at 22-26 oC, enables higher results of in vitro sperm viability in comparison with the two other methods. The absence of balance period of method 1 and the cooling curve utilized in protocol 2 affect negatively the parameters of sperm viability evaluated in the present study. In spite of the satisfactory results in vitro for the freezing of semen with the utilization of method 3, it presents low fertility rate (28.6%).

Sincronização de estro com CIDR reutilizado em cabras lactantes da raça Toggenburg tratadas com somatotropina bovina recombinante (r-bST)

The response of goats treated with r-bST in the estrous synchronization protocol was evaluated. Twenty-six Toggenburg females were divided in two treatments: T1 (n=13) treated with four injections of 250mg r-bST, at 14 days intervals, and T2 (n=13) treated with saline solution (control). The intravaginal device previously used by five days was inserted (day 0) one week after the last injection of r-bST and PGF2a (22.5µg) was injected in the animals of both treatments. The device was removed on day 6. All the females in estrus were bred by fertile bucks. The percentage of animals in estrus and the pregnancy rate were: 76.9 and 70, and 84.6 and 72.7% for goats in T1 and T2, respectively. The estrous cycle length; the interval from treatment to the onset of estrus; the ovulation number; and the intervals from the onset of estrus to ovulation, end of estrus to ovulation, and from device removal to ovulation were not affected by r-bST injection. The r-bST did not affect the diameter of the ovulatory follicles (P>0.05). Progesterone concentration showed similar values (P>0.05) among the goats of T1 and T2 during the treatment with device. The r-bST administration did not affect estrous synchronization.

149 EFFECT OF RESVERATROL ANALOGUE ON DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE EMBRYOS

Oxidative stress is one of the main effects of in vitro culture. Generation of reactive oxygen species (ROS) by embryos can be enhanced by the sub-optimal in vitro culture conditions and are associated with a delay in embryonic development. However, supplementation of culture medium with antioxidant agents can minimize the effects of ROS (Guérin et al. 2001 Hum. Reprod. Update 7, 175-189). Resveratrol is an example of a potent antioxidant, and modifications in its structure can improve its biological activity. This study evaluated the effect of AR33 (formula with patent pending), an analogue of resveratrol with high antioxidant activity, on embryo development. Bovine cumulus-oocyte complexes recovered from ovaries collected at the slaughterhouse were in vitro matured for 24h and oocytes were in vitro fertilized for 20h, both at 38.8°C under 5% CO2 in air and high humidity. Partially denuded presumptive zygotes were randomly distributed in 4 treatments (with 6 replicates): 0µM (control, n=347), 0.1µM (n=337), 0.5µM (n=277), and 2.5µM (n=343) of AR33. The base medium was SOFaa supplemented with 2.5% FCS and incubation conditions were 38.8°C under 5% CO2 in air and high humidity. Half of culture medium was renewed (feeding) at Day 3 and 5 post-fertilization. Cleavage was evaluated at Day 3 and blastocyst rates at Day 7 and 8 post-fertilization. Data were analysed by logistic regression considering the significance level of P<0.05. Values are shown as mean±SEM. Cleavage rate was higher (P<0.05) for 2.5µM (69.0±4.4%) than for 0, 0.1, and 0.5µM AR33 (62.1±2.0%, 60.7±5.9%, and 56.7±5.8%, respectively). At Day 7, the blastocyst rate was similar (P>0.05) among 0.1, 0.5, and 2.5µM (18.1±5.4%, 17.5±2.9%, and 19.4±3.3%, respectively) and all of them were higher (P<0.05) than 0µM AR33 (12.4±2.5%). At Day 8, there was again no difference (P>0.05) among 0.1, 0.5, and 2.5µM AR33 (21.0±5.0%, 18.4±2.1%, and 24.6±3.3%, respectively) but only 0.1 and 2.5µM showed higher (P<0.05) blastocyst rate than 0µM AR33 (15.2±2.5%). In conclusion, the synthetic analogue of resveratrol tested in this study can improve bovine embryo development in culture medium supplemented with 2.5% FCS under 5% CO2 in air. A concentration of 2.5µM AR33 can be a choice for further studies.

Perfil metabólico de touros da raça Nelore (Bos taurus indicus) confinados e tratados com somatotrofina bovina recombinante (r-bST)

This study was carried out to evaluate the effect of recombinant bovine somatotropin (r-bST) administration on profiles of blood metabolites of two different ages Nellore bulls. Sixteen bulls were randomly allotted in a factorial arrangement 2 x 2 (ages: youngs and adults; and r-bST dose: 0 and 500 mg) with four animals per treatment. The mean ages of the young and adult animals were 13.37 and 20.62 months, respectively. Four animals per treatment received saline solution or r-bST 500mg, every 14 days, totaling nine applications per animal during 120 days. The Bulls were fed corn silage and concentrated diet based on corn crumb and soybean meal, twice a day, in individual stalls. Blood was collected every three days for metabolic evaluation. The statistical analyses of the data were performed in three applications, considering three periods (1, 2 and 3). Non-esterified fatty acid (NEFA) concentrations were weekly analyzed. Serum cholesterol, total protein and glucose levels were affected either by period or the treatment (P 0.05).