Utilização do choque osmótico na avaliação da viabilidade de sêmen criopreservado de ovinos

O presente trabalho analisou o emprego do choque osmótico (HOST) com o uso da água deionizada (0 mOsmol), como método de avaliação do sêmen ovino descongelado e correlacionou os resultado obtidos com aqueles encontrados em diferentes técnicas de avaliação de sêmen. Para tanto, vinte amostras de sêmen criopreservado (20 reprodutores), foram avaliadas quanto aos parâmetros de cinética espermática pelo sistema computadorizado (IVOS 12, Hamilton Thorne Biosciences, Beverly, MA, EUA) e análise subjetiva. A análise da viabilidade das membranas espermáticas foi efetuada com a associação de sondas fluorescentes (PI, JC-1 e FITC-PSA). A integridade estrutural da membrana plasmática foi estudada com o teste supravital com eosina (EOS) e, por fim, a integridade funcional da membrana foi avaliada pelo choque osmótico, com a utilização da água deionizada nas seguintes proporções: uma parte de sêmen para 10 (HOST 10), 50 (HOST 50) e para 100 (HOST 100) partes de água. Após a diluição do sêmen, nas diferentes proporções, as amostras foram analisadas quanto ao percentual de espermatozoides reativos ao HOST. Os valores de positividade obtidos para o HOST 10 (33,1%), HOST 50 (32,8%) e HOST 100 (31,8%) não diferiram significativamente. O HOST 10 apresentou importante correlação positiva com a integridade da membrana plasmática pela EOS (r = 0,8; p < 0,05). Os HOST 50 e 100 demonstraram correlações positivas com subpopulações espermáticas com membrana plasmática íntegra pela fluorescência (r = 0,83 e r = 0,85; p < 0,01). Os resultados obtidos revelaram que o HOST com água deionizada pode fornecer informações complementares para a avaliação da viabilidade do sêmen ovino pós-descongelação.This study evaluated the use of hipoosmotic swelling test (HOST) with deionized water (0 mOsmol), as a method of post thaw ram semen evaluation and correlate their findings with different techniques of semen evaluation. Therefore, twenty semen samples of 20 different adult rams were assessed as for kinetic sperm parameters through computerized system (IVOS 12, Hamiton Thorn Biosciences, Beverly, MA, EUA) and subjective analysis. The sperm membranes viability was carried out by the association of fluorescent probes (propidium iodide, JC-1 and FITC-PSA). The structural integrity of the plasma membrane was also studied through supravital test with eosin and the functional integrity of membrane evaluated by doing the hipoosmotic swelling test with deionized water (0 mOsmol), in the following proportions: One part of semen for 10 (HOST 10), 50 (HOST 50) and 100 (HOST 100) parts of water. After semen dilution in the different proportions it was fixed in formalin-buffered saline and analyzed with regard to percentage of HOST reactive sperm (bent/coiled). The percentage of reaction obtained for HOST 10 (33,1%); HOST 50 (32,8%) and HOST 100 (31,8%) did not differ significantly. HOST 10 presented positive correlation with the plasma membrane integrity by the EOS (r = 0,80; p < 0,05). Positive correlations between HOST 50 and HOST 100 with sperm subpopulation with membrane integrity by fluorescence were observed (r = 0,83 and r = 0,85; p < 0,01). The findings suggest that the HOST with deionized water can provide additional information for post thawing ram sperm viability evaluation

Stress distribution in fixed mandibular prostheses fabricated by CAD/CAM and conventional techniques : photoelastic and strain gauge analyses

The aim of this study was to evaluate the distribution of stress in complete fixed mandibular prostheses with infrastructures (IE) fabricated with different materials and techniques, under compressive force. A model of an edentulous mandible, which received five 4x11 mm external hexagon implants between the mental foramens, was fabricated. The groups were divided into: Group I - IE in nickel-chromium with an acrylic resin occlusal coating; Group II ? IE in nickel-chromium with a ceramic occlusal coating; Group III ? IE milled in zirconia with a ceramic coating. For the photoelastic methodology, 70 N axial loads were applied in three regions. Photographic images were taken and analyzed according to the number of high-intensity fringes. For the strain gauge methodology, the measurement of stresses was performed in two distinct regions. The same compression tests described earlier were then performed. The registered stress values were grouped in tables and submitted to two-factor variance analysis (ANOVA) and the Tukey test with 5% significance. The results of the two methodologies demonstrated smaller stress values for Group I, when compared to the other groups. It was possible to conclude that the complete fixed prostheses, with infrastructures cast in metal and acrylic occlusal coating, demonstrated better biomechanical results

Progesterone Levels and Reproductive Parameters in the Periovulatory Period of Nellore Cows under FTAI protocol

Background: Fixed Time Artificial Insemination (FTAI) has achieved a significant evolution in the last 18 years, however, despite the progress achieved by modern FTAI programs, the conception rates obtained are still low. Therefore, this study aimed to evaluate the interrelation between progesterone levels in the periovulatory period and reproductive parameters of Nellore cows submitted to an FTAI protocol. Materials, Methods & Results: On a random day, called day 0 (D0), 57 cows received a P4 device associated with the intramuscular (IM) application of 2.0 mg of estradiol benzoate. On D9, the P4 devices were removed and then were administered 500 μg of cloprostenol sodium IM; 0.6 mg of estradiol cypionate IM and 300 IUI of Equine Chorionic Gonadotrophin IM. Blood samples were collected for the determination of serum P4 concentrations on D9 and D11 of the protocol. The evaluations of follicular diameter (DFOL), follicular wall area (AFOL) and the vascularization area of the follicle wall (VFOL) were carried out on D11 using B-mode ultrasonography examination and colour Doppler, and then the artificial inseminations were performed. The evaluation of the corpus luteum diameter (CLD), of the total corpus luteum area (CLA), of the area of corpus luteum vascularization (CLV) and blood sampling for determination of post-ovulatory P4 levels (Post-P4) were performed on D24. For the analysis of the P4 concentration the chemiluminescence method was used, with a sensitivity of 0.1 ng/mL. According to the P4 concentrations on D11, cows were divided into 2 groups, LOW LEVELS OF P4 and HIGH LEVELS OF P4. The diagnosis of pregnancy was performed using transrectal ultrasonography on D45, at this point the cows were divided into 2 groups, PREGNANT and NON-PREGNANT. The correlation between DFOL and P4 dosage on D11 was moderate, negative and significant and between the AFOL and the serum P4 levels on D9, was moderate, negative and significant. As for the other correlations between follicular and luteal parameters and serum P4 levels, these were low to moderate, negative and not significant. Cows in the LOW LEVELS OF P4 group had significantly larger diameter and follicular areas than the cows in the HIGH LEVELS OF P4 group, the other follicular and luteal parameters showed no statistical difference. Of the total 57 cows that were inseminated, 30 cows became pregnant. Cows in the PREGNANT group had serum P4 levels on D9 equivalent to that obtained by the NON-PREGNANT group. However, at D11 the cows that became pregnant presented significantly lower serum P4 levels than cows that did not become pregnant. Discussion: The results of the interrelation between follicular parameters and P4 levels obtained in the present study, pointed out that the lower the levels of P4, the higher the follicular parameters, corroborating with other authors. Thus, larger preovulatory follicles provided high ovulation rates. Periovulatory serum P4 levels did not significantly affect the morphofunctional parameters of the CL. Such findings may be justified by high periovulatory P4 levels resulting from less efficient luteolysis, exert a negative effect on the results of FTAI protocols, because progesterone inhibits the release of LH pulses. It is concluded that lower periovulatory P4 levels established a favourable condition for follicular development and fertility, however, morphofunctional parameters of the corpus luteum were not affected. Keywords: follicular development, fertility, conception rate, ovarian hemodynamics, synchronization. Título: Níveis de progesterona e os parâmetros reprodutivos no período periovulatório de vacas Nelore submetidas ao protocolo de IATF. Descritores: crescimento folicular, fertilidade, índice de concepção, hemodinâmica ovariana, sincronização

Effect of the Estrus Expression on Follicular and Luteal Morphofunctional Characteristics and Fertility in Bos indicus females Synchronized for FTAI

Background: Recent studies have been conducted with the aim of improving the fertility rates in the FTAI programs in beef females. The observation of the estrus expression constitutes an important indicator of fertility in zebu females. Therefore, this work has as an objective to evaluate the impact of the estrus expression on the follicular, luteal and fertility morphofunctional characteristics of Nelore females synchronized for FTAI.Materials, Methods & Results: Sixty five lactating female Nelore (Bos taurus indicus) were used. On a random day, denominated day 0 (D0), the 65 Nelore females received a progesterone-releasing device associated to the application of 2.0 mg of estradiol benzoate intramuscularly (IM). On D9, the progesterone-releasing intravaginal devices were removed and was administered 500 μg of cloprostenol sodium IM; 0.6 mg of estradiol cypionate IM and 300 UI of Equine Chorionic Gonadotropin IM. At this point, the animals were marked with a marking stick for the determination of the estrus expression. On D11 of the synchronization protocol, the animals were characterized in two groups: without estrus expression (WO/ ESTRUS) and with estrus expression (W/ ESTRUS). The evaluation of the follicle diameter (FOLD), of the follicle wall area (FOLA), of the follicle wall vascularization (FOLV) and the percentage of vascularization in the area of the preovulatory follicle wall (%FOLV) were conducted on D11 using B-mode ultrasonography and color Doppler and then the artificial inseminations were performed. The evaluation of the corpus luteum diameter (CLD), the total area of the corpus luteum (CLA), of the area of vascularization of the corpus luteum (CLV), of the percentage of vascularization of the in the area of the corpus luteum (% CLV) and the collection of blood for the evaluation of the serum levels of progesterone were carried out on D24. The analysis of the serum progesterone concentration were performed by the chemiluminescence method using the Access immunoassay systems Progesterone, with a sensitivity of 0.1 ng/mL. The diagnosis of gestation was carried out by transrectal ultrasonography on D45.  The animals of the WITH ESTRUS group presented superior FOLD, FOLA, FOV (P < 0.05) averages to those presented by the cows of the WITHOUT ESTRUS group. In relation to the luteal characteristics, in the WITH ESTRUS group the cows exhibited averages for CLD, CLA, CLV and P4 significantly higher than those of the WITHOUT ESTRUS group. However, the bovines of the WITH ESTRUS group presented a % CLV similar to the presented by the females of the WITHOUT ESTRUS group. As for the fertility rates, there was difference between the experimental groups WITH ESTRUS and WITHOUT ESTRUS.Discussion: The present work demonstrated the impact of the estrus expression on the follicular morphological parameters, therefore in accordance with other studies. These authors show a relation between the estrus expression and levels of estradiol, which is directly related to the sperm transport in the female reproductive tract, oocyte maturation and fertilization directly affecting the fertility of females submitted to the FTAI program. Similarly, there was an interrelation between the estrus expression and the luteal morphological parameters, corroborating with the findings of some researches, where were verified higher luteal morphological parameters associated to higher conception rates in cows that demonstrated estrus behavior in the FTAI. Therefore, the estrus detection can be used as a tool to direct matings in synchronization protocols, promoting the identification of the animals with a higher probability of conception, owing to better follicular and luteal hemodynamic conditions

Postoperative Pain Management in a Sheep Using Continuous Epidural Morphine

Background: Although ruminants experience pain like other species, diagnosis and treatment may be imprecise and challenging, especially because of lack of recognition and restraint of therapeutic resources. In addition to the restrictions inherent in the species, organic dysfunctions may further restrict the analgesic arsenal, making it necessary to find viable and effective alternative for pain control. The continuous administration of opioids in the epidural space has been highlightedas an analgesic resource in several species. However, until this moment, there are few information on this practice in ruminants. Therefore, the aim of this study was to describe the use of continuous epidural infusion of morphine in a sheep undergoing to urethrostomy with a history of chronic pain and urolithiasis.Case: A 3-year-old Santa Ines sheep, weighting 110 kg, was presented to the Veterinary Hospital of Federal University of Bahia with history of recurrent urolithiasis, lameness, severe pain and continuous use of nonsteroidal anti-inflammatory drug (NSAID). The findings of the physical examination, imaging and laboratory tests revealed possible abomasum inflammation, chronic laminitis, left humerus-radioulnar osteoarthritis and urethral urolithiasis with uremia. A clinical stabilization of the animal was performed, including an interruption of the use of the NSAID and the administration of fluid, urinary acidifier (ammonium chloride), histamine H2 receptor antagonist (ranitidine), cytoprotectant (sucralfate), antibiotic (norfloxacin) and dipyrone. After 3 days, urethrostomy was performed followed by the implantation of an epidural catheter (19G) in the lumbosacral space (L7 - S1) by means of a Tuohy needle (18G), after the surgical procedure. Administration of morphine (0.1 mg kg-1) through the catheter at 24-h intervals, associated with dipyrone (25 mg kg-1) intravenously (IV), every 8 h, over a 5-day period, was the postoperative analgesic protocol chosen. The data of heart rate (HR), respiratory rate (f),rectal temperature (T °C), ruminal movements (RM) and possible behavior related to pain were registered at intervals of four-hours until the fifth day after surgery. After 24 h postoperative, stability of the variables (HR, f, T °C) with increased RM concomitant to the interest for food was verified. On the fifth postoperative day, the animal was hydrated, visibly comfortable and with normal levels of serum creatinine.Discussion: Considering the initial clinical status involving renal insufficiency and significant gastric abnormalities, the use of NSAIDs was immediately disregarded. However, the need to relieved musculoskeletal and postoperative pain, has raised interest in the use of morphine, whose literature refers duration of up to 24 h in other species, with reduced adverse effects compared to systemic administration, due to the considerably lower dose. Dipyrone was incorporated into the protocol forproviding visceral analgesia and minimal effects on the organic functions. During the 5-days of evaluation, the signs of well-being presented by the animal associated to the stability of vital parameters, feeding and satisfactory ruminal activity attested to the viability of the protocol tested. It was concluded that the use of morphine, through the continuous epiduralroute, contributed to the relief of postoperative pain, reestablishment of the organic functions and patient welfare, being suitable as an analgesic alternative to NSAID use in sheep.Keywords: ovine, analgesia, opioid, epidural

PROPORÇÃO DO SEXO NA PRODUÇÃO IN VITRO DE EMBRIÕES BOVINOS

Objetivou-se avaliar a proporção do sexo utilizando espermatozoides provenientes de sêmen convencional ou sexado para fêmea em um programa de produção in vitro de embriões (PIVE) bovinos. Foram utilizados oócitos de 87 doadoras Bos taurus indicus da raça Nelore, obtidos por sistema de aspiração folicular transvaginal guiado por ultrassonografia. Os oócitos selecionados foram maturados in vitro, fertilizados com espermatozoides oriundos de sêmen convencional (grupo 1 – CONV) ou sêmen sexado para fêmea (grupo 2 – SEX) e os embriões produzidos foram então cultivados in vitro por sete dias. Após o cultivo, os mesmos foram envasados e inovulados em receptoras mestiças Bos taurus taurus x Bos taurus indicus. Foram transferidos 1.319 embriões, sendo 1.081 produzidos com sêmen convencional (CONV) e 238 com sêmen sexado de fêmea (SEX). O diagnóstico precoce de gestação e a sexagem fetal foram realizados por ultrassonografia transretal. A taxa de gestação total foi 37,38% (493/1.319), sendo 37,74% (408/1.081) no grupo CONV e 35,71% (85/238) no grupo SEX. A proporção do sexo no grupo CONV foi desviada (P=0,002) a favor dos machos (57,84% de machos e 42,16% de fêmeas) e no grupo SEX ocorreu um desvio na proporção (P=0,001) a favor das fêmeas (68,24% de fêmeas e 31,76% de machos). Concluiu-se que a utilização do sêmen convencional em PIVE bovinos elevou a proporção de produtos machos e apesar da utilização de sêmen sexado de fêmea ser uma alternativa para pré-selecionar o sexo dos produtos, ainda houve uma considerável taxa de machos quando se utilizou este tipo de sêmen

DIÂMETRO FOLICULAR E FERTILIDADE DE VACAS MESTIÇAS LEITEIRAS SUBMETIDAS A PROTOCOLO DE IATF COM AJUSTES NO PROESTRO

Objetivou-se comparar protocolos de sincronização com uma e duas aplicações de um luteolítico simultâneas ou não a gonadotrofina coriônica equina (eCG) sobre o diâmetro do folículo ovulatório (DFOL) e a taxa de concepção de fêmeas mestiças leiteiras cíclicas. Para tanto, no D0 foi iniciado o protocolo por meio da inserção de dispositivo de progesterona (P4) e aplicação de 2,0mg de benzoato de estradiol. No D7 receberam 12,5mg de dinoprost trometamina. No D9 executou-se remoção dos P4 e aplicou-se 0,6mg de cipionato de estradiol. Nesse momento, as fêmeas foram subdivididas nos seguintes tratamentos: Grupo Controle - 2,5mL de solução fisiológica, Grupo 2PGF - 12,5mg de dinoprost trometamina, Grupo eCG - 300UI de eCG, Grupo 2PGF+eCG - 300UI de eCG e 12,5mg de dinoprost trometamina. No D11 foi realizada ultrassonografia transretal (US) para determinar o DFOL e os animais foram inseminados. O diagnóstico de gestação foi realizado por US 55 dias após. Os dados obtidos foram avaliados pelo SPSS considerando P≤0,05. O DFOL e as taxas de concepção foram semelhantes entre os protocolos de sincronização. Conclui-se que em animais cíclicos a utilização de protocolos com uma única dose PGF2α e sem suporte gonadotrófico do eCG parece proporcionar adequada resposta folicular e índices de fertilidade satisfatórios

Efeito de um quelante de cálcio, um detergente e da lecitina de soja sobre a qualidade do sêmen caprino congelado-descongelado

The aim of this paper was verifying the effect of the Equex STM Paste and EDTA addition to a Tris-egg yolk extender, on the post-thaw goat sperm viability. Nine semen samples of two adult goats were collected by artificial vagina and cryopreserved. It was also objective of this study, to evaluate the utilization of a soybean lecithin based commercial extender (Bioexcell® - IMV, L'Aigle, French) for the goat semen freezing. They were formed five experimental groups: TRIS; TRIS+EDTA; TRIS+EQUEX; TRIS+EDTA+EQUEX e Bioexcell. After evaluation, the semen was diluted in the five extenders and packed in 0.25mL straws with 100 million of motile spermatozoa. The samples were cooled at 0,46°C/min to 5°C, submitted at 75min of equilibration time and frozen in liquid nitrogen vapour. The thawing was accomplished in 37ºC water bath for 50s. There were no differences (P>;0,05) on the means of post-thaw total and progressive sperm motility among the groups TRIS, TRIS+EQUEX and TRIS+EQUEX+EDTA. The Bioexcell group obtained the least (P<0,05) percentage of post-thawing total and progressive sperm motility. After the thermotolerance test, it was observed the greatest (P<0,05) rates of total and progressive sperm motility in the Equex STM groups (TRIS+EQUEX and TRIS+EQUEX+EDTA). Thus, it can be affirmed that the Equex addition promotes better maintenance rates in the pos-thaw sperm viability, when compared with the extenders that did not contain it.Nove amostras de sêmen de dois caprinos adultos, colhidas com a utilização de vagina artificial, foram submetidas a criopreservação, com o objetivo de verificar o efeito do Equex STM Paste e do EDTA, adicionados a um diluidor a base de Tris-gema de ovo, sobre a viabilidade espermática pós-descongelação. Também foi objetivo desse trabalho, avaliar a utilização de um diluidor comercial, a base de lecitina de soja (Bioexcell® - IMV, L'Aigle, França), para a congelação do sêmen caprino. Assim, foram formados cinco grupos experimentais: TRIS; TRIS+EDTA; TRIS+EQUEX; TRIS+EQUEX+EDTA e Bioexcell. Logo depois da avaliação, o sêmen foi diluído nos diferentes meios e envasado em palhetas de 0,25 mL, com dose inseminante de 100 x 10(6) espermatozóides. As amostras foram submetidas ao resfriamento, com uma curva média de 0,46°C/min, até atingirem a temperatura de 5°C, mantidas por mais 75min em tempo de equilíbrio e, posteriormente, congeladas em nitrogênio líquido. A descongelação foi realizada em banho-maria a 37ºC por 50s. Não houve diferença (P>;0,05) entre as médias de motilidade total e progressiva, em relação aos grupos TRIS, TRIS+EQUEX e TRIS+EQUEX+EDTA. O grupo Bioexcell obteve o menor (P<0,05) percentual de espermatozóides com motilidade total e progressiva após a descongelação. Após o teste de termoresistência, as melhores (P<0,05) taxas de motilidade total e progressiva foram observadas para os grupos com Equex STM (TRIS+EQUEX e TRIS+EQUEX+EDTA). Assim, pode-se afirmar que a adição do Equex promove uma maior manutenção das taxas de viabilidade espermática após a descongelação, quando comparado com os diluidores que não o continham

Efeito de um quelante de cálcio, um detergente e da lecitina de soja sobre a qualidade do sêmen caprino congelado-descongelado

Nove amostras de sêmen de dois caprinos adultos, colhidas com a utilização de vagina artificial, foram submetidas a criopreservação, com o objetivo de verificar o efeito do Equex STM Paste e do EDTA, adicionados a um diluidor a base de Tris-gema de ovo, sobre a viabilidade espermática pós-descongelação. Também foi objetivo desse trabalho, avaliar a utilização de um diluidor comercial, a base de lecitina de soja (Bioexcell® - IMV, L'Aigle, França), para a congelação do sêmen caprino. Assim, foram formados cinco grupos experimentais: TRIS; TRIS+EDTA; TRIS+EQUEX; TRIS+EQUEX+EDTA e Bioexcell. Logo depois da avaliação, o sêmen foi diluído nos diferentes meios e envasado em palhetas de 0,25 mL, com dose inseminante de 100 x 10(6) espermatozóides. As amostras foram submetidas ao resfriamento, com uma curva média de 0,46°C/min, até atingirem a temperatura de 5°C, mantidas por mais 75min em tempo de equilíbrio e, posteriormente, congeladas em nitrogênio líquido. A descongelação foi realizada em banho-maria a 37ºC por 50s. Não houve diferença (P>;0,05) entre as médias de motilidade total e progressiva, em relação aos grupos TRIS, TRIS+EQUEX e TRIS+EQUEX+EDTA. O grupo Bioexcell obteve o menor (P<0,05) percentual de espermatozóides com motilidade total e progressiva após a descongelação. Após o teste de termoresistência, as melhores (P<0,05) taxas de motilidade total e progressiva foram observadas para os grupos com Equex STM (TRIS+EQUEX e TRIS+EQUEX+EDTA). Assim, pode-se afirmar que a adição do Equex promove uma maior manutenção das taxas de viabilidade espermática após a descongelação, quando comparado com os diluidores que não o continham

UTILIZAÇÃO DO TESTE HIPOSMÓTICO PARA AVALIAR A EFICÁCIA DE DIFERENTES PROTOCOLOS DE CRIOPRESERVAÇÃO DO SÊMEN CAPRINO

Este estudo teve por objetivo comparar a eficácia de quatro protocolos de congelação do sêmen caprino (glicerol, glicerol+EDTA; etilenoglicol; etilenoglicol+EDTA), através da utilização do teste hiposmótico (HOST). O sêmen foi colhido de dois machos da raça Alpina, sexualmente maduros, diluído nos diferentes meios, congelado e armazenado em nitrogênio líquido. Após a colheita, 20ìL do sêmen fresco foram incubados com 01mL de solução hiposmótica (combinação de citrato de sódio e frutose em água destilada com osmolaridade de 125mOsmol), em banho-maria a 370C por 30 minutos. Este procedimento foi repetido após a descongelação e, em seguida, uma amostra foi colocada sobre lâmina/lamínula e avaliada em contraste de fase com mil vezes de aumento. Um total de 100 células foi contado, e as médias percentuais de espermatozóides com edema ou dobramento de cauda, após o HOST, foram – para o sêmen fresco, glicerol, glicerol+EDTA; etilenoglicol; etilenoglicol+EDTA –, respectivamente, 53,89; 16,90; 10,25; 52,64 e 57,54. PALAVRAS-CHAVE: Caprinos, criopreservação, sêmen, teste hiposmótic