Assessment of sedation and cardiorespiratory variables in sheep using different anesthetic protocols during artificial insemination / Avaliação da sedação e variáveis cardiorrespiratórias em ovinos submetidos a diferentes protocolos de anestesia durante a realização da inseminação artificial

Artificial insemination (AI) is an important technique in sheep breeding. Since it is an invasive procedure, sedation and analgesia are essential. In the present study, 75 Texel sheep, standardized for weight (kg) and age (months) were used. The animals were randomly allocated to five groups: Acepromazine Group (AG); Acepromazine and Butorphanol Group (ABG); Acepromazine and Morphine Group (AMG); Acepromazine and Meperidine Group (AMRG) and Saline group (SG). The following parameters were assessed: sedation score, agitation level, heart rate (HR); respiratory rate (f), rectal temperature (T°R), latency time and time to perform artificial insemination (AI). Assessments times were M-20, M0, M1 and M2. Significant differences were considered when p˂0.05. There were no significant differences for weight and age. HR increased by 30.6, 34.2 and 42.5% from M-20 to M2 for the AG, ABG and AMRG, respectively. At M0, the AMRG obtained higher values, reaching 41.4% above the other groups. For f there was a decline of 21.8 and 26.9% in M1 in relation to M-20 for the AMRG and AMG, respectively, and a decrease of 20 and 25% between M-20 and M2. A comparison between SG f values showed an increase of 106.3 and 68.8% between M-20 and M1 and M2, respectively. The f values obtained at M1 and M2 for the SG were higher than those of the other groups. Although there were no intergroup differences in sedation level, the values obtained are clinically relevant. There were intergroup differences in agitation level, whereby at M1, AMRG and AMG obtained a score of 2 and SG 0, while at M2, AMG scored 2 and SG 0. There were no statistical differences for latency and AI times. Thus, under the conditions of the present study, the AMRG protocol was the least safe among the options assessed. The pre-anesthetic medication protocols promoted mild sedation and did not reduce the time required for AI.

A inseminação artificial (IA) é um importante técnica para melhoramento genético em ovinos. Em função de ser uma técnica invasiva, a tranquilização e analgesia são fundamentais. No presente estudo foram utilizados 75 ovinos, da raça Texel, padronizados quanto a peso (kg) e idade (meses). Os animais foram distribuídos aleatoriamente em cinco grupos: Grupo Acepromazina (GA); Grupo Acepromazina e Butorfanol (GAB); Grupo Acepromazina e Morfina (GAMF); Grupo Acepromazina e Meperidina (GAME) e Grupo Salina (GS). Os parâmetros avaliados foram: escore de sedação, grau de agitação, frequência cardíaca (FC); frequência respiratória (f), temperatura retal (T°R), tempo de latência e tempo para realização da IA. Os momentos de avaliação foram: M-20, M0, M1 e M2. Diferenças significativas foram consideradas quando p˂0,05. Não houveram diferenças significativas para peso e idade. Com relação a FC observou-se um aumento de 30,6%, 34,2% e 42,5% de M-20 para M2 para os grupos GA, GAB e GAME, respectivamente. Entre grupos, observou-se que em M0 o grupo GAME apresentou valores superiores, chegando a um valor de 41,4% acima dos demais grupos. Para a f destaca-se uma diminuição de 21,8% e 26,9% em M1 com relação a M-20 para os grupos GAME e GAMF, respectivamente. Já entre M-20 e M2 para GAME e GAMF destaca-se uma diminuição de 20% e 25%, na devida ordem. Quando comparados os valores de f do grupo GS, observa-se que entre o M-20 e os momentos M1 e M2 houve um aumento de 106,3% e 68,8% respectivamente. Os valores de f obtidos nos momentos M1 e M2 para GS foram maiores que os demais grupos. Quanto ao grau de sedação não houveram diferenças entre os grupos, contudo os valores obtidos são de relevância clínica. Com relação ao grau de agitação, houveram diferenças entre grupos, em M1 os grupos GAME e GAMF pontuaram 2, enquanto GS pontuou 0, em M2 o grupo GAMF pontuou 2 e o grupo GS 0. Para o tempo de latência e tempo para realização da IA não houveram diferenças estatísticas. Assim, nas condições do presente estudo o protocolo em GAME apresentou-se como o menos seguro dentre as opções avaliadas. Os protocolos instituídos de medicação pré-anestésicas promoveram discreta sedação dos pacientes e não diminuíram o tempo para a realização da técnica de IA.

Negative pressure in the pre-freezing of ram semen

Background: The effect of controlled stress on cells has been widely studied and there is strong evidence that positivepressure improves the survival of cryopreserved gametes and embryos. Positive pressure resulted in higher motility offrozen boar semen, and increased bull semen cryotolerance. Looking for similar effects, we developed a piece of equipment (Nitrocooler) to apply negative pressure. The “Nitrocooler” increased the cryotolerance in bovine IVP blastocysts.Nevertheless, until now, negative pressure has not been tested in sperm cells. This study aimed to determine the effectsand the best negative pressure intensity to apply in pre-freezing ram semen.Material, Methods & Results: The ram semen obtained with artificial vagina was immediately diluted (1 + 1.5) in Trisegg yolk, and fractioned into 4 experimental groups, Control: Without treatment; P200: Submission to 200 mBar negativepressure; P500: Submission 500 mBar negative pressure; P800: Submission to 800 mBar negative pressure. Then, Tris-yolkglycerol was added to all groups to obtain a dilution of 1 + 3, and a sperm concentration of 180 million per straw. Thecooling and freezing procedure was identical for all groups. After thaw, progressive motility (PM) was assessed at zero,1, 2 and 3 h of TRT and after Percoll selection (PMPP). Acrosome integrity (ACI) was assessed by FITC PNA staining;plasmatic membrane integrity (PMI) by hypo-osmotic test, acrosome integrity post Percoll (ACIPP), membrane integritypost Percoll (PMIPP), and cleavage rate after heterologous IVF with bovine oocytes. Data were analyzed by t test (P <0.05). Just after thawing, control group showed higher PM (49%) than P200 (40.9%), P500 (38.9%) and P800 (38.9%),without differences among them. There were no differences between PM in the Control, P200, P500 and P800 groupsat 1 h, 2 h and 3 h of TRT, and in ACI...(AU)

Negative pressure in the pre-freezing of ram semen

Background: The effect of controlled stress on cells has been widely studied and there is strong evidence that positivepressure improves the survival of cryopreserved gametes and embryos. Positive pressure resulted in higher motility offrozen boar semen, and increased bull semen cryotolerance. Looking for similar effects, we developed a piece of equipment (Nitrocooler) to apply negative pressure. The “Nitrocooler” increased the cryotolerance in bovine IVP blastocysts.Nevertheless, until now, negative pressure has not been tested in sperm cells. This study aimed to determine the effectsand the best negative pressure intensity to apply in pre-freezing ram semen.Material, Methods & Results: The ram semen obtained with artificial vagina was immediately diluted (1 + 1.5) in Trisegg yolk, and fractioned into 4 experimental groups, Control: Without treatment; P200: Submission to 200 mBar negativepressure; P500: Submission 500 mBar negative pressure; P800: Submission to 800 mBar negative pressure. Then, Tris-yolkglycerol was added to all groups to obtain a dilution of 1 + 3, and a sperm concentration of 180 million per straw. Thecooling and freezing procedure was identical for all groups. After thaw, progressive motility (PM) was assessed at zero,1, 2 and 3 h of TRT and after Percoll selection (PMPP). Acrosome integrity (ACI) was assessed by FITC PNA staining;plasmatic membrane integrity (PMI) by hypo-osmotic test, acrosome integrity post Percoll (ACIPP), membrane integritypost Percoll (PMIPP), and cleavage rate after heterologous IVF with bovine oocytes. Data were analyzed by t test (P <0.05). Just after thawing, control group showed higher PM (49%) than P200 (40.9%), P500 (38.9%) and P800 (38.9%),without differences among them. There were no differences between PM in the Control, P200, P500 and P800 groupsat 1 h, 2 h and 3 h of TRT, and in ACI...

Comparative evaluation of pyrogens tests in pharmaceutical products

A comparison of methodologies for detection of pyrogens in pharmaceutical products was performed. The rabbit pyrogen test was optimized and the dose-response curve was obtained for the 2nd International Standard for bacterial endotoxins, establishing 13.81 EU/mL/kg as the concentration of endotoxin necessary to induce a temperature rise of 0.5ºC. The 0.5ºC cut-off was shown to give results that were more compatible with the pyrogenic doses for humans. The Limulus amoebocyte lysate test (LAL) was standardized with gel-clot and chromogenic endpoints, and used for the comparative evaluation of pharmaceutical products showing good agreement. The use of beta-glucan-reactive and non-reactive LAL reagents identified some products with false-positive results. The interference test was carried out and the specifications validated for some new products as the maximum valid dilution. The results emphasized the importance and limitations of the assays recommended for the evaluation of purity and quality control of parenteral medicinal products, improving the existing methodologies in the context of reduction and replacement in the use of animal models.

Realizou-se a comparação de metodologia para avaliação de pirogênios em produtos farmacêuticos. Otimizou-se o teste da hipertermia em coelhos elaborando a curva dose-resposta com o 2º Padrão Internacional de endotoxinas bacterianas, com base na qual determinou-se a concentração de 13,81 UE/mL por kg de peso corporal, necessária para produzir aumento de temperatura de 0,5ºC. Observou-se que o limite de 0,5ºC forneceu resultados comparáveis com as doses pirogênicas para o homem. Padronizou-se o teste do lisado de amebócitos do Limulus (LAL) com determinação do ponto final cromogênico e por geleificação, que foram utilizados para a avaliação de produtos farmacêuticos obtendo-se resultados concordantes. Avaliaram-se as respostas de reagentes LAL reativos e não-reativos a beta-glicanos, observando diferenças que poderiam reprovar amostras com base em resultados falso-positivos. Executou-se o teste de interferências, validou-se o procedimento e estabeleceu-se a máxima diluição válida para produtos farmacêuticos sem especificações farmacopéicas. Os resultados enfatizam a importância e as limitações dos ensaios preconizados para avaliação da pureza e controle da qualidade de produtos farmacêuticos parenterais, contribuindo para aprimorar as metodologias existentes no contexto da redução e substituição dos modelos animais.

Comparative evaluation of pyrogens tests in pharmaceutical products

A comparison of methodologies for detection of pyrogens in pharmaceutical products was performed. The rabbit pyrogen test was optimized and the dose-response curve was obtained for the 2nd International Standard for bacterial endotoxins, establishing 13.81 EU/mL/kg as the concentration of endotoxin necessary to induce a temperature rise of 0.5ºC. The 0.5ºC cut-off was shown to give results that were more compatible with the pyrogenic doses for humans. The Limulus amoebocyte lysate test (LAL) was standardized with gel-clot and chromogenic endpoints, and used for the comparative evaluation of pharmaceutical products showing good agreement. The use of beta-glucan-reactive and non-reactive LAL reagents identified some products with false-positive results. The interference test was carried out and the specifications validated for some new products as the maximum valid dilution. The results emphasized the importance and limitations of the assays recommended for the evaluation of purity and quality control of parenteral medicinal products, improving the existing methodologies in the context of reduction and replacement in the use of animal models.

Realizou-se a comparação de metodologia para avaliação de pirogênios em produtos farmacêuticos. Otimizou-se o teste da hipertermia em coelhos elaborando a curva dose-resposta com o 2º Padrão Internacional de endotoxinas bacterianas, com base na qual determinou-se a concentração de 13,81 UE/mL por kg de peso corporal, necessária para produzir aumento de temperatura de 0,5ºC. Observou-se que o limite de 0,5ºC forneceu resultados comparáveis com as doses pirogênicas para o homem. Padronizou-se o teste do lisado de amebócitos do Limulus (LAL) com determinação do ponto final cromogênico e por geleificação, que foram utilizados para a avaliação de produtos farmacêuticos obtendo-se resultados concordantes. Avaliaram-se as respostas de reagentes LAL reativos e não-reativos a beta-glicanos, observando diferenças que poderiam reprovar amostras com base em resultados falso-positivos. Executou-se o teste de interferências, validou-se o procedimento e estabeleceu-se a máxima diluição válida para produtos farmacêuticos sem especificações farmacopéicas. Os resultados enfatizam a importância e as limitações dos ensaios preconizados para avaliação da pureza e controle da qualidade de produtos farmacêuticos parenterais, contribuindo para aprimorar as metodologias existentes no contexto da redução e substituição dos modelos animais.