Adição de hidroxitolueno butilado (BHT) no diluidor ACP-106c para congelação de sêmen canino

Este estudo foi realizado para determinar o efeito de hidroxitolueno butilado (BHT) sobre a qualidade do sêmen canino congelado e descongelado, utilizando o diluidor à base de água de coco em pó (ACP-106c). Para tanto, foram realizadas quinze coletas de sêmen provenientes de cinco cães. O sêmen obtido foi diluído em ACP-106c acrescido de glicerol e gema de ovo. As amostras foram então transferidas para tubos contendo diferentes concentrações de BHT (0; 0,5; 1,0 e 2,0 mM). Em seguida, as amostras foram envasadas, congeladas e armazenadas em nitrogênio líquido. O sêmen coletado foi avaliado in natura quanto aos seguintes parâmetros: coloração, volume da fração espermática, motilidade total, vigor, concentração, morfologia e funcionalidade de membrana espermática. Após uma semana, as amostras foram descongeladas e avaliadas por meio de análise computadorizada, como também foram realizadas análises da funcionalidade de membrana e da morfologia espermática. A motilidade progressiva no grupo BHT 2,0 mM foi significativamente superior (P < 0,05) do que a do grupo BHT 0 mM (27,6 ± 11,7% vs. 19,0 ± 9,5%, respectivamente). Em todos os demais parâmetros avaliados, não houve diferença entre os grupos testados. Portanto, conclui-se que a adição do BHT ao diluidor ACP-106c não afetou a qualidade do sêmen canino pós-descongelação

USO DO EXTRATO DE ALOE VERA NA CRIOPRESERVAÇÃO DO SÊMEN SUÍNO

Egg yolk is used as a cryoprotective agent in semen freezing diluents for protection against thermal shock, despite the possibility of microbiological contamination. Authors search for Aloe vera, an alternative medium for the conservation of semen. The objective was to evaluate the use of the cryoprotectant Aloe vera in the cryopreservation of swine semen. We used 25 ejaculates, collected through the gloved hand technique. The samples were diluted in the coolant diluent (10.0% egg yolk, Aloe vera 10, 20 and 30%) at 17 °C, freezing diluent (refrigerant diluent + glycerol 6.0%) at 5 °C and stored in vales. The vanes were evaluated for vigor, motility, membrane functionality, vitality, acrosome integrity and assisted semen analysis (CASA). The data were evaluated for normality, then analyzed using ANOVA and multiple comparisons, the analyzes were performed with a significance level of 5.0%, using the program R3.4.0. Descriptive statistics were used for data on vigor, motility and CASA. In the evaluation of thawed semen for membrane functionality, acrosome integrity and vitality, the highest values were found for the egg yolk samples (p&lt;0.05), and among the samples that used Aloe vera, found significant differences between the different concentrations tested (p&gt;0.05). The data of vigor, motility and CASA, of the samples using Aloe vera presented values of 0.0. The results showed that the different concentrations of Aloe vera did not exert the desired cryoprotective effect on the sperm cell, so it is understood that further studies are needed on its action on semen conservation, as well as a possible interaction with swine spermatozoa. On the other hand, it is believed that additional studies on the cooling curve of the porcine semen are still necessary, aiming at its cryopreservation.A gema de ovo é utilizada como agente crioprotetor em diluentes de congelação de sêmen visando à proteção contra o choque térmico, apesar de apresentar uma possibilidade de contaminação microbiológica. Autores buscam no Aloe vera, um meio alternativo para a conservação do sêmen. O objetivo foi avaliar o uso do crioprotetor Aloe vera na criopreservação do sêmen de suíno. Foram utilizados 25 ejaculados, coletados através da técnica da mão enluvada. As amostras foram diluídas no diluente de refrigeração (gema de ovo 10,0%, Aloe vera 10, 20 e 30%) a 17 ºC, diluente de congelação (diluente de refrigeração + glicerol 6,0%) 5 ºC e armazenados em palhetas. As palhetas foram avaliadas quanto ao vigor, motilidade, funcionalidade da membrana, vitalidade, integridade de acrossoma e na análise de sêmen assistida (CASA). Os dados foram avaliados quanto à normalidade, em seguida analisados utilizando ANOVA e comparações múltiplas, as análises foram realizadas com nível significância de 5,0%, utilizando o programa R3.4.0. Utilizou-se estatística descritiva para dados de vigor, motilidade e CASA. Na avaliação do sêmen descongelado para a funcionalidade da membrana, vitalidade e integridade de acrossoma, os maiores valores foram encontrados para as amostras com a gema de ovo (p&lt;0,05), e entre as amostras que utilizaram o Aloe vera, não foram encontradas diferenças significativas entre as diferentes concentrações testadas (p&gt;0,05). Os dados de vigor, motilidade e CASA, das amostras utilizando o Aloe vera, apresentaram valores de 0,0. Os resultados mostraram que as diferentes concentrações de Aloe vera não exerceram o efeito crioprotetor desejado sobre a célula espermática, sendo assim, entende-se que são necessários maiores estudos sobre a sua ação na conservação de sêmen, bem como uma possível interação com os espermatozoides suínos. Por outro lado, acredita-se que ainda são necessários estudos suplementares sobre a curva de resfriamento do sêmen suíno, visando a sua criopreservação

Ram and Goat Semen Immunosexed and Diluted in Powdered Coconut Water-Based Preservation Medium (ACP101/102c).

Background: Sperm sexing aims to separate sperm populations in carriers of the “X” or “Y” chromosome. Currently, flow cytometry is a technique that allows greater accuracy; however, it causes structural changes in sperm, reduces viability, and has a high cost. As a result, other methods have been researched, including immunosexing, which uses monoclonal antibodies to detect sex-specific surface antigens. Thus, the objective of this study was to evaluate the immunosexing technique using a monoclonal antibody against sex-specific protein (HY) in the conservation of ram and goat semen in ACP101/102c.Materials, Methods & Results: Ejaculates from 5 rams and 5 goats were collected with the aid of an artificial vagina; they were evaluated and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro, RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP ram (ACP101/102c + 20% egg yolk + 7% glycerol) and ACP goat (ACP101/102c + 2.5% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated at 4°C, stabilized for 30 min, frozen in liquid nitrogen vapor (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were evaluated in natura (T1), after immunosexing (T2) and after thawing (T3) for sperm motility subjectively using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and morphologically defect spermatozoa were determined. In the evaluation of ram semen regarding sperm motility and IMP, no statistically significant differences were observed between treatments after sexing in the evaluation of absolute data (P > 0.05), with the difference being observed only between T1 and T2, and T3 (P 0.05). Regarding the evaluation of goat semen samples, the motility parameters were consistent with the technique submitted; however, the IMP data did not appear as expected, requiring further evaluation for a better assessment of the technique for this species.Discussion: The data obtained from ram semen submitted to the immunosexing protocol, regarding the absolute evaluation of motility and IMP, demonstrated that the non-sexed semen (T1) was superior to the sexed treatments (T2 and T3); however, it is noteworthy that freezing started with approximately 50% of the cells, since the immunosexing technique results in a loss of viability of approximately 50% of the sperm, which corresponds to the ratio of sperm carrying the X chromosome. In addition, when the data in this study were transformed into relative values, no statistical differences were observed, indicating that the immunosexing protocol, as well as the freezing protocol, did not significantly affect the quality of ram sperm cells. In relation to the immunosexing of goat semen, future studies should be conducted in vitro to define a more appropriate protocol for the species and, in addition, in vivo studies should be performed to prove the quality of the technique. It was concluded that the immunosexing process using a monoclonal antibody against sex-specific protein (HY) associated with the use of powdered coconut water diluent (ACP101/102c) in the cryopreservation of semen proved to be efficient in the in vitro evaluation of ovine species

Semen quality, testicular B-mode and Doppler ultrasound, and serum testosterone concentrations in dogs with established infertility

Retrospective examination of breeding records enabled the identification of 10 dogs of normal fertility and 10 dogs with established infertility of at least 12 months of duration. Comparisons of testicular palpation, semen evaluation, testicular ultrasound examination, Doppler ultrasound measurement of testicular artery blood flow, and measurement of serum testosterone concentration were made between the two groups over weekly examinations performed on three occasions. There were no differences in testicular volume (cm3) between the two groups (fertile right testis = 10.77 ± 1.66; fertile left testis = 12.17 ± 2.22); (infertile right testis = 10.25 ± 3.33; infertile left testis = 11.37 ± 3.30), although the infertile dogs all had subjectively softer testes compared with the fertile dogs. Infertile dogs were either azoospermic or when they ejaculated, they had lower sperm concentration, sperm motility, and percentage of morphologically normal spermatozoa than fertile dogs. Furthermore, infertile dogs had reduced sperm membrane integrity measured via the hypoosmotic swelling test. Infertile dogs had significantly lower basal serum testosterone concentrations (1.40 ± 0.62 ng/mL) than fertile dogs (1.81 ± 0.87 ng/mL; P < 0.05). There were subjective differences in testicular echogenicity in some of the infertile dogs, and important differences in testicular artery blood flow with lower peak systolic and end-diastolic velocities measured in the distal supratesticular artery, marginal testicular artery, and intratesticular artery of infertile dogs (P < 0.05). Notably, resistance index and pulsatility index did not differ between infertile and fertile dogs. These findings report important differences between infertile and fertile dogs which may be detected within an expanded breeding soundness examination

CRIOPRESERVAÇÃO DE SÊMEN EM ONÇAS PINTADAS DE CATIVEIRO: CRIOPRESERVAÇÃO DE SÊMEN EM ONÇAS PINTADAS DE CATIVEIRO

The jaguar is one of the main important animals of the Brazilian fauna and among all felines, the jaguar (Panthera onca) is only smaller than the lion (Panthera leo) and the tiger (Panthera tigris), soon being classified as the largest cat from all over the American continent. Despite all the recognition and importance, the species has undergone a significant reduction in the number of individuals. This species has been maintaining its conservation status as a vulnerable species for a long time, according to the Brazilian Ministry of the Environment, and close to threatened, according to the classification of the IUCN Red List. The main causes that lead to a decrease in the population of this species are related to the reduction in the number of natural prey such as deer, peccary and collared peccary, and mainly due to the expansion of urban areas, destruction of natural habitats, being hit by roads and illegal hunting. Such factors capable of reducing the population of these animals have been constantly worsening due mainly to ineffective public policies for the protection of endangered species. Against this background, researchers are working to develop strategies to protect the species. The conservation strategy of greater prominence and of quicker result in the short term is the reproductive biotechniques, since such techniques are able to increase the number of individuals in a short period of time. In reproductive biotechniques, semen cryopreservation (freezing) stands out, a technique in which samples can be stored under extremely negative temperatures for a long period of time and the material can also be moved to any location.A onça-pintada é um dos principais animais da fauna brasileira e dentre todos os felinos, a onça-pintada (Panthera onca) somente é menor que o leão (Panthera leo) e o tigre (Panthera tigris), logo sendo classificada como o maior felino de todo o continente americano. Apesar de todo o reconhecimento e importância, a espécie vem sofrendo significativa redução no número de indivíduos. Já há bastante tempo essa espécie vem mantendo seu status de conservação como espécie vulnerável, segundo o Ministério do Meio Ambiente, e próximo de ameaçada, segundo classificação da Lista Vermelha da IUCN. As principais causas que levam à diminuição da população desta espécie estão ligadas à redução no número de suas presas naturais como cervos, queixadas e catetos, e principalmente devido à expansão de áreas urbanas, destruição dos habitats naturais, atropelamentos em rodovias e caça ilegal. Tais fatores capazes de reduzir a população desses animais vem se agravando constantemente devido principalmente as políticas públicas ineficazes para a proteção de espécies ameaçadas. Na contramão desta situação, pesquisadores trabalham visando o desenvolvimento de estratégias com o intuito de proteger a espécie. A estratégia de conservação de maior destaque e de resultado mais rápido a curto prazo são as biotécnicas reprodutivas, uma vez que tais técnicas são capazes de aumentar o número de indivíduos em um curto espaço de tempo. Nas biotécnicas reprodutivas, destaca-se a criopreservação (congelamento) de sêmen, técnica na qual se permite armazenar amostras sob temperaturas extremamente negativas, por um longo período de tempo e também deslocar o material para quaisquer localidades

Ram and Goat Semen Immunosexed and Diluted in Powdered Coconut Water-Based Preservation Medium (ACP101/102c).

Background: Sperm sexing aims to separate sperm populations in carriers of the “X” or “Y” chromosome. Currently, flow cytometry is a technique that allows greater accuracy; however, it causes structural changes in sperm, reduces viability, and has a high cost. As a result, other methods have been researched, including immunosexing, which uses monoclonal antibodies to detect sex-specific surface antigens. Thus, the objective of this study was to evaluate the immunosexing technique using a monoclonal antibody against sex-specific protein (HY) in the conservation of ram and goat semen in ACP101/102c.Materials, Methods &amp; Results: Ejaculates from 5 rams and 5 goats were collected with the aid of an artificial vagina; they were evaluated and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro, RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP ram (ACP101/102c + 20% egg yolk + 7% glycerol) and ACP goat (ACP101/102c + 2.5% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated at 4°C, stabilized for 30 min, frozen in liquid nitrogen vapor (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were evaluated in natura (T1), after immunosexing (T2) and after thawing (T3) for sperm motility subjectively using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and morphologically defect spermatozoa were determined. In the evaluation of ram semen regarding sperm motility and IMP, no statistically significant differences were observed between treatments after sexing in the evaluation of absolute data (P &gt; 0.05), with the difference being observed only between T1 and T2, and T3 (P &lt; 0.05). Regarding the relative percentage and sperm morphology, no statistically significant differences were observed (P &gt; 0.05). Regarding the evaluation of goat semen samples, the motility parameters were consistent with the technique submitted; however, the IMP data did not appear as expected, requiring further evaluation for a better assessment of the technique for this species.Discussion: The data obtained from ram semen submitted to the immunosexing protocol, regarding the absolute evaluation of motility and IMP, demonstrated that the non-sexed semen (T1) was superior to the sexed treatments (T2 and T3); however, it is noteworthy that freezing started with approximately 50% of the cells, since the immunosexing technique results in a loss of viability of approximately 50% of the sperm, which corresponds to the ratio of sperm carrying the X chromosome. In addition, when the data in this study were transformed into relative values, no statistical differences were observed, indicating that the immunosexing protocol, as well as the freezing protocol, did not significantly affect the quality of ram sperm cells. In relation to the immunosexing of goat semen, future studies should be conducted in vitro to define a more appropriate protocol for the species and, in addition, in vivo studies should be performed to prove the quality of the technique. It was concluded that the immunosexing process using a monoclonal antibody against sex-specific protein (HY) associated with the use of powdered coconut water diluent (ACP101/102c) in the cryopreservation of semen proved to be efficient in the in vitro evaluation of ovine species