Gradiente de densidade coloidal equipure™ utilizado pré e pós criopreservação de sêmen equino
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Data
2023-06-26
Autores
Pereira, Raiza Rocha
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Universidade Estadual Paulista (Unesp)
Resumo
O objetivo do trabalho foi verificar a qualidade espermática após utilização de um protocolo modificado de EquiPureTM antes e depois da criopreservação do sêmen equino. Experimento 1, foram utilizadas coletas de 15 garanhões (n=26). Quatro grupos experimentais foram formados: Controle (centrifugação convencional); EG (centrifugação com EquiPureTM antes do congelamento); CT+EG (centrifugação com EquiPureTM após o sêmen ser descongelado); EG+EG (centrifugação com EquiPureTM antes e depois do sêmen ser congelado). No experimento 2 foram realizados testes de fertilidade com os grupos experimentais: controle (centrifugação convencional); EG (centrifugação com EquiPureTM antes do congelamento); CT+EG (centrifugação com EquiPureTM após o sêmen ser descongelado); EG+EG (centrifugação com EquiPureTM antes e depois do sêmen ser congelado) utilizando quatro ciclos de 8 éguas (n=32) e um garanhão considerado “Bad freezing”. As inseminações foram feitas após a ovulação, mediante deposição profunda do sêmen no corno uterino ipsilateral ao lado da ovulação. Para as inseminações oito palhetas foram descongeladas em banho maria a 37°C por 30 segundos com uma concentração de 100 milhões de espermatozoides por palhetas. Os grupos CT e EG tiveram uma dose fixa de 800 milhões de espermatozoides. A dose inseminante dos grupos CT+EG e EG+EG foram dependentes da recuperação espermática pós-centrifugação (v:v) pós descongelamento com EquiPureTM. Os parâmetros espermáticos analisados foram a cinética espermática (motilidade total, progressiva e espermatozoides rápidos) pela análise computadorizada do movimento espermático e estabilidade da membrana plasmática, geração de O2- mitocondrial, potencial mitocondrial e peroxidação lipídica por citometria de fluxo. As análises espermáticas dos grupos Controle e EG foram analisadas imediatamente após descongelação 37ºC por 30 segundos (M0). Os grupos CT+EG e EG+EG foram analisados logo após o procedimento de centrifugação (M0). Todos os grupos foram analisados pós-incubação a 37ºC por 60 minutos (M60). No M0 (P<0,05) o grupo EG+EG aumentou a motilidade total, motilidade progressiva, rápidos, potencial de membrana mitocondrial e estabilidade da membrana plasmática comparada com os outros grupos avaliados; O grupo CT+EG aumentou os parâmetros de rápidos, estabilidade da membrana plasmática, potencial de membrana mitocondrial e diminuiu a produção de ânion superóxido, em relação aos grupos controle e EG. Pós-incubação a 37ºC por 60 minutos(M60) o grupo EG+EG aumentou os parâmetros de motilidade total e motilidade progressiva, rápidos, alto potencial de membrana mitocondrial, e diminui a produção de O 2- comparado com outros grupos. O grupo EG teve diferença do grupo controle na motilidade progressiva e espermatozoides rápidos. O grupo CT+EG diferiu do controle na motilidade total e progressiva, rápidos e peroxidação lipídica. O grupo CT+EG diferiu do EG na motilidade progressiva e espermatozoides rápidos. Controle, EG e CT+EG não diferiram com relação ao alto potencial de membrana mitocondrial e produção de O2- . Os grupos não diferiram na estabilidade da membrana plasmática. As taxas de recuperação dos espermatozoides foram menores nos grupos CT+EG e EG+EG (34,9±2,2; 30,1±1,5, respectivamente). A taxa de fertilidade por ciclo foi maior no grupo EG+EG (62,5%, 5/8) quando comparada aos demais grupos CT, EG, CT+EG (25% 2/8; 25% 2/8; 25% 2/8, respectivamente) os outros grupos testados não tiveram diferença. Concluiu-se, que a utilização de EquiPureTM aumenta a cinética espermática pós-descongelamento do sêmen equino, e que selecionar os espermatozoides antes da criopreservação e pós descongelamento pode aumentar a taxa de prenhez de garanhão considerado “bad freezing”.
The aim of this study was to verify sperm quality after using a modified EquiPureTM protocol before and after cryopreservation of equine semen. In Experiment 1, samples from 15 stallions (n=26) were used. Four experimental groups were formed: control (conventional centrifugation); EG (centrifugation with EquiPureTM before freezing); CT+EG (centrifugation with EquiPureTM after semen is thawed); and EG+EG (centrifugation with EquiPureTM before and after semen is frozen). In experiment 2, fertility tests were performed with the following experimental groups: control (conventional centrifugation); EG (centrifugation with EquiPureTM before freezing); CT+EG (centrifugation with EquiPureTM after semen is thawed); EG+EG (centrifugation with EquiPureTM before and after the semen is frozen) using four cycles of 8 mares (n=32) and one stallion considered “Bad freezing”. The inseminations were performed after ovulation through deep deposition of semen in the ipsilateral uterine horn next to ovulation. For the inseminations, eight straws were thawed in a water bath at 37°C for 30 seconds with a concentration of 100 million spermatozoa per straw. The CT and EG groups had a fixed dose of 800 million sperm. The insemination doses of the CT+EG and EG+EG groups were dependent on post-centrifugation sperm recovery (v:v) after thawing with EquiPureTM. The sperm parameters analyzed were sperm kinetics (total, progressive, and fast motility) by computerized analysis of sperm movement and plasma membrane stability, mitochondrial O2- generation, mitochondrial potential, and lipid peroxidation by flow cytometry. Sperm from the control and EG groups were analyzed immediately after thawing at 37ºC for 30 seconds (M0). The CT+EG and EG+EG groups were analyzed right after the centrifugation procedure (M0). All groups were analyzed after incubation at 37ºC for 60 minutes (M60). At M0 (P<0.05) the EG+EG group increased total motility, progressive motility, rapids, mitochondrial membrane potential, and plasma membrane stability compared to the other evaluated groups. The CT+EG group increased the parameters of rapids, plasma membrane stability, mitochondrial membrane potential, and decreased superoxide anion production in relation to the control and EG groups. Post-incubation at 37ºC for 60 minutes (M60) the EG+EG group increased the parameters of total motility and progressive motility, had rapid, high mitochondrial membrane potential, and decreased O2- production compared to other groups. The EG group differed from the control group in terms of progressive motility and fast spermatozoa. The CT+EG group differed from the control in total and progressive motility, rapids, and lipid peroxidation. The CT+EG group differed from the EG in progressive motility and fast spermatozoa. Control, EG, and CT+EG did not differ with respect to high mitochondrial membrane potential and O2- production. The groups did not differ in plasma membrane stability. Sperm retrieval rates were lower in the CT+EG and EG+EG groups (34.9±2.2; 30.1±1.5, respectively). The fertility rate per cycle was higher in the EG+EG group (62.5%, 5/8) when compared to the other CT, EG, CT+EG groups (25% 2/8; 25% 2/8; 25% 2/8, respectively); the other groups tested showed no difference. It was concluded that the use of EquiPureTM increases the post-thawing sperm x quality parameters of equine semen and that selecting spermatozoa before cryopreservation and post-thawing can increase the pregnancy rate of stallions considered “bad freezing”.
The aim of this study was to verify sperm quality after using a modified EquiPureTM protocol before and after cryopreservation of equine semen. In Experiment 1, samples from 15 stallions (n=26) were used. Four experimental groups were formed: control (conventional centrifugation); EG (centrifugation with EquiPureTM before freezing); CT+EG (centrifugation with EquiPureTM after semen is thawed); and EG+EG (centrifugation with EquiPureTM before and after semen is frozen). In experiment 2, fertility tests were performed with the following experimental groups: control (conventional centrifugation); EG (centrifugation with EquiPureTM before freezing); CT+EG (centrifugation with EquiPureTM after semen is thawed); EG+EG (centrifugation with EquiPureTM before and after the semen is frozen) using four cycles of 8 mares (n=32) and one stallion considered “Bad freezing”. The inseminations were performed after ovulation through deep deposition of semen in the ipsilateral uterine horn next to ovulation. For the inseminations, eight straws were thawed in a water bath at 37°C for 30 seconds with a concentration of 100 million spermatozoa per straw. The CT and EG groups had a fixed dose of 800 million sperm. The insemination doses of the CT+EG and EG+EG groups were dependent on post-centrifugation sperm recovery (v:v) after thawing with EquiPureTM. The sperm parameters analyzed were sperm kinetics (total, progressive, and fast motility) by computerized analysis of sperm movement and plasma membrane stability, mitochondrial O2- generation, mitochondrial potential, and lipid peroxidation by flow cytometry. Sperm from the control and EG groups were analyzed immediately after thawing at 37ºC for 30 seconds (M0). The CT+EG and EG+EG groups were analyzed right after the centrifugation procedure (M0). All groups were analyzed after incubation at 37ºC for 60 minutes (M60). At M0 (P<0.05) the EG+EG group increased total motility, progressive motility, rapids, mitochondrial membrane potential, and plasma membrane stability compared to the other evaluated groups. The CT+EG group increased the parameters of rapids, plasma membrane stability, mitochondrial membrane potential, and decreased superoxide anion production in relation to the control and EG groups. Post-incubation at 37ºC for 60 minutes (M60) the EG+EG group increased the parameters of total motility and progressive motility, had rapid, high mitochondrial membrane potential, and decreased O2- production compared to other groups. The EG group differed from the control group in terms of progressive motility and fast spermatozoa. The CT+EG group differed from the control in total and progressive motility, rapids, and lipid peroxidation. The CT+EG group differed from the EG in progressive motility and fast spermatozoa. Control, EG, and CT+EG did not differ with respect to high mitochondrial membrane potential and O2- production. The groups did not differ in plasma membrane stability. Sperm retrieval rates were lower in the CT+EG and EG+EG groups (34.9±2.2; 30.1±1.5, respectively). The fertility rate per cycle was higher in the EG+EG group (62.5%, 5/8) when compared to the other CT, EG, CT+EG groups (25% 2/8; 25% 2/8; 25% 2/8, respectively); the other groups tested showed no difference. It was concluded that the use of EquiPureTM increases the post-thawing sperm x quality parameters of equine semen and that selecting spermatozoa before cryopreservation and post-thawing can increase the pregnancy rate of stallions considered “bad freezing”.
Descrição
Palavras-chave
Garanhão, Sêmen, Criopreservação, Gradiente de densidade coloidal, EquiPure, Congelamento, Freezing, Semen, Stallion