Criopreservação do sêmen de garanhões da raça crioula com baixo e alto escore de condição corporal e refrigeração de sêmen equino com diluentes a base de caseína.
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2020-06-30
Autores
Novello, Guilherme
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Universidade Estadual Paulista (Unesp)
Resumo
A criopreservação (resfrigeração e congelamento) de sêmen equino é uma biotecnologia amplamente utilizada na indústria de criação de cavalos. No entanto, vários fatores podem afetar a qualidade seminal e a fertilidade do sêmen criopreservado. Claramente, as individualidades dos garanhões associadas à capacidade de resfriamento ou congelamento do sêmen, diluentes de sêmen, sistema de transporte e processamento são bem descritas como associadas a alterações na qualidade seminal. Nos homens, a obesidade e a síndrome metabólica têm sido associadas a efeitos adversos nos parâmetros espermáticos. Com isso, o objetivo deste estudo foi testar diferentes diluentes de sêmen, dispositivos de resfriamento, processamento, bem como comparar a capacidade de congelamento de sêmen de garanhões com baixo escore de condição corporal e alto escore de condição corporal. Capítulo 2: Primeiramente, foram testados os parâmetros de cinética espermática e as taxas de recuperação embrionária do sêmen diluído e refrigerado com diferentes extensores à base de caseína (INRA 96 ou BotuSêmen Gold) com ou sem plasma seminal e armazenados em três diferentes dispositivos comerciais de refrigeração. No experimento 1, 45 ejaculados de nove garanhões foram coletados, avaliados e igualmente divididos entre os diluentes e depois diluídos para 50 milhões de espermatozoides/mL. Em seguida, o sêmen diluído foi armazenado em três containers de refrigeração passiva (Equitainer, Equine Express II e BotuFlex) por 48 horas. No experimento 2, os mesmos ejaculados diluídos no experimento 1 foram centrifugados com cushion, o sobrenadante foi descartado e o “pellet” foi ressuspenso a 100 milhões de espermatozoides/mL com seu respectivo extensor. O sêmen foi então refrigerado como no experimento 1. Nos dois experimentos, os parâmetros de cinética espermática, integridade da membrana plasmática e o alto potencial da membrana mitocondrial foram avaliados às 0, 24 e 48 horas após o resfriamento. Para o experimento 3, 12 éguas (n = 24 ciclos) foram inseminadas com sêmen de um garanhão refrigerado por 48 horas. O sêmen foi processado como descrito no experimento 1. O lavado de embrião foi realizado 8 dias após a ovulação. No experimento 1, o BotuSêmen Gold apresentou motilidade total e progressiva superior em relação ao INRA 96 (P <0,05). Não houve diferenças significativas entre os sistemas de refrigeração em nenhum dos experimentos. No experimento 2, os diluentes INRA 96 e BotuSêmen Gold apresentaram motilidade total e progressiva semelhante, no entanto o BotuSêmen Gold apresentou parâmetros superiores de velocidade espermática em todos os momentos. A recuperação embrionária foi idêntica para os dois extensores (50%). Finalmente, os resultados aqui obtidos sugerem que o BotuSêmen Gold é um diluente de sêmen equino a ser incluído nos testes frente ao INRA 96 na prática clínica. Em segundo lugar, foi avaliado o efeito do escore de condição corporal (ECC) na capacidade de congelamento de sêmen de garanhões da raça Crioula. Vinte garanhões foram alocados em 2 grupos de acordo com o ECC: os garanhões com ECC <7 foram caracterizados como Baixo-ECC (11/20) e os garanhões com ECC ≥7 como Alto-ECC (9/20). Foi obtida uma história clínica completa seguida de medidas morfométricas e de acúmulo de gordura subcutânea. As concentrações plasmáticas de glicose foram avaliadas após teste oral de tolerância a glicose. Para isso, os garanhões foram submetidos a jejum durante a noite e o xarope de milho foi administrado por via oral às 8h da manhã. Amostras de sangue foram coletadas para avaliação da glicose antes e após a administração oral de xarope de milho. Além disso, um ejaculado de cada garanhão foi coletado e diluído a 50 milhões de espermatozoides/mL com extensor à base de leite desnatado (BotuSêmen, Botupharma, Brasil). O sêmen foi centrifugado (600 × g / 10 min), o sobrenadante foi descartado e o “pellet” ressuspenso a 200 milhões de espermatozoides/mL com BotuCrio (Botupharma, Brasil) e submetido ao processo de criopreservação. A cinética espermática foi avaliada por CASA e integridade da membrana plasmática, ãnion superóxido, potencial mitocondrial, peroxidação lipídica e peróxido de hidrogênio pela citometria de fluxo em amostras de sêmen pós-descongelamento. O estudo morfométrico (peso corporal, ECC e circunferência do pescoço em 25% e 50%) foi superior em garanhões com alto-ECC (P <0,05). No entanto, não houve diferenças nos parâmetros espermáticos ou na concentração plasmática de glicose entre os grupos (P> 0,05). Em resumo, o alto ECC não afetou os parâmetros espermáticos e a capacidade de congelamento de sêmen de garanhões da Raça Crioula.
Cryopresrvation (e.g. cooling and freezing) of equine semen is a widely used biotechnology in the horse breeding industry. However, several factors can affect sperm quality and fertility of the cryopreserved semen. Clearly, stallion individualities associated with semen cooling- or freezing-ability, semen extender, transport system and semen processing are well described to be associated with changes in sperm quality. Of interest, in men the obesity and metabolic syndrome have been associated with adverse effects in sperm parameters. Therefore, the objective of this study was to test different semen extenders, cooling devices, semen processing, as well as to compare the semen freezing ability of stallions with normal body condition score and those characterized as obese. Chapter 2: Firstly, semen parameters and embryo recovery rates of cooled stallion semen extended with different casein-based extenders (INRA 96 or BotuSemen Gold) with or without seminal plasma and stored in three commercial cooling devices were tested. In experiment 1, 45 ejaculates from nine mature stallions were collected, assessed, and equally split between both extenders and then extended to 50 million sperm/mL. Then, the extended semen was stored in three passive cooling containers (Equitainer, Equine Express II, and BotuFlex) for 48 hours. In experiment 2, the same ejaculates extended in experiment 1 were cushion-centrifuged, the supernatant was discarded, and the pellets were resuspended at 100 million sperm/mL with their respective extender. Semen was then cooled and stored as in experiment 1. In both experiments, sperm motility parameters, plasma membrane integrity, and high mitochondrial membrane potential were assessed at 0, 24, and 48 hours post cooling. For experiment 3, 12 mares (n = 24 cycles) were bred with 48 hour–cooled semen from one stallion. Semen was processed as described in experiment 1. Mares had embryo flushing performed by 8-day post-ovulation. In experiment 1, BotuSemen Gold displayed superior total and progressive motility relative to INRA 96 (P<0.05). There were no significant differences between the types of containers in any experiment. In experiment 2, INRA 96 and BotuSemen Gold extenders had similar total and progressive motility, but BotuSemen Gold had superior sperm velocity parameters at all timepoints. Embryo recovery was identical for both extenders (50%). Finally, the results obtained herein suggest that BotuSemen Gold is a suitable alternative to be included in semen cooling tests against INRA 96 in clinical practice. Secondly, the effect of obesity in semen freezing ability of Crioulo stallions was evaluated. Twenty stallions were allocated into 2 groups according to their body condition score (BCS): stallions with BCS <7 were characterized as Low-BCS (11/20) and stallions with BCS ≥7 as High-BCS (9/20). A complete clinical history followed by morphometric and subcutaneous body fat measurements were obtained. Serum glucose concentrations were evaluated after oral sugar test. For this, the stallions were fasted overnight and corn syrup was orally administered at 8 am. Blood samples were collected for glucose assessment before and after oral administration of corn syrup. In addition, one ejaculated of each stallion was collected and extended at 50 million sperm/mL with skimmed-milk based extender (BotuSemen, Botupharma, Brazil). Semen was centrifuged (600 ×g/10 min), the supernatant discarded, the pellet resuspended at 200 million sperm/mL with BotuCrio (Botupharma, Brazil) and submitted to cryopreservation. Sperm kinetics were assessed by CASA and plasma membrane integrity, ROS, mitochondrial membrane potential and lipid peroxidation by flow cytometer in post-thawed semen samples. The morphometric study (e.g. body weight, BCS and neck circumference at 25% and 50%) were superior in obesity stallions (P<0.05). However, there were no differences in sperm parameters or serum glucose concentration between groups (P>0.05). In summary the high-BCS in Crioulo horse didn’t affect sperm parameters and freezing ability of Crioulo stallions.
Cryopresrvation (e.g. cooling and freezing) of equine semen is a widely used biotechnology in the horse breeding industry. However, several factors can affect sperm quality and fertility of the cryopreserved semen. Clearly, stallion individualities associated with semen cooling- or freezing-ability, semen extender, transport system and semen processing are well described to be associated with changes in sperm quality. Of interest, in men the obesity and metabolic syndrome have been associated with adverse effects in sperm parameters. Therefore, the objective of this study was to test different semen extenders, cooling devices, semen processing, as well as to compare the semen freezing ability of stallions with normal body condition score and those characterized as obese. Chapter 2: Firstly, semen parameters and embryo recovery rates of cooled stallion semen extended with different casein-based extenders (INRA 96 or BotuSemen Gold) with or without seminal plasma and stored in three commercial cooling devices were tested. In experiment 1, 45 ejaculates from nine mature stallions were collected, assessed, and equally split between both extenders and then extended to 50 million sperm/mL. Then, the extended semen was stored in three passive cooling containers (Equitainer, Equine Express II, and BotuFlex) for 48 hours. In experiment 2, the same ejaculates extended in experiment 1 were cushion-centrifuged, the supernatant was discarded, and the pellets were resuspended at 100 million sperm/mL with their respective extender. Semen was then cooled and stored as in experiment 1. In both experiments, sperm motility parameters, plasma membrane integrity, and high mitochondrial membrane potential were assessed at 0, 24, and 48 hours post cooling. For experiment 3, 12 mares (n = 24 cycles) were bred with 48 hour–cooled semen from one stallion. Semen was processed as described in experiment 1. Mares had embryo flushing performed by 8-day post-ovulation. In experiment 1, BotuSemen Gold displayed superior total and progressive motility relative to INRA 96 (P<0.05). There were no significant differences between the types of containers in any experiment. In experiment 2, INRA 96 and BotuSemen Gold extenders had similar total and progressive motility, but BotuSemen Gold had superior sperm velocity parameters at all timepoints. Embryo recovery was identical for both extenders (50%). Finally, the results obtained herein suggest that BotuSemen Gold is a suitable alternative to be included in semen cooling tests against INRA 96 in clinical practice. Secondly, the effect of obesity in semen freezing ability of Crioulo stallions was evaluated. Twenty stallions were allocated into 2 groups according to their body condition score (BCS): stallions with BCS <7 were characterized as Low-BCS (11/20) and stallions with BCS ≥7 as High-BCS (9/20). A complete clinical history followed by morphometric and subcutaneous body fat measurements were obtained. Serum glucose concentrations were evaluated after oral sugar test. For this, the stallions were fasted overnight and corn syrup was orally administered at 8 am. Blood samples were collected for glucose assessment before and after oral administration of corn syrup. In addition, one ejaculated of each stallion was collected and extended at 50 million sperm/mL with skimmed-milk based extender (BotuSemen, Botupharma, Brazil). Semen was centrifuged (600 ×g/10 min), the supernatant discarded, the pellet resuspended at 200 million sperm/mL with BotuCrio (Botupharma, Brazil) and submitted to cryopreservation. Sperm kinetics were assessed by CASA and plasma membrane integrity, ROS, mitochondrial membrane potential and lipid peroxidation by flow cytometer in post-thawed semen samples. The morphometric study (e.g. body weight, BCS and neck circumference at 25% and 50%) were superior in obesity stallions (P<0.05). However, there were no differences in sperm parameters or serum glucose concentration between groups (P>0.05). In summary the high-BCS in Crioulo horse didn’t affect sperm parameters and freezing ability of Crioulo stallions.
Descrição
Palavras-chave
Sêmen, Criopreservação, Escore de condição corporal, Diluente de sêmen equino, Sperm, Cryopreservation, Body condition score, Stallion semen extender