Assessment of sperm viability is an important component for evaluating stallion sperm quality. The flow cytometer is considered the standard in the assessment of sperm plasma membrane integrity (viability); however, this instrument is costly to purchase and use, and it requires an experienced technician to operate it. The growing practice of assisted reproductive technologies (ARTs) in the equine industry has increased the need for an accurate but cost-effective means of determining sperm membrane viability. The NucleoCounter® SP-100TM is reported to be an accurate, easy-to-perform, and an efficient stallion-side test for sperm membrane viability. To evaluate usefulness of the NucleoCounter® SP-100TM for assessing sperm membrane integrity, neat semen was subjected to four treatments with varying seminal plasma volumes and sperm concentrations. Sperm membrane viability was assessed immediately, and at 24 and 48 hours after cooled-storage using three methods: 1) flow cytometer utilizing the fluorescent vital stains SYBR-14/propidium iodide; 2) NucleoCounter® SP-100TM utilizing the fluorescent vital stain propidium iodide; 3) eosin-nigrosin stained air-dried smears of semen. Sperm motion characteristics (total and progressive motility) were assessed using a computer assisted sperm motion analyzer (CASMA) and results were compared to sperm membrane viability to determine the relationship between sperm membrane viability and motion characteristics. Results were compared statistically by: 1) analysis of variance (ANOVA); 2) linear regression analysis; 3) coefficient of variation on untransformed and transformed data (arc sine square root); and 3) the agreement of two instruments, by means of which the difference between measurements of the two instruments were plotted on the y-axis and the average of measurements from the two instruments were plotted on the x-axis. Results obtained with the NucleoCounter® SP-100TM agreed best with the flow cytometer, and least with eosin-nigrosin staining. Coefficients of variation were ≤ 5% for the three methods (transformed data). Sperm motion characteristics and sperm viability were similar among treatments at Time 0. At Times 24 and 48, sperm motion characteristics decreased at a more significant rate compared to viability in the treatments containing ≥ 50% seminal plasma, whereas differences among treatments were only significant at seminal plasma concentrations above 50% when only sperm membrane viability was considered.

As espécies reativas de oxigênio são fundamentais na fisiologia espermática. No entanto, um desequilíbrio entre a produção e a capacidade antioxidante caracteriza o estresse oxidativo (EO). O espermatozoide é extremamente suscetível ao EO pois, dentre outras características, a membrana plasmática é rica em ácidos graxos poli-insaturados responsáveis por promoverem a fluidez necessária em processos fisiológicos como motilidade e fertilização. Por outro lado, essas insaturações são mais facilmente oxidadas e vulneráveis à peroxidação lipídica. Em função desta susceptibilidade, estas células dependem fortemente de compostos presentes no plasma seminal (PS) para a proteção contra esse evento. Dessa forma, a carnosina, dipeptídeo presente no PS pode ser uma das responsáveis pela proteção contra o acúmulo do MDA. No entanto, durante a criopreservação do sêmen equino é necessário retirar o PS. Em estudo recente, verificamos que esta remoção, torna os espermatozoides sensíveis ao subproduto extremamente deletério da peroxidação lipídica, o malondialdeído (MDA). Como a carnosina é removida junto com o plasma seminal durante a criopreservação, foram desenvolvidos 2 experimentos sequenciais visando a melhora da qualidade do sêmen criopreservado com adição de carnosina. Amostras de sêmen de sete garanhões foram tratadas com concentrações crescentes de carnosina adicionadas ao diluidor (1mM, 50mM e 100mM). Após a descongelação, as amostras foram divididas retrospectivamente em grupos de alta congelabilidade (AC: motilidade maior que 30%) e baixa congelabilidade (BC: motilidade menor que 30%). Amostras tratadas com 50mM apresentaram menor porcentagem de células com lesão de membrana plasmática e, quando tratadas com 100mM, células com maior amplitude do deslocamento lateral de cabeça. Amostras controle BC apresentaram menor porcentagem de células com DNA íntegro em relação às amostras AC. No entanto, houve um leve aumento na porcentagem de células com DNA íntegro em amostras BC com 100mM, não diferindo das amostras AC. Por outro lado, amostras BC criopreservadas com 50mM apresentaram maiores porcentagens de células com escore calculado de potencial de membrana mitocondrial e mais suscetíveis ao EO em relação ao controle. Apesar da proteção parcial, a maior suscetibilidade à peroxidação lipídica torna-se um problema, especialmente pelo fato de que espermatozoides equinos são mais suscetíveis ao MDA. Um motivo para este efeito seria a afinidade da carnosina em reagir com açúcares, o que poderia influenciar negativamente a atividade mitocondrial e o status oxidativo, ao diminuir a produção de piruvato pela via glicolítica. Desta forma, no experimento 2, amostras BC foram tratadas com a combinação de carnosina (0 e 50mM) e piruvato (0 e 5mM) em arranjo fatorial 2x2. Verificou-se que o tratamento com piruvato (5mM) proporcionou menos células com baixa atividade mitocondrial. Por outro lado, a carnosina (50mM), promoveu maior motilidade total, progressiva e células rápidas. Houve uma tendência de aumento nas células com velocidade progressiva e atividade mitocondrial na combinação de tratamentos. Não houve diferença entre os grupos na suscetibilidade ao EO que, no entanto, correlacionou-se negativamente com células móveis, rápidas e integridade de membrana plasmática e acrossomal. Estes resultados indicam que subprodutos da peroxidação lipídica, sendo o principal deles o MDA, podem causar danos ao DNA, às mitocôndrias e à cinética espermática. Neste contexto, a carnosina (100mM) parece ter um leve efeito protetor ao DNA contra o acúmulo de MDA. Além disto, 50mM de carnosina parece auxiliar na manutenção da velocidade progressiva e atividade mitocondrial quando associada ao piruvato (5mM). Assim, a carnosina e o piruvato podem ser utilizados na prevenção de crioinjúrias em amostras de baixa congelabilidade. / Reactive oxygen species (ROS) plays a key role in the sperm physiology. However, an imbalance between ROS production and antioxidant capacity characterize the oxidative stress (OE). The spermatozoa are extremely susceptible to EO because, among other characteristics, the plasma membrane is rich in polyunsaturated fatty acids responsible for promoting fluidity necessary in physiological processes such as motility and fertilization. However, these unsaturations are more easily oxidized and vulnerable to lipid peroxidation. Due to this susceptibility, these cells strongly depend on compounds present in the seminal plasma (SP) to protect against this event. Thus, carnosine, a dipeptide present in SP of stallions, may be a key factor on the protection against MDA accumulation. Nevertheless, during the equine sperm cryopreservation process, SP is removed. In a recent study, we observed that seminal plasma removal led to an increased susceptibility of equine spermatozoa to extremely deleterious product of lipid peroxidation, malondialdehyde (MDA). As the carnosine is removed together with the seminal plasma during cryopreservation, two sequential experiments were developed aiming to improve the quality of stallion cryopreserved semen by means of carnosine therapy. Samples from seven stallions were treated with increasing concentrations of carnosine added to the extender (1mM, 50mM and 100mM) and submitted to cryopreservation. After thawing, samples were classified as high freezeability (HF: total motility greater than 30%) and low freezeability (LF: total motility lower than 30%). Samples treated with 50mM presented lower percentage of sperm showing plasma membrane damage and, when treated with 100mM, a greater amplitude of the lateral head displacement was observed. Untreated LF samples showed a lower percentage of cells showing intact DNA in relation to HF samples. By contrast, when LF samples were treated with 100mM, there was an increase in the percentage of cells with intact DNA, which was similar to the HF samples. On the other hand, LF samples cryopreserved with 50mM had a higher percentage of cells showing high calculated mitochondrial membrane potential score and increased susceptibility to OE in relation to the control. Despite the partial protection, the increased susceptibility to lipid peroxidation is a concern since equine spermatozoa is highly vulnerable to the MDA. Those results could be due to the affinity of carnosine to react with sugars, which could negatively influence mitochondrial activity and an oxidative state by decreasing pyruvate production. Hence, in experiment 2, LF samples were treated with a combination of carnosine (0 and 50mM) and pyruvate (0 and 5mM) in a 2x2 factorial arrangement. We observed that samples treated with pyruvate (5mM) had decreased percentage of cells with low mitochondrial activity. On the other hand, carnosine (50mM) increased total motility, progressive motility and fast cells. We also observed a tendency to increased progressive velocity and mitochondrial activity in the combination of treatments. There was no difference on sperm susceptibility to OE between treatments. However, this variable correlated negatively with the percentage of motile and rapid cells as well as those showing intact membrane and acrosome. These results indicate that the byproduct of lipid peroxidation (MDA) may cause damage to DNA, mitochondria and sperm kinetics. In this context, carnosine (100mM) appears to have a mild protective effect on DNA against the accumulation of MDA. Furthermore, 50mM of carnosine seems to improve progressive velocity and mitochondrial activity when associated with pyruvate (5mM). Thus, carnosine and pyruvate can be used on cryoinjuries prevention in low freezeability samples.

As espécies reativas de oxigênio são fundamentais na fisiologia espermática. No entanto, um desequilíbrio entre a produção e a capacidade antioxidante caracteriza o estresse oxidativo (EO). O espermatozoide é extremamente suscetível ao EO pois, dentre outras características, a membrana plasmática é rica em ácidos graxos poli-insaturados responsáveis por promoverem a fluidez necessária em processos fisiológicos como motilidade e fertilização. Por outro lado, essas insaturações são mais facilmente oxidadas e vulneráveis à peroxidação lipídica. Em função desta susceptibilidade, estas células dependem fortemente de compostos presentes no plasma seminal (PS) para a proteção contra esse evento. Dessa forma, a carnosina, dipeptídeo presente no PS pode ser uma das responsáveis pela proteção contra o acúmulo do MDA. No entanto, durante a criopreservação do sêmen equino é necessário retirar o PS. Em estudo recente, verificamos que esta remoção, torna os espermatozoides sensíveis ao subproduto extremamente deletério da peroxidação lipídica, o malondialdeído (MDA). Como a carnosina é removida junto com o plasma seminal durante a criopreservação, foram desenvolvidos 2 experimentos sequenciais visando a melhora da qualidade do sêmen criopreservado com adição de carnosina. Amostras de sêmen de sete garanhões foram tratadas com concentrações crescentes de carnosina adicionadas ao diluidor (1mM, 50mM e 100mM). Após a descongelação, as amostras foram divididas retrospectivamente em grupos de alta congelabilidade (AC: motilidade maior que 30%) e baixa congelabilidade (BC: motilidade menor que 30%). Amostras tratadas com 50mM apresentaram menor porcentagem de células com lesão de membrana plasmática e, quando tratadas com 100mM, células com maior amplitude do deslocamento lateral de cabeça. Amostras controle BC apresentaram menor porcentagem de células com DNA íntegro em relação às amostras AC. No entanto, houve um leve aumento na porcentagem de células com DNA íntegro em amostras BC com 100mM, não diferindo das amostras AC. Por outro lado, amostras BC criopreservadas com 50mM apresentaram maiores porcentagens de células com escore calculado de potencial de membrana mitocondrial e mais suscetíveis ao EO em relação ao controle. Apesar da proteção parcial, a maior suscetibilidade à peroxidação lipídica torna-se um problema, especialmente pelo fato de que espermatozoides equinos são mais suscetíveis ao MDA. Um motivo para este efeito seria a afinidade da carnosina em reagir com açúcares, o que poderia influenciar negativamente a atividade mitocondrial e o status oxidativo, ao diminuir a produção de piruvato pela via glicolítica. Desta forma, no experimento 2, amostras BC foram tratadas com a combinação de carnosina (0 e 50mM) e piruvato (0 e 5mM) em arranjo fatorial 2x2. Verificou-se que o tratamento com piruvato (5mM) proporcionou menos células com baixa atividade mitocondrial. Por outro lado, a carnosina (50mM), promoveu maior motilidade total, progressiva e células rápidas. Houve uma tendência de aumento nas células com velocidade progressiva e atividade mitocondrial na combinação de tratamentos. Não houve diferença entre os grupos na suscetibilidade ao EO que, no entanto, correlacionou-se negativamente com células móveis, rápidas e integridade de membrana plasmática e acrossomal. Estes resultados indicam que subprodutos da peroxidação lipídica, sendo o principal deles o MDA, podem causar danos ao DNA, às mitocôndrias e à cinética espermática. Neste contexto, a carnosina (100mM) parece ter um leve efeito protetor ao DNA contra o acúmulo de MDA. Além disto, 50mM de carnosina parece auxiliar na manutenção da velocidade progressiva e atividade mitocondrial quando associada ao piruvato (5mM). Assim, a carnosina e o piruvato podem ser utilizados na prevenção de crioinjúrias em amostras de baixa congelabilidade. / Reactive oxygen species (ROS) plays a key role in the sperm physiology. However, an imbalance between ROS production and antioxidant capacity characterize the oxidative stress (OE). The spermatozoa are extremely susceptible to EO because, among other characteristics, the plasma membrane is rich in polyunsaturated fatty acids responsible for promoting fluidity necessary in physiological processes such as motility and fertilization. However, these unsaturations are more easily oxidized and vulnerable to lipid peroxidation. Due to this susceptibility, these cells strongly depend on compounds present in the seminal plasma (SP) to protect against this event. Thus, carnosine, a dipeptide present in SP of stallions, may be a key factor on the protection against MDA accumulation. Nevertheless, during the equine sperm cryopreservation process, SP is removed. In a recent study, we observed that seminal plasma removal led to an increased susceptibility of equine spermatozoa to extremely deleterious product of lipid peroxidation, malondialdehyde (MDA). As the carnosine is removed together with the seminal plasma during cryopreservation, two sequential experiments were developed aiming to improve the quality of stallion cryopreserved semen by means of carnosine therapy. Samples from seven stallions were treated with increasing concentrations of carnosine added to the extender (1mM, 50mM and 100mM) and submitted to cryopreservation. After thawing, samples were classified as high freezeability (HF: total motility greater than 30%) and low freezeability (LF: total motility lower than 30%). Samples treated with 50mM presented lower percentage of sperm showing plasma membrane damage and, when treated with 100mM, a greater amplitude of the lateral head displacement was observed. Untreated LF samples showed a lower percentage of cells showing intact DNA in relation to HF samples. By contrast, when LF samples were treated with 100mM, there was an increase in the percentage of cells with intact DNA, which was similar to the HF samples. On the other hand, LF samples cryopreserved with 50mM had a higher percentage of cells showing high calculated mitochondrial membrane potential score and increased susceptibility to OE in relation to the control. Despite the partial protection, the increased susceptibility to lipid peroxidation is a concern since equine spermatozoa is highly vulnerable to the MDA. Those results could be due to the affinity of carnosine to react with sugars, which could negatively influence mitochondrial activity and an oxidative state by decreasing pyruvate production. Hence, in experiment 2, LF samples were treated with a combination of carnosine (0 and 50mM) and pyruvate (0 and 5mM) in a 2x2 factorial arrangement. We observed that samples treated with pyruvate (5mM) had decreased percentage of cells with low mitochondrial activity. On the other hand, carnosine (50mM) increased total motility, progressive motility and fast cells. We also observed a tendency to increased progressive velocity and mitochondrial activity in the combination of treatments. There was no difference on sperm susceptibility to OE between treatments. However, this variable correlated negatively with the percentage of motile and rapid cells as well as those showing intact membrane and acrosome. These results indicate that the byproduct of lipid peroxidation (MDA) may cause damage to DNA, mitochondria and sperm kinetics. In this context, carnosine (100mM) appears to have a mild protective effect on DNA against the accumulation of MDA. Furthermore, 50mM of carnosine seems to improve progressive velocity and mitochondrial activity when associated with pyruvate (5mM). Thus, carnosine and pyruvate can be used on cryoinjuries prevention in low freezeability samples.