DELETERIOUS EFFECT OF HIGH CARNOSINE CONCENTRATIONS IN EXTENDERS DURING SPERM CRYOPRESERVATION IN DOGS / EFECTO DELETÉREO DE ALTAS CONCENTRACIONES DE CARNOSINA EN DILUYENTES DURANTE LA CRIOPRESERVACIÓN ESPERMÁTICA EN PERROS

Cryopreservation is a key process among the canine reproductive biotechnologies. However, during sperm cryopreservation an excessive reactive oxygen species (ROS) generation occurs, leading to decrease in sperm quality. Therefore, several antioxidants were tested during sperm cryopreservation to prevent such effects, however the carnosine it has not used. Carnosine is a protein present in the seminal plasma, and unlike other antioxidants has the ability to remove products of lipid peroxidation (malondialdehyde), which are as harmful as ROS. Thus, the aim of this study was to evaluate the effects of different carnosine concentrations, during sperm cryopreservation in dogs. For this purpose, six dogs in reproductive age were used, and after sperm collection the samples were cryopreserved in Control (tris-citrate egg yolk extender), Carnosine 1mM, 50mM and 100mM groups. After thawing samples were analyzed by computer-assisted analysis of sperm motility, plasma membrane (eosin/nigrosin), acrosome integrity (fast green/rose Bengal), mitochondrial activity, DNA integrity and sperm resistance to oxidative stress (by TBARS). Decrease was observed in motility sperm kinetics (total and progressive motility) and reduced lipid peroxidation products in the group treated with 50mM and 100mM. On the other hand, 1mM was similar to control group. In conclusion, higher carnosine concentration (50 and 100mM) apparently promoted impairment in energy production and consequently was harmful to sperm kinetics. Thus, future studies must be performed using different carnosine concentrations and in association with substrates for glycolysis and oxidative phosphorylation.RESUMENLa criopreservación es un proceso clave entre las biotecnologías reproductivas en caninos. Sin embargo, durante la criopreservación espermática se da una generación excesiva de especies reactivas de oxígeno (ROS), lo que lleva a una disminución en la calidad espermática. Por lo tanto, varios medios de congelación utilizando antioxidantes para evitar tales efectos han sido evaluados, aunque la carnosina todavía no se ha utilizada. La carnosina es una proteína presente en el plasma seminal que a diferencia de otros antioxidantes tiene la habilidad de remover productos de la peroxidación lipídica (malondialdehído), que son tan dañinos como los ROS. Por lo tanto, el objetivo de este estudio fue evaluar los efectos de diferentes concentraciones de carnosina durante la congelación espermática en perros. Para este propósito se utilizaron seis perros en edad reproductiva y después de la colectar los eyaculados, las muestras fueron criopreservadas en un diluyente Control (tris, citrato, yema de huevo), Carnosina 1mM, 50mM y 100 mM. Después del descongelado, las muestras fueron evaluadas mediante el análisis computerizado de la motilidad, integridad de membrana plasmática (eosina / nigrosina), integridad del acrosoma (Fast - green / rosa de Bengala), la actividad mitocondrial (3’3 Diaminobenzidina), la integridad del ADN (SCSA) y la evaluación de la resistencia al estrés oxidativo (TBARS). Se observó una disminución en la cinética de los espermatozoides (motilidad total y progresiva) y una reducción de los productos de la peroxidación lipídica en los grupos tratados con 50 mM y 100mM de carnosina. Por otro lado, el grupo con 1 mM de carnosina fue similar al control. En conclusión, una alta concentración de carnosina (50 y 100mM) parece afectar la producción de energía del espermatozoide y por lo tanto es perjudicial para la cinética del espermatozoide. Por lo tanto, futuros estudios deben realizarse utilizando diferentes concentraciones de carnosina y en asociación con sustratos para la glucólisis y la fosforilación oxidativa

Effect of carnosine on the protection against cryoinjuries in semen of good and bad freezers\' stallions

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

Study of oxidative status during the chilling of dog sperm

O sêmen refrigerado de cães apresenta maior taxa de concepção, facilidade no transporte, pois dispensa a utilização de nitrogênio líquido e fácil aplicação na inseminação artificial pela via intravaginal em relação ao sêmen criopreservado. Entretanto, os espermatozoides sofrem uma modificação em sua estrutura lipídica ao passar da fase líquido-cristalina para fase gel levando a alteração na permeabilidade e resistência da membrana plasmática. A característica espermática que confere maior resistência à membrana plasmática é a alta concentração de ácidos graxos poli-insaturados (PUFAs), predominantemente o ácido docosahexaenoico. Por outro lado, os PUFAs tornam os espermatozoides mais suscetíveis à peroxidação lipídica. Neste contexto, avaliou-se a suscetibilidade de espermatozoides de cães refrigerados a 5ºC, durante 24 horas, após a incubação com as diferentes espécies reativas de oxigênio: superóxido (O2 •- ), radical hidroxila (OH•), soluções de peróxido de hidrogênio (H2O2) e o subproduto da peroxidação lipídica, malondialdeído (MDA). Além disso, objetivamos determinar a terapia antioxidante ideal para a refrigeração seminal de cães nas condições aqui apresentadas. Ejaculados provenientes de 10 animais hígidos (N = 10), entre 2 e 6 anos, foram coletados e refrigerados (50×106 espermatozoides/mL) durante 24 horas. Posteriormente, foram submetidos ao desafio oxidativo pela incubação com os sistemas geradores de superóxido, radical hidroxila, soluções de peróxido de hidrogênio e malondialdeído. As amostras foram analisadas quanto à cinética espermática (CASA Computer Assisted Sperm Analysis), integridade de membrana plasmática (Eosina-Nigrosina) e acrossomal (Fast-green Rosa-bengala), atividade mitocondrial (3, 3 diaminobenzidina), peroxidação lipídica (ensaio TBARS) e quanto à atividade enzimática da superóxido dismutase. Para a análise estatística, utilizou-se o teste LSD (Least Significant Difference), considerando-se nível de significância inferior a 0,05 para efeito significativo.Chilled dog spermatozoa has a higher conception rate, ease of transport, as it does not require liquid nitrogen and is easy to apply in artificial insemination via intravaginal compared to cryopreserved semen. However, spermatozoa undergo changes on lipid structure when passing from the liquid-crystalline phase to the gel phase, compromising the permeability and resistance of the plasma membrane. The sperm characteristic that confers greater resistance to the plasma membrane is the high concentration of polyunsaturated fatty acids (PUFAs), predominantly docosahexaenoic acid. On the other hand, PUFAs make sperm more susceptible to lipid peroxidation. In this context, the susceptibility of chilled dog spermatozoa at 5ºC for 24 hours after incubation with different reactive oxygen species was evaluated: superoxide anion (O2 •-), hydroxyl radical (OH• ), hydrogen peroxide solution (H2O2) and the by-product of lipid peroxidation, malondialdehyde (MDA). In addition, we aimed to determine the ideal antioxidant therapy for chilled dog spermatozoa under the conditions presented here. Ejaculates from 10 healthy animals (N = 10), aged between 2 and 6 years, were collected and chilled (50×106 sperm/mL) for 24 hours. Subsequently, were submitted to oxidative challenge by incubation with generating systems of superoxide anion, hydroxyl radical and solutions of hydrogen peroxide and malondialdehyde. Samples were analyzed for sperm kinetics (CASA - Computer Assisted Sperm Analysis), plasma membrane integrity (Eosin-Nigrosin) and acrossomal (Fast-green Rose-bengal), mitochondrial activity (3, 3 diaminobenzidine), lipid peroxidation (assay TBARS) and for the superoxide dismutase enzymatic activity. For statistical analysis, the LSD (Least Significant Difference) test was used, considering a significance level lesser than 0.05 for a significant effect

Practical methods to assess the effects of heat stress on the quality of frozen-thawed Belgian Blue semen in field conditions

The increased exportation of semen and embryos of double-muscled beef breeds to tropical and developing countries makes it important to investigate the reproductive capacity of these breeds in adapting to tropical conditions. The aim of this study was to evaluate the quality of Belgian Blue semen collected after there is heat-stress (HS; as a mimic of tropical condition) compared with non-heat stressed (NHS; as their comfort zone), using practical spermatozoa staining methods such that prevail in developing countries. There was screening of semen kinetics using CASA and evaluation of their DNA-, acrosome, plasma membrane-integrity, and mitochondrial activity. For each staining technique, there was evaluation of 12 frozen-thawed semen samples from six Belgian Blue bulls collected after there were HS and NHS conditions in Belgium. Mixed linear regression models were used to assess the effects of HS for each CASA variable and staining method outcome using the replicate nested with bull as a random effect. There were differences (P < 0.05) in values when there were semen collections following HS and NHS conditions for several post-thawing kinetic variables. Furthermore, the mean percentages of DNA-, acrosome-, and plasma membrane-integrity, as well as mitochondrial activity were greater (P < 0.05) when semen was collected following NHS compared with HS conditions. Conclusively, results indicated that when there was collection of semen following HS conditions, there were detrimental effects on the viability and quality of Belgian Blue semen which is an important consideration for the semen collection, processing, and evaluation in tropical countries