Cat epididymal semen cryopreserved with and without vitamin E: effect on sperm parameters and lipid peroxidation

The aims of this study were to investigate: 1) if the addition of α-tocopherol (vitamin E) in three concentrations (0.3, 0.6 and 0.9 mM) is able to preserve spermatozoa integrity after thawing and 2) the effect of α-tocopherol supplementation on lipid peroxidation. Fifty four domestic cats were used in this study constituting 18 pools (3 cats per pool). Each pool was submitted at four experimental groups: group 0 (control) – epididymal sperm were frozen with a commercial Botucrio® extender; group 0.3, group 0.6 and group 0.9 – the extender was supplemented with 0.3, 0.6 and 0.9 mM of α-tocopherol, respectively. Each semen sample was evaluated for motility, progressive forward motility, morphology, sperm viability (plasma membrane integrity-PMI), hypo-osmotic swelling test (HOST), before and after thawing. The evaluation of lipid peroxidation reaction by Thiobarbituric Acid Reactive Substances (TBARS) test was performed on thawed semen only. Results demonstrated that there was no significant difference between control and the three α-tocopherol groups with regards to motility and progressive motility after thawing (P > 0.05). As expected, in fresh samples viability was significantly higher than in all the cryopreserved groups in which there was no positive influence of any of the αtocopherol concentration used. Lipid peroxidation was higher in the supplemented groups 0.6 and 0.9 mM of α-tocopherol than in control and in 0.3 mM group. In conclusion, the addition of α-tocopherol to the commercial extender had no positive influence on reduction of lipid peroxidation. This topic deserves further investigations to better understand the effect of cryopreservation procedures on epididymal spermatozoa and to establish adequate strategies to counteract sperm cryodamages.

Cat epididymal semen cryopreserved with and without vitamin E: effect on sperm parameters and lipid peroxidation

The aims of this study were to investigate: 1) if the addition of α-tocopherol (vitamin E) in three concentrations (0.3, 0.6 and 0.9 mM) is able to preserve spermatozoa integrity after thawing and 2) the effect of α-tocopherol supplementation on lipid peroxidation. Fifty four domestic cats were used in this study constituting 18 pools (3 cats per pool). Each pool was submitted at four experimental groups: group 0 (control) epididymal sperm were frozen with a commercial Botucrio® extender; group 0.3, group 0.6 and group 0.9 the extender was supplemented with 0.3, 0.6 and 0.9 mM of α-tocopherol, respectively. Each semen sample was evaluated for motility, progressive forward motility, morphology, sperm viability (plasma membrane integrity-PMI), hypo-osmotic swelling test (HOST), before and after thawing. The evaluation of lipid peroxidation reaction by Thiobarbituric Acid Reactive Substances (TBARS) test was performed on thawed semen only. Results demonstrated that there was no significant difference between control and the three α-tocopherol groups with regards to motility and progressive motility after thawing (P > 0.05). As expected, in fresh samples viability was significantly higher than in all the cryopreserved groups in which there was no positive influence of any of the αtocopherol concentration used. Lipid peroxidation was higher in the supplemented groups 0.6 and 0.9 mM of α-tocopherol than in control and in 0.3 mM group. In conclusion, the addition of α-tocopherol to the commercial extender had no positive influence on reduction of lipid peroxidation. This topic deserves further investigations to better understand the effect of cryopreservation procedures on epididymal spermatozoa and to establish adequate strategies to counteract sperm cryodamages.(AU)

Efeito do estádio do ciclo estral e adição de hormônios ao meio de cultivo in vitro sobre a morfologia do complexo cumulus-oócito canino

Este estudo foi desenvolvido com o intuito de avaliar comparativamente o efeito do estádio do ciclo estral, bem como a adição de hormônios aos meios de cultivo in vitro sobre a morfologia do complexo cumulus-oócito canino. Os oócitos foram coletados de 30 cadelas hígidas submetidas à ovariosalpingo-histerectomia, divididas em dois grupos de acordo com a condição reprodutiva (fase folicular e anestro). A avaliação foi feita por microscopia eletrônica de varredura e transmissão, antes e após o período de cultivo in vitro por 72h em dois diferentes meios: meio controle - A (TCM199) e meio B: TCM199 adicionado de 10UI/mL de LH, 2?g/mL de progesterona (P4) e 2?g/mL de Estradiol(E2). Os resultados revelaram que o diâmetro oocitário não é influenciado pela fase reprodutiva, assim como a comunicação intercelular, e os aspectos morfológicos relacionados à forma e arranjo das células do cumulus, in vivo. A fase do ciclo estral exerceu influência sobre a morfologia do complexo cumulus-oócito, de tal forma que oócitos pertencentes ao grupo folicular apresentaram-se com maior grau de maturidade nuclear e citoplasmática do que oócitos do grupo do anestro. A suplementação hormonal afetou positivamente a maturação nuclear, mas não exerceu efeito sobre a maturação citoplasmática. Com relação à ultra-estrutura a presença de hormônios nos meios de cultivo foi benéficas tanto para a comunicação entre as células do cumulus como para a manutenção da integridade oocitária

This study was developed in order to comparatively evaluate the effects of stages of the estrous cycle, as well as the addition of hormones to culture media in vitro on the morphology of cumulus-oocyte complex canine. The oocytes were collected from 30 healthy bitches ovariosalpingo underwent hysterectomy were divided into two groups according to reproductive condition (follicular phase and anestrus). The evaluation was performed by scanning electron microscopy and transmission, before and after cultivation in vitro for 72h in two different media: control medium - A (TCM199) and medium B: TCM199 added 10IU/mL LH, 2?g / mL progesterone (P4) and 2?g/mL Estradiol (E2). The results revealed that the oocyte diameter is not influenced by reproductive stage, as well as intercellular communication, and morphological aspects related to the shape and arrangement of cumulus cells, in vivo. The estrous cycle phase influenced the morphology of cumulus-oocyte complex, such that follicular oocytes in the group presented with greater maturity than nuclear and cytoplasmic oocyte group of anestrus. The hormonal supplementation positively affected nuclear maturation, but had no effect on cytoplasmic maturation. Regarding the ultrastructural presence of hormones in culture media was beneficial both for communication between the cumulus cells as well as preserving the integrity of oocyte