Methodological standardizations in the cooling of canine semen

Abstract

The advancement of biotechnologies applied to animal reproduction has increased the demand for qualified services in artificial insemination in carnivores, particularly with the growing use of refrigerated semen. This biotechnology offers advantages such as reducing animal stress, facilitating storage, and presenting better fertilization rates compared to frozen semen. However, the lack of standardized protocols can affect the success of the technique and consequently reduce the fertility of the breeders. Over the past ten years, there has been progress in the quality of cryopreserved canine semen, with the use of different extenders, cryoprotectants, and cooling rates. Although the reduction in sperm quality is associated with storage time and temperature, the results still present better outcomes compared to freezing. With the use of established techniques in handling semen from domestic species, it is possible that refrigeration of canine semen could show considerable improvements. Thus, the removal of seminal plasma, primarily used in horses, is a promising technique that may enhance sperm quality after canine semen refrigeration. Additionally, the use of extenders can minimize damage to spermatozoa, but the concentration and dilution rate still need to be determined. So far, few studies and methodologies standardize and investigate the ideal dilution rates, the impact of storage at 5°C, and a 24-hour storage period. Therefore, the production of reactive oxygen species should be considered, with a need for more research to optimize the quality of refrigerated semen. Thus, this proposal aims to test the best sperm concentration and the influence of seminal plasma and centrifugation on canine semen refrigeration, under sperm kinetic parameters. For this purpose, ten healthy dogs will be used, with two ejaculates collected from each and divided into the following experimental groups: 1- Control (3:1 dilution, relative to the total volume); 2- Centrifugation for seminal plasma removal and dilution to 50 and 200 x 10^6 spermatozoa/mL; and 3- Centrifugation and removal of seminal plasma, with prior 2:1 dilution, following the same concentrations, and subjected to passive cooling (5°C) for 24 hours. The semen laboratory evaluations will include sperm kinetics and flow cytometer to assess cell membranes and the production of free radicals (oxidative stress). It is expected that the dilution and centrifugation for seminal plasma removal will promote better membrane integrity and sperm kinetics in semen cryopreservation.