Discriminant Analysis and Data Mining CHAID Decision Tree as Tools to Evaluate the Buffering Effect of Hydroxytyrosol on Reactive Oxygen Species in Rooster Sperm Cryopreservation

SIMPLE SUMMARY: For the conservation of genetic resources in avian species, semen freezing is very helpful. The disadvantage of this process is that spermatozoa suffer different types of damage. Exogenous antioxidants can be added to the cryopreservation extender to mitigate this damage. This study aimed to test whether the addition of different hydroxytyrosol (HT; an antioxidant derived from olive oil) concentrations produces beneficial effects in the sperm of a local avian breed (Utrerana roosters). For this purpose, different semen quality parameters, which fell under the following macro-areas were evaluated in both fresh and thawed semen: motility, morphology, membrane functionality, and flow-cytometry-related traits. At the statistical level, a descriptive analysis and a canonical discriminant analysis were performed, which allowed us to extract valuable information about the different studied variables. Lastly, a chi-squared automatic interaction detection (CHAID) decision tree (DT) was carried out and the reactive oxygen species (ROS) variable was found to have the highest power to discriminate between the different treatments according to the HT concentration. Low or no HT concentrations resulted in higher ROS values, and therefore, possible mechanical damage unrelated to plasma membrane peroxidation can be produced in the frozen–thawed rooster spermatozoa. ABSTRACT: Sperm cryopreservation is effective in safeguarding genetic biodiversity in avian species. However, during this process, spermatozoa are very susceptible to plasma membrane peroxidation in the presence of high concentrations of reactive oxygen species (ROS). To mitigate this effect, the addition of exogenous antioxidants, such as hydroxytyrosol (3,4-dihydroxyphenylethanol; HT), an antioxidant derived from olive oil, to the cryopreservation sperm diluent, could be useful. To verify this, a cryopreservation diluent was supplemented with different concentrations (0 μg/mL, 50 μg/mL, 100 μg/mL, and 150 μg/mL) of HT. For this, semen was collected in 10 replicates from 16 roosters of the Utrerana avian breed, and a pool was prepared with the optimum quality ejaculates in each replicate. After cryopreservation, spermatozoa were thawed and different in vitro semen quality parameters were evaluated. A discriminant canonical analysis (DCA) was carried out and revealed that total motility (TM; Lambda = 0.301, F = 26,173), hypo-osmotic swelling test (HOST; Lambda = 0.338, F = 22,065), and amplitude of lateral head displacement (ALH, Lambda = 0.442; F = 14,180) were the variables with the highest discriminant power. Finally, a chi-squared automatic interaction detection (CHAID) decision tree (DT) was performed excluding fresh semen samples and ROS was found to be the most valuable variable to discriminate between the different established freezing groups. Samples in the absence of HT or with low concentrations of this antioxidant showed less desirable ROS values in cryopreserved rooster semen. The present study could lead to the improvement of cryopreservation techniques for the genetic material of local poultry breeds and optimize the conservation programs of endangered native avian breeds.