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During the poultry sperm cryopreservation process, an excess of reactive oxygen species is generated resulting in oxidative stress which harms the quality of avian spermatozoa. To counteract this effect, the addition of exogenous antioxidants, such as Pectoliv-80A (a by-product of olive oil), to the cryopreservation diluent is interesting. For this purpose, 16 roosters belonging to the Utrerana avian breed were used. Six semen pools (from the 6 different replicates) were divided into 4 aliquots corresponding to different concentrations of Pectoliv-80A that were tested (0, 300, 400, and 500 μg/mL), and the cryopreservation process was carried out. To evaluate post-thawing semen quality, different parameters such as motility, membrane functionality, reactive oxygen species production, lipid peroxidation, and acrosome integrity were studied. A discriminant canonical analysis was used to determine both the differences between the Pectoliv-80A concentration groups and the discriminant power of the aforementioned parameter used for semen evaluation. Total motility and membrane functionality were reported to be the most discriminant variables for differentiating the different antioxidant enrichment groups and concluded that concentrations of 300 μg/mL showed the most desirable quality of post-thawing semen. The present study could lead to the optimization of both cryopreservation and quality evaluation techniques of the sperm of rooster species, that support the conservation program of endangered local breeds., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Various climatological and lunar cycle parameters have a direct impact on animal reproduction, and in the case of the avian species, spermatozoa are extremely sensitive to heat stress. These parameters could influence sperm freezability, which will ultimately affect post-thawing semen quality, being sperm motility in roosters a relevant indicator of this quality as it is highly related to fertility. Therefore, the objective of the present study is to determine which are the climatological and lunar cycle parameters that have a greater effect on sperm freezability in roosters. Methods: Sperm was obtained from 16 Utrerana breed roosters and a total of 27 replicates were performed. A pool was made with those ejaculates that met the minimum quality criteria for each replicate, and four freezing-thawing samples per replicate were analyzed. The straws were thawed, and sperm motility was evaluated, classifying the results obtained into four seminal quality groups according to the guidelines of the Food and Agriculture Organization of the United Nations (Group 1: Good, Group 2: Satisfactory, Group 3: Acceptable but undesirable and Group 4: Unsatisfactory). The following traits were recorded for each day of semen collection: maximum temperature, minimum temperature, maximum barometric pressure, minimum barometric pressure, maximum gust, wind direction, mean wind speed, sunshine hours, rainfall, moon phase, and percentage of illuminated lunar surface over the total area. Results: A discriminant canonical analysis was performed to determine which of these parameters offered the most information when classifying an ejaculate in each quality group, with minimum temperature, the new moon as moon phase, minimum barometric pressure, and rainfall being the most significant variables. Discussion: According to the results obtained, semen quality decreases when temperature and precipitation are lower, pressure is higher, and when there is a new moon phase. Therefore, these environmental conditions should be avoided for sperm collection and processing. [ABSTRACT FROM AUTHOR]

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. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: The use of olive oil by-products for caprine sperm cryopreservation offers an interesting opportunity to improve post-thawed sperm quality, as antioxidants such as hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG) could reduce lipid peroxidation. Therefore, this study provides evidence of the positive effect of the addition of HT, DHPG, or the mixture of both antioxidants in cryopreserved buck sperm. In addition, the application of Bayesian statistics for data analysis may enable quantifying the dimensionality of the real effect of antioxidants on sperm. The present study evaluates the effect of olive oil-derived antioxidants, hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG), on cryopreserved caprine sperm using Bayesian inference of ANOVA. For this proposal, sperm was collected, pooled and diluted in freezing media supplemented with different concentrations of HT, DHPG and the mixture (MIX) of both antioxidants. Sperm motility, viability, acrosome integrity, mitochondrial status, and lipid peroxidation (LPO) were assessed in fresh and frozen-thawed sperm samples. The results provided evidence that HT at low concentrations improves sperm motility and viability, and reduces the LPO. Contrastingly, DHPG and MIX exert a positive effect by reducing LPO values as concentration increases. Additionally, mitochondrial potential was reduced when samples were supplemented with HT at low concentrations and mixture of both antioxidants. Conclusively, the addition of olive oil-derived antioxidants (HT at 10 µg/mL and DHPG at 30 µg/mL) implements a protective effect in cryopreserved buck sperm. Bayesian analysis alternatives offer new possibilities to determine the repercussion of antioxidants on sperm, both quantitatively and qualitatively. [ABSTRACT FROM AUTHOR]