Crocin Improves the Quality of Cryopreserved Goat Semen in Different Breeds

Simple Summary In goat breeding, artificial insemination has a strong economic impact: It provides genetic improvement in intensive production systems and guarantees breed preservation in extensive systems. Sperm freezability is affected by several factors, such as breed species and freezing procedures. In addition, in goats, the seminal plasma enzyme phospholipase A reacts with egg yolk (component of semen extender), compromising sperm viability. Thus, seminal plasma is removed before freezing. This removal causes a decreasing level of antioxidants that prevent formation of toxic lipid peroxides with deleterious effects on fertility. Crocin is a water-soluble carotenoid that acts as an antioxidant, protecting cells and tissues against oxidation. Therefore, the aim of the present study was to assess the effect of crocin in the semen extender before cryopreservation. Five different goat breeds (Garganica, Jonica, Maltese Mediterranean Red and Saanen) were chosen to evaluate sperm qualitative characteristics, such as post-thawing sperm motility, viability, morphology and membrane integrity, as well as DNA fragmentation and intracellular ROS levels. The results highlighted that crocin supplementation in the extender decreased oxidative stress and improved sperm motility and the DNA integrity of frozen-thawed sperm in different breeds. The effect of crocin in the semen extender before cryopreservation was evaluated on sperm parameters of 20 bucks of five different breeds: Garganica (GA), Jonica (JO), Maltese (MA), Mediterranean Red (MR) and Saanen (SA). Semen samples were centrifuged, to remove seminal plasma, divided in two aliquots and diluted with Tris-egg-yolk-based extender, containing 0 (control group) and 1 mM crocin. Crocin concentration was established after a preliminary dose trial. On fresh and frozen-thawed sperm, motility, viability, morphology, membrane integrity, DNA fragmentation and ROS levels were evaluated. The freezing process led to a decrease (p< 0.05) in all the sperm parameters recorded, confirming the deleterious effect of cryopreservation on goat semen. The most interesting result regarding the inclusion of crocin in the extender before cryopreservation was as follows: Crocin significantly improved (p< 0.05) sperm motility in all breeds, except for Mediterranean Red, compared to the control group. Furthermore, 1 mM crocin reduced percentage of spermatozoa with DNA fragmentation with a marked decrement (p< 0.05) in Garganica and Saanen, as compared to the control group. Finally, intracellular ROS decreased (p< 0.01) in the crocin-treated sperm of all breeds, as compared to the control. In conclusion, supplementation of 1 mM crocin in the extender decreased oxidative stress, improving sperm motility and the DNA integrity of frozen-thawed sperm in different breeds.