Effect of heterologous blood plasma (HBP) on the freezing extender of equine sperm

Abstract

Sperm cryopreservation is essential for the conservation of genetic resources, but freezing can compromise its fertilizing capacity. Optimizing cryopreservation techniques is crucial for developing effective extenders. Platelet-rich plasma (PRP) is an innovative therapeutic approach, showing promising results. However, considering the difficulty of performing absolute platelet counts in the field, this study integrated heterologous blood plasma (HBP) into the cryopreservation extender for equine semen. The objective was to evaluate the efficacy of HBP, added to freezing extender, through analysis of sperm kinetic parameters, plasma membrane integrity (PMI), acrosomal membrane integrity (AMI), and mitochondrial membrane potential (MMP). The experiment was conducted at the Animal Andrology Laboratory of the Federal Rural University of Pernambuco, using five stallions (5 to 9 years old). Blood samples were collected in tubes containing 3.6% sodium citrate and subjected to centrifugation (100G/10 minutes). Supernatant was collected and centrifuged (1600G/20 minutes) and combined into a single tube and homogenized. Ejaculates were collected using an artificial vagina. For freezing, samples were diluted in Botucrio^Ⓡ, supplemented with HBP (0%, 1%, 2%, and 5%), and frozen in an automated system (TK 3000^Ⓡ). Statistical analyses were performed using GraphPad InStat software. No significant differences were observed in total motility parameter among the treated groups. However, the study revealed significant differences in lateral head amplitude (ALH) parameters, with the group treated with 5% HBP showing higher values than control group. Additionally, linearity (LIN) values were greater in the 1.25% and 5.0% groups compared to control group (P ≤ 0.05). Kinetic parameters such as LIN and WOB have been shown to have a high correlation with the progressivity of frozen-thawed sperm (Harris. et al. Reproduction. 2023). Conversely, the wobble index (WOB) demonstrated a significant increase compared to control group. Regarding acrosome membrane integrity (%), it was seen a positive effect of HBP in the groups with concentrations of 2.5% (65,12± 6.01) and 5% (65,64± 3.80) when compared to control group (61,44±7.17). These results can be attributed to the buffering effect of HBP, that prevents osmotic shock, decreasing the risk of crystallization during the cryopreservation process. It can be concluded that adding heterologous blood plasma to the freezing medium demonstrated satisfactory results in sperm kinetics, presenting linear sperm with an intact acrosome.