Different Methods for Seminal Plasma Removal and Sperm Selection on the Quality and Fertility of Collared Peccary Sperm

Abstract

Methods for seminal plasma (SP) removal and the selection of collared peccary sperm for fertilization were compared. The experiments evaluated the following: the (I) impact of centrifugation for SP removal before swim-up for sperm selection and (II) a comparison of different Percoll® gradient densities (PG 45–90% and PG 35–70%). Non-selected sperm served as the control. Sperm quality was assessed based on motility patterns, morphology, membrane functional integrity, viability, reactive oxygen species (ROS), glutathione (GSH), and DNA integrity. Subsequently, the most successful group in the previous experiment and washing by centrifugation (WC) were compared for motility patterns and fertilization using pig oocytes. Swim-up decreased motility and enhanced ROS compared to the control. Centrifugation before swim-up harmed integrity and viability compared to the control. PG 45–90% (96.8 vs. 69.7 vs. 40.7 µm/s) allowed for a better velocity average pathway (VAP), a better velocity straight line, and better linearity (LIN) than those of the control and PG 35–70% (88.4 vs. 56.0 vs. 27.3 µm/s). Thus, PG 45–90% was used for fertilization. PG 45–90% obtained a higher VAP, a higher amplitude of the lateral head, straightness, and higher LIN than those of the control and WC. Cleavage (25.2–26.3%) and morula (8.1–10.5%) rates did not differ between the groups. Therefore, PG 45–90% and WC were efficient in isolating collared peccary sperm capable of fertilizing pig oocytes.

Effect of Diluents and Storage Time on the Cryopreservation of Collared Peccary (Pecari tajacu) Semen after Cooling Storage in a Transport Container at 5 °C

“…Based on the results obtained in the sperm binding assay, it is possible to infer that peccary samples diluted, chilled, and cryopreserved in PRIMXCell ® Ultra could be used for in vitro fertilization, but this remains to be confirmed. This statement is supported by the fact that for peccaries, efficient methods of sperm selection using Percoll gradients for in vitro fertilization are already being developed and have been capable of generating heterologous embryos using porcine oocytes [ 48 ]. Moreover, this commercial extender contains some non-informed “bioactive” molecules whose main function is to potentiate the biosynthesis of platelet-activating factors (PAFs) [ 19 ], which stimulate motility, fertilizing potential, and fertility rates [ 49 ].…”

Catalase and superoxide dismutase supplementation to the diluent for cryopreservation of collared peccary semen

“…Initially, an aliquot of 50 μL of a solution of PBS and H 2 DCFDA [2.5 mL PBS and 0.5 μL (0.1 M) H 2 DCFDA] was homogenized in an aliquot of 50 μL of sperm suspension (1:1) and incubated at 37°C for 30 min in the dark. Then, the sperm suspension was subjected to two washes in PBS (50 and 20 μL) after centrifugation at 500 g for 5 min (Santos et al, 2023).…”

“…Leica. Kista, Sweden) in order to determine the percentage of viable spermatozoa with intracellular ROS, where the intensity of the fluorescence of the images was quantified using the ImageJ 1.45 s software (National Institutes of Health, Bethesda, Maryland, USA)(Santos et al, 2023). The background signal intensity was subtracted from the values obtained for the treatment images.…”

“…The background signal intensity was subtracted from the values obtained for the treatment images. From each sample, up to 100 sperm were selected for the quantification of fluorescence intensity.The group without antioxidants (control) were used to calibrate the measurements, and the measured value of each sperm was divided by the mean of the calibrator to generate relative expression levels [arbitrary fluorescence units (AFU)](Santos et al, 2023).…”