We verified the possibility of cooling peccary semen for 4, 24, and 48 h before cryopreservation, using different dilution media (TRIS + egg yolk (20%) and PRIMXcell Ultra). Ten ejaculates were divided equally into six aliquots and then diluted. Two aliquots were stored in a biological incubator (4 h), and the remaining aliquots were stored in a commercial container, the Botutainer^® (24 and 48 h), both at 5 °C. The samples were cryopreserved and then evaluated for kinetic parameters, functionality, integrity, mitochondrial activity, morphology, and sperm binding capacity. After thawing, samples diluted in TRIS showed total motility of 43.4 ± 6.8%, 48.4 ± 6.2%, and 38.6 ± 5.0% after cooling for 4, 24, and 48 h before cryopreservation, respectively. Such results are significantly greater than those achieved with the use of PRIMXcell diluent for 4 (8.3 ± 2.8%), 24 (4.7 ± 1.4%), and 48 h (4.8 ± 2.9%) storage (p < 0.05). Furthermore, TRIS provided better preservation of sperm membrane integrity when samples were cooled for 24 h (44.5 ± 4.7%) before cryopreservation compared to those samples diluted in PRIMXcell Ultra stored for 24 (25.7 ± 4.0%) and 48 h (25.2 ± 4.0%) before freezing (p < 0.05). In summary, we suggest TRIS diluent + egg yolk (20%) as an effective option to allow semen to cool for 24 or 48 h in a transport container before cryopreservation.

The cryopreservation of male gonadal tissue is critical to conserve genetic material and use it later via assisted reproduction. This study aimed to evaluate cryopreservation methods (slow freezing, SF; solid surface vitrification, SSV) as well as the optimal concentrations of intracellular cryoprotectants during the SSV of testicular tissue from prepubertal collared peccaries. Five pairs of testes were dissected on different days into small fragments (3 mm³) and allocated to a non-cryopreserved, a control group or one of three treatment groups: SF; SSV 3 M (1.5 M dimethyl sulfoxide [DMSO] plus 1.5 M ethylene glycol [EG]); or SSV 6 M (3 M DMSO plus 3 M EG). After one week of storage in liquid nitrogen, tissue samples were warmed and evaluated in terms of histology, viability, proliferative capacity potential, and DNA integrity. The scores for histological integrity and cellular damage for SF (2.08 ± 0.05 and 2.33 ± 0.07, respectively) were similar to the results found in SSV 6 M (1.93 ± 0.04 and 2.30 ± 0.07; p > 0.05). However, these scores were better when compared to SSV 3 M (1.87 ± 0.05 and 2.08 ± 0.06; p < 0.05). The percentage of cellular viability was around 57% after all preservation treatments (p > 0.05), which was lower than in the control group (88.8 ± 1.9%; p < 0.05). The SSV 6 M treatment was better than the other treatments regarding the proliferative capacity potential of spermatogonia cells (3.52 ± 0.03) (p < 0.05), although it was lower than in the control group (4.00 ± 0.12) (p < 0.05). Additionally, SSV 6 M led to the same DNA integrity (97.0 ± 0.7%) as in the control group (99.4 ± 0.3%). These collective findings suggest that the combination of SSV with 6 M cryoprotectants is the most efficient for the cryopreservation of testes from prepubertal collared peccaries.