The aim of this investigation was to determine the optimal storage medium for testicular hypothermic transportation and identify the ideal concentration for the application of the protective agent 5-aminolevulinic acid (5-ALA). Furthermore, this study aimed to explore the underlying mechanism of the protective effects of 5-ALA. First, we collected and stored mouse testicular fragments in different media, including Hank’s balanced salt solution (HBSS; n = 5), Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; n = 5), and alpha-minimum essential medium (αMEM; n = 5). Storage of testicular tissue in HBSS preserved the integrity of testicular morphology better than that in the DMEM/F12 group (P < 0.05) and the αMEM group (P < 0.01). Testicular fragments were subsequently placed in HBSS with various concentrations of 5-ALA (0 [control], 1 mmol l−1, 2 mmol l−1, and 5 mmol l−1) to determine the most effective concentration of 5-ALA. The 2 mmol l−1 5-ALA group (n = 3) presented the highest positive rate of spermatogonial stem cells compared with those in the control, 1 mmol l−1, and 5 mmol l−1 5-ALA groups. Finally, the tissue fragments were preserved in HBSS with control (n = 3) and 2 mmol l−1 5-ALA (n = 3) under low-temperature conditions. A comparative analysis was performed against fresh testes (n = 3) to elucidate the underlying mechanism of 5-ALA. Gene set enrichment analysis (GSEA) for WikiPathways revealed that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was downregulated in the 2 mmol l−1 5-ALA group compared with that in the control group (normalized enrichment score [NES] = −1.57, false discovery rate [FDR] = 0.229, and P = 0.019). In conclusion, these data suggest that using 2 mmol l−1 5-ALA in HBSS effectively protected the viability of spermatogonial stem cells upon hypothermic transportation.

Keywords: 5-aminolevulinic acid, hypothermic transportation, male fertility preservation, oxidative stress, testis transplantation

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