The effects of tissue sample size and media on short-term hypothermic preservation of porcine testis tissue

The objective of this study was to develop effective strategies for hypothermic preservation of immature porcine testis tissue to maintain structural integrity and cell viability. In Experiment 1, testes from 1-week-old piglets were used to study the effects of tissue sample size (as intact testes or fragments of 100-or 30 mg) and the use of one of 9 different media on hypothermic preservation of the testis tissue for 6 days. The examined media included: Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM), Leibovitz L15 (L15), L15 with fetal bovine serum (FBS, at 10%, 20% or 50%), HypoThermosol solution-FRS (HTS), Ham’s F12, and Media 199. On days 0, 3, and 6, testis tissues were digested to compare the cell survival rates. Tissue sections were also semi-quantitatively assessed to determine the efficiency of different preservation strategies. There was no effect of testis sample size (P > 0.05), but cell survival rates of testis cells isolated from preserved testis tissues changed depending on the media and day (P < 0.05). Testis tissue within HTS did not show morphological changes after 6 days. In Experiment 2, two of the top performing media (20% FBS-L15 and HTS) were selected for immunocytochemical detection of gonocytes. Proportions of gonocytes (%) in isolated testis cells, however, did not differ between the two media on days 0, 3, or 6. These results show that testis tissue can be maintained for 3 days at 4°C with high cell survival rate, and tissue morphology can be preserved for at least 6 days in HTS.