title: Reproductive stage-dependent effects of additional cryoprotectant agents for the cryopreservation of stallion germ cells.
abstract: The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1-1.5 yr, n=6) and post-pubertal (3-7 yr, n=5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell population, and viable population were assessed after 1 and 3 months of cryopreservation. The viable UTF1-positive population of pre-pubertal stallion germ cells was also measured using immunocytochemistry after 1 and 3 months of cryopreservation. As expected, the viability, cell population, and viable cell population were significantly reduced after 1 and 3 months of cryopreservation. At the pre-pubertal stage, the addition of trehalose or PEG to 10% DMSO did not show any effect on the viability, cell population, viable cell population, or viable UTF1-positive germ cells at either 1 or 3 months after cryopreservation. However, at the post-pubertal stage, the viable population was significantly higher in germ cells that were cryopreserved with 5% or 10% PEG, than in the cells cryopreserved with 10% DMSO only. In conclusion, PEG at 5% or 10% added to 10% DMSO serves as an optimal cryoprotectant agent for the cryopreservation of germ cells from post-pubertal stallions.
authors.affiliation: Department of Animal Science, Kyungpook National University, Sangju, 37224, Republic of Korea.
authors.forename: Heejun
authors.initials: H
authors.lastname: Jung
authors.affiliation: National Institute of Animal Science, RDA, 593-50 Sanrokbukro, Jeju, 63242, Republic of Korea.
authors.forename: Namyoung
authors.initials: N
authors.lastname: Kim
authors.affiliation: Department of Animal Science, Kyungpook National University, Sangju, 37224, Republic of Korea; Department of Animal Science and Biotechnology, Kyungpook National University, Sangju, 37224, Republic of Korea. Electronic address: mjyoonemail@gmail.com.
authors.forename: Minjung
authors.initials: M
authors.lastname: Yoon
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D006736:Horses / Q000502:physiology*; D008297:Male; D012662:Semen Preservation / Q000662:veterinary*; D012741:Sexual Maturation; D013094:Spermatozoa / Q000502:physiology*; D013234:Stem Cells / Q000187:drug effects* / Q000502:physiology
chemical_list: D003451:Cryoprotective Agents
keywords: Cryopreservation; Cryoprotectant; Germ cells; Stallion; Testes