Comparison of cryosurvival and spermatogenesis efficiency of cryopreserved neonatal mouse testicular tissue between three vitrification protocols and controlled-rate freezing |
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| Institution: | 1. Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, University of Mustafa Kemal, 31000, Hatay, Turkey;2. Pathology and Laboratory Medicine, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada;3. Division of Urology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada;4. The Hospital for Sick Children & Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada;1. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China;2. Laboratory of Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China;3. Department of Animal Sciences, Georg-August-Universität Göttingen, Göttingen, 37075, Germany;1. Laboratory on Animal Germplasm Conservation, Universidade Federal Rural do Semi-Árido – UFERSA, BR 110, Km 47, Costa e Silva, 59625-900, Mossoró, RN, Brazil;2. Laboratory of Manipulation of Oocytes and Preantral Follicles, Universidade Estadual do Ceará – UECE, Paranjana Ave, 1700, Itaperi, 60740-000, Fortaleza, CE, Brazil;3. Laboratory of Wild Animal Biology and Medicine, Faculty of Veterinary Medicine, Federal University of Pará, Belém, Pará, Brazil;4. Schothorst Feed Research, the Netherlands;1. Animal Development and Differentiation Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan;2. The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan;3. Transgenic Pig Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan;4. Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan;1. Laboratory of Reproduction, Center for Technology Transference of Zebu Dairy Cows-CTZL, Embrapa Cerrados, DF 180, Km 18, Brasília, Federal District, Brazil;2. Embrapa Genetic Resources and Biotechnology, Brasília, Federal District, Brazil;3. University of Brasília, Brasília, Federal District, Brazil;1. Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China;2. Department of Andrology, Center for Men''s Health, Department of ART, Institute of Urology, Urologic Medical Center, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China;3. Shanghai Key Laboratory of Reproductive Medicine, Shanghai, 200025, China;1. Department of Theriogenology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran;2. Theriogenology Association, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran;3. Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran |
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| Abstract: | Grafting of cryopreserved testicular tissue is a promising tool for fertility and testicular function preservation in endangered species, mutant animals, or cancer patients for future use. In this study, we aimed to improve the whole neonatal mouse testicular tissue cryopreservation protocols by comparing cryosurvival, spermatogenesis, and androgen production of grafted testicular tissue after cryopreservation with three different vitrification protocols and an automated computed controlled-rate freezing. Whole neonatal mouse testes were vitrified with various vitrification solutions (V1) 40% EG + 18% Ficoll + 0.35 M Sucrose, (V2) DAP 213 (2 M DMSO + 1 M Acetamid + 3 M PG), or (V3) 15% EG + 15% PG + 0.5 M Sucrose (total solute concentration V1:74.34%, V2:44.0%, and V3:49.22% wt/vol). Alternatively, neonatal testicular tissue was also frozen in 0.7 M DMSO +5% fetal bovine serum using controlled-rate freezing and compared to fresh grafted testicular tissue, sham grafted controls, and the vitrification protocol groups. Fresh (n = 4) and frozen-thawed (n = 4) testes tissues were grafted onto the flank of castrated male NCr Nude recipient mouse. The grafts were harvested after three months. Fresh or frozen-thawed grafts with controlled-rate freezing had the highest rate of tissue survival compared to other vitrified protocols after harvesting (p < 0.05). Both controlled-rate freezing and V1 protocol groups displayed the most advanced stages of spermatogenesis with elongated spermatids and spermatozoa in 17.6 ± 1.3% and 16.3 ± 1.9% of seminiferous tubules based on histopathological evaluation, respectively. Hosts of the testicular graft from controlled-rate freezing had higher levels of serum testosterone compared to all other vitrified-thawed graft groups (p < 0.05). This study shows that completed spermatogenesis from whole neonatal mouse testes were obtained when frozen with controlled-rate freezing and V1 vitrification solution and that testicular cryopreservation efficacy vary with the protocol and vitrification technique. |
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| Keywords: | Mouse testes Spermatogenesis Spermatozoa Grafting Cryopreservation Vitrification Testosterone |
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