title: A study on the vitrification of stage III zebrafish (Danio rerio) ovarian follicles.
abstract: Attempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation protocol for long-term storage still remains elusive. Fish oocytes offer some advantages when compared to embryos, which may help in improving the chances of cryopreservation. In the present study, a series of cryo-solutions were designed and tested for their vitrifying ability using different devices (0.25ml plastic straw, vitrification block and fibreplug™). Toxicity of vitrification solutions was evaluated by assessing follicle membrane integrity with trypan blue staining. In addition, the effect of vitrification protocol on stage III zebrafish ovarian follicles was investigated by measuring the cytoplasmic ATP content and the mitochondrial distribution and activity using JC-1 probe and confocal microscopy. After vitrification, follicles showed membrane integrity of 59.9±18.4% when fibreplug and V16(1.5M methanol+4.5M propylene glycol) solution were employed. When vitrified in V2(1.5M methanol+5.5M Me2SO) the membrane integrity decreased to 42.0±21.0%. It was observed that follicles located in the middle of the fragments were more protected from injuries and some of them showed good morphological appearance 2h post-warming. Mitochondria integrity of granulosa cells layer was clearly damaged by the vitrification protocol and ATP level in the follicles declined significantly after warming. Vitrification of zebrafish follicles in ovarian tissue fragments and its effect at sub-cellular level is reported here for the first time. Information gained from this study will help in guiding development of optimal protocol for cryopreservation of fish oocytes.
authors: Aquaculture Graduate Program, Nilton Lins University, Manaus 69058-040, Brazil; Institute of Biomedical and Environmental Science and Technology, University of Bedfordshire, Luton, Bedfordshire LU2 8DL, UK. Electronic address: godoyaqua@yahoo.com.br.
authors: Leandro Cesar
authors: LC
authors: Godoy
authors: Aquam Research Group, Department of Animal Science, Federal University of Rio Grande do Sul, Porto Alegre 91540-000, Brazil.
authors: Danilo P
authors: DP
authors: Streit
authors: Institute of Biomedical and Environmental Science and Technology, University of Bedfordshire, Luton, Bedfordshire LU2 8DL, UK.
authors: Tiziana
authors: T
authors: Zampolla
authors: Aquam Research Group, Department of Animal Science, Federal University of Rio Grande do Sul, Porto Alegre 91540-000, Brazil.
authors: Adriana
authors: A
authors: Bos-Mikich
authors: School of Applied Sciences, Bournemouth University, Dorset BH12 5BB, UK.
authors: Tiantian
authors: T
authors: Zhang
keywords: Aquaculture; Breeding program; Cryobanking; Fish gamete; Mitochondria