Development of in-vitro culture and cryopreservation protocol for zebrafish (Danio rerio) ovarian tissue fragments

5.00
Hdl Handle:
http://hdl.handle.net/10547/308923
Title:
Development of in-vitro culture and cryopreservation protocol for zebrafish (Danio rerio) ovarian tissue fragments
Authors:
Anil, Siji
Abstract:
Cryopreservation of fish ovarian tissue fragments can be a viable alternative to cryopreservation of oocytes and embryos. The ability to cryopreserve both maternal and paternal gametes would provide a reliable source of fish genetic material for scientific and aquaculture purposes. The main aim of the present study was to develop an in-vitro culture protocol and cryopreservation protocol for zebrafish ovarian tissue fragments. In-vitro culture protocol for the tissue fragments containing stage I and stage II follicles were developed and the growth assessment of follicles were evaluated using biomarkers. To develop the cryopreservation protocol using control slow cooling method, the effect on freezing medium, cryoprotectants and cooling rate on the tissue fragments were investigated. The in-vitro culture experiments showed that L-15 medium (pH 9) containing 100mIU/ml FSH along with 20% FBS was effective for tissue fragments containing stage I and II follicles to grow in-vitro. The growth of the ovarian follicle stages was confirmed by the level of expression of p450aromA and vtg1 gene. The optimal cryopreservation protocol for the ovarian tissue fragments was found as 2M methanol+ 20%FBS in 90% L-15 medium with the cooling rate of 4°C/min. Although the highest survival rate obtained for stage II follicles within the fragments was 68.2±1.9% and stage I follicles within the fragments was 55.4±2.3% using TB staining, it showed a significant decrease in their ATP levels. This is the first study carried out on the zebrafish ovarian tissue fragments. Study on cryopreservation of the ovarian tissue fragments and development of the in-vitro culture protocol and use of biomarkers for the ovarian tissue fragments were reported here for the first time. The outcomes of this study have provided useful information for future cryopreservation protocol development.
Citation:
Anil, S. (2013) 'Development of in-vitro culture and cryopreservation protocol for zebrafish (danio rerio) ovarian tissue fragments'. PhD thesis. University of Bedfordshire.
Publisher:
University of Bedfordshire
Issue Date:
2013
URI:
http://hdl.handle.net/10547/308923
Type:
Thesis or dissertation
Language:
en
Description:
A thesis submitted to the University of Bedfordshire in accordance with the requirements for the degree of Doctor of Philosophy
Appears in Collections:
PhD e-theses

Full metadata record

DC FieldValue Language
dc.contributor.authorAnil, Sijien
dc.date.accessioned2013-12-23T12:18:54Z-
dc.date.available2013-12-23T12:18:54Z-
dc.date.issued2013-
dc.identifier.citationAnil, S. (2013) 'Development of in-vitro culture and cryopreservation protocol for zebrafish (danio rerio) ovarian tissue fragments'. PhD thesis. University of Bedfordshire.en
dc.identifier.urihttp://hdl.handle.net/10547/308923-
dc.descriptionA thesis submitted to the University of Bedfordshire in accordance with the requirements for the degree of Doctor of Philosophyen
dc.description.abstractCryopreservation of fish ovarian tissue fragments can be a viable alternative to cryopreservation of oocytes and embryos. The ability to cryopreserve both maternal and paternal gametes would provide a reliable source of fish genetic material for scientific and aquaculture purposes. The main aim of the present study was to develop an in-vitro culture protocol and cryopreservation protocol for zebrafish ovarian tissue fragments. In-vitro culture protocol for the tissue fragments containing stage I and stage II follicles were developed and the growth assessment of follicles were evaluated using biomarkers. To develop the cryopreservation protocol using control slow cooling method, the effect on freezing medium, cryoprotectants and cooling rate on the tissue fragments were investigated. The in-vitro culture experiments showed that L-15 medium (pH 9) containing 100mIU/ml FSH along with 20% FBS was effective for tissue fragments containing stage I and II follicles to grow in-vitro. The growth of the ovarian follicle stages was confirmed by the level of expression of p450aromA and vtg1 gene. The optimal cryopreservation protocol for the ovarian tissue fragments was found as 2M methanol+ 20%FBS in 90% L-15 medium with the cooling rate of 4°C/min. Although the highest survival rate obtained for stage II follicles within the fragments was 68.2±1.9% and stage I follicles within the fragments was 55.4±2.3% using TB staining, it showed a significant decrease in their ATP levels. This is the first study carried out on the zebrafish ovarian tissue fragments. Study on cryopreservation of the ovarian tissue fragments and development of the in-vitro culture protocol and use of biomarkers for the ovarian tissue fragments were reported here for the first time. The outcomes of this study have provided useful information for future cryopreservation protocol development.en
dc.language.isoenen
dc.publisherUniversity of Bedfordshireen
dc.subjectC142 Reproductive Biologyen
dc.subjectcryopreservationen
dc.subjectzebrafishen
dc.subjectDanio rerioen
dc.titleDevelopment of in-vitro culture and cryopreservation protocol for zebrafish (Danio rerio) ovarian tissue fragmentsen
dc.typeThesis or dissertationen
dc.type.qualificationnamePhDen_GB
dc.type.qualificationlevelPhDen
dc.publisher.institutionUniversity of Bedfordshireen
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