Studies on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling and vitrification

2.50
Hdl Handle:
http://hdl.handle.net/10547/134933
Title:
Studies on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling and vitrification
Authors:
Guan, Mo
Abstract:
Cryopreservation of gametes provides a promising method to preserve fish genetic materials, which offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful cryopreservation of spermatozoa of about 200 fish species has been achieved, systematic studies on cryopreservation of fish oocytes have only recently been undertaken. The objective of the present studies was to use zebrafish as a model system to develop a cryopreservation protocol for fish oocytes and to develop reliable viability assessment methods for monitoring zebrafish oocyte viability both before and after cryopreservation. A simple and rapid enzymatic method for zebrafish oocytes isolation was developed and the investigations on cryopreservation of zebrafish oocytes using improved controlled slow cooling and vitrification were carried out. Oocyte viability following cryopreservation was investigated by ATP assay, oocyte viability molecular signature (OVMS) and cryomicroscopic observation in addition to staining methods. The optimum conditions for oocyte enzymatic separation were identified as 0.4mg/ml collagenase or 1.6mg/ml hyaluronidase treatment for 10min at 22ºC and this method can be used for oocytes at all stages. The use of sodium free medium (KCl buffer), fast warming and 4-step removal of cryoprotectants in an improved controlled slow cooling protocol significantly enhanced oocyte viability (67.5 ± 1.7%) when compared with a previous study (16.3 ± 2.3%) in this laboratory. Mixtures of cryoprotectants (methanol, Me2SO and propylene glycol), stepwise addition and removal of cryoprotectants in combination of a new vitrification system (CVA65 vitrification system) were used in vitrification studies. Oocyte survivals after vitrification assessed by trypan blue staining were relatively high (76.5 ± 6.3%) shortly after warming in KCl buffer. Furthermore, the result of ATP assay showed that ATP levels in oocytes decreased significantly after cryopreservation indicating the bioenergetic systems of oocytes were damaged. Cryomicroscopic observations demonstrated that Intracellular ice formation (IIF) is the main factor causing injuries during cryopreservation of zebrafish oocytes. The results provided by the present study will assist successful protocol design for cryopreservation of fish oocytes in the future.
Affiliation:
LIRANS Institute of Research in Applied Natural Sciences
Publisher:
University of Bedfordshire
Issue Date:
Mar-2009
URI:
http://hdl.handle.net/10547/134933
Type:
Thesis or dissertation
Language:
en
Description:
A thesis submitted to the University of Bedfordshire in partial fulfillment of the requirements for the degree of Doctor of Philosophy
Appears in Collections:
PhD e-theses

Full metadata record

DC FieldValue Language
dc.contributor.authorGuan, Moen
dc.date.accessioned2011-06-30T09:20:17Z-
dc.date.available2011-06-30T09:20:17Z-
dc.date.issued2009-03-
dc.identifier.urihttp://hdl.handle.net/10547/134933-
dc.descriptionA thesis submitted to the University of Bedfordshire in partial fulfillment of the requirements for the degree of Doctor of Philosophyen
dc.description.abstractCryopreservation of gametes provides a promising method to preserve fish genetic materials, which offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful cryopreservation of spermatozoa of about 200 fish species has been achieved, systematic studies on cryopreservation of fish oocytes have only recently been undertaken. The objective of the present studies was to use zebrafish as a model system to develop a cryopreservation protocol for fish oocytes and to develop reliable viability assessment methods for monitoring zebrafish oocyte viability both before and after cryopreservation. A simple and rapid enzymatic method for zebrafish oocytes isolation was developed and the investigations on cryopreservation of zebrafish oocytes using improved controlled slow cooling and vitrification were carried out. Oocyte viability following cryopreservation was investigated by ATP assay, oocyte viability molecular signature (OVMS) and cryomicroscopic observation in addition to staining methods. The optimum conditions for oocyte enzymatic separation were identified as 0.4mg/ml collagenase or 1.6mg/ml hyaluronidase treatment for 10min at 22ºC and this method can be used for oocytes at all stages. The use of sodium free medium (KCl buffer), fast warming and 4-step removal of cryoprotectants in an improved controlled slow cooling protocol significantly enhanced oocyte viability (67.5 ± 1.7%) when compared with a previous study (16.3 ± 2.3%) in this laboratory. Mixtures of cryoprotectants (methanol, Me2SO and propylene glycol), stepwise addition and removal of cryoprotectants in combination of a new vitrification system (CVA65 vitrification system) were used in vitrification studies. Oocyte survivals after vitrification assessed by trypan blue staining were relatively high (76.5 ± 6.3%) shortly after warming in KCl buffer. Furthermore, the result of ATP assay showed that ATP levels in oocytes decreased significantly after cryopreservation indicating the bioenergetic systems of oocytes were damaged. Cryomicroscopic observations demonstrated that Intracellular ice formation (IIF) is the main factor causing injuries during cryopreservation of zebrafish oocytes. The results provided by the present study will assist successful protocol design for cryopreservation of fish oocytes in the future.en
dc.language.isoenen
dc.publisherUniversity of Bedfordshireen
dc.subjectcryopreservationen
dc.subjectzebrafishen
dc.subjectoocyte viabilityen
dc.subjectC410 Applied Geneticsen
dc.subjectDanio rerioen
dc.titleStudies on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling and vitrificationen
dc.typeThesis or dissertationen
dc.contributor.departmentLIRANS Institute of Research in Applied Natural Sciencesen
dc.type.qualificationnamePhDen
dc.type.qualificationlevelDoctoralen
dc.publisher.institutionUniversity of Bedfordshireen
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