• Development and charaterisation of 3 dimensional culture models for zebrafish (Danio rerio) skeletal muscle cells

      Vishnolia, Krishan Kumar (University of BedfordshireUniversity of Bedfordshire, 2013-09)
      Zebrafish (Danio rerio) have been extensively used over the past two decades to study muscle development, human myopathies and dystrophies, due to its higher degree of homology with human disease causing genes and genome. Despite its unique qualities, zebrafish have only been used as an in-vivo model for muscle development research, due to the limitations surrounding lack of a consistent isolation and culture protocol for zebrafish muscle progenitor cells in-vitro. Using different mammalian myoblast isolation protocols, a novel and robust protocol has been developed to successfully isolate and culture zebrafish skeletal muscle cells repeatedly and obtain differentiated long multi nucleated zebrafish myotubes. Commitment to myogenic lineage was confirmed by immuno-staining against muscle specific protein desmin, and expression pattern of different genetic markers regulating myogenesis. In order to recapitulate the in-vivo bio-physiological environment for zebrafish skeletal muscle cells in-vitro, these cells were successfully cultured in tissue engineered three dimensional (3D) constructs based on fibrin and collagen models. Maturation of tissue engineered collagen and fibrin based constructs was confirmed using the basic parameters described in the literature i.e. collagen three times greater contraction from the original width (Mudera, Smith et al. 2010) and fibrin constructs tightly coiled up to 4mm of diameter (Khodabukus, Paxton et al. 2007). In-vitro characterisation of zebrafish skeletal muscle cells showed hypertrophic growth of muscle mass compared to hyperplasic growth in-vivo as suggested for fish species in literature (Johnston 2006), which is different from human and other mammals. Comparative analysis of zebrafish muscle cells cultured in monolayer against cultured in 3D tissue engineered constructs showed significant increase in fusion index, nuclei per myotube (two-fold) and myotubes per microscopic frame (two-fold). Cells cultured in tissue engineered construct closely resembled in-vivo muscle in terms of their unidirectional orientation of myotubes. These tissue engineered 3D zebrafish skeletal muscle models could be used for various purposes such as drug screening, effect of different temperature extremes, studying underlined pathways involved in human diseases; and with further refinements it would potentially replace the need for studies on live fish in these areas.
    • Development of cryopreservation techniques for early stages zebrafish (Danio rerio) oocytes

      Tsai, Sujune (University of BedfordshireUniversity of Bedfordshire, 2009-01)
      Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, the sensitivity to chilling and toxicity of cryoprotectants of early stage zebrafish ovarian follicles were studied before designing protocols for their cryopreservation using controlled slow cooling. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. In vitro culture method for early stage zebrafish ovarian follicles were also developed. The studies showed that stage I and II ovarian follicles are less sensitive to chilling than stage III follicles and methanol was the least toxic cryoprotectant. 4M methanol in potassium chloride (KCl) buffer was found to be the optimal cryoprotective solution and the optimum cooling rate was 4 °C/min for stage I and II follicles. Although the highest survivals after 2 h post-thawed incubation were 50.7 ± 4.0% for stage II ovarian follicles obtained with FDA+PI staining, ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell death. Furthermore, in vitro culture experiments showed that there was no growth for stage I and II ovarian follicles after cryopreservation, indicating that successful cryopreservation of early stage zebrafish ovarian follicles at liquid nitrogen still remains elusive. From in vitro culture study, 90% L-15 medium at pH 9.0 containing 10 IU/ml hCG was effective for in vitro culture of stage I and II ovarian follicles. Systematic study on cryopreservation of early stage fish ovarian follicles at liquid nitrogen temperature is reported ii here for the first time. The results will provide useful information on the future development of protocol design for successful cryopreservation of early stage fish ovarian follicles.
    • Development of in-vitro culture and cryopreservation protocol for zebrafish (Danio rerio) ovarian tissue fragments

      Anil, Siji (University of BedfordshireUniversity of Bedfordshire, 2013)
      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.
    • Development of new methods to assess the quality of zebrafish (Danio rerio) ovarian follicles

      Zampolla, Tiziana (University of BedfordshireUniversity of Bedfordshire, 2009-03)
      High quality fish oocytes are essential for in vitro maturation (IVM), in vitro fertilization (IVF) protocols, and for use in cryopreservation. It is important to develop methods for assessing oocyte quality for applications in aquaculture, the preservation of endangered species and managing fish models used in biomedical research. The lack of reliable methods of evaluating oocyte quality limits progress in these areas. The present study was undertaken to develop new methods to assess ovarian follicle viability and quality of stage III zebrafish (Danio rerio) ovarian follicles. The methods developed were then applied to study the impact of cryoprotectant and/or cryopreservation procedures. A vital staining procedure, not previously used with zebrafish oocytes, has been investigated. FDA-PI (Fluorescein diacetate-Propidium Iodide) staining was found to be a more sensitive then currently used viability tests and it could also be applied to all ovarian follicles developmental stages. Mitochondrial activity and distribution as biological markers was investigated with the mitochondrial membrane potentialsensitive dye JC-1- (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide). Confocal microscopy, Cryo-scanning and electron microscopy studies were undertaken to determine mitochondria distributional arrangement within the ovarian follicle. This provided new information on zebrafish ovarian follicle structure, and showed that mitochondria exhibited a contiguous distribution at the margin of the granulosa cell layer surrounding stage III zebrafish oocytes. Cryoscanning results showed a polygonal structure of the vitelline envelope, which is reported here for the first time with the mitochondrial distributional arrangement in the granulosa cell layer. Mitochondrial distribution and the evaluation of mitochondrial activity proved to be sensitive markers for ovarian follicle quality, providing more detailed information on cryoprotectant impact. The measurement of ATP levels, ADP/ATP ratio and mtDNA copy number were also undertaken following cryoprotectant exposure. These findings, together with the observation of mitochondrial distribution, suggested that even cryoprotectant treatments that are considered to have little or no toxicity can have a deleterious effect on mitochondrial activity, potentially compromising oocyte growth and embryo development. Therefore, a further optimization of the currently used protocol may need to be considered. The study of organelle distribution and organisation would support in vitro maturation and oocyte development fields, as well as their use as biological markers for quality determination. These findings will contribute to a better understanding of oogenesis/folliculogenesis processes in fish.
    • The effect of cryopreservation on the genome of fish reproductive cells

      Kopeika, Julia (University of BedfordshireUniversity of Bedfordshire, 2003-09)
      Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programs for endangered species. Many studies have been devoted to the mechanisms of cryodamage. However, in spite of growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells often remains poor. It is known that cryopreservation causes extensive damage to membrane, results in decreased metabolism of cells, and disturbs the bioenergetical processes of cells by damage to mitochondria. Nonetheless, it has not yet been identified clearly if cryopreservation causes some disruption in the genetic integrity of reproductive cells and the safety of this approach still needs to be confirmed. The present study was undertaken on the spermatozoa of weather loach (Misgurnus tassilis) and blastomeres cells of zebrafish (Danio rerio). It was shown that survival was decreased for embryos derived from sperm after cryoprotectant treatment or cryopreservation. Some evidence has emerged that this decrease is more likely to reflect some genetic instability caused by cryopreservation of sperm. The present study showed for the first time that the DNA repair system of oocytes was activated after fertilisation with cryopreserved sperm. The effect of DNA repair system was also studied. It was found that incubation of fertilised eggs in caffeine could reverse the detrimental effects of cryopreservation of loach sperm on subsequent embryo development. On the other hand incubation of fertilised eggs with 3-aminobenzamide - inhibitor of the poly (ADP-ribose) polymerase (PARP)- brought further decrease in the survival of embryos derived from cryopreserved sperm. The effect of individual donors of sperm and eggs on overall embryo survival was also studied and these investigations revealed significant differences between different donors. Effect of cryopreservation on zebrafish blastomeres was studied at the DNA molecular level. Mitochondrial DNA was sequenced after cryopreservation and increased level of frequency of the mutation was observed. This finding showed that cryopreservation might potentially increase the instability of mtDNA genome. The significance of these changes on the subsequent function of the cells is to be elucidated. Meanwhile this study suggests that it is important to be cautious in making judgements on the safety of cryopreservation techniques in reproduction.
    • Investigation of the effects of different cryopreservation parameters on the genome of 51/4 hpf zebrafish (Danio rerio) embryos

      Ahmed, Raju; University of Bedfordshire (University of BedfordshireUniversity of Bedfordshire, 2013-06)
      In recent years, numerous studies have linked cryopreservation with increased occurrence of mutations, DNA fragmentation and the event of apoptosis in biological objects. However, the evidence emerged from such studies is somewhat inconclusive. The current study, therefore, aimed to analyse the DNA damage response (DDR) from the cryopreserved cells in order to characterise the nature of the putative DNA damage. The study set out to investigate the effects of different cryopreservation parameters on the genome in terms of double strand breaks (DSBs), single strand breaks (SSBs), and various forms of sequence alteration using 5¼ hour post fertilisation (hpf) zebrafish (Danio rerio) embryos. The experimental conditions under which the investigation was carried out were short term chilling at 0˚C, treatment with two cryoprotective agents (CPA), namely, MeOH and Me2SO, and cooling to -35˚C. Assays for detecting DSB-activated DDR proteins and SSB-activated DDR proteins in 5¼ hpf zebrafish (Danio rerio) were developed and then utilised to investigate the occurrence of DSBs and SSBs in the genome of the embryos treated with the experimental conditions. The study then analysed the expression profiles of a set of genes unique to the base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) pathways as indicators of the occurrence of various forms of sequence alterations in the genome of the embryos treated with the experimental conditions. It was found that chilling and CPA treatment did not induce DSBs or SSBs but up-regulated the MMR and BER, respectively. CPA treatment also down-regulated the NER and the MMR mechanisms. Cooling, on the contrary, did not induce DSBs but induced SSBs in the genome, which were repaired when the embryos were provided with a recovery time. Cooling also up-regulated the NER and the BER mechanisms in the embryos. The overall finding of the study indicated that the experimental conditions increased the occurrence of various single stranded DNA lesions in the genome of the embryos. The present study provided important insights into how eukaryotic cells respond to different cryopreservation parameters, which will significantly enhance the current knowledge of the effects of cryopreservation on the genome of biological objects.
    • Investigations into the cryopreservation of zebrafish (Brachydanio Rerio) embryos

      Zhang, Tiantian (University of BedfordshireUniversity of Bedfordshire, 1994-06)
      Cryopreservation of fish embryos was studied using zebrafish embryo as a model system. Cryoprotectant toxicity, chilling sensitivity and embryo membrane permeability were investigated with a view to developing optimum protocols for cryopreservation of the embryos using controlled slow cooling, non-freezing storage and vitrification. Methanol was found to be the most effective cryoprotectant for controlled slow cooling and non-freezing storage of zebrafish embryos with 11 %heart beat stage embryos surviving after controlled slow cooling to -25°C. Zebrafish embryos were found to be very chilling sensitive with early developmental stages being the most sensitive to chilling injury. Embryo developmental stages after closure of the blastopore (>12-h), especially post heart beat stages were much more resistant to cryoprotectant toxicity and chilling injury. Heart beat stage (27-h) embryos proved to be the best embryo developmental stage for controlled slow cooling and non-freezing storage. Dechorionated embryos are more sensitive to cryoprotectant toxicity and chilling injury. The sensitivity to chilling injury of zebrafish embryos limited the use of controlled slow cooling and non-freezing storage for long term cryopreservation. The attempts at cryopreservation of zebrafish embryos using vitrification produced no embryo survival, although up to 32 % embryos remained morphologically intact immediately after vitrification. Poor cryoprotectant permeation, dehydration and consequently ice formation within the egg are probably the main factors on effecting embryo survival. The results of zebrafish embryo permeability studies demonstrated that the chorion of the embryos was permeable to water and cryoprotectants, whilst the vitelline (plasma) membrane was an effective permeability barrier. The inability to achieve sufficient penetration of the vitelline membrane by cryoprotectants poses severe problems for long term cryopreservation, which need to be overcome, possibly by permeabilisation of the vitelline membrane, before successful cryopreservation can be achieve.
    • Studies on cryopreservation of zebrafish (Danio Rerio) oocytes using controlled slow cooling

      Plachynta, Maksym (University of BedfordshireUniversity of Bedfordshire, 2007-02)
      Cryopreservation of fish germ cells has important applications in aquaculture, conservation of endangered species and human genomic studies. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. The objective of the present study was to develop successful cryopreservation protocol for zebrafish oocytes at temperature of liquid nitrogen (-196°C), or if unachieved, to investigate the limiting factors associated with fish oocytes cryopreservation. In this study, the effects of cryoprotectants exposure and enzymatic treatments on oocytes survival were studied, and new viability tests for zebrafish oocytes were developed. The effects of controlled slow cooling with different cryoprotective agents, in different freezing media and at different cooling rates on cryosurvival of zebrafish (D. rerio) oocytes were investigated. Cryomicroscopic observations on zebrafish oocytes were also carried out. Three reliable vital tests -trypan blue (TB) staining, ATP assay, and in vitro maturation followed by germinal vesicle breakdown observation (GVBD) were found suitable for assessment of oocytes viability. Vitellogenesis (stage III) was found to be the optimal developmental stage for cryopreservation. Methanol was found to be the best CPA for zebrafish oocytes. Combination of 4M methanol and 0.2M glucose in potassium chloride (KCI) buffer was found to be the optimal cryoprotective solution. Controlled slow cooling at 0.3°C/min rate, combined with seeding at -12.5°C and plunge to liquid nitrogen (LN) at-40°C were found to be the optimal conditions for cryopreservation of stage III oocytes. However, even with the optimal protocol, TB-assessed viability, Le. the ratio of oocytes with intact plasma membrane after cooling to -196°C was 19.6±8%. Furthermore, GVBD experiments showed that none of the cryopreserved oocytes can be matured in vitro, and their ATP levels were decreased dramatically, indicating that successful cryopreservation of fish oocytes at liquid nitrogen temperature still remains elusive. Cryomicroscopic observations demonstrated, that the damages of oocytes are associated with intracellular ice formation (lIF). IIF occurred simultaneously with extracellular ice formation (ElF) in nearly 100% of the cases, and formation of lethal hexagonal type of ice was observed. This study was the first systematic attempt to cryopreserve fish oocytes at liquid nitrogen temperature. The results provided will undoubtedly assist successful protocol design for cryopreservation of fish oocytes in the future.
    • Studies on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling and vitrification

      Guan, Mo (University of BedfordshireUniversity of Bedfordshire, 2009-03)
      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.
    • Studies on the effect of chilling on sox genes and protein expression in zebrafish (Danio rerio) embryos

      Desai, Kunjan; University of Bedfordshire (University of BedfordshireUniversity of Bedfordshire, 2012)
      In aquaculture, short term chilled storage has been used to transport brood stock fish embryos for genetic improvement programmes. It is therefore important to understand the effect of chilling on embryos at both developmental and molecular levels. In the present study, gene expression patterns in zebrafish embryos were studied before investigations were carried out on the effect of chilling on gene and protein expression in these embryos. The gene expression results obtained in different developmental stages using conventional PCR showed that, only sox genes were expressed throughout the tested developmental stages from 30% epiboly to 6 somites. Quantitative RT-PCR was then used to investigate sox gene expression patterns during chilling of 50% epiboly stage embryos at 0°C for up to 180 min and also after warming. Significant decreases in sox2 and sox3 expressions were observed when compared to those of controls following chilling whilst significant increases of expressions of the two genes were observed after warming in the embryos chilled for 30 and 60 min. Studies on the impact of cryoprotectant MeOH on sox genes and protein expression showed that 50% epiboly stage zebrafish embryos could tolerate chilling for up to 6 h with or without MeOH. It was observed that expression of all three sox genes were significantly decreased following chilling for 3 h at 0°C. However the degree of decrease was less pronounced in embryos chilled with different concentrations of MeOH. Significant increases in sox genes were observed in hatching stage embryos chilled with 1 M MeOH for 3h but subsequent sox2 and sox19a protein expression was not affected. The effect of long term chilling (18h) on sox gene and protein expression in 50% epiboly stage embryos was also investigated. Improved hatching rates (56% ± 5) were achieved when embryos were chilled with 1 M MeOH + 0.1 M sucrose. Results from gene expression studies showed a stable sox2 gene expression in 18 h chilled embryos in cryoprotectant mixture when compared to that of embryos chilled without cryoprotectant mixture. Similar patterns were observed when the expression of sox2 and sox3 protein was investigated. This is the first study carried out on the effect of chilling in early stage zebrafish embryos at the molecular level. The results obtained from the present study provided useful information on the molecular mechanisms of the effect of chilling on zebrafish embryos and will have important implications in designing chilled storage protocols for fish embryos.
    • Studies on the effect of cryopreservation on gene expression in zebrafish blastomeres

      Lin, Chia-Hsin; Institute of Research in the Applied Natural Sciences (University of BedfordshireUniversity of Bedfordshire, 2009-01)
      Cryopreservation is now a common practice in the fields of aquaculture, conservation and biomedicine. The success of cryopreservation is usually analysed in terms of cell survival, although there are other potential adverse effects that don’t necessarily result in cell death. These include DNA damage, which could result in altered gene expression. The aim of this study is to discover if cryopreservation has an impact on gene expression using zebrafish (Danio rerio) as the model organism. As the whole embryo cannot yet be successfully cryopreserved, isolated blastomeres from the embryos were used to investigate the impact of cryo-treatment on gene expression. This study sets out firstly to determine an optimum cryopreservation protocol for 50% epiboly blastomeres (Epiboly displaces the blastoderm margin to 50% of the distance between the animal and vegetal pole). Blastomeres had the highest survival level (70.2 ± 3.2%) when a mixture of 1.5 M dimethyl sulfoxide (DMSO) and 0.1 M sucrose were used as cryoprotectants. As quantitative analysis of gene expression using real-time PCR requires the use of a housekeeping gene as an internal control to normalize date, the second study aimed to identify and validate housekeeping genes for use in cryopreservation studies of zebrafish blastomeres. Seven potential housekeeping genes were analysed across a range of embryo stages and isolated blastomeres using the GeNorm and NormFinder software packages. Results indicated β-actin and EF1α housekeeping genes to be the most appropriate for cryopreservation studies on zebrafish embryos and blastomeres, and these housekeeping genes were used in the third study, the effect of cryopreservation on Pax gene expression. The results indicated that the trends (profile changes) in expression of Pax2a and Pax5 occurred to a lesser extent in frozen-thawed blastomeres than in fresh blastomeres whilst the opposite was true for Pax8. The trends in expression of Pax2b were delayed in frozen-thawed blastomeres compared to fresh blastomeres. Cryopreservation can therefore disrupt normal gene expression patterns in zebrafish embryos which could have a detrimental effect on embryo development. This is the first study on the stability of housekeeping and transcription factor genes in chilled and cryopreserved embryonic cells of the zebrafish. This work will significantly enhance future studies investigating the impact of cryopreservation on gene expression in zebrafish embryos.