Development of a Spermatogonial Germ Cell Transplantation Platform for Southern Bluefin Tuna (Thunnus Maccoyii)

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Development of a spermatogonial germ cell transplantation platform for southern bluefin tuna (Thunnus maccoyii)

Smith, Andre https://research.usc.edu.au/discovery/delivery/61USC_INST:ResearchRepository/12125983610002621?l#13126851420002621

Smith, A. (2014). Development of a spermatogonial germ cell transplantation platform for southern bluefin tuna (Thunnus maccoyii) [University of the Sunshine Coast, Queensland]. https://research.usc.edu.au/discovery/fulldisplay/alma99448779702621/61USC_INST:ResearchRepository Document Type: Thesis

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Please do not remove this page Development of spermatogonial germ cell transplantation platform for southern bluefin tuna (Thunnus maccoyii)

Andre N. Smith

B.Sc., Honours (Marine Biology)

Submitted in fulfilment of the requirement for the degree of Master of Science, University of the Sunshine Coast

Faculty of Science, Health, Education and Engineering (FoSHEE)

University of the Sunshine Coast

Locked Bag 4, Maroochydore DC, Queensland, 4558 Australia

January 2014 Abstract

Germ cell transplantation technology may become an important tool for the successful closed life-cycle aquaculture of southern bluefin tuna (Thunnus maccoyii) (SBT), to ease the fishing pressure on the wild stocks and develop a self-sustained industry. Currently, there are issues surrounding the care and maintenance of SBT broodstock, as they are large animals (up to 160kg) and reach sexual maturity at a late age (10 – 12 years). Germ cell transplantation has the potential to overcome the difficulties in maintaining large bodied broodstock, such as SBT, by transplanting donor-derived testicular cells (SBT) into a smaller bodied surrogate species such as yellowtail kingfish (Seriola lalandi) (YTK). YTK has been selected as a prospective surrogate host, as YTK larval development and spawning occurs at a similar temperature as SBT, and reaches sexual maturity at a younger size (3kg) and age (3 – 4 years) than SBT. Another reason why YTK has been selected as a potential surrogate for SBT is that they are a commercially important species and the aquaculture of this species is already well developed in South Australia, in close proximity to where SBT testis material is available.

The current study has laid the framework for a germ cell transplantation protocol for SBT by establishing some of the prerequisite knowledge required for this technology to progress. The first component of the study optimised an effective protocol to collect and dissociate SBT testis material to obtain a high number of viable testicular cells for transplantation was developed. This component also examined the effect of size and maturity of SBT on obtaining viable cell numbers. Secondly, the study confirmed an effective method to cryopreserve SBT testis material to ensure that SBT testicular cells were readily available year-round for germ cell transplantation. The study established the optimised rearing conditions for YTK in a small-scale rearing system in the third component. Lastly, the current study assessed the suitability of YTK as a surrogate host for SBT by successfully transplanting SBT testicular cells into YTK larvae, and then rear those transplanted larvae at low density in a small-scale system. This component successfully demonstrated that YTK supported the migration and colonisation of transplanted SBT testicular cells and therefore, YTK has the potential to act as a surrogate broodstock for SBT.

Declaration

I hereby declare that this thesis does not contain material which has been accepted for the award of any other degree or diploma in any university or institution, and to the best of my knowledge does not contain any copy or paraphrase of material previously published or written by any other person, except where due reference is made in the text of the thesis.

Andre N. Smith

University of the Sunshine Coast,

Maroochydore DC, Queensland,

January 2014

Acknowledgements

This research was funded by the Australian Seafood CRC and the Faculty of Science, Health, Education and Engineering at the University of the Sunshine Coast, Maroochydore, Queensland.

I would like to extend my sincere thanks to the many individuals and organizations that made this project possible. I would like to thank my principal supervisor, Professor Abigail Elizur. She not only exposed me to a whole new world of science, but did so in the nicest possible way, which included inviting me to stay at her home whilst I was spending time at the University on the Sunshine Coast. I would also like to thank my co-supervisor Dr Erin Bubner for her continuous guidance, support and for pushing me to strive to learn new ideas and techniques and improve as a scientist.

I must thank Cleanseas Tuna Ltd (CST), who were a key partner in the project and contributed largely to its success. I appreciate their outstanding support for research and development projects such as this one, and without their commitment, we would not have the opportunity to be able to do what we do as scientists. In particular, I would like to thank CST’s Chief Executive Officer, Dr Craig Foster for his support of the project and, Dr Bennan Chen, Mr David Poppi and Mr Adam Miller for their technical assistance throughout the project. I am also grateful to Mr Robert Staunton and the staff at Australian Tuna Fisheries for allowing me to join them on numerous commercial southern bluefin tuna harvests so I could sample their fish.

I am indebted to my fellow students and staff at the University of the Sunshine Coast for all their help. They have been instrumental in the successful completion of the project. In particular, I would like to thank Mr Ido Bar, Mr David Bright, Mr Jorge Amat-Fernandez, Dr Scott Cummins and Mrs Adi Bar for many late nights in the laboratory, many hours of mincing tuna testis and for opening up their homes to me while I visited the Sunshine Coast. I would also like to thank Dr. Nyugen Nyugen for his assistance with the statistical analysis of the project.

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Thank you also to Flinders University for hosting me as a visiting student at the Lincoln Marine Science Centre throughout the project and the wonderful staff at the Centre for providing a great support network and too much cake while completing the project.

Finally, I would like to thank my family and close friends for their encouragement, especially my wife, Sarah, for her continuous, unwavering love and support.

Contents

Abstract ...... i

Declaration ...... ii

Acknowledgements ...... iii

Contents ...... v

List of Tables ...... viii

List of Figures ...... viii

List of Abbreviations ...... x

1.0 Introduction ...... 1

1.1 Bluefin tuna industry ...... 1

1.2 Germ cell transplantation ...... 3

1.2.1 Cryopreservation of genetic material ...... 9

1.2.2 Small-scale larval rearing of marine finfish ...... 13

1.3 Thesis scope and outline ...... 16

2.0 Materials and Methods ...... 19

2.1 Small-scale larval rearing of YTK ...... 19

2.2 Dissociation of SBT testis material/preparation of SBT donor cells for transplantations ...... 21

The dissociation of SBT testis material was assessed and optimised in this section based on methods provided by Professor Goro Yoshizaki’s research group from the Tokyo University of Marine Science and Technology, for the dissociation of PBT testis material ...... 21

2.2.1 Collection of SBT testis material ...... 21

2.2.2 Optimisation of the dissociation protocol ...... 21

2.2.3 Assessment of the effect of size and reproductive maturity of SBT on testicular cell numbers and viability ...... 24

2.2.4 Histological analysis ...... 25

2.3 Cryopreservation of SBT testis material ...... 26

2.4 Transplantation of SBT testicular cells into YTK surrogate hosts ...... 27

2.4.1 Testicular cell preparation ...... 27

2.4.2 Germ cell transplantation ...... 28

2.4.3 Rearing of transplanted larvae ...... 29

2.4.4 Assessment of SBT donor testicular cells in recipient YTK larvae ...... 30

2.5 Statistical analyses ...... 30

3.0 Results ...... 31

3.1 Small-scale larval rearing of YTK ...... 31

3.2 Dissociation of SBT testis material ...... 32

3.2.1 Optimisation of the dissociation protocol ...... 32

3.2.2 Assessment of the effect of size and reproductive maturity of SBT on testicular cell number and viability ...... 34

3.3 Cryopreservation of SBT testis material ...... 39

3.4 Transplantation of SBT testicular cells into YTK surrogate host ...... 42

4.0 Discussion ...... 46

4.1 Small-scale larval rearing of YTK ...... 46

4.2 Dissociation of SBT testis material ...... 49

4.2.1 Optimisation of the dissociation protocol ...... 49

4.2.2 Assessment of the effect of size and reproductive maturity of SBT on testis cell numbers and viability ...... 51

4.3 Cryopreservation of SBT testis material ...... 52

4.4 Transplantation of SBT testicular cells into YTK surrogate hosts ...... 55

4.5 Future implications ...... 58

4.6 Conclusion ...... 59

5.0 References ...... 61

Appendix One ...... 66

vii

List of Tables

Table 2. Summary of treatment groups examined in experiment two of the cryopreservation trials ...... 27

Table 4. Summary of transplantation experiment where donor southern bluefin tuna (Thunnus maccoyii) cells were transplanted into recipient yellowtail kingfish (Seriola lalandi) larvae ...... 43

List of Figures

Figure 1. Step-by-step representation of zebrafish spermatogenesis from primordial germ cell (PGC) to spermatozoa throughout the three phase spermatogenesis process (adapted from Schulz et al. 2009). The three phases include the proliferative or mitotic phase, the meiotic or spermatocytary phase and the spermiogenic phase (SG). The cell types include the primordial germ cell (PGC), type-A spermatogonia (A- gonia), type-B spermatogonia (B-gonia), spermatocyte, spermatid and spermatozoa. The curved arrows indicate self-renewal of cells...... 5

Figure 2. Illustration of the distinct germ cell transplantation techniques currently available for use in fish using recipients at varying stages of development: A) primordial germ cell (PGC) transplantation in fish embryos; B) germ cell transplantation in newly hatched fish larvae and; C) germ cell transplantation in adult fish (adapted from Lacerda et al. (2012))...... 6

Figure 3. Typical interaction between cooling rate, solution injury, ice formation injury, and cell survival (adapted from Benson et al. (2012)). At low cooling rates “solution effects” are the dominant factor in cell damage, but as cooling rates increase and exposure time decreases, these effects are minimized. Conversely, at high cooling rates, intracellular ice formation is the dominant factor in cell damage, and as cooling rates are decreased, the likelihood of intracellular ice formation decreases. The combination of these two effects implies that there will be an inverted “U” shaped survival curve and an optimal cooling rate that minimizes both the solution effects and intracellular ice formation...... 12

Figure 4: Flow chart representing the steps involved in developing a germ cell transplantation framework for southern bluefin tuna (Thunnus maccoyii) (SBT) and the aims involved in the current project. Aim 1 involved optimising the dissociation protocol by examining the effect of different enzymes and various size and stage of reproductive maturity of SBT at different times of the year. Aim 2 examined a cryopreservation protocol for SBT testis material. Aim 3 investigated the optimised larval rearing conditions of yellowtail kingfish (Seriola lalandi) (YTK) in a small-scale system. Aim 4 investigated the suitability of YTK as a surrogate for SBT by transplanting SBT testicular cells into the peritoneal cavity of YTK larvae, then rearing those larvae to an appropriate age to assess the migration and colonisation of the transplanted testicular cells...... 18

Figure 5. Illustrative representation of the Percoll gradient process before (A) and after (B) centrifugation at 800g at 4oC for 30 minutes...... 24

Figure 6. Mean (±SE) survival of yellowtail kingfish (Seriola lalandi) larvae reared in a small-scale system (280 L tanks) at 1, 5 and 60 larvae.L-1 at 18 DPH, when stocked into the tanks at 0 DPH. Means with different superscripts are significantly different (P < 0.05) (n = 2)...... 31

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Figure 7. Mean (±SE) survival of yellowtail kingfish (Seriola lalandi) larvae reared in a small-scale system (280L tanks) presented with two different feeding regimes from 19 to 30 DPH. Means with different superscripts are significantly different (P < 0.05) (n = 3)...... 32

Figure 8. Mean number (± SE) of testicular cells produced from three different concentrations of southern bluefin tuna (Thunnus maccoyii) testis material (30, 50 and 100mg.mL-1) dissociated with collagenase/dispase (4/1200U mg.mL-1) or trypsin (0.0583mg.mL-1). Means with different superscripts are significantly different (P < 0.01) (n = 4)...... 33

Figure 9. Total number of testicular cells dissociated from different amounts of southern bluefin tuna (Thunnus maccoyii) testis material (100, 200, 300, 400, and 500mg.mL-1) with Collagenase/Dispase (2mg.mL- 1) or Trypsin (0.0583mg.mL-1) before (A) and after (B) a Percoll gradient...... 34

Figure 10. Cell number and viability of 1000mg of dissociated testis material collected from southern bluefin tuna (Thunnus maccoyii) of different weights (A, C) and fork lengths (B, D) during June to October 2012 and April and May in 2013 (n = 84)...... 35

Figure 11. Histological sections of testis material from southern bluefin tuna (Thunnus maccoyii) at stage 1 (A, B), stage 2 (C, D), stage 3 (E, F) and stage 4 (G, H) of reproductive maturity (SG = spermatogonia, SC = spermatocyte, ST = spermatid, SZ = spermatozoa, MSD = main sperm duct). Magnification x40 (A, C, E, G), x400 (B, D, F, H). Stages 1 and 2 are classed as immature and stages 3 and 4 are classed as mature...... 36

Figure 12. Testicular cell number and viability dissociated from 1000mg of testis material collected from southern bluefin tuna (Thunnus maccoyii) with different gonadosomatic index (A, C) and stage of reproductive maturation (B, D) sampled during June to October 2012 and April and May in 2013 (n = 84). .. 37

Figure 13. Testicular cell number (A) and viability (B) dissociated from 1000mg of testis material collected from southern bluefin tuna (Thunnus maccoyii) sampled during June to October 2012 and April and May in 2013 (n = 84)...... 39

Figure 14. Mean (± SE) number (A) and viability (B) of cells dissociated from 1000mg of cryopreserved and fresh southern bluefin tuna (Thunnus maccoyii) tuna testis material. Cell viability (B) was assessed at 0, 12 and 44 hours post dissociation (n = 4). Means with different superscripts are significantly different (P < 0.05)...... 40

Figure 15. Mean (± SE) number (A) and viability (B) of cells dissociated from 1000mg of cryopreserved southern bluefin tuna (Thunnus maccoyii) tuna testis material using three different cooling methods and two different concentrations of cryoprotectant agent (DMSO) (n = 4): 1) tissue stored directly into liquid nitrogen, 9% DMSO; 2) tissue stored at -80oC for 90 minutes (min) before transferred into liquid nitrogen, 5% DMSO; 3) tissue stored at -80oC for 90 min before transferred into liquid nitrogen, 9% DMSO and; 4) tissue stored at -20oC for 90 min before transferred into liquid nitrogen, 9% DMSO. Means with different superscripts are significantly different (P < 0.05). * denotes a zero value ...... 41

Figure 16. Incorporation of transplanted PKH26-labelled southern bluefin tuna (Thunnus maccoyii) testicular cells in the genital ridge of recipient yellowtail kingfish (Seriola lalandi) (YTK). Bright field (A, C) and fluorescent field (B, D) of the ventral view of the peritoneal cavity of non-transplanted YTK (A, B) and transplanted YTK (C, D) at 18 days post transplantation (29 DPH). Anterior end of each fish is located top right. White arrows indicate location of the genital ridges attached to the peritoneum. Yellow arrows indicate fluorescently labelled donor derived cells. Magnification x100 ...... 44

Figure 17. Incorporation of transplanted PKH26-labelled southern bluefin tuna (Thunnus maccoyii) testicular cells in the removed gonad of recipient yellowtail kingfish (Seriola lalandi) (YTK). Bright field (A, C) and fluorescent field (B, D) of removed gonad of non-transplanted YTK (A, B) and transplanted YTK (C, D) at 28 days post transplantation (39 DPH). Yellow arrows indicate fluorescently labelled donor derived cells. Magnification x400 ...... 45

List of Abbreviations

SBT Southern bluefin tuna M Molar

PBT Pacific bluefin tuna mM Millimolar

YTK Yellowtail kingfish sec Second

TAC Total allowable catch min Minute

DPH Days post hatch h Hour

Ltd Limited PBS Phosphate buffered saline F1 First generation offspring DMSO dimethyl sulfoxide

F2 Second generation L-15 Leibovitz’s-15 media offspring FBS Foetal bovine serum

PGC Primordial germ cell o C Degrees Celsius kg Kilogram µm Micrometre g Gram W Watt mg Milligram kW Kilowatt

L Litre % Percentage mL Millilitre U Standard Japanese unit

µL Microlitre CST Cleanseas Tuna

1.0 Introduction

1.1 Bluefin tuna industry

Tuna species belong to the Scombridae family of fishes and are large, pelagic predatory fish that are widely distributed throughout the oceans of the world (Ottolenghi 2008). Of the 33 species and sub-species that exist world-wide, none are as large or as highly valuable as the bluefin tuna species (Ottolenghi 2008; Boustany 2011). The three bluefin tuna species are the Atlantic bluefin tuna (Thunnus thynnus), Pacific bluefin tuna (Thunnus orientalis) (PBT) and southern bluefin tuna (Thunnus maccoyii) (SBT) and are all highly desired for the international sushi trade (Ottolenghi 2008; Boustany 2011). Early fisheries targeting bluefin tuna date back thousands of years; however, globalisation of fisheries in recent years has seen an increase in fishing pressure on wild stocks (Farwell 2001; Sawada et al. 2005; Findlay 2006; Ottolenghi 2008; Boustany 2011). Tuna species are captured using a number of different methods including purse seines, long-lines, traps, handlines, bait boats and sport fishing (Ottolenghi 2008). The global catch of SBT peaked in the early 1960’s (81,605 tonnes) and by the early 1980’s, some reports suggested that the SBT stock was under immense fishing pressure and was being overfished (Findlay 2006; Patterson et al. 2010). Although some industry members challenged the stock assessment reports, in 1983 the first Total Allowable Catch (TAC) of 21,000 tonnes was introduced in Australia. In the 1990’s, the cage culture or ranching of SBT was trialled by tuna fishermen in South Australia.

The ranching process is primarily located offshore of Port Lincoln, South Australia, where the farmed SBT are conditioned and fattened over a period of approximately six months and sold as a premium export product (Farwell 2001; Findlay 2006; Patterson et al. 2010). In 2005, SBT ranching comprised of more than 95% of Australia’s TAC, compared to 3% in 1991-92 (Findlay 2006). Although the wild SBT fishery has embarked on some managerial changes to aid in the sustainability of the industry, the World Conservation Union lists SBT as critically endangered. SBT is also a threatened taxon under the Victorian Flora and Fauna Guarantee Act 1988 and an endangered species under the New South Wales Fisheries Management Act 1994. In 2010, the

Australian Environment Protection and Biodiversity Conservation Act 1999 listed SBT as conservation dependant, which is defined in Section 179 (6) as: the species is the focus of a specific conservation program, the cessation of which would result in the species becoming vulnerable, endangered or critically endangered within a period of 5 years.

Bluefin tuna cage culture, which now comprises the majority of the bluefin tuna catch worldwide, involves purse seining the schools of tuna, transferring them to floating pens or cages, and fattening them for several months before harvesting them to increase their market value (Findlay 2006; Ottolenghi 2008). Tuna cage culture is well established in Mediterranean countries for Atlantic bluefin tuna, in Australia for SBT and in Japan and Mexico for PBT (Sawada et al. 2005). Because the prices for bluefin tuna in the sashimi market are much higher than for canned tuna, the emergence and globalization of this market greatly changed the economics of global bluefin fisheries and encouraged the overexploitation of bluefin tuna (Boustany 2011). Currently, the three bluefin species are among the most overexploited tuna species in the world (Boustany 2011).

More recently, there has been a large effort to establish closed life-cycle aquaculture of bluefin tuna to ease the fishing pressure on the wild stocks and develop a self- sustained industry. Research on closed life-cycle aquaculture of the PBT started in 1970 in Japan, with the first successful spawning broodstock in experimental sea-cages at Kinki University in 1979 (Sawada et al. 2005). Subsequently, the completion of the life-cycle of the PBT was achieved in the summer of 2002 when artificially hatched fish (F1 generation) spawned and produced healthy larvae (F2 generation) (Sawada et al. 2005). This was an important breakthrough in the technological development of sustainable tuna aquaculture (Sawada et al. 2005). The successful spawning of the Atlantic bluefin tuna in captivity soon followed with the first spawning obtained in Spain in 2004 after broodstock were hormonally induced with gonadotropin-releasing hormone analogue (GnRHa) (Mylonas et al. 2007). SBT were then successfully spawned in an onshore breeding facility of Cleanseas Tuna Ltd in Australia during the summers

2 of 2007/08 through 2012/13, with the offspring cultured to an early stage of development (Adam Miller, personal communication). The company has completed its fourth consecutive annual on-shore SBT spawning program, having improved the longevity of the controlled spawning period (Thomson et al. 2010). Despite these breakthroughs in the successful spawning of the bluefin tuna in the captive environment, there are still major challenges that need to be overcome. One of the main difficulties is the management of tuna broodstock (Takeuchi et al. 2009; Yazawa et al. 2010), as they are typically slow growing, long-lived and become sexually mature at a late age (Farley and Davis 1998; Mori et al. 2001; Findlay 2006; Ottolenghi 2008; Patterson et al. 2008; Patterson et al. 2010). New innovative approaches may be needed to facilitate the closed life-cycle aquaculture of bluefin tuna and overcome some of the obstacles faced with managing the broodstock. Technologies such as hormone therapy to positively regulate pubertal development in fish (Nocillado et al. 2007) and germ cell transplantation technology that produces donor derived gametes from a surrogate host (Takeuchi et al. 2003) could assist in the breeding of bluefin tuna species in captivity.

1.2 Germ cell transplantation

Recent research has reported that the entire world fishery resource will be depleted in about 40 years if environmental destruction and commercial fishing continue at the current pace (Worm et al. 2006). Technologies need to be urgently developed to protect endangered and valuable species whilst still preserving the environment. Raising endangered and valuable species in captivity is one solution. However, this involves some risks that have the potential to wipe out whole populations, as they are generally raised in captivity at high densities in ponds or tanks. Therefore, risks such as facility accidents, outbreaks of infectious diseases and the inability of farmed fish to adapt to their natural environments once released from captivity generally effects the whole tank or pond and thus, effecting the whole population (Okutsu et al. 2008). In addition, some endangered and valuable species cannot be easily raised and/or cultured in captivity. Technologies such as germ cell transplantation and the

3 cryopreservation of testicular cells may help to overcome these issues. This technology involves spawning the target species germ cells in captivity from small-bodied surrogate parents that are better suited to such conditions (Takeuchi et al. 2003; Okutsu et al. 2006 (a); Okutsu et al. 2008; Majhi et al. 2009; Takeuchi et al. 2009; Yazawa et al. 2010; Yoshizaki et al. 2011). Germ cell transplantation is achieved by transplanting donor-derived primordial germ cells (PGC), or differentiated germ cells from a donor species, into the peritoneal cavities of newly hatched surrogate species larvae. The germ cells migrate to form part of the surrogate gonad and then go on to produce functional gametes (i.e. oocytes and sperm) of the donor species (Takeuchi et al. 2003).

Germ cells are different from the somatic cells that compose the body in that they can give rise to a new generation (Starz-Gaiano and Lehmann 2001). Germ cells undergo meiosis and differentiate into highly specialised cell types, such as sperm, in a process called spermatogenesis (Starz-Gaiano and Lehmann 2001; Schulz et al. 2009). Spermatogenesis is a highly organised and coordinated process, in which spermatogonia proliferate and differentiate to form mature spermatozoa (Schulz et al. 2009) (Figure 1). The process can be split into three different phases; the proliferative or mitotic phase with different generations of spermatogonia, the meiotic phase with the formation of the spermatocytes and the spermiogenic phase with the spermatids emerging from meiosis and differentiating into motile, flagellated spermatozoa (Schulz et al. 2009) (Figure 1). As with many other organisms, fish germ cells emerge at a distant position from the origin of the somatic genital ridges and migrate to this site to make up a functional gonad (Starz-Gaiano and Lehmann 2001). If transplanted donor- derived germ cells can simulate the migratory path of the endogenous germ cells in the surrogate species, they have the capacity to proliferate and differentiate into functional gametes in the gonad, to then ultimately generate viable seed of the donor species (Takeuchi et al. 2003; Takeuchi et al. 2004).

Proliferative phase Meiotic phase SG phase

Spermatozoa B-gonia Spermatocyte Spermatid A-gonia

PGC Figure 1. Step-by-step representation of zebrafish spermatogenesis from primordial germ cell (PGC) to spermatozoa throughout the three phase spermatogenesis process (adapted from Schulz et al. 2009). The three phases include the proliferative or mitotic phase, the meiotic or spermatocytary phase and the spermiogenic phase (SG). The cell types include the primordial germ cell (PGC), type-A spermatogonia (A-gonia), type-B spermatogonia (B-gonia), spermatocyte, spermatid and spermatozoa. The curved arrows indicate self-renewal of cells.

To establish a germ cell transplantation method in fish, a technique for mass isolation of germ cells is necessary (Takeuchi et al. 2002). Takeuchi et al. (2002) developed a technique to purify a large number of viable rainbow trout (Oncorhynchus mykiss) germ cells and variations of this method have now been to developed for other salmonid species (Takeuchi et al. 2004) as well as other fish species such as medaka (Oryzias latipes) (Herpin et al. 2008), Nile tilapia (Oreochromis niloticus) (Lacerda et al. 2008), Nibe croaker (Nibea mitsukurii) (Takeuchi et al. 2009), chub mackerel (Scomber japonicas) (Yazawa et al. 2010) and PBT (Yazawa et al. 2013). Currently, methods have been developed for transplanting germ cells into surrogate species when they are: 1) an embryo; 2) a newly hatched fish larvae or; 3) an adult fish (Lacerda et al. 2012) (Figure 2). Each method has various advantages and disadvantages and the selection of a suitable developmental stage of the surrogate for transplantation depends on the objectives of the study, the type of donor species and the availability of surrogate species (Lacerda et al. 2012).

A) Primordial germ cell transplantation in fish embryos

Donor derived Donor gamete Adult donor fish Recipient embryo Larvae Adult fish germ cells production B) Germ cell transplantation in newly hatched fish larvae

Donor derived Donor gamete Adult donor fish Recipient larvae Adult fish germ cells production

C) Germ cell transplantation in adult fish

Donor derived Donor gamete Adult donor fish Adult recipient fish germ cells production

The first reported instance of intra-peritoneal transplantation of PGC into hatching fish embryo’s found that transplanted PGC were able to migrate towards and colonise the genital ridges of recipient larvae (Takeuchi et al. 2003). This was done in an allogenic environment (i.e. intra-species) using PGC isolated from the genital ridges of rainbow trout (O. mykiss) transplanted into the peritoneal cavity of rainbow trout larvae. One of the key factors in the successful migration and colonisation of transplanted PGC in surrogate hosts is that the immune system of the surrogate hatched larvae is relatively immature at the time of transplantation (Takeuchi et al. 2003). Thus, transplanted PGC will not be rejected by the surrogate’s immune system and can proliferate and mature in the xenogenic (inter-species) environment (Takeuchi et al. 2003). Following this, PGC isolated from the genital ridges of newly hatched rainbow trout were then transplanted into the peritoneal cavity of newly hatched Masu salmon (O. masou) larvae to assess the techniques suitability in the xenogenic environment. The study showed that PGC from the rainbow trout successfully migrated towards and colonised

6 the genital ridge of the recipient larvae and upon reaching adulthood, produced xenogenic donor-derived offspring (Takeuchi et al. 2004).

Although there has been some success with germ cell transplantation technology using PGC, there are various issues associated with it. Firstly, fish PGC can only be collected within a period of a few weeks before and after hatching and since there are only a small number of PGC, visualising and gathering sufficient numbers for transplantation is difficult (Okutsu et al. 2008). Secondly, transplanted recipients that reach sexual maturity produce more recipient derived gametes (i.e. its own oocytes and spermatozoa) than those that are donor-derived (Okutsu et al. 2008). Therefore, more recent studies have focused on developing the use of triploid surrogate host species that spawn only donor derived gametes to overcome these issues. Type-A spermatogonia has also been the focus of these studies due to their plasticity and sexual bipotency they can also be used in germ cell transplantation experiments, however unlike PGC, they can be found in testis material at high numbers at many developmental stage (Okutsu et al. 2006 (a); Okutsu et al. 2007). Furthermore, type-A spermatogonia act as stem cells, and retain the ability of self-renewal (Okutsu et al. 2006 (b)). Type-A spermatogonia are reported to undergo mitosis 6 – 16 times, followed by two consecutive cycles of meiosis; thus one founder spermatogonia can produce over 260,000 spermatozoa (Okutsu et al. 2006 (b); Nobrega et al. 2009; Schulz et al. 2009). The optimal stage of testis development to obtain the highest number of type-A spermatogonia, however, has not yet been determined and needs future research. Type-A spermatogonia cells are thought to act in a similar fashion to PGC in that they respond to signals produced by the genital ridge and migrate along it to form part of the primordial gonad (Okutsu et al. 2006 (a)). Testicular cells containing spermatogonia, isolated from adult rainbow trout and transplanted into newly hatched rainbow trout (i.e. allogenic environment) and masu salmon embryo’s (i.e. xenogenic environment), successfully colonised and differentiated into functional gametes and produced viable offspring (Okutsu et al. 2006 (a); Okutsu et al. 2007). The use of spermatogonia derived from adult testes to produce functional donor derived gametes in recipient larvae provides a powerful tool for germ cell transplantation technology

7 and an avenue for breeding commercially important fish species such as the bluefin tuna.

Transplantation of type-A spermatogonia into recipient larvae of marine species was thought to be a more difficult task compared to freshwater species, as marine fish have much smaller larvae and are therefore more fragile and difficult to handle (Takeuchi et al. 2009). However, allogenic transplantations have been successfully demonstrated by Takeuchi et al. (2009) and Morita et al. (2012) with the Nibe croaker (N. mitsukurii) and the Japanese yellowtail kingfish (Seriola quinqueradiata), respectively. The first xenogenic transplantation in marine species was reported by Yazawa et al. (2010) and showed recipient Chub mackerel (S. japonicas) successfully supported the colonisation, survival and proliferation of donor derived cells from the Nibe croaker. This finding was a significant step forward in the development of germ cell transplantation technology with marine species. It demonstrated that this technology can be applied in the xenogenic environment and also potentially with species from different taxonomic families.

The age of recipient larvae is an important factor to consider, as it effects the successful migration and colonisation of the donor derived PGC or type-A spermatogonia. Specifically, there appears to be a distinct and narrow window of opportunity in the developmental stages of recipient larvae when exogenous PGC can be incorporated into their gonads (Takeuchi et al. 2003; Takeuchi et al. 2009). Detailed histological examinations of early gonadal development of the recipient larvae is an important step in finding the suitable stage of development for transplantations (Takeuchi et al. 2003; Takeuchi et al. 2009). For example, a previous study found that recipient rainbow trout larvae lose their ability to guide donor PGC’s towards their genital ridges two weeks after endogenous PGC finish migrating (Takeuchi et al. 2003). There also appears to be a distinct and narrow window of opportunity to transplant donor-derived germ cells into Nibe croaker larvae. Donor cells successfully migrated and colonised in recipient Nibe croaker larvae of 4 and 5 mm in length when endogenous PGC were still migrating (Takeuchi et al. 2009). However, donor cells did

8 not colonise in larvae of 6 mm in length, when endogenous PGC had already started to proliferate (Takeuchi et al. 2009).

Yellowtail kingfish (S. lalandi) (YTK) has been identified as a potential surrogate host for SBT in Australia. Despite the fact that YTK belong to a different family class (Carangidae) than SBT (Scombridae), the two species have similar larvae culture requirements (i.e. water temperature), which is essential for successful migration and colonisation of transplanted germ cells. In addition, YTK become sexually mature at a younger age (approximately 2 to 3 years old) and smaller size (<15 kg) than SBT (approximately 12 years old and >100kg). Studies on YTK as a surrogate host for SBT investigated the potential window of opportunity for germ cell transplantation, and has established it to be between 7 and 10 days post hatch (DPH), based on detailed histological analysis of the migration and colonisation of the endogenous PGCs and taking into account the physical aspects of larvae (i.e. size, skin pigmentation) (Bubner 2011). Furthermore, S. lalandi and S. quinqueradiata have already shown to support the migration, colonisation and survival of intraperitoneal transplanted testicular cells in the allogenic environment (Bubner 2011; Morita et al. 2012) and S. quinqueradiata have successful produced donor derived offspring (Morita et al. 2012). YTK are also a commercially important species and therefore the aquaculture of this species is already well developed and larvae are readily available almost year-round for transplantation (Chen et al. 2007; Bubner 2011).

1.2.1 Cryopreservation of genetic material

Despite the fact that YTK larvae are readily available almost year round for transplantation, the availability of SBT testicular material is limited to the commercial harvest season that runs between April and October. Cryopreservation of SBT testis material could ease the pressure of having to coordinate the availability of donor tissue with the presence of suitable surrogate host larvae.

The history of gamete, embryo and tissue cryopreservation for genetic preservation extends back about six decades (Saragusty et al. 2011). Many advances have been made during this time and cryopreservation of genetic material has been used to

9 breed many species of animals spread to aquaculture (Fabbrocini et al. 2000; Saragusty et al. 2011). Cryopreservation of genetic material primarily allows for gametes to be available year-round to aid in the synchronisation of artificial insemination, transportation, storage and it may also help reduce the need to maintain large numbers of captive adults as broodstock (Fabbrocini et al. 2000; Gwo et al. 2005; Jensen et al. 2008). Other benefits associated with the cryopreservation of genetic material are the reduced risk of transmitting diseases, the possible production of hybrids with desirable characteristics and the creation of new lines for selective breeding (Fabbrocini et al. 2000; Gwo et al. 2005; Jensen et al. 2008). Cryopreservation is a procedure by which cells or whole tissues are suspended in a solution, cooled to very low sub-zero temperatures (usually -196oC in liquid nitrogen), stored for some period of time, then thawed and recovered to resume their normal function (Leibo and Pool 2011). In aquaculture its routine use is currently restricted to sperm as eggs cannot be preserved due to high yolk content.

The first theory of cryopreservation was established in the 1930’s by B.J. Luyet, but it was not until the accidental finding of the cryoprotective properties of glycerol by C. Polge’s research group in 1949 that the first successful cryopreservation of spermatozoa occurred (reviewed by Gosden (2011)). Two main problems arise for cells cooled below freezing temperature: 1) water crystallizes to form ice and; 2) salt concentrations rise (Watson 2000; Mocé and Vicente 2009; Gosden 2011). Thus, the key to successful cryopreservation of cells of every type and species is to avoid ice crystals forming inside the cells and reduce the side effects of freezing outside the cell (Watson 2000; Mocé and Vicente 2009; Gosden 2011). Polge’s research group had backed up Luyet’s findings, where very few spermatozoa regained motility after cooling to -79oC in fructose solutions. However, when one of Polge’s lab technicians accidently mixed up the fructose solution with glycerol, the researchers were amazed when virtually all the cells survived (reviewed by Gosden (2011)). Since this important finding, the cryopreservation of spermatozoa has been most successfully associated with human and bovine reproductive technologies but has also been applied to many other mammalian species (Woods et al. 2004; Gosden 2011; Saragusty et al. 2011;

Benson et al. 2012). The cryopreservation of spermatozoa is also now well established in a number of fish species and studies involving the cryopreservation of spermatozoa from more than 200 fish species have been published (Suquet et al. 2000; Routray et al. 2006).

Cryoprotective agents, such as glycerol, work simultaneously with cooling rates where, if the cooling rate is decreased, the osmotic imbalance caused by the lower temperature can be rectified by the exosmosis of water and the influx of the cryoprotective agent into the cell (Benson et al. 2012). This means that the slower cooling rate allows cells enough time to maintain the chemical potential of water between the inter and extracellular membrane, which decreases the likelihood of the formation of intracellular ice, which can damage the cell membrane. In contrast, if the cooling rate is too slow, the prolonged exposure to the cryoprotective agent can be toxic to the cell and cause irreversible membrane damage (Benson et al. 2012). Mazur and Miller (1976) suggested that the optimal cooling rate is that which is as fast as possible to avoid solution effects, but slow enough so that the cells can dehydrate sufficiently to avoid intracellular ice formation (Figure 3).

Solution injury Intracellular ice formation

Cell survival

Percentage (%) Optimal cooling rate

Slow cooling Rapid cooling Cooling rate

More recently, there has been an increase in research effort directed towards the cryopreservation of testicular tissue of various fish species containing abundant numbers of germ cells at various developmental stages (Woods et al. 2004). The development of a technique to cryopreserve testicular tissue would be beneficial for the development of germ cell transplantation technology as it would allow year round access to type-A spermatogonia cells for transplantation. Cryopreservation of testicular tissue would also remove the pressure of having to coordinate the availability of donor tissue with the presence of suitable surrogate host larvae, and allow the use of this technology to be applied in other parts of the world, as material can be transported (Fabbrocini et al. 2000; Gwo et al. 2005). Tissue cryopreservation can be more complex than cell cryopreservation, as different cell types are present

12 within the tissue that differ in characteristics, such as water and cryoprotectant permeability, and have different sensitivities to chilling and osmotic challenges.

A study by Kobayashi et al. (2003) reported the cryopreservation of genital ridges of rainbow trout using four different cryoprotectant agents at varying concentrations (dimethyl sulfoxide (DMSO), glycerol, 1,2-propanedial and ethylene glycol). They found the highest survival rate of frozen/thawed PGC were isolated from tissue cryopreserved with 1.8 M ethylene glycol as the cryoprotectant (Kobayashi et al. 2003). Transplantation of PGC from frozen/thawed genital ridges cryopreserved with ethylene glycol, subsequently successfully colonized and proliferated in the gonads of recipient (Kobayashi et al. 2003) demonstrating that their ability to colonise and proliferate was not compromised by the cryopreservation technique. A similar study by Lee et al. (2013) also examined the viability of type-A spermatogonia from rainbow trout when frozen with various cryoprotective agents, including 1.3 M DMSO, 1.3 M propylene glycol and 1.3 M glycerol. The study found that the viability of the type-A spermatogonia frozen in the presence of DMSO was significantly higher compared to the other cryoprotectants examined. The study also established that the colonisation and proliferation efficiencies of donor derived rainbow trout type-A spermatogonia in recipient gonads were not significantly different among those cryopreserved for periods of 1, 24, 939 days or cells prepared from fresh material. Among the various cryoprotectants tested for cryopreservation of fish spermatozoa and testis material, DMSO is thought to be a better cryoprotectant for most cells, probably because its entry and exit into cells is much faster than other cryoprotectants (Basavaraja and Hegde 2004). The demonstrated successful cryopreservation of testis material of these species opens up the possibility for this technology to be applied to many other commercially important and/or endangered species as well as the possibility to assist other research areas such as germ cell transplantation technology.

1.2.2 Small-scale larval rearing of marine finfish

Another important aspect for the success of germ cell transplantation technology is to optimise the survival of the transplanted larvae. Larval rearing of marine finfish is

13 typically done on a large scale, for both commercial and scientific purposes, which is when a large number of larvae are reared together in the same large tank (i.e. 8000L) (Hart et al. 1994). One reason that larval rearing is done on a large scale is due to the high number of problems that occur in small-scale system (Hart et al. 1994). Problems that can arise in small-scale systems include obtaining the ideal ratio between water volume to wall and floor area, to reduce the contact of larvae with tank walls, and the formation of surface oil films when using live feeds (Hart et al. 1994). Oily surface films have been attributed to failed initial swim bladder inflation (Trotter et al. 2003 (b)) and this failure to inflate swim bladder can result in reduced survival and growth of marine fish larvae (Chatain and Ounais-Guschemann 1990; Battaglene et al. 1994). The failure of fish larvae to inflate the swim bladder has also been attributed to abiotic factors such as lighting, salinity, turbidity and temperature (Doroshev et al. 1981; Chatain and Ounais-Guschemann 1990; Battaglene et al. 1994; Martin-Robichaud and Peterson 1998; Trotter et al. 2001; Trotter et al. 2003 (a); Trotter et al. 2003 (b); Partridge et al. 2011). Correct swim bladder inflation is essential for functional buoyancy control and swimming ability, which can have a direct effect on the successful transition between endogenous feeding (i.e. egg sac) to exogenous feeding (i.e. live feed) (Martin- Robichaud and Peterson 1998; Carton 2005) that is critical to their survival. Large-scale rearing systems also allow for a better buffering capacity in regards to changes in the environment (i.e. water temperature, pH, and salinity). However, sometimes researchers in particular have no choice but to rear larvae on a small-scale, low density environment (i.e. approximately 1 larvae.L-1), such as with germ cell transplantation when only a small number of larvae can be transplated at any one time. Therefore, it is extremely important to try and optimise the various conditions of small-scale rearing where possible.

Regardless of the fore-mentioned problems with small-scale systems, large variability in survival, growth and the quality of hatchery reared marine fish is a common and complex problem that occurs also in large scale systems. Mass mortalities can occur even when optimal abiotic factors (i.e. light intensity, turbidity, temperature, photoperiod and salinity) are provided (Moustakas et al. 2004; Carton 2005;

Battaglene et al. 2006; Blood and Laurel 2011; Partridge et al. 2011). The causes of larval mortality are many and previous studies have investigated the optimisation of abiotic factors such as light intensity, turbidity (Carton 2005), temperature (Blood and Laurel 2011), photoperiod (Partridge et al. 2011) and salinity (Moustakas et al. 2004) to increase larval survival in a number of marine fish species and must be considered when setting up any new system. It is well understood that nutrition is also a critical factor for proper larval development (Chen et al. 2006; Chen et al. 2007) and despite the recent progress in the quality of inert diets for fish larvae, feeding of most species of interest in aquaculture still rely on live feeds during the early life stages (Conceição et al. 2010). Live feeds such as rotifers (Brachionus placatilis) and Artemia sp. are widely recognised to be an excellent starter feed for marine larvae (Hung et al. 2002; Conceição et al. 2010). In Australian hatcheries, YTK are fed large strain rotifers at first feed, then fed Artemia sp. from around 12 DPH, before larvae are weaned onto artificial micro-compound diets (Chen et al. 2006; Conceição et al. 2010; Woolley et al. 2012). This weaning stage is considered an important step in the larval culture process and can have a significant impact on the growth and survival of the larvae (Chen et al. 2006; Soligo et al. 2011). There has been great interest in reducing the dependence of larval fishes on live prey and the ability of larvae to digest formulated feeds appears to increase with age and development of the associated organs of the digestive system (Gordon and Hecht 2002; Chen et al. 2006; Chen et al. 2007; Soligo et al. 2011). At present, there is limited information published on feeding regimes for YTK on both large and small-scales. Optimising feeding regimes could contribute to improved larvae survival, which would be beneficial when rearing larvae for both commercial and scientific purposes.

Larval stocking density is also a very important aspect to consider in larval rearing systems as it can have a direct effect on fish growth and survival. Optimum stocking densities can differ not only for individual species but also relative to the rearing method used (Hatziathanasiou et al. 2002; Kupren et al. 2011). For example, survival of post-larvae sea bass (35 DPH) was found to be higher in tanks with a stocking density of 5 and 10 larvae.L-1, compared to 15 larvae.L-1 and 20 larvae.L-1; but this was not the

15 case for fish growth that was actually promoted at higher stocking densities (Hatziathanasiou et al. 2002). However, a negative correlation was found between stocking density and growth in perch (Perca fluviatilis) (Baras et al. (2003)). Baras et al. (2003) also found that increasing the stocking density did not compromise growth and decreased the overall impact of cannibalism through several complementary mechanisms. The mechanisms were firstly, a postponed emergence of cannibalism, secondly, a lower proportion of cannibals in the population, and thirdly, a lower rate of cannibalism per capita as predation was complicated and less directed at high stocking density (Baras et al. 2003). These studies therefore highlight the importance of investigating optimum culture conditions on a species-specific basis where they have not already been established.

1.3 Thesis scope and outline

The main scope of this project was to develop a germ cell transplantation platform to establish surrogate broodstock technology for SBT. Firstly, methods to obtain a high number of viable SBT type-A spermatogonia cells for transplantation were examined. Specifically, different types and concentrations of dissociation enzymes and the dissociation of varying amounts of the testis material were tested. Secondly, the optimal size and maturity of SBT that produced the highest number of viable type-A spermatogonia for transplantation was examined. Specifically, three different size classes of SBT (< 27 kg, 27 – 41 kg, >41kg) were sampled once a month during the commercial harvest season with the prevalence of type-A spermatogonia in each size class determined. Thirdly, the cryopreservation of SBT testis material to enable year- round access to germ cells for transplantation was also examined. Specifically, the project assessed variations of the cryopreservation protocol developed by Professor Goro Yoshizaki’s research group from the Tokyo University of Marine Science and Technology, for the cryopreservation of PBT (Thunnus orientalis) testis material. To examine the suitability of YTK as a surrogate host for SBT, firstly, the small-scale rearing of YTK under three different stocking densities and two different feed regimes was explored. This was followed by the adoption of a procedure developed by

Professor Goro Yoshizaki’s research group (Takeuchi et al. 2003; Takeuchi et al. 2004; Yoshizaki et al. 2011) and Dr. Erin Bubner’s PhD (Bubner 2011) to transplant SBT type-A spermatogonia into YTK larvae. The study then examined the migration and colonisation of transplanted SBT type-A spermatogonia cells in the YTK larvae. An outline of the current study is presented in Figure 4.

Collect of testis material from SBT during commercial harvest

Aim 1: Optimise SBT Aim 2: Establish SBT

tissue dissociation SBT testis tissue testis tissue protocol dissociation cryopreservation technique

Prepare fresh SBT testicular cells for transplantation

Aim 3: Optimise Obtain suitable small-scale larval YTK larvae rearing of YTK

Transplant SBT germ cells into YTK larvae Aim 4: Establish the suitability of

YTK as a potential Rear transplanted YTK

surrogate for SBT larvae to an assessable age

Produce YTK surrogates

2.0 Materials and Methods

2.1 Small-scale larval rearing of YTK

The rearing of YTK larvae in a small-scale system was examined during two experiments that aimed to establish optimal larval stocking densities and feeding regimes for up to the first 30 DPH. The first experiment examined three different larval stocking densities (1 larvae.L-1, 5 larvae.L-1 and 60 larvae.L-1 with two replicate tanks per treatment group) and their effect on larvae survival. The availability of identical tanks (six 280L tanks) at the Cleanseas commercial hatchery restricted the amount of replicates to two in this instance. The second experiment examined two different feeding regimes with three replicate tanks: 1) fed solely Artemia and 2) weaned from Artemia to artificial dry food, and their effect on survival of YTK larvae from 18 DPH to 39 DPH.

Fertilized YTK eggs were sourced from Cleanseas Tuna Ltd, Arno Bay, South Australia. For hatching, approximately 100,000 YTK eggs were incubated in a single 425L incubator at 21oC for 60 h. Once the larvae hatched (i.e. 0 DPH), they were kept in the incubator for 24h before being moved into larvae rearing tanks. In the stocking density trial, the larvae were moved into six 280L rearing tanks at 1 DPH at the assigned stocking densities (1 larvae.L-1, 5 larvae.L-1 and 60 larvae.L-1). In the feed regime trial, the larvae were moved into a single 850L rearing tank at 1DPH (stocking density of 90 larvae.L-1), before they were subsequently moved into six 280L tanks at 18DPH (stocking density of 1 larvae.L-1).

All rearing tanks were part of a flow through system supplied with filtered seawater (sand and 25µm sock filtered) with a daily water exchange rate of 450% at 0 DPH, which increased to 600% by 30 DPH. One airstone was placed in each tank and dissolved oxygen (mg.L-1 and % saturation), water temperature and pH were monitored three times daily. Light intensity (Lux), salinity and ammonia were monitored at the start and end of the experiment. The tanks were cleaned daily using a siphon hose, the water outlet screens were cleaned every second day and the

19 surface of the water was skimmed up to five times daily to remove any build-up of foreign particles, dead rotifers, Artemia and excess protein. Two fluorescent tube lights (35W each) above each rearing tank provided a surface light intensity of 255 lux at the centre of each rearing tank and a 12 h light (0900 to 2100 h) and 12 h dark photoperiod was used. A 300W aquarium heater (280L tanks, Aqua One, Ingleburn, Australia), or a 2.5kW coil heater (850L tank, Hotco, Australia), were placed in the tanks to assist in maintaining constant temperature.

In both experiments one and two, microalgal paste (Nannochloropsis sp., Instant Algae, Reed Mariculture Inc., USA) was added to the rearing tanks to maintain a cell density of 4.86x105 cells.mL-1 between 1 and 4 DPH and increased to 9.71x105 cells.mL-1 at 5 DPH until the end of the experiments. Larvae were fed rotifers (Brachionus plicatilis) enriched with Spresso (INVE Aquaculture, Belgium) according to the manufacturer’s instructions from 3 to 18 DPH three times per day to a target density of 15 rotifers.mL- 1. Artemia was introduced into the YTK diet from day 11 onwards. The YTK larvae were fed a single feed of unenriched Artemia nauplii on 11 DPH and two feeds on 12 DPH to a target density of 0.1 and 0.2 individuals.mL-1, respectively. On 13 DPH until the end of the stocking density experiment, larvae were fed an enriched Artemia (Enriched with Spresso, INVE Aquaculture, Belgium) ration three times per day (0900, 1230 and 1630h) to a target density of 0.25 Artemia.mL-1 on 13 DPH increasing to 2 Artemia.mL-1 by 18 DPH.

In the feed regime experiment, YTK larvae from 18 to 30 DPH were fed either solely an Artemia diet or were weaned from Artemia onto dry feed (300 - 400µm Wean S, INVE, Belgium). In the tanks that were fed solely Artemia, the live feed was added to the tanks four times daily (0900, 1100, 1330 and 1630 h) to reach a target density of 2 Artemia.mL-1. Weaning of the larvae occurred between 19 and 26 DPH, where 20g of dry food was introduced to the larvae at 19 DPH and increased until they were then fed solely 100g of dry food from 27 to 30 DPH. From 19 to 27 DPH, the amount of Artemia feed decreased by 0.25 Artemia.mL-1 per day from 2 Artemia.mL-1 to zero Artemia.

2.2 Dissociation of SBT testis material/preparation of SBT donor cells for transplantations

2.2.1 Collection of SBT testis material

Testis material was collected from SBT during commercial harvests from sea-cages offshore of Port Lincoln, South Australia. The testis material was then transferred into approximately 100 mL of 10mM phosphate buffered solution (PBS) (Sigma, P4417), which was kept on ice until arriving back at the laboratory approximately four hours after collection.

2.2.2 Optimisation of the dissociation protocol

The testis material collected in Section 2.2.1 was removed from PBS and weighed with and without the mesenteric fat. It was then dissected into small pieces (between 300 and 1000mg), transferred into a glass cavity slide with small volume of PBS (between 2 and 3 mL) and minced with wecker scissors. Once the testis material had been minced to a paste-like consistency, it was transferred into a 15mL tube and rinsed twice in Leibovitz’s-15 (L-15) media (Gibco Invitrogen, 11415-064) containing 1% Penicillin/Streptomycin (Sigma, P0781). Rinsing consisted of pipetting the solution up and down gently in the 15mL tube to mix the cells before it was centrifuged at 200g for 5 min at 4oC. The supernatant was discarded between each rinse. The cells were then transferred into a 12 well plate with the corresponding dissociation media for incubation. Different dissociation enzymes, concentrations of enzymes and amount of minced testis material were examined, as described in Table 1.

Table 1. Summary of the different dissociation enzymes, concentrations of enzymes and amount of minced testis material examined in the three experiments involved in optimising the dissociation of southern bluefin tuna (Thunnus maccoyii) testis material. Exp Dissociation Concentration of Amount of Incubation Percoll Reps Enzymes enzyme minced testis temperature gradient tissue (mg.ml-1) and time 1 Collagenase/ A)2 mg.mL-1 /600U 30mg 2h at 30oC No 4 dispase B)4mg.mL-1 /1200U C)10mg.mL-1 /6000U

2 Collagenase/ 4mg.mL-1 /1200U 30, 50, and 2h at 30oC No 4 dispase or 100mg trypsin 0.0583 mg.mL-1

3 Collagenase/ 2mg.mL-1 /1000U 100, 200, 300, 3h at 25oC Yes 0 dispase or 400 and 500mg trypsin 0.0583 mg.mL-1 *Exp = Experiment, Reps = Replicates

Once the corresponding dissociation media was added to the rinsed tissue, as described above, the tissue was incubated according to Table 1. Whilst incubating, the cell suspension was gently mixed by pipetting slowly up and down every 30 min for 30 sec. Following the incubation, the dissociation media and tissue were filtered at 100 and 40 µm (Steriflip filter, Merck Millipore, Germany), sequentially. The cell suspension was then slowly pipetted into a 15mL centrifuge tube containing a Percoll gradient (the Percoll gradient separates cell types so a cell suspension enriched in type-A spermatogonia could be obtained) (Figure 5). The Percoll gradient was prepared by diluting Percoll Plus (GE Health, 17-5445-01) with PBS to 20% and 30% concentrations. Two mL of the 30% Percoll was pipetted into the 15mL centrifuge tube before 1mL of the 20% Percoll was very slowly pipetted on top (Figure 5A). One mL of the cell suspension was then slowly pipetted onto the 20% Percoll layer and the Percoll gradient was then centrifuged at 800 g for 30 minutes at 4°C (Figure 5B). The Spermatogonia enriched fraction was then carefully pipetted from within the 20% Percoll/PBS layer before performing two washes with 3mL of L-15 media containing 1%

Penicillin/Streptomycin. The cells were counted using a haemocytometer under a compound microscope (Olympus BX51) before and after the Percoll gradient, to assess the effectiveness of the gradient for each treatment group.

Cell suspension (1ml) A

20% Percoll/PBS (1ml)

30% Percoll/PBS (2ml)

B L-15 media (1ml)

Spermatogonia 20% Percoll/PBS (1ml) fraction 30% Percoll/PBS (2ml)

Figure 5. Illustrative representation of the Percoll gradient process before (A) and after (B) centrifugation at 800g at 4oC for 30 minutes.

The cells in the resulting cell suspension from the Percoll gradient was then rinsed twice in L-15 media containing 1% Penicillin/Streptomycin before being counted for each treatment group using a haemocytometer under a compound microscope (Olympus BX51).

2.2.3 Assessment of the effect of size and reproductive maturity of SBT on testicular cell numbers and viability

Testis material was sampled every month from SBT during their commercial production season, as described in Section 2.2.1. Material was collected from fish ranging in size from 17 to 71 kg (n = 84) between June and October, 2012 and between April and May, 2013 (see Table 1, Appendix One). Water temperature during the sampling period was obtained from Australian Southern Bluefin Tuna Industry Association, which was logged every 15 min from a sea cage offshore of Port Lincoln, South Australia. Mean daily water temperature (oC) ranged between 13.4 and 20.0oC during 2012 sampling period, and 17.6 and 19.9oC during 2013 sampling period (See Figure 1, Appendix 1).

A cross section of testis material was sampled from the middle of one lobe from each fish and placed in Bouins fixative (Sigma, HT10131) and stored overnight at 4oC. The following day, the sampled testis tissue was washed thoroughly in 70% ethanol and subsequently stored in 70% ethanol at 4oC until required for histological analysis (see Section 2.2.4). The remaining testis material was then divided into 1g pieces and minced and washed as described in Section 2.2.3. The testis material was then incubated in 2mL of 40mg.mL-1 Collagenase H (Roche, 11074059001) and 33.3mg.mL-1 Dispase II (Roche, 04942078001) in L-15 media containing 5% foetal bovine serum (FBS) (Invitrogen, 10437-077) and 1% DNase 1 (Roche, 11284932001) for 3h at 25oC. During the enzymatic dissociation, gentle pipetting was applied to physically disperse any remaining intact portions of the testis. The resultant cell suspension was filtered through 150µm and 50µm (Partec Celltrics) to eliminate non dissociated cell clumps before being washed two more times in L-15 media containing 1% Penicillin/Streptomycin. The cell suspension was then suspended in 1mL of L-15 media containing 1% Penicillin/Streptomycin and slowly pipetted into a 15mL centrifuge tube containing a Percoll gradient. Once the Percoll gradient was performed, the donor cells were then resuspended in 3mL of L-15 media containing 1% Penicillin/Streptomycin, 1% FBS and 1% DNase 1 and stored at 4oC for up to 48h.

At 0 and 24h after dissociation, the viability of the cells was assessed using the Trypan blue exclusion method (Kobayashi et al. 2007). Specifically, the resultant cell suspension for each fish was immediately incubated in the presence of Trypan Blue dye (Sigma, T8154) for 2 minutes prior to counting the cells on a haemocytometer. Cell survival was assessed by examining the cells ability to exclude the Trypan blue dye. If the cell was able to exclude the blue dye from its cell membrane, it was classed as a viable cell. Alternatively, if a cell appeared to have a blue cell membrane, it was classed as non-viable.

2.2.4 Histological analysis

The testis material stored in 70% ethanol was embedded in a paraffin wax and sectioned (7µm thick) using a microtome. These sections were stained with Harris

25 haematoxylin and eosin stains, permanently mounted using DePex (BDH Chemicals), examined and digitally imaged using a compound microscope (100 to 400× magnifications, Olympus BX51) equipped with a digital camera (Olympus, Camedia C- 7070). The stage of reproductive maturity was then assigned to each sample classified on an ordinal scale of 1 to 5 according to the criteria previously described in Bubner et al. (2012) for SBT.

2.3 Cryopreservation of SBT testis material

The cryopreservation of SBT testis material was assessed and optimised in two separate experiments. The first experiment directly examined the suitability of a protocol for SBT testis material, with one developed by Professor Goro Yoshizaki’s research group from the Tokyo University of Marine Science and Technology, for the cryopreservation of PBT testis material. This was done by examining the number and viability of cells isolated from dissociated that was fresh or cryopreserved. The second experiment examined different variations of the protocol for the cryopreservation of SBT testis material (see Table 2) and their effect on cell number and viability. For both experiments, testis material was collected from SBT, as described in Section 2.2.1.

In the first experiment, the freshly collected testis material from one SBT (SBT 1, Table 2, Appendix One) was divided into 500mg pieces that were minced separately in a glass cavity slide using wecker scissors. The minced tissue was then transferred into 2mL cryogenic vials with 1 mL of cryopreservation media (1.5% bovine serum albumin (Sigma, A2153) and 100mM Trehalose Dihydrate (Sigma, T9531) in Leibovitz’s L-15 media, 9% of the cryoprotectant dimethyl sulfoxide (DMSO) (Sigma, D4540)). The vials were then placed into pre-chilled (4oC) Biocell bio freezing vessels (Nihon Freezer Co. Ltd, Japan) and transferred into -80oC for 90min before being plunged into liquid nitrogen. The remaining testis material was stored at 4°C overnight in PBS.

In the second experiment, the testis material of four SBT (SBT 2 – 5, Table 2, Appendix 1) was again divided into 500mg pieces in the laboratory and minced separately in a glass cavity slide using wecker scissors. The minced tissue was then transferred into 2mL cryogenic vials with 1 mL of cryopreservation media (1.5% bovine serum albumin

26 and 100mM Trehalose Dihydrate in Leibovitz’s L-15 media) containing two different amounts of the cryoprotectant DMSO (5 and 9%). The first treatment was plunged directly into liquid nitrogen without the use of a Biocell. The remaining vials were then placed into chilled Biocell bio freezing vessels that were either transferred into -20 or - 80oC for 90min before being plunged into liquid nitrogen. A summary of the different treatment groups is shown in Table 2.

Table 2. Summary of treatment groups examined in experiment two of the cryopreservation trials Treatment Temp when in Time in Biocell Biocell Plunged Concentration of o number Biocell ( C) (min) into N2 cryoprotectant (%) 1 - - No Yes 9 2 -80 90 Yes Yes 5 3 -20 90 Yes Yes 9 4 -80 90 Yes Yes 9

After overnight storage in liquid nitrogen, the testis material was thawed at 15oC for 3min, washed once with Leibovitz’s L-15 media containing 1% penicillin/streptomycin that was pre-chilled to 4oC, and subsequently dissociated using the optimised protocol described in Section 2.2.3. Cell numbers and cell viability (At 0, 12 and 44h for experiment one and 0h for experiment 2) of the resultant cell suspension was assessed for each treatment group, as described previously.

2.4 Transplantation of SBT testicular cells into YTK surrogate hosts

Five transplantation experiments took place at Cleanseas Tuna’s Hatchery in Arno Bay, South Australia between August 2012 and September 2013.

2.4.1 Testicular cell preparation

Testicular cells for transplantation were prepared from testis material collected from SBT as described in Section 2.2.1. Following collection, the testis material was divided into 1g pieces and minced with wecker scissors in a glass cavity slide before being dissociated using the protocol described in Section 2.2.3. Following dissociation, the number of viable cells per 1g of testis material was counted using 15 µL of the cell

27 suspension on a Neubauer counting chamber after Trypan Blue exclusion and extrapolating the results. For visualisation, the remaining cells were then stained with the fluorescent membrane dye PKH26 (Sigma, PKH26GL). Approximately 1 million cells were suspended in 400µL of diluent C (an iso-osmotic aqueous solution provided with the PKH26 dye) with 4µL of dye and incubated for 5min. The suspension was then centrifuged at 100g for 5min at 4oC. The supernatant was discarded and 3mL of L-15 media containing 1% Penicillin/Streptomycin was then added to the cells. The resultant cell suspension was then washed an additional two times, by centrifuging the cell suspension two more times, discarding the supernatant and re-suspending the cells in L-15 media containing 1% Penicillin/Streptomycin. The cells were stored on ice for up to 48 h prior to transplantation.

2.4.2 Germ cell transplantation

Transplantation needles were prepared by pulling thin walled glass capillaries (Narishige, G-100) using an electric puller (Narishige, PC-10). The tips of the needles were sharpened with an electric grinder (Narishige, EG-400) until the opening of the needle reached 40µm. Recipient larvae (6 to 11 DPH) were sourced from stocks reared in 280, 3000 or 8000L tanks. For transplantation, the larvae were anaesthetized with 0.0075% ethyl 3-aminobenzoate methanesulfonate salt (Sigma, A5040) in seawater, which contained 0.1% bovine serum albumin. They were transferred onto a petri dish coated with 3% agar using a wide bore 3mL plastic transfer pipette. Donor cells were then transplanted into the anaesthetised larvae with a micromanipulator (Narishige, M33-01R) and microinjector (Narishige, IM-9B) attached to a dissection microscope (Olympus, SZH10). After transplantation, recipient larvae were transferred from the petri dish to a recovery tank filled with seawater, before being transferred into a larval 100 or 280 L rearing tank. Control larvae (i.e. non-transplanted larvae) were also stocked at this point into separate larval rearing tanks.

2.4.3 Rearing of transplanted larvae

Different larval rearing systems were used in each transplantation experiment, where various modifications were made overtime to enhance larvae survival post- transplantation (see Table 3).

Table 3. Summary of larval rearing conditions, origin and transplantation age of larvae for transplantation trials that took place at Cleanseas Tuna’s yellowtail kingfish (Seriola lalandi) hatchery in Arno Bay, South Australia between August 2012 and September 2013 Trial Date Origin of larvae Age (DPH) when Larval Rearing System type transplanted Tank Size 1 Aug 2012 280L tanks 6 – 11 280L Flow through 2 Sept 2012 280L tanks 6 – 11 280L Flow through 3 Nov 2012 280L tanks 6 – 11 100L Re-circulation 4 June 2013 3000L R&D tanks 6 – 10 100L Re-circulation 5 Sept 2013 8000L commercial tanks 6 – 10 100L Flow through

Larval rearing tanks were either part of a flow-through system, as described in Section 2.1, or part of a recirculation system where the out-going water was collected back into a biofilter. Specifically, in the recirculation system, the biofilter recirculated between an 850L sump tank, a 350L tank that contained small plastic beads that promoted the growth of ammonia stripping bacteria, and a protein skimmer to rid the water of excess protein. The biofilter’s 850L sump tank was heated by two coil heaters (2.5kW, Hotco, Australia). The larval rearing tanks were supplied with water from the biofilter’s 850L sump tank that flowed through a 5 and 1µm sock filter as well as an ultra-violet light filter. The exchange rate in the 100L larval rearing tanks was 1000% daily water exchange throughout the trial. Aeration, light regime, water quality monitoring, tank cleaning and maintenance, water temperature control for the transplantation trials were the same as the stocking density trial in Section 2.1. Nanopaste and live feeds (rotifers and Artemia) were added to all larval rearing tanks in the transplantation trials as described in Section 2.1. From 18DPH, the larvae were fed two types of artificial dry food (200 - 300µm Start L and 300 - 400µm Wean S, INVE,

Belgium) until the end of the experimental periods. The number of surviving larvae was assessed at the conclusion of the experiments.

2.4.4 Assessment of SBT donor testicular cells in recipient YTK larvae

Migration and colonisation of the PKH26 labelled SBT donor testicular cells in the surrogate host YTK gonad was evaluated by observing the transplanted fish under a fluorescent microscope (BX50, Olympus) at 18 and 28 days post transplantation (DPT) (29 and 39 DPH). To facilitate examination of the gonad in the body cavity of the larvae, the head and digestive organs were removed. The gonad was initially observed in the body cavity before fixing the larvae using a rapid fixative (Ufix, Sakura, Japan) for 3 min, followed by 2 rinses in PBS to assist the removal of the gonad. Once the gonad was removed, it was placed on a Frontier glass microscope slide (FRC-01, Matsunami, Japan) with a coverslip and further evaluated by observing it under a fluorescent microscope. Digital images of the migration and colonisation of the SBT donor cells were recorded using a digital camera (UC50, Olympus) attached to the microscope (Olympus BX51) and examined using image software (AnalySIS, Version 5, Olympus).

2.5 Statistical analyses

Results are presented as mean values ± standard error. Statistical analyses were performed using SPSS statistics package volume 20. A one-way analysis of variances (ANOVA) was used to analyse significant differences between means (P < 0.05) when analysing survival in the larval rearing of YTK experiments. However, when comparing mean survival of YTK larvae fed different feeding regimes, a general linear model was used. The data were transformed in this section after the assumption of the ANOVA failed when using the Levene’s parametric test. The data were then transformed and analysed using arsine square root (P < 0.05). A two-way analysis of variance (ANOVA) was used to analyse significant differences between means (P < 0.05) in the optimising of the dissociation of SBT testis material and the cryopreservation of SBT testis material. All correlation analysis between cell numbers, viability, size and reproductive maturity of SBT was performed using a general linear model (P < 0.05). All percentage data were transformed and analysed using log transformation (P < 0.05).

3.0 Results

3.1 Small-scale larval rearing of YTK

No significant difference in YTK larvae survival at 30 DPH was evident when the larvae were stocked at 1, 5 and 60 larvae.L-1 (df = 2, F = 0.328, P = 0.743) (Figure 6). Larvae survival trend was the highest when rearing tanks were stocked at 5 larvae.L-1 (3.64 ± 2.15%), followed by tanks stocked at 1 larvae.L-1 (2.68 ± 1.96%) and 60 larvae.L-1 (1.7 ± 0.44%), respectively (Figure 6).

6 a

5 a

3 a

Survival (%) 2

0 1 51 60 -1 Stocking density (larvae.L )

When examining two different feed regimes of YTK larvae survival, the survival of larvae that were stocked at 1 larvae.L-1 was significantly higher when fed solely Artemia from 18 to 30 DPH (20.00 ± 6.25%), compared to larvae that were weaned onto artificial dry food during the same period (4.49 ± 1.30%) (P = 0.041) (Figure 7).

a 25

10 b Survival (%) 5

0 Artemia Dry Food 1

Feeding regime

3.2 Dissociation of SBT testis material

3.2.1 Optimisation of the dissociation protocol

When examining different concentrations of the dissociation enzyme in Experiment 1, there was no significant difference in the mean number of cells dissociated when using 2, 4 or 10 mg.mL-1 of collagenase/dispase (7.0 x 104 ± 2.55, 9.0 x 104 ± 0.84, 4.4 x 104 ± 1.21 cells.mL-1, respectively) (df = 2, F = 1.843, P = 0.200). In Experiment 2, the interaction between enzyme type and amount of testis material was not significantly different between the treatment groups examined (F (1, 35) = 0.15, P = 0.701) (Figure 8). There was no significant difference in the number of cells produced when examining enzyme type (F (1, 35) = 3.75, P = 0.061); however, there was a significant difference in the number of cells produced when examining the amount of testis material dissociated (F (2, 35) = 22.27, P < 0.001) (Figure 8). Specifically, a significantly higher number of cells were produced when 100mg.mL-1 of tissue was dissociated compared to 30 or 50mg.mL-1 of testis material (P < 0.001) (Figure 8).

30 b Collangenase/Dispase b 25

)

4 Trypsin 20 (x10 -1 15 a a a 10 a Cells.mL 5

0 30 50 100 -1 Tess material (mg.mL )

In Experiment 3, where the amount of tissue dissociated was examined, the number of cells dissociated also generally increased as the amount of tissue dissociated increased (Figure 9). The highest number of cells were produced when 500 mg.mL-1 of testis material was dissociated with collagenase/dispase prior to the Percoll gradient, followed by 300 and 400 mg.mL-1 (Figure 9A). Before the Percoll gradient was performed, cell numbers ranged between 21.3 and 928.0 cells.mL-1 (x104) for collagenase/dispase and 28.1 and 461.0 cells.mL-1 (x104) for Trypsin (Figure 9A). Post Percoll, cell numbers ranged between 10.0 and 88.0 cells.mL-1 (x104) for collagenase/dispase and 1.0 and 11.0 cells.mL-1 (x104) for Trypsin (Figure 9A). When comparing the number of cells between groups after the percoll gradient, the highest number of cells were also produced 500 mg.mL-1 of testis material was dissoiated with collagenase/dispase, folowed by 300 and 400 mg.mL-1 (Figure 9B). Statistical analysis of this data was not performed due to an insufficient number of replicate groups.

A 1000 Collagenase/Dispase (Pre-Percoll)

) 800 4 Trypsin (Pre-percoll)

(x10 600 -1

400

Cells.mL 200

0 100 200 300 400 500

B 100 Collagenase/Dispase 90 (Post-Percoll) 80

) Trypsin (Post- Percoll) 4 70 60 (x10

-1 50 40 30 20 Cells.mL 10 0 100 200 300 400 500 -1 Tess material (mg.mL )

Figure 9. Total number of testicular cells dissociated from different amounts of southern bluefin tuna (Thunnus maccoyii) testis material (100, 200, 300, 400, and 500mg.mL-1) with Collagenase/Dispase (2mg.mL-1) or Trypsin (0.0583mg.mL-1) before (A) and after (B) a Percoll gradient.

3.2.2 Assessment of the effect of size and reproductive maturity of SBT on testicular cell number and viability

The total cell number (cells.mL-1) generally increased with increasing SBT weight and fork length, with moderate positive correlation evident between the variables respectively (r = 0.34 and 0.29 respectively, n = 84, P < 0.01) (Figure 10A, B). Cell viability was quite variable across the range of SBT weight and fork length examined

(Figure 10C, D); however, moderate positive correlations were evident in both cases (r = 0.35 and 0.32 respectively, n = 84, P < 0.01) (Figure 10C, D).

) 4 A B 3000 (x10 -1 2000

1000 Cells.mL

0 100 C 80 D 60 40 20

Cell Viability (%) 0 0 20 40 60 80 80 100 120 140

Fish Weight (kg) Fork Length (cm)

Of the 62 SBT sampled for histological analysis, 26 were classified as stage 1 of reproductive maturation (Figure 11A, B), 28 as stage 2 (Figure 11C, D), seven as stage 3 (Figure 11E, F) and one was as stage 4 (Figure 11G, H).

A B

MSDè

SGè

çST SCè è C D

SCè MSDè SZè

STè E F

MSDè SZè

SCè

G H

SZè

MSDè SCè

Total cell numbers (cells.mL-1) obtained in the dissociation experiments generally increased with increasing gonadosomatic index (GSI) and stage of reproductive maturation, with a strong and moderate positive correlations evident between the variables, respectively (r = 0.68 and 0.32 respectively, n = 84, P < 0.01) (Figure 12A, B). A moderate positive correlation was also detected between cell viability and GSI (r = 0.26, n = 84, P < 0.01) (Figure 12C); however, there was no statistical correlation evident between cell viability and stage of reproductive maturation (P = 0.33) (Figure 12D).

3500

A B )

4 3000 2500 (x10 -1 2000 1500

Cells.mL 1000 500 0

100 C D 80 (%) 60 40 Viability

20 Cell 0 0.0 0.1 0.2 0.3 0.4 0 1 2 3 4 5 Gonadosomac Index Reproductive Classification

Cell numbers ranged between 1.8 x 104 cell.mL-1 in April 2013 to 3.27 x 106 cell.mL-1 in September 2012 (Figure 13). There was a significant difference in cell numbers dissociated between different months during the commercial SBT harvest season (df = 6, F = 3.641, P = 0.003). Specifically, testis material that was dissociated in September had significantly higher cell numbers than June, July and August in 2012 and April and May in 2013 (P = 0.010, 0.010, 0.011, 0.009, and 0.007, respectively) but not October, 2012 (P = 0.058) (Figure 13A). There was a significant difference in cell viability of the cells dissociated between different months during the commercial SBT harvest season (df = 6, F = 4.378, P = 0.001). Specifically, testis material that was dissociated in September had significantly lower cell viability than June, July and October in 2012 (P = 0.021, 0.002, and 0.013, respectively) but not October, 2012 and April and May, 2013 (P = 0.114, 0.883, and 0.846, respectively) (Figure 13B).

1000 A

)

4 800 b

(x10 600 -1 400

Cells.mL 200 a a a a a 0 100 a a

B a 90 ab ab ab 80

70 b 60 50 Cell Viability (%) 40 June ‘12 May ‘13 Oct ‘12 July ‘12 Sept ‘12 April ‘13 Aug ‘12

Time of sampling

3.3 Cryopreservation of SBT testis material

In the first experiment that directly examined the suitability of a cryopreservation protocol developed for PBT, the mean number of testicular cells dissociated from fresh tissue (98.94 x 104 ± 3.50 mL-1) was significantly higher compared to the number of cells dissociated from the cryopreserved tissue (11.63 x 104 ± 11.70 mL-1) (df = 1, F = 51.140 P < 0.001) (Figure 14A). The viability of the cells prepared from fresh and cryopreserved tissue decreased from 0 to 12 h and 12 to 44 h after dissociation. There

39 was a significant difference between fresh tissue and cryopreserved tissue at 0, 12 and 44 h (P = 0.002) (Figure 14B). However, the only significant difference detected was between cells prepared from fresh tissue at 0, 12 and 44 h and cryopreserved tissue at 44 h (P = 0.002, 0.011 and 0.013, respectively) (Figure 14B).

120 A b 100

) 4 80 (x10 -1 60

40 Cells.mL 20 a

0 B 100 0 hr 12 hr 44 hr b b 90 ab b 80 ab 70 60 a 50 40 30 Cell Viability (%) 20 10 0 Cryopreserved Fresh

SBT tess material storage type

In the second experiment that examined different concentrations of DMSO in the cryopreservation media and different cooling regimes, the number of cells dissociated from Treatment 3 (i.e. tissue cooled at -80oC for 90 min in 9% DMSO) (21.17x104 ± 9.63

40 mL.-1) was significantly higher compared to all other treatment groups (P = 0.030, 0.041 and 0.031 respectively) (Figure 15A). Cell viability showed a similar trend, with cell viability also significantly higher in Treatment group 3 compared to all other groups (P < 0.001, P = 0.001 and P < 0.001, respectively) (Figure 15B).

40 35 A b 30 25 20 15

Cells.mL-1 (x104) 10

5 a a 0 * 100 90 B b 80 70 60 50 40 a 30 Cell Viability (%) 20 10 * * 0 1 2 3 4 Treatment Group

3.4 Transplantation of SBT testicular cells into YTK surrogate host

Approximately 12,000 YTK larvae were transplanted with donor SBT testicular cells during five different transplantation experiments conducted in 2012 and 2013 (Table 4). Mean body weight and GSI of donor SBT across all five experiments (n = 14) was 57.0kg ± 2.9kg and 0.145 ± 0.013, respectively. Survival of the transplanted larvae ranged from 0% in Experiment 1 at 18 DPH to 9.2% in Experiment 4 at 64 DPH (Table 4). The presence of PKH26 labelled donor cells were observed in the developing gonad of transplanted YTK in Experiments 2, 4 and 5 (Table 4). Specifically, PKH labelled cells that displayed red fluorescence were observed in the developing gonad in the peritoneal cavity of the transplanted larvae (Figure 16) and in the developing gonad after removal from the peritoneal cavity (Figure 17). This suggests that transplanted donor testicular cells had successfully migrated and colonised in the recipient’s gonads. These cells were seen in the developing gonads of 50% of the transplanted larvae in Experiment 2 (n = 2), 33.3% (n = 12) and 37.5% (n = 8) of recipient larvae in Experiment 4 and 16.7% (n = 6) in Experiment 5 (Table 4).

Table 4. Summary of transplantation experiment where donor southern bluefin tuna (Thunnus maccoyii) cells were transplanted into recipient yellowtail kingfish (Seriola lalandi) larvae Exp. Number of Age (DPH) at Survival (%) Survival (%) Observation Successful transplanted transplantation of of control of donor cells colonisation larvae transplanted larvae in larvae and migration larvae of donor cells 1 845 7 -8 0 (18DPH) 0 n/a n/a 1041 8 – 9 0 (18DPH) 4.5 n/a n/a 805 9 – 10 0 (18DPH) 0 n/a n/a

2 1050 8 - 9 0.2 (30DPH) 0.06 Yes 50% 1093 8 - 9 0.1 (30DPH) 10 No 0 1030 8 - 9 0 (30DPH) 10 No n/a

3 1010 6 – 7 0 (18DPH) n/a n/a n/a 1210 9 – 10 0 (18DHP) n/a n/a n/a 150 11 0 (18DPH) n/a n/a n/a

4 680 6 – 7 9.1 (64DPH) 15.14 Yes 33.3% 1000 9 – 10 9.2 (64DPH) 14.60 Yes 37.5%

5 850 6 – 7 0 (30DPH) 1.65 n/a n/a 1321 9 – 10 0.9 (30DPH) 6.96 Yes 16% n/a = not applicable

A B

C D

A B

C D

4.0 Discussion

Germ cell transplantation could be a way to overcome current issues associated with the management of bluefin tuna broodstock (Takeuchi et al. 2009; Yazawa et al. 2010). It would allow smaller bodied, faster maturing surrogates, which are already a well- established aquaculture species, to produce bluefin tuna eggs and sperm. The current study examined the suitability of YTK as a potential surrogate host for the SBT. To address this question, the current study examined a number of aspects associated with development of surrogate technology, with a focus on small-scale larval rearing of YTK larvae, the optimisation of the dissociation and cryopreservation of SBT testis material. To further assist in forming a platform for germ cell transplantation technology for SBT, the current study transplanted SBT testicular cells into YTK larvae, and reared those larvae to an age appropriate to assess the migration and colonisation of the transplanted SBT testicular cells (Figure 4).

4.1 Small-scale larval rearing of YTK

In recent research, one of the options for suitable surrogates for germ cell transplantation technology have been larvae between 6 and 11 DPH (Lacerda et al. 2012). The reason for this is that surrogates are typically transplanted within a small window of opportunity, (typically between 6 and 11 DPH) when the endogenous cells are migrating within the recipient. These recipient fish lose their ability to guide donor germ cells towards their genital ridges after the endogenous germ cells finish migrating (Takeuchi et al. 2003). Survival of transplanted larvae is critical to be able to assess their suitability as a surrogate. However, mass mortalities of larvae can occur in rearing systems even when optimal abiotic factors are provided (i.e. light intensity, turbidity, temperature, photoperiod and salinity) (Moustakas et al. 2004; Carton 2005; Battaglene et al. 2006; Blood and Laurel 2011; Partridge et al. 2011). Therefore, the current study examined different larval stocking densities and feeding regimes of YTK in a small-scale larval rearing system, with the overall aim of optimising survival of

46 transplanted larvae in future experiments. The survival of the YTK larvae reared in Cleanseas Tuna’s commercial YTK hatchery is on average 10% to 30 DPH when stocked between 90 and 100 larvae.L-1 (Bennan Chen, personal communication). The survival of YTK larvae in the current study was lower in all three treatments of the stocking density experiment than the survival of YTK that are stocked at commercial stocking densities (Survival between 1.7 and 3.6 in the current study compared to an average of 10% survival between 90 and 100 larvae.L-1 at Cleanseas Tuna).

Stocking density in marine aquaculture is considered a very important factor, as it can have a direct negative impact on the growth rate and survival of fish (Baras et al. 2003; Zarski et al. 2011). High stocking densities have shown to alter fish behaviour, metabolism and immunology due to a heightened stress response in the animals under these conditions (Bolasina et al. 2006). The current study examined the effect of three different stocking densities (1, 5 and 60 larvae.L-1) on larvae survival; however, there was no statistical difference between the three groups (Figure 6). This suggests that survival was not significantly effected at these densities (1, 5 and 60 larvae.L-1) and therefore may not be a limiting factor when trying to optimise survival of YTK larvae following germ cell transplantation. The study by Kupren et al. (2011) also found no significant effects of stocking density of Ide (Leuciscus idus) and Chub (Leuciscus cephalus) larvae mortality when examining eight density variants from 50 to 400 larvae.L-1 (at the interval of 50 larvae.L-1). It must be acknowledged that due to constraints on the availability of identical tanks at the Cleanseas hatchery in Arno Bay, only two replicate tanks were able to used for this experiment, thus, limiting the strength of the statistical analysis.

Another aspect which can impact on larval survival is the feed regime. There has been great interest in reducing the dependence of larval fishes on live feed in aquaculture as it is very costly due to the time and labour involved in their production (Gordon and Hecht 2002; Chen et al. 2006; Chen et al. 2007; Soligo et al. 2011). However, inadequate weaning from live feeds to an artificial diet can have a significant impact on larvae survival and growth (Chen et al. 2006; Soligo et al. 2011). Soligo et al. (2011)

47 found that the specific growth rate of the common snook (Centropomus undecimalis) was significantly higher in larvae that were weaned from Artemia to a commercial artificial dry food diet compared to larvae fed a continuous Artemia diet. However, survival of the larvae was not significantly different between the two treatments. The feed regime experiment examined in this study, found that survival of YTK larvae fed the Artemia diet only had significantly higher survival (20.0%) than larvae that were weaned onto the artificial diet between (4.5%) (Figure 7). The results therefore suggest that providing transplanted YTK larvae with live feed as a food source rather than weaning them onto artificial dry feed may help transplanted larvae survive to an age where the migration and colonisation of transplanted germ cells can be assessed. However, acceptable growth rates of marine fish larvae cannot be sustained solely by using live feed (Soligo et al. 2011). Therefore, to be able to rear transplanted larvae to maturity, they would need to be weaned when the larvae outgrow live feed as a sustainable food source.

The study highlighted the difficulties of rearing larvae on a small-scale, with high variability in larvae survival in both density and feed regime experiments. Low swim bladder inflation was most likely a major contributor to the low survival observed in the experiments conducted here as the larvae were reared from 1DPH in the small- scale system, where achieving high rates of swim bladder inflation is quite difficult (Bennan Chen, personal communication). Successful swim bladder inflation is essential for functional buoyancy control and swimming ability, which can have a direct effect on the successful transition between endogenous feeding (i.e. egg sac) to exogenous feeding (i.e. live feed) that is critical to larvae survival (Martin-Robichaud and Peterson 1998; Carton 2005). Low swim bladder inflation could have been effected by the difficulty in obtaining an ideal ratio between water volume, tank wall and floor area in the small-scale larval rearing system, which effects the surface tension and that the larvae need to break to inflate their swim bladder. In addition, the formation of surface oil films when using live feeds is difficult to effectively remove in small-scale systems (Hart et al. 1994) and have also been attributed as the cause of low rates of initial swim bladder inflation in many marine fish species (Trotter et al. 2003 (b)), which can

48 reduce survival and growth of marine fish larvae (Chatain and Ounais-Guschemann 1990; Battaglene et al. 1994). Cleanseas Tuna successfully achieve swim bladder rates above 95% in their commercial production of YTK, and therefore, for the later implantation trials we have sourced the larvae from their commercial operation and strongly recommend doing so if possible for future germ cell transplantation work.

The study by Duray et al. (1997) also highlighted the difficulties in rearing larvae on a small-scale when they reported significantly lower survival of grouper larvae in an experimental system (7.4% survival in 500L tanks) compared to a larger, commercial sized rearing system (19.8% survival in 3000L tanks). Survival in this study was lower than that typically observed in commercially reared YTK. However, large-scale rearing, as in commercial rearing, of finfish larvae is sometimes not an option in some forms of research, such as germ cell transplantations due to the low numbers of experimental fish. Therefore, it was important for the current study to investigate the rearing conditions of YTK in a small-scale system as experimental fish numbers in germ cell transplantation trials will be determined by the amount of transplantations that are performed. This is due to the transplantations being a highly technical and time consuming task.

4.2 Dissociation of SBT testis material

4.2.1 Optimisation of the dissociation protocol

The next step was to optimise a protocol to dissociate SBT testis material to then produce a cell suspension rich in type-A spermatogonia that was viable for transplantation. To optimise the dissociation protocol of SBT testis material for transplantation, different dissociation enzymes, concentrations of enzymes and the proportion of minced testis material to dissociation media were examined. Although there is limited literature on the optimisation of the dissociation process itself, previous studies have reported the use of either combined enzymes collagenase/dispase or trypsin for the dissociation of testis material (Takeuchi et al.

2009; Morita et al. 2012). The use of trypsin as the dissociation enzyme produced 9.4 x104 type-A spermatogonia cells when dissociating 1mg.mL-1 of testis material from 3 month old nibe croaker (Takeuchi et al. 2009). When 100mg.mL-1 of testis tissue from 10 month old Japanese yellowtail was dissociated using the combined dissociation enzyme collagenase/dispase, approximately 106 testicular cells were produced.

The results from the experiments conducted in the current study found no significant difference in cell numbers when examining the two dissociation enzymes collagenase/dispase and trypsin when dissociating 30, 50 and 100mg.mL-1 of SBT testis tissue. However, there was a significant difference in cell numbers when larger amounts of tissue were dissociated. There was also no significant difference in cell numbers between the three different working concentrations (2, 4 and 10 mg.mL-1) of the enzyme collagenase/dispase when dissociating 30mg of testis material. Significantly higher number of testicular cells were however produced when 100mg.mL-1 of tissue was dissociated with either trypsin or collagenase/dispase compared to 30 and 50 mg.mL-1. The fact that larger amounts of dissociated testis tissue produced higher amounts of cells was further supported when cell numbers dramatically increased as the amount of testis material increased. Statistical analysis of this data was not performed due to lack of replication; however, the results clearly indicate a trend of increasing cell numbers with increasing tissue amount. The results of this experiment also showed that the dissociation enzyme collagenase/dispase was a more effective enzyme than trypsin before and after the Percoll gradient was applied when dissociating larger amounts of testis tissue. These results differ to those when using a smaller amount of tissue, where no significant difference was found between the two dissociation enzymes. This may have been related to the larger amount of tissue being dissociated between the two experiments and that the dissociation enzyme collagenase/dispase may be more effective with large amounts of testis tissue compared to trypsin. The results also showed that cell numbers dropped dramatically after the Percoll gradient was applied in each treatment, which was expected as the Percoll gradient acts as a cell separation technique by using density gradient centrifugation and removes cells considered not typical of type-A spermatogonia size.

In summary, the results established a protocol that enhanced the number and viability (i.e. up to 1 million cells.mL-1) of testicular cells dissociated from SBT testis material, available for transplantations into potential surrogate hosts. This method enabled us to transplant a maximum of 1321 recipient larvae over two days using 10.5x106 cells. Generally, time was the limiting factor in regards to the number of transplantations that could be carried out rather than the amount of cells available. Previous germ cell transplantation research has stated that a 15nL injection, transplants around 20,000 testicular cells into the recipient larvae (Morita et al. 2012).

4.2.2 Assessment of the effect of size and reproductive maturity of SBT on testis cell numbers and viability

The SBT commercial harvest season typically runs between April and October, which is the timeframe that fresh SBT testis tissue is available to sample. The next stage of the current study examined the quantity and quality of testicular cells dissociated from different sizes and stages of reproductive maturity of SBT that were sampled every month of the commercial harvest season. The aim of this was to assist in forming a protocol that would consistently be able to produce a cell suspension rich in type-A spermatogonia suitable for germ cell transplantation.

The results showed SBT weight and fork length ranged from 16.7 and 71.0 kg and 92 and 145 cm, respectively. Both SBT weight and fork length had a positive correlation with increasing cell numbers and viability which ranged from 1.8 and 3268.0 cells.mL-1 (x104) and 19.6 and 98 % viability (Figure 10). One of the reasons for this could is that a larger testis breaks down and dissociates easier than a smaller testis. The difference in consistency between small and large testis was also clearly noticeable when mincing the testis material prior to dissociating, with larger testes proving to be much easier to mince with the wecker scissors than the smaller testes.

In addition, increasing GSI and stage of reproductive maturation also had a positive correlation with increasing cell number (Figure 12). GSI was also positively correlated with cell viability. Ideally, these factors, along with fish weight and fork length should be targeted when sampling SBT testis material to obtain testicular cells for

51 transplantation. The reality of sampling at sea with time constraints and harsh weather conditions, assessing the GSI and using gross observations to assess approximate stage of reproductive maturation could prove to be difficult, but possible provided there were enough resources (i.e. adequate equipment, enough personnel). Previous studies on the stage of reproductive maturity of production SBT revealed that 92% of fish that were sampled were immature (either stage 1 or stage 2 of reproductive maturity), and therefore, it is rare to find a mature testes during the limited commercial harvest period (Bubner et al. 2012). The sampling of SBT testis material at sea could be eased by solely targeting large fish, which would be amiable downstream for germ cell transplantation technology.

The investigation into sampling SBT at different times of the commercial harvest found that SBT testis material sampled in September produced significantly higher amount of testicular cells than the majority of the other months (Figure 13A). However, SBT testis material sampled in September also produced significantly lower viability than June, July, and October in the same year (Figure 13B). However, SBT were only sampled over one set of months during an SBT commercial harvest season as thus, there was insufficient amount of replications in sampling SBT testis material to confirm this result. The significantly lower viability of the cells during September suggests there might have been an artefact associated with the processing of the cells, rather than a significant difference between the months. Further sampling of SBT testis material should take place over future commercial harvest seasons to clarify the preferred time of year to sample and dissociate SBT testis material.

4.3 Cryopreservation of SBT testis material

As stated previously, SBT testis material is only available during the commercial harvest season, which means that its availability is limited throughout the year. Cryopreservation technology could assist in supplying a readily available source of SBT testis material for germ cell transplantations year-round. The results of the present study showed that SBT testis material can be successfully cryopreserved, thawed and dissociated to produce viable testicular cells for transplantation. The first experiment

52 assessed the suitability of a cryopreservation protocol developed for PBT. The results showed that cryopreserved SBT testis material did produce viable testicular cells, however, the number of cells was lower than that of fresh SBT testis tissue (i.e. tissue that was not cryopreserved) (Figure 14). This may be due to two main issues that arise for cells cooled below freezing temperature: 1) water crystallizes form ice, which in turn, raise salt concentrations and can rupture cells, 2) the toxicity of the cryoprotective agent after prolonged exposure can be toxic to the cell and cause irreversible membrane damage (Watson 2000; Mocé and Vicente 2009; Gosden 2011). When the viability of cells was assessed overtime using the Trypan blue exclusion method, the viability of the testicular cells dissociated from the cryopreserved tissue did not significantly decrease up to 44 h post-thawing and dissociation; however, the viability of these cells was significantly lower at this time compared to the viability of the cells dissociated from the fresh tissue at 0, 12 and 44 h post-thawing. This suggests that testicular cells dissociated from cryopreserved SBT testis material degrades faster compared to testicular cells that are dissociated from fresh SBT testis material. Among the various factors that affect the success of a cryopreservation method, is the balance between the bio-toxicity of the cryoprotectant and the protection afforded during the freezing and thawing steps (Fabbrocini et al. 2000). The results shown in this study suggest that fresh SBT testis material would be preferred for transplantation trials because significantly more cell numbers can be dissociated from fresh testis tissue compared to cryopreserved testis tissue. However, in the instance where fresh SBT testis material is not available, cryopreserved testis material may be an adequate alternative. The study by Lee et al. (2013) found that thawed type-A spermatogonia derived from cryopreserved rainbow trout testis material did not lose their transplantability after 939 days of cryopreservation. Functional eggs and sperm were successfully derived from frozen testicular germ cells through the transplantation of those germ cells into sterile triploid rainbow trout hatchlings. This is an encouraging finding that gives confidence about future transplantation of SBT testicular cells that are cryopreserved using the current cryopreservation protocol. The viable SBT testicular cells produced from cryopreserved tissue indicates that the cryoprotective

53 agent DMSO was effective in protecting the cells against damage whilst freezing. Further improvements to the cryopreservation protocol may assist in obtaining a larger amount of viable SBT testicular cells and would be worthy of investigation in the near future. This would reduce the time required to prepare the cells for transplantation as it eliminates the reliance on fresh tissue collected at sea as the cryopreserved tissue would be readily available. Cryopreservation could also assist in allowing year-round access to material to obtain testicular cells for transplantation as SBT testis material is only available throughout the SBT commercial harvest season (April until October), especially while the commercial rearing of YTK allows for the availability of YTK larvae almost year-round. Cryopreservation of testicular tissue would also remove the pressure of having to coordinate the availability of donor tissue with the presence of suitable host larvae, and allow the use of this technology to be applied in other geographical locations such as the University of the Sunshine Coast, as cryopreserved material can be easily transported.

The second of the cryopreservation experiments aimed at optimising the established cryopreservation protocol by; slowing the cooling rate to eliminate the formation of ice crystals, reduce the toxicity of the cryoprotectant, and plunging the SBT testis material straight into liquid nitrogen. The results show that the original protocol developed by Professor Goro Yoshizaki’s research group yielded significantly higher cell numbers and viability than all other treatments (Figure 15). This protocol successfully produced viable SBT testicular cells post-thawing, by storing SBT tissue at -80oC for 90 min before transferred into liquid nitrogen, and using a cryopreservation media containing 9% DMSO. Plunging the testis tissue straight into liquid nitrogen rather than into -80oC in a Biocell to control the cooling rate yielded zero cells even though the cryopreservation media containing 9% DMSO was used. This is most likely due to the formation of ice crystals in the cell membrane which can rupture and cause serious damage to the cells (Woods et al. 2004; Mocé and Vicente 2009; Gosden 2011). This indicates that controlling the cooling rate has a decisive role in determining the survival of cryopreserved and thawed cells.

Cooling the testis material at a slower rate by using a -20oC freezer instead of a -80oC freezer did produce a small amount of testicular cells. However, the amount of cells was significantly lower than the original protocol, which could be due to the slower cooling rate allowing the toxicity of the DMSO to damage the cells (Woods et al. 2004). DMSO is widely used in for the cryopreservation techniques and the concentration of this cryoprotectant varies among species type and the type of tissue or cells being cryopreserved. The current study aimed at decreasing the risk of the toxicity of DMSO on the cells whilst freezing, by reducing the concentration of the cryoprotectant in the cryopreservation media. Previous studies on the cryopreservation of sperm found that sperm quality after freezing-thawing improved when the concentration of cryoprotective agents was lowered from 8 to 4% (Mocé and Vicente 2009). However, this was not the case in the present study: when a lower concentration of DMSO was used here (from 9% to 5%), as it may not have offered enough protection against intracellular ice formation during freezing. Although DMSO at higher concentrations are potentially toxic to cells, previous studies have shown that DMSO concentrations between 12.0 and 17.5% have been used successfully to cryopreserve sperm (Mocé and Vicente 2009). Future studies to further optimise the cryopreservation of SBT testis material could examine higher DMSO concentrations. Although the results of the current study showed that SBT testis material can be successfully cryopreserved and thawed to produce viable testicular cells, such cells have not yet confirmed to successfully migrate and colonise in transplanted surrogate hosts, which would need to be confirmed to establish the true success of the cryopreservation technique.

4.4 Transplantation of SBT testicular cells into YTK surrogate hosts

The previous components of this study (small-scale larval rearing of YTK, dissociation and cryopreservation of SBT testis material) lead into the key segment, which investigated the suitability of YTK as a surrogate broodstock for SBT. During 5 transplantation experiments (between 2012 and 2013), over 12,000 YTK larvae were transplanted with SBT testicular cells. The current study has confirmed that intraperitoneally transplanted testicular cells from SBT (Scombridae) can be

55 incorporated into the developing gonads of recipient YTK larvae (Carangidae). Specifically, PKH26-labelled SBT donor testicular cells that were transplanted into the peritoneal cavity of YTK recipients between 6 and 11 DPH were observed to be incorporated into the genital ridges of YTK recipient larvae when examined at 21 and 28 DPT. The findings confirm the ability of the transplanted SBT testicular cells to migrate towards the genital ridge of the YTK recipient was not suppressed by the xenogenic environment. The colonisation rate was between 33.3 and 37.5%, which was low compared to other studies, where 70% colonisation was observed in chub mackerel (Scomber japonicus) recipients in the xenogenic environment and 92.6% in the Japanese Yellowtail (S. quinqueradiata) in the allogenic environment (Yazawa et al. 2010; Morita et al. 2012). However, the success reported in the current research represents a significant achievement and a big step forward in determining the suitability of YTK as a surrogate host for SBT.

The initial three experiments had very poor survival (<0.2%) of transplanted YTK larvae. In these three experiments, the control larvae had a survival rate that ranged from 0% and 10%. The fourth transplantation experiments achieved better survival by sourcing larvae from Cleanseas Tuna’s YTK production or R&D larval rearing, which successfully achieves swim bladder inflation above 95% (Bennan Chen, personal communication). The survival rates of this experiment achieved survival up to 9%. The poor survival rate of the transplanted and control larvae in the initial three experiments could have been attributed to a number of factors including: water temperature fluctuations, inadequate water flow and water dynamics, poor quality eggs or larvae, low swim bladder inflation, bacterial blooms in larval rearing tanks and the potential stress of the transplantation process on the larvae. However, poor swim bladder inflation in the YTK larvae is the most probable major contributor to the poor survival. Out of the five transplantation experiments, the two experiments that achieved the highest larvae survival were achieved when larvae were sourced from the industry partner Cleanseas Tuna Ltd (Experiments 4 and 5). These YTK larvae were considered superior quality compared to those reared by the research group in the other experiments (Experiment 1, 2, and 3), with swim bladder inflation in excess of 95% (Bennan Chen, personal

56 communication). This highlights the importance of sourcing good quality YTK larvae for such experiments. It was not possible to source YTK larvae from Cleanseas Tuna’s production run for the initial three experiments as the availability of SBT testis material and YTK larvae did not align. The optimal survival rate of the transplanted YTK larvae in this study were similar to previous germ cell transplantation research on the Japanese Yellowtail (S. quinqueradiata), where the survival of transplanted larvae was 8.0 and 10.9% for larvae of 4.8 and 5.2mm in length, respectively (Morita et al. 2012). The survival rate of the Japanese Yellowtail did increase with the size of the larvae, with survival rates increasing to 15.9 and 29.6% for larvae 5.6 and 6.0mm in length, respectively (Morita et al. 2012). Increasing the survival rate of transplanted YTK larvae are paramount if future research is to build on the findings of this study. Higher survival rate of transplanted recipients may be achieved by transplanting larvae at an older age (>11 DPH). However, they would need to be <15 DPH, as previous histological analysis of YTK gonads found that at 15 DPH endogenous PGC had settled at the genital ridge and had started to become enclosed by somatic cells. This development demonstrates that larvae at this age may no longer be suitable for transplantation as the primordial gonad had started to develop in the larvae (Bubner 2011). Although the study by Morita et al. (2012) found higher survival in the Japanese Yellowtail when the larvae were transplanted at a longer length (suggesting an older age), the incorporation rate of the donor cells into the recipient gonad decreased with the length of the transplanted larvae.

Antibiotics have been shown to result in a five-fold increase in survival with increased growth and less intestinal bacterial growth in the striped trumpeter larvae (Battaglene et al. 2006). While not recommended as a general strategy for commercial larval rearing, antibiotics could make a difference in the survival of larvae in small scale rearing associated with transplantation work in YTK and other species.

While the survival of the transplanted larvae was low, the current study resulted in approximately 110 YTK surrogate broodstock. These fish are now over 7 months old. Molecular analysis on the YTK surrogates is currently being carried out by Mr Ido Bar

57 from the University of the Sunshine Coast. Preliminary results of Polymerase Chain Reaction (PCR) analysis found that gonads sampled from 5 month old transplanted YTK surrogates contained SBT DNA. This provides evidence that the transplanted SBT testicular cells have colonised within the YTK gonads. In-situ hybridisation analysis are currently taking place to provide visual evidence of the transplanted SBT testicular cells within the YTK gonad to determine if they have actually proliferated or only migrated and colonised. These fish are now being held by Cleanseas Tuna and in another 2 years’ time, when the YTK surrogates reach sexual maturity, we will be able to assess whether any SBT derived sperm or eggs are produced.

4.5 Future implications

To implement a traditional genetic breeding program for SBT, a 12 year generation time would be required to select for improved traits such as; improved growth rates, improved resistance to disease and parasites, and enhanced post-harvest product quality. Using YTK as a surrogate host for SBT would greatly decrease the generation time needed to select for desirable traits. The successful transplantation of testicular cells derived from SBT with desirable traits into YTK larvae, could well result in the production of SBT displaying those desirable traits within three years. This means that over the course of one generation of SBT under a traditional genetic breeding program (12 years), surrogate broodstock technology could achieve 2-3 generations in improvements in selected lines. From an economic and product quality perspective, germ cell transplantation technology holds great potential for aquaculture production.

As highlighted previously, germ cell transplantation technology has the potential to greatly reduce the generation time of specific aquaculture species. In the current study, if the transplanted YTK indeed carry SBT germ cells to sexual maturity, there is real potent to reduce the generation time of SBT from 12 to 3 years. This could further be reduced by the transplantation of donor testicular cells into adult YTK. This was reported by Lacerda (2012) who reported donor derived spermatozoa within 9 weeks transplantation into the adult Nile tilapia (Oreochromis niloticus). However, this

58 technique is still in the research stage, and has yet to achieve viable donor derived gametes.

Another approach to enhance the germ cell transplantation procedure is the sterilisation of the surrogate host species so that only offspring of the donor species are produced. Recent studies by Okutsu et al. (2007) investigated xenogenic transplantation of rainbow trout (Oncorhynchus mykiss) spermatogonia into sterile triploid masu salmon (Oncorhynchus masou) larvae. This study resulted in offspring populations consisting only of the donor trout that were generated through the fertilisation of eggs and sperm from surrogate salmon parents. The benefits of using a sterile surrogate host are producing solely donor-derived offspring which results in less time, money and energy being wasted in raising non-target offspring (Okutsu et al. 2007). The sterilisation of YTK surrogates could result in an increase in seed production of SBT, as gonad production would be solely generating donor-derived gametes (Okutsu et al. 2007). This could potentially be applied to future germ cell transplantation research in SBT and assist in closing the life-cycle for aquaculture. Currently the University of the Sunshine Coast Genecology research group is investigating the conditions for the generation of YTK triploids. If proliferation of SBT donor cells can be confirmed in the YTK surrogate host, future transplantations could be carried out into triploid YTK larvae.

4.6 Conclusion

This study has established that the key components that form a platform for germ cell transplantation technology for SBT can be achieved. The methods examined and outlined here established the dissociation and cryopreservation SBT testicular material which will enable year-round access to transplantable testicular cells for transplantation. In addition, the current study also demonstrated that the somatic microenvironment of the YTK larval gonads supported the survival of testicular cells derived from SBT. This highlights the potential for the application of this technology is potentially applicable between a donor species (SBT – Scombridae) and recipient species (YTK – Carangidae) that are of different families (Yazawa et al. 2010). In the

59 current study, it is still unknown if the migrated cells will proliferate and form viable gametes. However, it is clear that this research constitutes a major breakthrough that could assist in closing the life-cycle of SBT for aquaculture and could potentially replace the need to maintain large, late maturing bluefin tuna broodstock in captivity, which is a major challenge for the sustainable aquaculture of SBT. In this way, the study has made a significant contribution to the challenge of achieving the sustainable aquaculture of SBT.

5.0 References

Baras, E., P. Kestemont, et al. (2003). "Effect of stocking density on the dynamics of cannibalism in sibling larvae of Perca fluviatilis under controlled conditions." Aquaculture 219(1-4): 241-255. Basavaraja, N. and S. N. Hegde (2004). "Cryopreservation of the endangered mahseer (Tor khudree) spermatozoa: I. Effect of extender composition, cryoprotectants, dilution ratio, and storage period on post-thaw viability." Cryobiology 49(2): 149-156. Battaglene, S. C., S. McBride, et al. (1994). "Swim bladder inflation in larvae of cultured sand whiting, Sillago ciliata Cuvier (Sillaginidae)." Aquaculture 128(1–2): 177-192. Battaglene, S. C., D. T. Morehead, et al. (2006). "Combined effects of feeding enriched rotifers and antibiotic addition on performance of striped trumpeter (Latris lineata) larvae." Aquaculture 251(2–4): 456-471. Benson, J. D., E. J. Woods, et al. (2012). "The cryobiology of spermatozoa." Theriogenology 78(8): 1682-1699. Blood, D. M. and B. J. Laurel (2011). "The effects of temperature on hatching and survival of northern rock sole larvae (Lepidopsetto polyxystra)." Fishery Bulletin 109(3): 282 - 291. Bolasina, S., M. Tagawa, et al. (2006). "Effect of stocking density on growth, digestive enzyme activity and cortisol level in larvae and juveniles of Japanese flounder, Paralichthys olivaceus." Aquaculture 259(1–4): 432-443. Boustany, A. (2011). Bluefin Tuna: The state of the science. Ocean Science Division. Washington, DC., PEW Environmental Group. Bubner, E. (2011). Towards the captive breeding of the southern bluefin tuna (Thunnus maccoyii), Flinders University, Adelaide, Australia. PhD. Bubner, E., J. Farley, et al. (2012). "Assessment of reproductive maturation of southern bluefin tuna (Thunnus maccoyii) in captivity." Aquaculture 364–365(0): 82-95. Carton, A. G. (2005). "The impact of light intensity and algal-induced turbidity on first-feeding Seriola lalandi larvae." Aquaculture Research 36(16): 1588-1594. Chatain, B. and N. Ounais-Guschemann (1990). "Improved rate of initial swim bladder inflation in intensively reared Sparus auratus." Aquaculture 84(3–4): 345-353. Chen, B. N., J. G. Qin, et al. (2007). "Deleterious effects of food restrictions in yellowtail kingfish Seriola lalandi during early development." Aquaculture 271: 326-335. Chen, B. N., J. G. Qin, et al. (2006). "Ontogenetic development of the digestive system in yellowtail kingfish Seriola lalandi larvae." Aquaculture 256: 489-501. Conceição, L. E. C., M. Yúfera, et al. (2010). "Live feeds for early stages of fish rearing." Aquaculture Research 41(5): 613-640. Doroshev, S. I., J. W. Cornacchia, et al. (1981). "Initial swim bladder inflation in the larvae of physoclistous fishes and its importance for larval culture." Rapports et Proces-verbaux des Réunions. Conseil International pour l'Éxploration de la Mer 178(495 - 500). Duray, M. N., C. B. Estudillo, et al. (1997). "Larval rearing of the grouper Epinephelus suillus under laboratory conditions." Aquaculture 150(1–2): 63-76.

Fabbrocini, A., S. Lubrano Lavadera, et al. (2000). "Cryopreservation of seabream (Sparus aurata) spermatozoa." Cryobiology 40: 46-53. Farley, J. H. and T. L. O. Davis (1998). "Reproductive dynamics of southern bluefin tuna, Thunnus maccoyii." Fishery Bulletin 96: 223-236. Farwell, C. J. (2001). Tunas in captivity. Tuna: physiology, ecology and evolution. B. A. Block and E. D. Stevens. San Diego, California, Academic Press. 19: 391-412. Findlay, J. (2006). Southern Bluefin Tuna Fishery. Fishery Status Reports 2005. K. McLoughlin, Australian Bureau of Agriculture and Resource Economics and Sciences: 85 - 94. Gordon, A. K. and T. Hecht (2002). "Histological studies on the development of the digestive system of the clownfish Amphiprion percula and the time of weaning." Journal of Applied Ichthyology 18(2): 113-117. Gosden, R. (2011). "Cryopreservation: a cold look at technology for fertility preservation." Fertility and Sterility 96(2): 264-268. Gwo, H. H., T. S. Weng, et al. (2005). "Development of cryopreservation procedures for semen of Pacific bluefin tuna Thunnus orientalis." Aquaculture 249: 205-211. Hart, P. R., W. G. Hutchinson, et al. (1994). "An experimental system for small-scale experiments with marine fish and crustaceans." Aquacultural Engineering 13(4): 251-256. Hatziathanasiou, A., M. Paspatis, et al. (2002). "Survival, growth and feeding in early life stages of European sea bass (Dicentrarchus labrax) intensively cultured under different stocking densities." Aquaculture 205(1–2): 89-102. Herpin, A., P. Fischer, et al. (2008). "Sequential SDF1a and b-induced mobility guides Medaka PGC migration." Developmental Biology 320: 319-327. Hung, L. T., N. A. Tuan, et al. (2002). "Larval rearing of the Asian Catfish, Pangasius bocourti (Siluroidei, Pangasiidae): alternative feeds and weaning time." Aquaculture 212(1–4): 115-127. Jensen, N. R., M. D. Zuccarelli, et al. (2008). "Cryopreservation and methanol effects on burbot sperm motility and egg fertilization." North American Journal of Aquaculture 70: 38-42. Kobayashi, T., Y. Takeuchi, et al. (2007). "Generation of viable fish from cryopreserved primordial germ cells." Molecular Reproduction and Development 74: 207-213. Kobayashi, T., Y. Takeuchi, et al. (2003). "Cryopreservation of trout primordial germ cells." Fish Physiology and Biochemistry 28: 479-480. Kupren, K., D. Zarski, et al. (2011). "Effect of stocking density on growth, survival and development of asp Aspius aspius (L.), ide Leuciscus idus (L.) and chub Leuciscus cephalus (L.) larvae during initial rearing under laboratory conditions." Italian Journal of Animal Science 10(3): 178-184. Lacerda, S., G. Costa, et al. (2012). "Germ cell transplantation as a potential biotechnological approach to fish reproduction." Fish Physiology and Biochemistry: 1-9. Lacerda, S. M. S. N., S. R. Batlouni, et al. (2008). "Germ cell transplantation in tilapia (Oreochromis niloticus)." Cybium 32(2): 115-118. Lee, S., Y. Iwasaki, et al. (2013). "Generation of functional eggs and sperm from cryopreserved whole testes." Proceedings of the National Academy of Sciences.

Leibo, S. P. and T. B. Pool (2011). "The principal variables of cryopreservation: solutions, temperatures, and rate changes." Fertility and Sterility 96(2): 269-276. Majhi, S. K., R. S. RHattori, et al. (2009). "Germ cell transplantation using sexually competent fish: an approach for rapid propagation of endangered and valuable germlines." PLoS ONE 4(7): 1-8. Martin-Robichaud, D. J. and R. H. Peterson (1998). "Effects of light intensity, tank colour and photoperiod on swimbladder inflation success in larval striped bass, Morone saxatilis (Walbaum)." Aquaculture Research 29(8): 539-547. Mazur, P. and R. H. Miller (1976). "Permeability of the human erythrocyte to glycerol in 1 and 2 M solutions at 0 or 20 °C." Cryobiology 13(5): 507-522. Mocé, E. and J. S. Vicente (2009). "Rabbit sperm cryopreservation: A review." Animal Reproduction Science 110(1–2): 1-24. Mori, M., T. Katsukawa, et al. (2001). "Recovery plan for an exploited species, southern bluefin tuna." Population Ecology 43(2): 125-132. Morita, T., N. Kumakura, et al. (2012). "Production of donor-derived offspring by allogeneic transplantation of spermatogonia in the yellowtail (Seriola quinqueradiata)." Biology of Reproduction 86(6): 1 - 11. Moustakas, C. T., W. O. Watanabe, et al. (2004). "Combined effects of photoperiod and salinity on growth, survival, and osmoregulatory ability of larval southern flounder Paralichthys lethostigma." Aquaculture 229(1–4): 159-179. Mylonas, C. C., C. Bridges, et al. (2007). "Preparation and administration of gonadotropin- releasing hormone agonist (GnRHa) implants for the artificial control of reproduction maturation in captive-reared Atlantic bluefin tuna (Thunnus thynnus thynnus)." Reviews in Fisheries Science 15: 183-210. Nobrega, R. H., S. R. Batlouni, et al. (2009). "An overview of functional and stereological evaluation of spermatogenesis and germ cell transplantation in fish." Fish Physiology and Biochemistry 35: 197-206. Nocillado, J. N., B. Levavi-Sivan, et al. (2007). "Temporal expression of G-protein-coupled receptor 54 (GPR54), gonadotropin-releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus." General and Comparative Endocrinology 150: 278-287. Okutsu, T., S. Shikina, et al. (2007). "Production of trout offspring from triploid salmon parents." Science 317: 1517. Okutsu, T., K. Suzuki, et al. (2006 (a)). "Testicular germ cells can colonize sexually undifferentiated embryonic gonad and produce functional eggs in fish." Proceedings of the National Academy of the Sciences 103(8): 2725-2729. Okutsu, T., Y. Takeuchi, et al. (2008). "Spermatogonial transplantation in fish: production of trout offspring from salmon parents." Fisheries for Global Welfare and Environment 5th World Fisheries Congress: 209-219. Okutsu, T., A. Yano, et al. (2006 (b)). "Manipulation of fish germ cell: visualization, cryopreservation and transplantation." Journal of Reproduction and Development 52(6): 685- 693.

Ottolenghi, F. (2008). Capture-based aquaculture of bluefin tuna. Capture-based aquaculture. Global overview; FAO Fisheries Technical Paper, no. 508. A. Lovatelli and P. F. Holthus. Rome, Italy, FAO: 169-182. Partridge, G. J., D. D. Benetti, et al. (2011). "The effect of a 24-hour photoperiod on the survival, growth and swim bladder inflation of pre-flexion yellowfin tuna (Thunnus albacares) larvae." Aquaculture 318(3–4): 471-474. Patterson, H., G. Begg, et al. (2010). Southern Bluefin Tuna Fishery. Fishery Status Report 2010. J. Woodhams, I. Stobutzki, S. Vieira, C. R. and G. Begg, Australian Bureau of Agriculture and Resource Economics and Sciences: 359 - 367. Patterson, T. A., K. Evans, et al. (2008). "Movement and behaviour of large southern bluefin tuna (Thunnus maccoyii) in the Australian region determined using pop-up satellite archival tags." Fisheries Oceanography 17(5): 352-367. Routray, P., A. K. Choudhary, et al. (2006). "Cryopreservation of dead fish spermatozoa several hours after death of Indian major carp, Labeo rohita and its successful utilization in fish production." Aquaculture 261: 1204-1211. Saragusty, J., R. Hermes, et al. (2011). "Mammalian reproduction out of cryopreserved cells and tissues: current state of the art and future options." International Zoo Yearbook 45(1): 133-153. Sawada, Y., T. Okada, et al. (2005). "Completion of the Pacific bluefin tuna Thunnus orientalis (Temminck et Schlegel) life cycle." Aquaculture Research 36: 413-421. Schulz, R. W., L. R. de Franca, et al. (2009). "Spermatogenesis in fish." General and Comparative Endocrinology doi: 10.1016/j.ygcen.2009.02.013. Soligo, T. A., A. S. Garcia, et al. (2011). "Weaning of the common snook (Centropomus undecimalis); early juveniles rearing laboratory using commercial and experimental diets." Boletim Do Instituto De Pesca 37(4): 367-374. Starz-Gaiano, M. and R. Lehmann (2001). "Moving towards the next generation." Mechanisms of Development 105(1–2): 5-18. Suquet, M., C. Dreanno, et al. (2000). "Cryopreservation of sperm in marine fish." Aquaculture Research 31: 231-243. Takeuchi, Y., K. Higuchi, et al. (2009). "Development of spermatogonial cell transplantation in Nibe croaker, Nibea mitsukurii (Perciformes, Sciaenidae)." Biology of Reproduction 81: 1055- 1063. Takeuchi, Y., G. Yoshizaki, et al. (2002). "Mass isolation of primordial germ cells from transgenic rainbow trout carrying the green fluorescent protein gene driven by the vasa gene promoter." Biology of Reproduction 67: 1087-1092. Takeuchi, Y., G. Yoshizaki, et al. (2003). "Generation of live fry from intraperitoneally transplanted primordial germ cells in rainbow trout." Biology of Reproduction 69: 1142-1149. Takeuchi, Y., G. Yoshizaki, et al. (2004). "Surrogate broodstock produces salmonids." Nature 430: 629-630. Thomson, M., M. Deichmann, et al. (2010). Recent developments in southern bluefin tuna larval and juvenile rearing. Towards the Sustainable Aquaculture of Bluefin Tuna. S. Miyashita, K. Takii, W. Sakamoto and A. Biswas. Kinki University, Japan.

Trotter, A. J., S. C. Battaglene, et al. (2003 (b)). "Effects of photoperiod and light intensity on initial swim bladder inflation, growth and post-inflation viability in cultured striped trumpeter (Latris lineata) larvae." Aquaculture 224(1–4): 141-158. Trotter, A. J., P. M. Pankhurst, et al. (2001). "Swim bladder malformation in hatchery-reared striped trumpeter Latris lineata (Latridae)." Aquaculture 198(1–2): 41-54. Trotter, A. J., P. M. Pankhurst, et al. (2003 (a)). "Effects of temperature on initial swim bladder inflation and related development in cultured striped trumpeter (Latris lineata) larvae." Aquaculture 221(1–4): 141-156. Watson, P. F. (2000). "The causes of reduced fertility with cryopreserved semen." Animal Reproduction Science 60–61(0): 481-492. Woods, E. J., J. D. Benson, et al. (2004). "Fundamental cryobiology of reproductive cells and tissues." Cryobiology 48(2): 146-156. Woolley, L. D., G. J. Partridge, et al. (2012). "Mortality reduction in yellowtail kingfish (Seriola lalandi) larval rearing by optimising Artemia feeding regimes." Aquaculture 344 - 349: 161 - 167. Worm, B., E. B. Barbier, et al. (2006). "Impacts of biodiversity loss on ocean ecosystem services." Science 314(5800): 787-790. Yazawa, R., Y. Takeuchi, et al. (2010). "Chub mackerel gonads support colonization, survival, and proliferation of intraperitoneally transplanted xenogenic germ cells." Biology of Reproduction 82: 896-904. Yazawa, R., Y. Takeuchi, et al. (2013). "The Pacific bluefin tuna (Thunnus orientalis) dead end gene is suitable as a specific molecular marker of type A spermatogonia." Molecular Reproduction and Development: n/a-n/a. Yoshizaki, G., K. Fujinuma, et al. (2011). "Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources." Comparative Biochemistry and Physiology Part D: Genomics and Proteomics 6(1): 55-61. Zarski, D., K. Kupren, et al. (2011). "The effect of initial larval stocking density on growth and survival in common barbel Barbus barbus (L.)." Journal of Applied Ichthyology 27(5): 1155- 1158.

Appendix One

Table 1: Details of southern bluefin tuna (Thunnus maccoyii) sampled for maturation assessments during commercial harvests between June and October, 2012 and April and May, 2013. Fish Month Weight Length Gonad GSI Size Maturation Cell count Viability sampled (kg) (cm) weight Class class (cells.mL-1) (%) without fat (g) 1 June ‘12 59.6 142 40.63 0.068 L 3 67.5 92.4 2 June ‘12 33.7 119 18.84 0.056 M 2 42.0 90.8 3 June ‘12 43.7 131 23.47 0.054 L 15.0 89.4 4 June ‘12 40.6 128 20.67 0.051 M 2 35.0 92.3 5 June ‘12 36.0 117 14.72 0.041 M 2 48.0 92.5 6 June ‘12 26.3 108 4.02 0.015 S 10.0 76.7 7 June ‘12 25.4 105 6.30 0.025 S 1 6.0 89.0 8 June ‘12 29.7 112 12.53 0.042 M 1 13.0 85.4 9 June ‘12 42.1 127 25.53 0.061 L 26.0 89.7 10 June ‘12 20.9 100 5.70 0.027 S 2 15.0 94.9 11 June ‘12 23.9 103 7.16 0.030 S 1 8.0 79.3 12 June ‘12 55.1 145 52.55 0.095 L 4 108.0 88.4 13 July ‘12 25.0 103 4.89 0.020 S 8.0 87.2 14 July ‘12 41.8 127 22.12 0.053 L 70.0 95.1 15 July ‘12 31.7 117 14.38 0.045 M 2 27.3 93.9 16 July ‘12 46.5 128 26.47 0.057 L 2 100.0 95.7 17 July ‘12 22.1 104 3.38 0.015 S 1 9.8 90.0 18 July ‘12 22.6 104 4.02 0.018 S 1 10.8 91.0 19 July ‘12 43.0 127 15.41 0.036 L 1 41.8 94.9 20 July ‘12 31.7 113 9.06 0.029 M 1 22.8 91.9 21 July ‘12 36.5 123 12.06 0.033 M 1 7.3 89.8 22 July ‘12 42.7 124 17.01 0.040 L 1 19.5 94.6 23 July ‘12 23.3 103 7.24 0.031 S 2 6.0 96.4 24 July ‘12 41.5 126 20.45 0.049 L 41.5 92.7 25 Aug ‘12 37.9 120 17.13 0.045 M 2 69.3 90.4 26 Aug ‘12 22.9 109 7.39 0.032 S 1 6.8 79.0 27 Aug ‘12 47.9 132 26.05 0.054 L 86.5 91.0 28 Aug ‘12 58.0 144 36.32 0.063 L 1 20.5 90.2 29 Aug ‘12 34.5 117 20.82 0.060 M 2 142.0 72.9

30 Aug ‘12 28.6 109 8.95 0.031 M 13.5 85.5 31 Aug ‘12 18.3 93 3.91 0.021 S 1 9.5 95.0 32 Aug ‘12 46.8 131 20.39 0.044 L 23.8 88.7 33 Aug ‘12 30.5 111 4.03 0.013 M 1 5.0 80.4 34 Aug ‘12 16.7 92 3.68 0.022 S 1 2.5 75.4 35 Aug ‘12 46.4 129 27.93 0.060 L 28.8 84.7 36 Aug ‘12 57.7 142 50.10 0.087 L 2 57.3 80.0 37 Sept. ‘12 19.5 100 5.10 0.026 S 1 249.3 48.9 38 Sept. ‘12 30.3 118 13.93 0.046 M 40.0 30.9 39 Sept. ‘12 35.0 116 12.00 0.034 M 1 46.3 19.6 40 Sept. ‘12 24.8 109 8.19 0.033 S 2 30.0 26.6 41 Sept. ‘12 26.5 108 9.45 0.036 S 71.0 64.3 42 Sept. ‘12 31.7 120 15.15 0.048 M 2 40.0 86.8 43 Sept. ‘12 38.9 122 28.83 0.074 M 2 195.8 92.5 44 Sept. ‘12 25.6 105 8.91 0.035 S 23.5 83.9 45 Sept. ‘12 65.1 139 78.53 0.121 L 2 3268.0 87.1 46 Sept. ‘12 71.0 145 79.66 0.112 L 2 2740.0 91.1 47 Sept. ‘12 67.2 144 71.60 0.107 L 2 364.0 90.8 48 Sept. ‘12 45.4 129 38.33 0.084 L 2 932.0 91.8 49 Oct ‘12 19.83 100 5.26 0.027 S 2 249.3 48.9 50 Oct ‘12 50.37 132 63.76 0.127 L 3 3268.0 87.1 51 Oct ‘12 44.68 129 22.60 0.051 L 2740.0 90.2 52 Oct ‘12 26.73 109 7.92 0.030 S 30.0 83.5 53 Oct ‘12 34.43 116 12.06 0.035 M 1 40.0 96.0 54 Oct ‘12 54.63 140 106.60 0.195 L 3 364.0 89.4 55 Oct ‘12 25.22 107 7.04 0.028 S 1 71.0 87.8 56 Oct ‘12 28.52 112 11.77 0.041 M 1 46.3 98.0 57 Oct ‘12 48.9 136 128.86 0.264 L 3 932.0 95.4 58 Oct ‘12 31.79 116 10.14 0.032 M 1 40.0 80.6 59 Oct ‘12 30.27 113 23.67 0.078 M 195.8 96.1 60 Oct ‘12 24.79 103 10.84 0.044 S 23.5 84.4 61 April ‘13 45.4 125 22.01 0.048 L 2 34.3 83.7 62 April ‘13 17.2 94 2.55 0.015 S 2 6.3 82.5 63 April ‘13 35.8 118 10.21 0.029 M 7.0 55.5 64 April ‘13 20.3 100 6.23 0.031 S 1 1.8 55.6 65 April ‘13 29.8 112 9.62 0.032 M 2 7.0 83.8

66 April ‘13 30 111 11.31 0.038 M 3 22.0 93.0 67 April ‘13 31.2 117 12.85 0.041 M 1 3.0 55.6 68 April ‘13 20.6 99 3.96 0.019 S 1 8.5 73.0 69 April ‘13 43.6 129 27.17 0.062 L 3 4.5 93.8 70 April ‘13 32.1 118 9.74 0.030 M 1.8 55.6 71 April ‘13 43 126 23.23 0.054 L 3 54.3 91.1 72 May ‘13 35 121 15.61 0.045 M 2 9.3 86.5 73 May ‘13 24.2 104 5.78 0.024 S 1 7.0 78.6 74 May ‘13 21.7 103 6.47 0.030 S 1 5.0 46.5 75 May ‘13 41.7 126 25.95 0.062 L 2 74.5 92.8 76 May ‘13 26.7 103 8.96 0.034 S 1 36.3 90.4 77 May ‘13 35.2 119 16.57 0.047 M 20.8 89.2 78 May ‘13 30.2 114 12.22 0.040 M 2 19.8 90.9 79 May ‘13 31.8 115 11.14 0.035 M 2 27.3 88.4 80 May ‘13 23.7 102 5.91 0.025 S 27.0 72.2 81 May ‘13 37.4 119 115.27 0.308 M 40.5 91.3 82 May ‘13 39.5 121 18.66 0.047 M 20.5 38.0 83 May ‘13 41.3 123 15.81 0.038 L 2 10.8 53.6 84 May ‘13 45.3 129 18.23 0.040 L 2 8.5 66.5

20 19 C) o 18 * 17 16 15

Temperature ( 14 13 1/06/12 2/07/12 2/08/12 2/09/12 3/10/12 3/11/12 28/04/13 29/05/13 Date

Figure 1. Mean (± SE) daily water temperature (oC) logged from sea cages near Port Lincoln, South Australia between June and October in 2012 and April and May in 2013. * Denotes missing data.

Table 2. Summary of southern bluefin tuna (Thunnus maccoyii) sampled for cryopreservation trials Fish Experiment Length (cm) Weight Gonad weight (g) GSI Maturation stage (kg) 1 1 148 70.9 61.21 0.086 2 2 2 126 42.8 24.51 0.057 2 3 2 129 43.0 26.38 0.061 2 4 2 139 56.6 48.72 0.086 2 5 2 148 70.9 131.22 0.185 4 *denotes gonad weight without fat, GSI = Gonadosomatic index