Does aquaculture diet composition influence the nutrition retention and condition of wild-caught Acanthopagrus butcheri broodstock?

Submitted by

Jesse Isaiah Newton

This thesis is presented for the degree of the Bachelor of Science Honours

College of Science, Health, Engineering and Education, Murdoch University, 2020 Cover Image. A wild Acanthopagrus butcheri captured from the Murray River region of the Peel-Harvey Estuary and used for the feed trial conducted in this study.

Photograph by Jesse Newton. Abstract Aquaculture is an essential industry for global food security, with production surpassing that obtained from wild capture fisheries. Future development of this profit- driven industry to meet the needs of a rapid growing population is dependent on the improved efficiency and sustainability of feeding methods. The health and nutrition of broodstock is crucial as it directly influences larval development and survival. Wet diets of whole fish and cephalopods are often fed to broodstock, however, they are abundant in long-chain highly unsaturated fatty acids and high rancidity making them prone to oxidation while in storage. Oxidation makes feed products rancid and degrades reproductive organs in fish, namely the livers. This leads to smaller spawns and reduction in larval survival. Aquaculture production is often limited by poor larval performance as a direct influence of inadequate knowledge of broodstock nutritional requirements. Due to the variance in nutritional requirements at a species level, investigative research is required.

Acanthopagrus butcheri, an important recreational fishing species in Australia, fits the criteria for diversification of small-scale aquaculture production outlined by the Food and Agricultural

Organisation of the United Nations. This study aims to investigate the impacts of aquaculture diets on the physiological health of wild caught A. butcheri broodstock, by measuring the transfer of dietary nutrients from the following commercial aquaculture diets, frozen squid and pilchards,

Breed-M broodstock conditioning diets and C2, a powdered larval diet combined with gelatine into pellets. Breed-M and C2 provided superior fatty acid profiles and antioxidant defence, while pilchards had high rancidity values which led to oxidation of liver tissue. Livers from wild caught fish reflected the lowest oxidation values, despite also containing minimal levels of antioxidants.

Breed-M and C2 provide antioxidants and fatty acids that are excessive of A. butcheri requirements, however wet fish diets of pilchards result in rapid degradation of valuable broodstock, negatively impacting growth and the physiological condition of reproductive organs.

This study provides insight into the dietary requirements of finfish broodstock and shows the implications of insufficient broodstock nutrition in aquaculture production. Acknowledgements I would like to thank my supervisory team of Drs. Gavin Partridge, Ben Roennfeldt, Alan

Cottingham and James Tweedley for your advice, help and support throughout. The time dedicated to assisting this project from its beginning to the submission of this thesis has been invaluable to me and I am extremely appreciative of what has been a steep learning experience. Having the input from each of your areas of expertise has enabled me to learn from multiple perspectives and for that I am grateful.

Many thanks go to Dr. Ben Roennfeldt, who almost single handedly designed and built the aquaculture facility! Your Bream expertise was invaluable, especially when you caught double my weekly catch in one day to get us started.

This project could not have worked without the assistance from Dr. Gavin Partridge and the team at Australian Centre for Applied Aquaculture Research (ACAAR) who helped leading up to and throughout the trial, producing the C2 feed, assisting with sample analysis and allowing me to utilise their facilities.

Thanks go to the Peel-Harvey Catchment Council and John Tonkin College for their collaboration and support of this project and to Tackleworld Miami, for assisting in the capture of our broodstock and engaging the local community so they could take part in fishing for our broodstock.

Thank you to the Department of Primary Industries and Regional Development

Diagnostic Laboratory Services, for allowing me to work in the lab so I gain experience and a better understanding of the analyses and also PathWest and Vetpath laboratories for working to process our samples as fast as they did during such a restricting and uncertain time.

A special thanks to Charlie, you kept me connected to the outside world when you helped with late night tank cleaning and water changes and made me feel somewhat normal, when you were still processing mussels in the lab next to me beyond midnight.

To all of the other volunteers that helped me along the way, I would not have been able to complete this project without your assistance. The journey of transforming a dusty storage shed to a successful aquaculture facility, the road trips to catch broodstock (many unsuccessful) and the very long final day, helping to complete the trial. These all took many hands and minds and I appreciate the time you lended me.

To Hayley, your unwavering support, encouraging words and constant belief in me through the past 18 months has been invaluable. Theres noone else I would have rathered to dissect fish with until 3am and I wouldn’t have had clean clothes or a proper meal for the final six months if it wasn’t for you. Table of Contents 1 Introduction...... 1 1.1 Aquaculture ...... 1 1.2 Aquaculture industry importance ...... 2 1.3 Australian aquaculture ...... 4 1.4 Sparid aquaculture ...... 5 1.5 Broodstock as key to global aquaculture ...... 6 1.6 Link between nutrition, body condition and reproductive physiology ...... 8 Dietary fatty acids ...... 9 Antioxidants ...... 10 Blood biochemistry...... 11 1.7 Acanthopagrus butcheri ...... 13 1.8 Aims and objectives ...... 15 2 Materials and methods ...... 17 2.1 Aquaculture system ...... 17 2.2 Fish capture and handling ...... 20 2.3 Feed trial ...... 21 2.4 Sample preparation ...... 26 2.5 Condition analysis ...... 27 2.6 Liver analysis ...... 28 2.7 Statistical analysis ...... 29 3 Results ...... 30 3.1 Nutritional composition of diets ...... 30 3.2 Fish condition ...... 36 3.3 Blood analysis ...... 41 3.4 Liver analysis ...... 48 4 Discussion ...... 57 4.1 Condition analyses ...... 57 4.2 Blood biochemistry...... 60 4.3 Liver analyses ...... 63 5 Future Directions ...... 66 6 Conclusions ...…………………………………………………………………… 68 7 References...... 70 8 Appendix ...... 80 1 Introduction

1.1 Aquaculture Fishing for sustenance by humans has been documented as far back as 100,000 years ago (Walter et al., 2000), with fish consumption being historically important, stemming from religious beliefs and extending to consumption as a luxury food item

(Fabinyi, 2012; Pauly, 2018). Aquaculture is a broad term for a variety of practices involving the culture of aquatic animals and plants for human uses and consumption, usually in contained or semi-contained facilities (Landau, 1992). Aquaculture practices date back to evidence of Aboriginal fish trapping (~ 8,000 YA), with people manipulating the land to capture and hold fish in dams and ponds for harvesting at their convenience

(Jordan, 2012; McNiven, 2017). There are also early records of controlled growth of cyprinids, being spawned and reared over 2500 years ago in China, with suggestions it has been ongoing for at least twice that time (Landau, 1992).

In modern times, the term aquaculture predominantly refers to large scale commercial production and is thus a profit driven industry (Stickney, 2017). Seafood production, comprising both wild capture fisheries and aquaculture, was estimated at 200 million tonnes in 2015, with the contribution from aquaculture first surpassing that of wild fisheries in 2013 (Figure 1.1) (Hannah Ritchie, 2020). Aquaculture production has spanned across ~600 species, with majority falling into the main groups of finfish, molluscs, crustaceans and cephalopods (Figure 1.1) (FAO, 2018; Garibaldi, 1996).

1 a)

b)

Figure 1.1. World seafood production: a) comparison of aquaculture and wild capture fisheries from 1960 to 2015 b) the major aquatic groups from 1961 to 2013. Reproduced from Hannah Ritchie (2020).

1.2 Aquaculture industry importance Aquaculture is an essential production industry in the food, stock enhancement and aquarium sectors, accounting for 53% of all aquatic animal production. Food production is the largest aquaculture sector (88%), with demand stemming from various cultures and socio-economic standings (FAO, 2018).

2 Seafood is the fastest growing protein production industry worldwide, with an annual growth of 3.2% between 1961 – 2015, which exceeded the annual population growth for that same period (1.6%) (FAO, 2018). In 2015 fish accounted for 17% of all human protein consumption, with that number predicted to increase due to increased reliance on aquaculture to feed an ever growing population, predicted to surpass 9.5 billion people by 2050 (United Nations, 2019; FAO, 2018). While aquaculture growth has been rapid, the potential for further expansion of the industry, utilising suitable coastal habitats, could provide over 100 times the current global seafood demand (Gentry et al.,

2017). Consumption of seafood is known to have a range of health benefits for humans, namely being the only readily available source of long chain omega-3 highly unsaturated fatty acids, eicosapentaenoic acid (EPA) and docosaheptaenoic acid (DHA) and essential micronutrients such as iron, zinc and selenium (Hicks et al., 2019; Kok et al., 2020). Omega-

3 fatty acids DHA and EPA are proven to be critical in reduction of occurrence of cardiovascular disease while micronutrients rich in seafoods have been provem essential for brain function and sight development (Mehmood and Mateen, 2020; Schilling, 2019).

Although consumer surveys have relayed a preference for wild-caught seafood products over aquaculture (Davidson et al., 2012), consumers are becoming increasingly conscious about the source and impact of their consumption in environmental and ethical respects (Banovic et al., 2019). Aquaculture, provided the correct production methods are undertaken, can sustainably achieve high intensity food production (Folke and Kautsky,

1992; Tschirner and Kloas, 2017). In order to achieve sustainable production, improved efficiency is needed, particularly with regard to the unsustainable use of feed resources such as fish oil, fish meal and soybean product (Tschirner and Kloas, 2017). The impacts of feeding high protein fish meals and fish oils sourced from wild stocks, can be equally damaging to ecosystems as the overexploitation of higher trophic level species 3 (Tzankova, 2004). In order to improve the efficiency and sustainability of commercially fed diets, more species-specific research is required on aquatic animals shown to have aquaculture production potential (Izquierdo et al., 2001).

1.3 Australian aquaculture In Australia, aquaculture is the fastest growing primary industry, with fish production at 97,000 tonnes in 2016 projected to increase to 151,000 tonnes by 2030

(FAO, 2018). The Australian seafood industry is one of Australia’s largest food exports

(Parish and Freeman, 2011) with production from both fisheries and aquaculture having an estimated increase of 4% to $3.3billion in 2019-20 (Mobsby and Curtotti, 2019).

Australia holds a reputation for reliable supply of high-quality product and takes advantage of the close proximity to Asia, where seafood demand is fast growing (Kidane,

2007; Mobsby and Curtotti, 2019).

Australian aquaculture production is predominantly in marine products such as salmonids, abalone rock lobster and tuna, accounting for 85% of total fisheries export value, showing the dependence the Australian industry has on a small variety of products.

It is estimated, thatmarine production could reach between 16 – 24 million tonnes if just

1% of Australia’s potential area was developed (Figure 1.2) (Gentry et al., 2017).

4

Figure 1.2. Current marine aquaculture production vs potential production. a. Current marine aquaculture fish production. b. Potential marine aquaculture if 1% of the suitable area in each country was utilised for low-density aquaculture (Produced from Gentry et al., 2017).

1.4 Sparid aquaculture The Sparidae (Bream or Porgies) which belong to the order perciformes includes around 115 species (Pavlidis and Mylonas, 2011). The family dominates marine recreational fisheries, making up 12% of worldwide capture (Freire et al., 2020) and are economically important worldwide, with 45 species being harvested in wild capture fisheries (Basurco et al., 2011). Aquaculture of sparids is dominated by marine species,

5 with most cultured in sea cages in coastal waters The vast majority of commercial sparid aquaculture production occurs in Asian countries (~55%), e.g. China, Japan, Taiwan and

South Korea, and Mediterranean countries (~44%), e.g. Greece, Turkey, Spain and Italy

(Basurco et al., 2011). Limited commercial aquaculture also occurs in the Dominican

Republic, United Arab Emirates and Oman (Basurco et al., 2011). Although 11 species are grown commercially, global sparid aquaculture is dominated by two species, Sparus aurata (Gilt-head sea bream) and Pagrus major (red seabream) (Basurco et al., 2011).

Between 1986 and 2006, global aquaculture production of sparids saw an annual increase of 10%, from 36,000 metric tonnes to 244,000 metric tons (Basurco et al., 2011).

In this same period, the value of this aquaculture product grew only 6% annually, due to saturation of the market with product that well exceeded demand, a result of poor planning and understanding of the market (Basurco et al., 2011). The decline in the value of these species can also be attributed to seasonal changes in the market. As farms utilise cage aquaculture, they produce fish in large quantities as the season permits, rather than at a steady rate, reducing the unit price due to market saturation. Considering these experiences, sparid aquaculture could benefit greatly with development of small-scale recirculating aquaculture systems, allowing controlled spawns and feeding for a timed release of product into the market for maximum economic yield (Liu et al., 2016). Such systems have been highlighted as a solution to achieving sustainable production of fish to supply a growing human population (FAO, 2018).

1.5 Broodstock as key to global aquaculture Broodstock, i.e. sexually-mature adults, provide hatcheries with larvae. By controlling conditions and inducing spawning on demand, larvae can be produced at the time of year that best meets market demand for the given product (Joseph et al., 2017).

Commercial aquaculture grow out operations generally purchase larvae from hatcheries,

6 however some larger companies run their own broodstock and spawning operations.

Properly conditioned broodstock will produce healthy larvae at an effective spawn rate should they be given optimum nutritional diets in the months prior to spawning. This is the ultimate challenge for hatcheries. However, broodstock nutrition is often underappreciated in production and is thus lacking in studies for specific dietary requirements (Izquierdo et al., 2001; Utting and Millican, 1997).

Quality broodstock are difficult to acquire, with individuals being wild caught, they need to fill criteria to produce offspring of the best possible quality. Thus, maintaining broodstock at optimum health for multiple years is essential for successful/productive hatchery operations (Stieglitz et al., 2017).

One of the recurring issues in aquaculture is mass mortality of animals in the early life stages. This is largely attributed to poor nutrition provisions in first feeds and a lack of essential nutrient transfer from broodstock, often if they have had a poor conditioning period or allocated incorrect nutrients from whole fish and in artificial diets (Hu et al.,

2018). Artificial diets developed for broodstock are heavily reliant on fish meal and fish oil to provide the correct protein and energy demands, most importantly for those culturing high trophic level fish that generally have a higher demand (Dam et al., 2019).

Aquaculturists often aim at maximum feeding efforts of broodstock, as insufficient feed leads to reduced fecundity and issues in early life stages of offspring (Izquierdo, 1997;

Izquierdo et al., 2001). However, high fish meal and fish oil content diets often results in wasted nutrients, therefore the quality of a diet must be specifically designed for each species. While the basic levels of protein, lipids and fats may be known at a family level, each species requires specific nutrition composition, to ensure efficiency of feeding for sustainability and maximised production (Hardy, 2010).

7 1.6 Link between nutrition, body condition and reproductive physiology In any aquaculture program, feeding of stock is often the most important detail, due to the high cost of diets and their substantial influence on the quality and quantity of product (Kortner et al., 2011; Xu et al., 1994). Diets of fish have long been designed to raise larvae to a market size that exhibits desirable taste, texture, smell and visual appearance both externally and of the flesh. Each product depends on a specific market, with various end results required. Broodstock diets such as Breed-M are aimed at providing nutrition suitable for adult fish to reach optimal condition for reproduction of healthy eggs with the aim for high hatch rates and healthy development. Larval diets, such as C2, are developed for the critical early life stages, including the highest quality protein sources to maximise survival to reach growout stage for human consumption, the quality of the stock as a result of nutrition will lead to economical gain (Kankainen et al., 2012).

Many aquaculture producers, use thawed, whole bait fish as the sole diet for their stock, including broodstock (Ottolenghi, 2008). This method is conducted with the belief that it is the most natural feed and replicates accurately, the nutrition of wild fish and is also a cheaper alternative than formulated feeds (Lochmann et al., 2009). Although this may contribute a large portion of fish nutrition in the wild, frozen bait fish have been shown to degrade rapidly, going rancid leading to loss of essential fatty acids and valuable vitamins due to oxidation processes (Cho, 1990; Wood and Hintz, 1971).

Synthesizing diets that can be sustainably produced and result in optimal growth, development and reproduction an inhibitory factor for the aquaculture industry. Fish meals and oils are relied upon as the primary source of high quality nutrient provisions in diets (Tacon and Hasan, 2007). Diets supplemented or replaced by alternate meals and oils have had little success in producing the same product quality for human consumption

(Bowyer et al., 2012), with plant-based proteins yielding impaired fish health and growth rates due to the distinguishable differences of amino acid and fatty acid composition from

8 fish oil compared to other oil sources (Le Boucher et al., 2012; Shahidi and Wanasundara,

1998).

Growth in aquatic animals is caused by the production of muscle, fat, epithelial and connective tissue (Engrola et al., 2018). For this to occur, the diet must provide energy, which is majorly contained within protein, carbohydrates and lipids (De Silva and

Anderson, 1994). Proteins are combinations of amino acids, with essential amino acids being those that organisms are unable to synthesize at all or at insufficient rates required for utilisation and so need to be supplemented by diets (De Silva and Anderson, 1994).

The amino acid requirements of fish are particularly high compared to other vertebrates, and amino acid composition is an important factor in development and growth, particularly in early life stages (Engrola et al., 2018).

Carbohydrates are a relatively cheap energy source and are an essential dietary component with protein and lipids, however most species have very limited requirements due to being a less digestible energy source (De Silva and Anderson, 1994; Engrola et al.,

2018; Naylor et al., 2000). Lipids are easily digested and utilised as energy, however, surplus lipids are wasted and result in fat retention in the liver and muscle. This is often found in cultured fish when compared to wild fish (Rodríguez-Barreto et al., 2012).

Dietary fatty acids

Success of diets are dependent on the fatty acid profile provided, with requirements varying for species. The fatty acid ratios and their interactions with other dietary contents such as vitamins E and B and micronutrients can also impact nutrition utilisation of finfish

(Edouard Bourre and Marc Paquotte, 2008).

Lipids, or fatty acids are burned and used for basic energetic functions such as movement, the individual fatty acids and their combinations play specific crucial roles in

9 the growth and reproduction (Rodríguez-Barreto et al., 2012; Tocher, 2003). Long chain polyunsaturated fatty acids (PUFA) are critical dietary constituents, with omega-3 PUFAs being a focal provision from diets due to the inability of fish to synthesize the required concentrations, if at all (Izquierdo, 2004). It is well documented that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are two of the most important omega-3 fatty acids, as they are essential for the development of the brain and eyes (Brodtkorb et al.,

1997). These fatty acids must also be provided at the correct ratio, with DHA having a greater impact on reproductive success and therefore requiring a higher ratio (Harel et al.,

2002). DHA and EPA are also essential in broodstock diets, with the correct ratio producing substantially more eggs than diets containing insufficient levels of DHA. The inclusion of omega-3 highly unsaturated fatty acids (HUFAs) in broodstock diets is important in subsequent larval development, as insufficent level can cause larval rearing problems (Ling et al., 2006).

Antioxidants

Antioxidants are essential in aquaculture diets, providing protection against oxidative stress often stemming from dietary intake, with requirements increasing during reproduction

(Izquierdo et al, 2001). Many physiological processes are dependent on vitamin B1

(Thiamine and itsphosphorylated forms). Thiamine is essential in nerve function and processing carbohydrates, and fish with deficiencies show poor appetite, muscle atrophy, convulsions, loss of equilibrium, edema (inflammation and swelling), poor growth and increased sensitivity to physical disturbance or light (Brown et al., 1998). Thiamine deficiencies are caused by dietary intake of thiaminase, and have been linked to earlymortality syndrome, (EMS) in lake trout (Salvelinus namaycush), causing high death rates of individuals between hatching and first feeding (Fitzsimons et al, 2001; Houde,

2015).

10 Aquaculture broodstock diets often contain very high lipid levels, with long chain

(LC) HUFAs, such as EPA and DHA, being the most desirable for their connection to reproductive success. These highly unsaturated fatty acids are also the most succeptible to oxidation (Lembke and Schubert, 2014), also known as rancidity. Rancidity yields thiobarbituric acid reactive substances (TBARS) as a byproduct. Measuring of TBARS therefore indicates rancidity of feeds and livers as a portion of oxidised tissue (St. Angelo et al., 1996). Iodine in feeds are formed by the reaction of peroxides in fats and oils with iodide ions, the measurement of which provides evidence of rancidity as a peroxide value.

These peroxides lead to degradation of essential feed components such as fatty acids and animal tissue once consumed (Jaswir et al., 2009). Selenium, a naturally occuring antioxidant, has been reported to work in conjunction with other antioxidants, to reduce lipid oxidation, which leads to muscle atrophy and disease in finfish (Hilton, 1989).

Studies have reported benefits, such as improved flesh colour to extended shelf life from dietary supplementation of selenium (Buentello et al., 2016; Le et al., 2014).

Vitamin E is another antioxidant, and constitutes a small group of related tocopherols, which in conjunction with selenium, are effective protectors against lipid oxidation of polyunsaturated fatty acids (Di Mascio et al., 1991), while also showing evidence for raising the immunity of finfish in captivity (Montero et al., 1999; Wang et al., 2006).

Blood biochemistry

Blood samples can be extracted from live fish without causing extensive physical damage, a valuable method particularly with broodstock which are too valuable to be sacrificed to check overall stock health. By extracting plasma from blood samples, parameters such as alanine aminotransferase, lipase and triglycerides can be measured, giving indications of stock health in relation to their nutrition, infection and injury (Percin

11 and Konyalioglu, 2008).

Blood serum chemistry can be significantly impacted by trace metals and alanine aminotransferase (ALT) is used as a biomarker for toxic effects of such metals as well as tissue degradation in liver (Fırat and Kargın, 2010; Knudsen et al., 2016) Alanine aminotransferase is an enzyme primarily found in the liver, and high levels can be indicative of liver damage, often in relation to tissue oxidation (Knudsen et al., 2016;

Yamada et al., 2006).

Triglycerides are a type of lipid found in the tissues of animals (Oishi et al., 2020).

When excess lipids are not processed immediately for basic metabolic functions, they are converted to triglycerides which are stored in the liver and tissue of fish and are able to be detected in blood serum (Habte-Tsion et al., 2013). Successful storage of lipids as energy reserves in the liver are important functions of fish and can be impacted by the nutritional input of diets. Blood serum has been shown to have depleted triglycerides in alignment with lipid accumulation in livers, and therefore provide a valuable testing parameter of stock health (Oishi et al., 2020). Triglycerides reflect different levels in fish based on sex, with females typically reporting higher triglycerides prior to spawning, with depleted levels below males post-spawn (Suljević et al., 2017).

Lipase is the enzyme produced in the pancreas that performs an essential role in digestion and the processing of dietary lipids (Pezzilli et al., 1993). When the pancreas is overworked from processing highly fatty diets, it can become inflamed and excrete excess levels into the blood, therefore high total serum lipase, with high triglycerides, are indicative of damage to the pancreas, often due to oxidation from diets excessively high in lipids.

The glutamate dehydrogenase (GLDH) enzyme is essential for metabolism and can be used as a biomarker for liver damage. Similar to ALT, unusually high levels indicate

12 oxidative stress but with a higher magnitude of elevation and is not affected by skeletal muscle damage (Jaeschke et al., 2012; Li et al., 2011).

Urea is a type of nitrogenous waste produced with the consumption of diets with high fatty acid composition and due to its toxicity, is excreted. Ammonia is the most abundant nitrogenous waste in fish, followed by urea (Toni et al., 2017) and is measured to indicate function of the liver, kidney and gills as they are key in the regulation of urea

(Hazon et al., 2003).

Total serum protein is often measured to determine the immune function of fish in response to medication and nutrition (Mehrabi et al., 2012). Unusual high levels of serum total protein in fish can indicate a positive immune response whereas decreased levels can indicate oxidative stress (Zhang et al., 2018; Zhang et al., 2017). Protein levels are likely to fluctuate during spawning seasons, with increased protein levels prior to spawns and subsequent reduced levels post-spawn (Suljević et al., 2017).

Cholesterol levels in the serum of teleosts are likewise related to sex, with females having higher levels than males, required as a precursor of sex steroid hormones. Levels fluctuate according to the spawning season, with higher amounts required for higher metabolic activities i.e. reproduction (Sharma et al., 2017). Serum cholesterol levels are highly dependent of nutrition, having a positive correlation with diets high in fatty acids, particularly saturated fatty acids (Nayak et al., 2019).

1.7 Acanthopagrus butcheri Acanthopagrus butcheri (Black Bream) live in isolated populations in estuaries situated in Australia’s south west from New South Wales to Western Australia and is a highly important sparid caught by recreational and commercial fishers (Malseed and

Sumner, 2001; Ryan et al., 2015).Black bream are solely estuarine species (Potter et al.,

13 2015), completing its life cycle within the estuary (Norriss et al., 2002) despite estuarine conditions being particularly harsh for spawning, egg development and larval life due to their dynamic physico-chemical environment and anthropogenic degradation (Potter and

Hyndes, 1999; Tweedley et al., 2016). The isolated nature of these populations has led to variance in the genetics and physiology of separate populations driven by the hydrology of their estuarine environments, which are markedly different and prone to further rapid changes (Chaplin et al., 1997; Cottingham et al., 2018).

Acanthopagrus butcheri is tolerant to a wide range of salinities from freshwater to hypersaline conditions and is adapted to environments of fluctuating temperature

(Tweedley et al., 2019). Despite this tolerance, their ideal growth and health is experienced in a salinity of 24ppt (Haddy and Pankhurst, 2000; Partridge and Jenkins, 2002). Estuaries provide adequate habitat for A. butcheri, with preferences of overhanging banks, fallen tree branches and mangroves within deep pools of low to moderate salinity (Norriss et al.,

2002).

Seagrass beds provide a shelter for juvenile A. butcheri and are the habitat for multiple sources of prey, such as shellfish, amphipods, copepod nauplii, worms, crustaceans, small fish of other species and algae. (Chuwen et al., 2007). Their diets change frequently with growth as they are opportunistic feeders, so specific diets vary according to their environment, and it is suggested that consumption of plant matter is circumstantial due to their feeding behaviour, preferring to eat gastropods and bivalves from the benthos and surfaces opposed to hunting within the water column (Chuwen et al., 2007; Poh et al., 2018). Their diets are markedly variable in the wild between estuaries and across seasons, evidence of the opportunistic nature of their feeding (Tweedley et al.,

2019). Thus, determining the specific dietary requirements for the purpose of recreating ideal nutrition in aquaculture projects presents a complex challenge.

14 Aquacultured sparid species produce large quantities of buoyant eggs, with those settling considered infertile and often removed from the system to prevent water quality deterioration and the spread of funguses (Izquierdo, 1997). Sparid eggs are approximately

0.8 to 1.1mm in diameter and larvae hatch at an early life stage, 1 to 2.5 days post spawn.

Larvae rely on a large yolk sac up until first exogenous feeding 3 to 5 days post hatch and are highly vulnerable to the abiotic and biotic functions of their environment in these early stages (Pavlidis and Mylonas, 2011). Temperature regulation is critical in hatcheries of sparid species, with temperatures outside of the preferred range of 15°C to 22°C in temperate species resulting in increased occurrences of abnormalities and high mortality rates during the hatch times and early larval stages (Norriss et al., 2002).

1.8 Aims and objectives The aim of this research was to investigate three aquaculture broodstock diets namely; a common aquaculture diet comprising pilchards and squid (control), a commercially available conditioning diet (LANSY Fish Breed-M Concentrate Powder) and a powdered marine larval diet (C2), fed to Acanthopagrus butcheri over a four and a half month period encompassing the spawning season to compare the transfer of the essential fatty acids EPA and DHA, and antioxidants, thiamine and Vitamin E into the livers.

The current study will investigate the hypothesis that aquaculture diets provide insufficient antioxidants, namely thiamine and vitamin E, leading to oxidation of feeds and subsequent damage of liver tissues of the broodstock. These will be tested by measuring the parameters malondialdehyde and thiobarbituric acid reactive substances in the livers and diets.

Blood serum will be tested to elucidate the value of parameters as biomarkers for physiological health of A. butcheri in response to their nutrition. The blood serum

15 parameters to be tested are alanine aminotransferase, glutamate dehydrogenase, lipase, cholesterol, urea, triglycerides and protein.

16 2 Materials and methods

2.1 Aquaculture system A recirculating aquaculture system (RAS) was designed and built at Murdoch

University specifically for a feed trial of diets for wild-caught Acanthopagrus butcheri broodstock (Figure 2.1). Full strength seawater was sourced from a bore used by the

Australian Centre for Applied Aquaculture Research (ACAAR) in Fremantle and delivered to the facility by road as required. Seawater was pumped into four 1000 L

Intermediate Bulk Containers (IBCs) and diluted, using freshwater treated with sodium thiosulphate to remove chlorine, to a salinity of 20-25ppt.

The RAS comprised thirteen IBCs, twelve as culture experimental tanks and one as a sump (Figure 2.1). To enable gravity return of water to the sump, culture tanks were raised on plastic pallets. Wastewater and sediment were drawn from the base of the culture tanks. In the case of all the female and mixed sex tanks (see 2.3 Feed Trial), wastewater passed through egg collection baskets, before returning to the sump, for tanks with males that did not have egg collection baskets this wastewater went directly from the standpipe into the sump.

As water entered the sump, it trickled through a multilayer mechanical filtration basket containing fine filter wool, a coarse sponge, and later through the trial, carbon and zeolite layered in filter wool. Water trickled through the filter basket and settled in the sump, before being pumped into the pressurized sand filter. Water pumped through the sand filter then passed through an ultraviolet sterilizer, before rising into two, 200 litre plastic drums half filled with plastic biological filter balls. Water was dispersed over the bio-balls with PVC spray bars and flowed out of the bottom of the drums back to the tanks by gravity (Figure 2.1).

17 Each culture tank had a flow of approximately 800 L h-1, regulated by ball valves at each tank inlet. Water entered the system from above the water level, breaking the surface for increased aeration.

Running along the water inlet pipes was aeration PVC piping, pumped from above the filtration area, with one air-stone running in each tank, sitting just above the tank floor, directly next to the standpipe to create an up draw of particulate matter to improve its extraction. Each tank was fitted so the connection of a second air-stone could be made quickly if required. The recirculating aquaculture system was filled with saltwater three weeks prior to the addition of broodstock, with the heater placed in the sump and set to

20º Celsius, water and air pumps running. Ammonia chloride was added three times within the first week to trigger biological cycling and the establishment of nitrifying

+ bacteria. Water quality parameters, Total ammonia nitrogen (NH3/NH4 ), Nitrite (NO2),

-1 Nitrate (NO3), pH, Temperature, Salinity and Dissolved Oxygen (DO; mg L and % saturation) were recorded daily, beginning three weeks prior to the addition of A. butcheri broodstock and continuing for the duration of the feed trial. Unionised ammonia (NH3) was calculated from pH, temperature (°C), salinity and total ammonia nitrogen measurements following Eagle (2010).

Temperature and DO were recorded using an Oxyguard dissolved oxygen meter and salinity was checked using a refractometer. The remaining parameters were analysed using API colour reagent test kits. Throughout the trial DO levels ranged between

10.19 mg L-1 and 6.45 mg L-1.

Daily water temperature remained stable from July to the beginning of November

(~19 ºC) before increased air temperature led to water temperature rise, reaching a peak of

29 ºC in December (Appendix 1).

18 Daily water exchanges (~1000L) were used to maintain optimal water quality throughout

-1 the trial.TAN remained < 0.5 mg L while the unionised ammonia (NH3) portion remained< 0.01 mg L-1 (Appendix 2a).Nitrite peaked at 4 mg L-1 on 5th September, then remained ≤ 1 mg L-1 and nitrate showed a similar pattern (Appendix 2.b; 2.c).

Figure 2.1. Recirculating aquaculture system floorplan designed for the feed trial of Acanthopagrus butcheri broodstock and the culture of their larvae.

19 2.2 Fish capture and handling Adult A. butcheri were collected from the Murray River; one of the main tributaries of the Peel-Harvey Estuary. Fish were captured by rod and line fishing from a small boat in July 2019. During capture circle hooks were used to prevent fish being hooked in the throat and gut, and only mouth-hooked fish were retained for the experiment. All broodstock were between 250 and 350 mm total length and thus mature (Cottingham et al., 2018).

Their sex was determined based on the presumption that males would produce milt when gentle pressure was applied along the belly, towards the anus. As this method does not always produce milt in males, this was checked again using the same technique during the pre-trial weigh and measure to increase accuracy (this provided 100% accuracy confirmed by the final dissections).

During capture selected fish were stored on the boat in an insulated plastic container containing aerated estuary water. Water was regularly exchanged in and out of the cooler box to reduce fluctuations in temperature and nitrogenous waste build up. Up to twelve individuals were caught and transported in each trip to ensure transport containers were not overstocked. On arrival to the boat ramp, fish were transferred from the container to large plastic fish bags, with three fish per bag. Bags contained new estuary water and pure oxygen (ratio 1:3 by volume, respectively); these were secured with elastic bands, rested in containers to prevent rolling or puncture of the bag and immediately taken to the aquaculture facility at Murdoch University. Captured broodstock were held, with males and females contained separately in sex specific tanks without feed for up to three weeks before the trial. This period without feed was due to the time taken to capture the number of wild broodstock required for the trial and quantity limitations for transport.

20 A small number of wild-caught broodstock were euthanised and dissected to

remove their livers (see 2.6. Liver Analysis), in order to obtain baseline liver parameters

to compare with feed trial results.

2.3 Feed trial All broodstock were externally tagged for identification (Fig. 2.2.). Six broodstock

were randomly assigned to each tank. Of the twelve tanks, three contained females only,

three males only and the remaining six tanks a mix of each sex. This selection was chosen

to provide genetic diversity of broodstock in the ‘mixed sex’ tanks for the stock

enhancement project being conducted. ‘Male only’ and ‘female only’ tanks for each

treatment provided stock that would not be induced to spawn by hormone injection, aimed

at giving performance and condition data not influenced by the expenditure of energy

from spawning.

Figure 2.2 A. butcheri broodstock with external identification tag.

Each tank of fish was fed one of three experimental diets; a control diet comprising pilchards and squid at a 1:1 ratio, a commercially available conditioning diet in a powder form, combined with water to make pellets (Breed-M) and a formulated powdered larval diet, combined with gelatine to make large pellets (C2) (Table 2.1).

21 Table 2.1. Tank allocation of Acanthopagrus butcheri broodstock for a feed trial of aquaculture diets, control, Breed-M and C2.

Tank # Diet ♂ ♀ Label 1 C2 0 6 Female 2 C2 3 3 Mix 3 Breed-M 0 6 Female 4 Control 3 3 Mix 5 Control 0 6 Female 6 Breed-M 3 3 Mix 7 Breed-M 3 3 Mix 8 C2 3 3 Mix 9 Control 3 3 Mix 10 Breed-M 6 0 Male 11 C2 6 0 Male 12 Control 6 0 Male

22 The control diet was selected based on an aquaculture industry standard of whole squid and pilchards that are used as an economical diet to maintain broodstock while they are not in spawning condition. Squid and pilchards were kept frozen at -18°C and then thawed and cut into ~10 mm cubes to cater for the small mouth size of A. butcheri. Squid and pilchard were fed at a 1:1 ratio and all parts were used.

The second trial diet was Breed-M, which is used worldwide as a conditioning diet and is formulated to produce high quality eggs and larvae (Aquaculture, 2019). The

Breed-M powder was mixed with water to produce the optimum size and texture for feeding, i.e. a ration of Breed-M to water of 53:47 and rolled into 1g round pellets. Breed-

M was mixed once every five days and kept refrigerated at 4°C and the pellets rolled each morning prior to feeding.

The third broodstock diet was made using a formulated larval diet named C2 in powder form, which was combined with gelatine to create a larger pellet. This diet (C2) was formulated by ACAAR to enhance growth at the most critical stage of growth and development, and thus contain the best quality of ingredients. C2 powder was combined with a gelatine and water mix at a 1:1 ratio, refrigerated and cut into 10mm cubes. Fresh

C2 pellets were made each week and stored at 4°C.

Each of the diets were analysed prior to the feed trial to determine nutrient composition (Table 2.2). Samples of whole pilchard, whole squid, C2 in gelatine pellets and Breed-M pellets, were sent to Agrifood Technology, Department of Primary

Industries and Regional Development Diagnostic Laboratory Services (DDLS), Marine and Freshwater Research Laboratory (MAFRL) and PathWest Laboratory Medicine

Western Australia (methods outlined in 2.6. Liver Analysis).

23 Table 2.2. Nutritional and rancidity parameters analysed at the various laboratories for the four diet components; pilchards and squid (control), Breed-M pellets and C2 gelatine pellets.

Agrifood Technology DDLS MAFRL PathWest Protein TBARS Moisture Vitamin B (thiamine) Fat total MDA Sodium Carbohydrates Vitamin E (tocopherol) Potassium Ash Free fatty acids Calcium Moisture Phosphorus Energy Magnesium Free fatty acids Iron Acid value Zinc Peroxide value Manganese Selenium Copper Boron Chromium Cobalt Nickel Molybdenum

24 Broodstock were fed on an energy basis. As the energy values varied for each of the diets, the control diet was used as the base level for daily energy requirements, due to its common use in industry. Bait pilchards provided 497kj 100g-1 and Squid, 339kj 100g-1 therefore feeding was provided at a 1:1 ratio providing energy at 418kj 100g-1.

Fish were fed the energy equivalent of 2.5% of their body weight per day on the control diet. This percentage was based on the 2% body weight fed to A. butcheri in aquaculture grow out practices (Sarre et al., 2003), and was raised by 0.5% with the aim of providing sufficient nutrition to successfully condition for healthy spawning, and curb potential aggression resulting from hunger (Ariyomo and Watt, 2015; Håstein, 2004).

푑푎푖푙푦 푓푒푒푑 (푘푗) = 푡푎푛푘 푏푖표푚푎푠푠 (푔) × 2.5% × 4.18 kj 푔−1

Feed was given each morning and any remaining feed in the afternoon was removed and recorded, to gather an approximate total weight of feed consumed.

Broodstock from each of the mixed tanks were injected with Ovaprim to induce spawning one and a half months into the feed trial. Egg collection buckets were placed in the collection baskets for each of the mix tanks. Eggs were collected for a week and gently released into a larval tank, larvae were kept isolated in tanks according to the treatment diet of the broodstock.

At the completion of the four and a half month feed trial, the broodstock were removed from the tanks and euthanised in 80L tubs containing 40mg/L of Aqui-S anaesthetic, i.e. double the recommended dosage for sedation, and monitored for gill movement to ensure mortality had occurred prior to being prepared for dissection.

25 2.4 Sample preparation Once deceased fish were patted down with paper towel to remove excess water, weighed (g) to two decimal places and measured for total length (mm). Approximately

5mls of blood was then extracted from each fish using a 10ml syringe and 1 inch needle, coated in lithium heparin, to prevent blood from coagulating or clotting. Blood was injected from the syringe into lithium heparin coated vacutainers, inverted twice and then placed on bubble wrap over ice in a foam cooler box. Blood samples were sent to DDLS where plasma was collected, before being sent to Vetpath for biochemistry testing of the following parameters; alanine aminotransferase (ALT), glutamate dehydrogenase

(GLDH), lipase (DGGR) (Kook et al., 2014), cholesterol (Chol.), triglycerides (TRIG), urea and total protein.

Each fish was then placed in a zip lock bag and labelled with the corresponding treatment, tank number, sex and tag number. Bagged fish were placed in an ice slurry for dissection. Each individual broodstock was dissected to remove the liver and gonads, which were weighed to two decimal places before being frozen at -80°C for analysis (see

2.6. Liver Analysis).

26 2.5 Condition analysis Performance and condition indices were used to depict the growth and physical performance of the broodstock for each of the experimental aquaculture diets. All fish in the trial were included in the condition analyses. This included fish from the mixed tanks that were induced for spawning, which was considered for possible influence on results and accounted for in the analysis (2.7. Statistical Analysis). Condition analyses were calculated for weight gain, Specific growth rate (SGR), apparent feed conversion ratio on a dry weight basis (DFCR), apparent protein efficiency ratio (PER), apparent energy efficiency ratio (EER), gonadosomatic index (GSI), hepatosomatic index (HSI) using the following formulas (Elfeki et al., 2018., Laining, 2019).

푊푒푖푔ℎ푡 푔푎푖푛 = 푓푖푛푎푙 푤푒푖푔ℎ푡 (푔) − 푖푛푖푡푖푎푙 푤푒푖푔ℎ푡 (푔)

ln[푓푖푛푎푙 푤푒푖푔ℎ푡 (푔)] − ln[푖푛푖푡푖푎푙 푤푒푖푔ℎ푡 (푔)] 푆퐺푅 (% 퐷푎푦−1) = ( ) × 100 푡

Where ln is the natural logarithm and t is time in days. Specific growth rate thus gives the percentage weight gained per day. SGR was calculated for individual fish.

푓푒푒푑 푐표푛푠푢푚푒푑 [푔]

퐷퐹퐶푅 = 푓푖푠ℎ 푤푒푖푔ℎ푡 푔푎푖푛 [푔]

Dry weight food conversion ratio given in this formula, is units of feed (dry weight basis) input to units of growth output. As food is fed to tanks with multiple fish, DFCR is calculated per tank of fish rather than per fish.

27 푓푖푠ℎ 푤푒푖푔ℎ푡 푔푎푖푛 [푔]

퐸퐸푅 = 푒푛푒푟푔푦 푐표푛푠푢푚푒푑 [푘퐽]

푓푖푠ℎ 푤푒푖푔ℎ푡 푔푎푖푛 [푔]

푃퐸푅 = 푝푟표푡푒푖푛 푐표푛푠푢푚푒푑 [푔]

푔표푛푎푑 푤푒푖푔ℎ푡 [푔]

퐺푆퐼 = × 100 푡표푡푎푙 푓푖푠ℎ 푤푒푖푔ℎ푡 [푔]

푤푒푡 푤푒푖푔ℎ푡 푙푖푣푒푟 퐻푆퐼 = ( ) × 100 푡표푡푎푙 푤푒푖푔ℎ푡 푓푖푠ℎ

2.6 Liver analysis Liver samples were required to be pooled due to the large tissue requirements for the testing of total thiamine relative to the size of livers of Acanthopagrus butcheri. Livers were pooled of the same sex from the same diet, using two or three livers to make a total weight of 10 grams as required by PathWest. Allowing for two results from each sex in each treatment. Baseline A. butcheri liver samples that were analysed prior to the feed trial were also included in analysis as “Wild” (n = 2 females and 6 males).

Liver analyses was conducted only on broodstock kept in sex only tanks due to budgeting. Spawning has significant impacts on the physiology of fish, therefore fish in the mixed tanks which were induced to spawn, were omitted.

Liver samples, frozen at -80ºC, were analysed for thiamine, fatty acids, vitamin E

(McMurray and Blanchflower, 1979; McMurray CH, 1980) and the lipid oxidation parameter, thiobarbituric acid reactive substances (TBARS) (Association, 2012; Jo and

28 Ahn, 1998). Thiamine (Icke, 1980) was analysed by PathWest’s vitamin assay laboratory and the remainder was analysed at Department of Primary Industries and Regional

Development Pathology Laboratory Services (DDLS).

2.7 Statistical analysis All statistical analyses were conducted following the principal: Analysis of

Variance was tested using an interactive model. Where no interaction was detected, an additive model was used.

Weight gain, SGR, FCR, PER, EER, GSI and HSI were tested using three factor analysis of variance (three-way ANOVA), testing for significant difference (P < 0.05) between treatment and tank type (male, female and mixed tanks) and interactions. Where significant differences were detected, a posthoc Tukey’s test was employed to determine which treatments were statistically different. Statistical analysis was conducted using R

Studio (RStudio, 2015).

Vitamin E, TBARS and MDA of livers were analysed for differences using two- way ANOVA with factors sex and treatment. Where differences were detected, a posthoc

Tukey’s test was conducted to determine between which treatments the differences occurred. Due to initial thiamine testing of baseline (Wild) A. butcheri yielding innacurate results, only one thiamine liver concentration value was obtained, and therefore was not included in statistical analysis.

Graphs showing the mean values for each of the condition indices, blood serum and liver parameters for males and females fed each of the three diets were compiled using

Microsoft Excel 2016.

29 3 Results

3.1 Nutritional composition of diets

Protein levels in the Breed-M and C2 diets (> 30 g 100 g-1) were high compared to the control diet of squid and pilchard (< 22 g 100 g-1) which had a much higher moisture content (Table

3.1).

Major antioxidant levels were greatest in the Breed-M and C2 feeds. For example, selenium was twice as high in Breed-M (1.5 mg kg-1) and C2 (1.8 mg kg-1) than the control diet (0.83 mg kg-1). Vitamin B (thiamine) levels varied markedly among diets, being greatest in Breed-M (150 µg g-1) followed by C2 (25.3 µg g-1) and very low in the control diet components, i.e. 0.85 and 0.5 µg g-1 in pilchards and squid respectively. Vitamin E concentration was similarly low in the control diet (P: 3.9 mg kg-1, S: 22.6 mg kg-1) in comparison to Breed-M (396.2 mg kg-1) and C2 (499.6 mg kg-1).

Trace mineral levels in Breed-M and C2 feeds far exceeded those recorded from pilchards and squid. Iron, zinc and manganese were particularly low in the control diet, with C2 providing the highest concentrations of most trace minerals with the exception of copper, which was highest in pilchards (40 mg kg-1).

-1 Peroxide values of pilchards (14.5 mEq 02 kg fat ) were almost twice as high as the

-1 -1 formulated feeds Breed-M (5.6 mEq 02 kg fat ) and C2 (6.6 mEq 02 kg fat ), with free fatty acids at least three times the levels in pilchard than Breed-M and C2 (Table 3.1).

Rancidity values were highest in pilchards and least in the squid with MDA concentrations of 134.8 and 4.1 µmol kg-1 respectively. Breed-M and C2 diets were similarly low (14.4 and 31.2 µmol kg-1 respectively) compared to pilchards.

30 Table 3.1. Summary of analysis on feed nutrition and rancidity values for three aquaculture diets, Control (Pilchard and Squid), Breed-M and C2. (*not detected) (AR; as received).

Analyte Pilchard Squid BreedM C2 Proximate composition (g 100 g-1) Wet Weight Moisture 70.7 79.0 49.6 46.3 Crude protein 22.8 16.1 31.2 35.5 Fat Total 2.5 0.6 7.3 6.8 Carbohydrate 1.0 2.6 5.1 4.9 Ash 3.0 1.7 6.8 6.5

Dry Weight (%) Crude protein 77.8 76.7 61.9 66.1 Fat Total 8.5 2.9 14.5 12.7 Carbohydrate 3.4 12.4 10.1 9.1 Ash 10.2 8.1 13.5 12.1

Energy (kj 100 g-1 AR) 497.0 339.0 888.0 939.0

Free fatty acids (AR) Acid value (mgKOH g-1) 33.20 0.00* 10.10 4.30 Free fatty acids (as oleic acid) (%) 16.70 0.00* 5.10 2.20 -1 Peroxide value (meq 02 kg fat ) 14.50 0.00* 5.60 6.60

Rancidity values (AR) TBARS (mg kg-1) 8.80 0.50 3.90 2.20 MDA (µmol kg-1) 134.80 4.10 14.44 31.20

Vitamins (AR) Vitamin B (Thiamine) (µg g-1) 0.85 0.50 150.00 25.30 Vitamin E (mg kg-1) 3.90 22.60 396.21 499.60

Minerals (g kg-1 AR) Sodium 2.50 4.20 5.30 5.50 Potassium 5.70 3.00 4.90 5.50 Calcium 9.90 0.30 8.70 12.00 Phosphorus 8.60 2.60 8.00 13.00 Magnesium 0.63 5.70 1.00 1.60

Trace Minerals (mg kg-1 AR) Iron 43.00 4.00 66.00 330.00 Zinc 30.00 19.00 61.00 110.00 Manganese 1.40 0.50 13.00 27.00 Selenium 1.10 0.56 1.50 1.80 Copper 1.20 40.00 7.50 13.00 Boron 1.10 1.70 2.50 2.90 Chromium 0.03 0.03 0.28 0.52 Cobalt 0.02 0.01 0.04 0.32 Nickel 0.02 0.05 0.22 0.32 Molybdenum 0.04 0.018 0.18 0.10 NB: Control diet is a 1:1 wet weight ratio pilchard and squid, Breed-M powder is combined with water at 53:47 and C2 larval diet combined with gelatine (1:1 weight).

31 Breed-M had the highest saturated (1561.9 mg 100 g-1), mono-unsaturated (520.5 mg 100 g-1) and poly-unsaturated (2197.3 mg 100 g-1) fatty acid concentrations, while C2 had similarly high concentrations in each of saturated, mono-unsaturated and poly- unsaturated (1397.9, 446.8, and 1759.8 mg 100 g-1 respectively. Pilchard saturated (929.0 mg 100 g-1) and poly-unsaturated (973.5 mg 100 g-1) fatty acid concentrations were almost equal, while squid had less than half the concentration of saturated (216.1 mg 100 g-1), mono-unsaturated (29.5 mg 100 g-1) and poly-unsaturated (465.4 mg 100 g-1) fatty acids than each other dietary component (Table 3.2).

DHA (C22:6n3) was highest in Breed-M (948 mg 100 g-1), substantially higher than

C2 (624 mg g-1), which contained a slightly higher amount than pilchards (570 mg g-1).

Squid contained the least of both DHA and EPA (307 and 124 mg g-1) and its control diet counterpart had a likewise low level of EPA (171 mg g-1) while C2 and Breed-M (585 and

391 mg g-1) provided more substantial amounts (Table 3.2).

32 Table 3.2. Summary of fatty acid profiles for three aquaculture diets, Control (Pilchard and Squid), Breed-M and C2.

Analyte (as received) Pilchard Squid Breed-M C2 Analysed fatty acids (mg 100 g-1) Saturated Fatty Acids C8:0 Caprylic 0.00 0.00 0.00 0.00 C10:0 Capric 0.36 0.00 0.52 1.39 C11:0 Undecanoic 0.35 0.00 0.00 0.00 C12:0 Lauric 4.86 0.00 3.61 4.49 C13:0 Trisdecanoic 3.57 0.00 2.75 1.64 C14:0 Myristic 160.12 15.78 232.01 279.29 C15:0 Pentadecanoic 33.47 2.51 30.42 16.61 C16:0 Palmitic 491.66 157.82 998.51 827.83 C17:0 Margaric 63.29 8.77 30.88 26.25 C18:0 Stearic 139.38 27.46 207.01 189.48 C20:0 Arachidic 9.01 0.00 11.88 7.44 C21:0 Henicosanoic 3.58 0.00 8.39 2.12 C22:0 Docosanoic 6.21 0.00 8.11 2.78 C23:0 Tricosanoic 13.11 3.71 21.18 20.61 C24:0 Tetracosanoic 0.00 0.00 6.58 0.00 Total Saturated Fatty Acids 928.97 216.05 1561.85 1397.93

Mono-unsaturated Fatty Acids C14:1n5 Myristoleic 0.58 0.29 2.94 5.46 C15:1 Pentadecenoic 3.72 0.57 0.00 0.38 C16:1n7 Palmitoleic 77.56 4.92 221.84 211.54 C17:1 Heptadecenoic 2.66 0.55 27.83 35.18 C20:1 Eicosenoic 8.02 23.13 213.50 136.58 C22:1n9 Docosenoic 3.53 0.00 23.60 34.98 C24:1 Tetracosenoic 18.54 0.00 30.78 22.66 Total Mono-unsaturated Fatty Acids 114.61 29.46 520.50 446.78

Poly-unsaturated Fatty Acids C18:2 cis 48.38 1.69 491.91 209.86 C18:3n6 Gamma Linolenic 3.51 0.00 4.33 8.48 C18:3n3 Alpha Linolenic 39.27 1.08 75.85 72.16 C18:4n3# Steridonic 43.06 1.23 94.67 146.31 C20:2 8.24 2.39 12.95 7.77 C20:3n6 Dihomo-y-linolenic 7.43 0.00 8.11 10.06 C20:4n6 Arachidonic 36.00 15.46 54.57 36.34 C20:3n3 Eicosatetraenoic 4.69 7.91 9.45 11.18 C20:5n3 Eicosapentanaeoic 170.70 123.73 390.91 585.06 C22:2 0.00 0.00 0.00 0.00 C22:4n6# 17.21 2.07 44.80 1.53 C22:5n3# Docosapentaenoic 24.58 2.60 61.92 46.69 C22:6n3 Docosahexaenoic 570.42 307.26 947.84 624.42 Total Poly-unsaturated Fatty Acids 973.49 465.42 2197.29 1759.86

Trans saturated C18:1cis+trans 119.62 15.63 695.59 513.77 C18:2 trans 1.17 0.00 0.55 3.79 NB: Control diet is a 1:1 wet weight ratio of pilchard and squid, Breed-M powder combined with water at 53:47 ratio respectively and C2 larval diet combined with gelatine (1:1 weight).

33 The concentration of DHA (C22:6n3) was higher than EPA (C20:5n3) in each dietary component, with the highest DHA:EPA ratio in pilchards (3.3:1) followed by squid (2.5:1) and Breed-M (2.4:1). C2 was the closest to an even ratio with EPA content being slightly lower than DHA (1.07:1). DHA was highest in Breed-M (948 mg 100 g-1), substantially higher than C2 (624 mg g-1) and pilchards (570 mg g-1). Conversely squid contained the least amount of DHA and EPA (307 and 124 mg g-1) compared to the pilchard EPA content (171 mg g-1), while C2 and Breed-M (585 and 391 mg g-1) provided more substantial amounts (Figure 3.1).

1

0.9

0.8

0.7

0.6 0.5 C22:6n3 (DHA) 0.4 Proportion C20:5n3 (EPA) 0.3 0.2 0.1 0 Pilchards Squid Breed-M C2 Diet

Figure 3.1. Dietary levels of essential fatty acids docosahexaenoic acid (C22:6n3) and eicosapentaenoic acid (C20:5n3) for three aquaculture diet components, control (Pilchard and Squid), Breed-M and C2.

34 Fish in each tank were fed the equivalent of 4.18 kj g-1 according to 2.5% of their body weight, accurate within 10kj for the entire trial, with adjustment for mortality. Fish on the control diet consumed a total of 26.18 kg of feed, compared to 12.10 kg and

13.43 kg for Breed-M and C2, respectively.

Table 3.3. Overall feed summary for Acanthopagrus butcheri broodstock subject to a feed trial of three aquaculture diets. Total feed input summary

Control Breed-M C2 Initial Biomass 7,849.55 7,089.35 8,936.77 Final Biomass 7,663.34 7,819.03 9,676.39 Initial n 24 24 24 Final n 22 21 22 Initial Weight Average (g) 327.06 (± 15.6) 295.39 (± 17.0) 372.37 (± 31.5) Final Weight Average (g) 348.33 (± 17.0) 372.33 (± 15.2) 439.84 (± 29.7) Weight change (g) 21.27 76.94 67.47 Weight Fed (g) 26,177.66 12,098.78 13,433.56 Dry Weight Fed (g) 6,583.68 6,097.79 7,213.82 Energy Fed (kj) 109,422.62 107,437.17 126,141.13 Protein Fed (g) 5,091.56 3,774.82 4,768.91

35 3.2 Fish condition

3.2.1 Growth Fish fed the control diet displayed particularly low growth over the 137 days of the trial (7.91% weight gain ± 2.20 SE) compared to those fed Breed-M (17.17% ± 2.17) and

C2 (18.31% ± 2.27). A three-way ANOVA demonstrated that the extent of weight gain differed with treatment (P = 0.002). Differences were detected for tank type (P = 0.025) but posthoc Tukey’s test revealed differences between females from mixed tanks and females from sex only tanks were not significantly different (P = 0.10) to α. Differences between sex were not significantly different ( P = 0.66) and no interactions were detected.

A three-way ANOVA conducted using the variance in mean weight gain showed statistically significant differences (P < 0.001) between treatments, with no interaction between sex and diet. Posthoc Tukey’s test revealed differences in mean weight gain were significant between Breed-M and control (P = 0.016) and C2 and control (P < 0.001), whereas differences between Breed-M and C2 were not detected (P = 0.9).

Dry weight food conversion ratios among treatments differed significantly (P =

0.006), with values for the control being higher than those for Breed-M (P = 0.02) and

C2 (P = 0.008) which did not differ from each other. Differences between sex were not detected (P = 0.9).

Specific growth rates were significantly different between treatment (P < 0.001), with those differences being due to the pairwise comparison between the control and each of Breed-M (P = 0.007) and C2 (P = 0.001) which did not differ from each other.

36

Table 3.4. Acanthopagrus butcheri body condition indices for females and males fed one of three aquaculture diets, control (squid and pilchard), Breed-M and C2 of all fish in male only, female only and mixed sex tanks (mean ± standard error) (* = significant to α [0.05]).

Control Breed-M C2 Female (n=12) Male (n=12) All Control Female (n=12) Male (n=12) All Breed-M Female (n=12) Male (n=12) All C2 (n=24) (n=24) (n=24) Mean Weight Gain (g) 26.39 ± 8.69 17.89 ± 9.20 22.14 ± 6.25 54.61 ± 8.07 44.18 ± 8.86 49.40* ± 5.96 58.68 ± 10.21 75.14 ± 12.73 66.91* ± 8.16

Mean Weight Gain (%) 9.25 ± 3.49 6.57 ± 2.78 7.91 ± 2.20 18.45 ± 3.16 15.89 ± 3.08 17.17* ± 2.17 14.05 ± 2.27 22.58 ± 3.63 18.31* ± 2.27

Food Conversion Ratio 94.16 ± 31.83 34.94 ± 4.96 67.51 ± 24.08 20.04 ± 8.19 12.38 ± 1.63 16.38* ± 4.33 13.66 ± 3.54 11.02 ± 4.03 12.40* ± 2.62

Specific Growth Rate 0.06 ± 0.02 0.05 ± 0.02 0.05 ± 0.01 0.12 ± 0.02 0.11 ± 0.02 0.12* ± 0.01 0.11 ± 0.02 0.14 ± 0.02 0.13* ± 0.02

38

3.2.2 Feed efficiency Breed-M (P = 0.02) and C2 (P < 0.001) had significantly higher energy efficiency ratios than the control diet (Table 3.5). Protein efficiency ratios yielded the same differences between the control diet and Breed-M (P = 0.001) and C2 (P < 0.001) showing treatment diets working around four times the efficiency.

The values for the GSI of A. butcheri broodstock were not found to be dependent on the aquaculture diets (P = 0.4) or sex (P = 0.8) with no significant difference detected.

Variance was detected between tank type (P = 0.01) and a posthoc Tukey’s showed evidence of differences between female only tanks to male only tanks (P = 0.01) and mixed sex tanks (P = 0.04).

For hepatosomatic index, an interaction between sex and treatment was detected (P

= 0.02), however a posthoc Tukey’s test detected no honestly significantly different interactions. Differences were found between tank type however, with HSI of fish from female only tanks higher than those from mixed tanks (P = 0.001) and male only tanks

(P < 0.001).

39

Table 3.5. Feed efficiency indices for female and male Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (pilchard and squid), Breed-M and C2 (mean ± standard error) (* = significant to α [0.05]).

Control Breed-M C2 Female Male All Control Female Male All Breed-M Female Male All C2 (n= 12) (n= 12) (n= 24) (n= 12) (n= 12) (n= 24) (n= 12) (n= 12) (n= 24) Energy Efficiency Ratio 0.006 ± 0.00 0.005 ± 0.00 0.007 ± 0.00 0.012 ± 0.00 0.010 ± 0.00 0.011* ± 0.00 0.014 ± 0.00 0.013 ± 0.00 0.014* ± 0.00 Protein Efficiency Ratio 0.121 ± 0.04 0.105 ± 0.05 0.113 ± 0.04 0.353 ± 0.06 0.276 ± 0.05 0.314* ± 0.05 0.371 ± 0.11 0.355 ± 0.06 0.363* ± 0.07 Gonadosomatic Index 2.81 ± 0.55 1.99 ± 0.61 2.40 ± 0.58 2.81 ± 0.84 1.58 ± 0.20 2.22 ± 0.64 2.07 ± 0.63 1.37 ± 0.19 1.69 ± 0.39 Hepatosomatic Index 1.31 ± 0.09 0.96 ± 0.03 1.13 ± 0.09 1.32 ± 0.12 1.15 ± 0.07 1.24 ± 0.10 1.36 ± 0.07 1.11 ± 0.05 1.22 ± 0.07

40 3.3 Blood analysis Alanine aminotransferase (ALT) levels in the plasma of A. butcheri appeared relatively similar across treatment diets, with the exception of females fed the Breed-M diet, due to one particularly high reading of 178 U L-1 (Figure 3.2). The mean ALT of the other female broodstock fed Breed-M was 22.25 U l-1, similarly low as the other treatments of males and females. This similarity was reflected with a two-way ANOVA showing no evidence of a difference between either sex (P = 0.3) or treatment (P = 0.6).

120

100

80

1 - 60

U U L 40

20

0 Control Control Breed-M Breed-M C2 Female C2 Male Female Male Female Male

Diet/Sex

Figure 3.2. Alanine aminotransferase levels in plasma for female and male Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (pilchard and squid) (n = 2f, 2m), Breed-M (n = 2f, 2m) or C2 (n = 2f, 2m) (mean ± standard error).

41 Glutamate dehydrogenase (GLDH) showed a similar pattern as ALT, with one exception from the group of females fed Breed-M showing abnormally high levels, at 150

U L-1, excluding this, lowered the average from 44.33 U L-1 to 6.83 U L-1. Once again, no significant differences were detected between sex (P = 0.4) and treatment (P = 0.6) for

GLDH (Figure 3.3).

90

80

70

60

1 - 50

U U L 40

30

20 10 0 Control Control Breed-M Breed-M C2 Female C2 Male Female Male Female Male

Diet/Sex

Figure 3.3. Glutamate dehydrogenase in the plasma of female and male Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (pilchard and squid) (n = 2f, 2m), Breed-M (n = 2f, 2m) or C2 (n = 2f, 2m) (mean ± standard error).

42 Lipase activity in plasma of broodstock fed the Breed-M (F: 7.75 ± 0.5, M: 7.75 ±

0.5) and control diets (F: 7.00 ± 0.4, M: 7.00 ± 0.0) were very similar, with the C2 diet

(F: 8.75 ± 0.8, M: 7.65 ± 2.3) yielding just slightly higher means than Breed-M, with the exception of one male fed C2 having particularly high lipase activity measured at 26 U L-

1 (Figure 3.4). However, the lipase levels were not found to differ significantly with sex

(P = 0.46) or treatment (P = 0.18).

18

16

14

12

1 - 10

U U L 8

6

4 2 0 Control Control Breed-M Breed-M C2 Female C2 Male Female Male Female Male

Diet/Sex

Figure 3.4. Total serum lipase activity in plasma of female and male Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (pilchard and squid) (n = 2f, 2m), Breed- M (n = 2f, 2m) or C2 (n = 2f, 2m) (mean ± standard error).

43 Cholesterol levels in the blood plasma of A. butcheri broodstock differed with sex

(P < 0.001), with females showing elevated levels compared to the males from their respective treatment diets (Figure 3.5). Cholesterol levels between diets were not found to be significantly different (P = 0.4).

18

16

14

12

1 - 10 8

n.mol n.mol L 6

4 2 0 Control Control Breed-M Breed-M C2 Female C2 Male Female Male Female Male

Diet/Sex

Figure 3.5. Plasma cholesterol levels of female and male Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (pilchard and squid) (n = 2f, 2m), Breed-M (n = 2f, 2m) or C2 (n = 2f, 2m) (mean ± standard error).

44 Urea concentration in plasma of A. butcheri broodstock was very similar from those fed the control and C2 diets, with those fed the Breed-M diet having lower levels in plasma of both males and females (Figure 3.6). A two-way ANOVA provided evidence of reduced urea in broodstock fed the Breed-M diet (P = 0.06). Urea in the plasma of both the control and C2 groups was slightly lower in females, however this was not a substantial or statistically significant difference (P = 0.4). Tukey HSD test revealed differences between Breed-M and control (P = 0.1) and Breed-M and C2 (P = 0.08) to not be conclusive with the small sample size.

70

60

50

1 - 40

g g L

m 30

20 10

0 Control Control Breed-M Breed-M C2 Female C2 Male Female Male Female Male

Diet/Sex

Figure 3.6. Urea concentration of plasma from female and male Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (pilchard and squid) (n = 2f, 2m), Breed- M (n = 2f, 2m) or C2 (n = 2f, 2m) (mean ± standard error).

45 Triglyceride levels in the plasma of males fed the C2 diet were substantially higher

(6.3 n mol-1 ± 0.6) than any other group, with control having the lowest concentrations for both males (2.43 n.mol L-1 ± 0.7) and females (2.38 n.mol L-1 ± 0.6). Females fed the

C2 diet however, recorded substantially lower triglycerides in plasma at 2.6 n mol-1 (±

0.3). Differences were found to be significantly different dependent on sex (P = 0.03) and diet (P = 0.03), with the differences occurring between diets C2 and control (P = 0.03).

An interaction effect model detected no interactions.

8

7

6

1 - 5

4

n.mol n.mol L 3 2

1 0 Control Control Breed-M Breed-M C2 Female C2 Male Female Male Female Male

Diet/Sex

Figure 3.7. Triglycerides in plasma of Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (pilchard and squid) (n = 2f, 2m), Breed-M (n = 2f, 2m) or C2 (n = 2f, 2m) (mean ± standard error).

46 A two-way ANOVA showed total protein measurements from the plasma of A. butcheri showed significant variance (P = 0.05), with a posthoc Tukey’s test detecting a significant difference between the C2 (50.0 g L-1 ± 1.6) and control (44.1 g L-1 ± 1.6) diets

(P = 0.04).

Although plasma from males contained lower total protein levels than females in each diet, this trend was not pronounced enough to generate a significant difference (P =

0.17) (Figure 3.8).

60

50

40

1 - 30

g L 20

10

0 Control Control Breed-M Breed-M C2 Female C2 Male Female Male Female Male

Diet/Sex

Figure 3.8. Total protein in the plasma of Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (pilchard and squid) (n = 2f, 2m), Breed-M (n = 2f, 2m) or C2 (n = 2f, 2m) (mean ± standard error).

47 3.4 Liver analysis

3.4.1 Vitamins A two-way ANOVA revealed that the thiamine levels in the livers of fish fed different treatment diets were significantly different (P < 0.001). Differences were also detected between males and females (P = 0.02), although no interaction was detected. A posthoc Tukey’s test detected significant differences between the control (1.0 mg kg-1 ± 0.007) and Breed-M diets (5.0 mg kg-1 ± 0.54) (P < 0.001) as well as the control and C2 diets (3.4 mg kg-1 ± 0.64) and control (P = 0.02) (Figure 3.9). Although thiamine in the wild A. butcheri does not qualify for statistical analysis (n = 1), it appearred much lower than Breed-M and C2.

7

6

5

1 - 4

3 mg mg kg

2

1

0 Control Control Breed-M Breed-M C2 C2 Male Wild Wild

Female Male Female Male Female Female Male

Treatment

Figure 3.9. Thiamine (vitamin B) concentration in livers of Acanthopagrus butcheri brood stock fed one of three aquaculture diets: control (pilchard and squid) (n = 2f, 2m), Breed-M (n = 2f, 2m) or C2 (n = 2f, 2m) in comparison to wild caught stock (n = 1) (mean ± standard error).

48 Variation in vitamin E concentration was detected between treatment (P < 0.001) but sex had no influence (P = 0.34). Posthoc Tukey’s test detected significant differences, with the control diet having evidently lower concentrations than Breed-M (P = 0.001) and

C2 (P < 0.001). Wild fish were also found to have significantly lower concentrations than

Breed-M (P < 0.001) and C2 (P < 0.001) (Figure 3.10).

450 400 350

300

1 - 250

200 mg mg kg 150 100 50 0 Control Breed-M C2 Wild Treatment

Figure 3.10. Vitamin E (tocopherol) concentration in livers of Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (pilchard and squid) (n = 4), Breed-M (n = 4) or C2 (n = 4) in comparison to wild caught stock (n= 8) (mean ± standard error).

49 3.4.2 Rancidity values Thiobarbituric acid reactive substances (TBARS) concentrations in livers of A. butcheri were significantly different between sex (P = 0.013) and treatment (P = 0.08).

Males recorded higher TBARS than females across all treatments with male fish fed the control diet having the highest TBARS readings (6.7 mg kg-1 ± 0.7) (Figure 3.11). Female fish fed the control diet were also higher than the other treatments, however with highly variable results (14.22 U mol kg-1 ± 7.97). Females fed C2 had the lowest TBARS readings (1.6 mg kg-1 ± 0.4), excluding the wild fish, which were lower for both females and males (1.25 ± 0.15 and 1.5 ± 0.15 mg kg-1 respectively). TBARS were significantly lower than fish fed the control for those fed Breed-M (P = 0.09) and C2 (P = 0.02). Wild fish

(P < 0.001) were also found to be statistically lower than control.

The TBARS rancidity values for the aquaculture diets were reflected closely by malondialdehyde (MDA) concentration for each sex and diet, with only a slight variation in MDA level in female fish fed C2, having a relatively lower MDA reading. Wild fish, however, had relatively higher MDA concentrations in comparison to TBARS (Figure

3.12). MDA was significantly lower in fish fed C2 (P = 0.039) in comparison to those from the control diet, however differences between Breed-M (P = 0.18) were not found to be statistically significant. Wild fish (P = 0.02) were found to have significantly lower

MDA readings than those fed the control diet.

50

8.00

7.00 6.00

1 5.00 - 4.00

mg mg kg 3.00

2.00

1.00 0.00 Control Control BreedM BreedM C2 C2 Male Wild Wild Female Male Female Male Female Female Male

Treatment/Sex

Figure 3.11. Thiobarbituric acid reactive substances (mean ± standard error) detected in livers of Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (pilchard and squid) (n = 4), Breed-M (n = 4) or C2 (n = 4) in comparison to wild caught stock (n = 2f, 6m).

51 30.00

25.00

20.00 1 -

15.00

µmol kgµmol 10.00

5.00

0.00 Control Control BreedM BreedM C2 C2 Male Wild Wild Female Male Female Male Female Female Male

Treatment/Sex

Figure 3.12. Malondialdehyde in the livers of Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (squid and pilchard) (n = 4), Breed-M (n = 2f, 2m) or C2 (n = 2f, 2m) in comparison to wild caught stock (n = 2f, 6m) (mean ± standard error).

52 3.4.3 Fatty acids

Total omega-3 long chain polunsaturated fatty acid content in the livers of A. butcheri broodstock was highest in those fed the C2 diet. Livers from C2 fed fish also had the highest

EPA (C20:5n3#) (279.66 mg 100 g-1) content, despite having a lower DHA (731.17 mg 100 g-1) content than fish fed on Breed-M (793.27 mg 100 g-1) and control (841.69 mg 100 g-1) diets (Figure 3.13). The ratio of DHA:EPA in A. butcheri is highly skewed to DHA in all diets, with control being the highest (~6.2:1), Breed-M similarly high (~5:1) andC2thelowest (~2.6:1) (Figure 3.).

53

1600

1400

1200

1

- 1000 800 C22:6n3 (DHA

600 C22:5n3# mg mg 100g 400 C20:5n3 (EPA) 200 C20:3n3 0 C18:4n3# C18:3n3

Diet

Figure 3.13. Cumulative total (mean concentration) of long chain omega-3 polyunsaturated fatty acids of livers from Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (pilchard and squid) (n = 4), Breed-M (n = 4) or C2 (n = 4) in comparison to wild caught stock (Wild).

1

0.9

0.8 0.7

0.6 0.5 C22:6n3 (DHA) 0.4 Proportion C20:5n3 (EPA) 0.3

0.2 0.1 0 Control Breed-M C2 Wild Diet

Figure 3.14. Proportion of DHA and EPA concentration (mean) in livers of Acanthopagrus butcheri broodstock fed one of three aquaculture diets, control (squid and pilchard) (n = 4), Breed-M (n = 4) or C2 (n = 4).

54 The concentration of EPA in the liver was significantly different among diets (P <

0.001) but not sex (P = 0.86). Those fed the C2 diet had levels significantly higher than those fed the Breed-M (P < 0.001) and control diets (P < 0.001) compared to wild caught fish (P < 0.001).

350

300

250

1 - 200

150

mg 100 g 100

50

0 Control Control Breed-M Breed-M C2 C2 Male Wild Wild

Female Male Female Male Female Female Male

Treatment

Figure 3.15. Eicosapentaenoic acid (EPA) concentrations in livers of Acanthopagrus butcheri fed one of three aquaculture diets, control (pilchard and squid) (n = 2f, 2m), Breed-M (n = 2f, 2m) or C2 (n = 2f, 2m) in comparison to wild caught stock (Wild) (mean ± standard error).

55 No significant differences were detected in DHA between sex (P =0.4) or treatment

(P = 0.3). Liver DHA concentration for Breed-M, C2 and Wild stock was higher in males, while females fed the control diet had > 1000 mg g-1 compared to the males at 666 mg g-1

(Figure 3.15).

1200

1000

800 1 - 600

mg mg 100 g 400

200

0 Control Control Breed-M Breed-M C2 C2 Male Wild Wild

Female Male Female Male Female Female Male

Treatment

Figure 3.16. Docosahexaenoic acid (DHA) concentrations in livers of Acanthopagrus butcheri fed one of three aquaculture diets, control (pilchard and squid) (n = 4), Breed-M (n = 4) or C2 (n = 4) in comparison to wild caught stock (n = 2F and 2M) (mean ± standard error).

56 4 Discussion Understanding the impacts diets have on the physiological condition and performance of fish is essential in increasing aquaculture production and lowering costs.

Knowledge of specific nutrition is vital in production, as the chemical processes in digestion and dietary nutrient utilisation is intricate and can differ at the species level. The critical stage in the growth and development of fish is in their egg and larval stage, which is supplemented by broodstock diets (Sofia, 2018). Broodstock nutrition is therefore essential in aquaculture and requires more in-depth research to determine optimal nutrition (Izquierdo, 2001). The sparid Acanthopagrus butcheri is an important recreational and commercially targeted species in Australia and presents potential for small scale food production with increased knowledge of optimal nutrition requirements.

The aim of this study was to investigate three broodstock diets namely; a common aquaculture diet comprising pilchards and squid (control), a commercially available conditioning diet (LANSY Fish Breed-M Concentrate Powder) and a powdered marine larval diet (C2), fed to wild-caught

A. butcheri over a four and a half month period encompassing the spawning season to compare the transfer of the essential fatty acids EPA and DHA, and antioxidants, thiamine and Vitamin E into the livers. The results revealed the detrimental impacts of feeding rancid, frozen, whole fish to broodstock and provided evidence that developing broodstock feeds is required to facilitate the maintenance of long-term health of broodstock and their offspring.

4.1 Condition analyses While fish growth is traditionally not of importance in broodstock, growth and condition indices revealed numerous characteristics of the experimental diets that would prove vital in selection of nutrition for broodstock. Due to the opportunistic feeding nature of A.

57 butcheri, combined with the degraded condition of the Peel-Harvey Estuary (Valesini et al., 2019), condition of wild caught fish may be suboptimal. Broodstock were also help for up to three weeks without food before the initial weigh and measure, while this is not detrimental to the health of fish (Pérez‐Sánchez et al., 1994), it is expected that any lost condition would be made up for during the feed trial, resulting in a positive weight gain.

In the experiment, the A. butcheri broodstock fed the formulated diets of Breed-M and C2 experienced significantly higher weight gains than those fed the control diet of squid and pilchard. As all fish were fed the same amount of energy, the improved weight gain of the fish fed on the formulated diets could reflect the higher fatty acid content and superior profile. It should be noted that some individuals recorded weight loss during the trial; for example, one tank belonging to the control treatment recorded an overall weight loss (mean ~3g). This could, in part, be attributed to natural hierarchies and aggression which was observed during this trial, which has been reported by other authors such as

Renn et al., (2016). There was a major variation in weight change of individuals ranging from a 12% weight loss (control) to a 39% gain (C2). This was likely a result of the aggression displayed around feeding time, with dominant fish being observed chasing other fish away from the surface.

Feed efficiency ratios (FER) were highly variable, with the control diet yielding the highest values indicating the high feed requirements to growth yield in comparison to the

Breed-M and C2 diets. All FER values were found to be inefficient, with food intake resulting in relatively poor growth. Although this weight gain was particularly poor in fish fed the control diet of squid and pilchards. This was most likely influenced by the high amount of moisture in wet diets, with the broodstock consuming more food to achieve the same energy intake. Feed efficiency has also been found to have a positive correlation to lipid content feeds (Moutinho et al., 2017), supporting this study’s findings with the lipid composition of the pilchard and squid being particularly low compared to

58 the formulated diet. While gonadosomatic index (GSI) is commonly used to assess reproductive stage and condition of broodstock (Yadav, 2016; Fontoura et al., 2018), it is not effective for assessment of species that spawn multiple times in a breeding season such as A. butcheri (Brewer et al., 2008; Sarre et al., 2000). GSI was used to assess the final condition of the gonads as some fish stocked in mix sex tanks had been induced to spawn. Although statistical analysis revealed no significance difference, results did suggest females generally have higher GSIs than males albeit it is variable. A small variance in the visual display of the data, suggests that C2 may produce generally lower

GSIs, however the data extracted from this trial were not conclusive, likely due to the variance in their spawning times and due to the small sample sizes available for testing.

Measurements of gonads prior to spawning may convey the impact these diets have on

GSI, before they can expend energy on reproduction. GSI has been reported to respond similarly to the liver, conditioning according to the season (Akel and Moharram, 2007).

In place of GSI, HSI is an effective index for assessing spawning condition as it changes markedly according to seasons (Akel and Moharram, 2007).Values of the

Hepatosomatic index (HSI) only differed significantly with tank type. Tanks containing only female fish had significantly higher HSI values than those with only males or mixed sex tanks. It has been reported that HSI is lowest immediately after spawning in multiple species such as Limanda limanda (Common Dab) and Mugil curema (White Mullet)

(Htun-Han, 1978; Albieri et al., 2010; Saleh and Ali, 2019). This is important support that the mixed sex tanks spawned in response to hormonal induction, expending energy stores from their livers. This highlights the importance for optimal liver condition as it is essential for nutrient storage for reproductive activity. Fish from the male only tanks also had significantly lower HSIs, while other fish species present distinct differences of HSI between sex (Jan and Ahmed, 2016), analysis from this trial did not detect significant differences. Male fish were conditioned to spawn from the beginning

59 of the trial, with many producing milt during weigh and measure. This may suggest that males actively release their sperm without the presence of females.

Due to the feeding nature of A. butcheri, where food is crushed in their mouth before consumption (Sarre et al., 2000), there were likely discrepancies in the volume of food consumed by the fish to what was fed. Feeding using thismethod likely reduced the nutrient intake of fish being fed, particularly with Breed-M, as the feed was observed dissolving in the water out of their gills during mastication. The C2 feed also crumbled as fish ground the pellets, however, most of the feed remained combined in granules allowing the fish to see and consume them later, therefore this was less likely to have had a significant impact on the results.

4.2 Blood biochemistry Biochemical analysis of the blood plasma was used to assist in the identification of tissue damage in response to oxidation from the diets. Parameters of triglycerides, urea and cholesterol provided indicators of the impacts of the nutrition provisions.

The mean alanine aminotransferase (ALT) levels recorded from the plasma of A. butcheri across all treatments was 25.39 U L-1, excluding one female from the Breed-M treatment, recording 178 U L-1. This high ALT level could be a negative response to liver oxidation, as the enzyme is often produced in excess as a direct response to liver damage

(Yamada et al., 2006). Reference levels in the confamilial sparid Sparus auratus ranged from 8-427 U L-1 with a mean of 97 U L-1 (Canfield et al., 1994). Reference levels for another sparid Sparidentex hasta ranged from 239-506.5 U L-1 with a mean of 398.6

(n=143) (Mozanzadeh et al., 2015). Measuring the results of the A. butcheri in reference to the previously stated species would suggest a normal and expected range of ALT, however the distinct difference of one individual would suggest that 178 U L-1 is not expected for this species. While this result is not evidently a result of nutrition from this trial, further investigation into ALT levels for A. butcheri may assist in the potential health 60 implications of this blood serum parameter.

Glutamate dehydrogenase (GLDH) were not affected by the diets consumed by A. butcheri in this study. The same individual that recorded high ALT also had an extremely high GLDH reading. This is likely indicative of oxidative stress to the fish. As blood biochemistry was not analysed prior to the feed trial, blood biochemistry results are not consistent across species for comparison and GLDH results were not affected by the diet there is a likelihood of this being evidence of pre-existing liver damage.

Blood from A. butcheri broodstock fed the C2 diet contained higher levels of total serum lipase than broodstock of Breed-M and the control diets. This difference was marked in the male fish, with females only showing slightly increased levels than the other groups. Increased lipase was likely a response to higher adipose fat in fish fed the

C2 pellets and may not be reflected in females due to their higher fatty acid requirements and utilisation prior to spawning (Zheng et al., 2019).

Cholesterol levels in teleosts have been shown to fluctuate by season and vary from males to females (Sharma et al., 2017). Analysis of the cholesterol in the A. butcheri broodstock reflected this, with males having significantly lower cholesterol levels than females across all treatments. Serum cholesterol fluctuates in response to nutrition, with high dietary nutrients leading to an increase and is also a precursor to sex hormones and is required in larger amounts leading up to spawning season (Sharma et al., 2017).

The quantity of urea in plasma can provide indications of physiological health of stock, as uncharacteristically high levels indicate damage or failure in the kidney, gills and/or liver (Sharma et al., 2017; Zulfiqar et al., 2020). Studies of blood biochemistry for several marine species of teleost, i.e.sparids such as Dicentrarchus labrax, Scophthalmus maximus and Sparus auratus showed plasma urea levels in the range of 44-59 mg L-1.

Urea levels recorded in the wild caught A. butcheri broodstock had urea levels

61 between 30-70 mg L-1, with differences detected between diets, however with a posthoc test, these were not statistically conclusive. Breed-M appeared to be lower and larger sample sizes would provide a clearer outcome.

Triglycerides are often found in high concentrations when quality nutrition is readily provided. Concentrations in the serum of A. butcheri were shown to be significantly higher in males fed the C2 diet in comparison to any other diet of males or females, however females fed C2 had particularly low triglyceride levels. Male and female fish often show different serum levels reflected as a response to reproduction.

Females record higher triglyceride levels prior to spawning, and subsequent lower levels post-spawning (Suljević et al., 2017). Low triglycerides were also indicative of inadequate nutrition, as reflected by fish exposed to starvation (Sharma et al., 2017).

Uncharacteristically low serum protein is often reflected in fish affected by starvation and stress and is reported in fish that are immunocompromised (Maqsood et al., 2009; Zhang et al., 2017). Extreme high levels of total serum protein indicate liver tissue damage, signalling nutritional deficiencies that have caused liver damage (Bayne and Gerwick, 2001; Sharma et al,. 2017). Total serum protein was found to be lower in broodstock fed the control diet than those fed C2. This provides evidence that the pilchards and squid fed to broodstock are negatively impacting their physiological health.

Considering the results of blood biochemistry, further testing of A. butcheri aquaculture diets would provide valuable insights into the health effects of these diets on broodstock. Reference blood serum data will allow for accurate conclusions of physical health of broodstock with baseline and limiting parameter values. Results from total serum protein and triglycerides provided an indication of the damage being caused by whole fish diets fed to broodstock in a short period of exposure. Blood plasma parameters are highly obtainable, with the ability to extract blood from large fish on a repeatable basis without

62 sacrificing them and can provide feedback on the physiology of important reproductive organs in response to nutritional inputs.

4.3 Liver analyses Healthily functioning livers are essential in fish that are intended for reproduction, with their main roles of digestion and breaking down of feed matter assisting in the growth and development in highly critical phases of their offspring, from development of gametes to the utilisation of egg yolks before first exogenous feeding (Migaud et al., 2013). It is therefore of high importance to assess the physiological response of livers, to broodstock nutrition.

Thiamine and vitamin E levels were both higher in treatment diets Breed-M and C2 than the control diet of frozen pilchards and squid, with the presence of these antioxidants included in the manufactured diets to prevent oxidation causing physiological stress in fish (Peng et al., 2009). These are essential inclusions in nutrition of fish, due to the requirement of large proportions of long chain unsaturated fatty acids, which are highly volatile to oxidation (Wander et al., 1998). Thiamine is a highly effective antioxidant, however the presence of the thiaminase enzyme breaks thiamine down, removing its affect as an antioxidant (Shefat, et al., 2018), this occurs in most forage fish such as pilchards and the reduced growth and conditioning of fish fed the pilchard and squid are likely reflective of this deficiency.

Vitamin E is especially effective as an antioxidant in presence of selenium (Gatlin

III et al., 1986; Jaramillo Jr et al., 2009). Selenium was substantially higher, in diets C2 and Breed-M, than the control components pilchard and squid. The degree of tissue oxidation as a response to the insufficient supplementation of these antioxidants in squid and pilchards was tested by measuring malondialdehyde and thiobarbituric acid reactive substances, with high levels indicating proportion of tissue degraded as a result of oxidising feed (Tsikas, 2017). With a small number of livers 63 analysed due to cost, differences between these rancidity parameters did not reveal a significant difference according to the statistical cut-off, however the values recorded may suggest with a longer exposure time and a more robust sample size, a clearly higher oxidation rate may be identified in the livers of fish fed wet diets of pilchard and squid, especially in comparison to C2, which had substantial antioxidant defence incorporated in the diet. As squid had particularly low levels of all nutritional components, this would likely offset the detrimental impacts of pilchards, deflecting identification of the issues with this feed product.

The high rancidity values and low antioxidant contents recorded prior to the feed trial would suggest a negative impact of the pilchard diet, with the oxidation expected to continue for the duration of the product shelf life. Pilchards have been proven to oxidise in frozen storage after just one month (Fitz-Gerald and Bremner, 1998). This is reinforced by the peroxide value in pilchards being more than twice as high as Breed-M and C2 and free fatty acids three times as high.

Essential fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are well documented to be the most important lipids for broodstock reproduction success

(Agh et al., 2020; Luo et al., 2015). While DHA and EPA are highly important, they are highly vulnerable to oxidation and require sufficient antioxidants to combat degradation of feed components (Jin et al., 2017).DHA is required in higher amounts than EPA with a recommendation to feed teleosts at a rate of 1.5:1 (Henrotte et al., 2010). The A. butcheri broodstock fed C2, were provided a 1.07:1 ratio, DHA:EPA, particularly high for EPA, whereas pilchard (3.3:1), squid (2.5:1) and Breed-M (2.4:1) delivered particularly low levels of EPA.

Breed-M contained the highest level of dietary total fatty acids, however livers from fish fed C2 had the highest amount of omega-3 essential fatty acids, while maintaining

64 EPA at the highest ratio, closest to general recommendations for fish (Jin et al., 2017).

This possibly indicates better absorption due to a more complete fatty acid profile, however further investigation into the species requirement would provide better indication of the most suitable fatty acid profile. While high levels of fatty acids immediately improve reproduction and growth, it has also been reported that higher ratios of DHA:EPA can lead to lower antioxidant values therefore leading to tissue oxidation and reduced future production (Jin et al., 2017). Observations during dissections showed fat was retained around the internal tissues of many of the broodstock fed C2. This would indicate overuse of fatty acids within the diet.

The differences in TBARS were not found to be statistically different between treatments, however, the prevalence of TBARS were influenced by sex. As the sample sizes were particularly small, and there does appear to be higher TBARS from the control diet. Thus, a feed trial with more subjects and longer exposure to the diets may provide more definitive evidence on the oxidation of livers for A. butcheri fed these diets.

Oxidation levels may also have been impacted by the high levels of copper (40 mg kg-1) detected in pilchards. High levels of copper have been found to damage livers of fish and impair growth (Watanabe et al., 1997), although research into the dietary limits of copper in marine fish is limited and varies from freshwater fish due to mineral uptake from seawater and subsequent higher tolerance levels (Dang et al., 2009).

65 5 Future directions Reference levels of blood biochemistry for wild A. butcheri would provide a better insight into the impacts dietary intake is having on wild caught broodstock. Most parameters are sex and even season dependent, so utilising them as biomarkers for fish physiology to a species level would require reference levels and larger sample sizes for each sex in order to accurately identify nutrition respondent parameters. Blood serum biomarkers may also help with wild A. butcheri populations, using the parameters to analyse nutrition as an indication of the estuary system health.

In order to ultimately define the impacts aquaculture nutrition has on A. butcheri broodstock, further investigation into the specific requirements of fatty acids and their ratios are needed. DHA and EPA ratios vary to each species and the inclusion of other fatty acids even in small amounts can influence the integrity of the feed product, altering the transfer of nutrients from feed, to liver and tissue and subsequently to offspring.

The importance of broodstock come down to their production of large spawns with viable larvae. In order to get a full picture of the performance of broodstock, aquaculture diets require an in-depth analysis into the subsequent performance of their larvae. The impacts of rancid feeds with inadequate nutritional supplementation of fatty acids and

66 antioxidants is likely to have escalating effects on broodstock performance over time, therefore long term trials on the larval yield, as well as studies on the survival from hatch to first feed, will provide a better understanding of the negative impacts traditional diets can incur.

Investigation of more micronutrients may help in understanding the intricacy of fish nutrition. e.g. Taurine has been found to have significant improvements in growth for select fish species, however, taurine production is species specific and some species do not experience altered growth rates with its addition (Engrola et al., 2018).

67 6 Conclusions Condition indices provided a good baseline for diet selection criteria with formulated diets

Breed-M and C2 providing better growth and conditioning. HSI provided a valuable analysis of which individuals were spawning, with mixed tank fish having livers that had likely been spent of nutrients. Financial restrictions prevented analysis on all fish, however testing the livers of spawned fish may provide valuable information regarding nutrient utilisation in the spawning process. Blood biochemistry testing provided some indications of stock health that aligned with the feed and liver analyses, reflecting the fatty acid rich formulated diets through triglycerides and retained protein in plasma of fish fed C2.

Breed-M and C2 had limited differences when statistically tested, despite differences observed in data. This is a result of small sample sizes due to the logistical and financial restrictions of such a study in an honours budget for a pilot study. The results suggest further comparisons may clarify the drawbacks and benefits of each diet. From the results of the conducted analyses, further development of both C2 and Breed-M is required to cater to the dietary requirements of A. butcheri and currently the cheaper option of the two, Breed-M, would likely be the logical choice of hatchery managers for this and like species. With the evidence of high rancidity values in the pilchard diet, feeding to valuable broodstock is not recommended. Frozen bait fish used to feed to broodstock are higher in initial rancidity than formulated diets despite the formulated diets having higher rates of more volatile fatty acids which are more prone to oxidation.

68 This is evidence of a direct response to the higher levels of antioxidants in the formulated diets, vitamin E, thiamine and selenium. Breed-M contained the most fatty acids in the diet, however absorption rate was less than C2. This could point to breakdown of nutrients in the feed due to oxidisation, however no evidence of significant tissue damage would suggest the clouding of the water expelled from the gills during feeding is playing a part in loss of nutrients in the water column. Not only is this wasteful, but it leads to poor water quality which in turn can harm stock.

It is worth noting that aquaculture is a profit driven industry and cost of feeding is often the largest operating expense. While frozen fish are initially cheaper than formulated feeds, they are proven to be an insufficient diet for long term stock health.

Broodstock, if not in prime physical condition, produce sub optimal spawns, with smaller yields and higher larval mortality rates. With low antioxidant levels combined with volatile ingredients, frozen fish are likely to continually degrade the tissue of valuable broodstock, leading to declines in reproductive performance over the years. The economic benefits of maintaining healthy broodstock should be an important consideration in selecting nutrition.

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79 8 Appendix

45 Air Temp 40 Water Temp

35

30 25 20

15 Temperature (ºC) Temperature 10 5 0 07 08 09 10 11 Date

Appendix 1. Daily water temperature record of the recirculating aquaculture system and maximum air temperatures recorded at Jandakot weather station Australian Bureau Meteorology (2020) for the duration of the trial.

80 0.50 0.006 0.45 NH4+ 0.005

a) 0.40

)

)

NH3 1 -

1 0.35 0.004 - 0.30 0.003

0.25 L (mg

(mg L (mg 3 3

4 4

0.20 0.002 NH

NH 0.15 0.10 0.001 0.05 0.00 0.000 07 08 09 10 11 Date

5 b)

4 NO2

1

- 3 L

mg mg 2 1 0 07 08 09 10 11 Date

200 c) 150 NO3

1 - 100

mg L 50

0 07 08 09 10 11 Date

Appendix 2. Daily water quality record of the recirculating aquaculture system for duration of + the A. butcheri broodstock feed trial: a) unionised ammonia (NH3) and ammonium (NH4 ), b) Nitrite (NO2) and c) Nitrate (NO3).

81