Thesis (PhD) -- tellenbosch University, 2014. / ENGLISH ABSTRACT: This study is part of the continuing global research effort dedicated to finding alternative aquafeed ingredients, which not only replace fish meal and fish oil as sources of essential nutrients in aquafeeds, but also provide specialist functional properties when included in feeds. Due to constraints in supply of fish meal and fish oil originating from wild capture fisheries, the continually growing aquafeed industry requires new sources of raw materials for the production of high quality feeds. The aim of the study was to investigate the specialist functional properties of feed ingredients (with emphasis on immune-stimulation potential) derived from fish processing waste after enzymatic hydrolysis by endogenous proteolytic enzymes (autolysis). Further aims were to (i) quantify effects of these feed ingredients on production performance of two species relevant to the South African aquaculture industry, namely Mozambique tilapia Oreochromis mossambicus and South African abalone Haliotis midae, (ii) compare performance to commercially available enzymatically produced feed ingredients, and (iii) separate the functional effects of these fish processing waste derived feed ingredients from the acid used to preserve them against bacterial spoilage during the autolysis process. Oil recovered after autolysis of rainbow trout viscera proved to be an attractive feed ingredient due to favourable effects on the non-specific cellular immune function of both Mozambique tilapia and South African abalone. However, in South African abalone, increased immune function due to inclusion of fish oil was accompanied by a significant decrease in production performance. The inclusion of hydrolysed proteins, obtained by autolysis of fish waste, in aquaculture feeds also improved non-specific immunity and survival of Mozambique tilapia significantly – independently of the preserving acid – although the same was not observed for South African abalone. Production performance was dependent on dietary hydrolysed protein inclusion levels in both species; excessive inclusion resulted in decreased production performance. The performance of dietary hydrolysed protein from autolysis and those from commercial production processes were significantly different, possibly as a result of different raw material origins and production processes. It is further shown that formic acid can contribute to improved water stability in abalone feeds, a novel mode of action not previously described. The study concludes that the simple autolysis process for processing of fish waste can provide aquafeed ingredients with immune stimulatory potential, which can contribute to improved production performance in the Mozambique tilapia and the South African abalone. The result can contribute to improved sustainability of the aquafeed industry, through substitution of fish meal and fish oil derived from capture fisheries with processed fish waste components. / AFRIKAANSE OPSOMMING: Hierdie studie vorm deel van die voortdurende en wêreldwye soeke na nuwe akwakultuur voerbestanddele wat nie net vismeel en –olie kan vervang as bron van noodsaaklike voedingstowwe nie, maar wat ook gespesialiseerde funksionele eienskappe openbaar wanneer dit in akwavoere ingesluit word. As gevolg van beperkings in die voorsiening van vismeel en –olie afkomstig vanaf wilde visserye, word die voortdurend groeiende akwavoer bedryf genoodsaak om nuwe grondstowwe te benut vir die vervaardiging van hoë kwaliteit voere. Die doelwit van die studie was om moontlike gespesialiseerde funksionele eienskappe (met spesifieke klem op potensiële immuun-stimulasie) van voerbestanddele te ondersoek wat herwin is vanaf reënboogforel proseseringsafval, na ensiematiese hidrolise d.m.v. endogene ensieme teenwoordig in die afval (outolise). Verdere doelwitte was om (i) effekte op produksie prestasie van hierdie bestanddele te kwantifiseer in twee akwakultuur spesies relevant tot die Suid- Afrikaanse bedryf (naamlik die Mosambiekse kurper Oreochromis mossambicus en die Suid- Afrikaanse perlemoen Haliotis midae), (ii) om produksie prestasie te vergelyk met kommersieel beskikbare voerbestanddele voorberei d.m.v. ensiemtegnologie, en (iii) om die moontlike effekte van die voerbestanddele te skei van die van die sure gebruik om die bestanddele te preserveer tydens die outolitiese proses. Daar is bevind dat olie herwin na outolise van reënboogforel ingewande ‘n goeie voerbestanddeel is wat gunstige effekte op die nie-spesifieke, sellulêre immuniteit van beide Mosambiekse kurper en Suid-Afrikaanse perlemoen gehad het. In die Suid-Afrikaanse perlemoen het verbeterde immuunfunksie gepaard gegaan met ‘n verswakking in produksie prestasie. Die gehidroliseerde proteïen komponent van ge-outoliseerde prosesseringsafval het ook beduidende verbetering in nie spesifieke immuniteit en oorlewing van Mosambiekse kurper tot gevolg gehad - onafhanklik van die preserverende suur - maar dieselfde is nie in die Suid-Afrikaanse perlemoen waargeneem nie. Produksie prestasie was afhanklik van die insluitingsvlakke van gehidroliseerde proteïen in beide spesies en dit is bevind dat oormatige insluiting produksie nadelig beïnvloed. Prestasie van proteïen afkomstig van outolise en die van ‘n kommersiële produksieproses het beduidend verskil, moontlik as gevolg van verskillende grondstowwe en prosesseringstegnieke. Daar is verder vir die eerste keer getoon dat mieresuur ‘n beduidende verbetering in waterstabiliteit in sekere perlemoenvoere teweeg kan bring. Die studie kom tot die slotsom dat die eenvoudige outolise proses funksionele akwavoerbestanddele kan produseer wat kan bydra tot ‘n verbetering in produksie prestasie in die Mosambiekse kurper en Suid-Afrikaanse perlemoen. Die resultaat kan bydra tot verbeterde volhoubaarheid van die akwavoer bedryf, deur vismeel en –olie afkomstig van wilde visserye, te vervang.

A gradient of sea temperatures is created along the South African coastline by the confluence of the cold Benguela Current on the West coast with the warm Agulhas Current on the East coast. This temperature gradient allows for an assortment of species to occupy the variety of microenvironments occurring in this area. Amongst these species is commercially important South African abalone, Haliotis midae, which although being capable of existing across this wide range of temperatures grows larger on the cooler West coast. Abalone reared on the warmer East coast however, experience greater mortalities especially during the more thermally variable summer months. The aim of the study was thus to assess the zone of tolerance for H. midae by exposing abalone to fluctuating temperatures in an attempt to model environmental temperature instability, a scenario which may likely be worsened by global climate change. Animals from the West and East coasts were exposed to two thermal treatments of fluctuating temperatures with the first group being kept at 16°C±2 and the second group kept at 16°C±4. The control group was maintained at a constant 16°C indicating that the mean temperature experienced by all three groups was 16°C. Oxygen consumption, nitrogen excretion and O:N ratio were assessed at the organismal level to give an indication of metabolic rate, amount of protein excreted and type of metabolic substrate utilized respectively. At the biochemical level, D-lactate accumulation was quantified to indicate whether metabolism was proceeding aerobically or anaerobically. Heat shock protein 70 (Hsp70) expression and degree of carbonylation were analyzed at the proteomic level with Hsp70 also being assessed at the transcriptomic level. All biological responses were measured at days 1, 3, 7 and 14 of the two week exposure. Oxygen consumption rates were significantly elevated on day 14 when comparing treatment group animals to control group animals of the same biogeographic region. P < 0.05 for both treatment groups from the West coast, while P < 0.001 for the East coast treatment groups. The ammonia excretion rates of the West coast animals were significantly lower than those of the controls at day 14 with P < 0.001 for both treatment groups, while ammonia excretion rates were elevated in East coast animals at day 14, although not significantly. Trends similar to those seen for ammonia excretion rates were exhibited by O:N ratios. West coast animals showed lower than control O:N ratios at day 14 (P < 0.01 for both treatment groups) while East coast animals displayed higher than control values (P < 0.05 only for the 16°C±2 group) at day 14. D-lactate, having been detected only for the West coast animals, showed no significant differences but large degrees of variation were noted on days 1 and 7. Carbonylation was evident for animals from both biogeographic regions with baseline carbonyl accumulation for East coast animals being greater (non-significantly) than that of the West coast animals. The hsp70 gene expression remained low for both biogeographic groups with West coast animals appearing to show slight elevations in expression at days 1 and 7, days which also displayed high degrees of variability. The West coast animals appeared to be better suited to coping with the thermal fluctuations, as they not only transiently reduced oxygen consumption rate to reduce ROS production, but also utilized the assistance of the D-lactate pathway possibly to maintain metabolism, both of which were not observed in the East coast animals. Although West coast abalone seemed to have slightly elevated hsp70 expression (suggestive of a repair response) when compared to their East counterparts, both groups of abalone were shown to have incurred notable amounts of protein damage (i.e. carbonylation). This suggests impairments in both protective and repair responses for animals from both biogeographic regions. The lack or attenuation of physiological responses noted in East coast abalone may be due to limitations in thermal adaptation but subsequent studies are required to confirm this notion. The information obtained from this study may assist in providing an insight into the mechanisms responsible for thermal limitation in H. midae and how this species is likely to respond to future periods of thermal instability which may be worsened by global climate change. An understanding of the processes leading up to limitations may potentially assist the abalone aquaculture industry in altering culturing practices early on to support optimal performance in abalone. / M.Sc. University of KwaZulu-Natal, Durban 2014.

An understanding of species specific water quality requirements is essential for efficient production of aquaculture products, an aspect not well documented for the land-based culture of the South African abalone, Haliotis midae. In order for the industry to remain competitive in international markets, efficient use of water supplies and the development of water reuse technology is needed. This study assessed the changes in water quality between tanks in a tiered serial-use raceway in relation to accumulated biomass and water flow and estimated the flow index (FI) (L h⁻¹ kg⁻¹) at which growth becomes significantly affected. The effect of dietary protein level, supplementation of pure oxygen and addition of sodium hydroxide (NaOH⁻) on water quality and fundamental production parameters in the serial-use raceways was also assessed. The serial-use raceways were used as a tool to create a range of water quality conditions at which the growth, feed conversion ratio (FCR) and condition factor (CF) of "cocktail" size (60 – 70 mm) H. midae could be monitored. The metabolic activity of the abalone resulted in a deterioration in water quality between tanks in series. pH (r² = 0.99; p < 0.001) and dissolved oxygen concentration (r² = 0.99; p < 0.001) were positively correlated with flow index (pH = 7.38 FI°·°² ; dissolved oxygen = 6.92 FI°·°⁴), while free ammonia nitrogen (FAN) (r² = 0.99, p < 0.001) and nitrite (NO²⁻ - N) (r² = 0.93, p < 0.001) were negatively correlated with flow index (FAN = 8.02 FI⁻°·⁷¹). Nitrite concentrations increased over time indicating colonisation of Nitrosomonas bacteria on the basket surfaces. A flow index of 7.2 – 9.0 L h⁻¹ kg⁻¹ was estimated as the minimum to avoid significant reductions in weight and shell length gain and increases in FCR values. Total ammonia nitrogen (TAN) and FAN concentrations were significantly correlated to dietary protein (P) (t = 6.63, p < 0.0001 and t = 6.41, p < 0.0001, respectively) and flow index (t = 5.42, p < 0.0001 and t = 3.9, p < 0.0002, respectively) and could be estimated using the models TAN = 9.73 P – 110.3 log (FI), and FAN = 0.132 P – 1.10 log (FI). Mean FAN concentrations were 67 and 41 % lower in tanks fed a diet containing 22 and 26 % protein respectively, when compared to tanks fed a 33 % protein diet. Supplementation with pure oxygen (103 ± 8 % saturation) improved shell length gain (t = 3.45, p = 0.026) in abalone exposed to high FAN (2.43 ± 1.1 μg L⁻¹) and low pH (7.6 ± 0.13), relative to a treatment with no oxygen supplementation (92 ± 6 % saturation). Addition of a sodium hydroxide solution resulted in elevated mean pH in treatment raceways when compared to control raceways. The increased pH resulted in significantly higher weight gain (g abalone⁻¹) (F₁·₁₂ = 4.51; p = 0.055) and shell length gain (mm abalone⁻¹) (F₁·₁₂ = 4.56; p = 0.054) at an α-error level of < 5.5 %. In two trials, weight gain and shell length gain were significantly correlated to pH (p < 0.001), and multiple regression of pH, dissolved oxygen and FAN consistently revealed pH to be the best predictor of growth. It is therefore suggested that decreasing pH is the first limiting water quality variable for abalone in serial-use raceways. As a decrease in water pH is linked to respiration by the abalone and subsequent increase in dissolved carbon dioxide (CO₂) concentration, future studies should examine the effects of CO₂ on H. midae metabolic rate, calcification rate and health. The results of this study will contribute toward our understanding of the specific water quality requirements for H. midae in commercial aquaculture systems, and influence the design and management procedures for abalone water reuse systems.

According to farm records, cultured Haliotis midae (50-70 g.abalone⁻¹) were growing 10% slower in winter when compared to summer. This reduction in growth rate also coincided with enlarged gonads. Initial trials showed that there were differences in mean monthly growth rates ranging from 1.97 – 5.14 g abalone⁻¹ month⁻¹, and gonad bulk index (GBI) also varied between months (GBI range: 26.88 ± 12.87 to 51.03 ± 34.47). The investment of energy into gonad tissue growth did not compromise whole body growth as the abalone continued to gain weight throughout the reproductive periods, probably due to gonadal growth. Growth of this size class of abalone was not influenced by water temperature or day length, suggesting favourable on-farm culture conditions (regression analyses, p > 0.05). There is no need to implement a seasonal dietary regime. Cultured H. midae were fed artificial diets with different protein sources, including only soya, only fishmeal, a combination of soya and fishmeal, and these were compared to kelp-fed abalone. Kelp-fed abalone grew slower than those fed artificial feeds (p>0.05). Gonad growth was the greatest when soya meal was included in the diet (average GBI: 74.91 ± 23.31), while the average gonad size of abalone fed the fishmealbased diet had gonads which were 38% smaller, and kelp-fed abalone had gonads which were 75% smaller than those of the abalone fed on diets containing soya meal. The increased gonad mass in abalone fed on diets including soya meal could be attributed to phytoestrogenic activity, as a result of the presence of isoflavones found in the soya plant; this remains to be tested. The use of soya in brood stock diet development is advised. The influence of dietary protein to energy ratio (1.41 – 2.46 g MJ⁻¹) on growth and gonad size was tested. Protein and energy levels within the ranges tested (22 and 33% protein; 13.5 and 15.6 MJ kg⁻¹) did not interact to influence growth rates of cultured H. midae. GBI increased from 50.67 ± 4.16 to 83.93 ± 9.35 units as a function of dietary protein to energy ratio (y = 42.02 x⁰·⁸¹; r² = 0.19; regression analysis: F₁¸₃₈ = 8.9; p = 0.005). In addition, protein level influenced gonad size, with gonad growth being greater in abalone fed the high protein diet (factorial ANOVA: F₁¸₃₂ = 7.1, p = 0.012). Canning yields were reduced by 7% when the protein content was increased, while increasing the quantity of dietary energy improved canning yields by ~ 6% (one-way ANOVA: F₁¸₂₈ = 14.4, p= 0.001). The present study provided evidence that although growth rates are varying seasonally, reproductive investment is not hindering weight gain. Gonad growth can be influenced if desired by farms, depending on the level of soya inclusion, as well as the protein to energy ratio in the diet. Monthly variation in growth and gonad size, as well as the influence of diet on gonad growth were highlighted, and the implications for farm application and further research were discussed.

Abalone, Haliotis midae, farmers in South Africa that feed formulated diets reported a periodic drop in abalone growth during periods of increased gonad development. A large drop in abalone biomass was noticed after presumed spawning events. This study was aimed to determine the effect of diet and sex-sorting on gonad development in abalone. Experiments were conducted on a commercial abalone farm from July 2012 to the end of June 2013. Isonitrogenous and isoenergetic diets were formulated with two protein sources. A fishmeal and soybean meal (S-diet) diet and a fishmeal only (F-diet) diet were fed to abalone (50 - 70 g abalone⁻¹) over 12 months. Weight and length gain, gonad bulk index (GBI), visceral index (%) and meat mass index (%) were determined monthly and seasonally. A histological study on the female gonads was conducted. This study also included an experiment to test the effect of sex-sorting (70 - 80 g abalone⁻¹) on growth and body composition with treatments including males (M), females (F) and equal numbers of males and females (MF). Weight gain and length gain were faster in S-diet-fed abalone (RM-ANOVA, F ₍₁, ₁₆₎ = 7.77, p = 0.01; F ₍₁, ₆₉₎ = 49.9, p < 0.001, respectively). Gonad development was significantly affected by the inclusion of soybean meal with S-diet-fed abalone showing higher GBI-values than F-diet-fed abalone (RM-ANOVA, F ₍₁, ₃₃)= 16.22, p = 0.0003). Male abalone had higher GBI-values than females (RM-ANOVA, F ₍₁, ₃₃₎ = 39.87, p < 0.0001). There was no significant difference in average feed conversion ratio (FCR) between diets over time (RM-ANOVA, F ₍₁, ₂₁₎ = 0.008, p = 0.97). However, average FCR-values were significantly highest between November 2012 and March 2013, the presumed spawning season. The visceral mass (gut and gonad) as a proportion of whole mass (visceral index, %) was significantly higher in abalone fed the S-diet (RM-ANOVA; F ₍₁, ₆₉₎ = 68.06, p < 0.0001). There was no difference in meat mass index (%) between diets for both male and female abalone (RM-ANOVA; F ₍₇, ₂₄₈₎ = 0.80, p = 0.60; F ₍₇, ₂₄₁₎ = 1.7, p = 0.11,respectively). Meat mass index significantly decreased from September 2012 to February 2013 coinciding with the period of high GBI-values. The distribution of oocyte maturity stages differed between diets. The majority of oocytes within S-diet-fed abalone were fully mature stage 8 oocytes compared to a majority of stage 7 oocytes in F-diet-fed abalone. Histology corroborated peaks in GBI-values for abalone fed both diets. There was no significant difference in growth, GBI, visceral index (%) and meat mass index (%) between abalone sorted into monosex and mixed-sex populations. Thus, the presence of the opposite sex did not have an effect on growth and gonad mass in H. midae. The phytoestrogens daidzin, glycitin, genistin, daidzein, glycitein and genistein were present in soybean meal and only traceable amounts were found in the F-diet. This study provided evidence that soybean meal present in formulated feed affected growth and gonad development in H.midae. The difference in the distribution of the maturity stages of oocytes was affected by diet. Sex-sorting abalone into monosex and mixed-sex populations had no influence on weight and length gain and gonad development.

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2009. / An 84-day study was conducted to find a suitable diet and feeding level for the culture of South African abalone (Haliotis midae) in Namibia. Two experimental diets, namely, a seaweed diet (SWD) Laminaria pallida (macrophytic algae) and a formulated diet (FD) (macro-algae), for use in abalone (Haliotis midae) feed development, were evaluated. The animals used in this study were juveniles (24.33 ± 3.14 mm shell length; 2.72 ± 0.83 g live weight, mean ± SE) and sub-adults (58.07 ± 10.33 mm shell length and 41.96 ± 20.61 g live weight, mean ± SE). The nutrient profile of the SWD and FD displayed no differences in the protein and carbohydrate levels. Crude protein levels ranged from 4.91 to 17.68% (dry matter (DM) basis). The lipid levels in the FD (0.25%) were almost 0.56% lower than that in the SWD (0.76%). The feed conversion ratio (FCR) and protein efficiency ratio (PER) for the sub-adult abalone ranged from 2.80 to 10.90 and 0.10 to 0.40, respectively. The juvenile abalone fed on the FD yielded significantly lower (P < 0.05) FCRs (0.8) and higher PERs (1.20) than their counterparts fed on the SWD. A similar trend was observed for the sub-adult abalone although the differences were not significant (P > 0.05). The relative growth rate (RGR) of juvenile fed on the FD was 25% lower compared to those fed on the SWD, while that of the sub-adult abalone fed on the FD was 29% lower compared to the abalone fed on the SWD. From the daily growth rate (DGR) in terms of daily body weight (DGRBW) calculated after the 84-day period, repeated-measures ANOVA (RANOVA) indicated no interaction between time period and diet. Although slightly lower, the DGRBW for the juvenile abalone fed on the SWD diet (0.033 g/day) did not differ significantly from the DGRBW of abalone fed on the FD (0.079 g/day). In contrast, sub-adult abalone fed on the SWD exhibited significantly higher DGRBW compared to those fed on the FD. Although the abalone fed on the FD was slightly higher in nutritional content, there was no significantly difference (P > 0.05) in the nutritional profile of the abalone soft body tissue fed on either the SWD or FD. There was no significant (P > 0.05) difference in preference when comparing the aroma of the abalone meat samples fed on either the SWD or FD. However, there was a significant difference (P < 0.05) in the consumers’ preference in terms of flavour for the abalone sample fed on the FD. The trained taste panel results indicated that there was no difference in the aroma and flavour of the abalone fed on the different diets (P > 0.05). This study showed that cultured juvenile H. midae, readily accepted a FD, producing high consumption and survival rates. The FD still warrants further refinement and testing for it to become a more effective mariculture feed with commercial potential.

Abalone poaching is a major problem in South Africa. The South African abalone, Haliotis midae, rates as an extreme example of high levels of illegal harvesting. The research aimed at examining the role of the different role players in preventing poaching of the species in the East London area of the Eastern Cape Province of South Africa and determining the challenges the officers faced as there was an increase in poaching in the area. To achieve this aim, the research techniques including questionnaires, interviews and focus groups were used. Fisheries compliance and enforcement faces challenges of the illegal harvesting of abalone since 1994. In the past 18 years (1994-2012), and more specifically in the past nine years (2004-2012), poaching of abalone has increased at an alarming rate along the East London coastline. The failure of the state to issue fishing rights and conduct effective sea-based compliance, combined with the incentives to fish abalone created the conditions for rapid emergence of illegal harvesting. The uncontrolled fishing had a dramatic effect on the stock, and the average size of abalone decreased significantly. The Eastern Cape Province abalone cultivation industries were developed due to the decline in harvesting of abalone. Government departments such as the Department of Agriculture, Forestry and Fisheries; the Department of Economic Development, Environmental Affairs and Tourism; the South African Police Services together with the Eastern Cape Parks and Tourism Agency have conducted joint operations to combat the illegal harvesting of abalone. These operations have led to many arrests of abalone poachers along the East London coastline. The quantity of confiscated abalone has increased from 2007 to 2011. The positive results achieved by the departments during joint operations showcase robust efforts to eradicate the environmental transgression in the East London Coastline. Joint operations are encouraged by all the departments to save the species for future generations. Workshops involving different stakeholders had to take place and the policies in place must have a bottom-up approach where communities are involved.