Dietary biodiesel-derived crude glycerol in gilthead seabream juveniles (Sparus aurata): effects on growth performance and metabolism

In the European Union the turn towards renewable energy sources has increased the production of biodiesel from rapeseed oil, leaving glycerol (also known as glycerin) as a valuable by-product. For every litre of biodiesel produced, approximately 79 g of crude glycerol are generated. As the biodiesel production grows, the quantity of crude glycerol generated will be considerable and its utilization will become an urgent topic. One possibility is the use of crude glycerol on animal feeds. Glycerol has been evaluated as a dietary energy source for several farm animals, including fish. A study was undertaken to assess the effect of dietary biodiesel-derived glycerol (from rapeseed oil) on the overall growth performance, digestive capacity and metabolic nutrient utilization in juvenile gilthead seabream fed a low fishmeal level diet. Two practical diets were formulated to be isonitrogenous (crude protein, 45.4% DM), isolipidic (18.5% DM) and isoenergetic (gross energy, 21.3 kJ/g DM). The control diet (CTRL) was formulated with intermediate levels of marine-derived proteins (19%). In the same basal formulation, 5% glycerol (GLY) was incorporated at the expenses of wheat. Each dietary treatment was tested in triplicate tanks over 63 days, with 20 gilthead seabream (Sparus aurata), with a mean initial body weight (IBW) of 27.9  0.12 g. At the end of the trial, fish fed the CTRL diet reached a final body weight of 84.3  2.2 g (more than 3-fold increase of initial body weight). Fish fed the GLY diet showed a significantly higher (P<0.05) growth, expressed in terms of final body weight and specific growth rate. Voluntary feed intake was similar between the two treatments, but both feed efficiency and protein efficiency ratio were significantly improved (P<0.05) in fish fed the GLY diet. Dietary glycerol had no effect (P>0.05) on the apparent digestibility of protein. In comparison to the control treatment, dietary glycerol significantly improved (P<0.05) protein and fat retention. Activities of digestive enzymes were significantly affected by the various dietary treatments. Fish fed the GLY diet showed an enhanced activity of alkaline phosphatase (ALP) and pepsin, while activities of lipase and leucine-alanine peptidase (LAP) were little affected by dietary glycerol. Fish show the ability to use crude glycerol as a dietary energy substrate.

Nutrition and farming practices as modulators of quality in gilthead seabream (Sparus aurata)

Gilthead seabream is the most important farmed species in the Mediterranean, and knowledge on how common farming practices impact its quality is limited. As such, this Thesis aimed to evaluate how gilthead seabream flesh quality is affected by some of these practices. In Chapter 2, the influence of nutritional factors was evaluated, specifically the high replacement of traditional marine-derived ingredients, both fishmeal and fish oil, with vegetable sources. We have seen that the vegetable-based diets tested did not greatly impact seabream flesh quality, although some alterations were seen in the fatty acid profile of the muscle. However, and despite having caused no alterations in flesh texture, vegetable ingredients reduced the amount of sulphated glycosaminoglycans in the extracellular matrix, affected muscle pH and reduced the activity of proteolytic enzymes. Throughout this Thesis, we measured for the first time the activity of proteolytic enzymes in seabream muscle, and cathepsin B was found to play a pivotal role in post-mortem muscle degradation. In Chapter 3, we evaluated the effect of harvesting and slaughter stress on seabream quality, and contrary to what is seen in most farmed species, our results show that gilthead seabream muscle structure is highly resistant to changes caused by stressful events. Nonetheless, considering that welfare is an increasingly important quality criterion, the use of a zero-withdrawal anaesthetic as a rested harvest technique or even slaughter method could prove valuable to the industry. In Chapter 4, we used maslinic acid as a dietary supplement, to modulate the muscle’s energetic status pre-mortem. As a finishing strategy, maslinic acid failed to increase levels of glycogen and ATP in the muscle. However, supplementation resulted in higher muscle fibre diameter and lower cathepsin B activity, and maslinic acid is likely to be useful to promote growth in this species. In general our Thesis has generated new knowledge to a major challenge facing the aquaculture industry, which is to find a compromise between the trends towards intensive rearing and consumer demand for healthy, high quality seafood being ethically acceptable and having a low impact on the environment.

Cloning of the Bone Gla Protein gene from the teleost fish Sparus aurata (gilthead seabream). Molecular organization, developmental appearance and evolutionary implications

Tese de Doutoramento, Biologia Molecular, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2001

Resposta fisiológica e bioquímica ao jejum na dourada (sparus aurata)

Dissertação de Mestrado, Aquacultura, Unidade de Ciências e Tecnologias dos Recursos Aquáticos, Universidade do Algarve, 1997

Controle hormonal da diferenciação e inversão sexual na dourata Sparus aurata (L.)

Tese de Doutoramento, Biologia Marinha, Unidade de Ciências e Tecnologias dos Recursos Aquáticos, Universidade do Algarve, 2001

Contamination of farmed gilthead sea bream (Sparus aurata, Linnaeus, 1758) by the parasite Amyloodinium ocellatum: a proteomic approach

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Uniersidade do Algarve, 2015

Relationship between photoperiod, plasma concentration of ionic calcium and the histology of the prolactin-secreting cells of the rostral pars distalis of the pituitary gland, the corpuscles of stannius and the ultimobranchial gland in the goldfish, Carassius auratus L. /

The relationship between photoperiod, plasma concentration of ionic calcium and the histology of the prolactin-secreting cells of the rostral pars distalis of the pituitary gland, the Corpuscles of Stannius and the Ultimobranchial gland were investigated. Neither the plasma concentration of ionic calcium nor histologically apparent prolactin cell activity could be correlated with photoperiod. Some evidence of a photoperiodic effect on both the Corpuscles of Stannius and the Ultimobranchial gland was obtained. The expected reciprocal relationship between the activity of these glands was not obvious at the histological level . Quantitative and qualitative analysis at the light microscope level revealed, however, that the hormone prolactin-secreting eta cells of the rostral pars distalis and the hypocalcin-secreting cells of the Corpuscles of Stannius may be arranged in a lamellar pattern comprized of synchronous bands of cells in like-phase of a secretory cycle consisting of four stages - synthesis, storage, release and reorganization. Such synchronized cell cycles in these glands have not heretofore been described in literature. It is suggested that the maintenance of at least 255? of the cells in any one phase of the cycle ensures a supply of the required hormone at all times.

Ionic regulation in goldfish, Carassius auratus, acclimated to constant and diurnally-cycling temperature conditions

The effects of a diurnal sine-wave temperature cycle (250 +- 5° C) on the wa terI-e etc r o1 yt est a t us 0 f gol df1' Sh , Carassius auratus, was assessed through determination of Na+, K+, Mg2+, Ca2+, Cl- and water content in plasma, Red blood cells and muscle tissue. Animals were also acclimated to o 0 0 static temperatures (20 C, 25 c, 30 C) corresponding to the high, low and mid-ooint temperatures of the cycle. All groups were sampled at 03:00, 09:00, 15:00 and 21:00 hr. Hemoglobin content and packed cell volume, as well as electrolyte and 'water levels were determined for each animal and red cell ion concentrations and ion : hemoglobin ratios estimated. Cycled animals were distinct from those at constant temperatures in several respects. Hematological parameters were elevated above those of animals at constant temperature and were, on a diurnal basis, more stable. Red blood cell electrolyte levels varied in an adaptively appropriate fashion to cycle temperatures. This was not the case in the constant temperature groups_ Under the cycling regime, plasma ion levels were more diurnally stable than those of constant temperature fish. Although muscle parameters in cycled fish exhibited more fluctuation than was observed in plasma, these also tended to be relatively more stable than was the caseErythrocytic data are discussed in terms of their effects on hemoglobin-oxygen affinity while plasma and muscle observations were considered from the standpoint of overall water-electrolyte balance. In general, cycled fish appeared to be capable of stabilizing overall body fluid composition, while simultaneously effecting adaptively-appropriate modifications in the erythrocytic ionic microenvironment of hemoglobin. The sometimes marked diurnal variability of water-electrolyte status in animals held at constant temperature as opposed to the conservation of cycled fish suggests that this species is, in some fashion, programmed for regulation in a thermally-fluctuating environment. If this interpretation is valid and a phenomenon of general occurrence, some earlier studies involving constant acclimation of eurythermal species normally occupying habitats which vary in temperature on a daily basis may require reconsideration. at constant temperature.

The effects of temperature on nickel tocicity in goldfish (carassius auratus L.)

Although it is widely assumed that temperature affects pollutant toxicity, few studies have actually investigated this relationship. Moreover, such research as has been done has involved constant temperatures; circumstances which are rarely, if ever, actually experienced by north temperate, littoral zone cyprinid species. To investigate the effects of temperature regime on nickel toxicity in goldfish (Carassius auratus L.), 96- and 240-h LCSO values for the heavy metal pollutant, nickel (NiCI2.6H20), were initially determined at 2DoC (22.8 mg/L and 14.7 mg/L in artificially softened water). Constant temperature bioassays at 10°C, 20°C and 30°C were conducted at each of 0, 240-h and 96-h LCSO nickel concentrations for 240 hours. In order to determine the effects of temperature variation during nickel exposure it was imperative that the effects of a single temperature change be investigated before addressing more complex regimes. Single temperature changes of + 10°C or -10°C were imposed at rates of 2°C/h following exposures of between 24 hand 216 h. The effects of a single temperature change on mortality, and duration of toxicant exposure at high and low temperatures were evaluated. The effects of fluctuating temperatures during exposure were investigated through two regimes. The first set of bioassays imposed a sinewave diurnal cycle temperature (20.±.1DOC) throughout the 10 day exposure to 240-h LeSO Ni. The second set of investigations approximated cyprinid movement through the littoral zone by imposing directionally random temperature changes (±2°C at 2-h intervals), between extremes of 10° and 30°C, at 240-h LC50 Ni. Body size (i.e., total length, fork length, and weight) and exposure time were recorded for all fish mortalities. Cumulative mortality curves under constant temperature regimes indicated significantly higher mortality as temperature and nickel concentration were increased. At 1DOC no significant differences in mortality curves were evident in relation to low and high nickel test concentrations (Le., 16 mg/L and 20 mg/L). However at 20°C and 30°C significantly higher mortality was experienced in animals exposed to 20 mg/L Ni. Mortality at constant 10°C was significantly lower than at 30°C with 16 mg/L and was significantly loWer than each of 2DoC and 39°C tanks at 20 mg/L Ni exposure. A single temperature shift from 20°C to 1DoC resulted in a significant decrease in mortality rate and conversely, a single temperature shift from 20°C to 30°C resulted in a significant increase in mortality rate. Rates of mortality recorded during these single temperature shift assays were significantly different from mortality rates obtained under constant temperature assay conditions. Increased Ni exposure duration at higher temperatures resulted in highest mortality. Diurnally cycling temperature bioassays produced cumulative mortality curves approximating constant 20°C curves, with increased mortality evident after peaks in the temperature cycle. Randomly fluctuating temperature regime mortality curves also resembled constant 20°C tanks with mortalities after high temperature exposures (25°C - 30°C). Some test animals survived in all assays with the exception of the 30°C assays, with highest survival associated with low temperature and low Ni concentration. Post-exposure mortality occurred most frequently in individuals which had experienced high Ni concentrations and high temperatures during assays. Additional temperature stress imposed 2 - 12 weeks post exposure resulted in a single death out of 116 individuals suggesting that survivors are capable of surviving subsequent temperature stresses. These investigations suggest that temperature significantly and markedly affects acute nickel toxicity under both constant and fluctuating temperature regimes and plays a role in post exposure mortality and subsequent stress response.

L'Sparus

L’Sparus –el nom prové de la denominació en llatí de l'orada– és un petit submergible de color carabassa que té la forma d’un torpede. No és més llarg que la cama d’una persona adulta, però disposa de l’espai necessari per encabir els mecanismes que l’orienten, el propulsen i li proporcionen energia. L’Sparus és un robot autònom, perquè navega tot sol i no necessita cap intervenció humana per localitzar objectes submergits, és capaç de veure-hi o sentir-hi. La combinació de senzillesa i efectivitat li han proporcionat la victòria en la competició SAUC-E, que, organitzada per l’OTAN, ha tingut lloc el mes de juny a La Spezia, a Itàlia

Low allozyme variation in snapper, Pagrus auratus, in Victoria, Australia

The population genetic structure of snapper, Pagrus auratus (Bloch and Schneider), in Victoria was investigated using six polymorphic allozyme loci. Fish were sampled from four sites in Victoria and single locations in South Australia, Western Australia and New Zealand. Although there were distinct genetic differences between the snapper populations from each of the Australian states and New Zealand, only minor and largely insignificant differences were detected among Victorian populations. The results are consistent with previous genetic and tagging studies that indicate no mixing between snapper stocks in Victoria and Spencer Gulf in South Australia. This justifies separate management of the snapper fisheries in these regions. The low levels of polymorphism and heterozygosity in Victorian snapper suggest an isolation by distance model of population structure rather than one of discrete subpopulations.

Effect of cage colour and light environment on the skin colour of Australian Snapper Pagrus auratus (Bloch & Schneider, 1801)

A two-factor experiment was performed to evaluate the effects of cage colour (black or white 0.5 m³ experiment cages) and light environment (natural sunlight or reduced level of natural sunlight) on the skin colour of darkened Australian snapper. Each treatment was replicated four times and each replicate cage was stocked with five snapper (mean weight=351 g). Snapper exposed to natural sunlight were held in experimental cages located in outdoor tanks. An approximately 70% reduction in natural sunlight (measured as PAR) was established by holding snapper in experimental cages that were housed inside a 'shade-house' enclosure. The skin colour of anaesthetized fish was measured at stocking and after a 2-, 7- and 14-day exposure using a digital chroma-meter (Minolta CR-10) that quantified skin colour according to the L*a*b* colour space. At the conclusion of the experiment, fish were killed in salt water ice slurry and post-mortem skin colour was quantified after 0.75, 6 and 22 h respectively. In addition to these trials, an ad hoc market appraisal of chilled snapper (mean weight=409 g) that had been held in either white or in black cages was conducted at two local fish markets. Irrespective of the sampling time, skin lightness (L*) was significantly affected by cage colour (P<0.05), with fish in white cages having much higher L* values (L*≈64) than fish held in black cages (L*≈49). However, the value of L* was not significantly affected by the light environment or the interaction between cage colour and the light environment. In general, the L* values of anaesthetized snapper were sustained post mortem, but there were linear reductions in the a* (red) and b* (yellow) skin colour values of chilled snapper over time. According to the commercial buyers interviewed, chilled snapper that had been reared for a short period of time in white cages could demand a premium of 10–50% above the prices paid for similar-sized snapper reared in black cages. Our results demonstrate that short-term use of white cages can reduce the dark skin colour of farmed snapper, potentially improving the profitability of snapper farming.

Effect of carotenoids and background colour on the skin pigmentation of Australian snapper Pagrus auratus (Bloch & Schneider, 1801)

Three 2-factor experiments were conducted to determine the effects of background colour and synthetic carotenoids on the skin colour of Australian snapper Pagrus auratus. Initially, we evaluated the effects on skin colour of supplementing diets for 50 days with 60 mg kg⁻¹ of either astaxanthin (LP; Lucantin^®Pink), canthaxanthin (LR; Lucantin^® Red), apocarotenoic acid ethyl ester (LY; Lucantin^® Yellow), selected combinations of the above or no carotenoids and holding snapper (mean weight=88 g) in either white or black cages. In a second experiment, all snapper (mean weight=142 g) from Experiment 1 were transferred from black to white, or white to white cages to measure the short-term effects of cage colour on skin L*, a* and b* colour values. Skin colour was measured after 7 and 14 days, and total carotenoid concentrations were determined after 14 days.

Cage colour was the dominant factor affecting the skin lightness of snapper with fish from white cages much lighter than fish from black cages. Diets containing astaxanthin conferred greatest skin pigmentation and there were no differences in redness (a*) and yellowness (b*) values between snapper fed 30 or 60 mg astaxanthin kg⁻¹. Snapper fed astaxanthin in white cages displayed greater skin yellowness than those in black cages. Transferring snapper from black to white cages increased skin lightness but was not as effective as growing snapper in white cages for the entire duration. Snapper fed astaxanthin diets and transferred from black to white cages were less yellow than those transferred from white to white cages despite the improvement in skin lightness (L*), and the total carotenoid concentration of the skin of fish fed astaxanthin diets was lower in white cages. Diets containing canthaxanthin led to a low level of deposition in the skin while apocarotenoic acid ethyl ester did not alter total skin carotenoid content or skin colour values in snapper.

In a third experiment, we examined the effects of dietary astaxanthin (diets had 60 mg astaxanthin kg⁻¹ or no added carotenoids) and cage colour (black, white, red or blue) on skin colour of snapper (mean weight=88 g) after 50 days. Snapper fed the astaxanthin diet were more yellow when held in red or white cages compared with fish held in black or blue cages despite similar feed intake and growth. The skin lightness (L* values) was correlated with cage L* values, with the lightest fish obtained from white cages. The results of this study suggest that snapper should be fed 30 mg astaxanthin kg⁻¹ in white cages for 50 days to increase lightness and the red colouration prized in Australian markets.

Effects of cage netting colour and density on the skin pigmentation and stress response of Australian snapper Pagrus auratus (Bloch & Schneider, 1801)

The unnaturally dark pigmentation of cultured Australian snapper Pagrus auratus can be improved through dietary astaxanthin supplementation and by holding fish in tanks with a white background. The practical application of these  laboratory-based findings was examined with two experiments to establish if the advantages of transferring fish to light coloured tanks before harvest could be achieved on-farm using white cages and to determine the effects of fish density on skin colour. For the first experiment, snapper (mean TL=29.7 cm) were transferred from a commercial snapper sea cage to black or white netted cages and fed diets supplemented with unesterified astaxanthin (supplied as Lucantin^® Pink, BASF) at 0 or 39 mg kg⁻¹ for 42 days. Skin colour was measured using the CIE L* (black–white), a* (green–red), b* (blue–yellow) colour scale. Snapper held in white netting cages became significantly lighter (higher L* ) than snapper held in black cages; however, values were not as high as previous laboratory-based studies in which snapper were held in white plastic-lined cages. Snapper fed astaxanthin displayed significantly greater a*and b* values, and total carotenoid concentrations after 42 days. In addition, total carotenoids were higher in fish from black than white cages. The second experiment was designed to investigate whether density reduced the improvements in skin colour achieved by holding fish in white coloured cages and whether cage colour affected stress. Snapper (mean weight=435 g) were acclimated to black cages and fed 39 mg kg⁻¹ astaxanthin for 44 days before transferring to black or white plastic-lined cages at 14 (low), 29 (mid) or 45 (high) kg m⁻³ for 7 days after which time skin colour, plasma cortisol and plasma glucose concentrations were measured. Skin lightness (L* ) was greater in snapper transferred to white plastic-lined cages with the lightest coloured fish obtained from the lowest density after 7 days. Density had no effect on plasma cortisol or glucose levels after 7 days, although plasma cortisol was elevated in snapper from black cages. For improved skin colouration we recommend feeding unesterified astaxanthin at 39 mg kg⁻¹ for approximately 6 weeks and transferring snapper to white plastic-lined cages or similar at low densities for short periods before harvest rather than producing fish in white netting sea cages subject to biofouling.