byConstantinos C Mylonas, Project Coordinatorand KritonGrigorakis, New product development (HCMR, Greece), Hanna Rosenfeld, Reproduction & Genetics–Grey mullet leader (NCM-IOLR, Israel), William Koven, Nutrition and Larval & Grow out husbandry-Grey mullet leader(NCM-IOLR, Israel), Luis Guerrero, New product development leader (IRTA, Spain), Rocio Robles, Dissemination Leader (CTAQUA, Spain; actual affiliation Testing Blue SL, Spain)

 

Another one of the species included in the EU-funded DIVERSIFY project (see April issue of International Aquafeed), which ran between 2013 and 2018 was the Grey mullet (Mugil cephalus).

Farming of grey mullet has been practiced for centuries, but production of this potentially invaluable source of animal protein in Europe has been small and non-intensive (Nash &Koningsberg, 1981; Pillay, 1993).

It is a euryhaline species, found throughout the world (Oren, 1981) and is a rapid-growing, omnivorous teleost that can be reared over the wide geographical and temperature range of the Mediterranean basin (Crosetti,2015). Therefore, it can be an excellent candidate for the enhancement of aquaculture in earthen ponds, coastal lagoons, and deserted salinas that exist throughout the EU Mediterranean countries.

During the autumn and winter months adults migrate to the sea in large aggregations to spawn. When juveniles are 16–20 mm, they migrate to inshore waters and estuaries, where they can be collected for farming operations during late August to early December.

Most of the flathead grey mullet fry used in commercial aquaculture are collected from the wild, especially in the Eastern and Southern Mediterranean, Saudi Arabia and Gulf States and South East Asia. Cultured flathead grey mullet is generally grown semi-intensively in polyculture ponds that can include common carp, grass carp, silver carp, Nile tilapia, milkfish and European seabass. Although growth has been reported highest in lower salinity water, they can be successfully reared in fresh water, brackish water and sea water.

Full-scale commercial production of grey mullet in monoculture is still in its infancy. Induced spawning and production of fry on a limited scale for aquaculture has been reported in Italy, Israel and Egypt. Hatchery produced juvenile females have been grown to 1.9 kg in two years on a fishmeal-containing pelleted feed.

The development of a fishmeal-free feed will reduce the cost of fish production and will be more sustainable and environmentally friendly. This means grey mullet would be more acceptable to an increasingly aware consumer public that demands sustainability and lower environmental impact.

Moreover, grey mullet aquaculture has the advantage of providing not only affordable whole fish and fillets, but also fish roe (bottarga), a high value product (>100€ kg-1), which market is expanding around the Mediterranean.

Therefore, grey mullet has considerable economical potential as a species that provides an inexpensive source of sustainable, high quality protein, product diversification, and a value-added product such as bottarga.

A market for grey mullet is well established in the Mediterranean where Egypt alone consumed more than 129,000 million
tonnes (MT) in 2015 (Soliman et al 2015). In addition, the European market for grey mullet is likely to increase in the coming years, due to the demand from established and newly immigrant families originating from
North Africa, Middle East and Asia.

Currently, the industry is a capture-based aquaculture, relying almost exclusively on the capture of wild fry (ca 1,000,000,000). It is now recognised that this approach severely reduces natural fisheries and is unsustainable where regulation of this practice is expected in the near future.

However, the future growth of the grey mullet aquaculture is limited by a number of bottlenecks, which will be addressed in DIVERSIFY. Firstly, controlling the reproductive cycle and improving egg quality via broodstock management and nutrition is necessary not only for the production of robust larvae, but also for producing high value bottarga.

Secondly, development of a larval rearing protocol is necessary to reduce early mortalities, size dispersion as well as increasing metamorphic synchrony, which will lead to a supply of high-quality juveniles. Finally, development of a sustainable, economical, fishmeal-free grow out feed is needed, which would perform well under different environmental conditions of temperature, pond type, and water quality, thus broadening the geographical range of grey mullet aquaculture in Europe.

The DIVERSIFY project has addressed these important bottlenecks with a coordinated research effort in reproduction, larval nutrition and husbandry, and grow out of the species. The combination of biological, technological and socioeconomic research activities developed in DIVERSIFY are expected to support the diversification of the EU aquaculture industry and help in expanding production, increasing aquaculture products and development of new markets.

 

Grey mullet in the DIVERSIFY Project:

Reproduction

Lacking the natural spawning environment, captive grey mullet fail to reproduce spontaneously, largely due to a failure to undergo complete gametogenesis. In this respect, and within the framework of DIVERSIFY, considerable progress was made by optimising hormonal treatments for alleviating reproductive dysfunctions among captive grey mullet broodstocks.

Bio-potent yeast (Pichia pastoris) produced recombinant gonadotropins (r-FSH and r-LH) that were used as therapeutic agents in a series of in vitro and in vivo assays. The best performing treatment consisted of r-FSH and a dopamine antagonist (metoclopramide) that were co-injected during the onset of the reproductive season.

The latter treatment demonstrated synchronised gonadal development within and between sexes, giving rise to stimulated spermatogenesis in males and follicle growth and maturation in females. Further spawning induction trials that timed the administration of GnRHa and metoclopramide with advanced stages of gamete maturation were relatively successful.

A basic breeding unit, comprising a single female and three males, was found to facilitate synchronisation and in turn increase fertilisation rate. Nevertheless, our results highlight an episodic fertilisation rate ranging between 0-to-98 percent and point to a future need to fine tune and optimise the hormone-based breeding protocol for captive grey mullet.

Broodstock diet containing fish oil (FO), which is relatively rich in n-3 long chain polyunsaturated fatty acids (LC-PUFA), positively affected hatching success and larvae survival. The established breeding protocol for captive grey mullet could
be effectively applied during natural as well as artificially shifted spawning seasons.

Over several consecutive spawning seasons, tens of millions of high-quality eggs were produced giving rise to mass production of robust fingerlings. A shipping protocol for grey mullet eggs was also established specifying the optimised conditions including egg developmental stage (gastrula) and packing density for short term (≤ 11 h) and long term (26 h) shipments.

The assessment of the effects of captivity on first sexual maturity of wild-caught and hatchery-produced grey mullet indicated that: (1) the rearing conditions established allow for a growth rate equivalent to that of wild grey mullet from the Mediterranean Sea; (2) the reduction of the rearing density from 90-to-45 fish per m3 has no effect on grey mullet growth and sexual maturity and (3) hatchery-produced grey mullet have a good potential to develop ovaries spontaneously up to a condition useful for bottarga production.

Furthermore, the effects of fish origin (wild vs. domesticated) and culture conditions on advanced and spontaneous development of gonads, which exhibit the required criteria of high quality bottarga (i.e. minimal size of 100g, bright yellowish color and chewy texture), were assessed.

They indicated that (1) the traditional grey mullet farming procedure in freshwater ponds could be applicable, and also an advantage, for roe production; (2) Domestication appears to have a favorable effect on the spontaneous development of mullet ovaries characterised by a condition useful for bottarga production and (3) pigment-enriched diets can enhance the roe coloration to meet the criteria for high quality bottarga (roe).

However, two stumbling blocks that may impair the profitability of grey mullet farming for bottarga production are (1) extended grow out to a minimum of three years and (2) relatively low percentages (20- 50%) of females developing ovaries at the appropriate size (≥ 100g).

Future studies, therefore, should focus on genetic improvement programs giving rise to advanced sexual maturity and spontaneous ovarian development in captive grey mullet females.

 

Nutrition

The results suggest that grey mullet >89 dph grown in low salinity (15‰) have the capability to synthesise DHA from shorter carbon chain precursors while there is little or no biosynthesis of LC-PUFA in fish exposed to high salinity (40‰). This follows as grey mullet juveniles in nature would be moving to the lower salinity waters of river mouths and estuaries, which are characterised by an environment less rich in LC-PUFA and more abundant in smaller chain PUFA precursors.

Low salinity upregulated the gene expression of ∆6 desaturase, the rate-limiting enzyme of LC-PUFA biosynthesis) but was independent of DHA dietary level. On the other hand, both low salinity and DHA level upregulated the gene expression of elongase.

The two transcription factors, sterol regulatory element binding protein (SREBP1) and peroxisome proliferator activated receptors (PPAR) are involved in the regulation of fatty acid biosynthesis.

Although both SREBP1 and PPAR expression were highest in 15‰ water, PPAR expression was inversely regulated by dietary DHA at both salinities, while SREBP1 was inversely regulated by DHA only in the low salinity. These findings suggest that dietary levels of DHA can be decreased when feeding older juvenile mullet, provided that the salinity is reduced to levels found in estuarine waters.

This would translate to a significant savings for farmers as the purchase of feed for the grow-out of fish to market weight can represent 60 percent of production costs and DHA is costly as a feed ingredient.

The β-amino sulfonic acid taurine plays an array of critical roles that promote fish growth and survival. An increasing number of marine teleosts have demonstrated an essential dietary requirement for this nutrient as they lack the enzyme cysteine sulfinic acid decarboxylase (CSD), a key component in the taurine synthesis pathway.

DIVERSIFY found that CSD is synthesised by juvenile grey mullet in the absence of dietary taurine and that the expression of this key gene increases with increased levels of dietary taurine until one percent where CSD expression decreases rapidly possibly due to a negative feedback mechanism.

The increased taurine in the blood circulation of the liver, due to higher dietary taurine, may stimulate increased endogenous taurine synthesis within liver cells to reduce osmotic pressure across the membrane and prevent cell shrinkage and changes in intracellular hydro-mineral balance.

Cholesterol 7 alpha-hydroxylase (CYP7a1) is the key enzyme in the synthesis of bile salts and was not affected by increased levels of dietary taurine. This suggests that endogenous taurine synthesis was sufficient for bile salt synthesis.

Taken together, it appears that grey mullet juveniles have the capacity for endogenous taurine synthesis that may be sufficient for cell volume homeostasis and bile salt production, but may fall short in optimising skeletal muscle function and growth, thereby requiring a minimum of 0.5 percent of taurine in the diet.

In grey mullet broodstock the mobilisation of energy reserves in terms of lipids and proteins was quite similar between wild and captive mature females. Moreover, in fatty acids and fatty acid groups, there were no conspicuous differences, independent of age, between female gonads from domesticated and wild captive broodstock fed fish oil-based diets or broodstock fed soybean oil-based diets.
This suggests a gonadal biosynthetic capability for biosynthesis of LC-PUFA from shorter chain precursors. Nevertheless, when comparing the FA and lipid class profiles between female and male gonads, there were highly marked differences.

In female gonads, the TA, TAG, wax and sterol esters were higher compared to male gonads while the male gonads had higher quantities of the PL phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine, as well as cholesterol compared to female gonads.

Noteworthy were also the very high levels of DHA in the male gonads compared to the female gonads. Interestingly, the male gonads from the soybean-fed group were higher in DHA than the fish oil group despite the fact that soybean oil does not contain this essential fatty acid.

The fish oil diet resulted in better egg hatchability, as well as larval tolerance of food deprivation and improved swim bladder inflation. These benefits may be due to another fish oil component, possibly carotenoids.

Fish acceptability of the developed DIVERSIFY grey mullet diet appeared enhanced by replacing poultry meal with fish meal, suggesting that the inclusion of other nutrients may be necessary in order to maintain a fish meal free diet.

The fatty acid profiles of the tissues generally resembled those of the diets. Feeding the developed diet resulted in fish displaying a more balanced lipid profile than fish fed the commercial carp diet. For instance, the fillets from the DIVERSIFY diet were poorer in 18:2n-6, but also exhibited a higher absolute content of n-3 LC PUFA eicosapentaenoic acid and docosahexaenoic acid (EPA+DHA, respectively).

On the other hand, the female gonads, unlike the flesh, displayed a selective retention of the essential fatty acids EPA, DHA and arachidonic acid (ARA) independent of dietary regime. The surprisingly high levels of ARA in the tissues compared to the poor amount supplied by the diets highlights the physiological relevance of this FA in this species' reproductive performance and suggests the potential capacity for its endogenous production from the 18:2n- 6 precursor.

The sensorial analysis found no differences in selected sensory categories between the fish fed the carp diet and the DIVERSIFY diet.

The use of excessive levels of soybean in fish diets can cause inflammatory responses in the distal intestinal epithelium, which affects fish health, reduces intestinal nutrient absorption and somatic growth. Inflammation is frequently associated with oxidative stress and the up regulation of the genes involved in the innate anti- oxidation system.

In the DIVERSIFY studies, there was no indication of inflammation. In fact, digestive tract samples from all fish exhibited healthy tissue with no signs of disease and presumably oxidation stress. Although there was a significant improvement in the performance of fish fed the diet that included poultry meal instead of increased soybean meal, it was likely due to a taurine deficiency.

Taken together, the results suggest that there is a significant improvement in grey mullet juvenile performance when using animal-based proteins, such as poultry meal, at about 13 percent DW diet. On the other hand, this advantage may be modulated by the supplementation of essential amino acids such as methionine and taurine.

 

Larval husbandry

The studies on grey mullet larval husbandry determined that the most effective concentration of microalgae daily added to the larval rearing tanks of grey mullet was 0.4 x 106 cells ml-1 of Nannochloropsisoculataor 0.023x106cells ml-1 of Isochrysisgalbana, in terms of larval growth and survival.

These microalgal concentrations, although differing between these species, both provided the same level of turbidity of 1.19 NTU. Turbidity is considered a factor that facilitates

prey recognition and larval consumption by providing a contrasting background.

On the other hand, further studies revealed that the dominant factor defining the benefit of algal tank supplementation was the biochemical composition of the microalgae, which contain unidentified compounds common to both, Isochrysisgalbanaand Nannochloropsisoculata, that promote larval growth and survival.

Although algal supplementation to the larval rearing tanks did not affect the ontogeny of brush border and pancreatic digestive enzymes, there were dramatic changes in enzyme activity as a function of age and the transition from strictly carnivorous larvae to omnivorous juveniles.

Alkaline phosphatase activity, a marker for brush border absorption, was ca. eight times higher and α-amylase activity increased 5.3 times in 79 dph mullet compared to 40 dph individuals. In addition, gut maturation occurred around 61 dph.

The results suggest that aquaculture feeds at this developmental stage should include not only considerable protein but also higher levels of starch or other low-cost amylolytic energetic compounds compared to starter feeds fed to younger grey mullet or the juvenile stages of carnivorous species.

From these studies, the clear benefits of microalgal addition at species-specific concentrations to the larval rearing tanks of grey mullet were shown. Further studies also highlighted that the use of lyophilised microalgae was just as effective as the use of live microalgae, in terms of tank turbidity as well as larval rotifer consumption, swim bladder inflation, growth and survival.

Interestingly, the use of lyophilised microalgae enhanced the maturation of the intestine more rapidly in grey mullet fry, suggesting earlier weaning onto a dry prepared diet is possible, when using this dried alga. Taken together, the results of this study showed that using lyophilised algae would be a significant saving in time, labour and infrastructure and may have expressed a growth advantage in older fish and is recommended in the larva rearing of grey mullet.

From these studies it was shown that juveniles are producing increasing amounts of amylase at the same time that protease activity is decreasing at an age when they are migrating to lower salinity estuarine waters.

This begs the question whether weaning diets should be designed for a carnivorous, herbivorous or omnivorous mode of feeding. The results showed that fish performance was best, in terms of growth, survival, feed efficiency and gut maturation when fed an omnivorous diet.

Furthermore, the high amylase and maltase activity in the omnivorous diet would provide glucose as an energy substrate, which could be protein sparing resulting in improved growth. These results continue to support the use of high carbohydrate-low protein diets to wean juvenile grey mullet, which would be more economical.

The results of the larval studies were implemented in the development of a grey mullet larval rearing protocol, which was tested in six m3 semi-commercial V-tanks in Israel. In the 2017 season, 78,704 juveniles were produced as a result of the production protocols. This did not include the juveniles harvested for experimental tasks that year within the framework of DIVERSIFY.

This meant that the entire juvenile production for 2017 was ca. 200,000 fish and survival was 20 percent from egg to 60 dph, which makes commercial juvenile production of grey mullet a reality.

 

Grow out husbandry

Fish meal substitution between 50 and 75 percent by a mixture of different plant protein sources (corn gluten, wheat gluten and soy protein concentrate) in wild grey mullet fry weaned onto compound diets did not affect good growth performance and survival.

The proximate composition, pancreatic and intestinal enzyme activity confirm the capacity of this species to digest plant protein sources at early life stages. These results indicated that weaning diets for wild grey mullet harvested for restocking aquaculture ponds and on-growing may be formulated with a high level of fish meal replacement by alternative plant protein sources.

Moreover, it seems plausible that fry of this species may accept and use satisfactorily compound diets with a complete fishmeal substitution by plant protein sources. Diets with a 50 and 75 percent of fish meal replacement by plant protein sources were 15.5 and 23.6 percent less expensive than the fish meal diet, which is very relevant considering that feed costs account for >50 percent of the production costs in aquaculture production.

Three separate experiments tested the effect of stocking grey mullet at different densities (4, 6, 10, 12, 29, 55 and 286 fish per m2) in a range of cement and polypropylene tanks. The results showed that increasing the fish stocking density above six fish per m2 can lead to decreased growth in an increasing segment of the population resulting in larger numbers of smaller fish.

This may be a result of higher stress among cohorts from increasing competition for the same food sources. In future studies, the effect of increased ration size, use of extruded and not pelleted diets as well as the number of meals per day (simulate continuous feeding) will be employed.

This should reduce the number of slower growing, smaller fish in the population and increase the efficiency of grow-out. The effect of different stocking densities during grow-out was tested in Greece (4 and 6 individuals per m2), Spain (0.5 and 1.0 fish per m2 , and Israel (1 and 2 fish per m2).

Generally, poor growth was reported in all countries with no significant effect of density or observed differences in proximate and fatty acid analyses. Spain did report a trend of improved growth and feed efficiency in the lower stocking density treatment, while this inclination was muted in the Greece and Israel trials.

The generally poor performance of the grey mullet in the Greek, Spanish and Israeli trials can be due to a number of factors. Certainly, attempting to grow mullet in full strength seawater (40%), which was the case in the Israeli trial, is not going to deliver the best growth.

This is because a significant amount of energy will be channeled into osmoregulation instead of building tissue. However, a major impediment is likely the extruded diet, which remains not sufficiently attractive to the fish as they appear to prefer the primary productivity of the pond to the more nutrient dense feed.

Moreover, in earthen ponds the mullet are likely using sediment to aid mashing of the plant material in the gizzard for better digestion and absorption. In order to improve the feasibility of intensive monoculture of this species, the dietary formula of the current grey mullet feed must be improved.

 

Socioeconomics and new product development

Grey mullet is a fish species that is known only in limited areas of Europe. In the areas where pond aquaculture is a common practice, the species is well recognised by consumers and it is included in the local restaurants of the regions.

Nowadays there is a new generation of chefs trying to promote the consumption of grey mullet due to the association of this species with pond culture in areas with high natural value and in some cases, using culture practices in accordance with the respect to the surrounding ecosystem.

Grey mullet benefits from the high primary productivity and special features of this ecosystem. With regard to aspects of its final product features, the grey mullet has its own individualities.

It is commonly sold as a whole in a range of sizes from 300g up to two kilogrammes. The bigger specimens are used to produce the product known as 'bottarga', which is the salted and dried female roe. The species is well known, especially in Middle East countries and North African communities. With the work developed in the DIVERSIFY project, the high filleting yield of the species has been confirmed (usually exceeding 40%), which is a very promising feature when considering filleting or further processing. The total proximate composition of the products developed (protein, lipid, moisture, inorganic and carbohydrates content), the energy contents of the selected products, the quantitative nutritional value in aspects of fatty acids and the sensory profile of each of them have been determined.

As expected, processing had an effect on both the proximate composition and fatty quality of the products when compared to the raw fillet tissue. However, the effect depended on the processing method used as well as the inclusion of additional materials (such as olive oil) during the product formulation.

The lipid content of the fillet ranges from

As with other fish species, grey mullet contains high omega-3 polyunsaturated fatty acids, although it contents depends on the dietary history of the fish. In absolute concentrations, the input that the consumer gets by consuming grey mullet is subjected to the total fillet lipid contents and therefore, it is also very variable.

The omega3/omega6 ratio which is an important nutritional health indicator exceeds the minimum-required ratio of two, therefore indicating a food with high health benefits.

Concerning product development from the DIVERSIFY species, new product concepts, generated combining information of the market perceptions and the technical limitations and the economical prospect efficiencies, were submitted to a quantitative screening.

For the grey mullet, smoked fillets and fillets in olive oil were the two prioritised products, both of them processed forms of medium degree of processing.

Regarding sensory properties, grey mullet processed products exhibited unique sensory profiles. The processed products showed a more complex sensory profile, with more attributes than the unprocessed cooked fillet of the species. The developed characteristics of the processed products in their majority were connected to the added materials and/or the processing method.

The sensory analysis of the two products created from grey mullet. The smoked grey mullet fillets are mainly characterised by a smoky aroma, salty taste and sardine flavour and a fibrousness texture. The grey mullet fillets in olive oil are characterised by salty and sardine aroma, canned tuna flavour, and a fibrous and secondary greasy texture.

Finally, we evaluated the correlation between the fish dietary history (e.g. dietary fat and protein levels, fat sources, etc.) or other rearing parameters (e.g. rearing system, temperature, or density) and the end-product quality. Results from DIVERSIFY indicated that filleting yields and protein contents did not seem to be influenced significantly by rearing and dietary histories at the grow out stage.

In addition, basic information regarding the packaging of the food products, conservation conditions, preliminary product shelf life and consumer handling/cooking specifications were provided as well.

The technical feasibility suggested that it was possible to produce these products at an industrial scale, which was corroborated by the presence of other similar products in the market.

The results of the consumer test carried out with the fish products developed with grey mullet have shown the strong influence of having the product information in advance on the consumer acceptance degree. The two products prepared with grey mullet, grey mullet fillet preserved in olive oil and grey mullet smoked fillet showed an overall a good acceptance by consumers in all the countries participating in the test (Spain, Italy, Germany, UK and France).

Market research has identified the market potential for grey mullet and indicated a low to medium market impact for the fish market and aquaculture market based on the relatively easy processing of this species and a few high-margin products that can be created.

There is already market demand for bottarga and grey mullet in the Mediterranean basin countries, so market penetration can be done relatively easily by just emphasising that grey mullet is now available all over Europe. Buyers from supermarkets are always interested in new speciesthatcanincreasetheirmarketshareinspecificbuyingsegments.

Grey mullet can be attractive as fresh and as frozen product. Fresh locally produced in the EU is for their retail margins much more attractive than frozen meat from another continent.

In conclusion, the grey mullet is a very promising species in aspect of its end product quality. Besides the bottarga which is a well-established market delicacy, grey mullet can be utilised for commercialisation of its nutritious flesh and additionally it can create additional highly accepted/valued processed forms.

A technical production manual has been produced for grey mullet and can be downloaded from the project's website at www. diversifyfish.eu.

A technical production manual has been produced for grey mullet and can be downloaded from the project's website at www.diversifyfish.eu.

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