Impact of the partial replacement of fish meal with a chloroplast rich fraction on the growth and selected nutrient profile of zebrafish (

In recent years increasing aquaculture production combined with a high variance in availability and cost of fishmeal and fish oil commodities, has led to a need to identify alternative source materials for protein and the polyunsaturated fatty acids (PUFAs) which fish oil is prized for containing. Chloroplasts are the organelles in plants’ leaves where many of the valuable nutrients, fatty acids (FAs), amino acids, vitamins and pigments, are synthesised. Chloroplasts could be incorporated into fish diets either retained in, or liberated from, plant cells. In this study zebrafish were fed with seven different diets individually; fish was fed with reducing fishmeal levels (10, 20 or 50%) by either spinach leaf powder (SLP) or a chloroplast rich fraction (CRF) prepared by an established method to recover chloroplasts. Both SLP and CRF had a positive impact on growth, taste response, whole fishFA composition, and carotenoid profile. Fish fed with CRF diets showed significantly (P≤0.05) greater α-linolenic (C18:3 n-3) and hexadecatrienoic (C16:3) acids content than those of SLP and the control. Hexadecanoic acid (C16:3) is a unique FA in the galactolipids of the chloroplast; its presence in zebrafish tissues proves that zebrafish digest and absorb chloroplast galactolipids. Lutein profile of eggs produced by zebrafish fed with CRF diet was significantly (P≤0.05) higher than that of SLP and control. Alterations in egg colour were also noted, warranting further investigations of diet impacts on fish fecundity, embryo fertility, hatch rate and larval survival.


Introduction
Fish and its products can be consumed in various forms and are a good source of proteins, fats, vitamins and minerals (1). The aquaculture industry has grown in recent years to provide approximately 54% of world fish and seafood in 2016 (2), with e 70% of fish produced using aquaculture feeds in 2012 (3). Many of these feeds include fishmeal, as a source of high quality protein, and fish oil, which is high in unsaturated FAs, (4) to provide the necessary nutritional profiles for target species. Fishmeal and fish oil are produced from capture fish and fish byproducts (3,5), therefore the availability of both is affected by catch rates and raw material availability.
As world aquaculture fish production has increased (from 39 million tonnes in 2011 -54 million tonnes in 2016 (2), this puts extraordinary pressure on the supplies of fishmeal and fish oil. There is consequently a need to identify cheaper and more sustainable alternatives to both protein and oil materials, at least partially.
Zebrafish (Danio rerio) are now established as an excellent model organism for study in areas such as developmental biology, toxicology, genetics and medicine (6)(7)(8)(9). They have also been used as model for studying lipid metabolism under the microscope (10).
Zebrafish share a high degree of amino acid sequence and functionality with mammals including humans. They also possess characteristics, including simple husbandry, gregarious behaviour, small adult size, rapid growth, development and maturation, Please do not adjust margins Please do not adjust margins high fecundity and productivity, which make them an ideal model species for pilot feed studies on novel and difficult to source or synthesis ingredients (11,12). SLP, and CRF were investigated as dietary ingredients in zebrafish to determine whether they could partially substitute for fishmeal fish feed.
Chloroplasts are a type of semi-autonomous organelle found in plant leaves but also in other organisms like algae. They are responsible for photosynthesis, the process where sunlight energy is converted into chemical energy. It is in chloroplasts where valuable macro and micro-nutrients including protein (~50-60%), lipid (35-40%), and lipid soluble pigments such as chlorophylls, carotenoids (e.g. provitamin A and lutein) and tocopherols/vitamin E (5-10%) are synthesised (13)(14)(15)).
Use of CRF as an ingredient facilitates the use of plants that are not conventionally consumed by humans or fish in feed manufacture adding valuable nutrients at the same time. The nutrient profile of CRF from green plant species shows it is rich in proteins, lipids, essential FAs, vitamins and minerals, which could be used as potential functional ingredients in human or animal food formulations (16,17).
The major FA (>60%) in chloroplasts is the omega-3 FA known as α-linolenic acid, mainly in the form of galactolipids. Although ALA can be provided to fish by various plant sources, like vegetable oils, the most abundant source of ALA in the biosphere is galactolipids that make up the majority of the thylakoid membranes of chloroplasts. In our study the increase in fish-body ALA content can be attributed to the digestion and update of ALA from the galactolipids in the SLP and CRF material.
A portion of this FA is converted by zebrafish into long chain PUFAs, found in fish lipids (18).  (19). Consumption of fish oils also contributes to energy supplies and assists in the proper absorption of fat soluble vitamins namely A, D, E, and K through digestion (20).  (16), carried out in our laboratory, and their relevant compositional analysis to this study is depicted in Table 2.

Preparation
All ingredients required were thoroughly mixed using a Hobart food mixer andwater was added until fell between the two. All diets were refrigerated at +4 º C for the duration of the study.

Subjects and Husbandry
All fish were bred in-house at The Institute of

Feeding and Sampling
Fish were hand fed 4% body weight using Sarstedt Total Feed Intake (TFI) = total feed given -waste output. Net production = Mass at end of study period -mass at start of study period. (22) SGR demonstrates the growth achieved per day during a time period of which subjects are fed the test diet, calculated as follows: Where: Ln = natural log W 1 = Initial weight W 2 = Final weight t 1 = Starting time point (day one) t 2 = End time point (final day number) (23)

Diet Palatability and Acceptance
Feeding observations were carried out weekly to assess fish response and feed acceptance. The amount (%) of feed (4% body weight of all fish in the tank) that remained at 20 second time intervals during the five minute period post feed introduction was estimated to the nearest 10%. Observers were trained to recognise the amount of feed remaining in a tank.

Zebrafish Spawning
Each of the seven diets was fed to two tanks, one male and one female tank, six fish per tank. Feed was given daily ad libitum until satiation. After three weeks of feeding the fish were bred by mixing males and females together (1:1 male: female ratio) one evening in medium (3 L) Aquatic Habitat zebrafish tanks, measuring 11.5 cm x 25 cm x 15 cm. A mesh net with a 2 mm aperture was placed in the tank to create a false raised base with a gradient to prevent cannibalism of the eggs. As soon as the lights came on the next day the flow to the tank was switched off and the net was raised slightly to stimulate spawning. After two hours the fish were removed and returned to all male or all female housing. Eggs were collected, transferred to sample jars then snap frozen with liquid nitrogen; samples were stored at -80 o C. Spawning was repeated every three days until sufficient eggs had been collected for the required sample size. Fish were then euthanized using a Home Office approved Schedule 1 method. Fish were also stored at -80 o C until further analyses.

Ethical Issues
The work carried out here was done so under the Establishment Licence for the University of

Fatty Acid Composition
The FA content of the zebrafish was determined by esterification of total lipid extracts, obtained from The oven temperature was maintained at 120 °C for 1 min, and then increased to 250 °C at a ramp of 5° min -1 . The final temperature of 250 °C was held for 2 min. Detection was conducted using a mass spectrometer and identification of individual FA was achieved using a mass spectrum library and its comparison of retention times to FAME standards.
The percentage content of each FA was calculated and the concentrations (mg mL -1 ) were determined using the response factor of the internal standard.

Zebrafish Retinol Analysis
The analysis and detection of all trans retinol in the whole zebrafish for test trials and controls was based

Zebrafish and Eggs
Carotenoids were extracted as section 2.8.3, but the detection program of the HPLC was different and was based on Garner, Neff (27). Briefly, two mobile

Growth, Performance and Palatability
Both the CRF and SLP materials had a positive impact on growth (Fig.1). No significant differences (P≥0.05) were found in growth between the control diet and any of the experimental diets, except for the CRF10 and SLP10 diets, which showed significantly (P≤0.05) increased growth compared with the control (Fig.1). The only significant differences between the control diet and any experimental diet for FCR was a decrease in both Assessment of diet consumption rate indicates that inclusion of both the CRF and SLP materials has a positive impact on diet taste or appearance. Fig.3 shows that all experimental diets were consumed at A B Damaged chloroplasts Figure 2 Representative microstructure of intact CRF (A) and SLP with partially damaged CRF dots (B). Optical microscopy with differential interference contrast microscopy (DIC) was used (Leitz Diaplan Microscope). A drop of each sample (initially dissolved with water) was placed on a glass slide, topped with a cover slip and allowed to dry before being placed under the microscope. All images were taken using a 100X objective lens. A digital camera was attached to the microscope to capture images of the samples. The scales of the images were calibrated against a glass mounted graticule (1 mm, 0.01 mm division from Graticules Ltd, Tonbridge).

Fatty Acid Composition
The FA composition of the trial zebrafish is presented in Table ( The ratio of ALA to EPA (4:1) concentration (mg g -1 ) for CRF diets (by average) in zebrafish body post-feed analysis was calculated from Table 3.
Values were in line with that of zebrafish, fed a commercial meal i.e. 4:1 of ALA to EPA for the non-transgenic sample of a previous study by Yoshizaki,Kiron (37). Table 3   diet. In both cases this increase in ALA appears to be dose dependent.

Carotenoid Content in Zebrafish (Whole)
Lutein and beta-carotene are the major carotenoids in CRF and SLP whilst astaxanthin is the prominent carotenoid in many fish species (mainly marine fish) Similarly, whilst the red colour of pepper is due to capsanthin, the pink red coloration of crustaceans is accounted for by astaxanthin.  In freshwater fish, β-carotene is converted into retinoic acid and in some cases into retinol, whilst lutein is converted into anhydrolutein, which, in turn, is cleaved to dehydroretinol (vitamin A 2 ), the common form of vitamin A found in most fresh water fish (45). From our data it seems likely that βcarotene was not detected in zebrafish because it is converted into retinol and retinoic acid.

Zebrafish Egg Carotenoids
It was noted that feed composition has a significant impact on fish egg colour (Fig.4) Figure 5 Zebrafish (Casper strain) and its egg pigment variation due to carotenoid accumulation in response to carotenoid-rich diets. This revealed that carotenoids have essential role in the embryonic development of fish (9).

Retinol Content in Whole Zebrafish
The  (52). When the importance of retinoids in the reproduction of zebrafish was examined using retinoid rich and deficient diets, reduction of whole body retinoids by 68% in females and 33% in males was obtained with the retinoid deficient diet. In addition, females fed with the retinoid deficient diets produced significantly fewer eggs than that contained retinoid or the control (52).

Conclusion
Inclusion of the SLP and CRF materials into zebrafish feed formulations with reductions in fishmeal has proven successful.

Conflicts of Interest
There are no conflicts to declare.