Stéphane Frouel, Julie Castier and Maxime Hugonin of Mixscience, France, report on a trial run in Portugal to evaluate the potential of a commercial phytogenic to improve the resistance and performance of rainbow trout Oncorhynchus mykiss challenged by Yersinia ruckeri

Effect of a commercial phytogenic solution on Rainbow Trout

Oncorhynchus mykiss) challenged by Yersinia ruckeri

by Stéphane Frouel, Aquaculture Project Manager, Julie Castier, RID Project Manager and Maxime Hugonin, Aquaculture Product Manager, Mixscience, France

Aquaculture is the fastest growing food-producing sector in the world, contributing to one-third of the global food fish production.

The nutritional benefits of fish consumption have a positive link on protein availability, which helps to decrease poverty rates in developing countries.

Rainbow trout Oncorhynchus mykiss is one of the most important farmed aquatic species. It is assumed by experts that the world's consumption of rainbow trout hit 950,000 tonnes annually at the beginning of this year.

The majority of this volume is being provided by aquaculture, with global sales volume of farmed trout reaching roughly 830,000 tonnes. The continued relatively high cost of salmon has seen rainbow trout establish itself as a cheaper alternative fish, offering many of the same health benefits.

The specialists predict a compound annual growth rate for worldwide rainbow trout market of approximately five percent over the next 10 years.

Nevertheless, this trout industry is facing continuous challenges and diseases that significantly affect the profitability and quality of farm production.

A serious disease in Salmonids

Among main bacterial diseases, Enteric Red Mouth (ERM) disease is a serious septicemic bacterial disease of Salmonid fish species. It is caused by Yersinia ruckeri, a gram-negative rod-shaped enterobacterium that has a wide host range, broad geographical distribution and causes significant economic losses.

This disease gets its name from the subcutaneous haemorrhages that appear at the corners of the mouth and tongue, with other clinical signs include exophthalmia, darkening of the skin, splenomegaly and inflammation of the lower intestine with accumulation of thick yellow fluid. The bacteria enters the fish via the secondary gill lamellae and from there, it spreads to the blood and internal organs. To date, the main solution to this issue remains the use of antibiotics thanks to their easy use and rapid effects in curative treatment.

Unfortunately, the use and abuse of chemicals raises public health concerns such as antibiotic resistance and adverse effects on the environment. This in turn has in some cases lead to these remedies earning aquaculture production and its associated products a negative public image. In order to explore alternatives active research is currently ongoing which will reduce their use.

Nutrition and feed additives with specific antibacterial and/or immune-modulatory properties are increasingly important to supporting the industry with many of these challenges.

Among these alternatives, which includes solutions such as phytogenics, interesting benefits have been found such as growth performance improvement, feed efficiency optimisation and disease resistance.

One of them, developed by Mixscience, France, has proven its efficiency from laboratory to field scale in different aquatic species (Hugonin et Frouel, 2019). In this study, authors sought to explore its efficacy on the resistance of rainbow trout (Oncorhynchus mykiss) to an infection by Yersinia ruckeri.

Moreover, in order to extend the application to the full value chain of the trout industry and to ensure that the product could be applied either at the farm or in the feedmill through the extrusion process, the product was tested either directly in the extruded feed or top dressed in the feed after the process. The resulting bio-efficacies were then compared.

Trout trial

The trial was conducted in indoor facilities in Portugal, with rainbow trout (Oncorhynchus mykiss) originated from a commercial farm the experimental species under testing. The fish were transferred to the experimental facilities by a duly authorised carrier and kept in sanitary quarantine for three weeks.

No significant mortality or pathological signs were observed in association to their transport. During this quarantine period, the fish were maintained and fed with a standard commercial trout diet.

The trial had three distinct experimental phases: an adaptation period, a feeding period and a challenge period as described in Figure 1.The trial comprised a total of four dietary treatments, all based on a single basal formulation.

This basal formulation contained 20 percent of marine derived proteins (fishmeal and fish hydrolysate) and plant ingredients such as soy protein concentrate, wheat gluten, corn gluten meal, soybean meal, whole peas and wheat. A blend of fish oil and rapeseed oil was used as the main lipid source. This basal diet was supplemented with crystalline essential amino acids and an inorganic phosphate source to avoid any nutritional deficiencies. All diets were isonitrogenous (44 percent crude protein), isolipidic (22 percent crude fat) and isoenergetic (22.5MJ/kg).

This diet served to feed both the non-challenged positive control [PC] and the challenged negative control [NC] treatments. This basal formula was also further supplemented with the test additive at five kg/tonne [Phytogenic EX], with the product being incorporated in the mash (pre-extrusion).

In order to compare extrusion and top-dressing applications, one diet containing the basal formula was also top dressed with the test additive at five kg/tonne [Phytogenic TD] after extrusion.

Diets were manufactured using extrusion and all of the powder ingredients, including the phytogenic (Phytogenic EX), were mixed accordingly to target formulations in a double-helix mixer and ground (below 400µm) in a micropulveriser hammer mill. Diets were manufactured by means of a pilot-scale twin-screw extruder with a screw diameter of 55.5mm and temperature ranging from 106- to 111ºC. Feeds were dried in a vibrating fluid bed dry.

In the case of the treatment with top dressed phytogenic (Phytogenic TD), following drying, the oils and the product were applied by vacuum coating. The suspension of oils and additives were sprayed onto the pellets under vacuum (800mbar) for approximately two minutes.

Phase I - Growth performance trial

Stock of rainbow trout with a mean initial body weight (IBW) of 26.8 ± 1.6g were fed one of the four experimental diets during 35 days. Fish grew in quadrangular PVC tanks (volume: 750L) supplied with recirculated freshwater.

Throughout the trial, average water temperature was 15 ± 0.3ºC and average dissolved oxygen level was 6.4 ± 0.5mg/L. Fish were subjected to a controlled photoperiod regime of 12:12 (light:dark). Fish were hand fed to visual satiety, with three meals per day (09:00, 14:00 and 17.00) during week days and twice a day (09:00 and 14:00) during weekends. Utmost care was taken to avoid feed wastage and allow a precise quantification of feed intake and the fish were group weighed at the start and at the end of the trial.

Phase II - Disease challenge with Yersinia ruckeri

After 35 days of feeding, fish from each dietary treatment were subjected to a strong disease challenge with Yersinia ruckeri, isolated from rainbow trout in Portugal. Bacteria were routinely cultured at 22˚C in tryptic soy broth (TSB) or in tryptic soy agar (TSA) supplemented with NaCl to a final concentration of one percent (w/v).

Both cultures were stored at -80˚C in TSB supplemented with 15 percent (v/v) glycerol. To

prepare the inoculum for injection into the fish peritoneal cavities, 100µL of stocked bacteria were cultured overnight at 22˚C on TSA. The bacteria were collected exponentially from the TSA and re-suspended in sterile TSB. Based on previous challenge data, the intended bacterial concentration to kill 20-30 percent of the fish (LD50) was obtained by absorbance reading and adjustment against its growth curve to 4 x 106 colony forming units (CFU)/ml. Bacterial concentration was confirmed by plating the resulting cultures on TSA plats and counting of the colony forming units (CFU/ml).

After the 35 days of feeding period, 80 fish per dietary treatment with an average body weight (100 ± 5g), were transferred to the challenge room and intraperitoneally injected with 100µl Yersinia ruckeri (3.6 x 106CFU/ml). The only exception were fish from the PC treatment, which were intraperitoneally injected with an identical volume of Hanks' Balanced Salt Solution (HBSS). Thereafter, groups of 20 intraperitoneally injected (Yersinia or HBSS) fish were randomly distributed among 60L aquaria.

During the infection challenge period, each dietary treatment was tested in quadruplicate. Fish mortality was recorded for 17 days, with the weight of every dead fish noted. Although showing a reduced appetite, the fish were fed with the corresponding diet during the challenge period. Water rearing conditions were kept identical to those recorded during the growth performance phase (temperature: 15 ± 0.5°C; dissolved oxygen > 6.0mg/L).

Impact of the phytogenic on performance

No mortality occurred during the feeding period before the set-up of the challenge indicating that the phytogenic had no adverse effect under normal conditions.

At the end of the 35 days experimental feeding period (Phase I), the rainbow trout showed a 3.9-fold increase of initial body weight in best performing treatments. Dietary treatments had no significant effect on final body weight (FBW) and specific growth rate (SGR) (P<0.05).

Therefore, neither the phytogenic supplementation nor the feed application mode (pre- or post-extrusion) seemed to affect performance criteria in rainbow trout [see Table 1].

the absence of a statistical significance and within a very acceptable range, the treatment with product applied by top-coating at 5kg/tonne (Phytogenic TD) tended to show a slight increase of FCR, and consequently a lower PER, than Phytogenic EX, in which the same product dose was applied in the mash (pre-extrusion). Although not considered as problematic, this aspect should be assessed during a longer feeding period.

Effect of the phytogenic on disease resistance

After the challenge, the total mortality of fish from the PC treatment (not infected with Yersinia ruckeri, but injected with HBSS) was low and reached only 3.8 ± 2.5 percent.

The initial target of the challenge was 20-30 percent of mortality. In the present trial, the challenge was stronger than expected since the total mortality observed in the challenged fish was 42.5 ± 6.5 percent [see Figure 2].

Dietary supplementation with phytogenic at a dose equal to 5 kg/ton showed a strong protective effect against an infection with Yersinia ruckeri in rainbow trout. In comparison to fish fed with the NC diet, those fed with Phytogenic TD and Phytogenic EX showed a significant relative reduction of mortality (ranging from 41 to 47 percent respectively). The additive quickly stabilized the impact of Yersinia since mortality did not evolve eight days post challenge whereas it continuously increased in NC treatment.

Both in mash (pre-extrusion) and top-coating (post-extrusion) application modes were found effective suggesting that the phytogenic does not lose its bio-efficacy over the extrusion process.

Effective control

This study evaluated the protective effect of a commercial phytogenic on the growth performance and resistance of rainbow trout (Oncorhynchus mykiss) to an infection by Yersinia ruckeri.

A secondary objective was also to compare the impact of extrusion process on bio-efficacy of the actives contained in the product by comparing extrusion or top-dressing applications.

We concluded that this feed additive provides efficient control for trout under challenged conditions, whatever the mode of application. These results consolidated the already demonstrated efficacy of the product in different aquatic species and confirm that this phytogenic can be considered as an holistic approach to control the use of antibiotics in all aquaculture systems.

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