Determining the incidence of granulomatosis in meagre (Argyrosomus regius): modular effect of the nutritional balance of micronutrients in diet

Abstract

El cultivo tradicional de especies como la dorada (Sparus aurata) o la lubina (Dicentrarchus labrax) han alcanzado su madurez, forzando a la búsqueda de nuevas especies que sean capaces de cubrir otros nichos de mercado. La corvina (Argyrosomus regius) es una especies de rápido crecimiento que ha sido propuesta como un buen candidato para la diversificación de la acuicultura. Sin embargo, la salud es uno de los puntos más críticos en su producción, siendo la granulomatosis sistémica la enfermedad más frecuente, llegando a afectar hasta al 100 % de la población de corvina cultivada. El objetivo de esta Tesis fue evaluar el efecto de nutrientes antioxidantes, como son la vitamina E y C, el uso de diferentes secuencias de alimentación durante el cultivo larvario, diferentes densidades de cultivo y la suplementación de los minerales manganeso, selenio y zinc, sobre la aparición de la granulomatosis sistémica en larvas y juveniles de corvina. En esta Tesis, se ha observado que los granulomas pueden comenzar a aparecer en larvas de tan solo 20 días de vida, sin embargo, su aparición se puede evitar con la correcta secuencia de alimentación y suplementación en la dieta, al menos hasta 44 días de vida. La incidencia de granulomas en juveniles es muy alta y la suplementación de vitamina E y C en la dieta puede retrasar o impedir la aparición de nuevos granulomas, pero no se pueden eliminar los ya existentes, siendo necesario actuar en los primeros estadios de desarrollo. Los resultados muestran que un alta suplementación en la dieta de las vitaminas antioxidantes E y C tienen una influencia en la incidencia de esta enfermedad, lo que sugiere que puede ser mediada mediante factores nutricionales.

The farming of traditional aquaculture species such as sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) has reached their maturity and has forced the search of new species which can cover other market niches. Meagre (Argyrosomus regius) is one of the fast-growing species proposed as a candidate for marine finfish diversification on commercial aquaculture in the Mediterranean and Eastern Atlantic coasts. Nevertheless, one of the most critical points in meagre production is the fish health, considering the systemic granulomatosis as the more frequent disease in this species. The objective of the present Thesis was to study the effect of antioxidant nutrients, such as vitamin E and C (Chapters 3, 5 and 6), different feeding sequences (Chapter 4), stocking densities (Chapter 6) and supplementation of manganese, selenium and zinc (Chapter 6), on the appearance of systemic granulomatosis in meagre larvae and juvenile. In juvenile and adult meagre, the incidence of systemic granulomatosis can affect up to 100 % of the population, so, firstly, an experiment was carried out to elucidate the effect of the supplementation with vitamins E, C and K on the incidence of granulomas in juvenile meagre (Chapter 3). Juvenile meagre were fed six diets with different levels of vitamin E, C and K (150/20/0, 150/20/23, 300/70/0, 300/70/23, 450/230/0 and 450/230/23 mg kg-1, respectively). In this study, a molecular approach was carried out, by studying the expression of several genes related to oxidative stress (sod, cat and gpx) and immune system (cox-2 and tnfα). Supplementation with 450 mg kg-1 vitamin E and 230 mg kg-1 vitamin C strongly induced the expression of antioxidant enzymes and immune genes, in an attempt to reduced deleterious effect of reactive oxygen species. The incidence of granulomas in liver and heart was reduced when high levels of antioxidant vitamins E and C were added to the diet. An immunostaining for actin showed the presence of actin around some of the granulomas and the observation of irregular aggregated of cells around the blood vessels, what could suggest a possible origin of the granulomas in the blood vessels. The results of this experiment showed that the incidence of granulomas in juvenile meagre is very high (86-100 %), and an adequate supplementation of vitamin E, C and K can retard and avoid the appearance of new granulomas. However, the granulomas already present cannot be eliminated, being necessary to act in earlier stages. For that reason, an additional study was carried out in meagre larvae (Chapter 4). Meagre larvae were fed four different feeding regimes as follows: RS and RO (rotifer enriched with Easy DHA Selco or Ori-Green from 3-30 dph, respectively), AS and AO (rotifer enriched with Easy DHA Selco or Ori-Green from 3-21 dph and Artemia enriched with Easy DHA Selco or Ori-Green from 12-30 dph, respectively). It was observed that meagre larvae could be weaned from enriched rotifer to inert diet after 20 dph, but the lack of Artemia in the feeding sequence lead to poor survival and growth probably because of a high energy cost/benefit related to the small size of the prey, indicating that is necessary an intermediate feeding between rotifers and microdiet. Lipid peroxidation, as indicated by TBARS value, was higher in fish fed without Artemia in the diet. The results showed that granulomas first appeared in meagre larvae at 20 dph when fed rotifers only. Conversely, a reduced appearance of granulomas and lipid peroxidation occurs when Artemia is included in the feeding sequence. In the light of these results, a new experiment was planned (Chapter 5), where meagre larvae were reared until 30 dph following the best feeding sequence obtained in the previous study and then, fed from 30 to 44 dph with five different diets with graded levels of vitamin C and E (40/100, 200/500, 400/1,000 and 800/500, respectively) and a fifth diet with 400/1,000 and substitution of fish oil by krill oil. Despite the fact that antioxidant vitamins did not affect growth performance, the appearance of granulomas was avoided when the microdiet was supplemented with 800 mg kg-1 of vitamin E and 2,000 mg kg-1 of vitamin C. This mitigation was simultaneous with the reduction of TBARS content in larvae, which were indeed highly correlated with the appearance of granulomas (R2=0.892, y=0.0446x+0.0756). The results of this study showed that the occurrence of systemic granulomatosis seemed to be associated to the larvae peroxidation status, so that high dietary levels of vitamin E and C (800 and 2,000 mg kg-1, respectively), reduced lipid peroxidation and completely prevented the appearance of granulomas in meagre larvae at 44 dph. At this point in the Thesis, it was observed that the systemic granulomatosis can first appear in very early stages of life (20 dph), but with the adequate feeding sequence and vitamins supplementation in the microdiet it can be avoided, almost until 44 dph. In the last study it the effect of different levels of vitamin C (100, 600, 1,200 and 3,200 mg kg-1), minerals Se, Zn and Mn (1.5 mg kg-1, 200 mg kg-1 and 40 mg kg-1, respectively) and two different densities (3.20 and 6.20 kg m-3) on the appearance of systemic granulomatosis in juvenile meagre was evaluated (Chapter 6). Supplementation of vitamin C and E and minerals Mn, Zn, and Se did not affect meagre growth in terms of final weight, length, FCR and SGR, with growth parameters only affected by the stock density, being lower in fish reared at a high density (6.20 kg m-3). High supplementation of vitamin C (1,200-3,200 mg kg-1) significantly reduced the percentage of granulomas in any tissue, but only when juvenile meagre were reared at 3.20 kg m-3). The high stock density can disturb the balance between the production of ROS and its removal, and this imbalance can in turn lead to a status of oxidative stress which may cause different diseases and lesions. In the present study, the levels of TBARS were affected by the stock density, being higher in fish farmed at high density. Therefore, the present results suggest that the high stock density is a stressful factor that can lead to oxidative stress, lipid peroxidation and promote the appearance of granulomas. The present results show that high levels of vitamin C (1,200-3,200 mg kg-1 C) and low stock density (3.20 kg m-3) favour the growth of juvenile meagre, reducing the lipid peroxidation indicators and decreasing the incidence of granulomas, which confirms that this pathology is mostly triggered by the deficiency of antioxidant nutrients, particularly vitamin C. These studies have shown that the granulomas can begin to appear in meagre larvae at 20 dph, but they could be avoided with the correct feeding sequence and supplementation of vitamins, almost until 44 dph. The incidence of granulomatosis in juvenile meagre is high and the supplementation of vitamin E and C in the diet can retard and avoid the appearance of new granulomas but the granulomas already present cannot be eliminated, being necessary to act in earlier stages. High stock density is a stressful factor that can reduce growth, increase the appearance of systemic granulomatosis and oxidative stress. The results show that the combination of high dietary content of antioxidant vitamins E and C can have an influence on the incidence of the granulomatosis in meagre, which suggests that this pathology could be mediated by nutritional factors.