Microbial and nutritional aspects on the production of live feeds in a fish farming industry

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Aquaculture is an enterprise in constant development, in particular relating to its effect on the environment and also the quality of its products. It represents a valid alternative to traditional fishing, facing the increasing demand for fish
  37 O RIGINAL   ARTICLE Microbial and nutritional aspects on the production of live feeds in a fish farming industry A. DE DONNO, F. LUGOLI, F. BAGORDO, S. VILELLA, A. CAMPA, T. GRASSI, M. GUIDODepartment of Biological and Environmental Sciences and Technology, (Di.STe.B.A.), University of Salento, Lecce, Italy J PREV MED HYG 2010; 51: 37-43 Introduction In the last few years the aquaculture industry has assumed a very important role as an alternative to traditional fish-ing; an actual necessity to limit the environmental impact of commercial fishing, the abolition of some techniques and the restriction of the activity to allow for biological repopulation, has made fish breeding an enterprise in ex-pansion and with an outlook to develop with time [1].For these reasons, in Italy and in the other European countries, new productive techniques, especially in the artificial reproduction and feed sectors, as well as inno-vative technology of breeding are developing. Moreo-ver, strategies for the control of the quality, hygiene and the food safety are in rapid evolution.The production process to rear the marine fish strains, like sea bass, is very complex and the quality of the final product depends on several factors [2]. Therefore, for the optimization of the fish production, the indus-try must concern itself with all the productive phases, checking the quality of the products in all stages of production (live food, larvae, final product).In particular, the availability of high quality juvenile stock, together with the environment of breeding, is of great importance, since it can significantly influence the quality of the final product. A larval feed of good quality is a fundamental requirement in order to get a commercial fish of good quality [3]. The successes that occur all over the world in the induced reproduction of sea strains are due mainly to the possibility to offer live Key words Fish-farming • Microbial quality • Fatty acid prey to the larvae, in particular Rotifers (  Brachionus  plicatilis ) and Artemia (  Artemia salina ) [4].The production of live feeds in hatcheries represents a very critical phase in the breeding of many different spe-cies of fish because, it is technically possible to produce a feed of a high nutritional value, its hygienic quality is very low [5-7]. Many bacteria are able to surpress the growth of Rotifers and Artemia or to provoke an unexpected mortality [8]. In other cases the harmful effects occur in the larval stages, shown in low growth rates and low survival rates [9, 10]. Although the greater part of the bacteria is not pathogenic for Rotifers and Artemia, the risk of a potential accumulation end/or transfer of the bacteria along the trophic chain still exist.The Rotifers represent the principal vectors of the bacteria [11, 12]. In fact, even when the breeding of Rotifers is carried out while respecting the hygienic norms [13, 14], the elevated culture density (billion of Rotifers) and the food used for the growth of the live feeds constitute an elevated organic load, which is quickly colonized by bacteria [15-18].Usually the aerobic bacterial population varies from 10 7  to 10 12  ufc/g in the live feeds and from 10 4 to 10 7  CFU/ ml in the culture medium [14].The Rotifers can concentrate until 10 5  CFU/individuos in their intestine and such bacterial accumulation could srcinate from the diet rather than from inside multipli-cation [15]. In the culture medium the bacterial concen-tration remains around 10 7  CFU/ml. Summary  Aquaculture is an enterprise in constant development, in  particular relating to its effect on the environment and also the quality of its products. It represents a valid alternative to traditional fishing, facing the increasing demand for fish  products.To guarantee to the consumer a product of high nutritional, organoleptic and hygienic quality, it is fundamental to monitor every phase of the fish farming industry, isolating the potential risk points.For this reason there has been a rapid evolution of productive technique, particularly in the technology, artificial reproduction and feed sectors.The aim of this research has been the monitoring of the evolution of certain microbial and nutritional quality indexes (total micro-bial counts and lipid analysis on suspensions of Rotifers and  Artemia, used as live feed) in the larval phase of the productive cycle of the farm raised fish, in an intensive system. The study has shown an increment in the total microbial counts in the fish  farming industry within the production of Rotifers and Artemia, more evident in the suspensions of Rotifers.  In addition the study has demonstrated that the maintenance phase, in the enrichment protocol, can reduce the EPA and DHA content. The results confirm the importance of microbial and nutritional control of the live feeds before they get supplied to fish larvae.  A. DE DONNO ET AL. 38 Under conditions of low food quality, the bacteria, fed on by the Rotifers, could compensate for the food deficit [19]. The Vibrio anguillarum  causes a crash in Rotifer crops [20, 21]. Balompapueng et al. [22] discovered bacterial strains such as Flavobacterium ,  Aeromonas  and Vibrio sp . resulted toxic for the Rotifer population. Comps and Menu [23] hypothesized that the infectious diseases (vi-ral and mycotic) were in related to the low productivity of Rotifer cultures and to an abnormal mortality rate.At the moment, most of the rearing systems use cul-ture stock to produce live feed for their larvae. From a microbiological point of view, these systems can be very unpredictable, especially because after the rearing water disinfection there isn’t any microbial control in those initial stages of the Rotifer culture.In this context, the hygienic management of the larval rearing systems represents an issue of extreme impor-tance, for it can strongly influence the larval survival and the hygienic quality of the final product. The feeding of the larvae of rearing fish strains with live feeds represents a delicate and critical phase, especially considering the nutritional health of the fish. The use of live feed guarantees the availability of food with high nutritional characteristics, particularly under the lipidic profile. The lipids represent a nutritional class that is very important for the fish. In fact the lipids develop the energetic, functional and plastic faculties of the fish. In particular, the polyunsaturated fatty acids (PUFA) above all the highly unsaturated fatty acids (HUFA) (with a high number of carbon atoms), especially series ω  3, are very important for the growth and reproduction of the adult fish; besides this they increase the survival rate of the larval forms [24-29]. In terms of this, the essential fatty acids eicosapentaenoic (20:5 ω   3 or EPA) and do-cosahexaenoic (22:6 ω   3 o DHA) are very important, since they represent the precursors of fatty acids and of molecules of high biological interest. Many organisms are able to convert the linolenic acid (18:3 ω   3) in EPA and DHA, however, sea fish are apparently incapable of it (Watanabe 1982) and therefore the presence of HUFA is fundamental in their diet. Although it is true that the freshwater fish are able to carry out this conversion, even for them the presence of EPA and DHA in the feed is impor-tant, probably because such an ability is not particularly efficient in maintaining optimal levels of growth. The larval forms seem to have a greater need for HUFA, probably because they have an elevated metabolism. It is very difficult to quantify exactly, the necessary quan-tity of HUFA, as it varies in relation to the larval stages, to the rearing strains and to the type of zooplankton [28, 30- 32]. Since the nutritional quality of the zooplankton is not always suited to the demands of the rearing animals, tech-niques of enrichment are often used. The Rotifers and the Artemia are fed with integrated diets using different protocols in accordance with the necessi-ties and costs [33].The aim of the present experimental work has been to estimate and to compare different protocols of enrich-ment employed within an intensive rearing system of bass and bream, in order to discover which is the safer, from a microbial point of view, to be employed within the phases of production of live feeds. Moreover, the nutritional characteristics, particularly the lipidic pro-file, were studied in order to understand the influence of different used protocols on the live feeds quality.For this reason the microbial charges in Rotifer and Artemia samples, which came from this rearing system, were evaluated. Materials and methods S󰁡󰁭󰁰󰁬󰁩󰁮󰁧 The microbial investigation on live samples of Rotif-ers and Artemia was preceded by a preliminary phase (about six months) of standardization of the methods. The experimental plan used for this investigation is shown in the Figure 1, for the Rotifers, and in the Figure 2, for the Artemia. The Rotifers were initially fed with crops of phyto-plankton, yeasts and specific commercial products, and enriched with compounds with a high content of poly-unsaturated fatty acids, 12 hours before their administra-tion to the fish larvae. This is a complex production that begins with a sterile algal monoculture in small volumes, culminating in the productions of Rotifers, in great volumes. To the final crop, different protocols of treatment were applied (symbolically indicated s in Fig. 1 with A, B and C) that correspond to common products used in commerce (Selco, HD Culture Selco - ®  INVE - and Rotimac TM  – BioMarine Aqua Fauna Inc.Hawborne,California, USA). In the next phase three different enrichment Fig. 1.  Experimental plan used for the microbiology analysis on the Rotifers.  TOTAL MICROBIAL COUNT AND FATTY ACID PROFILES OF CULTURED ARTEMIA AND ROTIFERS 39 protocols were tested, EASY DHA, FITO and DHA PROTEIN SELCO - ®  INVE - (indicated with E, F e G in Figure 1). For the maintenance phase two different treatments were analyzed; a thermal treatment and a treatment with phytoplankton (T and F in Fig. 1).The production of Artemia, consisted firstly of an incubation period, then the hatching of the cysts, followed by an enrichment phase, which used three different protocols (SPARI, SERRA-NI and SUPER SELCO - ® INVE - respectively indicated in Figure 2 with SP, SE and SS). The maintenance phase was performed through thermal treatment in a refrigerator for 12 hours.The analyses were conducted in the month of Novem-ber. Both the typologies of live feed Total Microbial Counts (TMC) were determined in three sample unit. The collections for the microbial investigations were per-formed in each phase and for each protocol of treatment. The samples were taken using sterile containers, trans-ported in refrigerated containers (4°C) and subjected to analysis within 24 hours of their collection. T󰁯󰁴󰁡󰁬 M󰁩󰁣󰁲󰁯󰁢󰁩󰁡󰁬 C󰁯󰁵󰁮󰁴 (TMC) E󰁶󰁡󰁬󰁵󰁡󰁴󰁩󰁯󰁮 This parameter represents the cultivable viable micro-bial biomass. This analysis assesses the total number of viable microbial cells in 1 ml of sample and gives a gen-eral assessment of the hygienic quality of a considered sample. The assessment was carried out on suspensions of water + Rotifers (approximately 200 individuals per millilitre) or Artemia (approximately 1000 individuals per millilitre).For each suspension scalar dilutions from 10 -1  to 10 -7  were prepared. Each dilution, with the srcinal suspension, was seminated in double on Plate Count Agar (PCA, Biokar Diagnostic cod. BK 144HA, Beauvais, France) with 2% NaCl. The plates were incubated at 25°C for 48 hours. A󰁮󰁡󰁬󰁹󰁳󰁩󰁳 󰁯󰁦 󰁬󰁩󰁰󰁩󰁤󰁳 Four different samples of Artemia were analyzed, the first three were subjected to three different enrichments (SP, SE, SA) while the fourth sample, SE after the en-richment, was subjected to the maintenance phase for 12 hours at 4°C. Also for the Rotifers, four different samples were analyzed, the first three were subjected to three different enrichments (E, F, G) while the fourth sample was subjected to the maintenance phase for 8 hours at 4°C and then to the enrichment.The analysis of lipids was conducted treating the sam-ples with a mixture of chloroform/methanol [34] and the lipid extract was then submitted to the following transesterification through heat treatment (90°C for twenty minutes) with benzene and bore trifluorure in metabolic solution. With the resultant mixture of esters of fatty acids the gas-chromatographic analysis was conducted through gas-chromatography Hewlett Pack-ard GC System, HP6890 with column HP 5890. The method used for the separation was a programmed ramp of which the oven column started at a value of 150°C for 4 minutes, increasing by 4°C every minute to reach a final temperature of 250°C which was maintained for 30 minutes. The total duration of the chromatographic run, being about 60 minutes. The temperature of the injector and detector was fixed at 250°C.Helium was used as the transport gas in the column (mobile phase) with a constant flow of 1 ml/minute. The identification of the fatty acids was carried out through the comparison of the retention times of the peaks that emerged through the gas-chromatography, relating the our sample to a known standard. The standard mixture used was the “Mixture 37” (Supelco - Bellefonte, PA).The abundance of the single fatty acids has been ex-pressed as a percentage of the total identified fatty acids, through integration of the peaks revealed by the flame ionization detector with an integrator connected to the gas-cromatograph. S󰁴󰁡󰁴󰁩󰁳󰁴󰁩󰁣󰁡󰁬 A󰁮󰁡󰁬󰁹󰁳󰁩󰁳 The data were elaborated statistically using the software “Statgraphics ®  Plus”. For the TMC the mean, median, standard deviation, maximum and minimum were calculated. Besides this, to individualize the eventual differences and significance between the different phases and different treatments the variance analysis was subjected to an ANOVA test and a Student-Newman-Keuls post-hoc test, at a 95% confidence level.The data from the analysis was plotted in a Box-and-Whisker diagram, that illustrates the distribution in quartiles, the maximum, the minimum, the mean and the median of the values relating to the parameter TMC in each phase or treatment. Results T󰁯󰁴󰁡󰁬 M󰁩󰁣󰁲󰁯󰁢󰁩󰁡󰁬 C󰁯󰁵󰁮󰁴 (TMC) E󰁶󰁡󰁬󰁵󰁡󰁴󰁩󰁯󰁮 󰁯󰁦 R󰁯󰁴󰁩󰁦󰁥󰁲󰁳 The data, expressed as decimal logarithms of the TMC, recorded in the different phases of the work of the Rotif-ers, have been statistically elaborated and shown in the Figure 3, plotted in a Box-and-Whisker diagram. Fig. 2.  Experimental plan used for the microbiology analysis on the Artemia.  A. DE DONNO ET AL. 40 The TCM shows an increasing mean trend with initial values (phase 1) equal to 3,68 Log cfu/ml and final val-ues (phase 6) of 5,67 Log cfu/ml.The analysis of the variance (ANOVA) shows a sig-nificant difference between the TCM of the various phases at a 95,0% confidence level (p < 0,05). To determine the statistically significant mean, the Mul-tiple Range Test was used, in accordance with the Student-Newmann-Keuls procedure. The significantly different TMC values have been underlined between phase 1, the group composed of the phases 2, 3 and 4 and the group composed of the phases 5 and 6. In terms of the data relating to the various treatments used in each phase (Fig. 4), statistically significant dif-ferences (p < 0,05) have been found only between the treatments of the phase 5. In particular the treatment 5/F (FITO Enrichment Protocol) shows TMC values significantly lower than the treatments 5/E (EASY DHA Enrichment Protocol) and 5/G (DHA PROTEIN SELCO Enrichment Protocol). T󰁯󰁴󰁡󰁬 M󰁩󰁣󰁲󰁯󰁢󰁩󰁡󰁬 C󰁯󰁵󰁮󰁴 (TMC) E󰁶󰁡󰁬󰁵󰁡󰁴󰁩󰁯󰁮 󰁯󰁦 A󰁲󰁴󰁥󰁭󰁩󰁡 The data, expressed as decimal logarithms of the TMC, recorded in the different phases of the work of the Ar-temia, have statistically been elaborated and are shown in Figure 5, plotted in a Box-and-Whisker diagram. The TCM shows an increasing mean trend with initial values (phase 1) equal to 5,35 Log cfu/ml and final val-ues (phase 3) of 5,98 Log cfu/ml.The analysis of the variance (ANOVA) records a signif-icant difference between the TCM of the various phases at a 95,0% confidence level (p < 0,05).Also in this case the Multiple Range Test in accordance with the procedure of Student-Newmann-Keuls was used to determine the statistically significant mean. Each phase has shown significantly different TMC mean values, at a 95% confidence level (p < 0,05). The analysis of the vari-ance and the Multiple Range Test applied to the different treatments in each phase (Fig. 6), highlights that only the treatment with SP (SPARI Enrichment Protocol) shows TMC values statistically higher (p < 0,05) compered to those found in SE (SERRANI Enrichment Protocol) and SS (SUPER SELCO Enrichment Protocol). Fig. 3.  Box-and-Wisker Plots of TMC in the different phases of Rotifers production. Phases: 1 = Inoculation; 2 = Release; 3 = Cul-ture Start; 4 = Culture End; 5 = Enrichment; 6 = Manteinance. Fig. 4.  Box-and-Wisker Plots of TMC in the different treatments,  for each phases of Rotifers production. Phases: 1 = Inoculation; 2 = Release; 3 = Culture Start; 4 = Culture End; 5 = Enrichment; 6 = Maintenance. Protocols of treatment: A = Selco; B = HD Culture Selco - ® INVE; C = Rotimac TM  – BioMarine Aqua Fauna Inc.Hawborne,California, USA; E = EASY DHA; F = FITO; G = DHA PROTEIN SELCO - ®  INVE; T = thermal. Fig. 5.  Box-and-Wisker Plots of TMC in the different phases of Artemia production. Phases: 1 = Start Enrichment; 2 = End En-richment;; 3 = Manteinance. Fig. 6.  Box-and-Wisker Plots of TMC in the different treatments,  for each phases of Artemia production. Protocols of treatment: SE = SERRANI; SP = SPARI; SS = SUPER SELCO - ®  INVE.  TOTAL MICROBIAL COUNT AND FATTY ACID PROFILES OF CULTURED ARTEMIA AND ROTIFERS 41 E󰁶󰁡󰁬󰁵󰁡󰁴󰁩󰁯󰁮 󰁯󰁦 󰁴󰁨󰁥 󰁦󰁡󰁴󰁴󰁹 󰁡󰁣󰁩󰁤󰁳 󰁰󰁲󰁯󰁦󰁩󰁬󰁥 The analysis of lipids conducted on the samples of the rotifers (Figure 7 and Figure 8) has not shown particu-lar differences in the fatty acids profile following the protocols of enrichment, except a minor percentage of acid docosahexaenoic (DHA) in treatment F. The results obtained by the analysis of the Artemia (Figs. 9, 10) show a higher percentage of saturated fatty acids (SFA) relative to the treatment SE although it’s not far from the other treatments in the polyunsaturated fatty acids profile (PUFA) recording other than having an elevated quantity of eicosapentaenoic acid (EPA). The SP enrich-ment protocoll has shown a higher percentage of PUFA, especially in the DHA quantity.Very different data was obtained from the maintenance protocol: in some cases similar percentages were re-corded before and after the treatment, in others, an in-crease of the saturated fatty acid content was observed for the Rotifers, while for the Artemia it was the mo-nounsaturated fatty acid quantity. A diminution in the EPA quantity was observed after the maintenance phase both for the Rotifers and for the Artemia. A reduction of the DHA content was also recorded after the same phase, but only for the Rotifers. Discussion and conclusions Both in the case of the Rotifers and of the Artemia a progressive increase of the total microbial charge was recorded in the various phases of production, always starting from values that were on average low. This result was more evident for the Rotifers, whose charge moved from 3.68 Log cfu/ml to 5.67 Log cfu/ml, in comparison to the Artemia that from an initial charge of 5.35 Log cfu/ml a final charge of 5.98 Log cfu/ml was recorded. In accordance with the results recorded in lit-erature, we can say that the values found don’t highlight any particularly critical situations. In fact, usually the density of aerobic bacteria in the culture water ranges from 1 x 10 4  (4 Log) to 1 x 10 7  (7 Log) cfu/ml [12, 35]. In particular the Rotifers culture water the bacterial concentration are in the order of 10 7  cfu/ml [36].The highest increase of microbial charge found in the production of the Rotifers could possibly be attributed to the higher number of phases involved in the same production process. The results related to the various protocols adopted in the Rotifer production industry showed statistically sig-nificant differences (p < 0,05) between the treatments of the enrichment phase. Especially the enrichment with phytoplankton (Protocol F) recorded TCM values significantly lower than the EASY DHA (Protocol E) treatments and than the DHA PROTEIN SELCO (Pro-tocol G) treatment.Analogous studies on samples of Artemia have pointed out that the enrichment with the commercial product DHA SELCO, containing the antimicrobial substances, represents a valid alternative to enrichment with phy-toplankton; nevertheless it’s clearly specified that its antimicrobial properties are inferior to those of the phyto-plankton [37]. The nutritional benefits of the algae, on the other hand, is extensively documented and their potential Fig. 8.  Percentage of acids eicosapentaenoic (EPA) and acid docosahexaenoic (DHA). Fig. 9.  Fatty acids profile of Artemia in the different protocols of enrichment. Fig. 10.  Percentage of acids eicosapentaenoic (EPA) and acid docosahexaenoic (DHA). Fig. 7.  Fatty acids profile of Rotifers in the different protocols of enrichment.
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