Immune Responses and Resistance against Vibrio parahaemolyticus Induced by Probiotic Bacterium Arthrobacter XE-7 in Pacific White Shrimp, Litopenaeus vannamei

A 63-d feeding experiment was conducted to determine the effects of dietary supplementation of probiotic bacterium Arthrobacter XE-7 on immune responses and resistance against Vibrio parahaemolyticus in the Pacific white shrimp, Litopenaeus vannamei . The probiotic bacteria were administered orally at four different doses of 0, 10⁶, 10⁸, and 10¹⁰ colony-forming unit (CFU)/g feed for shrimp. On Day 50, the shrimp were challenged with Vibrio parahaemolyticus by bath. On Days 7, 21, 49, and 63, six shrimp per tank were sampled to take intestine and hemolymph. With increasing dietary supplementation of probiotic bacteria, shrimp mortality decreased from 63.16% (the control) to 55.9% (10⁶ CFU/g feed), 51.75% (10⁸ CFU/g feed), and 51.78% (10¹⁰ CFU/g feed), respectively. Vibrio counts in intestine of shrimp fed probiotic bacterium Arthrobacter XE-7 was generally lower than that in the control shrimp ( P  < 0.05). The probiotic bacteria generally increased the immune parameters in shrimp, that is, total hemocyte counts, percentage phagocytosis, respiratory burst activity, and serum phenoloxidase activity. The results showed that probiotic bacterium Arthrobacter XE-7 can be used as probiotic in shrimp feed.     

Effects of the probiotic, Lactobacillus acidophilus, on the growth performance, haematology parameters and immunoglobulin concentration in African Catfish (Clarias gariepinus, Burchell 1822) fingerling

This experiment was carried out to evaluate the effects of the probiotic, Lactobacillus acidophilus , on the growth performance, haematology parameters and immunoglobulin concentration in African catfish Clarias gariepinus fingerling. Two experimental diets were formulated to contain 35 g kg⁻¹ crude protein and 10 g kg⁻¹ lipids accordingly and fed three times daily for 12 weeks to 25 C. gariepinus fingerlings per fibreglass tank in 12 replicates each. The control diet was prepared with no probiotic supplementation whereas the second diet was prepared supplemented with a probiotic, L. acidophilus , containing about 3.01 × 10⁷ colonies/g of diet. The results show that growth performance [specific growth rate (SGR) and relative growth rate (RGR)], nutrient utilization [protein efficiency ratio (PER) and feed conversion ratio (FCR)] and survival were significantly ( P <0.05) higher in fish maintained on the probiotic-supplemented diet compared with those on the control diet. Haematology parameters (packed cell volume, haemoglobin, erythrocyte sedimentation rate, red blood cell and white blood cell, total serum protein, Ca²⁺, Mg²⁺, Cl⁻, glucose and cholesterol) and total immunoglobulin concentrations were also significantly better in fish fed the probiotic-supplemented diet than in the control. Although the water quality parameters monitored were better in the fish fed the probiotic-supplemented diet than in the control, the parameters were not significantly different ( P >0.05). From the results of this experiment, we conclude that L. acidophilus can be used as a probiotic agent in African catfish culture, to enhance fish health, survival and better feed efficiency and growth performance.     

Growth, stress tolerance and non-specific immune response of Japanese flounder Paralichthys olivaceus to probiotics in a closed recirculating system

ABSTRACT:   Effects of probiotics on growth, stress tolerance and non-specific immune response in Japanese flounder Paralichthys olivaceus were evaluated in a closed recirculating system. Survival and growth of flounder treated by supplying commercial probiotics either in the diet (the probiotic diet group), or into the rearing water (the water supply group), were higher compared to the untreated group (the control group). Water quality parameters, pH, NH₄-N, NO₂-N and PO₄-P showed lower concentration in the probiotic diet group compared with the control group and the supply group. Plasma lysozyme activity in the probiotic diet group and the water supply group was significantly higher ( P  < 0.05) than that in the control group. In heat shock stress tests, flounder in the probiotics-treated groups showed greater heat tolerance (measured by 50% lethal time, LT50) than the control group. Pathogen challenge tests with Vibrio anguillarum (2 × 10⁷ c.f.u./mL) resulted in significantly higher survival in the probiotics-treated groups than the control group. Results indicated that probiotics supplied in the rearing water and the diet of fish enhanced the stress tolerance and the non-specific immune system of Japanese flounder, providing them a higher resistance against stress conditions and pathogens.     

Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum), were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10¹, 10³ or 10⁵ plaque forming units (pfu) L⁻¹ of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish L⁻¹ (low density) or 140 fish L⁻¹ (high density), and the tank flow rate was 250 mL⁻¹ min. Virus exposure began at 6 days before hatch and continued until fish were 44 days old. Cumulative per cent mortality, analysis of survival and hazard functions, and discrete-time event analysis were used to explore the patterns of survival and mortality. In eggs and fish exposed to IPNV, mortality significantly greater than in the 0 pfu L⁻¹ exposure did not occur until IPNV concentration was 10⁵ pfu L⁻¹ at low fish density and 10³ pfu IPNV L⁻¹ at high fish density. These results suggest that in the natural aquatic environment, where rainbow trout densities are likely to be considerably lower than in this study, mortality resulting from infection with IPNV will very likely not occur when ambient concentrations of virus are ≤10³ pfu IPNV L⁻¹. In aquaculture rearing units, trout density is likely to be as high or higher than the densities used in this study. Therefore, continuous inputs of virus at concentrations greater than 10¹ pfu L⁻¹ may result in IPN epidemics in aquaculture facilities.     

Biotransformation of tuna waste by co-fermentation into an aquafeed ingredient

Dried skipjack tuna ( Katsuwonus pelamis) waste (red meat, gills, viscera, fins, etc.) were mixed with 25% wheat flour and inoculated with a starter culture of Lactobacillus plantarum National Collection of Industrial Microorganisms (NCIM) 2912 (10⁸–10⁹ cells mL⁻¹) and Bacillus licheniformis MTCC 6824 (10⁷–10⁸ cells mL⁻¹). Changes in the nutritional quality (crude protein, crude fat, crude ash, crude fibre and nitrogen-free extract and aminoacids) were monitored during a fermentation period of 14 days. The proximate analysis showed significant changes in the composition of L. plantarum -fermented tuna (LPFT) and B. licheniformis -fermented tuna (BLFT) from the unfermented raw materials. Fermentation of tuna waste has resulted in a significant ( P <0.05) increase in the protein content of tuna waste between days 6 and 12. All the amino acid contents in BLFT increased during fermentation, whereas, in LPFT the levels of serine, histidine, tyrosine, methionine, cystine and phenylalanine contents were decreased. A marginal increase in calcium and phosphorus levels was recorded in the fermented products. The results of the study suggest that LPFT or BLFT can be used as a novel aquafeed ingredient for different fish species.     

Bioencapsulation of erythromycin using adult brine shrimp, Artemia franciscana (Latreille)

Live adult brine shrimp, Artemia franciscana (Latreille), were enriched with erythromycin to determine if Artemia could accumulate therapeutic levels for subsequent feeding to young fish. Three trials were conducted to determine the erythromycin incorporation and survival rates of enriched Artemia when fed either liposomes containing erythromycin or various erythromycin suspensions. Erythromycin concentration in Artemia fed a liposome suspension was low (∼ 5 μg mL⁻¹) relative to Artemia fed the direct suspension (> 100 μg mL⁻¹) over the same time period. When enriched with suspensions up to 1 g erythromycin L⁻¹ sea water for 14 h, Artemia survival was not significantly affected ( P > 0.05) relative to controls. Using a suspension of 1 g L⁻¹, tissue erythromycin concentrations of 109 ± 16 μg erythromycin mL⁻¹  Artemia homogenate (mean ± SEM) were achieved after 12 h. Concentrations above 170 μg mL⁻¹ were obtained using suspensions of 2–5 g L⁻¹, but Artemia survival significantly ( P < 0.05) decreased.     

Utilization of monounsaturated fatty acid (18:1n-9, oleic acid) by freshwater prawn Macrobrachium rosenbergii (de Man) juveniles

The utilization of oleic acid as an energy source and the effects of oleic acid levels and/or dietary soy bean lecithin (SBL) on oleic acid utilization, growth and survival, and lipid class and fatty acid compositions of freshwater prawn, Macrobrachium rosenbergii (de Man), juveniles were determined.
Increase in levels of dietary oleic acid from 10 to 80 g kg⁻¹ significantly ( P ≤ 0.05) reduced growth, survival and feed conversion efficiency of M. rosenbergii juveniles during the 40-day feeding period. Inclusion of 20 g kg⁻¹ SBL had no significant effect ( P ≥ 0.05) on growth and survival, nor was there any interactive effect between dietary SBL and oleic acid levels.
Body fatty acid profile of prawns reflected dietary fatty acid quantity. The fatty acid composition of prawns fed diets containing 80 g kg⁻¹ oleic acid had tremendously high proportions of oleic acid. Polar lipids, mostly phosphatidylcholine (PC), constituted the bulk of the extracted total lipids. Prawns fed with SBL had significantly ( P ≥ 0.05) higher PC content.
Oleic acid was metabolized for energy by M. rosenbergii juveniles at the same rate regardless of dietary level of SBL and/or oleic acid. Expired ¹⁴CO₂ accounted for half of the ingested radioactivity 48 h after feeding with labelled diets. No significant difference in the amount of ¹⁴CO₂ expired by prawns fed the labelled test diets was found. Per cent radioactivity ingested and absorbed into the body was also not significantly different in prawns of the different dietary treatments.     

Infectious pancreatic necrosis virus: experimental infection of goldsinny wrasse, Ctenolabrus rupestris L. (Labridae)

The use of wrasse as cleaner fish in farming of Atlantic salmon, Salmo salar L., is now widespread in Scotland, Ireland and Norway, but little is known about the susceptibility of these fish to the common pathogens of cage-cultured salmon. The susceptibility of goldsinny wrasse, Ctenolabrus rupestris L., to infectious pancreatic necrosis virus (IPNV) was investigated. Captive-bred C. rupestris were infected with IPNV-Sp (Shetland strain) using a bath challenge method. Two infection doses were used, low (4.1 × 10⁵ pfu mL⁻⁻¹) and high (2.4 × 10⁶ pfu mL⁻⁻¹), with two replicates for each experiment. Bathing times were 1 h for the low dose and 5 h for the high dose. A maximum prevalence of infection (30%) was seen 2 weeks post-infection in replicate 1 of the low dose experiment and was accompanied by low tissue titres. The tissue titres dropped to an undetectable level by 4 weeks post-infection. In the high dose experiment, a high prevalence of infection was seen along with moderate titres in tissues as well as a marked pathological response. Both prevalence and intensity declined rapidly and the fish recovered. In the high dose experiment, faecal titres followed the same pattern as those in the tissues. The implications for the use of wrasse in the farming of Atlantic salmon are discussed.     

Selection of probiotic bacteria for use in shrimp larviculture

Three candidate probiotics, Bacillus foraminis, Bacillus cereus biovar toyoi and Bacillus fusiformis , were isolated from hydrogen-producing fermented solution and identified using 16S rRNA sequence analysis. Bacillus foraminis and B. cereus biovar toyoi exhibited strong antagonism against Streptococcus iniae and Photobacterium damselae subsp. piscicida in in vitro co-culture for competitive exclusion assay and then were conducted in the larviculture system of Penaeus monodon reared from zoea 1 to postlarva 1. The daily addition of B. cereus biovar toyoi resulted in significantly deleterious effects on survival ( P <0.01) whereas the daily addition of B. fusiformis showed highest survival rate (88.7±0.7%) but no statistically significant difference from control (73.3±12.1%). Bacillus fusiformis was continuously applied in the larviculture system of Litopenaeus vannamei . Administration of B. fusiformis significantly increased survival ( P <0.01) in both treatments added daily (87.9±1.7%) and every other day (54.7±1.2%), respectively, at a concentration of 10⁵ CFU mL⁻¹ over control (41.2±1.3%).     

Effect of water temperature on the growth performance and digestive enzyme activities of Chinese longsnout catfish (Leiocassis longirostris Günther)

The present study was carried out to investigate the influence of water temperature on the growth performance and digestive enzyme (pepsin, trypsin and lipase) activities of Chinese longsnout catfish. Triplicate groups of Chinese longsnout catfish (35.6±0.48 g, mean±SE) were reared at different water temperatures (20, 24, 28 and 32 °C). The feeding rate (FR), specific growth rate (SGR) and feed efficiency ratio (FER) were significantly affected by water temperatures and regression relationships between water temperature and FI, SGR as well as FER were expressed as FR=−0.016 T ²+0.91 T −10.88 ( n =12, R ²=0.8752), SGR=−0.026 T ²+1.39 T −17.29 ( n =12, R ²=0.7599) and FER=−0.013 T ²+0.70 T −8.43 ( n =12, R ²=0.7272). Based on these, the optimum temperatures for FR, SGR and FER were 27.66, 26.69 and 26.44 °C respectively. The specific activities of digestive enzymes at 24 or 28 °C were significantly higher than that at 20 or 32 °C. In addition, there was a significant linear regression between FR or SGR and specific activities of pepsin and lipase, which indicated that pepsin and lipase played important roles in regulating growth through nutrient digestion in Chinese longsnout catfish.     

Quantitative dietary threonine requirement of juvenile Pacific white shrimp, Litopenaeus vannamei (Boone) reared in low-salinity water

An 8-week feeding trial was conducted to determine the threonine requirement of juvenile Pacific white shrimp Litopenaeus vannamei (Boone) in low-salinity water (0.50–1.50 g L⁻¹). Diets 1–6 were formulated to contain 360 g kg⁻¹ crude protein with fish meal, wheat gluten and pre-coated crystalline amino acids with six graded levels of l -threonine (9.9–19.0 g kg⁻¹ dry diet). Diet 7, which was served as a reference, contained only intact proteins (fish meal and wheat gluten). Each diet was randomly assigned to triplicate groups of 30 shrimps (0.48±0.01 g), each four times daily. Shrimps fed the reference diet had similar growth performance and feed utilization efficiency compared with shrimps fed the diets containing 13.3 g kg⁻¹ or higher threonine. Maximum specific growth rate (SGR) and protein efficiency ratio were obtained at 14.6 g kg⁻¹ dietary threonine, and increasing threonine beyond this level did not result in a better performance. Body compositions, triacyglycerol and total protein concentrations in haemolymph were significantly affected by the threonine level; however, the threonine contents in muscle, aspartate aminotransferase and alanine aminotransferase activities in haemolymph were not influenced by the dietary threonine levels. Broken-line regression analysis on SGR indicated that optimal dietary threonine requirement for L. vannamei was 13.6 g kg⁻¹ dry diet (37.8 g kg⁻¹ dietary protein).     

An experimental investigation on aspects of infectious salmon anaemia virus (ISAV) infection dynamics in seawater Atlantic salmon, Salmo salar L.

This study investigated infection dynamics of infectious salmon anaemia virus (ISAV) by conducting two experiments to examine minimum infective dose and viral shedding of ISAV. In terms of minimum infective dose, the high variability between replicate tanks and the relatively slow spread of infection through the population at 1 × 10¹ TCID₅₀ mL⁻¹ indicated this dose is approaching the minimum infective dose for ISAV in seawater salmon populations. A novel qPCR assay incorporating an influenza virus control standard with each seawater sample was developed that enabled the quantity of ISAV shed from infected populations to be estimated in values equivalent to viral titres. Viral shedding was first detected at 7 days post-challenge (5.8 × 10⁻² TCID₅₀ mL⁻¹ kg⁻¹) and rose to levels above the minimum infective dose (4.2 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹) on day 11 post-challenge, 2 days before mortalities in ISAV inoculated fish started. These results clearly demonstrate that a large viral shedding event occurs before death. Viral titres peaked at 7.0 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹ 15 days post-infection. These data provide important information relevant to the management of ISA.     

Changes in the nutritional parameters of muscles of the common carp (Cyprinus carpio) and the silver carp (Hypophthalmichthys molitrix) following environmental exposure to cyanobacterial water bloom

The present study evaluated the effect of naturally developing cyanobacteria on the composition of muscles of two commercially important freshwater fish species. Fish were exposed to cyanobacterial biomass including Microcystis aeruginosa and Microcystis ichthyoblabe for 4 weeks. Then, they were transferred to dechlorinated potable water without any cyanobacteria for another 4-week period, thus modelling their preparation for consumers. Samples of muscles were collected every week during exposure and subsequent stay in dechlorinated potable water. The cyanobacterial water bloom of 3.9–6 × 10⁵ cells mL⁻¹ (133–383 μg g⁻¹ of total MC DW) induced statistically significant effects only in the content of fatty acids ( P <0.05; P <0.01) in the common carp ( Cyprinus carpio ), while all studied parameters including the content of dry matter and fat ( P <0.01), proteins ( P <0.05), fatty acid composition ( P <0.05; P <0.01) and some amino acids ( P <0.05) were affected in the silver carp ( Hypophthalmichthys molitrix ). This study has shown that cyanobacteria in the environment of commercially produced fish may decrease the dietetic value of fish muscles.     

Effect of Customized Probiotics on the Physiological and Immunological Responses of Juvenile Western King Prawns (Penaeus latisulcatus Kishinouye, 1896) Challenged with Vibrio harveyi

Juvenile western king prawn P. latisulcatus were fed 10⁵ colony-forming units (CFU)/mL of two probiotics Pseudomonas synxantha and P. aeruginosa for 28 days. P. latisulcatus were then challenged with V. harveyi at 0 (control), 10³, 10⁵, and 10⁷ CFU/mL. During the seven days of challenge, disease resistance of the probiotic-fed prawns was compared with that of prawns not fed probiotics. The immunological responses of the prawns did not improve during the challenge period in terms of total haemocyte count, hyalinocyte, semi-ganulocyte, granulocyte, clotting time, bacteraemia, and intestinal bacterial load. Overall, when prawns were challenged with V. harveyi, the LT₅₀ values got shorter as V. harveyi concentration increased. LT₅₀ values of prawns fed probiotics were significantly longer (P < 0.05) than those not fed probiotics. At a V. harveyi concentration of 10³ CFU/mL, the 100% survival of the prawns fed probiotics was three times more likely than those of the prawns not fed probiotics.     

The effectiveness of commercial probiotics in northern white shrimp <Emphasis Type="Italic">Penaeus vannamei</Emphasis> ponds

ABSTRACT:   The effectiveness on water quality, population density of bacteria, and shrimp production in ponds treated with commercial probiotics was tested in Penaeus vannamei ponds in Hai-yan, China. Six ponds with replicates for treatment and control were used. Results showed that the probiotics could improve the population density of beneficial bacterial flora, reduce concentrations of nitrogen and phosphorus, and increase yields of shrimp. The average counts of Bacillus sp., ammonifying bacteria, and protein mineralizing bacteria were found to be significantly higher in treated ponds compared to control ponds ( P  < 0.05). In control ponds, an increase in presumptive vibrios was observed and the average density was up to 2.09 × 10³ cfu/mL, whereas that was only 4.37 × 10² cfu/mL in treated ponds ( P  < 0.05). The use of probiotics also significantly increased dissolved oxygen ( P  < 0.05) and reduced dissolved reactive-phosphorus, total inorganic nitrogen and chemical oxygen demand ( P  < 0.05). An average of 8215 ± 265 kg shrimp/ha was obtained in treated ponds with a feed conversion ratio (FCR) of 1.13 ± 0.05 and survival rate of 81.00 ± 6.25% compared with 4985 ± 503 kg shrimp/ha, 1.35 ± 0.12 and 48.67 ± 3.51%, respectively, in control ponds. This indicates that the addition of the commercial probiotics had a noticeable influence on water quality of shrimp ponds and shrimp production.     

Production Response of Freshwater Prawns Macrobrachium rosenbergii to Increasing Amounts of Artificial Substrate in Ponds

Abstract.— The response of freshwater prawns Macrobrachium rosenbergii to increasing amounts of artificial substrate was evaluated in ponds. Juvenile prawns (0.24 ± 0.13 g) were stocked into nine 0.04-ha ponds at 74,000/ha. Three control ponds received no artificial substrate while artificial substrate in the form of horizontal strips of polyethylene "construction fence" was added to the six treatment ponds to produce 40% or 80% increases in available surface area. Increasing availability of surface area produced a direct linear increase ( P < 0.05, r ²= 0.89) in total production with no significant change in average weight ( P > 0.05). There was an inverse linear relationship between available surface area and feed conversion ratios ( P < 0.01, r ² = 0.66) likely indicating increased availability of natural foods or reduced stress among animals. There was a direct linear increase in the percentage of females which achieved sexual maturity ( P < 0.01, r ²= 0.71) as the amount of added substrate was increased. Size and number of other sexual morphotypes were not significantly affected. These responses are consistent with those that would be expected if stocking densities were decreased. These data indicate that prawn production increases in direct relation to the amount of added substrate while utilizing feed more efficiently. The effect of substrate orientation on its functionality should be evaluated to allow further increases in substrate inclusion amounts for additional production intensification.     

Effects of dietary organic acids on growth, nutrient digestibility and gut microflora of red hybrid tilapia, Oreochromis sp., and subsequent survival during a challenge test with Streptococcus agalactiae

A 14-week feeding trial was conducted to determine the effects of dietary organic acids. The experimental diets were added with 0, 1, 2 or 3 g kg⁻¹ of a novel organic acid blend or with 2 g kg⁻¹ of potassium diformate and fed to triplicate groups of red hybrid tilapia ( Oreochromis sp.). Upon completion, tilapia were challenged by immersion with Streptococcus agalactiae . There was no significant difference ( P >0.05) in the growth, feed utilization and nutrient digestibility among treatment groups despite a trend towards improved results with fish fed organic acid-supplemented diets. Diet pH decreased, causing a reduction in the digesta pH of the stomach and gut. Total bacteria per gram of faeces were significantly ( P <0.05) reduced from 1.81 × 10⁸ colony-forming units (CFU) (control group) up to 0.67 × 10⁸ CFU in the fish fed organic acid diets. A similar trend was observed for adherent gut bacteria. Cumulative mortality of fish fed no organic acids was higher compared with fish fed organic acid-supplemented diets at 16 days post challenge. The data showed that dietary organic acids can exert strong anti-microbial effects and have the potential to exert beneficial effects on growth, nutrient utilization and disease resistance in tilapia.     

Effects of dietary phosphorus and lipid levels on utilization and excretion of phosphorus and nitrogen by rainbow trout (Oncorhynchus mykiss). 2. Production-scale study

In a 8-week production-scale experiment at a commercial trout farm, the effects of dietary lipid level and phosphorus level on phosphorus (P) and nitrogen (N) utilization of rainbow trout (initial mean weight 99 g) were assessed. A low-phosphorus, high-lipid experimental diet (457 g protein, 315 g lipid, 9.1 g P  kg^–1 dry diet) was compared with a commonly used commercial diet (484 g protein, 173 g lipid, 13.6 g P  kg^–1 dry diet). P and N budgets were constructed using data from the production-scale experiment and digestibility data for the two diets. In addition, orthophosphate and ammonia-N waste were measured in effluent over one 24-h period. Relative to the commercial diet, the experimental diet resulted in significantly increased feed efficiency ratio, N retention and P retention, and substantially reduced dissolved, solid and total P waste (g kg^–1 dry feed). Although N retention resulting from the experimental diet was higher, this was attributable to higher N (protein) digestibility of the experimental diet. Solid N waste (g kg^–1 dry feed) resulting from the experimental diet was substantially lower, but dissolved N waste (g kg^–1 dry feed) was not significantly different relative to the commercial diet. Mean effluent orthophosphate production (mg day^–1 kg^–1 fish) of fish fed the experimental diet was substantially lower than that of fish fed the commercial diet ( P  < 0.05), but effluent ammonia-N production (mg day^–1 kg^–1 fish) was not significantly affected by dietary treatment.     

Encapsulation capacity of Artemia nauplii with customized probiotics for use in the cultivation of western king prawns (Penaeus latisulcatus Kishinouye, 1896)

The encapsulation capacity of Artemia nauplii with customized probiotics Pseudomonas synxantha and Pseudomonas aeruginosa for use in the cultivation of western king prawns (Penaeus latisulcatus) was investigated. Seven trials were conducted to investigate this encapsulation capacity in terms of Artemia survival and probiotic load in Artemia. Newly hatched Artemia nauplii at 250 nauplii mL^?1 were fed individual probiotics at 0, 10³, 10⁵ and 10⁷ colony‐forming units (CFU) per millilitre, and mixtures of these two probiotics (10⁵ CFU mL^?1) at 30:70, 50:50 or 70:30 v/v in a medium of ozonated water (OW), tryptone soya broth (TSB), and a mixture of these media. The appropriate medium for encapsulation of probiotics by Artemia nauplii was the mixture of OW and TSB at 75:25 v/v; whereas, the use of OW or TSB alone was not effective. Artemia nauplii most effectively encapsulated the customized probiotics at 10⁵ CFU mL^?1. The results indicates that the encapsulation of Artemia nauplii is optimized by using a combination of P. synxantha and P. aeruginosa at 50:50 v/v in a media mixture of OW and TSB at 75: 25 v/v. Artemia should be harvested at 48 h when survival is still high (78%) and the probiotic load in Artemia is high (3 × 10⁴ CFU nauplius^?1).     

An in vitro method to determine phosphorus digestibility of rainbow trout Oncorhynchus mykiss (Walbaum) feed ingredients

An in vitro method was developed to assess the digestibility of phosphorus in 12 plant and animal feed ingredients for rainbow trout Oncorhynchus mykiss (Walbaum). The method simulates the gastrointestinal tract of the rainbow trout with regard to pH and gastrointestinal enzymes. Phosphorus solubility was measured after acid digestion (pH 3) with and without gastric enzymes, after alkaline digestion (pH 9) with and without intestinal enzymes, and after a two-step process involving acid and alkaline digestion. Commercially available digestive enzymes from mammals were compared with digestive enzymes from rainbow trout. Correlating in vitro digestibility with in vivo digestibility showed that acid digestion with both commercial enzymes ( r ²=0.98, P  < 0.05) and trout enzymes ( r ²=0.94, P  < 0.05) predicted the in vivo digestibility of animal feed ingredients. Alkaline digestion with both enzyme systems (commercial r ²=0.79; trout r ²=0.74, P  < 0.05) or without ( r ²=0.82, P  < 0.05) enzymes predicted the in vivo digestibility of ingredients from animal byproducts but not those from plant products. The in vitro digestibility with two enzyme steps (acid and alkaline) predicted in vivo digestibility of plant and animal ingredients ( r ²=0.79 for commercial enzymes and r ²=0.74 for trout enzymes) better than did one-step acid or alkaline digestion.