Folate in eggs and developing larvae of Atlantic halibut, Hippoglossus hippoglossus L.

Folate mobilization from the yolk compartment during larval development was studied by analysing the folate concentration in whole body, embryo and yolk in a single batch of Atlantic halibut, Hippoglossus hippoglossus L., eggs and larvae that showed successful fertilization and development. There was a net loss of approx. 50% of folate from yolk during endogenous feeding. Further, only 23% of the decrease in yolk folate was retained in the larval body. The data suggest a need for folate for metabolic and growth purposes during embryogenesis of approximately 2 μg g^?1 weight gain. Relative to these data and published folate requirement for cold‐water species, batches of egg from 16 Atlantic halibut brood fish contained variable and, for some batches, critically low levels of folate. This may constitute a potential problem for larval development until start feeding.     

A formulated diet for Atlantic halibut (Hippoglossus hippoglossus, L.) larvae

A diet for Atlantic halibut-larvae was formulated taking into account the fact that marine-fish larvae have a limited ability to assimilate protein and lipid. Dietary protein consisted of a free amino-acid premix (7.2% of crude protein), predigested-squid mantle (7.2%), squid mantle (8.6%) and cod-muscle mince (77.0%). Lipid sources were soyabean lecithin (33% of crude lipids), crude phospholipids extracted from cod roe (10%) and sardine oil (57%). Larvae were weaned onto the experimental diet at wet-body weights of 0.07, 0.10 or 0.16 g, respectively. The experimental diet was fed for 31, 25 or 17 days, respectively, and the experiment was terminated on the same calendar day for all groups. A control group was fed with Artemia nauplii enriched with DHA Selco™ from 0.07 g. Survivals ranged from 78% in larvae transferred at 0.10 g to 96% in those transferred at 0.16 g and in the control group. Daily specific-growth rates (SGR) were 3.1 ± 0.07, 3.3 ± 0.11 and 2.2 ± 0.01% day⁻¹ in larvae transferred at 0.07, 0.10 and 0.16 g, respectively, while growth in the control group was 5.1% day⁻¹. It was concluded that weaning of Atlantic-halibut larvae is feasible from 0.7 g (approximately 20 days post first-feeding) when the formulated diet contains predigested protein and ample amounts of phospholipids.     

Feed intake, growth and feed conversion efficiency of Atlantic halibut, Hippoglossus hippoglossus (L.)

The individual food intake of each fish in each of four groups of Atlantic halibut, Hippoglossus hippoglossus (L.) (mean weight: 422 g) was monitored by direct observation over a period of 21 days. Gross feed conversion efficiency (= growth·feed intake^?1), net feed conversion efficiency and maintenance ration were estimated by regression analysis. Specific growth rates were found to be linearly related to weight-specific consumption at a temperature of 8–9°C: growth = 1.922· feed intake ? 0.242. Maintenance ration was 0.126% of body weight day^?1. The gross feed conversion efficiency increased asymptotically with increasing feed intakes and growth rates, and was found to approach 1.9 at high growth rates (0.5 on a dry weight basis). One feeding per day seemed to be sufficient for maximum food intake and growth rate.     

Weaning of Atlantic halibut Hippoglossus hippoglossus L. using formulated diets with various levels of ascorbic acid

Atlantic halibut larvae (120 mg) were weaned to formulated diets with different supplementations of ascorbate- poly-phosphate, ApP (300, 2000 and 3000 mg ascorbic acid (AA) equivalents kg⁻¹ diet). The experiment lasted for 50 days with cofeeding of enriched Artemia and formulated diets during the first 30 days. During the last 20 days, only formulated diets were offered to the fish. One control group was fed only Artemia (770 mg AA kg⁻¹ dry weight) during the entire experimental period. The specific growth rate during the 50 days was ≈ 4.5% day⁻¹ and the mean weights in all dietary groups were ≈ 1 g when the experiment was terminated. No differences in mean weight and mortality were observed between the groups fed formulated diets and that fed Artemia during the experiment. The fish size in the groups fed formulated diets ranged between 0.10 and 3.05 g and this differed from the Artemia group where the size ranged between 0.35 and 1.35 g. Dietary levels of ApP had no positive effect on growth and survival. The retention of AA was significantly higher in the groups fed high dietary levels of ApP. Apparently, the bioavailability of high dietary levels of ApP appeared to be low for young halibut. After stressing the fish using a high-salinity challenge test, no significant difference in survival occurred among the dietary groups. Cortisol levels in plasma recorded 3 h post stress was significant lower in the Artemia group compared with the groups fed the formulated diets.     

Effects of protein hydrolysate in weaning diets for Atlantic cod (Gadus morhua L.) and Atlantic halibut (Hippoglossus hippoglossus L.)

The study aims to test whether predigested dietary protein enhances the utilization of formulated diets at weaning, and also whether it stimulates intestinal maturation. In this study, Atlantic cod [ Gadus morhua L.; 41 days posthatch (dph)] and Atlantic halibut [ Hippoglossus hippoglossus L.; 63 days postfirst feeding (dpff)] were weaned onto diets with graded levels of protein hydrolysate. By increasing the inclusion of dietary protein as hydrolysate from 0 to 400 g kg⁻¹, cod increased the rate of survival from 7 ± 1% to 18 ± 2% (82 dph; regression, P =  4*10⁻⁷). In halibut, the survival rate decreased from 57 ± 9% to 22 ± 7% as the inclusion of protein in the form of hydrolysate increased from 0 to 450 g kg⁻¹ (119 dpff; regression, P =  8*10⁻⁵). Growth was not affected in any of the species. Results in specific activities of the intestinal enzymes leucine aminopeptidase (LAP) and alkaline phospatase (AP) supported the results in survival in halibut and partly also in cod, by showing increased activities in groups with increased survival ( anova , P <  0.05). The lower optimal level of hydrolysed protein in halibut than in cod is suggested mainly because of a slower feeding practice in halibut, which allows more extensive nutrient leaching before ingestion.     

The effects of feeding frequency on growth of juvenile Atlantic halibut, Hippoglossus hippoglossus L.

Four experiments were conducted to investigate the effects of feeding frequency on growth of juvenile Atlantic halibut, Hippoglossus hippoglossus L. Fish (22–75 g) fed three (3 ×) or five times per day (5 × day^?1) under constant light and temperature (13±1°C) consumed significantly more feed than fish fed 1 × day^?1 but by the end of the experiment only fish fed 5 × day^?1 were heavier and had greater specific growth rates (SGR). Under simulated winter conditions (9L:15D, 5±1°C), halibut (～300 g) fed every other day consumed more feed, had a greater SGR and final weight compared with fish fed every third day. Feed conversion ratios were not different among treatment groups in any of the experiments. These results suggest that growth rates may be improved by feeding juvenile halibut more than 1 × day^?1.     

Effect of predigested protein on growth and survival of Atlantic halibut larvae (Hippoglossus hippoglossus L.)

In this study Atlantic halibut (Hippoglossus hippoglossus L.) larvae (0.12 ± 0.04 g) were, from day 40 post first feeding, offered six diets in which 10% or 30% of the dietary protein was hydrolysed with (a) pepsin (P), (b) pepsin + trypsin (PT) or (c) pepsin + trypsin + chymotrypsin (PTC). In addition, a diet without hydrolysed protein was offered, and enriched Artemia was fed as control. The amount of soluble protein increased progressively with the enzyme treatments P, PT and PTC and with higher inclusion levels of hydrolysed protein. Survival was highest among the larvae offered Artemia (83 ± 0%) or the diet 10P (10% pepsin hydrolysed protein; 67 ± 4%). The diet 10P supported survival significantly better than the more hydrolysed diets 10PTC, 30P, 30PT and 30PTC, but not significantly better than the non‐hydrolysed diet and 10PT. Specific growth rate (SGR) was 1.76 ± 0.20 in average for all groups of larvae and was not significantly affected by the diets. Still, the larvae offered pepsin hydrolysed diets tended to have better growth (2.10 ± 0.05 SGR; P < 0.06) than the larvae offered the other hydrolysed diets. The larvae offered the formulated diets did not differ in chemical composition.     

Egg yolk nutritional constituents as indicators of egg quality in Atlantic halibut (Hippoglossus hippoglossus L.)

Variation in oocyte quality affects sustainability of finfish aquaculture products including Atlantic halibut (Hippoglossus hippoglossus L.). To have an insight into role of major egg yolk constituents in the oocyte quality, egg fatty acid (FA), amino acid (AA) and folate contents were related to normal blastomere symmetry, and fertilization and hatching success. Significant correlations were found between dihomo‐γ‐linolenic acid (DGLA, 20:3n6), palmitic acid (PA, 16:00) and docosapentaenoic acid (DPA, 22:5n3), and normal blastomere symmetry and survival success. Egg concentrations of myristic acid (MA, 14:0), oleic acid (OA, 18:1n9), stearidonic acid (SA, 18:4n3) and eicosadienoic acid (EDA, 20:2n6) explained 56 % variation in fertilization success. OA and EDA explained 70 % of variation in blastomere symmetry. Palmitoleic acid (POA, 16:1n7) and linolenic acid (LNA, 18:3n3) concentrations explained 57% of variation in hatching success. Egg valine concentrations correlated with fertilization rates, and aspartic acid and leucine correlated with normal blastomere symmetry. Alanine and valine concentrations explained together 45% of variation in fertilization. Glutamic acid and tyrosine concentrations explained 68% of variation in normal blastomere symmetry while serine, arginine and valine explained 36% of variation in hatching. These FAs and AAa may be potential indicators of oocyte quality in Atlantic halibut.     

Nutrient composition and metamorphosis success of Atlantic halibut (Hippoglossus hippoglossus, L.) larvae fed natural zooplankton or Artemia

Atlantic halibut larvae were fed docosohexanoic acid- (DHA) selco enriched Artemia (RH-cysts) or wild zooplankton in duplicate tanks from first-feeding and 60 days onward. The zooplankton were collected from a fertilized sea water pond and consisted mainly of different stages of Eurytemora affinis and Centropages hamatus . There were no differences in survival, or in growth during the first 45 days of feeding, between larvae fed the two prey items, but the larvae fed Artemia showed much higher incidence of malpigmentation and impaired eye migration than larvae fed zooplankton. The prey organisms contained similar amounts of dry matter and protein, but Artemia was higher in lipid and glycogen than the zooplankton. Larvae fed Artemia were higher in both glycogen and lipid than the zooplankton-fed larvae towards the end of the feeding period. There were large differences between the prey organisms in the concentrations of essential fatty acids (% of total fatty acids) which was reflected in the fatty acid composition of the larval body. It is concluded that the macronutrient composition of Artemia in the present study was probably within the optimal range for promotion of growth and survival in young Atlantic halibut. The concentration of n-3 HUFA, and especially DHA, is however, very much lower in enriched Artemia than in copepods, and may be one of the factors triggering developmental errors in Atlantic halibut.     

Atlantic halibut (Hippoglossus hippoglossus) vitellogenin: induction,isolation and partial characterization

Vitellogenin (VTG) synthesis was induced by repeated injections of estradiol-17 in juvenile Atlantic halibut (Hippoglossus hippoglossus). VTG eluted as a large, phosphoprotein containing peak on DEAE-Sephacel chromatography of plasma from estradiol-17 treated juvenile and mature female, but not mature male halibut. A purification procedure for Atlantic halibut VTG was developed, where VTG was precipitated with MgCl₂, EDTA and distilled water, and the precipitated protein submitted to anion exchange chromatography on DEAE-Sephacel. Precipitated VTG eluted as a broad, partly dissociated peak on DEAE-Sephacel, when chromatography was run at 4°C, but the protein appeared intact when analysed both by SDS PAGE and native PAGE. DEAE-Sephacel chromatography at room temperature resulted in an irregular elution pattern and a dissociated protein fraction, as analysed by SDS PAGE. Biochemical characterization of VTG showed that the molecular mass of the monomer was ca 160 kDa, as estimated by SDS-PAGE. The total lipid content was 19.8% w/w, with 64%, or 12.7% of the total weight, as phospholipid. Protein bound phosphorus constituted 0.62% w/w of halibut VTG. Plasma dilution curves from mature and maturing female halibut were parallel with a dilution curve from halibut egg yolk homogenate in an homologous RIA. Plasma from mature male, but not juvenile halibut crossreacted with the VTG antiserum.     

A study of the susceptibility of Atlantic halibut,Hippoglossus hippoglossus (L.), to viral haemorrhagic septicaemia virus isolated from turbot,Scophthalmus maximus (L.)

The susceptibility of Atlantic halibut, Hippoglossus hippoglossus (L.), to viral haemorrhagic septicaemia virus (VHSV) was tested. Juvenile halibut of approximately 5 g weight were subjected to challenge by intraperitoneal injection, cohabitation and immersion to a VHSV isolate from an outbreak of the disease in turbot, Scophthalmus maximus (L.). The intraperitoneal injection gave the highest mortality rate of 28% after 50 days. The cohabitee group suffered 19% mortality rate and the immersion group only 2%. Control groups included turbot exposed either by intraperitoneal injection or immersion which suffered mortality rates of 93 and 50%, respectively. The results suggest that halibut are markedly less susceptible to VHSV than turbot.     

Growth, feed conversion efficiency and growth heterogeneity in Atlantic halibut (Hippoglossus hippoglossus) reared at three different photoperiods

Juvenile Atlantic halibut, Hippoglossus hippoglossus (initial weight (SD) 191.3 (±44.7)) g, were reared for 99 days at a constant temperature of 11°C and subjected to three different light regimes from 13 September to 21 December: continuous light (LD24:0), simulated natural photoperiod of Bergen (60°25′N, LDN) and constant 20 h light:4 h dark (LD20:4). The fish reared on the different photoperiod regimes differed in their growth patterns as juveniles exposed to long days, i.e. LD20:4 and LD24:0, exhibited faster growth than those experiencing a natural photoperiod. The LD20:4 group showed the highest average specific growth rate (0.72% body weight day^?1), whereas fish on LDN displayed the lowest average specific growth rate (0.60% body weight day^?1). The final mean weights of the LD20:4 and the LD24:0 groups were 15% and 12% higher than those of the LDN group. Dividing the duration of the experiment into three time periods shows that the LD20:4 and LD24:0 had a higher feed conversion efficiency (FCE) as compared with the LDN group during the first and the last period, while a reversed situation was observed in the second period. Our data indicate a larger variation in growth rates among individuals in the best‐performing groups (here LD20:4 and LD24:0). This may indicate that formation of size hierarchies is more pronounced in groups with more homogenous growth (here LDN). Overall, our findings indicate that extended light regimes result in faster growth and better feed conversion in juvenile Atlantic halibut. In line with findings on other flatfish species, this supports the concept that constant long day:short night or continuous light regimes should be used by the farmer in order to maximize growth and improve feed conversion in Atlantic halibut.     

Growth, feed conversion and chemical composition of Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.) fed diets supplemented with krill or amphipods

The effects of partial replacement of fish meal (FM) with meal made from northern krill (Thysanoessa inermis), Antarctic krill (Euphausia superba) or Arctic amphipod (Themsto libellula) as protein source in the diets for Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.) on growth, feed conversion, macro‐nutrient utilization, muscle chemical composition and fish welfare were studied. Six experimental diets were prepared using a low‐temperature FM diet as control. The other diets included northern krill where 20, 40 or 60% of the dietary FM protein was replaced with protein from northern krill, and two diets where the FM protein was replaced with protein from Antarctic krill or Arctic amphipod at 40% protein replacement level. All diets were iso‐nitrogenous and iso‐caloric. Atlantic salmon grew from 410 g to approximately 1500 g during the 160 day experiment, and Atlantic halibut grew from 345 g to 500–600 g during the 150 day experiment. Inclusion of krill in the diets enhanced specific growth rate in salmon, especially during the first 100 days (P < 0.01), and in a dose–response manner in halibut for over the 150 day feeding period (P < 0.05). Feed conversion ratio did not differ between dietary treatments, and no difference was found in dry matter digestibility, protein digestibility and fish muscle composition. Good growth rates, blood parameters within normal ranges and low mortalities in all experimental treatments indicted that fish health was not affected either Atlantic salmon or Atlantic halibut fed the various zooplankton diets.     

Pigmentation and eye migration in Atlantic halibut (Hippoglossus hippoglossus L.) larvae: new findings and hypotheses

Atlantic halibut juveniles, which have been fed Artemia during larval development, frequently demonstrate malpigmentation and impaired eye migration. This is in contrast to the high percentage of normally developed larvae fed copepods, reared under similar conditions. Nutrition is therefore an important component influencing larval development. Analyses of the nutrient composition of Artemia and copepods show that Atlantic halibut larvae fed Artemia probably receive sufficient amounts of vitamin A by converting canthaxanthin, while iodine may be deficient, possibly leading to interrupted thyroid hormone synthesis. An unbalanced fatty acid composition, such as high levels of arachidonic acid and low levels of docosahexaenoic acid, can be another limiting factor in Artemia. Vitamin A, fatty acids and thyroid hormones have all been shown to affect pigmentation in flatfish. They are ligands to nuclear receptors, thyroid hormone receptors, retinoic acid receptors, retinoic X receptors and peroxisomal proliferator‐activated receptors, which are members of the superfamily of steroid hormone receptors. The receptors interact with each other to promote gene expression that modulates proliferation and differentiation of cells. Our hypothesis is that these interactions are important for development during flatfish metamorphosis. Very little data exist on the topic of impaired eye migration. However, energy limitation, iodine deficiency and an unbalanced fatty acid composition have been proposed as possible explanations. Here, we review the literature on development of pigment cells and the possible mechanisms behind the effects of vitamin A, fatty acids and thyroid hormone on pigmentation and eye migration during development of Atlantic halibut larvae.     

Successful early weaning of Atlantic halibut (Hippoglossus hippoglossus L.) in small shallow raceway systems

Small raceways were used in a weaning experiment with Atlantic halibut (Hippoglossus hippoglossus L.) larvae. The size of the tanks was 1.0 × 0.4 m with a 1‐ to 2‐cm water level. Duplicate larval groups were transferred to the raceways from circular first feeding tanks at 0.07, 0.10 and 0.16 g wet weight, while recommended weaning size of this species is 0.2–0.3 g. During the first 7 days of weaning, Artemia was used as a food supplement in combination with the formulated dry feed. Thereafter only dry feed was used. The dry feed used in this experiment was produced by a special heat technique. The 0.07, 0.10 and 0.16 g larval groups were evaluated after 31, 25 and 17 days respectively (same date) The corresponding average survival was 81.4%, 78.0% and 96.6% and the specific daily growth rate was 3.18%, 3.17% and 2.38% respectively. In the Artemia control group, a survival rate of 96.0% and a growth rate of 5.28% was achieved. To evaluate the weaning success, the groups were followed in a 22‐day post‐weaning period on a commercial dry diet. Higher growth rates, 5.8–6.9%, were then obtained in all experimental groups, except control. The survival here averaged approximately 80% in the three experimental groups, but showed some differences between replicates. One hundred per cent survival was achieved during weaning in the former Artemia group. From the start of weaning to the end of the post‐weaning period, the survival rates averaged 64% for the 0.07 and 0.10 g groups, approximately 80% for the 0.16 g group and 96% in the Artemia control group. Higher variance (CV) through the experiment and highest growth of the 75% quartiles of the fish groups compared with the 50% and 25%, indicated suppressed growth of the smaller fish. The successful weaning at these small sizes considerably reduced the live food period. Based on the present knowledge of the energetic demands of this species, it is calculated that weaning at 0.07 g compared with 0.25 g will reduce the amount of Artemia needed by at least 60%. No differences in pigmentation or degree of completed eye migration were detected between groups, indicating that this is determined at earlier developmental stages.     

A correlation between phototactic response and first-feeding of Atlantic halibut (Hippoglossus hippoglossus L.) larvae

The correlation between positive phototaxis and feeding incidence at first‐feeding for groups of Atlantic halibut larvae ranging in age from 210 to 240 degreedays, post hatch, was examined. Phototactic response was measured as the fraction of larvae that responded by horizontal swimming towards a light source, and the median travel distance for the responding fraction of larvae. Within the ranges investigated, larval age, size or proportion of deformed larvae had no significant effect on the phototactic response, or on the feeding incidence (deformed larvae excluded from analysis). The fraction of larvae responding phototactically and the median travelling distance for responding larvae were significantly correlated. Feeding incidences after 24 and 48 h were also significantly correlated with both measures of phototactic response. The possibility of using phototactic response in quality assessment is discussed.     

Evaluation of Calanus finmarchicus copepod meal in practical diets for juvenile Atlantic halibut (Hippoglossus hippoglossus)

The copepod, Calanus finmarchicus, has potential as a new ingredient in practical feeds for marine fish. Two experiments were conducted to evaluate the nutritional value of C. finmarchicus meals when fed to juvenile Atlantic halibut. The first study determined protein, lipid and energy digestibility coefficients of four C. finmarchicus meals prepared under different processing conditions. The second study evaluated growth and nutrient utilization efficiency of juveniles fed diets containing varying proportions of fish meal, plant meals and C. finmarchicus meal. Moisture, ash, protein, lipid and gross energy contents of the C. finmarchicus meals were 28–93, 74–138, 505–648, 123–269 g kg⁻¹, and 21–26 MJ kg⁻¹, respectively. Protein, lipid and energy digestibilities of C. finmarchicus meals were 91–99%, 90–95% and 90–99%. The digestibilities significantly decreased for the Calanus meals processed at higher temperatures. During the growth study, halibut fed a diet containing 240 g kg⁻¹ C. finmarchicus meal had significantly higher weight gain and growth rate than all other groups. Nitrogen and energy retention efficiencies ranged between 35–45% and 33–43%, and were significantly higher for fish fed 160–240 g kg⁻¹ C. finmarchicus meal than for fish fed plant protein and control diets. The results indicate that growth and nutrient utilization efficiency are improved in Atlantic halibut fed diets supplemented with C. finmarchicus meal.     

The problem of meeting dietary protein requirements in intensive aquaculture of marine fish larvae, with emphasis on Atlantic halibut (Hippoglossus hippoglossus L.)

Atlantic halibut (Hippoglossus hippoglossus) achieve a mature gastrointestinal tract approximately 2 months after first feeding (12 °C). The immature digestion may be the reason that compound diets fail to sustain growth and survival in first feeding halibut larvae and in larvae of other marine fish species. On the other hand, larvae fed with live feeds are capable of extraction of sufficient quantities of nutrients to sustain high growth rates. A lower availability of the protein in formulated diets compared with live prey is considered to be an important reason for the low performance of formulated diets. One approach to increase dietary protein availability is supplementation of pre‐digested proteins. Experiments using tube fed individual larvae show that halibut larvae are able to utilize hydrolysed protein more efficiently than intact protein. However, Atlantic halibut in culture did not respond well to dietary supplementation of hydrolysed protein, in contrast to some other species. One reason may be extensive leaching of pre‐hydrolysed proteins from the microparticulate feed. Atlantic halibut are slow feeders and may thus suffer more from nutrient leaching than species eating more rapidly. Feed formulation techniques affect dietary protein leaching, and in this paper, different techniques and their impact on feed properties are described. Microbound diets are most widely used in larval rearing, but show high rates of nutrient leaching. Lipid‐based capsules seem to have the best potential to prevent leaching, however, they are not able to deliver a complete diet. The high need for improvements in larval feed formulation techniques are clearly stated, and some suggestions are given. Among these are production of complex particles, where small lipid‐based capsules or liposomes containing the low molecular weight water‐soluble nutrients are embedded. In such feed particles the water‐soluble molecules are protected from leaching. Techniques for delivery of water‐soluble nutrients that are needed in large quantities, i.e. free amino acids or hydrolysed and water‐soluble protein, remain to be developed.     

A comparison of retinol, retinal and retinyl ester concentrations in larvae of Atlantic halibut (Hippoglossus hippoglossus L.) fed Artemia or zooplankton

Atlantic halibut (Hippoglossus hippoglossus L.) larvae were fed enriched Artemia or zooplankton in duplicate tanks from 0 to 60 days after first‐feeding. Both diets and the larvae were analysed for vitamin A (VA) in order to confirm earlier findings, in which Artemia fed larvae had lower levels of VA compared with larvae fed zooplankton. Furthermore, we wanted to investigate the composition of the retinoids in the larvae. The results showed that Artemia and zooplankton contains low levels of VA, probably too low to sustain the assumed requirement. Nevertheless, larvae fed Artemia had the same level of retinal and retinol as larvae fed zooplankton. We found a significant lower level of retinyl esters in larvae fed Artemia. The total VA level was lower in larvae fed Artemia only at the end of the feeding trial after the onset of metamorphosis. Our conclusion is that feeding Artemia to Atlantic halibut larvae is not likely to cause VA deficiency.