The proteolytic digestive activity and growth during ontogeny of Parachromis dovii larvae (Pisces: Cichlidae) using two feeding protocols

The proteolytic digestive activity and growth of Parachromis dovii larvae during the ontogeny were evaluated in a recirculation system using two feeding strategies during a 28-day period. Larvae were reared using two feeding protocols (three replicates each): (A) Artemia nauplii (at satiation), fed from exogenous feeding [8 days after hatching (DAH)] until 15 DAH followed by nauplii substitution by formulated feed (20 % day^?1) until 20 DAH and then formulated feed until 28 DAH; (B) formulated feed (100 % BW daily) from exogenous feeding until 28 DAH. Levels of acid (pepsin type) and alkaline digestive proteases as well as growth and survival of larvae were measured along the feeding period. Survival was high and similar between treatments: 98.9 ± 0.0 for Artemia, 97.3 ± 0.0 % for formulated feed. The specific growth rate for length and weight was higher in larvae fed with Artemia nauplii than in larvae reared with formulated feed: 3.4 ± 0.1 versus 1.8 ± 0.1 % day^?1 for body length (P = 0.009) and 12.2 ± 0.1 versus 6.5 ± 0.3 % day^?1 for body weight (P = 0.002). The acid and alkaline proteolytic activity was detected, in both treatments, from the beginning of the experiment, at 8 DAH. The total enzymatic activity (U larva^?1) for acid and alkaline proteases was higher in larvae reared with Artemia after 12 DAH, whereas the specific enzymatic activity was similar for both enzyme types in the two treatments. The results suggest that P. dovii larvae were capable to digest formulated diets from the beginning of exogenous feeding and that they could be reared with formulated feeds. However, the formulated feed used should be nutritionally improved because of the poor growth obtained in this research.     

Effects of early weaning strategies on growth,survival and digestive enzyme activities in cobia (<Emphasis Type="Italic">Rachycentron canadum</Emphasis> L.) larvae

The effects of weaning strategies of cobia (Rachycentron canadum L.) larvae to commercial microdiets, either from rotifers or from Artemia, on growth, survival and enzymatic digestive capacity, were investigated. In the first experiment, cobia larvae were weaned from rotifers by co-feeding with a microdiet (Otohime) from 8, 13 or 20 days post-hatching (dph). The larvae in the control treatment were fed rotifers (2–12 dph), Artemia nauplii from 7 dph, and co-fed with the microdiet from 20 dph. In the second experiment, the larvae were weaned from Artemia, which was fed to the larvae from 7 dph, by co-feeding with a microdiet (NRD) from 8, 13 or 18 dph. The larvae in control treatment were fed rotifers, then Artemia to the end of the experiment (28 dph). Weaning of cobia larvae onto a microdiet directly from rotifers significantly reduced growth, survival and digestive capacity of the larvae and did not lead to larval acceptance of the microdiet, compared to those weaned from Artemia in the first experiment. Early weaning of cobia larvae onto NRD microdiet (on 8 or 13 dph) from Artemia in the second experiment also reduced growth, survival rate and gut maturation index, compared to those fed live feed. With available microdiets, weaning of cobia larvae could start from Artemia at around 18 dph in order to obtain comparable growth, survival and gut maturation to larvae fed live feed.     

Weaning of bullseye puffer (Sphoeroides annulatus) from live food to microparticulate diets made with decapsulated cysts of Artemia and fishmeal

Two experiments were carried out to test microparticulate diets forweaning hatchery-produced larvae and juveniles of bullseye pufferSphoeroides annulatus. The diets were formulated with differentprotein sources: diet 1 with a combination of decapsulated cysts ofArtemia and fishmeal, and diet 2 with a combination offishmeal, squid, tuna gonad and shrimp meal. In the first experiment60-days-old fish were weaned with the microdiets over five days. Fishsurvival after 11 weeks of feeding was 92% for diet 1, 85%for diet 2, and 95% for the control fish fed Artemianauplii. Once it was determined that bullseye puffer can be adequatelyreared with artificial dry diets, diet 1 was used to test earlier timesfor weaning to reduce the period of Artemia feeding. In thesecond experiment, three different times were tested for initiation ofweaning in sibling fish larvae, i.e., at 29, 34, and 39 days post-hatch.Small differences in weight, length and survival were found amongweaning treatments after 23 days of feeding. When weaned at day 29post-hatch, fish larvae grew from an initial weight of 38.4 mg andlength of 11.1 mm to a final weight and length of 405.7 mg and 25.1 mmrespectively. Final survival in this treatment was 49.3%. Thereduced period of Artemia feeding would provide an economicalalternative for the species to take into consideration for its cultureat commercial scale.     

Preliminary study on the activity of protease enzymes in Persian sturgeon (Acipenser persicus Borodin, 1897) larvae in response to different diets: effects on growth and survival

The specific activity of alkaline protease, trypsin and pepsin‐like enzymes was measured in yolk sac stage Acipenser persicus larvae and over a 1‐month feeding experiment using live Artemia nauplii (ND), formulated feed (FD) or mixed food (MD). Artemia nauplii group larvae showed significantly higher growth and survival during the first 15 days while FD larvae showed the lowest growth and survival. At day 30, MD larvae exhibited significantly higher growth than the ND group. Alkaline protease activity showed the lowest activity on day 15; the highest activity was observed in the MD group larvae. Pepsin‐like activity showed a drastic increase from day 1 to 5 in all treatments, but remained stable throughout the next 25 days, with the lowest and the highest activity in the FD treatment on day 10 and in the MD treatment on day 30 post‐feeding respectively. Trypsin‐like activity in group ND remained almost the same from day 5 to 30, whereas in groups MD and FD, it decreased significantly from day 10 to 30. The contribution of the naupliar proteases was moderate but effective. Additionally, better performance in Artemia fed sturgeon larvae may also be due to the structure and digestibility of proteins and the food intake stimulation by the nauplii.     

Stable carbon (δ13C) and nitrogen (δ15N) isotopes as natural indicators of live and dry food in Piaractus mesopotamicus (Holmberg, 1887) larval tissue

This study proposed the use of the stable isotope technique to track the type of food utilized by pacu Piaractus mesopotamicus larvae during their development, and to identify the moment when the larvae start using nutrients from the dry diet by retaining its carbon and nitrogen atoms in their body tissues. Five‐day‐old pacu larvae at the onset of exogenous feeding were fed Artemia nauplii or formulated diet exclusively; nauplii+formulated diet during the entire period; or were weaned from nauplii to a dry diet after 3, 6 or 12 days after the first feeding. δ¹³C and δ¹⁵N values for Artemia nauplii were ?15.1‰ and 4.7‰, respectively, and ?25.0‰ and 7.4‰ for the dry diet. The initial isotopic composition of the larval tissue was ?20.2‰ and 9.5‰ for δ¹³C and δ¹⁵N respectively. Later, at the end of a 42‐day feeding period, larvae fed Artemia nauplii alone reached values of ?12.7‰ and 7.0‰ for δ¹³C and δ¹⁵N respectively. Larvae that received the formulated diet alone showed values of ?22.7‰ for δ¹³C and 9.6‰ for δ¹⁵N. The stable isotope technique was precise, and the time at which the larvae utilized Artemia nauplii, and later dry diet as a food source could be clearly defined.     

Proteolytic Activity in California Halibut Larvae (Paralichthys californicus)

The digestive tract of many marine fish larvae undergoes numerous morphological and functional changes during ontogeny that can substantially influence larval survival under culture conditions. Increasing our knowledge of the digestive capacity and nutritional requirements of the larvae of new candidate species for aquaculture will aid in the development of optimal feeding protocols and greatly improve production under hatchery conditions. In this study, we assess the proteolytic capacity of California halibut (Paralichthys californicus) larvae using biochemical and histological analyses. Newly hatched larvae were reared in a semiclosed recirculating system and fed with highly unsaturated fatty acid (HUFA)–enriched rotifers from hatching until 19 d posthatch (dph) and HUFA‐enriched Artemia nauplii thereafter. Total and specific activity of trypsin and leucine‐aminopeptidase (LAP) and acid and alkaline protease activities were assessed throughout development using spectrophotometric techniques. Trypsin‐like activity and LAP and alkaline protease activities were detected shortly after hatching and before the opening of the mouth. Acid protease activity was not detected until 36–40 dph, concomitant with the development of the gastric glands. The specific activity of trypsin and LAP showed two distinct peaks at 8 and 20 dph. The second peak coincided with the shift from rotifers to Artemia. Hence, newly hatched California halibut larvae possess alkaline proteolytic activity before first feeding. Based on the digestive capacity evaluated in this study and the timing of the development of the functional stomach, we propose that California halibut can be adequately weaned to formulated microdiets around 36 dph.     

Histological evaluation of the elimination of Artemia nauplii from larval rearing protocols on the digestive system ontogeny of shi drum (Umbrina cirrosa L.)

The influence of the absence of Artemia nauplii from larval diet protocols on growth and digestive system ontogeny was studied using histological techniques in the shi drum (Umbrina cirrosa). One group of larvae was reared using the standard intensive rearing protocol, which offers a combination of enriched rotifers (Brachionus plicatilis), Artemia spp. nauplii and artificial diet (Std-group). Another group was reared using the same protocol, but without the offering of Artemia nauplii (group No-Artemia). The ontogenesis of the digestive system from hatching to metamorphosis was a very rapid process, and there were no differences between the two feeding regimes in the temporal appearance of the various components of the digestive system. The first organised presence of the hepatic and pancreatic tissue appeared at 2–3 d after hatching (dah), suggesting that these organs function from a very early developmental stage. In the No-Artemia larvae between 13 and 29 dah there was a reduction in the height of enterocytes in the intestinal mucosa, a progressive flattening of the primary intestinal folds in the anterior and posterior intestine and a decrease in lipid stores in the liver, suggesting a period of relative starvation. However, by the end of the study at 41 dah, there were no significant differences in body length, intestinal morphology or liver lipid stores between larvae reared under the two feeding regimes. The study suggests that the diet may influence the maturation and/or function, but not the ontogeny of the digestive system. Furthermore, the rapid differentiation of the digestive system in shi drum and the prompt recovery of the No-Artemia larvae from the symptoms of starvation by 29 dah, indicate a plasticity during ontogenesis and the ability of larvae to adapt to artificial diets at very early developmental stages.     

Evaluation of partial replacement of live and fresh feeds with a formulated diet and the influence of weaning <Emphasis Type="Italic">Panulirus ornatus</Emphasis> phyllosomata onto a formulated diet during early ontogeny

We have evaluated the potential of a formulated diet as a replacement for live and fresh feeds for 7-day post-hatch Panulirus ornatus phyllosomata and also investigated the effect of conditioning phyllosomata for 14–21 days on live feeds prior to weaning onto a 100% formulated diet. In the first trial, the highest survival (>55%) was consistently shown by phyllosomata fed a diet consisting of a 50% combination of Artemia nauplii and 50% Greenshell mussel, followed by phyllosomata fed 50% Artemia nauplii and 50% formulated diet and, thirdly, by those receiving 100% Artemia nauplii. The second trial assessed the replacement of on-grown Artemia with proportions of formulated diet and Greenshell mussel that differed from those used in trial 1. Phyllosomata fed a 75% combination of formulated diet and 25% on-grown Artemia and 50% on-grown Artemia and 50% Greenshell mussel consistently showed the highest survival (>75%). Combinations of Greenshell mussel and formulated diet resulted in significantly (P < 0.05) reduced survival. In trial 3, phyllosomata were conditioned for 14, 18 or 21 days on Artemia nauplii prior to weaning onto a 100% formulated diet, which resulted in survival rates that were negatively related to the duration of feeding Artemia nauplii. In the final trial, phyllosomata were conditioned for 14 days on live on-grown Artemia prior to weaning onto one of three formulated diets (one diet with 44% CP and two diets with 50%). Phyllosomata fed a 44% CP diet consistently showed the highest survival (>35%) among all treatments, while those fed a 50%-squid CP diet showed a significant (P < 0.05) increase in mortality at day 24. The results of these trials demonstrate that hatcheries can potentially replace 75% of live on-grown Artemia with a formulated diet 7 days after hatch. The poor performance associated with feeding combinations of Greenshell mussel and formulated diet, and 100% formulated diet as well as conditioning phyllosomata for 14–21 days on live feeds prior to weaning onto a formulated diet highlights the importance of providing Artemia to stimulate feeding.     

Breeding and larval rearing of the rabbitfish,Siganus guttatus (Bloch)

Females of Siganus guttatus reared to sexual maturity in canvas tanks were induced to spawn by using human chorionic gonadotropin (HCG, Ayerst) at 500 IU/fish or about 2 IU/g body weight. The amount of HCG used depended on the initial mean egg diameter; the smaller the diameter, the more HCG was used. Fish with oocytes characterized by germinal vesicle migration (mean egg diameter ≥ 0.47 mm) spawned without HCG injection. Fertilization and hatching rates for both treated and untreated fish were more than 90%. The larvae were reared to metamorphosis using rotifers from day 2–17, rotifers + newly hatched Artemia nauplii from day 18–20 and rotifers + newly hatched Artemia nauplii + artificial feed from day 21–35. In addition, Isochrysis galbana was introduced to the rearing tanks from day 1–10 and Chlorella sp. and/or Tetraselmis sp. from day 1–35. Survival rates of larvae tended to be lower as the broodstock became older.     

Changes in digestive enzyme activities during larval development of leopard grouper (Mycteroperca rosacea)

The leopard grouper is an endemic species of the Mexican Pacific with an important commercial fishery and good aquaculture potential. In order to assess the digestive capacity of this species during the larval period and aid in the formulation of adequate weaning diets, this study aimed to characterize the ontogeny of digestive enzymes during development of the digestive system. Digestive enzymes trypsin, chymotrypsin, acid protease, leucine–alanine peptidase, alkaline phosphatase, aminopeptidase N, lipase, amylase and maltase were quantified in larvae fed live prey and weaned onto a formulated microdiet at 31 days after hatching (DAH) and compared with fasting larvae. Enzyme activity for trypsin, lipase and amylase were detected before the opening of the mouth and the onset of exogenous feeding, indicating a precocious development of the digestive system that has been described in many fish species. The intracellular enzyme activity of leucine–alanine peptidase was high during the first days of development, with a tendency to decrease as larvae developed, reaching undetectable levels at the end of the experimental period. In contrast, activities of enzymes located in the intestinal brush border (i.e., aminopeptidase and alkaline phosphatase) were low at the start of exogenous feeding but progressively increased with larval development, indicating the gradual maturation of the digestive system. Based on our results, we conclude that leopard grouper larvae possess a functional digestive system at hatching and before the onset of exogenous feeding. The significant increase in the activity of trypsin, lipase, amylase and acid protease between 30 and 40 DAH suggests that larvae of this species can be successfully weaned onto microdiets during this period.     

Evaluation of weaning performance of California halibut (Paralichthys californicus) larvae using growth,survival and digestive proteolytic activity

This study evaluated weaning success of California halibut, Paralichthys californicus, larvae onto a microdiet at various stages of development utilizing growth, survival and digestive enzyme activity. Weaning onto a microdiet was evaluated at 16, 26, 36 and 46 days posthatch (dph). Alkaline and acid proteases and leucine aminopeptidase activities were measured after weaning and compared between the weaned treatment and Artemia‐fed controls. Survival was significantly lower in the microdiet‐fed treatments compared to the control groups. Growth was significantly reduced in all weaning treatments compared to the control, except for the 46 dph group. No differences in enzyme activities were detected between treatment and diet at 16 and 26 dph; however, activities were higher for the microdiet‐fed larvae at 36 and 46 dph. This study demonstrates that California halibut larvae possess a differentiated and effective digestive system early in development and can be weaned with relative success (>40% survival) before completion of the metamorphosis (i.e., 36 dph). The lack of weaning success at an early date cannot be entirely because of the absence of a functional stomach but could be related to, among other factors, the low‐microdiet ingestion rates observed and higher leaching of smaller microdiets.     

Optimisation of Artemia feeding rate for carp larvae (Cyprinus carpio L.)

Carp (Cyprinus carpio) larvae were fed on measured numbers of Artemia nauplii, and daily growth of the larvae monitored for a period of 10 days in order to determine the effect of varying feeding levels. A 34% reduction in specific growth rate was observed over the experimental period. At a temperature of 24 ± 0.5° C, carp larvae were found to require 200–250% of their body weight of Artemia nauplii per day for optimal growth and food conversion during the first five days of feeding, reducing to 100–120%/day over the following five days. The significance of these results is discussed in relation to potential economies of Artemia cyst use in hatcheries.     

Establishing larval feeding regimens for the Forktail Blenny Meiacanthus atrodorsalis (Günther, 1877): effects of Artemia strain,time of prey switch and co‐feeding period

This study aimed to establish feeding strategies covering the whole larval period of the forktail blenny, Meiacanthus atrodorsalis, based on the standard hatchery feeds of rotifers and Artemia. Three purposely designed experiments were conducted to determine the appropriate times and techniques to transition larvae from rotifers onto Artemia nauplii of a Great Salt Lake (GSL) strain, and a specialty AF strain, as well as subsequent transition onto enriched metanauplii of GSL Artemia. With a 3‐day co‐feeding period, larvae adapted well to a transition from rotifers to newly hatched GSL Artemia nauplii as early as 5 days posthatching (DPH), and as early as 3 DPH when fed the smaller AF Artemia nauplii. However, prolonging the rotifer‐feeding period up to 11 DPH did not negatively affect survival. Larvae fed Artemia nauplii of the AF strain showed 17–21% higher survival, 24–33% greater standard length and body depth, and 91–200% greater dry weight, after 20 days relative to those fed nauplii of the GSL strain. Meanwhile, enriched Artemia metanauplii of the GSL strain were shown to be an acceptable alternative to AF Artemia nauplii for later larvae, producing similar survival and growth when introduced from 8 DPH. Based on our findings, we recommend feeding M. atrodorsalis larvae rotifers as a first food between 0 and 2 DPH, introducing AF Artemia nauplii from 3 DPH, followed by enriched GSL Artemia metanauplii from 8 DPH onward, with a 3‐day co‐feeding period between each prey change.     

Development of digestive enzymes in larvae of Mayan cichlid Cichlasoma urophthalmus

The development of digestive enzymes during the early ontogeny of the Mayan cichlid (Cichlasoma urophthalmus) was studied using biochemical and electrophoretic techniques. From yolk absorption (6?days after hatching: dah), larvae were fed Artemia nauplii until 15?dah, afterward they were fed with commercial microparticulated trout food (45% protein and 16% lipids) from 16 to 60?dah. Several samples were collected including yolk-sac larvae (considered as day 1 after hatching) and specimens up to 60?dah. Most digestive enzymes were present from yolk absorption (5?C6?dah), except for the specific acid proteases activity (pepsin-like), which increase rapidly from 8?dah up to 20?dah. Three alkaline proteases isoforms (24.0, 24.8, 84.5?kDa) were detected at 8?dah using SDS?CPAGE zymogram, corresponding to trypsin, chymotrypsin and probably leucine aminopeptidase enzymes, and only one isoform was detected (relative electromobility, Rf?=?0.54) for acid proteases (pepsin-like) from 3?dah onwards using PAGE zymogram. We concluded that C. urophthamus is a precocious fish with a great capacity to digest all kinds of food items, including artificial diets provided from 13?dah.     

Changes in the digestive enzymes and hormones of gilthead seabream larvae (Sparus aurata, L. 1758) fed on Artemia nauplii enriched with free lysine

Variations in digestive enzymes and hormones during the larval development of gilthead seabream (Sparus aurata) fed on live prey (Artemia nauplii) enriched with free lysine were investigated for 16 days (from day 24 to day 40). Prior to initiation of the experiment, newly hatched larvae were transferred from incubators to fiber glass tanks (300 l) with black walls and fed the same diet until day 24. Each experiment was performed in triplicate. The amount of free lysine in the Artemia nauplii was increased by adding a 5.3 mM free amino acid solution to the culture water during a 16-h enrichment period. Larvae were sampled four times at four-day intervals, for 16 days. Larvae fed on the control group had lower growth than those fed on the lysine group at the end of the study but the difference was not significant (P > 0.05). The highest trypsin activity and lap/leu–ala peptidase ratios were observed in the control group (P < 0.05). Significant differences between bombesin activities of the treatment groups were not found in the fifth minute after the start of feeding (P > 0.05) but were significant in the fifteenth minute (P < 0.05). Significant differences between the CCK levels of the treatment groups were found (P < 0.05). In conclusion, three main ideas are presented in this study. First, Artemia nauplii can successfully be enriched with free lysine. Second, bombesin (GRP) and cholecystokinin (CCK) activity can be stimulated by free lysine. Third, the mechanisms controlling adaptation of the activity of the trypsin to the amount of dietary protein were not activated until day 40.     

Bioencapsulated probiotics increased survival, growth and improved gut flora of turbot (Psetta maxima) larvae

Bacterial diseases are probably the major cause of mortality in aquaculture. The use of probiotics is one of the solutions to manage bacterial infections, by preventing the colonization and exclusion of pathogenic bacteria. The effect of two selected probiotics in turbot (Psetta maxima) larvae was evaluated. Turbot larvae were reared on three different dietary treatments consisting of newly hatched Artemia nauplii from day 16 to 34 post-hatch. Groups A and B were fed with Artemia-containing probiotics FAA and FAB, respectively, and group C was control group. Results indicated that the addition of probiotics increased survival and promoted turbot growth, showing that they were beneficial for the aquaculture of turbot. Moreover, turbot fed with bioencapsulated probiotics attained higher percentages of survival during its development.     

The use of squid protein hydrolysate as a protein source in microdiets for gilthead seabream Sparus aurata larvae

In the present study, the use of predigested proteins as an ingredient of microdiets offered to gilthead seabream larvae was tested. The protein source (freeze-dried squid powder) was hydrolysed with protease (trypsin and pancreatin). Different levels of raw squid protein and hydrolysate (100% protein, 50% protein/50% hydrolysate, 100% hydrolysate) were added to the microdiets to produce a dietary protein level of 65%. For comparison, cofeeding of Artemia nauplii and microdiet as well as microdiet supplemented with pancreatin were also offered to the larvae. The final average dry weights of 32-day-old larvae were 1.65 ± 0.04 mg, 1.38 ± 0.06 mg and 1.13 ± 0.1 mg, respectively, for larvae cofed 0%, 50% and 100% hydrolysate microdiets and Artemia nauplii. Survival of larvae was not affected by protein source. The survival of larvae cofed Artemia nauplii and microdiet was significantly higher than that of larvae fed exclusively on microdiet (68% and 80%, respectively). These results suggest that the use of hydrolysate (at 50% greater) as a protein source in diets for seabream larvae is not to be recommended.     

Feeding Larval Red Drum on Microparticulate Diets in a Closed Recirculating Water System1

A feeding protocol was developed for red drum larvae based on combining a commercial microparticulate diet (Kyowa Fry Feed) with live prey (rotifers) in a closed, water reuse system. In five trials, growth and survival were measured on larvae reared on a combination of live and microdiet for 1–5 d and then microdiet alone. Results in each trial were compared to control larvae reared on live rotifers Brachionus plicatilis and brine shrimp nauplii Artemia salinas. The most satisfactory combination was feeding live food and microdiet together for the first five days and then completely discontinuing live prey, eliminating the need to feed brine shrimp to the larvae. Growth rates of larvae fed progressively larger sizes of the microdiet were as good as larvae reared on live prey. Both groups metamorphosed to the juvenile stage at less than one month. Survival rates on the five day live food and microdiet combination were a remarkable 60% from egg to the juvenile stage. The successful weaning of red drum to microdiets paves the way to produce a semipurified diet to test nutrient requirements of larval fish.     

Rearing of pike-perch larvae using formulated diets – first success with starter feeds

The present study evaluated the performance of two commercial diets: AgloNorse (AN) and BioKyowa (BK), and two experimental, formulated diets based on casein (C) or casein plus casein hydrolysates (CH) in rearing of pike‐perch larvae (Sander lucioperca L.). All fish were 5 day old and control group was fed live Artemia nauplii. Fish were sampled periodically for histological comparison of morphological changes in the digestive tracts. Survival of fish fed Artemia nauplii, BK and AN was similar: 54.4%, 50.8% and 52.4%, respectively, while the fish fed formulated diets C and CH showed considerably lower survival: 28.4% and 21.6% respectively. After 5 weeks of rearing, the average body mass of fish ranged from 212±32 mg in Artemia fed group to 53.8±6.8 mg in C diet fed group. A considerable vacuolization of supranuclear zone in enterocytes of posterior intestine was observed in the larvae fed commercial diets. No anomalies in liver development were found. Hepatocytes of fish fed BK diet showed larger glycogen storage areas, compared with those occupied by lipids. The highest zymogen accumulation of pancreatic cytoplasm was observed in fish fed Artemia. In fish fed C and CH diets, anomalies in digestive system development were indicated by lower and less numerous intestinal folds, smaller hepatocytes, retarded development of gastric glands, and in CH group – also local fatty degeneration of liver.