Antioxidant effects of nanoencapsulated lipoic acid in tissues and on the immune condition in haemolymph of Pacific white shrimp Litopenaeus vannamei (Boone, 1931)

The antioxidant and oxidative damage responses and immune parameters in shrimp, Litopenaeus vannamei, reared in clear water (CW) and in a biofloc system (BFT) were evaluated. The animals were given feed supplemented with either nanocapsules containing the antioxidant lipoic acid (NCLA) or empty nanocapsules (NC). Antioxidant and oxidative stress analyses were evaluated by measuring glutathione S‐transferase activity (GST), reduced glutathione (GSH) concentration and lipid peroxidation (TBARS) in the gills, hepatopancreas and muscle. The immune parameters were then determined by the differential hemocyte count in the haemolymph. The final weight of the shrimp reared in both CW and BFT and treated with NCLA increased (p < .05). The shrimps reared in BFT showed increased GST activity in the gills and hepatopancreas after NCLA treatment (p < .05). Higher GSH levels were observed in the hepatopancreas with NCLA supplementation in CW and BFT (p < .05). Decreased levels of TBARS were observed in the gills and muscles of shrimp supplemented with NCLA and maintained in CW (p < .05). A decrease in the percentage of hyaline hemocytes and an increase in granular hemocytes were observed in shrimp treated with NCLA in CW and BFT (p < .05). The antioxidant treatment induced positive responses in terms of the zootechnical, biochemical and immunological parameters.     

The role of açaí (Euterpe oleracea Mart. 1824) as a chemoprotective agent in the evaluation of antioxidant defence,oxidative damage and histology of juvenile shrimp Litopenaeus vannamei (BOONE, 1931) exposed to ammonia

This study investigated the effects of the use of the inclusion of açaí on the diet of shrimp Litopenaeus vannamei on antioxidant and histopathological responses after exposure to ammonia. The shrimps were fed two experimental diets: control and with 10% of açaí inclusion (W/W), for 35 days. Afterwards, the organisms were exposed at four concentrations of ammonia (0.01‐control; 0.26; 0.48 and 0.91 mg NH₃‐N L^?1) for 96 hr. The total antioxidant capacity (ACAP) of the gills decreased significantly in both diets when exposed to ammonia, whereas in the muscle, the açaí promoted an increase in ACAP. Concomitantly, lipid peroxidation levels increased in the gills and decreased in muscle. After exposure to ammonia, glutathione‐S‐transferase activity increased in hepatopancreas in shrimps fed with açaí facilitating the detoxification of lipid peroxidation by‐products, and the concentration of protein sulfhydryl groups decreased in the gills and muscle of the shrimp of the control diet, evidencing protein damage, an unobserved response in shrimps that received the açaí diet. Histopathological changes decreased in açaí‐fed shrimps about the control diet after exposure to ammonia. It is concluded that açaí mitigated ammonia‐induced histopathological changes, improved the antioxidant defence system (gills and muscle) and attenuated the lipid damage in the muscle.     

Effect of dietary L‐carnitine supplementation on growth performance and lipid metabolism in Rhynchocypris lagowski Dybowski fed oxidized fish oil

Lipid content of a diet is very susceptible to oxidation, which has many negative effects on farmed animals. Therefore, this study studied the protective effect of L‐carnitine (LC) on fish body stimulated by oxidized fish oil (OFO) from lipid metabolism. Lipid content of the diet was replaced by OFO in 0, 100 and 400 meq/kg. L‐carnitine was added to the diet in two levels, 500 and 1,000 mg/kg, giving a total of seven experimental diets. A total of 735 healthy Rhynchocypris lagowski Dybowski with an initial weight of 4.48 ± 0.14 g after 2‐week adaptation randomly divided into 15 glass aquariums. Fish were fed satiated three times daily. After 8 weeks, biometry was done to evaluate growth performance, and hepatopancreas and muscle samples were taken for biochemical analysis. The result showed that feeding with OFO had negative growth. However, in fish received both OFO and LC, growth indices improved slightly (p > .05). Feeding with OFO and LC, the content of EPA, DHA and PUFA in the muscle of R. lagowski was significantly higher than that in the control group (p < .05), which reached the maximum value in the OFO100 + LC500 group. The content of SFA, MUFA, ∑n‐6 and PUFA in hepatopancreas increased significantly (p < .05), and the content of SFA reached the maximum in OFO100 + LC500 group. Feeding with OFO increased hepatopancreas total cholesterol, triacylglycerol, HDL/LDL ratio, FAS and ACCα that involved in lipid synthesis enzymes, while reduced HL and HSLα enzyme activity and gene expression that associated with lipid decomposition. Dietary LC moderated the effects of OFO on lipid metabolism. According to the result of the present study, it can be argued that feeding of R. lagowski with OFO has negative effects on growth performance and lipid metabolism, whereas LC dosages used in this study have increased the oxidation rate of fatty acids in the hepatopancreas of R. lagowski and improved the accumulation of fat in hepatopancreas cells induced by oxidized fish oil.     

Essential oil of Lippia alba in the diet of Macrobrachium rosenbergii: Effects on antioxidant enzymes and growth parameters

The juveniles of Macrobrachium rosenbergii (0.060 ± 0.004 g and 2.018 ± 0.071 cm) were fed during 60 days with diets containing different doses of Lippia alba essential oil (EOLA) (0.0—control, 1.0, or 2.0 ml EOLA/kg diet; in triplicate, with 20 prawns/replicate). After the experimental period, were verified the survival, growth parameters and the antioxidant enzymes, lipid peroxidation and Na/K‐ATPase activities in hepatopancreas and gills, respectively, of the animals. There were no significant differences on survival, growth parameters and gill Na/K‐ATPase activity. However, glutathione peroxidase and glutathione‐S‐transferase activities were lower in hepatopancreas of prawns fed with 1.0 ml EOLA/kg diet compared to the control. Moreover, the activity of the enzyme superoxide dismutase was higher in hepatopancreas of prawns that received 2.0 ml EOLA/kg diet compared to those fed with 1.0 ml EOLA/kg diet. Although the addition of both EOLA doses has not improved the survival and growth parameters of M. rosenbergii, these doses contributed to decrease lipid peroxidation. Additionally, the dose of 2.0 ml EOLA/kg diet contributed for increasing the antioxidant enzymes activities in the hepatopancreas, improving antioxidant status, and therefore, it can be recommended as diet supplementation for M. rosenbergii.     

Effects of α‐ketoglutarate on growth performance,antioxidant capacity and ammonia metabolization against chronic carbonate alkalinity stress in Songpu mirror carp (Cyprinus carpio Songpu)

A 3 × 2 factorial experiment was conducted to investigate the interaction between carbonate alkalinity and dietary α‐ketoglutarate (AKG) levels on the growth performance, antioxidant capacity and ammonia metabolization of Songpu mirror carp (Cyprinus carpio Songpu). Each diet (0%, 1% AKG) was randomly allotted to 0 mmol/L, 15 mmol/L, 30 mmol/L carbonate alkalinity groups with three replicate aquaria. The weight gain rate (WGR) significantly increased in the 1% AKG group and significantly decreased with increasing carbonate alkalinity (p < .05). Crude ash was significantly affected by the interaction between carbonate alkalinity and dietary AKG levels and significantly increased in the 1% AKG group (p < .05). A significant interaction between carbonate alkalinity and dietary AKG levels on superoxide dismutase (SOD) activities in hepatopancreas was observed (p < .05). The increasing carbonate alkalinities significantly decreased SOD activities in the hepatopancreas and foregut, catalase (CAT) activities in the hepatopancreas, foregut, midgut and hindgut and glutathione (GSH) contents in the hepatopancreas and foregut (p < .05). Despite AKG addition, the blood ammonia content still increased with increasing carbonate alkalinities. The gene expressions of AQP3, Rhag, Rhcg2 and Na⁺/K⁺‐ATPase (NKA) in the gills and glutamine synthase (GS) in the brain were significantly upregulated with increasing carbonate alkalinities (p < .05). In the 1% AKG group, GS gene expression significantly upregulated in the brains, whereas AQP3, Rhag, Rhcg2 and NKA gene expressions significantly downregulated in the gills (p < .05). In summary, the 1% AKG addition can enhance the gene expression of ammonia metabolization and improve the antioxidant capacity of Songpu mirror carp with chronic carbonate alkalinity stress.     

Response of Pacific white leg shrimp (Litopenaeus vannamei) on exposure to aflatoxin in feed

A research was accomplished to find the response of Litopenaeus vannamei to different levels of aflatoxin in feed. Two hundred live L. vannamei were collected from a shrimp breeding station located on the south of Tiab, in Hormozgan province. Aflatoxin contaminated diets with 0.18, 96.21, 184.74, 711.35, 977.11 and 1605.61 µg/kg were prepared and shrimps in six experimental groups with three replicates in each group for 28 days were fed with these diets. The results showed that activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) in haemolymph and histological alternation index (HAI) value of hepatopancreas in treatement groups were significantly higher than control group (p < 0.05). Decreased total protein and fat in haemolymph and increased protein in muscle of treatment shrimps were observed compared with the control group (p < 0.05). The HAI value of 977.11 and 1605.61 μg/kg groups were significantly higher than the control and other groups (p < 0.05). Histological study results showed that calculated HAI (more than 100) in these groups as a result of necrosis lesion observed in hepatopancreas. Moderate to severe alteration was recognized in group fed by 711.35 µg/kg aflatoxin. Minor aflatoxin levels (0.18, 96.21 and 184.74 µg/kg) caused inappreciable tissue alteration. This study demonstrated body composition, haemolymph parameters and histology of hepatopancreas were affected in the aflatoxin‐contaminated feed when the concentration was over 955.14 µg/kg.     

Using stable isotope analysis to assess the relationship among dietary protein sources,growth, nutrient turnover and incorporation in Nile tilapia (Oreochromis niloticus)

Nile tilapia (1.20 g) were fed for 56 days in five treatments (FM100 containing only fish meal [FM] as a protein source; soybean meal (SBM) replace 25%, 50%, 75% and 100% of FM protein in the diets of FM75, FM50, FM25 and FM0, respectively) to assess the effects of dietary protein sources on growth, turnover half‐life of nitrogen and carbon stable isotopes (δ¹³C and δ¹⁵N) in muscle, isotope discrimination factors (Δ¹⁵N and Δ¹³C), and the relative contribution of protein sources to muscle growth. Results showed that the final body weight, weight gain and specific growth rate of fish fed diet FM25 were significantly higher than other treatments (p < .05). By day 35, fish under all treatments reached isotopic equilibrium, and the equilibrium values consistently increase as a function of stable isotope values in diets. Growth accounted for most isotopic changes in muscle and resulted in significantly shorter half‐lives of nitrogen and carbon in FM25 (p < .05). The Δ¹⁵N of FM25 and Δ¹³C of FM0 were significantly lower than other treatments (p < .05). The proportional contributions of SBM protein were significantly higher than their respective levels in FM75 and FM25 (p < .05), indicating a preferential incorporation of nitrogen from the SBM in muscle tissue.     

Dietary protein requirement of the high altitude's representative teleost juveniles Schizopygopsis younghusbandi (Cypriniformes: Cyprinidae)

A 60‐day trial was conducted to investigate the effect of dietary protein on growth, whole‐body composition, hepatopancreas enzymes, digestion and absorption in the juveniles of Schizopygopsis younghusbandi. Six graded levels of dietary protein (200.0, 248.7, 303.5, 351.2, 395.8 and 449.3 g/kg diet) were formulated and assigned to triplicate groups of 60 fish (8.16 ± 0.02) for each aquarium. Results showed a significantly increased specific growth rate (SGR) in fish fed protein containing 351.2 g/kg diet (p < .05). Besides, intestinal ratio (IR), intestinal somatic index (ISI) and hepatosomatic index (HSI) were decreased and the condition factor (CF) was increased with dietary protein up to 351.2 g/kg diet, then altered reversely. Fish fed the optimal dietary protein showed the highest crude protein calcium, phosphorous and lowest crude lipid contents of the whole body in fish. Additionally, plasma ammonia content (PAC), and activities of GOT and GPT were enhanced by dietary protein levels (p < .05). The digestive enzymes of hepatopancreas were generally increased with the quadratic response to dietary protein levels. Optimal dietary protein level increased the intestinal enzyme activities of Na⁺, K⁺‐ATPase (NKA), alkaline phosphatase (AKP), gamma‐glutamyl transpeptidase (γ‐GT) and creatine kinase (CK). Based on the 2‐slope broken‐line model analysis of PWG, dietary protein requirement was determined to be 349.6 g/kg diet.     

Effects of polysaccharide from fermented wheat bran on growth performance,muscle composition,digestive enzyme activities and intestinal microbiota in juvenile common carp

The present study evaluated the effect of dietary polysaccharide from fermented wheat bran on growth performance, muscle composition, digestive enzyme activities and intestinal microbiota of juvenile common carp (Cyprinus carpio L.). Four diets supplemented with 0 (D0), 1.0 (D1), 2.0 (D2) and 4.0 (D3) g/kg fermented wheat bran polysaccharide (FWBP) were prepared. Fish were randomly assigned into four groups, each group having four replicates (30 fish/replicate) and fed with experimental diets for 8 weeks. Compared with the D0 group, the D3 group had higher final body weight and weight gain (p < .05). The amylase activity in the intestine and hepatopancreas of D1 and D2 groups and pepsin and trypsin activities in the hepatopancreas of D2 and D3 groups were significantly higher than D0 group. Significantly increased muscle protein and lipid content while reduced moisture content was observed in D1 group; additionally, D3 group had significantly lower muscle lipid content. The population of Bifidobacterium spp. in the intestine digesta of D1 and D2 groups was significantly increased, whereas lactic acid bacteria (LAB) and Escherichia coli were unaffected. Collectively, dietary FWBP supplementation could enhance digestive enzyme activities and Bifidobacterium population and change the muscle composition and improve growth performance of common carp.     

Effect of serotonin injection on the reproductive parameters and haemolymph methyl farnesoate level in the narrow‐clawed crayfish Pontastacus leptodactylus (Eschscholtz, 1823)

The effects of serotonin injection (1, 5 and 10 µg/g body weight) on spermatozoal production, vas deferens index (VDI), hepatosomatic index (HSI), testicular index (TI), gonado‐somatic index (GSI), ovarian egg number, total protein, methyl farnesoate (MF) levels of haemolymph, amino acid and fatty acid profiles of hepatopancreas were evaluated in Pontastacus leptodactylus. In male P. leptodactylus, the highest spermatozoal number (9.3 × 10⁶ ± 1.2) and MF level (0.66 ± 0.06 ng^?1 ml) were observed after injection of 10 µg/g of serotonin (p < .05). Serotonin injection, however, did not significantly affect the reproductive parameters in female P. leptodactylus (p > .05). It was also found that serotonin injection caused an increase in the level of MF in male P. leptodactylus but a reduction in female P. leptodactylus (p < .05). Therefore, a sex‐dependent response to serotonin could exist for MF synthesis in P. leptodactylus. Serotonin injection did not affect the total protein, amino acid and fatty acid profiles in the hepatopancreas of male P. leptodactylus (p > .05). It can be concluded that serotonin injection affects the reproductive parameters such as spermatozoal production, VDI, TI, GSI, HSI and haemolymph MF levels in P. leptodactylus.     

Dietary calcium requirement of red swamp crayfish (Procambarus clarkia)

To investigate dietary calcium requirement of red swamp crayfish (Procambarus clarkia), six semi‐purified diets were formulated to contain different concentrations of calcium (2.7(control group), 6.1, 11.9, 17.6, 23.5 and 29.1 g/kg calcium). Each diet was hand‐fed to triplicate of 15 crayfish with average initial body weight (6.22 ± 0.87) g for 8 weeks. The results showed that weight gain rate (WGR) significantly increased and feed conversion ratio (FCR) significantly decreased from 11.9 to 23.5 g/kg groups (p < .05). Protease activities in intestine and hepatopancreas and parathyroid hormone concentrations in serum significantly decreased with increasing dietary calcium levels (p < .05), while calcium and phosphorus contents in exoskeleton, calcium content in muscle and calcitonin concentrations in serum significantly increased (p < .05). The activities of lipase and amylase in intestine and hepatopancreas, serum alkaline phosphatase and total vitamin D concentrations in serum had significant increase as dietary calcium content increased up to 11.9–17.6 g/kg (p < .05). The inorganic phosphorus content in 29.1 g/kg group was significantly lower than those in other groups (p < .05). Broken‐line model analysis based on WGR and quadratic curve model analysis based on FCR showed that optimal dietary calcium requirement of red swamp crayfish ranged from 12.7 to 17.1 g/kg.     

Influence of dietary black soldier fly (Hermetia illucens Linnaeus) pulp on growth performance,antioxidant capacity and intestinal health of juvenile mirror carp (Cyprinus carpio var. specularis)

This study was conducted to investigate whether dietary supplementation of black soldier fly (Hermetia illucens Linnaeus) pulp (BSFP) affects growth performance, antioxidant capacity and intestinal health of juvenile mirror carp (Cyprinus carpio var. specularis). A total of 270 juvenile mirror carp (13.68 ± 0.02 g) were randomly allotted to five dietary treatments, BSFP0, BSFP25, BSFP50, BSFP75 and BSFP100, in which BSFP was included in the basal diet at 0, 43.7, 87.3, 131 and 174.7 g/kg, respectively. Then, fish were fed to apparent satiation for 8 weeks. Fish growth performance and nutrient utilization were not different among the five groups (p > .05). Increasing BSFP dietary content significantly decreased whole‐body lipid content but increased kidney index (p < .05). The general relative intestine length was significantly higher in the BSFP100 group than the BSFP0 group (p < .05). Increasing BSFP dietary content significantly increased serum catalase activity and decreased malonaldehyde content (p < .05). The intestinal villus height, villus area and muscle layer thickness were significantly lower in the BSFP100 group than the BSFP0 group (p < .05). No significant differences in the activity of intestinal trypsin, lipase and amylase were observed among all groups (p > .05). In conclusion, this study indicated that BSFP at the level below 131 g/kg could be added in diet of juvenile mirror carp without any negative effect on growth performance and intestinal health. Dietary BSFP inclusion at low levels decreased the whole‐body lipid content and increased the antioxidant activity of juvenile mirror carp.     

Evaluation of some haemolymph biochemical properties and F‐cell prevalence in hepatopancreas of white leg shrimp (Litopenaeus vanammei) after fed diets containing apple cider vinegar and propionic acid

The present study aimed to evaluate the effect of dietary ACV (apple cider vinegar) and PA (propionic acid) supplementation on some biochemical properties of haemolymph and fibrillar cell (F‐cell) number in the hepatopancreas of shrimp. Five different diets supplemented with 0%, 1%, 2%, 4% ACV and 0.5% PA diets were fed to Litopenaeus vanammei. Some biochemical parameters of haemolymph (cortisol, alkaline phosphatase, albumin, urea, uric acid, creatinine, phosphorus, total hemocyte count) and prevalence of F‐cell in hepatopancreas were examined after 60 days feeding trial. The albumin concentration and total hemocyte count in 2% and 4% ACV and PA groups were significantly higher than those of other groups (p < .05). Moreover, the highest phosphorus concentration was detected in shrimp fed 4% ACV and PA experimental diets (p < .05). No significant differences in haemolymph cortisol, urea, uric acid, creatinine, alkaline phosphatase and tubule area were observed among the experimental groups (p > .05). 2% ACV group showed more F‐cell number in hepatopancreas than other groups while the reduction in F‐cell number was observed in the PA group. Based on these observations, the tested ACV and PA diets remarkably exhibited positive impacts on nutritional value and they may be a potential for adding to shrimp diet.     

Effect of natural pigment sources on colouration of red zebra cichlid (Maylandia estherae Konings, 1995)

This study was done to assess the effect of the colouration of the red zebra cichlid (Maylandia estherae), using beet root red (Beta vulgaris ruba) and henna (Lawsonia inermis). The fish with an average weight of 1.21 ± 0.69 g was placed in 12 aquariums. Fifteen fish were placed in each aquarium. The fish were fed with diets which were without carotenoid supplement (AK), containing astaxanthin (AA), beet root red (AP) and henna (AI). Except the AK, each group contained 50 mg/kg carotenoid additives. As a result of the experiment, total carotenoid values between groups were not significant (p > .05). The redness (a*) value of groups was significant (p < .05), and the values of brightness (L*) and yellowness (b*) were not significant (p > .05). Samples were kept in the freezer at ?20°C for 120 days to see whether the fish kept their colour. All groups decreased compared with the 50th day. This difference in groups except the AP was significant (p < .05). The FCR, SGR and weight gain% was not significant (p > .05). The survival was 97.28%, and it was the same in all groups. The study concluded that natural carotene sources with lower costs had similar effects with astaxanthin.     

Effect of dietary bile acids on growth,body composition,lipid metabolism and microbiota in grass carp (Ctenopharyngodon idella)

To investigate the effects of dietary bile acids (BA) on growth and metabolism of lipid in grass carp (Ctenopharyngodon idella, C. idella) at high dietary lipid level, a basal diet (50 g kg^–1 lipid, 5L group) was supplemented with 20 g kg^–1 soybean oil (70 g kg^–1 lipid, 7L group); then, 0.06 g/kg BA was added in 7L diet to form the third diet (7L+BA group). The 96 C. idella (69.86 ± 6.24 g) were divided into three groups (duplicate per group) and fed three diets, respectively, for 8 weeks, and then, growth and lipid metabolism were determined. Results showed that growth of fish in 7L+BA group was significantly higher than 5L and 7L groups. The lipid level in whole body, hepatopancreas and muscle of grass carp in 7L+BA group were significantly lower than 7L group. Relative expression of lipid catabolism genes in hepatopancreas and muscle of 7L+BA group was significantly higher than 5L group. The amount of microbiota in intestine of fish in 7L+BA group was significantly higher than the other two groups. The present results indicated that BA in 7L diet improved growth of fish by increasing protein synthesizing, decreasing lipid content in fish body and by regulating amount of microbiota in intestine of fish.     

Dietary exposure to melamine and cyanuric acid induced growth reduction,oxidative stress and pathological changes of hepatopancreas in Pacific white shrimp

This study examined the effects of dietary melamine (MEL) and cyanuric acid (CYA) singly and in combination on growth, nutrient utilization, immunological responses, oxidative stress, and histological changes in Pacific white shrimp. Seven experimental isonitrogenous (35%) and isolipidic (8%) diets were formulated, namely diet 1 (a control diet without MEL and CYA); diets 2–5 (with MEL and CYA at 2.5?+?2.5, 5?+?5, 7.5?+?7.5 and 10?+?10 g kg^?1 diet); diet 6 (with only MEL at 10 g kg^?1 diet) and diet 7 (with CYA alone at 10 g kg^?1 diet). The shrimp with initial body weight 2.37?±?0.02 g were fed with these diets for 10 weeks. The results indicate that all the diets with MEL and CYA singly or in combination had adverse effects on growth and nutrient utilization relative to the control diet (p?<?0.05). Total protease and trypsin activities were significantly lowered by all diets containing MEL (p?<?0.05). Haemolymph parameters, including total hemocyte count, phenoloxidase (PO) activity, respiratory burst, and lysozyme activity, were significantly decreased (p?<?0.05) in shrimp receiving MEL alone (10 g kg^?1 diet) and at high combination dosages (10?+?10 g kg^?1 diet). Moreover, MEL and CYA induced oxidative stress, damaged hepatopancreas, decreased antioxidant responses, increased lipid peroxidation, and caused abnormality of hepatocytes.

     

Fish protein hydrolysate in diets of turbot affects muscle fibre morphometry,and the expression of muscle growth‐related genes

The study was conducted to investigate the effects of fish protein hydrolysate (FPH) in diets for turbot on growth performance, muscle fibre morphometry, and the expression of muscle growth‐related genes. A control diet (FPH0) contained 0 g/kg FPH, and four experimental diets were formulated replacing fishmeal with FPH at levels of 45 (FPH4.5), 120 (FPH12), 180 (FPH18) and 300 (FPH30) g/kg. Fish fed the FPH12 and FPH18 diets had higher specific growth rate (SGR) than fish fed the FPH0 diet (p < .05), and a quadratic regression was found between SGR and dietary FPH level (p = .001, R² = .677). Cross‐section area (CSA) and the length of sarcomere in the FPH12 group increased compared with the control group (p < .05), and a quadratic regression was observed between CSA and dietary FPH level (p = .006, R² = .574) and between sarcomere length and dietary FPH level (p = .018, R² = .788). An appropriate level of FPH down‐regulated myostatin 2 gene expression and up‐regulated proliferating cell nuclear antigen gene expression, while the expression of myogenic regulatory factors was not affected by dietary treatments (p > .05). To conclude, an appropriate level of FPH may improve muscle growth by regulating the expression of muscle growth‐related genes, and muscle microstructure and ultrastructure.