Avian riboflavin deficiency causes reliably reproducible peripheral nerve demyelination and, with vitamin supplementation, rapid remyelination.

Riboflavin deficiency produces severe peripheral neve demyelination in young, rapidly growing chickens. While this naturally-occurring vitamin B2 deficiency can cause a debilitating peripheral neuropathy, and mortality, in poultry flocks, it can also be a useful experimental animal model to study the pathogenesis of reliably reproducible peripheral nerve demyelination. Moreover, restitution of normal riboflavin levels in deficient birds results in brisk remyelination. It is the only acquired, primary, demyelinating tomaculous neuropathy described to date in animals. The only other substance that causes peripheral nerve demyelination similar to avian riboflavin deficiency is tellurium and the pathologic features of the peripheral neuropathy produced by this developmental neurotoxin in weanling rats are also described.

Effects of riboflavin deficiency on the lipid metabolism of duck breeders and duck embryos.

This study aimed to evaluate the effects of dietary riboflavin deficiency (RD) on the lipid metabolism of duck breeders and duck embryos. A total of 40 female 40-wk-old white Pekin duck breeders were randomly divided into 2 groups, received either RD diet (1.48 mg riboflavin/kg) or control diet (16.48 mg riboflavin/kg, CON) for 14 wk. Each group consisted of 20 duck breeders (10 replicates per group, 2 birds per replicate), and all experiment birds were single-caged. At the end of the experiment, reproductive performance, hepatic riboflavin, hepatic flavin mononucleotide (FMN), hepatic flavin adenine dinucleotide (FAD), hepatic morphology, hepatic lipid contents, and hepatic protein expression of duck breeders and duck embryos were measured. The results showed that the RD had no effect on egg production and egg fertility but reduced egg hatchability, duck embryo weight, hepatic riboflavin, FMN, and FAD status compared to results obtained in the CON group (all P < 0.05). Livers from RD ducks presented enlarged lipid droplets, excessive accumulation of total lipids, triglycerides, and free fatty acids (all P < 0.05). In addition to excessive lipids accumulation, medium-chain specific acyl-CoA dehydrogenase expression was downregulated (P < 0.05), and short-chain specific acyl-CoA dehydrogenase expression was upregulated in maternal and embryonic livers (P < 0.05). RD did not affect maternal hepatic acyl-CoA dehydrogenase family member 9 (ACAD9) expression, but duck embryonic hepatic ACAD9 expression was reduced in the RD group (P < 0.05). Collectively, dietary RD conditioned lower egg hatchability and inhibited the development of duck embryos. Increased accumulation of lipids, both maternal and embryo, was impaired due to the reduced flavin protein expression, which caused inhibition of hepatic lipids utilization. These findings suggest that abnormal duck embryonic growth and low hatchability caused by RD might be associated with disorders of lipid metabolism in maternal as well as embryos.

Impact and consequences of dietary riboflavin deficiency treatment on flesh quality loss in on-growing grass carp (Ctenopharyngodon idella).

Fish is among the cheapest and most promising sources of animal protein. The main edible portion of fish is muscle. This study explored the impact of dietary riboflavin on fish flesh quality and showed the possible role of muscle antioxidant defense in flesh quality in relation to dietary riboflavin. On-growing grass carp (initial average weight of 275.82 ± 0.57 g) were fed diets containing graded levels of riboflavin (0.63, 1.95, 3.98, 6.02, 7.96, and 10.04 mg kg-1 diet) for eight weeks. The results indicated that compared with the optimal riboflavin levels (3.98 and/or 6.02 mg riboflavin per kg diet), riboflavin deficiency treatment (0.63 mg riboflavin per kg diet) significantly reduced the muscle nutrients, including the protein, lipid, flavor amino acid, and total essential amino acid contents. Furthermore, the muscle shear force, pH value, and hydroxyproline concentration were reduced, while the muscle cooking loss and lactic acid content increased (P < 0.05). Compared with optimal riboflavin levels, the riboflavin deficiency treatment increased the reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl contents, while riboflavin treatments of 3.98-10.04 mg riboflavin per kg diet showed the lowest ROS and MDA contents (P < 0.05). Compared with the optimal riboflavin levels, the riboflavin deficiency treatment decreased the activities of copper/zinc superoxide dismutase (CuZnSOD), glutathione reductase (GR), catalase (CAT), and glutathione peroxidase (GPx), and reduced the glutathione (GSH) content (P < 0.05). Furthermore, the relative mRNA levels of antioxidant enzymes, including CuZnSOD, CAT, GR and GPx, and antioxidant-related signaling molecules, including NF-E2-related factor 2 (Nrf2) and casein kinase 2, were down-regulated, while those of Kelch-like ECH-associated protein 1b were up-regulated (P < 0.05). Collectively, the present study indicates that riboflavin deficiency treatment reduces the flesh quality, partly due to inhibition of the antioxidant defense through the Nrf2 signaling pathway, while optimal riboflavin levels reverse these negative effects.

Transcriptional profiling of liver in riboflavin-deficient chicken embryos explains impaired lipid utilization, energy depletion, massive hemorrhaging, and delayed feathering.

BACKGROUND: A strain of Leghorn chickens (rd/rd), unable to produce a functional riboflavin-binding protein, lays riboflavin-deficient eggs, in which all embryos suddenly die at mid-incubation (days 13-15). This malady, caused by riboflavin deficiency, leads to excessive lipid accumulation in liver, impaired ß-oxidation of lipid, and severe hypoglycemia prior to death. We have used high-density chicken microarrays for time-course transcriptional scans of liver in chicken embryos between days 9-15 during this riboflavin-deficiency-induced metabolic catastrophe. For comparison, half of rd/rd embryos (n = 16) were rescued from this calamity by injection of riboflavin just prior to incubation of fertile eggs from rd/rd hens. RESULTS: No significant differences were found between hepatic transcriptomes of riboflavin-deficient and riboflavin-rescued embryos at the first two ages (days 9 and 11). Overall, we found a 3.2-fold increase in the number of differentially expressed hepatic genes between day 13 (231 genes) and day 15 (734 genes). Higher expression of genes encoding the chicken flavoproteome was more evident in rescued- (15 genes) than in deficient-embryos (4 genes) at day 15. Diminished activity of flavin-dependent enzymes in riboflavin-deficient embryos blocks catabolism of yolk lipids, which normally serves as the predominant source of energy required for embryonic development. CONCLUSIONS: Riboflavin deficiency in mid-stage embryos leads to reduced expression of numerous genes controlling critical functions, including ß-oxidation of lipids, blood coagulation and feathering. Surprisingly, reduced expression of feather keratin 1 was found in liver of riboflavin-deficient embryos at e15, which could be related to their delayed feathering and sparse clubbed down. A large number of genes are expressed at higher levels in liver of riboflavin-deficient embryos; these up-regulated genes control lipid storage/transport, gluconeogenesis, ketogenesis, protein catabolism/ubiquitination and cell death.

Intestinal immune function, antioxidant status and tight junction proteins mRNA expression in young grass carp (Ctenopharyngodon idella) fed riboflavin deficient diet.

This study investigated the effects of riboflavin on intestinal immunity, tight junctions and antioxidant status of young grass carp (Ctenopharyngodon idella). Fish were fed diets containing graded levels of riboflavin (0.63-10.04 mg/kg diet) for 8 weeks. The study indicated that riboflavin deficiency decreased lysozyme, acid phosphatase, copper/zinc superoxide dismutase, glutathione reductase and glutathione peroxidase activities, and contents of complement component 3 and reduced glutathione in the intestine of fish (P < 0.05). Meanwhile, riboflavin deficiency increased reactive oxygen species, malondialdehyde and protein carbonyl contents and catalase activity (P < 0.05) in the intestine of fish. Furthermore, real-time polymerase chain reaction analysis was used to investigate mRNA expression patterns and found that the mRNA levels of interleukin 10 and transforming growth factor ß1, Occludin, zonula occludens 1, Claudin-b and Claudin-c, inhibitor protein κBα, target of rapamycin, ribosomal S6 protein kinase 1 and NF-E2-related factor 2, copper/zinc superoxide dismutase, glutathione peroxidase and glutathione reductase were decreased (P < 0.05) in the intestine of fish fed riboflavin-deficient diet. Conversely, the mRNA levels of tumor necrosis factor α, interleukin 1ß, interleukin 8, nuclear factor kappa B p65, Ikappa B kinase ß, Ikappa B kinase γ, Kelch-like-ECH-associated protein 1b, p38 mitogen-activated protein kinase, myosin light chain kinase and Claudin-12 were increased (P < 0.05) in the intestine of fish fed riboflavin-deficient diet. In conclusion, riboflavin deficiency decreased immunity and structural integrity of fish intestine. The optimum riboflavin level for intestinal acid phosphatase activity of young grass carp was estimated to be 6.65 mg/kg diet.

Dietary riboflavin deficiency decreases immunity and antioxidant capacity, and changes tight junction proteins and related signaling molecules mRNA expression in the gills of young grass carp (Ctenopharyngodon idella).

This study investigated the effects of dietary riboflavin on the growth, gill immunity, tight junction proteins, antioxidant system and related signaling molecules mRNA expression of young grass carp (Ctenopharyngodon idella). Fish were fed six diets containing graded levels of riboflavin (0.63-10.04 mg/kg diet) for 8 weeks. The study indicated that riboflavin deficiency decreased lysozyme and acid phosphatase activities, and complement component 3 content in the gills of fish (P < 0.05). Moreover, riboflavin deficiency caused oxidative damage, which might be partly due to decrease copper, zinc superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione-S-transferase activities and reduced glutathione content in the gills of fish (P < 0.05). Furthermore, the relative mRNA levels of antimicrobial peptides (liver expressed antimicrobial peptide 2 and Hepcidin), anti-inflammatory cytokines (interleukin 10 and transforming growth factor ß1), tight junction proteins (Occludin, zonula occludens 1, Claudin-c and Claudin-3), signaling molecules (inhibitor of κBα, target of rapamycin and NF-E2-related factor 2) and antioxidant enzymes (copper, zinc superoxide dismutase and glutathione reductase) were significantly decreased (P < 0.05) in the gills of fish fed riboflavin-deficient diet. Conversely, the mRNA levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 8, interferon γ2, and interleukin 1ß), signaling molecules (nuclear factor kappa B p65, IκB kinase ß, IκB kinase γ, Kelch-like-ECH-associated protein 1b and myosin light chain kinase) and tight junction protein Claudin-12 were significantly increased (P < 0.05) in the gills of fish fed riboflavin-deficient diet. In addition, this study indicated for the first time that young fish fed a riboflavin-deficient diet exhibited anorexia and poor growth. In conclusion, riboflavin deficiency decreased growth and gill immunity, impaired gill antioxidant system, as well as regulated mRNA expression of gill tight junction proteins and related signaling molecules of fish. Based on percent weight gain, gill lysozyme activity and reduced glutathione content, the dietary riboflavin requirements for young grass carp (275-722 g) were estimated to be 5.85, 7.39 and 6.34 mg/kg diet, respectively.

Selective vulnerability of peripheral nerves in avian riboflavin deficiency demyelinating polyneuropathy.

Riboflavin (vitamin B2) deficiency in young chickens produces a demyelinating peripheral neuropathy. In this study, day-old broiler meat chickens were fed a riboflavin-deficient diet (1.8 mg/kg) and killed on posthatch days 6, 11, 16, 21, and 31, while control chickens were given a conventional diet containing 5.0 mg/kg riboflavin. Pathologic changes were found in sciatic, cervical, and lumbar spinal nerves of riboflavin-deficient chickens from day 11 onwards, characterized by endoneurial oedema, hypertrophic Schwann cells, tomacula (redundant myelin swellings), demyelination/remyelination, lipid deposition, and fibroblastic onion bulb formation. Similar changes were also found in large and medium intramuscular nerves, although they were less severe in the latter. However, by contrast, ventral and dorsal spinal nerve roots, distal intramuscular nerves, and subcutaneous nerves were normal at all time points examined. These findings demonstrate, for the first time, that riboflavin deficiency in young, rapidly growing chickens produces selective injury to peripheral nerve trunks, with relative sparing of spinal nerve roots and distal nerve branches to muscle and skin. These novel findings suggest that the response of Schwann cells in peripheral nerves with riboflavin deficiency differs because either there are subsets of these cells in, or there is variability in access of nutrients to, different sites within the nerves.

Novel fibroblastic onion bulbs in a demyelinating avian peripheral neuropathy produced by riboflavin deficiency.

The finding of novel fibroblastic onion bulb-like structures in peripheral nerves is reported for the first time in avian riboflavin deficiency. Day old broiler meat chickens were fed a riboflavin deficient diet (1.8 mg/kg) and were killed on postnatal days 6, 11, 16, 21 and 31, whereas control chickens were fed a conventional diet containing 5.0 mg/kg riboflavin. The fibroblastic onion bulb-like structures were found in sciatic and brachial nerves from day 11 onwards and consisted of long cytoplasmic processes of hypertrophied fibroblasts surrounding demyelinated, remyelinated and normally myelinated axons. The fibroblast cytoplasmic processes often enveloped more than one nerve fibre to produce a unique compound-like onion bulb structure. These onion bulb-like structures occurred early in the course of segmental demyelination at the same time as tomacula formation and became increasingly more prominent in the later stages of demyelination and remyelination. The molecular basis of formation of these unique structures requires further study as to the basis of the attraction of the fibroblast processes to nerve fibres associated with myelinating Schwann cells. The model may also be useful in investigating the role of endoneurial fibroblasts in endoneurial fibrosis as the early fibroblastic response in the onion bulbs is distinct from the more usual fibroblastic deposition of collagen in end-stage peripheral nerve disease.

Avian riboflavin deficiency: an acquired tomaculous neuropathy.

The finding of tomacula, focal areas of sausage-shaped hypermyelination in peripheral nerves, is reported for the first time in avian riboflavin deficiency. Day-old, meat-type chickens were fed a riboflavin-deficient diet (1.8 mg/kg) and were killed on postnatal days 6, 11, 16, and 21, while control chickens were fed a conventional diet containing 5.0 mg/kg riboflavin. Tomacula were found in sciatic and brachial nerves from day 11 onward, became more frequent and prominent with increasing time, and preceded the onset of segmental demyelination.

Early paranodal myelin swellings (tomacula) in an avian riboflavin deficiency model of demyelinating neuropathy.

INTRODUCTION: Disruption of the complex architectural and molecular organization of the paranodal region of myelinated peripheral nerve fiber may initiate the evolving time dependent process of segmental demyelination. In support of this notion was the finding of focal paranodal myelin swellings (tomacula) due to redundant folding of myelin sheaths, early in the time course of an avian riboflavin deficiency model of demyelinating neuropathy. METHODS: Newborn broiler meat chickens were maintained either on a routine diet containing 5.0 mg/kg riboflavin (control group) or a riboflavin-deficient diet containing 1.8 mg/kg riboflavin. Riboflavin concentrations in the liver were measured at postnatal day 11. Peripheral nerves were morphologically examined at days 6, 11, 16 and 21 using light and electron microscopy and teased nerve fiber techniques. RESULTS: Riboflavin-deficient chickens showed signs of a neuropathy from days 8 and pathological examination of peripheral nerves revealed a demyelinating neuropathy with paranodal tomacula formation starting on day 11. Paranodal tomacula consisted of redundant myelin infoldings or outfoldings, increased in size and frequency after day 11. After day 16, the paranodal swellings showed prominent degenerative changes accompanied by an increased frequency of myelinated fibers showing demyelination. CONCLUSION: Tomacula due to redundant myelin folds are generally considered a remyelination phenomenon, yet in this avian riboflavin deficiency model of demyelination, the paranodal tomacula occurred early in the course of demyelination.

Peripheral neuropathy of dietary riboflavin deficiency in racing pigeons.

An occurrence of peripheral neuropathy in nine 14- to 55-day-old racing pigeons was documented. The predominant clinical signs were diarrhea, and leg and wing paralysis. Grossly, there was discoloration and swelling of all the peripheral nerve trunks. Microscopic lesions comprising swelling, fragmentation and demyelination of myelin sheaths, and proliferation of Schwann cells, were seen in the peripheral nerves of all birds examined. These changes were associated with moderate to severe swelling, fragmentation, atrophy and loss of axons. The peripheral nerve lesions in these cases were similar to those of dietary riboflavin deficiency in chickens. An analysis of the diet given to the pigeons indicated that the riboflavin concentration was only 0.9 mg/kg feed.

Defective Down syndrome in chicks is not caused by riboflavin deficiency in breeders.

1. Riboflavin-deficient practical and semi-purified diets were fed to broiler breeder hens. 2. Yolk and albumen riboflavin concentrations were depressed by the deficient diets and cases of curled toes were seen in the chicks. 3. The few instances of defective down did not resemble cases seen in field outbreaks of a condition involving depressed hatchability and a defective down syndrome. 4. It is concluded from observations in this experiment and in the field outbreaks that there is a condition in breeding chickens causing depressed hatchability and defective down that is not attributable to riboflavin deficiency.

Riboflavin requirement of broiler chicks fed a corn-soybean diet.

Two 21-day battery experiments were conducted with day-old broiler chicks fed a corn-soybean meal diet to re-evaluate the riboflavin requirement for optimum performance. In each experiment a total of 384 birds was assigned to six dietary treatments and given 0, 0.9, 2.0, 2.8, 3.6, or 4.4 mg of supplemental riboflavin/kg of feed. The corn-soybean meal diet was formulated to contain 22% CP and 3,200 kcal of ME/kg of feed. The analyzed riboflavin content of the unsupplemented diet was 2.6 mg/kg. A severe leg paralysis (inability to walk and to move one or both legs) was first observed between 10 to 12 days of age in birds receiving no supplemental riboflavin. Besides leg paralysis, high mortality and poor growth were the main signs of riboflavin deficiency observed. A minimum requirement for growth of 3.6 mg of riboflavin/kg of feed was determined using the broken-line technique. However, in order to prevent signs of leg paralysis, the minimum requirement of 4.6 mg riboflavin/kg of feed was suggested. Based on the maximum feed intake (834 g) observed for birds to reach maximum body weight (593 g) at 21 days of age, a minimum intake of approximately 6.5 micrograms riboflavin/g body weight was needed for maximum performance.

Peripheral neuropathy associated with dietary riboflavin deficiency in the chicken. I. Light microscopic study.

Chickens fed a riboflavin-deficient diet from hatching had leg weakness and paralysis as early as 12 days of age. Signs worsened through day 16; after 35 days, recovery was evident. Sciatic nerves from affected chickens were enlarged. Significant microscopic lesions were confined to peripheral nerves and included tissue separation (suggesting interstitial edema), Schwann cell swelling, perivascular leukocytic infiltration, and segmental demyelination accompanied by accumulation of osmiophilic debris in Schwann cell cytoplasm. Axon degeneration was present, but was not a primary lesion. Acid phosphatase enzyme activity of Schwann cells was increased in affected nerves. These results demonstrate that dietary riboflavin deficiency causes a demyelinating peripheral neuropathy in young, rapidly growing chickens.

Peripheral neuropathy of dietary riboflavin deficiency in chickens.

A strain of rapidly growing meat-type chickens was fed a diet deficient in riboflavin from 1-40 days of age. Diminished growth rate, progressive gait abnormality and reluctance to move were noted beginning on day 8. Neurologic abnormalities were related to peripheral neuropathy characterized by Schwann cell hypertrophy and degeneration with cytoplasmic lipid droplets' and segmental demyelination. Lesions were initially detected on day 10, and in concert with clinical signs became more profound between days 14 and 21. Sequestration of myelin debris within Schwann cells was common. Other features of the neuropathy included the presence of endoneurial edema and axonal degeneration involving small numbers of fibers. Remyelination of peripheral nerve fibers in birds on the deficient diet was occasionally seen on day 10, became progressively more prominent, and was marked by day 37. There was an associated, variable but incomplete, clinical improvement evident in later stages of the study. Liver concentrations of riboflavin in deficient birds were significantly reduced on day 13 but not on day 26. This neuropathy may be related to diminished tissue levels of the riboflavin-based coenzymes flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN) leading to reduced cellular energy levels and profoundly affecting Schwann cells at some critical point in growth.

Nutritional deficiency anemias in nonhuman primates.

The purpose of this report is to review past studies in which anemias, occurred spontaneously in nonhuman primates due to feeding inadequate diets or were induced by feeding diets deficient in a nutrient. Included is a review of anemias induced by deficiencies of iron, niacin, pyridoxine, pantothenic acid, protein, riboflavin, cyanocobalamin, folic acid, ascorbic acid and alpha-tocopherol. The anemia induced by deficiency of each nutrient is discussed with emphasis on the major clinical signs as well as peripheral blood and bone marrow pathology. Results of supplementation of the diet following induction of deficiency states are discussed also. Whenever applicable, a discussion is included of the use of nonhuman primates as animal models for studies simulating parallel nutritional deficiencies in man.

Involvement of water-soluble vitamins in diseases of swine.

The various roles of the water-soluble vitamins (including choline and vitamin C) in diseases of swine are outlined. The most important role is in the prevention of deficiency disease; another important role is in relation to the immune response. Deficiency signs relating to each vitamin are described and the metabolism of each vitamin is outlined. Recent estimates of requirements are set out, together with suggestions on supplementation of practical diets for swine.

Biomicroscopic and histologic pathology of the eye in riboflavin deficient rainbow trout (Salmogairdneri).

Quadruplicate lots of 35 fingerling rainbow trout (mean body weight 11.2 g) were fed for 20 weeks a complete diet or one deficient in riboflavin. Five fish from each low were examined for ocular lesions each week. At the end of the experiment eyes of three fish form each lot were examined histologically. Beginning with the 14th week, epithelial changes were present in the cornea; they appeared as thickening of the stroma and endothelium and resulted in clouding. Opacity of the lens originated in the posterior subcapsular cortex and progressed to lens fiber autolysis and liquefaction.

Embryonic death in mink due to riboflavin deficiency.

Riboflavin deficiency has been produced experimentally in mink by adding to the feed 10--20 mg galactoflavin--anti-B2 vitamin--per animal daily during the pregnancy. The experiment included 44 standard female mink (Table II). Riboflavin deficiency led to embryonic death; none of the mated females in the deficient group delivered kits. At the expected time of birth, six of the females were subjected to uterectomy or investigative laparatomy, which showed remnants of embryos that were mostly decomposed (Figs. 1 and 2). In a parallel group, four of six females on the same diet with the same doses of galactoflavin, but with 50--100 mg riboflavin added daily, delivered normal litters (Table II). Five standard male mink were placed on the same experimental diet supplemented with 30 mg galactoflavin per animal daily from 15/12 to 18/3. The fertility of these males was not influenced in a negative way.

Riboflavin requirement of channel catfish fingerlings.

Channel catfish fingerlings were fed purified diets containing five levels (0, 20, 40, 80, and 120 mg/kg) and six levels (0, 3, 8, 20, 40, and 80 mg/kg) of riboflavin in separate 20 and 12 week feeding studies. The dietary riboflavin level required to provide maximal growth in channel catfish fingerlings was found to be approximately 9 mg/kg of diet. All fish fed unsupplemented diets (riboflavin content less than 0.1 mg/kg diet) demonstrated a short body dwarfism which was the result of arrested longitudinal growth of the vertebrae. This abnormality did not occur in groups fed 3 mg/kg or higher levels of riboflavin. No histological abnormalities were recognized in the liver, kidney, lateral muscle, gastrointestinal tract, gills, or bones of deficient fish.