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DSM in Animal Nutrition & Health


Properties and Metabolism

Riboflavin exists in three forms in nature. The forms are free dinucleotide riboflavin and the two coenzyme derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Riboflavin is an odorless, bitter, orange-yellow compound that melts at about 280°C. The molecular structure of riboflavin is shown in Illus. 8-1. Riboflavin is only slightly soluble in water, but readily soluble in dilute basic or strong acidic solutions. It is quite stable to heat in neutral and acid but not alkaline solutions; very little is lost in cooking. Aqueous solutions are unstable to visible and ultraviolet light; instability is increased by heat and alkalinity. Riboflavin plays a key role in problems related to light sensitivity and photodegradation of milk and dairy products. Both light and oxygen have been found to induce riboflavin degradation (Becker et al., 2003). When dry, riboflavin is appreciably less affected by light.


Illustration 8-1


Riboflavin covalently bound to protein is released by proteolytic digestion. Phosphorylated forms (FAD, FMN) of riboflavin are hydrolyzed by phosphatases in the upper gastrointestinal tract to free the vitamin for absorption. Free riboflavin is absorbed by mucosal cells via an active saturable transport system in all parts of the small intestine. Transport of flavin by blood plasma is known to involve both loose association with albumin and tight associations with some globulins (McCormick, 1990). A genetically controlled riboflavin binding protein is present in serum and eggs. There is a hereditary recessive disorder in chickens, renal riboflavinuria, in which the riboflavin-binding protein is absent (White, 1996). Eggs become riboflavin-deficient and embryos generally do not survive beyond the fourteenth day of incubation (Clagett, 1971; Rivlin, 2006). Also, if riboflavin-binding protein is in excess it can diminish riboflavin availability to the chicken embryo (Lee and White, 1996). Presumably the lack of the specific vitamin transport protein prevents adequate transfer of dietary riboflavin to the developing fetus and riboflavin losses occur via maternal urine. In addition to poultry, specific binding proteins have been found in serum from pregnant cows and rats, human fetal blood, and uterine secretions in the pig. Hepatic cells from deficient animals have a relatively greater maximal absorption uptake of riboflavin (Rose et al., 1986). Hepatic cell riboflavin absorption occurs via facilitated diffusion. In mucosal cells, riboflavin is phosphorylated to FMN by the enzyme flavokinase (Rivlin, 2006; 2007). The FMN then enters the portal system, where it is bound to plasma albumin, transported to the liver, and there converted to FAD. Rivlin (2006) suggested there may be physiological mechanisms in pregnancy that facilitate transfer of riboflavin from maternal stores to the fetus in a manner that is fundamentally similar to that in the laying hen. Riboflavin is efficiently transferred to the fetus. Animals do not appear to have the ability to store appreciable amounts of riboflavin, with the liver, kidneys and heart having the greatest concentrations. The liver, the major site of storage, contains about one-third of the total body riboflavin. Intakes of riboflavin above current needs are rapidly excreted in urine, primarily as free riboflavin. Minor quantities of absorbed riboflavin are excreted in feces, bile and sweat.



Riboflavin is required as part of many enzymes involved with metabolism of carbohydrate, fat and protein. Riboflavin in these coenzyme forms (FMN and FAD) are called flavoproteins and act as intermediaries in the transfer of electrons in biological oxidation-reduction reactions. If levels of riboflavin are low, the respiration process becomes less efficient, and 10% to 15% more feed is required to meet energy needs (Christensen, 1983). The enzymes that function aerobically are called oxidases, and those that function anaerobically are called dehydrogenases. The general function is in oxidation of substrate and generation of energy (i.e., ATP). By involvement in the hydrogen transport system (cytochrome system), flavoproteins function by accepting and passing on hydrogen. Flavoproteins assist in the generation of ATP. Collectively, the flavoproteins show great versatility in accepting and transferring one or two electrons with a range of potentials. Many flavoproteins contain a metal (e.g., iron, molybdenum, copper, zinc), and the combination of the flavin and metal ion is often involved in the adjustments of these enzymes in transfers between single- and double-electron donors. Xanthine oxidase contains the metals molybdenum and iron. It converts hypoxanthine to xanthine and the latter to uric acid. It also reacts with aldehydes to form acids, including the conversion of retinal (vitamin A aldehyde) to retinoic acid. Flavoproteins may accept a hydrogen ion (H+) directly from the substrate, thus catalyzing oxidation of the substrate, or it may catalyze the oxidation of some other enzyme by accepting a hydrogen ion from it, for example, from the niacin-containing coenzymes, nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH). More than 100 flavoprotein enzymes may be arbitrarily classified into three groups:

  1. NADH2 dehydrogenases–reduced pyridine nucleotide is a substrate, and the electron acceptor is either a member of the cytochrome system or some other acceptor besides oxygen.
  2. Dehydrogenases–accept electrons directly from the substrate and can pass them to one of the cytochromes.
  3. Oxidases (true)–accept electrons from the substrate and pass them directly to oxygen (O2 is reduced to H2O2); they cannot reduce cytochromes.


Riboflavin functions in flavoprotein-enzyme systems to help regulate cellular metabolism, although they are also specifically involved in metabolism of carbohydrates. Riboflavin is also an essential factor in amino acid metabolism as part of amino acid oxidases. These enzymes oxidize amino acids, which results in the decomposition of the amino acids, yielding ammonia and a keto acid. Distinct oxidized D-amino acids (prosthetic group FAD) and L-amino acids (prosthetic group FMN) are produced. In addition, riboflavin plays a role in fat metabolism (Rivlin, 2006), and an FAD flavoprotein is an important link in fatty acid oxidation. This includes the acyl-coenzyme A dehydrogenases, which are necessary for the stepwise degradation of fatty acids. An FMN flavoprotein is required for synthesis of fatty acids from acetate. Thus flavoproteins are necessary for both degradation and synthesis of fatty acids.

Riboflavin and other vitamins play an important role in skin development, tensile strength and healing rates (Ward, 1993). A deficiency in riboflavin can slow epithelialization of wounds by 4-5 days (Lakshmi et al., 1989), reduce collagen content by 25%, and decrease tensile strength of wounds by 45%. Riboflavin deficiency can increase skin homocysteine by 2-4 fold, which ultimately impairs the cross-link formation of collagen (Lakshmi et al, 1990). Marginal field deficiencies of riboflavin could increase skin tears and cause longer healing times, and ultimately increase costly downgrades.

Among the enzymes that require riboflavin is the FMN-dependent oxidase responsible for conversion of phosphorylated pyridoxine (vitamin B6) to a functional coenzyme. Riboflavin deficiency also results in a decrease in the conversion of the vitamin B6 coenzyme pyridoxal phosphate to the main vitamin B6 urinary excretory product of 4-pyridoxic acid (Kodentsova et al., 1993). Riboflavin deficiency has an effect on iron metabolism, with less iron absorbed and an increased rate of iron loss due to an accelerated rate of small intestinal epithelial turnover (Powers et al., 1991, 1993).

Riboflavin co-enzyme Q10, and niacin are associated with poly (ADP-ribose) which functions in post-translational modification of nuclear proteins. The poly ADP-ribosylated proteins function in DNA repair, replication and cell differentiation (Premkumar et al., 2008).



Riboflavin requirements vary with heredity, growth, environment, age, activity, health, other dietary components and synthesis by host. Poultry species have a requirement between 1.8 and 4 mg per kg (0.45 and 1.8 mg per lb) of diet (NRC 1994). The NRC (1994) requirement for broiler chicks is reported as 3.6 mg per kg (1.6 mg per lb) of feed. However, Ruiz and Harms (1988) suggest that to prevent signs of leg paralysis in broilers fed a corn-soybean diet, the minimum requirement of 4.6 mg riboflavin per kg (2.0 mg per lb) of feed is needed. Olkowski and Classen (1998) likewise suggest a higher than NRC requirement for broilers at a level of 5 mg per kg (2.27 mg per lb). They suggest that the current recommended allowance of 3.6 mg per kg (1.6 mg per lb) is not sufficient for (1.59 mg per lb) modern breeds of broiler chickens. Ruiz and Harms (1989) also reported that 3.5 mg per kg of added riboflavin was needed to optimize the growth of turkey poults fed a corn-soybean meal diet. However, it took additional riboflavin to eliminate signs of deficiency—poor feathering and paralysis of one or both legs. Where sufficient data are available, studies indicate that riboflavin requirements decline with animal maturity and increase for reproductive activity. Chicks receiving diets only partially deficient in riboflavin may recover spontaneously, indicating that the requirement rapidly decreased with age (Scott et al., 1982). However, Deyhim et al.(1992a) reported that 3.6 ppm of dietary riboflavin for growing broilers was satisfactory through four weeks, but that benefits were obtained by exceeding the 3.6 ppm recommendation through eight weeks. Increased dietary fat or protein increases requirements for riboflavin in rats and chickens. It was assumed that high urinary riboflavin excretion during periods of negative nitrogen balance for a number of species was a reflection of impaired riboflavin utilization or retention. However, Turkki and Holtzapple (1982) suggested, in studies with rats, that the effect of protein on riboflavin requirement is related to rate of growth and not to protein intake per se. Microbial synthesis of riboflavin has been shown to occur in the gastrointestinal tract of a number of animal species and thus affects requirements. However, utilization of this endogenously synthesized riboflavin varies from species to species. Within a single species, utilization depends on diet composition and incidence of coprophagy. Carbohydrates such as starch, cellulose, or lactose are absorbed slowly and therefore exposed for longer times to the intestinal bacteria, resulting in an increased riboflavin synthesis. Dextrose, fat or protein as chief dietary constituents decrease intestinal production, thereby increasing dietary riboflavin requirements. Antibiotics, such as tetracycline, penicillin, and streptomycin, reduce the requirements of several animal species for riboflavin or might stimulate microorganisms that synthesize riboflavin. They may inhibit microorganisms in the gut that compete for riboflavin. Alkaline dietary pH appears to increase the level of riboflavin required in chick diets (Donaldson, 1986).

Temperature extremes apparently have an effect on riboflavin requirement. According to studies with pigs, riboflavin requirement is substantially higher at low than at high environmental temperatures (Seymour et al., 1968). At environmental temperatures below 11°C (52°F), feed required per unit of gain increased, and rate of gain decreased with decreasing temperature in pigs fed minimum levels of riboflavin. On the contrary, Onwudike and Adegbola (1984) report riboflavin requirements to be higher for chickens in a tropical environment. A dietary level of 4.1 mg per kg (1.9 mg per lb) riboflavin was adequate for egg laying with 5.7 mg per kg (2.6 mg per lb) for hatchability, compared to 2.1 and 3.6 mg per kg (1.0 and 1.7 mg per lb), respectively, for the NRC (1994) requirement. When exposed to chronic heat stress, the riboflavin requirement of broilers was in excess of 7 mg per kg (3.2 mg per lb) to prevent deficiency symptoms (Whitehead, 2000). Other factors which can influence riboflavin requirements have been reviewed (Ward, 1993). When requirement comparisons between animals residing at different environmental temperatures are calculated, total feed intakes must be considered.



Riboflavin is synthesized by green plants, yeast, fungi and some bacteria. Rapidly growing green leafy vegetables and forages, particularly alfalfa, are good sources, and the leaves have the highest content of the vitamin. Cereals and their by-products have a rather low riboflavin content, in contrast to their high thiamin content. Oilseed meals are fair sources, whereas grains should not be relied on as the sole source of riboflavin.

Riboflavin is one of the more stable vitamins, but can be readily destroyed by ultraviolet (UV) rays or sunlight. Appreciable amounts may be lost upon exposure to light. Up to one half the riboflavin content is lost in cooking eggs and pork chops in light and most of the vitamin in milk stored in clear glass bottles is also destroyed (McDowell, 2000). The riboflavin content of the milk of cows or goats is many times higher than that of their feed because of rumen synthesis. Human milk contains about 0.5 mg riboflavin per liter, while the content of cow’s milk is three times as high (i.e., 1.7 mg per liter). Milling of rice and wheat results in considerable loss of riboflavin since most of the vitamin is in the germ and bran, which are removed during this process. About one-half the riboflavin content is lost when rice is milled. Whole wheat flour contains about two-thirds more riboflavin than white flour (McDowell. 2000).

Riboflavin is more bioavailable from animal products than plant sources. Flavin complexes in plants are more stable to digestion and thus less digestible than animal sources. Singh and Deodhar (1992) concluded that milk factor(s) enhanced intestinal uptake of riboflavin in rat intestine. Bioavailability of riboflavin was less for chicks fed corn-soybean than for those fed purified amino acid diets; riboflavin bioavailability in the corn-soybean meal diet was 59.1% (Chung and Baker, 1990). Fermentation significantly increases the proportions of riboflavin present in the free form, resulting in a greater bioavailability of the vitamin in curd than in milk.

Riboflavin is commercially available as a crystalline compound produced by chemical synthesis or fermentation. It is available to the feed, food and pharmaceutical industries as a high-potency, USP or feed-grade crystalline powder, spray-dried powders, or dry dilutions. Riboflavin 5’-phosphate sodium salt is available for applications requiring a water-dispersible source of riboflavin. High-potency, USP or feed-grade crystalline powders are electrostatic, hygroscopic and dusty, and thus do not flow freely and show poor distribution in feeds. In contrast, dilutions of riboflavin (such as spray-dried powders or other concentrated, dry dilution products) or inclusion in a premix reduce electrostaticity and hygroscopicity for better flowability and distribution in feeds (Adams, 1978).



Only a few of the feedstuffs fed to poultry contain enough riboflavin to meet the requirements of young growing poultry (McDowell, 2000). A decreased rate of growth and lower feed efficiency are common signs of riboflavin deficiency in all species affected. Typical clinical signs often involve the eye, skin and nervous system. The most critical requirements for riboflavin are those exhibited by the young chick and the breeder hen. The characteristic sign of riboflavin deficiency in the chick is “curled-toe” paralysis. It does not develop, however, in a total riboflavin-free diet or when the deficiency is very marked, because the chicks die before it appears. Chicks are first noted to be walking on their hocks with their toes curled inward (Illus. 8-2 and 8-3). Deficient chicks do not move about, except when forced to do so, and their toes are curled inward both when walking and when resting on their hocks (Scott et al., 1982). Legs become paralyzed, but the birds may otherwise appear normal. An approximately 10% incidence of curled-toe paralysis was observed among birds fed a diet with no added riboflavin (1.5 mg per kg or 0.68 mg per lb) (Bootwalla and Harms, 1990).


Illustration 8-2: Riboflavin Deficiency


Left photo courtesy of M.L. Scott, Cornell University


Illustration 8-3: Riboflavin Deficiency in Poultry


Two-week-old chick with symptome of vitamin B2 deficiency: curled toe, paralysis of extremities.


Changes in the sciatic nerves produce the curled-toe paralysis in growing chicks. There is a marked enlargement of sciatic and brachial nerve sheaths with sciatic nerves reaching a diameter four to six times normal size. Histologic examinations of affected nerves show definite degenerative changes in myelin sheaths, which when severe may pinch the nerve, producing a permanent stimulus that causes the curled-toe paralysis (Scott et al., 1982). When the curled-toe deformity is long standing, irreparable damage has occurred in the sciatic nerve and administration of riboflavin no longer cures the condition. Retarded growth, splay and hock-resting postures and leg paralysis, rather than curled-toe paralysis, have been reported in some studies as the predominant signs of riboflavin deficiency in chicks (Ruiz and Harms, 1988; Chung and Baker, 1990). Turkeypoults and pheasants exhibit clinical signs similar to those of the chick, whereas ducks and geese are more likely to have a bowing of the legs in conjunction with perosis (NRC, 1994). In the poult, a dermatitis appears in about eight days; the vent becomes encrusted, inflamed and excoriated; growth is retarded or completely stopped by about the seventeenth day; and deaths begin to occur about the twenty-first day. However, when poults were fed a corn-soy diet analyzed to contain 2.7 mg per kg (1.2 mg per lb) of naturally occurring riboflavin, the only signs exhibited were a paralysis of one or both legs, poor feathering, poor growth and, finally, mortality (Ruiz and Harms, 1989). In the duckling, diarrhea and cessation of growth are generally associated with riboflavin deficiency. Other signs of riboflavin deficiency are growth retardation (Illus. 8-4 and 8-5), diarrhea after 8 to 10 days, and high mortality after about three weeks. When chicks are fed a diet deficient in riboflavin, their appetite is fairly good but they grow very slowly and become weak and emaciated. There is no apparent impairment of feather growth; on the contrary, main wing feathers often appear to be disproportionally long. Increased hematocrit, increased mean corpuscular volume, decreased mean hemoglobin concentration and a marked heterophil leucocytosis appeared in the chick prior to neurological manifestations (NRC, 1994). Perhaps as a result of mitochondrial dysfunction, riboflavin-deficient rats require 15% to 20% more energy intake than do control animals to maintain the same body weight (Rivlin, 2007).


Illustration 8-4: Riboflavin Deficiency


Courtesy of N. Ruiz and R. Harms, University of Florida


Illustration 8-5: Riboflavin Deficiency


A & B courtesy of N. Ruiz and R. Harms, University of Florida


In laying poultry, hatchability of incubated eggs is first reduced and subsequently egg production is decreased, roughly in proportion to degree of deficiency. Embryonic mortality has two typical peaks (the fourth and twentieth days of incubation) and often an intermediate peak on the fourteenth day. Embryos that fail to hatch from eggs of hens receiving low-riboflavin diets are dwarfed and exhibit pronounced micromelia; some embryos are edematous. The down fails to emerge properly, thus resulting in a typical abnormality termed “clubbed down,” which is most common in neck areas and around the vent. The nervous systems of these embryos show degenerative changes much like those described in thiamin-deficient chicks. When dietary riboflavin provided to breeder hens was decreased from 9.7 to 1.7 mg per kg (4.4 to 0.77 mg per lb) embryo mortality increased to 83.3% and hatchability to 3.1%; however, decreasing riboflavin from 9.7 to 7.0 or 4.4 mg per kg (4.4 to 3.2 or 2.0 mg per lb) had no effect on these variables (Flores-Garcia and Scholtyssek, 1992). Naber and Squires (1993b) reported that the riboflavin of egg albumen is a sensitive measurement to determine if riboflavin had been added to the diet of laying chickens. Hens fed a riboflavin-deficient diet laid eggs with low concentrations of the vitamin within four days. Therefore, the correlation between egg albumen riboflavin content and feed riboflavin content is high. Chicks fed a diet only marginally deficient in riboflavin often recover spontaneously. The condition is curable in the early stages, but in its acute stage it is irreversible (NRC, 1994). There is increasing evidence that vigor and livability of the baby chicks are directly tied to amount of riboflavin in the hen’s diet (Hoffmann-La Roche, 1969). Ruiz and Harms (1988) observed that riboflavin deficiency is more severe in modern strains of chicks and poults than in those used 40 to 50 years ago, perhaps due to the faster growth rate and improved feed conversion of the modern broiler.


Fortification Considerations

Riboflavin is one of the vitamins most likely to be deficient for poultry. Riboflavin fortification levels should be adjusted, especially to offset the reduction or exclusion of riboflavin-rich ingredients such as milk fermentation and fish by-products and dehydrated alfalfa from computerized best-cost poultry formulations. Poultry diets based on grains and plant protein sources are often borderline to deficient in riboflavin. Only a few feedstuffs fed to poultry contain enough riboflavin to contribute to the requirements for growth and reproduction. Poultry in confinement become more dependent on adequate vitamin (including riboflavin) and best-cost feed formulation (i.e., using principally corn and soybean meal) limits the number of riboflavin-rich feed ingredients. The greater the variety of feed ingredients, the lower the chance of vitamin and trace element deficiencies for animals and humans alike. Riboflavin is remarkably stable during heat processing; however, considerable loss may occur if foods are exposed to light during cooking and some losses occur in diets fed to animals out of doors. Only the portion of the vitamin in the feed exposed to light would be destroyed. Therefore, this may be of little significance as only the top layer of concentrate mixtures in automatic feeders would be affected. In dry form, riboflavin is extremely resistant to oxidation—even when heated in air for long periods. While it has been shown that field-cured alfalfa hay exposed to moisture can lose a significant amount of its riboflavin content in a relatively short time, under normal circumstances riboflavin has good stability when added to mixed feeds (Hoffmann-La Roche, 1969). Riboflavin is quite stable in multivitamin premixes (Frye, 1978). Naber and Squires (1993a) reported that the average albumen riboflavin content of eggs determined in their random survey was 4.05 µg per g. The efficiency of transferring riboflavin from the diet to whole egg declined with increasing dietary levels of supplemental riboflavin. However, the transfer appeared to be 46% efficient when diets were supplemented with up to 4.4 mg per kg (2.0 mg per lb) of riboflavin. Thus, the potential to fortify eggs with riboflavin appears high. Optimum vitamin nutrition, with higher concentrations of dietary vitamins, fed to laying hens results in higher levels of riboflavin and other vitamins in both poultry meat and eggs (Hernandez et al., 2002; Pérez-Vendrell et al., 2003b). Squires and Naber (1993a) noted depressed levels of both yolk and albumen levels of riboflavin if the diet was supplemented with less than 4.4 mg per kg (2.0 mg per lb) of the vitamin. In breeding hens, low albumen riboflavin content had an immediate effect on embryonic development and, ultimately, hatchability of fertile eggs. Therefore, for both commercial and breeding Leghorn hens, it was determined that supplementation of riboflavin should be no lower than 4.4 mg per kg (2.0 mg per lb). Based on these data, it may be speculated that the fortification needs of broiler breeders and turkey breeders would be much higher than the level reported for Leghorn hens.

Cook (1992) reported significant performance improvements in turkeys when riboflavin and (or) thiamin were increased to levels that exceed current industry averages. Similarly, the riboflavin requirement for broilers was reported to be higher than the industry average used today (Teeter and Deyhim, 1993). Some commercial turkey operations have adopted the practice of feeding higher levels of riboflavin (also vitamins C and E) to protect against pale, soft, and exudative syndrome (PSE) in meat.

There is controversy on the concept of removing vitamins and trace mineral supplementation from poultry and other species’ diets some time prior to slaughter. Skinner et al. (1992) reported that removal of vitamins and trace minerals from broiler diets did not impact performance. However, Teeter and Deyhim (1993) detected reduced performance and carcass variables when the same period was examined. Teeter and Deyhim (1996) reported reduced performance, carcass variables and increased mortality for both poultry and swine receiving inadequate vitamin supplementation. Deyhim et al. (1996) withdrew vitamins and trace minerals for 21 days in broiler diets during heat stress and found 37% less riboflavin in the Pectoralis major muscles. Such effects have the potential to impact consumer perception of poultry meat as wholesome and should be considered when vitamin withdrawal is being contemplated.


Vitamin Safety

A large body of evidence has accumulated that treatment with riboflavin in excess of nutritional requirements has very little toxicity either for experimental animals or for humans (Rivlin, 2006). There are no reports of riboflavin toxicity studies in poultry. Most data from rats suggest that dietary levels between 10 and 20 times the requirement (possibly 100 times) can be tolerated safely (NRC, 1987). When massive amounts of riboflavin are administered orally, only a small fraction of the dose is absorbed, the remainder being excreted in the feces. Lack of toxicity is probably because the transport system necessary for the absorption of riboflavin across the gastrointestinal mucosa becomes saturated, limiting riboflavin absorption (Christensen, 1973). Also, capacity of tissues to store riboflavin and its coenzyme derivatives appears to be limited when excessive amounts are administered.