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DSM in Animal Nutrition & Health

Folic Acid

Properties and Metabolism

Folacin is the generic descriptor not only for the original vitamin, folic acid, but also for related compounds that qualitatively show folic acid activity (Belic and Friesecke, 1979). The pure substance is designated pteroylmonoglutamic acid. Its chemical structure contains three distinct parts: consisting of glutamic acid, a para-aminobenzoic acid (PABA) residue and a pteridine nucleus (Illus. 12-1). The PABA portion of the vitamin structure was once thought to be a vitamin. Research has shown that if the folic acid requirement of the organism is met, there is no need to add PABA to the diet.

 

Illustration 12-1

12Illustration_12-1_FolicAcid

Much of the folic acid in natural feedstuffs is conjugated with varying numbers of extra glutamic acid molecules. Polyglutamate forms, usually containing three to seven glutamyl residues linked by peptide bonds of folic acid, are the natural coenzymes, being most abundant in every tissue examined (Wagner, 1984). These folic acid glutamates appear to be a biologically inactive storage form. Synthesized folic acid, however, is the monoglutamate form. It has been concluded that there are more biologically active forms of folic acid than any other known vitamin. Folic acid is a yellowish-orange crystalline powder, tasteless and odorless, and insoluble in alcohol, ether and other organic solvents. It is slightly soluble in hot water in the acid form but quite soluble in the salt form. It is fairly stable to air and heat in neutral and alkaline solution, but unstable in acid solution. From 70% to 100% of folic acid activity is destroyed on autoclaving at pH 1 (O’Dell and Hogan, 1943). It is readily degraded by light and ultraviolet radiation. Cooking can considerably reduce the folic acid activity of food.Polyglutamate forms of folic acid are digested via hydrolysis to pteroylmonoglutamate prior to transport across the intestinal mucosa. The enzyme responsible for the hydrolysis of pteroylpolyglutamate is a carboxypeptidase known as folate conjugase (Baugh and Krumdieck, 1971). Most likely, several conjugase enzymes are responsible for hydrolysis of the long-chain folate polyglutamates to the monoglutamates, which are then taken up by the mucosal cell (Rosenberg and Newmann, 1974). Pteroylmonoglutamate is absorbed predominantly in the duodenum and jejunum, apparently by an active process involving sodium. Kesavan and Noronha (1983) suggest from rat results that luminal conjugase is a secretion of pancreatic origin and that the hydrolysis of polyglutamate forms of folic acid occurs in the lumen rather than at the mucosal surface or within the mucosal cell.

Dietary folates, after hydrolysis and absorption from the intestine, are transported in plasma as monoglutamate derivatives, predominantly as 5-methyl-tetrahydrofolate. The monoglutamate derivatives are then taken up by cells in tissues by specific transport systems. There the pteroylpolyglutamates, the major folic acid form in cells, are built up again in stepwise fashion by an enzyme, folate polyglutamate synthetase. Polyglutamates serve to retain folic acid within the cells because only the monoglutamate forms are transported across membranes and only monoglutamates are found in plasma and urine (Wagner, 1995). Tactacan et al. (2010b) report that there is a folic acid transport system in the entire intestine of the laying hen. Uptake of folic acid in the cecum raises the likelihood of absorption of bacterial-derived folic acid. The same would be suggested for swine, as a large pool of available folic acid exists in the large intestine (Kim et al., 2004). In piglets, the folic acid content of feces was 301.3 nmol per day, representing 36% of their dietary folic acid intake. Piglet fecal folic acid was high in 5-methyl-tetrahydrofolate, an available folic acid form.

Specific folate-binding proteins (FBPs) that bind folic acid mono- and polyglutamates are known to exist in many tissues and body fluids including the liver, kidney, small intestinal brush border membranes, leukemic granulocytes, blood serum and milk (Tani and Iwai, 1984). The amount of folate-binding protein secreted by the endometrium during pregnancy was not affected by giving sows daily intravenous infusions of iron and tetrahydrofolate (Vallet et al., 1999). Physiologic roles of these FBPs are unknown, although they have been suggested to play a role in folic acid transport analogous to the intrinsic factor in the absorption of vitamin B12. In swine, sequence variation in the secreted folate binding protein gene is associated with differences in various factors affecting litter size, including ovulation rate, fertilization rate or embryonic survival, and uterine capacity (Vallet et al., 2005). Studies showed that about 79% to 88% of labeled folic acid is absorbed, and that absorption is rapid since serum concentrations usually peak about two hours after ingestion. Serum folate levels for gilts fed a single meal containing varying amounts of supplemental folic acid confirm that maximal levels are obtained within two hours post-feeding (Harper et al., 1991). Kokue et al. (1998) reported that supplemental synthetic folic acid competes with the reduced folates in the intestinal mucosa for the absorption pathway. The mean availability of folic acid in seven separate food items was found to be close to 50%, ranging from 37% to 72% (Babu and Skrikantia, 1976). Folic acid is widely distributed in tissues, largely in the conjugated polyglutamate forms. Urinary excretion of folic acid represents a small fraction of total excretion. Fecal folic acid concentrations are quite high, often higher than intake, representing not only undigested folic acid but, more importantly, the considerable bacterial synthesis of the vitamin in the intestine. Bile contains high levels of folic acid due to enterohepatic circulation, with most biliary folic acid reabsorbed in the intestine.

 

Functions

Folic acid, in the form 5, 6, 7, 8-tetrahydrofolic acid (THF), is indispensable in transfer of single-carbon units in various reactions, a role analogous to that of pantothenic acid in the transfer of two-carbon units (Bailer and Gregory, 2006; Bailey, 2007). The one-carbon units can be formyl, forminino, methylene or methyl groups. Some biosynthetic relationships of one-carbon units are shown in Figure 12-1. These one-carbon units are generated primarily during amino acid metabolism and are used in the metabolic interconversions of amino acids and in the biosynthesis of the purine and pyrimidine components of nucleic acids, which are needed for cell division. The important physiological function of THF consists of binding the single-carbon (C1) units to the vitamin molecule and thus transforming them to “active formic acid” or “active formaldehyde” so that these are interconvertible by reduction or oxidation and transferable to appropriate acceptors. Folic acid polyglutamates work at least as well as or better than the corresponding monoglutamate forms in every enzyme system examined (Wagner, 1984). It is now accepted that the pteroylpolyglutamates are the acceptors and donors of one-carbon units in amino acid and nucleotide metabolism, while the monoglutamate is merely a transport form.

 

Figure 12-1: Folic Acid Metabolism Requiring Single-Carbon Units

12Figure_12-1_Folic_Acid_Metabolism_Requiring_Single-Carbon_Units

Adapted from Scott et al., (1982) and McDowell (1989)

 

Specific reactions involving single-carbon transfer by folic acid compounds are:

  1. purine and pyrimidine synthesis, 
  2. interconversion of serine and glycine, 
  3. glycine-carbon as a source of C1 units for many syntheses,
  4. histidine degradation, and
  5. synthesis of methyl groups for such compounds as methionine, choline and thymine.

In rats an adequate supply of folic acid and related methyl donors can benefit fetal development directly by improving lipid-metabolism in fetal as well as maternal tissues (McNeil et al., 2009). Futhermore, folic acid is a vitamin from the B-complex involved as a co-factor of thymidilate synthetase, which is an essential enzyme for DNA and RNA synthesis (Davis and Nicol, 1988). 

Purine bases (adenine and guanine), as well as thymine (a pyrimidine base), are constituents of nucleic acids. With a folic acid deficiency, there is a reduction in the biosynthesis of nucleic acids essential for cell formation and function. Hence, deficiency of the vitamin leads to impaired cell division and alterations of protein synthesis; these effects are most noticeable in rapidly growing tissues such as red blood cells, leukocytes, intestinal mucosa, embryos and fetuses. In the absence of adequate nucleoproteins, normal maturation of primordial red blood cells does not take place and hematopoiesis is inhibited at the megaloblast stage. As a result of this megaloblastic arrest of normal red blood cell maturation in bone marrow, a typical peripheral blood picture results and is characterized by macrocytic anemia. White blood cell formation is also affected, resulting in thrombopenia, leukopenia and old, multi-lobed neutrophils. Because of the relationship of folic acid to neural tube defects, the U.S. Public Health Service has recommended that pregnant women consume at least 0.4 mg of folic acid daily to reduce the risk of spina bifida or other neural tube defects developing during pregnancy (Anonymous, 1996).

Vitamin B12 is necessary in reduction of one-carbon compounds in the oxidation stage of formate and formaldehyde. In this way, it participates with folic acid in the biosynthesis of labile methyl groups. The metabolism of labile methyl groups plays an important role in the biosynthesis of methionine from homocysteine and choline from ethanolamine. Folic acid has a sparing effect on the requirements of choline. The critical role of both folic acid and vitamin B12 on choline synthesis is discussed in the choline section.

Additionally, folic acid has been reported to help maintain the immune system. The immune system was severely inhibited by folic acid deficiency in rats (Kumar and Axelrod, 1978), which is probably mediated through a reduction in DNA synthesis, resulting in impaired nuclear division. Grieshop et al. (1998) reported that gestational folic acid supplementation in sow diets enhanced the secondary postnatal immune response of the piglets to a sheep red blood cell challenge.

 

Requirements

Various animal species differ markedly in their requirements for folic acid. The previous swine NRC (1988) publication suggests a dietary requirement of 0.3 mg per kg (0.14 mg per lb) for all classes of growing swine. However, based on new research information, the latest NRC (1998) recently increased their dietary folic acid requirement for gestating and lactating sows from 0.3 mg to 1.3 mg per kg (0.14 to 0.59 mg per lb). Folic acid requirements for monogastric species would be dependent on degree of intestinal folic acid synthesis and utilization by the animal. Lutz and Stahly (1997) evaluated the folic acid requirement of high lean growth pigs (8.6 to 23 kg; 19 to 51 lbs body weight) and reported that 0.31 mg folic acid per kg (0.14 mg per lb) of feed was adequate to support optimal growth and body nutrient accretion. Animals that practice coprophagy, would have a lower dietary need for folic acid, as feces are a rich source of the vitamin (Abad and Gregory, 1987). It has been suggested that part of the pig’s folic requirement could be met by coprophagy (Agricultural Research Council, 1981; McDowell, 2000). However, increased use of flooring that reduces the animals’ access to feces in commercial swine operations diminishes the importance of folic acid of fecal origin as a dietary source of the vitamin. Even though deficiencies can be produced with special diets, it has generally been reported that corn, soybean meal and other common feedstuffs in a practical swine diet should provide ample folic acid under most conditions. Self-synthesis of folic acid is dependent on dietary composition. For poultry, some research has indicated higher folic acid requirements for very high protein diets or when sucrose was the only source of carbohydrates (Scott et al., 1982). Keagy and Oace (1984) reported that dietary fiber had an effect on folic acid utilization; xylan, wheat bran and beans stimulated folic acid synthesis in the rat, reflected as higher fecal and liver folic acid. The levels of antibacterials added to the feed will affect microbial synthesis of folic acid. Sulfa drugs, which are commonly added to swine diets, are folic acid antagonists. In the chicken, sulfa drugs have been shown to increase the requirement (Scott et al., 1982) for this vitamin. Moldy feeds have also been shown to contain antagonists (i.e., mycotoxins) that inhibit microbial intestinal synthesis of folic acid in swine (Purser, 1981).

Folic acid requirements are dependent on the form in which it is fed and concentrations and interrelationships of other nutrients. Deficiencies of choline, vitamin B12, iron and vitamin C all have an effect on folic acid needs. Research by Johnson et al. (1950) revealed that baby pigs require both vitamin B12 and folic acid for hematopoiesis. Most folic acid in feedstuffs for swine is present in the conjugated form and the young pig is fully capable of utilizing it. Folic acid requirements are related to type and level of production. The more rapid the growth or production rates, the greater the need for folic acid because of its role in nucleic acid synthesis. Given the rapid growth rate until weaning and the stressful and disruptive period immediately following weaning, Letendreet al. (1991) investigated whether folic acid injection might influence their folate status and growth performance in piglets. The authors concluded folic acid injections increase serum concentrations and hepatic reserves of folates. However, the effect on folate status was not associated with an increase in growth performance or an influence on hematologic indices in piglets from two to 10 weeks of age. Letendre et al. (1991) suggested that even though serum folates decrease following weaning of piglets, detrimental effects on growth performance are not apparent.

The folic acid content of feed ingredients commonly used in swine diets, plus bacterial synthesis in the intestinal tract, appears adequate to meet the needs of all classes of swine. However, numerous reports have shown the benefits of folic acid supplementation in increasing fertility (Ensminger et al., 1951; Easter et al., 1983; Matte et al., 1984a, b; Tremblay et al., 1986; Lindemann and Kornegay, 1989; Tremblay et al., 1989; Matte et al., 1990a) and growth rate (Lindemann and Kornegay, 1986a) for swine consuming corn-soybean meal diets.

Folic acid deficiency in polytocous (giving birth to several offspring at one time) species such as the rat has been reported to decrease the weight of conceptuses, placentas, brain tissue and DNA concentration in the brain. Various researchers (Morgan and Winick, 1978; Thenen, 1979) have reported reduced litter size. Habibzadeh et al. (1986) reported that supplementation of folic acid in guinea pigs decreased embryo mortality and increased the number of live fetuses at 36 days of gestation.

Possible effects of folic acid on growth performance, attainment of puberty and reproductive capacity of gilts have been investigated (Matte et al., 1992). Long-term administration of folic acid did not affect age at puberty or reproductive capacity of gilts although there was some indication that 15 mg per kg (6.8 mg per lb) of dietary folic acid influenced growth performance of gilts by the end of the growing period.

Tissue synthesis during gestation is both rapid and intense (Matte and Girard, 1990). An increased supply of nutrients, including folic acid, is essential for enhanced metabolism during gestation, including that required for growth and development of conceptuses and placental structures (Pond and Houpt, 1978) and increased uterine secretory activity. Harper et al. (1989) recently reported that the number of live fetuses and percentage of fetal survival increased with 2.0 mg supplemental folic acid per kg (0.91 mg per lb) of diet in gilts fed a corn-soybean meal diet. Fetal weight and length, placental weight and length, empty uterine weight and amniotic and allantoic fluid volume, however, did not respond to folic acid supplementation.

Ensminger et al. (1951) observed that folic acid supplemented at the rate of 2.1 mg per kg (0.95 mg per lb) of diet improved reproductive performance in sows; no further improvement was noted with the addition of 211 mg per kg (96.0 mg per lb) PABA (a constituent of folic acid). Injection of folic acid at mating and nine days post-mating (Otelet al., 1972) was reported to have increased litter size from 8.3 to 10.0 pigs per litter. Easter et al. (1979; 1983) evaluated the addition of biotin, pyridoxine, thiamin and folic acid to corn-soybean meal gestating primiparous sow diets. Crossbred gilts received their respective treatment diets from three days post-mating until parturition, whereupon all gilts received a common lactation diet. Folic acid was supplemented to the basal diet at the rate of 0.2 mg per kg (0.09 mg per lb). Total pigs born per litter, pigs born alive per litter and pigs weaned per litter increased approximately 0.5 pig.

It has been shown that the folic acid requirement is elevated in pregnant women and that serum folates fluctuate during pregnancy. In humans, serum folates may reflect short-term dietary changes (Rothenberg et al., 1974). Matte et al. (1984a) measured post-weaning serum folate levels in sows throughout the reproductive cycle (weaning; mating; and day 15, day 30, day 60, day 90 and day 110 of gestation) to identify possible critical periods. These researchers observed a biphasic decrease in sow serum folate levels (Figure 12-2), first at mating and then at day 60 of gestation, suggestive of a possible mid-gestation folic acid deficiency. Natsuhori et al. (1994) compared the plasma concentrations of tetrahydrofolic acid (THF) versus N5-methyltetrahydrofolate (5MF) throughout the life cycle of pigs, including birth until two to four years of age and gestating and lactating sows. These authors indicated the principal component of plasma folates in newborn piglets is 5MF. However, the levels of 5MF dropped as the piglets nursed and grew, while the THF levels gradually increased. Conversely, during pregnancy, levels of THF declined while 5MF concentrations appeared to remain constant. They reported that during lactation THF levels increased, but not to the same levels as were present before gestation. Their study verified the decrease in plasma folates during pregnancy. The authors indicated this decrease is mainly due to the decrease of THF concentrations and suggested the loss of maternal folate is a result of supply to fetuses via the placenta. The decrease in serum folate concentration observed in normal human pregnancy (Hall et al., 1976) was suggested to be due to increased plasma volume. However, in sows the largest increase in plasma volume occurs during the last trimester of gestation.

12Figure_12-2_Concentration_of_Serum_Folates_in_Sows_Throughout_the_Reproductive_Cycle

Additionally, Matte et al. (1984b) and Matte and Girard (1989) reported that sow serum folate levels were responsive to intramuscular injection of 15 mg folic acid administered at weaning, first day of behavioral estrus, weekly during the first four weeks of gestation and every two weeks for the following eight weeks. This injection schedule maintained serum folate levels at day 60 of gestation, whereas folate concentrations decreased approximately 50% in sows that did not receive an injection. An improvement in reproductive performance (i.e., increased numbers of pigs born per litter and pigs born alive per litter) was observed in sows receiving the injectable folic acid compared with controls. This improvement was most pronounced for sows fed a flushing level (ad libitum intake) of feed compared with those fed a normal level of feed per day (1.8 kg or 4.0 lb) from weaning to mating and approximately the same to both groups thereafter. These results (Matte et al., 1984b) may suggest that folic acid was involved in a reduction of embryo mortality induced by a flushing treatment. Bazer et al. (1968) reported that flushing, although able to increase ovulation rate, is often associated with elevated embryo mortality. Therefore, the subsequent benefit of prolificacy at parturition is unrealized (Etienne et al., 1976). In another study utilizing injection of folic acid, when 25 mg was injected at the time of breeding and at one week later, the authors indicated that the benefits of folic acid were realized at a time when the sows’ serum folate levels were low and the requirements for folic acid in rapidly developing embryos would be high (Friendship and Wilson, 1991). If only parities 3 to 5 were analyzed, the litter size was improved by one pig per litter over that of control sows. Tremblay et al. (1986) observed that sow serum folate concentrations could be maintained by dietary folic acid to the same degree as that observed in sows injected with folic acid (Matte et al., 1984a). It was determined by regression analysis that about 4.3 mg supplemental folic acid per kg (1.95 mg per lb) of diet was necessary. Matte et al.(1990a) reported that sow serum folate concentration increased linearly when sows were supplemented with either 5 or 15 mg folic acid per kg (2.3 or 6.8 mg per lb) of diet. A level of 15 mg per kg (6.8 mg per lb) of dietary folic acid was necessary to maintain serum folate concentrations at eight weeks of gestation to those levels observed at mating. Additionally, litter size at weaning was greater in sows supplemented with 15 mg folic acid per kg (6.8 mg per lb) of diet. Tremblay et al. (1989) reported that the addition of 5 mg folic acid per kg (2.3 mg per lb) of diet increased survival rate of fetuses during early gestation and tended to increase the number of fetuses presumably living at day 30 of gestation when associated with high ovulation rates. Sows were assigned at weaning to two levels of folic acid (0 or 5 mg per kg) and three treatments to stimulate ovulation (restricted feed intake, ad libitum feed intake or pregnant mare serum gonadotropin [PMSG] injection).

Pharazyn and Aherne (1987) reported that supplementation of 0.45 mg folic acid per kg (0.20 mg per lb) to a complex diet containing an estimated 0.45 mg folic acid per kg (0.20 mg per lb) fed solely from day 109 of gestation throughout lactation produced no difference in litter size or litter weights either at birth or at weaning at day 28. Similarly, Gannon and Leibholz (1988) reported no significant difference in litter size or birth weight of pigs from sows supplemented with 1.0 mg folic acid per kg (0.45 mg per lb) of diet. The supplemental folic acid was fed for six weeks before mating and throughout gestation. Lindemann and Kornegay (1989), however, reported an increase in pigs born and weaned and a possible improved conception rate in a three-parity study in which sows received either 0 or 1.0 mg supplemental folic acid per kg (0.45 mg per lb) of diet. An interaction between folic acid supplementation and parity was observed indicating that the response to folic acid may have increased with each successive parity, from parity one to three. This is in agreement with O’Connor et al. (1989) who suggested that 0.6 mg of folic acid per kg (0.28 mg per lb) of diet may be insufficient for gestating sows. Thaler et al. (1989) observed an increase in litter size at parturition following the addition of 1.7 or 6.6 mg of dietary folic acid per kg (0.77 or 3.0 mg per lb). These authors reported a much greater improvement in litter size at birth and day 21 for sows fed the lower level of folic acid during the course of this two-parity study; however, reproductive potential of sows was relatively low in this study (7.8 pigs per litter in control sows), possibly masking the effect of the higher supplementation level. In contrast, a large study involving five different universities failed to show a benefit in reproductive performance when folic acid was fed throughout gestation at levels up to 4 ppm (Harper et al., 1994). However, in a Fuchs et al. (1995) study, with litter size ranging from 10.2 to 11.2 pigs per litter and when 3 and 5 mg of folic acid per kg were supplied in the feed to pregnant sows, embryo mortality and the number of stillborn piglets were decreased. Matte et al. (1992) investigated the folic acid requirements of gestating and lactating gilts. As expected, during gestation and lactation, addition of folic acid increased concentrations of serum folate. Although numerically higher in gilts receiving 15 mg per kg, litter size at parturition and weaning was not affected by folic acid addition. Growth of piglets and total litter weight from birth to eight weeks of age increased linearly with the level of folic acid (0, 5 or 15 mg per kg) in the gestation diet. The authors concluded that 15 mg per kg of folic acid was necessary to avoid the drop in concentration of serum folate in gestating sows and influenced some performance traits such as total litter weight up to eight weeks post-parturition. There was no effect of folic acid supplementation during lactation on reproductive performance. However, supplementation during both gestation and lactation maximized piglet growth. The authors concluded that greater than 5 mg of folic acid is required for maximum sow reproductive performance, but were not sure if 15 mg was too much or too little for reproducing swine.

Although the specific action of folic acid’s involvement in enhancing various reproductive parameters is unknown, it has been suggested (Scherf and Scott, 1989) that folic acid may act through its influence on the transfer of single-carbon units, which is necessary for purine and pyrimidine base synthesis and hence nucleic acid synthesis. The rate of cell proliferation during fetal development is great and the concentration of intracellular ribonucleic acid is highly correlated with embryonic survival. Although number of live fetuses at day 45 + 3 of gestation was not affected by supplying 2 ppm of dietary supplemental folic acid, Harper et al. (1996) determined that fetal pig weight, length, protein and RNA content were increased over controls. The authors suggested that these effects, which took place by day 45 of gestation and are associated with placental and fetal growth, may promote improved litter size at birth. The response to folic acid supplementation on the RNA and DNA contents of 25-day embryos is influenced by the breed of sows studied (Guay et al., 1998).

Matte et al. (1986) have also suggested that folic acid may be involved with an embryo growth factor. Matte et al. (1993) indicated that maternal folic acid can be transferred to fetuses in utero. In addition, Matte et al. (1996) reported a significant increase in total uterine prostaglandin E2 (PGE2) on day 12 and day 15 and a numerical (60%) increase in PGF2 for sows fed 15 mg folic acid from two weeks prior to estrus through either 12 or 15 days after mating. Although a more precise mechanism remains to be elucidated, Matte et al. (1996) concluded that the influence of folic acid during early gestation appears to be partially related to modifying the uterine environment. In particular, folic acid may affect the exocrine secretions of prostaglandins, which may reduce the maternal immune response to conceptuses and improve their chances of survival. Giguere et al. (1999) reported that the frequency of sows having elevated allantoic PGE2 was higher in nulliparous sows that received 15 ppm of supplemental folic acid.

More research is needed to determine folic acid requirements for swine and the conditions that elicit the greatest response.

 

Sources

Folic acid is widely distributed in nature, almost exclusively as THF acid derivatives, the stable ones having a methyl or formyl group in the 5-position and generally possessing three or more glutamic acid residues in glutamyl linkages. Only limited amounts of free folic acid occur in natural products, with most feed sources containing predominantly polyglutamyl folic acid. Folic acid is abundant in green leafy materials and organ meats. Soybean, other beans, nuts, some animal products and citrus fruits are good sources. Cereal grains, milk and eggs are generally poor sources of the vitamin.

A considerable loss of folic acid (50% to 90%) occurs during cooking or processing of foods. Folic acid is sensitive to light and heating, particularly in acid solutions. Under aerobic conditions, destruction upon heating is significant in most folic acid forms, with reduced folic acid more stable in foods because of relatively anaerobic conditions and because folic acid is protected from light (Brody et al., 1984).

Folic acid bioavailability in a variety of foods was found to generally exceed 70% (Clifford et al., 1990). Bioavailability of monoglutamate folic acid is substantially greater than polyglutamyl forms (rtilifford et al., 1990; Gregory et al., 1991). The availability of food folic acid may range from 30% to 80% that of the monoglutamate form, being generally less well utilized from plant-derived foods than from animal products. Bioavailability of orally administered 5-methyl folic acid and 5-formyl folic acid compared to folic acid were found to be equal for rats (Bhandari and Gregory, 1992).

Crystalline folic acid, produced by chemical synthesis, is available for feeds, foods and pharmaceuticals. Several lines of evidence indicate higher bioavailability of added folic acid than naturally occurring folates in many foods (Gregory, 2001). Although folacin is only sparingly soluble in water, the sodium salt is quite soluble and is used in injections as well as feed supplements (McGinnis, 1986; Tremblay et al., 1986). Spray-dried folic acid and dilutions of USP crystalline folic acid are the most widely used product forms in animal feeds. Synthetic folacin supplements are highly available, natural forms are approximately 50% less available.

 

Deficiency

Folic acid deficiency has been produced experimentally in many animal species, with a macrocytic anemia (megaloblastic anemia) and leucopenia (a reduced number of white blood cells) a consistent finding. Tissues that have a rapid rate of cell growth or tissue regeneration, such as epithelial lining of the gastrointestinal tract, epidermis and bone marrow, are most affected (Hoffbrand, 1978). Until recently folic acid deficiency in swine had only been produced by the simultaneous feeding of sulfa drugs. Deficiencies were not observed when young pigs were fed only purified diets or natural diets low in folic acid alone (Johnson et al., 1948), indicating that intestinal synthesis was adequate to meet needs. Feeding a purified diet containing 2% sulfasuxidine to weanling pigs resulted in reduced gains and alopecia (Cartwright and Wintrobe, 1949). The pigs also developed a mild normochromic, normocytic anemia. In bone marrow there was a decrease in the ratio of leukocytes to erythrocytes and an increase in the number of immature nucleated red blood cells. Positive response was obtained after supplementation with folic acid. Cunha et al.(1948) found that folic acid was needed for normal hematopoiesis with eight-week-old pigs fed a purified diet for 21 weeks with sulfasuxidine. A normocytic anemia resulted that was prevented by folic acid, whereas a more severe anemia was produced by using a crude folic acid antagonist. A combination of folic acid and biotin was more effective than folic acid in counteracting the anemia. Lindemann and Kornegay (1986b) reported that a combination of the antibiotic mixture ASP 250 (includes chlortetracycline, sulfamethazine and penicillin) and folic acid in a corn-soybean meal diet increased gains and feed consumption, with no effect of either fed alone. More severe deficiency signs that responded to folic acid supplementation were induced by feeding diets containing a sulfonamide and a folic acid antagonist (Welch et al., 1947). Under such circumstances, pigs became listless, had a reduced growth rate and developed diarrhea. Hematologic manifestations were severe macrocytic anemia, leukopenia with a more marked reduction in the number of polymorphonucleocytes and mild thrombocytopenia. Cartwright et al. (1952) reported a combined folic acid and vitamin B12 deficiency for pigs receiving a purified soybean protein diet that included a folic acid antagonist. Growth rate was reduced and macrocytic anemia, leukopenia and neutropenia developed with erythroid hyperplasia of the bone marrow. Folic acid supplementation immediately resulted in a normal blood and bone marrow profile, but growth was decreased and blood parameters subsequently relapsed.

In addition to sulfa drugs and other folic acid antagonists, moldy feeds can increase the need for the vitamin. In seven swine feeding trials involving more than 1,000 pigs fed corn with mold infestation, additional folic acid increased growth rate up to 15% and improved feed efficiency up to 9% (Purser, 1981). Folic acid supplementation was of no value when normal corn was fed.

Recently, inadequate folic acid has been associated with suboptimal reproductive performance of sows. A dramatic decrease in serum folic acid concentrations was observed during early and mid-gestation which may, in part, be associated with embryonic mortality (Matte et al., 1984a). In a separate trial, folic acid was administered intramuscularly according to a schedule that maintained serum folic acid concentrations at approximately the same level between weaning and 60 days of gestation (Matte et al., 1984b). Average live litter size was 12 piglets per litter for sows receiving folic acid and flushing treatments as compared with 10.5 piglets for sows without any treatment. In another study, addition of 5.0 mg folic acid per kg (2.3 mg per lb) of diet improved survival rate of fetuses during early gestation, 62.2% versus 55.1% compared to those not receiving folic acid (Tremblay et al., 1989). Lindemann and Kornegay (1989) found the number of matings required per female farrowing was less with folic acid supplements (1.07 versus 1.16 for controls). Friendship and Wilson (1991) suggested that folic acid from natural feedstuffs, plus the current low level of supplemental folic acid typically used, may not be sufficient to maximize reproductive performance of sows. These authors utilized a 400-sow commercial herd experiencing small litter size in their experiment. When 25 mg of folic acid was injected at the time of breeding and at one week later, and if only parities 3 to 5 were analyzed, the litter size was improved by one pig per litter over that of the control sows.

The effect of folic acid and glycine (to provide a methyl group to folic acid) supplementation was studied on embryo development in early pregnancy (Guay et al., 2002). Folic acid was found to be adequate during the first 25 days of gestation, but in multiparous Yorkshire-Landrace sows, the folic acid + glycine supplement appeared to optimize embryo development. Additional research is required to determine if supplemental dietary folic acid will reduce embryonic death loss under differing management systems to improve overall efficiency of production (i.e., to optimize pig survival and litter size).

 

Fortification Considerations

Folic acid needs for livestock may be supplied by complex practical diets (Maynard et al., 1979). Also, for most species including swine, substantial quantities of folic acid are provided through microbial synthesis (Aufreiter et al., 2011). Nevertheless, field observations have been made of diets that provide insufficient folic acid. Green forage is an excellent source of folic acid. Supplementation of folic acid would be most needed when animals are in confinement without access to fresh or preserved green forages. The successful treatment of field cases of folic acid deficiency and improved reproductive performance observed with supplemental folic acid have demonstrated that commercial feeds not supplemented with folic acid do not always supply adequate quantities of the vitamin to swine. Folic acid supplementation is also of importance when swine receive diets that contain folic acid antagonists such as sulfa drugs and grains contaminated with toxin-producing molds. Depending on a given year and climatic and harvesting conditions, a large percentage of the United States corn crop may contain some mold contamination. Therefore, folic acid supplementation should have a positive effect in many commercial swine operations. This would likely be an even more important consideration in developing tropical countries or regions where climates favoring mold growth are optimized. Individual responses to folic acid supplementation to counteract mold effect will obviously vary with the class of livestock being fed, species of mold present and the levels of toxin encountered (Bhavanishkankar et al., 1986). Although the research with reproducing female swine is limited, a pattern due to folic acid supplementation is evident. There is a consistent increase in total and live pigs born when reproducing females receive supplemental folic acid during gestation (Ensminger et al., 1951; Easter et al., 1983; Matte et al., 1984a, b; Tremblay et al., 1986; Lindemann and Kornegay, 1989; Tremblay et al., 1989; Matte et al., 1990a). The increase has occurred at dietary supplementation levels from 0.2 mg per kg (0.09 mg per lb) (Easter et al., 1983) to 15 mg per kg (6.9 mg per lb) (Matte et al., 1990b) as well as with folate injections throughout the first 12 weeks of gestation (Matte et al., 1984b). It seems apparent and logical that supplementation must occur in early gestation. Late gestational or lactational supplementation has been without effect on reproductive performance (Pharazyn and Aherne, 1987). However, Matte et al. (1992) indicated that supplementation of 15 mg folic acid per kg (6.8 mg per lb) of diet during both gestation and lactation maximized growth of piglets in their study. The response to folic acid appears to be greater in conditions of increased ovulation (e.g., sows versus gilts, flushed versus non-flushed), which suggests that there may be breed differences in response to folic acid supplementation (Lindemann, 1988). Harper et al. (2003) observed that sows fed diets supplemented with folic acid showed a tendency for larger litter size at day 45 of gestation. These results are further evidence that the requirement for additional folic acid in corn-soybean meal sow diets is small, and a response in sow reproductive performance will not be observed in all cases.

Gadient (1986) considered folic acid to be very sensitive to heat and light, slightly sensitive to moisture and insensitive to oxygen. Folic acid can be lost during storage of premixes, particularly at elevated temperatures (Frye, 1978). After three months of room temperature storage, 43% of the original folic acid activity was lost. Verbeeck (1975) found folic acid to be stable in premixes without trace minerals but that there may be as much as 50% loss in a premix with trace minerals kept at room temperature for three months. Adams (1982) reported only 38% retention of folic acid activity in a premix without trace minerals after three weeks at 45°C. However, he reported 57% retention of activity after three months at room temperature.

 

Vitamin Safety

Folic acid generally has been regarded as a nontoxic vitamin (NRC, 1998). No adverse responses to the ingestion of folic acid have been documented in swine. Fuchs et al. (1995) reported that supplying a shock dose of 30 mg per kg every seven days, which provides much greater levels than the requirement for folic acid, had no negative effect on changes of selected biochemical and hemologic indices in pregnant sows.