| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,2
* Department of Animal Science and Centre de Recherche en Biologie de la Reproduction (CRBR),Laval University, Ste-Foy, Quebec, Canada, G1K 7P4 and
and
Dairy and Swine Research andDevelopment Centre, Agriculture and Agri-Food Canada, Lennoxville, Quebec, Canada, J1M 1Z3
2 Correspondence:
2000 route 108, CP 90 Lennoxville, Quebec, Canada J1M 1Z3. (phone: (819) 565-9171; fax: (819) 564-5507; E-mail:
mattej{at}em.agr.ca). Contribution no. 739.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
Key Words: Crossbreds • Folic Acid • Meishan • Parous Rates
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
The catabolism of glycine provides a methyl group to folic acid for DNA synthesis and methylation of homocysteine (Bässler, 1997). This pathway, the glycine cleavage system (GCS), is catalyzed by a complex of four proteins. One of these proteins, T-protein, requires tetrahydrofolate as a cofactor and induces the formation of ammoniac and 5, 10-methylene-tetrahydrofolate (Kikuchi, 1973). In sows, glycine is the most abundant amino acid in the oviduct and uterine fluids during diestrus (Iritani et al., 1974), and in the allantoic fluid on d 30 of gestation (Wu et al., 1995). Therefore, the metabolic provision of glycine to uterus should be adequate for an optimal folate response.
Crossbreeding between Meishan, known for their higher litter size and embryo survival (Terqui et al., 1990), and occidental genotypes has been used to develop prolific Sino-occidental lines of sows. However, little information exists on vitamin metabolism in these genotypes in early pregnancy (Vallet et al., 1999). Taking into account the role of folic acid in the control of prolificacy in occidental breeds, the present work was undertaken to determine if different levels of prolificacy either by nulliparous status or by genetic origin are linked to folic acid metabolism.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
) without the supplementation of folic acid and glycine. Heat detection was done twice a day, between 0800 and 0900 and between 1600 and 1700, by introducing a boar into the pen. On their first estrus (d -21) (monitored from at least 2 wk after arrival), sows were allocated (Table 2) to the following two treatments: basal diet (Control) or basal diet supplemented with 15 ppm folic acid + 0.6% of glycine (folic acid+glycine). Glycine was added to prevent any possible short-term suboptimal provision of this amino acid especially for local uterine metabolism pool (Iritani et al., 1974; Wu et al., 1995). On the second estrus, sows were inseminated twice with commercial semen (pooled semen from three Duroc boars of proven fertility; CIPQ Inc., St-Lambert, Canada), 12 and 24 h after estrus detection. The first day of estrus detection was considered as d 0 for the experiment. Dietary treatments were imposed up to slaughter on d 25 of pregnancy.
|
|
Laboratory Analysis
Concentrations of serum folates and vitamin B12 were measured by a validated procedure described by Tremblay et al. (1986) and Bilodeau et al. (1989) (Quantaphase II, folate radioassay and vitamin B12 radioassay; Bio Rad Laboratories (Canada) Ltd., Montreal, Qc, Canada). Radioassay of folates (Bio-Rad, Quantaphase II, folate radioassay) overestimates by 400% the quantities of tetrahydrofolates, which is the principal form of circulating folates in pigs (Anonymous, 1992). Nevertheless, concentrations of plasma tetrahydrofolate and 5-methyl-tetrahydrofolate (the other principal form of circulating folates in pigs) were correlated with radioassay folates (tetrahydrofolate: r = 0.724, P = 0.0001, and 5-methyl-tetrahydrofolate: r = 0.824, P = 0.0001). Furthermore, when the cross-reactivity of each folate form was used in regression analysis, the analysis shows a good relation between radioassay folates and the summation of folates determined by HPLC (R2 = 0.90, P = 0.0001) (Guay et al., unpublished data). Interassay CV were 4.5 and 4.2% for folates and vitamin B12 respectively.
Relative total folate binding capacity was measured according to a modification and validation by Giguère et al. (1998), using I125 folic acid (Quantaphase II, folate radioassay; Bio Rad Laboratories) of the procedure described by O’Connor and Picciano (1993). Briefly, 0.1 mL of plasma plus 0.5 mL of potassium phosphate (0.1 M, pH 2.5) were incubated 15 min, and free folates were removed by adding 1 mL of a solution of 2.5% hemoglobin-coated charcoal (in potassium phosphate 0.1 M pH 7.4). The sample was vigorously mixed for 5 min and centrifuged 10 min at 1,000 x g. The supernate was neutralized using 0.05 mL of NaOH (1.0 M) and 0.2 mL potassium phosphate (0.7 M, pH 7.4). After addition of 250 µL of I125 folic acid and 10 µL of folic acid (0.9 µM), the sample was incubated for 60 min at room temperature. Concentration of folic acid to be used in the assay was determined using a saturation study (Giguère et al., 1998). Free I125 folic acid and folic acid were removed by adding 0.5 mL of hemoglobin-coated charcoal. The supernate was counted using a gamma counter (1474 Wizard gamma counter; Wallac Oy, Turku, Finland).
Relative TFBC was evaluated by the recovered percentage of the total radioactivity introduced in the tube after removal of free I125 folic acid, multiplied by the total amount of folates introduced in the tube and divided by the total volume of serum used for the assay.
 |
The amount of folic acid provided by the addition of the I125 folate was considered as negligible (Giguère et al., 1998). Inter- and intraassay CV were 7.1 and 4.1% for TFBC.
Concentration of folates in allantoic fluid was determined directly using a radioassay kit as described by Matte et al. (1993). Interassay CV was 4.0%. Concentration of vitamin B12 in allantoic fluid was measured with a radioassay (Quantaphase, B12 radioassay; Bio Rad Laboratories). Interassay CV value was 5.8%; validation tests showed satisfactory parallelism (CV among dilutions of 4.8%) between 100 and 300 µL of sample and recovery tests (mean: 101%). Concentrations of amino acids were determined with an LKB 4400 amino acid analyzer (LKB Biochrom Ltd., Cambridge, England). Samples were prepared according to Mondino et al. (1972). Briefly, 25 mg of 5-sulphosalicylic acid at 4°C was added to 1 mL of allantoic fluid and incubated for 1 h at 4°C. The supernate was removed after high-speed centrifugation (3000 x g) for 15 min at 4°C and kept at -20°C until analysis.
Concentrations of folates in embryo homogenates were determined by the method described by Matte et al. (1993). Interassay CV was 7.3%. Deoxyribonucleic acid content was determined by a colorimetric assay (Labarca and Paigen, 1980), and protein content was estimated by a Coomassie brillant blue G-250 method (Bio-Rad assay, Mississauga, ON, Canada) using BSA standards; intraassay CV were 4.7 and 1.6% for DNA and protein content, respectively. Homocysteine content was measured by HPLC (Melnyk et al., 1999) with some modifications after sample preparation according to Malinow et al. (1989). For the determination of total homocysteine, 100 µL of H20, 300 µL of 9 M urea solution, 50 µL n-amyl alcohol, and 50 µL of a freshly prepared 10% NaBH4 solution containing 0.1 N NaOH were added to 0.2 g of embryo homogenate. After gentle agitation, the solution was incubated in a water bath at 50°C for 30 min. After incubation, 500 µL of perchloric acid was added, and the sample was centrifuged for 7 min at 12,000 x g. After centrifugation, the supernate was filtered through a 0.45-µm pore filter (Syringe Filter Titan, PVDF 0.45 µM, 13 mm; SRI Inc., Eatontown, NJ). The extracts of embryo homogenates were directly injected onto the column using a Beckman autosampler model 507E (Beckman Instrument (Canada) Inc., Mississauga, ON, Canada), and homocysteine was separated by HPLC coupled to a Beckman solvent delivery system (model 126; Beckman Instrument (Canada) Inc.). A reversed-phase C18 SphereClone column (5 µm; 250 x 4.60 mm; Phenomenex (Torrance, CA) was used. Isocratic elution was performed using a mobile phase that consisted of 10 mM sodium phosphate monobasic, 0.03 mM ion-pairing reagent OSA, 0.5% acetonitrile (vol/vol), adjusted to pH 2.7 with 85% phosphoric acid. Analyses were performed at room temperature at a flow rate of 1.5 mL•min-1. Homocysteine was detected by a 5200A Coulochem II EC detector (ESA Inc., Chelmsford, MA). Peak area analysis for homocysteine was provided by GOLD Nouveau software (Beckman Instrument (Canada) Inc.). Validation assay showed satisfactory parallelism (CV, between 0.1 and 0.3 g, was 9.5%) and recovery tests (mean: 101%). Intraassay CV was 6.3%.
Homocysteine content of the endometrial tissues was measured from samples prepared according to Young et al. (1994). Reduction of homocysteine was performed using a procedure similar to Malinow et al. (1989) for plasma while a quantitation by HPLC was conducted as described for the embryo homogenates. Validation tests showed satisfactory parallelism (CV between 0.5 and 1.5 g was 10.3%) and recovery tests (mean: 100.4%). Intraassay CV was 3.3%.
Statistical Analysis
Effects of folic acid+glycine treatments and groups of sows were analyzed using the Mixed procedure of SAS (SAS Inst., Inc., Cary, NC) according to a 2 x 3 factorial arrangement in a random design with groups of sow (multiparous and nulliparous YL and ML) and dietary supplements (0 or 15 ppm folic acid+glycine) as main effects. The model was: Yij = µ + Bi + Fj + (Bi x Fj) + eij, where Yij = dependent variable, Bi = groups of sows, Fj = folic acid+glycine supplement, and eij = residual error. Preplanned single-df contrasts were used to compare 1) genotype effects between multiparous ML and YL sows, and 2) nulliparous effects between multiparous and nulliparous YL sows. For serum concentrations of relative TFBC, the time of pregnancy (d 0 and 25) was added to the model as a third factor and was analyzed using the repeated option of the Mixed procedure with the autoregressive option, and then sow was considered as a random effect and included in statistical analysis. The same procedure was followed for serum concentrations of folates and vitamin B12 on d 0, 8, 16, 25 of pregnancy. In this last case, initial values at the treatment allocation (d -21) were used as covariables. For concentrations of serum folates and vitamin B12 as well as content of allantoic folates and vitamin B12 in the allantoic fluid, a logarithm transformation was performed to normalize the experimental errors.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
) but not fetal weight, whereas litter size, fetal weight, and number of CL in multiparous sows were not affected by genotype; however, litter weight tended to be higher in ML than in YL multiparous sows (P < 0.06; Table 2). The genotype of sows and nulliparous status had no effect on embryo composition (Table 2).
During the gestation period, multiparous YL sows had higher serum concentrations of folates and vitamin B12 than nulliparous YL sows (P < 0.01; Figure 1). Multiparous YL sows had higher concentrations of serum B12 than multiparous ML sows, but ML sows had higher concentrations of serum folates and TFBC than multiparous YL sows (P < 0.01; Figure 1 and Table 3). In this last case, the decrease of serum folates and TFBC during gestation was more marked in multiparous ML sows than in multiparous YL sows (genotype x time, P < 0.05; Figure 1 and Table 3).
|
|
).
In the allantoic fluid, the amounts of glutamic acid, aspartic acid, serine, alanine, proline, taurine, and urea were lower in multiparous YL than in nulliparous YL sows (P < 0.05; Table 4), whereas the quantities of tyrosine were higher in multiparous YL than nulliparous YL sows (P < 0.01; Table 4). Multiparous YL sows had larger amounts of allantoic folates, vitamin B12, essential amino acids, arginine, tyrosine, and glutamine as well as ammonia and ornithine than multiparous ML sows (P < 0.05; Table 4).
|
). However in multiparous YL sows, the dietary supplement of folic acid+glycine led to opposite effects on embryo DNA and proteins compared with multiparous ML sows (genotype x folic acid+glycine, P < 0.05; Table 2). In fact, within multiparous ML group, the results showed that folic acid+glycine supplement tended to decrease embryo DNA and protein (P = 0.06), whereas within multiparous YL sows, embryo DNA tended to be increased by supplementation of folic acid+glycine (P = 0.06) but not embryo protein (P > 0.1). Within nulliparous YL sows, the folic acid treatment had no effect on embryo DNA and proteins (P > 0.1). Finally, the supplement of folic acid+glycine tended to increase embryo contents in folates (P = 0.06; Table 2) and significantly decreased the quantity of homocysteine in embryo of all groups of sows (P < 0.05; Table 2).
During the gestation period, the folic acid+glycine treatment increased the concentrations of serum folates and TFBC (P < 0.05, Figure 1 and Table 3) but no effect was noted on concentrations of vitamin B12. In the case of serum folates, the effect of folic acid+glycine supplements was particularly marked in nulliparous YL sows (nulliparous x folic acid+glycine, P < 0.05).
In the endometrium, concentrations of DNA and proteins were not affected by the folic acid+glycine treatment (Table 2). However, for concentrations of endometrial homocysteine, the supplement of folic acid+glycine treatment had opposite effects in nulliparous and multiparous YL sows (nulliparous x folic acid+glycine, P < 0.05; Table 2).
In allantoic fluid, the folic acid+glycine supplement did not alter (P > 0.05) the amount of folates, vitamin B12, essential or nonessential amino acids, except for serine and histidine that tended to be decreased by folic acid+glycine supplement (P = 0.06; Table 4).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
The basal concentrations of serum folates in nulliparous YL sows were lower than in multiparous YL sows. Such results are in agreement with Natsuhori et al. (1996), who reported higher concentrations of plasma folates in adult pigs compared with 6-mo-old pigs. Matte et al. (1993) also observed a reduction in the concentrations of folates in the peripheral plasma of sows during the growing period. During the first 6 mo of life, the intensive growth of the gilt is sustained by cellular hypertrophy and hyperplasia (Whittemore et al., 1988) and requires folates, an essential cofactor for the synthesis of DNA, RNA, and proteins (Herbert and Das, 1976). Despite lower concentrations of serum folates observed in nulliparous YL sows, TFBC was not affected by parity of sows. Folates bound to folate binding proteins are very efficiently transported to the liver, but not to other tissues, such as uterine and fetal tissues (Fernandes-Costa and Metz, 1979). Possibly in nulliparous YL sows, high levels of TFBC allow an effective recycling of folates to the liver to accelerate the remethylation and reduction of folates (Steinberg, 1984) and thus sustain the intensive growth of these animals. This intensive growth could also have modified the amino acids profiles and altered uterine amino acid transfer to the conceptus such as seen in the present experiment.
Genotype Effects
Multiparous ML sows had higher concentrations of serum folates and TFBC than multiparous YL sows. Although multiparous ML sows and YL sows had different numbers of parity, this difference may have a limited effect on folate metabolism. In fact, previous studies using multiparous occidental sows (two parities and more) noted no significant difference for concentrations of circulating folates within multiparous group (Tremblay et al., 1986; Tremblay et al., 1989b; Harper et al., 1994). However in sheep, prolific breeds, such as Romanov and Finnsheep, also have greater concentrations of serum folates than nonprolific breeds such as Suffolk (Girard et al., 1996). These higher concentrations of serum folates in sows from prolific breeds could be due to a greater efficiency of folate absorption (Darcy-Vrillon et al., 1988), a lower utilization of folates associated with suboptimal vitamin B12 status (Scott and Weir, 1981), a greater efficiency of the enterohepatic cycle (Steinberg et al., 1979), or to higher serum concentrations of TFBC (Natsuhori et al., 1991). Furthermore, in ML sows, concentrations of serum folates decreased by 29% between d 0 and 8 of gestation and then remained stable until d 25 of gestation. In multiparous YL sows, the decrease was less pronounced and more progressive between d 0 and 25. These differences between genotypes were similar to those observed between breeds of ewes (Girard et al., 1996), where concentrations of serum folates declined more markedly in prolific than in nonprolific breeds in early pregnancy. The reduction of serum concentrations of TFBC was also more marked between d 0 and 25 in ML than in YL multiparous sows. This rapid decline in serum concentrations of folates and TFBC in multiparous ML sows suggests an important utilization of folates in early pregnancy such as observed by Matte and Girard (1999). This period is characterized by an intensive transfer of folates into the uterine lumen before 15 d of gestation (Matte et al., 1996).
Multiparous ML sows had lower total content of some essential amino acids and arginine in allantoic fluid than multiparous YL sows. Despite higher concentrations of serum folates and TFBC, multiparous ML sows had a lower concentration of folates in allantoic fluid than multiparous YL sows. In early pregnancy, fetuses of Meishan sows develop at a slower rate than those of occidental sows and differ in the development of their fetal erythropoiesis system (Pearson et al., 1998). Taking into account the key role of allantoic fluid in fetal nutrition in early pregnancy (Buhi et al., 1983), a limited transfer of nutrients from the sow toward the fetus might be a factor for the delayed development of Meishan fetuses during the first 90 d of gestation (Wilson et al., 1998).
Meishan sows are generally recognized for larger litter size and higher embryo survival rate than occidendal breeds of sows (Terqui et al., 1990). The present genotype effect on folate metabolism and transfer of nutrients to the fetus, without significant effect on prolificacy, might be linked to the fact that the multiparous ML sows were not pure Meishan but crossbred animals. In such crossbreeding, the performance, in terms of ovulation rates and litter size, is usually intermediate between the two original breeds (Lee et al., 1995).
Effects of Folic Acid plus Glycine
Supplementation of folic acid to sows has been reported to increase litter size and embryo survival (Matte al., 1984b; Lindemann and Kornegay, 1989; Tremblay et al., 1989a), but other studies have shown limited response to folic acid supplement for litter size and embryo survival in multiparous (Harper et al., 1994; 1996) and nulliparous sows (Lindemann and Kornegay, 1989; Matte et al., 1993). In the present experiment, irrespective of genotype or nulliparous, the dietary supplement of folic acid+glycine did not affect litter size or embryo survival, in spite of marked nulliparous status and genotype differences for concentrations of vitamin B12 and folates. For nulliparous effect, the lack of effect contrasts with a previous report (Lindemann and Kornegay, 1989), which showed that the response to a folic acid supplement in terms of litter size increases in third parity sows but not in nulliparous sows. Although some variation has been reported among experiments for the performance responses to folic acid, Lindemann (1993) from a review of literature concluded that the likelihood of positive responses of folic acid, if the response is not real, is 1/2,048 and therefore that folic acid supplement had real effects on litter size and embryo development. In present experiment, one could suggest that the addition of dietary glycine had interfered with the folic acid effects on litter size and embryo development. However, such a possibility appears unlikely since, in supplemented sows, the reduction of allantoic contents in serine and histidine, other methyl donors, along with decrease of embryo homocysteine did not suggest any alteration of the overall methylation metabolism (Bässler, 1997).
According to Lindemann (1993), the physiological basis for the role of folic acid on pig reproduction remains to be determined. Rosenquist and Finnell (2001) proposed two main hypotheses for the impact of folates on embryo development. In the first hypothesis, folates would act directly on the embryo for normal function and cellular proliferation. For the second hypothesis, the folate effect would be indirect and through the methionine-homocysteine metabolism; a low folate status increases homocysteine and could induce abnormal embryo development. In present experiment, in spite of significant increase of embryo content of folates and a decrease of embryo content of homocysteine, the folic acid+glycine supplement did not affect significantly litter size and embryo survival. At pharmacological concentrations (mMol), homocysteine is known to induce dysmorphogenesis of the heart and neural tube in avian and rat embryos (VanAerts et al., 1994; Rosenquist et al., 1996). However, the present results suggest that at physiological concentrations (nMol), changes in embryo homocysteine were not sufficient to induce modifications in the embryo development, embryo survival, or litter size on d 25 of gestation. Nevertheless, folic acid+glycine supplement tended to increase embryo DNA content in multiparous YL sows but not in nulliparous YL as well as multiparous ML sows. In previous studies, folic acid supplement has increased total embryo concentrations of DNA and proteins in multiparous occidental sows (Tremblay et al., 1989a; Harper et al., 1996). Therefore, the present results suggest that needs of folic acid and glycine to optimize litter size and embryo survival appear to be met in nonsupplemented multiparous ML and multiparous and nulliparous YL sows during first 25 d of gestation. However in multiparous YL sows, embryos would require folic acid plus glycine supplement to optimize the embryo development but not in multiparous ML and nulliparous YL sows.
Although the supplement of folic acid+glycine increased the concentrations of serum folates, irrespective nulliparous status or genotype of sows, the effect was more marked in nulliparous YL sows. Such a difference in the accumulation of folates suggests that either multiparous sows use the folic acid supplement more rapidly in early pregnancy (Matte and Girard, 1999), that nonsupplemented multiparous sows have optimal folate status, or that multiparous sows are less efficient in absorption of folates contained in the folic acid supplement. These results also could be indicative of a reduced utilization of folic acid supplements in nulliparous YL sows, a condition that has been associated with a "methyl trap" (Scott and Weir, 1981). In this case, there is an accumulation of folates and homocysteine in plasma due to a suboptimal status in vitamin B12, an essential factor for the activity of the enzyme methionine synthase (Bässler, 1997). In the present experiment, concentrations of serum vitamin B12 were lower in nulliparous YL than in multiparous YL sows, leading to a suboptimal status of vitamin B12. In multiparous ML sows, in spite of lower concentrations of serum vitamin B12 than in multiparous YL sows, folic acid+glycine supplement did not induce a significant accumulation of serum folates as observed for nulliparous YL sows. Such a phenomenon was possibly due to high basal levels of serum folates (fasting concentrations) observed in multiparous ML sows. Nevertheless in multiparous ML sows, higher endometrial concentrations of homocysteine than in multiparous YL sows, in spite of high concentrations of serum folates, and low concentrations of vitamin B12 suggest a possible suboptimal status of vitamin B12 as observed in other species (Bässler, 1997). However in spite of possible suboptimal status of vitamin B12 in multiparous ML and nulliparous YL sows, there is not evidence that level of vitamin B12 may limit responses to folic acid+glycine supplement for embryo survival or litter size during first 25 d of gestation.
Conclusion
The nulliparous status and genotype of sows affect systemic and uterine metabolisms of folates and vitamin B12. However, the effects of folic acid+glycine supplementation on embryo development, litter size, embryo survival, and some aspects of folate metabolism suggest that the basal folate status of multiparous ML sows and multiparous and nulliparous YL sows was adequate during first 25 d of gestation but, in multiparous YL sows, folic acid+glycine supplement appears to optimize embryo development.
|
Implications |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
|
Footnotes |
|---|
Received for publication July 24, 2001. Accepted for publication March 22, 2002.
|
Literature Cited |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Lessard, M. Dupuis, N. Gagnon, E. Nadeau, J. J. Matte, J. Goulet, and J. M. Fairbrother Administration of Pediococcus acidilactici or Saccharomyces cerevisiae boulardii modulates development of porcine mucosal immunity and reduces intestinal bacterial translocation after Escherichia coli challenge J Anim Sci, March 1, 2009; 87(3): 922 - 934. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Simard, F. Guay, C. L. Girard, A. Giguere, J.-P. Laforest, and J. J. Matte Effects of concentrations of cyanocobalamin in the gestation diet on some criteria of vitamin B12 metabolism in first-parity sows J Anim Sci, December 1, 2007; 85(12): 3294 - 3302. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. F. Harper, J. W. Knight, E. Kokue, and J. L. Usry Plasma reduced folates, reproductive performance, and conceptus development in sows in response to supplementation with oxidizedand reduced sources of folic acid J Anim Sci, March 1, 2003; 81(3): 735 - 744. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) = 15 ppm folic acid + 0.6% glycine.