An Assessment of Dietary Folic Acid Levels During Gestation and Lactation on Reproductive and Lactational Performance of Sows: A Cooperative Study'

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1 Published December 11, 2014 An Assessment of Dietary Folic Acid Levels During Gestation and Lactation on Reproductive and Lactational Performance of Sows: A Cooperative Study' A. F. Harper233, M. D. Lindemann2, L. I. Chiba4, G. E. Combs5, D. L. Handline, E. T. Kornegay2, and L. L. Southern' S- 145 Committee on Nutritional Systems for Swine to Increase Reproductive Efficiency ABSTRACT Crossbred female swine (n = 393) were used in a multiparity study at five experiment stations to evaluate the effects of dietary supplementation of folic acid (FA) on serum folates status and reproductive performance. The dietary treatments were a corn-soybean meal basal diet (calculated FA,.34 ppm) supplemented with 0, 1, 2, or 4 ppm FA. Experimental diets were fed continuously from a minimum of 21 d before first mating throughout the entire study. At one station, blood samples for radioimmunoassay determination of serum folates concentration were collected by vena cava puncture at mating, d 55 of gestation, d 110 of gestation, and at weaning. Stage of reproduction and dietary FA supplementation affected (P <.005) serum folates concentrations. Serum folates declined from mating to d 55, remained low at d 110, and returned to higher levels at weaning. Linear increases (P <.001) in serum folates with increasing level of dietary FA were observed at each reproductive stage. Over the course of the study, reproductive performance criteria including total pigs born, live pigs at birth and d 21, and individual pig and litter weight at birth and d 21 were not affected ( P >.lo) by inclusion of FA in the diet. The number of days postweaning to estrus also was not affected by FA treatment. Under the conditions of this experiment, increasing level of FA in the diet had a pronounced effect in attenuating decreased serum folates concentration during gestation but was without benefit to reproductive performance. Key Words: Folic Acid, Serum, Reproductive Performance J. Anim. Sci : Introduction Folic acid and related metabolites, collectively termed folacin ( FA), constitute an essential, watersoluble vitamin for swine ahd many other mammalian 'Appreciation is expressed to Carl S. Akey, Inc., Lewisburg, OH for providing the vitamin-mineral premix; to Hoffmann-La Roche, Inc., Nutley, NJ for supplying the folic acid premix, for analyzing diet samples for folic acid content, and for partial support of the trials at Virginia; to the Virginia Pork Industry Board and the National Pork Producers Council for partial support of analytical costs; and to the John Lee Pratt Nutrition Program for supplying a graduate fellowship for the senior author. 'Dept. of Anim. and Poult. Sci., Virginia Polytechnic Inst. and State Univ., Blacksburg T0 whom correspondence should be addressed.: Tidewater Agric. Res. and Ext. Ctr., Suffolk, VA Dept. of Anim. and Dairy Sci., Auburn Univ., Auburn, AL Dept. of Anim. Sci., Univ. of Florida, Gainesville 'Dept. of Anim., Dairy, and Vet. Sci., Clernson Univ., Clemson, SC Dept. of Anim. Sci., Louisiana State Univ., Baton Rouge Received July 23, Accepted May 3, and avian species. The current National Research Council (NRC, 1988) suggested dietary requirement for FA is.3 ppm for all classes of swine. This level reflects the approximate endogenous level of FA found in typical corn-soybean meal diets. Recent studies suggest that FA supplementation may be of particular importance in the nutrition of reproducing gilts and sows. Matte et al. (1984a) observed a major decline in serum folates concentration of sows from mating to d 60 of gestation, suggesting a greater metabolic demand for FA during early to mid-gestation. Furthermore, serum folates status of reproducing sows has been altered by periodic i.m. injections of FA (Matte et al., 1984a,b) or by dietary supplementation (Tremblay et al., 1986; Thaler et al., 1989). Folic acid supplementation by continuous dietary inclusion of 1 ppm of FA (Lindemann and Kornegay, 1989) or 1.65 ppm of FA (Thaler et al., 1989) has resulted in improvements in litter size at birth. In contrast, Easter et al. (1983) reported only slight, nonsignificant improvements in litter size in gilts fed diets supplemented with.2 ppm of FA, and Gannon and Liebholtz (1988) reported no improvement with a l-ppm dietary supplementation in a commercial sow herd. 2338

2 ~ SUPPLEMENTAL FOLIC ACID AND SOW PERFORMANCE 2339 Table 1. Stations involved in the study Supplemental No. of folate females No. of levels, started reproductive Weaning Station PPm on study Breed cycles age, d Auburn 0, 1, 2, 4 66 Crossbred Clemson 0, 1, 2, 4 39 Crossbred Florida 0, Crossbred 7 21 Louisiana 0, 2 59 Crossbred Virginia 0, 1, 2, 4 93 Crossbred This study represents a coordinated effort of five research stations to determine the effects of dietary FA supplementation over an inclusion range of 0 to 4 ppm on serum folates concentration and sow reproductive performance over four consecutive parities. Experimental Procedures The study involved five research stations and an initial allotment of 393 female swine. A summary of the number of animals, treatments used, and other pertinent information relative to the individual stations is given in Table 1. At each station the females (primiparous at Clemson and Virginia and both primiparous and multiparous at Auburn, Florida, and Louisiana) were randomly allotted to treatments from outcome groups based on breeding weight, parity, and ancestry. Table 2. Composition of basal dietsa Ingredient 8 Ground yellow corn Soybean meal (44% CPIb Dicalcium phosphatec 1.51 Ground limestone 1.16 Salt.50 vitamin-trace mineral premixd.25 Calculated analysis (as fed) CP, % Lysine, %.60 Ca, %.81 P, %.60 ~~ ~ ~ ~ athe basal diet was formulated on an as-fed basis. The appropriate amount of the folic acid premix was added to attain desired final diet folate levels. bwhen 48% soybean meal was used the soybean meal was decreased and the grain increased by 1.15 percentage units. When defluorinated phosphate (DFP) was used the levels of DFP and limestone were adjusted to provide.81% Ca and.6% P with minor adjustments of corn and soybean meal to maintain.60% lysine. dsupplied (per kilogram of diet): 11,000 IU of vitamin A 550 IU of vitamin DQ; 44 IU of vitamin E; 5.5 mg of menadione dimethylprimidinol bisulfate (MPB); 33 pg of vitamin BIZ; 6.6 mg of riboflavin; 22 mg of d-pantothenic acid; 27.5 mg of niacin; 770 mg of choline chloride;.22 mg of d-biotin, 125 mg of Zn; 125 mg of Fe; 40 mg of Mn; 10 mg of Cu; 1.26 mg of I; and.3 mg of Se. The dietary treatments were a corn-soybean meal basal diet (Table 2) supplemented with either 0, 1, 2, or 4 ppm of FA at the Auburn, Clemson, and Virginia stations and 0 or 2 ppm of FA at the Florida and Louisiana stations. The basal diet was formulated to meet or exceed the nutrient requirements of breeding swine (NRC, 1988) and was calculated to contain.34 ppm of folacin. Analysis of the diets for FA content indicated that the experimental diets were correctly prepared at each station (Table 3). The females were fed their respective diets continuously from a minimum of 21 d before the initial first breeding through all consecutive parities. Diets were prepared in meal form. During breeding and gestation, the diets were fed once daily at a level of 1.81 kglsow except during periods of seasonally cold temperatures, when stations were allowed the option for sows to be fed 2.27 kgld. Sows were allowed feed on an ad libitum basis during lactation. Water was available on an ad libitum basis during gestation and lactation. Throughout gestation, the females were housed in enclosed breeding swine barns in individual crates (Clemson) or penned by treatment group with up to six animals per pen (Louisiana and Virginia) or penned by treatment group in outdoor sand lots that were essentially devoid of vegetation (Florida and Auburn). All sows were individually fed, except at Louisiana where sows were group-fed during gestation. The indoor pens were equipped with partially Station Table 3. Analyzed folacin content of ' experimental dietsab Supplemental folate level, ppm Auburn Clemson Florida Louisiana.ll Virginia aeach value is the mean of 7 to 16 analyzed values throughout the study. bmicrobiological folacin assays conducted in the laboratory of Hoffmann-La Roche, Nutley, NJ.

3 2340 HARPER slotted floors and nipple waterers. Females were moved into crates in a clean farrowing barn on d 109 or 110 of gestation and remained there throughout lactation. Pigs were weighed, ear-notched, and processed per station guidelines within 24 h of birth. Iron injections and vaccination schedules were according to station protocol. Crossfostering of pigs from large litters (> 12 pigs) was performed within 48 h after farrowing when another sow on the same dietary treatment was available. Due to the wide range of weaning ages at the various stations (19 to 35 d), litter traits beyond d 21 of lactation were not considered in the assessment of reproductive performance. Creep feed was made available at three stations, but consumption prior to d 21 was negligible. Gilts and sows were weighed at breeding, d 110 of gestation, within 24 h postpartum, at d 21 of lactation, and at weaning. Response variables for sow performance included gestation weight gain (breeding to d 110), lactation weight change (weight within 24 h after farrowing to weaning), lactation feed intake, number and weight of total pigs born per litter, the number and weight of pigs alive at birth and d 21, and days postweaning to estrus. Serum Folates Determination. At the Virginia station, blood samples were collected from the sows by anterior vena cava puncture within 24 h of mating, at d 55 and 110 of gestation, and on the day of weaning for determination of serum folates concentration. The samples taken at mating and during gestation were collected between 0700 and 0900 after a 24-h fast. At weaning, samples were collected when the sows were removed from the farrowing crate and moved to the breeding facility. At collection, blood was placed in sterile glass tubes that had been lightly coated with crystalline ascorbic acid. The blood was allowed to clot under refrigeration, and the serum was obtained by centrifugation. Aliquots of the serum were stored frozen in polypropylene tubes at -80 C until they were assayed for serum folates concentration. The method of serum folates assay employed a human serum folate radioassay (Quantaphase Folate@, Folate Radioassay, Bio-Rad Laboratories, Hercules, CA), which has been previously adapted for use with swine serum by Matte et al. (1984a). The range of assay sensitivity for sow serum was 8 to 160 ng/ml. Statistical Analysis. Certain data were deleted when they were found to be unbalanced across treatments. Removed from the analysis were sow and litter data beyond four parities on study, data from sows beyond six actual parities, and data from females that contributed only one litter prior to culling. Each sow and litter were considered an experimental unit. The data were analyzed using the least squares GLM procedures of SAS (1988). For the performance data, the model included the effects of treatment, study parity, station, treatment x parity interaction, treatment x station interaction, and the treatment x parity ET AL. x station interaction. Treatments were separated into linear and quadratic effects using orthogonal polynomials for unequally-spaced treatments. Because three participating stations incorporated all four FA treatments and two stations only used two treatments (0 and 2 ppm), two analyses were conducted, one including five stations with two treatments and one including three stations with four treatments. These separate analyses revealed no additional information in panty or treatment responses; therefore, the single pooled analysis with unbalanced treatments across stations was used. For analysis of the serum folate data, a nested design was used in which sow within treatment was the error term for treatment effects and sow within treatment x parity was the error term for parity and treatment x parity effects. The residual error term was used to test sampling time, treatment x sampling time, parity x sampling time, and treatment x parity x sampling time effects. Significant treatment effects on serum folate concentration were separated into linear and quadratic components. Results and Discussion Parity Effects on Sow and Litter Performance. The parity x station x treatment interaction was not significant (P >.lo); therefore, only the effects of parity across all dietary treatments are presented (Table 4). In this case, Parities 1 through 4 represent the reproductive cycle that occurred during this experiment (not biological reproductive cycle) because some multiparous sows were allotted to treatments at the Auburn, Florida, and Louisiana stations. Occasional station x parity interactions existed (P <.05), as would be expected with stations starting both primiparous or multiparous animals, but these interactions demonstrated only known parity effects on reproduction and are not presented. With advancing parity, sows exhibited progressively less weight gain during gestation (linear effect, P <.001) and a linear trend for less weight loss during lactation ( P <.08). Less weight loss during lactation corresponded with a linear increase ( P <.OOl) in feed intake during lactation with advancing parity. Less weight gain during gestation and less weight loss during lactation with advancing parity is in agreement with previous reports of a two-parity study (Michel et al., 1980) and a four-parity study (Esbenshade et al., 1986). Advancing parity also had a pronounced effect on litter size and weight (Table 4). Linear increases in number of pigs born alive (P <.02) and in litter weight at birth and d 21 ( P <.001) were observed. Individual pig weight at birth also increased linearly (P <.005) with advancing parity during the study. Increases in litter size and weight with advancing parity has been well documented (reviewed by Clark

4 ~ SUPPLEMENTAL FOLIC ACID AND SOW PERFORMANCE 2341 Table 4. Least squares means of parity number during the study on sow body weight changes, lactation feed intake, and reproductive performancea Panty during the study Criterion SEM Sow data Breeding wt, kgb Gestation gain, kgb Lactation loss, kg Lactation feed intake to d 21, kg/db Days postweaning to estrus' Litter size Total born Born alived Day Pig wt, kg Birth, all pigs' Birth, live pigs' Day OB Litter wt, kg Birth, all pigsb Birth, live pigsb Day 21b A9 aall P c.05 are noted; means are weighted by the number of observations with a total of 993 litter observations in the study. Number of observations for each parity were 399, 303, 204, and 87 for Parities 1, 2, 3, and 4, respectively. kinear effect of parity (P <.OOI). CLinear effect of parity (P c.005). kinear effect of parity (P <.02). and Leman, 1986) and maximum litter sizes can be expected in Parities 3 through 6 (Yen et al., 1987). Serum Folates. The FA treatment x parity x sampling time interaction for sow serum folates concentration was not significant; therefore, the data are presented as overall dietary treatment effects over the four parities studied (Figure 1). Effects of FA treatment on serum folates concentration were highly significant ( P <.005). There was a marked decrease in serum folates concentration from mating to d 55 of gestation for all FA treatment groups. The magnitude of this decrease ranged from ng/ml for the unsupplemented sows to ng/ml for the sows on the 4 ppm supplemental FA diets, resulting in a significant FA treatment x sampling time interactions (P c.01). From d 55 to 110, serum folates concentration for the unsupplemented sows seemed to decline further, whereas that for the 4 ppm FA group increased slightly. From d 110 to weaning serum folates concentration increased in a nearly parallel manner for all four treatment groups. Based on studies with rats (Clifford et al., 1990), serum folates concentration is considered a reliable indicator of folate nutritional status and serum folates determination is used in a diagnosis of human folate deficiency. To ensure a reliable serum assessment in this study, the sows were fed the same amount at a similar time daily before mating and during gestation, and samples were collected after a 24-h fast. However, serum folates concentration may have been slightly elevated at mating because 4 to 7 d before mating, the multiparous sows were consuming a three- to fourfold greater quantity of feed while in lactation. Indeed, further analysis indicated that primiparous sows that had been restrictively fed 1.81 kg/d for at least 21 d before mating produced serum folate values at mating I E w 60 i 50 - a 2 40 E 2 30 rl) m Mating Day 55 Day 1 IO Weaning Reproductive Stage AddedFA --to- ---c- 1 m an Figure 1. Serum folates concentration at four stages of the reproductive cycle in Virginia station sows fed different levels (0, 1, 2, and 4 ppm) of supplemental folic acid. Values are least squares means k SE with a total of 1,050 observations. The effect of reproductive stage was significant (P <.005]. Effect of FA supplementation was linear (P <.001) at each reproductive stage. +

5 2342 HARPER approximately 10 ng/ml lower than those produced by multiparous sows (data not shown). But, even in primiparous sows, serum folates concentration was approximately 10 ng/ml less at d 55 of gestation than at mating. The decline in serum folates concentration from mating to mid-gestation is consistent with an original report by Matte et al. (1984a). In that study, serum folates declined from 68.5 ng/ml at mating to a low point of 39.6 ng/ml at d 60 of gestation in sows fed diets containing no supplemental FA. Corresponding values for the control sows in this study were 53.3 ng/ ml at mating and 35.7 ng/ml at d 55 (Figure 1). The higher values for the original report may be explained in part by the higher calculated folacin content of the diet used (.6 ppm of FA), which was fed at a higher rate (2 kg/d). Other workers also have reported lowered serum or plasma folates during mid-gestation (Tremblay et al., 1986; O Conner et al., 1989; Thaler et al., 19891, but differences in experimental protocols make study comparisons difficult. For example, in the original report by Matte et al. (1984a), separate sow groups were used to represent different phases of the reproductive cycle. In the Thaler et al. (1989) study, serum samples during gestation were collected 3 h postprandial, a time when serum folates are likely to be temporally elevated due to rapid feed consumption. Serum folates also were relatively low at d 110 of gestation and substantially increased at weaning for all FA treatment groups. Elevated serum folates at weaning have been reported in other studies (Matte et al. 1984a; Tremblay et al., 1986; Thaler et al., 1989) and probably reflect the three- to fourfold increase in daily feed intake during lactation compared with ET AL. gestation. The fact that lactating sows were given free access to feed and not fasted comparably to gestating sows before sampling also may have contributed to elevated serum folates at weaning time. The results also show that the serum folates status of sows can be altered with dietary FA supplementation. Linear increases ( P <.OO l) in serum folates with increasing FA in the diet were observed at mating, d 55, d 110, and weaning (Figure 1). The lowest concentration observed for the 4-ppm FA treatment (50.0 ng/ml at d 55) was similar to the highest concentration for the unsupplemented controls (53.3 ng/ml at mating). Therefore, FA supplementation attenuated the decline in serum folates concentration associated with mid- to late gestation. Folic Acid Treatment Effects on Sow and Litter Performance. The station x FA treatment interaction was not significant (P >.lo) for the sow and litter traits measured. Therefore, the FA treatment effects are pooled across stations. Sow weight changes, lactation feed intake, and return to estrus interval were not significantly affected (P >.lo) by FA supplementation level (Table 5). There was a numerical trend (quadratic, P <.12) for increased weight gain during gestation with FA supplementation. Sows receiving the 1-ppm supplemental FA level gained 2.58 kg more weight during gestation than the unsupplemented sows. The biological significance of the observed increase in gestation weight gain is not apparent, but Easter et al. (1983) reported a similar finding in gestating gilts supplemented with.2 ppm of FA. Other studies have shown no effect of FA supplementation on gestation weight gain (Lindemann and Kornegay, 1989; Thaler et al., 1989; Table 5. Least squares means of dietary folic acid effects on sow body weight changes, lactation feed intake, and reproductive performancea Supplemental folate level, ppm Criterion SEM Sow data Gestation gain, kg Lactation loss, kg Lactation feed intake to d 21, kgld Days postweaning to estrus Litter size Total born Born alive Day Pig wt, kg Birth, all pigs Birth, live pigs Day Litter wt, kg Birth, all pigs Birth, live pigs Day ameans are weighted by the number of observations with a total of 993 litter observations in the study. Number of observations per folic acid treatment were 366, 144, 352, and 131 for the 0, 1, 2, and 4 ppm treatments, respectively.

6 SUPPLEMENTAL FOLIC ACID AND SOW PERFORMANCE 2343 Matte et al., 1990). Greater body weight loss during lactation with FA supplementation has been reported by Thaler et al. (1989) and Matte et al. (19901, apparently as a result of larger litter sizes being nursed in the FA-treated sows. Dietary FA did not affect (P >.lo) total or live litter size at birth or at d 21 of lactation. Furthermore, individual pig weight and total litter weight across dietary treatments were not different at birth or at d 21 of lactation. Several studies under a variety of experimental conditions have shown improvements in litter size with periodic i.m. injections of FA or FAcontaining compounds (Otel et al., 1972; Matte et al., 1984b; Friendship and Wilson, 1990). Other studies have demonstrated increased litter size at birth (= 1 pig/litter) with continuous dietary supplementation of 1 ppm of FA (Lindemann and Kornegay, 1989) or 1.65 ppm of FA (Thaler et al., 1989). Lindemann (1993) performed a comprehensive review of the literature involving FA supplementation of sows. Although not all reports indicated a significant positive response, the preponderance of data supported FA supplementation of sow diets. This cooperative study attempted to further clarify the merit of adding FA to sow diets and to determine the most appropriate supplementation level. Most reproductive traits evaluated were not affected. Using first-litter gilts, Easter et al. (1983) reported that supplementation of the diet with.2 ppm of FA from d 3 after mating throughout gestation had no significant effect on litter size at birth or at weaning. Gannon and Liebholtz ( 1988) also reported no improvement in litter size when the gestation diet at a commercial pig farm was supplemented with 1 ppm of FA. The reasons for inconsistent litter size responses to FA supplementation among different studies are not clear. It has been well documented that dietary folic acid is critical for normal reproduction and fetal development in such litter-bearing species as rats (Tagbo and Hill, 1977) and guinea pigs (Habibzadeh et al., 1986); this is presumably due to the role of FA as an essential cofactor for nucleic acid synthesis (Stokstad and Koch, 1967; Herbert and Das, 1976) in rapidly developing products of conception. Results from Tremblay et al. (1989) indicate that FA supplementation (5 ppm) may increase survival rate and protein concentration of fetal pigs at d 34 of gestation. Furthermore, the decline in serum folates concentration during pregnancy in sows suggests increased metabolic demand for FA. These factors taken together have led to the hypothesis that FA supplementation is necessary for maximization of litter size in swine. Little is known about storage and folate homeostasis in swine, but it may be that body stores of folate coupled with minor quantities found naturally in feedstuffs result in a deficiency condition during pregnancy that is, at most, marginal. If so, repeated responses to FA supplementation would be unlikely. It seems that FA nutritional status of the sow is not a dominant limiting factor for maximization of litter size under all circumstances. Implications These results corroborate previous findings that serum folates concentration in gilts and sows are decreased during mid-gestation. Furthermore, this decrease in serum folates can be attenuated by folic acid supplementation of the diet over an inclusion range of 0 to 4 ppm. However, a litter size response to folic acid supplementation will not be elicited under all circumstances. Such was the case under the experimental conditions of this cooperative study. Literature Cited Clark, L. R, and A. D. 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