W. C. Weldon3, A. J. "%din4, 0. A. MacDougald, L. J. Johnston5, E. R. Miller and H. A. Tucker Michigan State University6, E. Lansing 48824 ABSTRACT

Thirty-two gilts were used to evaluate the effects of increased dietary energy and CP during late gestation on mammary development. On d 75 of gestation, gilts were assigned randomly in a 2 x 2 factorial arrangement to adequate (5.76 Mcal m / d ) or increased (10.5 Mcal m / d ) energy and adequate (216 g CP/d) or increased (330 g CP/d) protein. On d 105 of gestation, gilts were slaughtered and total mastectomies were performed. Mammary tissue was separated into mammary parenchymal and mammary extraparenchymal stromal tissue and analyzed for DNA, RNA, protein and lipid. No interactions between dietary energy and protein level were detected (P > .20). When adjusted for number of mammary glands and maternal BW (weight of the sow less the weight of the fetuses), mammary parenchymal weight was 27% greater (P < .03)in gilts fed adequate energy than in gilts fed increased energy, but mammary extraparenchymal stroma weight was unaffected by dietary energy level. Total mammary parenchymal DNA was 30% greater in gilts fed adequate energy than in gilts fed increased energy (P < .03).Total mammary parenchymal RNA (P < .02) and total mammary parenchymal protein (P < .02) also were greater in gilts fed adequate energy than in gilts fed increased energy. Dietary protein level did not affect mammary variables measured, except that increased dietary protein tended to reduce mammary extraparenchymal stromal weight (P < .09). Increased dietary protein between d 75 and d 105 of gestation did not benefit mammary development, but increased dietary energy was detrimental to development of mammary secretory tissue. Key Words: Mammary Development, Gilts, Nutrition J. Anim. Sci. 1991. 69:194-200

Introduction

The ability of the sow to produce nutrients for its offspring is dependent on her ability to secrete milk. If milk production of sows is to

lAchowledgement is made to the Michigan Agric. Exp. Sta. for support of this research. %'%eauthors gratefully acknowledge Pa0 Ku for his assistance in the laboratory and Judy Lentz and Kelly Voorhies for their help in preparing this manuscript. 3Current address: Univ. of Nebraska-Lincoln, Anim. Sci. Dept. %o whom reprint requests should be sent. 'Current address: West Central Exp. Sta., Univ. of Minnesota. +kept. of ~ n i m .sci. Received January 11, 1990. Accepted June 14, 1990.

be maximized, feeding strategies during gestation must provide for proper development of the mammary gland, as well as of the sow and her fetuses. Minimal development of the mammary gland of gilts occurs between mating and d 50 of gestation, followed by a fivefold increase in mammaq tissue between d 50 and d 100 of gestation, as measured by total mammary DNA (Hacker and Hill, 1972). Total mammary DNA can be used as an estimate of mammary cell number, and, therefore, mammary development (Tucker, 1987), because DNA per mammary cell is constant during pregnancy and lactation (Tucker and Reese, 1962). Kensinger et al. (1982) found that concentration of DNA in the mammary gland increases more than threefold between d 75 and d 90 of

194

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EFFECTS OF INCREASED DIETARY ENERGY AND PROTEIN DURING LATE GESTATION ON MAMMARY DEVELOPMENT IN GILTS'J

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NUTRITION AND MAMMARY DEVELOPMENT IN GILTS

Materials and Methods

Animals. Thirty-two gilts, purebred Yorkshires and crossbreds that were at least onehalf Yorkshire, were mated during a 4-d period in four separate groups. Gilts were bred on their first estrus after removal from the finishing area. After an entire group was mated, gilts were weighed and moved to an environmentally controlled building and housed in individual stalls. Gilts were fed 1.82 kg/d of a diet formulated to meet NRC (1979) requirements for protein, energy and all other nutrients (adequate protein, adequate energy; Table 1) to d 75 of gestation.

Treatments. On d 75 f 2 of gestation, gilts (average weight 137 kg) were assigned, in a completely randomized design, to one of the four dietary treatments (eight per treatment). Three additional gilts were slaughtered at d 75 of gestation to quantify mammary development at the time when dietary treatments were imposed. Gilts received either adequate or increased CP (216 or 330 gld, respectively) and adequate or increased energy (5.76 or 10.5 Mcal ME/d, respectively) in a 2 x 2 factorial arrangement (Table 1). Dietary protein level was manipulated by changing concentrations of corn and soybean meal. Dietary energy level was changed in a similar fashion, by addition of cornstarch. Daily allotment of feed per gilt also was altered to achieve dietary intakes of CP and energy; daily intake of supplemental vitamins and minerals was held constant across all treatments. Lysine intake was held constant across treatments of similar protein level by the addition of L-lysine.HC1. Slaughter. On d 105 f 2 of gestation, animals were electrically stunned and exsan-

TABLE 1. COMPOSITION OF EXPERIMENTAL DIETS Diet Item Ingredient, % Corn Soybean meal, 44% CP Mono-dicalcium phosphate Ground limestone. Vitam&TMpremixa vitamin E-Se premixb salt

Adequate protein Adequate energy

85.4 10.1 1.85 1.22

.so SO

SO

Cornstarch L-lysineHC1 Total calculated analyses ME, McavLg CP, % Lysine, % Ca, %?

P,% supplied daily/glIt Feed,kgld ME,Mcal/d CP, gld Lysine, gJd

100.0 3.14 11.9

Adequate protein Increased energy

72.2 1.30 .93 .76 .35 .30 .29 23.6 .20 100.0

.70

3.34 6.9 .29 .50 .41

1.84 5.76 216 9.09

3.15 10.50 218 9.11

SO .&I

Increased protein Adequate energy

68.6 27.0 1.57 1.23 .62

SO SO

Increased protein Increased energy

93.2 4.31 .48 .99 .28 .28 .27 .24

100.0 3.12 17.9 .94 .84

.70 1.84 5.16

330 17.37

100.0 3.21 10.1 .53 .47 .39 3.27 10.50 330 17.37

Tomposition per kilogram premix: vitamin A, 661,380nr; vitamin D, 132,276 N, menadione, .66g, riboflavin, .66 g, niacin 3.53 g, d-pantothenic acid, 2.64 g, choline chloride, 88.18g; vitamin B12. 3.96 mg, Zn,7.5 g; Mu, 7.5 g, I, .ll g; Cu, 2 g; Fe, 12 g. bComposition per kilogram premix: vitamin E, 3,310 Iu; Se, 19.8 mg.

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gestation. Therefore, the period between d 75 and d 90 is a critical period in development of milk secreting tissue. Currently, little is known about the effects of nutrition on mammary development during pregnancy. This study was designed to determine the effects of increased dietary energy and protein during late gestation on the development of the mammary gland of gilts.

196

WELDON ET AL. TABLE 2. CHARACTERISTICS OF MAMMARY GLANDS OF GILTS AT TWO DIFFERENT STAGES OF GESTATION

15

No. of gilts Avg wt, kg Parenchymal wt, g Extraparenchymal stroma,g Parenchymal DNA, mg/g Total parenchymal DNA, g Parenchymal RNA, mg/g Total Parenchymal RNA, g Parenchymal protein, mg/g Total parenchymal protein. g Parenchymal lipid, mg/g Total parenchymal lipid, g

3 133.2 478 410 .71 .38

.64 .36 27.4 13.4 446 216

guinated. After exsanguination, total mastectomies were performed and the mammary glands were immediately frozen in a mixture of dry ice and acetone. Glands were stored at -21°C until analyses were performed. Individual fetuses were counted, weighed, and euthanatized. Dissection. Frozen mammary glands were cut into 1.5cm slices. Right-side glands were separated into mammary parenchymal and extraparenchymal stromal tissue, free of skin. Tissues could be distinguished by differences in color. Tissues were weighed and stored at -21°C until they were homogenized. All tissues from right-side mammary glands were immersed in liquid N for 10 min and forced through a 1.25-cm grid. The frozen crumbles then were homogenized in a Waring blender at a high speed at -21°C until all tissues was a fiie powder. The powdered sample was mixed and a representative sample was obtained from the frozen powdered tissue. Chemical Analyses. Mammary parenchymal and extraparenchymal stromal tissues were analyzed for DNA, RNA and lipid as described by Tucker (1964). Protein was determined using the method of Lowry et al. (1951) with bovine serum albumin as the standard. Statistical Analyses. Data were subjected to analysis of variance using procedures of SAS (1982). The data were analyzed as a factorial with protein and energy intakes as main effects. Breed of sow, period of breeding and the number of fetuses were included in the original model as covariates. Because these latter terms did not account for a significant

SD

105

SD

5 .o 88 109 .35 .22 .ll .03 5.8 5.2 79 75

32 153.4 1,012 686 2.87 2.91 3.52 3.64 146.0 147.6 140 138

10.4 275 158 .68 .97 .63 1.20 29.3 51.6 2.0 30

portion of the variation, .they were removed from the model used for the final analysis of variance. Results and Discussion

Characteristics of total mammary tissue of gilts at d 75 and d 105 are listed in Table 2. These data support the contention that the period between d 75 and d 105 of gestation is a major period of mammary development in swine. Gilts killed on d 75 were used as a reference point and were not included in the statistical analyses. There was no evidence of an interaction (P > .20) between dietary energy and protein level for the traits measured. Therefore, only main effects are reported. Mammary data are expressed per functional mammary gland (all teats associated with secretory tissue) per kilogram maternal BW (weight of the sow less the weight of the fetuses) to account for differences in mammary number and maternal BW. Mammary Development. Mammary parenchymal weight was 27% greater for gilts fed adequate energy than for gilts fed increased energy (Table 3). These results agree with previous results form prepubertal heifers (Sjersen et al., 1982) and lambs (Johnson and Hart, 1985) and indicate that the weight of the mammary parenchyma is reduced by feeding increased levels of energy. The amounts of protein or energy fed did not affect nucleic acid concentration of the mammary parenchymal tissue (Table 3).

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Day of gestation Item

197

NUTRITION AND MAMMARY DEVELX)PMENT IN GETS TABLE 3. EFFTCTS OF DIETARY PROTEIN AND ENERGY INTAKE ON THE CONSTITUENTS OF THE MAMMARY PARENCHYMAL TISSUE OF GILTS&

Adequate

Increased

sow wt, kg 149.5 158.2 No. of mammary glands/@td 6.94 7.13 Constituents of mammary parenchymal tissue Weight, g" 1.12 .88 DNA, mg/g 2.91 2.88 RNA, mP/g 3.64 3.48 RNA/DNA 1.28 1.28 protein, &g 137.2 115.0 149.7 Lipid, mg/g 135.9 Total DNA, mg 3.22 2.48 Total RNA, mg 4.w 3.09 Total protein, g .16 .IO Total lipid, g .15 .13

pe

Protein

Adequate

Increased

Energy

Protein

SE

156.1 7.00

151.6 7.06

.a?

.20 .75

2.43 .14

1.05 2.86 354 127 118.1 136.6 2.99 3.76 .13 .14

.95 2.83 3.58 1.29 134.1 149.0 2.71 3.41 .13 .14

.28 .87 .87 .77 .17

.07 .15 -15 .05 7.92 5 .00 .22 .28 .01 .01

.34

.a? .54 .47 .95

.06 .06 .03 .02 .02 .15

.09 .40 .38 .85 .79

b a t a on the constituents of mammary tissue are expressed per functional gland per kilogram maternal BW. based on right-side mammary glands. 'Probability level. dNo. of right-side mammayy glands associated with secretory tissue. %immed weight of parenchymal tissue.

Therefore, the 23% decrease (P< .03) in total DNA with extra energy reflects a difference in total amounts of secretory tissue. These results are consistent with the results from prepubertal heifers fed high or low planes of nutrition (Sjersen et al., 1982). Protein concentration tended to be lower and lipid concentration tended to be higher in the mammary parenchyma (Table 3) of gilts fed increased energy than in that of gilts fed adequate energy. Therefore, gilts fed increased energy had more (P < .06) fatty tissue in the mammary parenchyma (15 vs 14%). Protein concentration in the mammary parenchyma was not affected by dietary protein level. In contrast, lipid concentration in the gland tended (P < .09) to be increased by increased dietary protein. Feeding increased dietary energy resulted in reduced (P < .03)total DNA, RNA and protein of the mammary parenchymal tissue (Table 3). There was no difference in total lipid because gilts fed increased energy had less parenchymal tissue with a higher concentration of lipid than gilts fed adequate energy. The effects of increased dietary energy intake on total DNA, RNA and protein observed in this study are similar to results observed in prepubertal heifers (Sjersen et al., 1982) and sheep (Johnsson and Hart, 1985). Although these cited studies were performed

during the prepubertal growth period, the factors involved may be similar. Heifers (Sinha and Tucker, 1969a) and rats (Sinha and Tucker, 1969b) both undergo an allometric mammary growth phase just prior to puberty. The allometric growth phase appears to be related to the secretions of the ovary, because ovariectomy abolishes this allometric phase (Cowie, 1949). Also, this allometric growth phase coincides with the development of estrogen receptors in the mammary gland of mice (Muldoon, 1979). Therefore, it has been postulated that ovarian secretions, primarily estrogens, play a key role in stimulating this phase of growth. Prepubertal allometric growth of the mammary gland has not been studied in swine, but a similar phenomenon occurs in the development of porcine mammary glands during pregnancy. There is little change in the DNA content of the mammary gland up to d 50 of gestation (Hacker and Hill, 1972; Kensinger et al., 1982). However, at approximately d 75 of gestation, rapid proliferation of mammary tissue occurs. Maximal DNA content occurs at d 90 to d 100 of pregnancy (Hacker and Hill, 1972; Kensinger et al., 1982). It has been suggested that the allometric mammary growth during pregnancy in swine also is signaled by estrogen secretion, primarily from the placental tissue (Kensinger et al., 1982; DeHoff et al.,

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Energy Item

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WELDON ET AL.

TABLE 4. EFIWTS OF DIETARY PROTEIN AND ENERGY INTAKE ON THE CONSTITUENTS OF MAMMARY EXTRAPARENCHYMAL STROMAL TISSUE IN GILTS*

Pd

Rotein

Adequate

Increased

Adequate

Increased

Energy

Protein

SE

Weight, 8 DNA, mg/g RNA, mg/g RNA/DNA Protein, mg/g Lipid, mg/g Total DNA, mg Total RNA, mg Total protein, g Total lipid, g

.68 .I12 .349 3.23 3 1.70 565 .07 .23

.67 .116 .354 3.12 27.06 598 .08

.72 .lo7 ,342 3.26 28.61 575 .08 .23

.63 .118 .361 3.12 30.15 589 .07 .23 .a2 .37

.92 .60 .89 .95 .12

.09 .36 .62 .77 .59 .77 .71

.04 .009 26.91 .19 2.04 19.1 .22 .01 .001 .03

.a2 .39

24

.a2 .40

.a2 .41

24

.91 .94 .12 .74

.48 .34 .25

%ata on the constituents of mammeyl tissue are expressed per functional gland per kilogram maternal BW. %sed on right-side mammary glands. 'See Table 3 for average gilt weights and average number of mammary glands/gdt. dRobability level. "Weight of non-secretory tissue less skin, muscle and lymph nodes.

1986). Thus, control of prepubertal allometric growth and allometric growth during pregnancy in swine may be similar and interactions between plane of nutrition and the hormonal control of mammary growth may be comparable. In ruminants, high dietary energy decreases serum growth hormone concentrations (Sjersen et al., 1982; Johnsson et al, 1985). These decreases in growth hormone have been associated with reduced total DNA content of the mammary gland, indicating that it has a detrimental effect on mammary development. Injections of growth hormone in prepubertal lambs allowed to consume feed ad libitum restored DNA in the mammary gland to levels similar to that of animals fed a restricted diet (Johnsson et al, 1986). Total mammary parenchymal DNA was highly correlated (R = .95) to plasma growth hormone concentrations. Hence, growth hormone concentrations may have significant effects on the magnitude of the prepubertal growth phase in ruminants. During pregnancy, plasma concentrations of growth hormone remain constant in swine; therefore, growth hormone is not thought to be mammogenic in the pig during pregnancy (DeHoff et al., 1986), but the effects of different plasma concentrations of growth hormone have not been established. During fasting, growth hormone concentrations in the pig increase, with the length of fasting being related inversely to blood glucose concentration (Machlin et al., 1967). Diet-induced

differences in growth hormone concentrations may occw, they may play a role in enhancing mammary development in pregnant gilts fed adequate levels of dietary energy. Dietary protein level had no effect on total DNA, RNA, protein or lipid in the parenchymal tissue (Table 3). Chew et al. (1984) reported that high protein levels had adverse affects of total mammary DNA in prepubertal rats. In that study, rats fed to meet their protein requirement had greater total mammary DNA than rats fed either high or low protein. These data support speculation that the effects of protein on mammary development are small unless diet become either severely restricted or are in great excess of the requirement. Treatments in the present experiment, however, consisted of adequate and increased protein levels. Our results indicate that excess protein is of no benefit and may, in fact, reduce total DNA in the mammary parenchyma, although this effect was not significant (P > .20). Protein levels suggested by NRC (1979) appear sufficient to meet the amino acid requirements of gilts for mammary growth. Components of the mammary extraparenchymal stromal tissue (Table 4) were not affected by dietary treatment. These results agree with data previously reported for prepubertal heifers by Sjersen (1981). It is not surprising that dietary protein and energy levels had no effect on the composition of the mammary extraparenchymal stroma, because this tissue was composed mainly of lipid (56 to

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Energy Item

NUTRITION AND MAMMARY DEVELOPMENT IN GILTS

Implications

Feeding a high level of energy during periods of rapid mammary growth in swine impaired development of the mammary gland in gilts. This reduction in mammary cell

number may reduce the amount of milk produced by the sow during lactation for nourishment of the offspring. Llterature Cited

chew,B. P.,C. S. Zamora and T. S. Tanaka. 1984. The effects of dietary protein on mammary gland development in rats. J. Dairy Sci. 67(Suppl. 1):126 (Abstr.). Cowie. A. T. 1949. The relative growth of the mammary gland in normal, gonadectomized and adrenalectomized rats. J. Endocrinol. 6:145. DeHoff, M. A., C. S.Stoner, F.W. Bazer, R. J. Collier, R. R. Kraeling and F.C. Buommo. 1986. Temporal changes in steroids, prolactin and growth hormone in pregnant and pseudopregnant gilts during mammogenesis and lactogenesis. Domest. Anim. Endocrinol. 3:95. Hack. R. R. and D. L.W. 1972. Nucleic acid content of mammary glands of virgin and pregnant gilts. J. Dairy Sci. 55:1295. Johnsson, I. D. and I. C. Hart. 1985. &pubertal mammogenesis in sheep. 1. The effect of level of nutrition on growth and mammary development in female lambs. Anim. Prod. 41:323. Johnsson. I. D., I. C., Hart, A. D. Simmonds and S. V. Morant. 1985. Repubertal mammogenesis in sheep. 2. The effects of level of nutrition on the plasma concentrations of growth hormones, insulin and prolactin at various ages in female lambs and their relationship to mammary development. Anim. Prod. 41:333. Johnsson, I. D., I. C. Hart and A. Turvey. 1986.prepubertal mammogenesis in sheep. 3. The effects of restricted feeding on daily administration of growth hormone and bromocryptine on mammary growth and morphology. Anim. Prod. 4253. Kensinger, R. S., R. J. Collier,F.W. Bazer, C. A. Ducsay and H. N. Becker. 1982. Nucleic acid, metabolic and histological changes in gilt mammary tissue during pregnancy and lactogenesis. J. Anim. Sci. 541247. Lowry, 0.H.. N. J. Rosebrough, A. L. Farr and R J. Randell. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265. Machlin, L. J., M. Horino, F.Hertelendy and D. M. Kipnis. 1967. Plasmagrowthhormone and insulin levels in the pig. Endocrinology 82369. Muldoon, T.G. 1979. Mouse maommy tissue estrogen receptors: ontogeny and heterogeneity. In: T. H. Hamilton, J. H. Clark and W. A. Sadler (Ed.) Ontogeny of Receptors and Reproductive Hormone Action. Raven Press, New York. NRC. 1979. Nutrient Requirements of Swine (8th Ed.). National Academy Press, Washington, DC. SAS. 1982. SAS User's Guide: Statistics. SAS Inst., Inc., Gary, NC. Sinha, Y. N., and H. A. Tucker. 1969a. Mammary development and pituitary prolactin level of heifers from birth to puberty and during the estrous cycle. J. Dairy Sci. 52507. Sinha, Y. N. and H. A. Tucker. 1969b. Relationship of pituitary prolactin and LH to mammary and uterine growth of pubertal rats during the estrous cycle. Proc. SOC. Exp. Biol. Med. 131:908. Sjersen, K. 1981. Mammary development in relation to plane of nutrition and serum hormone concentrations

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60%). One might expect nutritional factors to affect the weight of the fatty tissue associated with the mammary gland, but not the composition of adipose gain. Mammary extraparenchymal stroma weight was not affected by increased energy, but gilts fed increased protein tended to have less weight of stroma. Neither dietary energy nor protein level affected total DNA, RNA, protein, lipid or the concentrations of these components in the extraparenchymal stroma (Table 4). This is in agrement with the findings of Sjersen (1981) that plane of nutrition had no effect on DNA, RNA, or lipid content in the mammary extraparenchymal stroma of prepubertal heifers. The Sow and Conceptus. There was no effect of dietary energy or protein level on the number or weight of fetuses. Protein level had no effect on weight of sows at d 105 flable 2). Sows fed increased energy during gestation were heavier (158.2 vs 149.4 kg; P = .02)at d 105 of gestation than sows fed adequate energy. In conclusion, the NRC (1979) requirements for pregnant gilts are sufficient to allow normal development of the gilt, her mammary glands and the fetuses up to d 105 of gestation. A high level of dietary energy during the period of maximal mammary development in swine (d 70 to d 105 of gestation) reduced total mammary parenchymal DNA. This reduction in DNA reflects a reduced mammary cell number flucker, 1987), which may impair subsequent lactational performance. Increased dietary protein is of no benefit to mammary development. Effects of nutrition on mammary development before d 75 and subsequent to d 105 have yet to be determined. Few data exist for the effects of diet on development of mammary tissue during pregnancy. More information is needed to understand better the mechanisms by which nutrition influences mammary development during pregnancy and the extent to which mammary development influences milk production.

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gland. Roc. Soc. Exp. Biol. Med. 16218. Tucker. H. A. 1987. Quantitative estimates of marmflary growth during various physiological states: A review. J. Dairy Sci. 70:1958. Tucker, H. A. and R. P. Reece. 1962.Nucleic acid estimates of mammary tissue and nuclei. Roc.SOC.Exp. Biol. Med. 111:639.

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in pre- and post-pubertal heifers. B . D . Dissertation. Michigan state univ., East Lansing. Sjersen, K., J. T. Huber, H. A. Tucker and R. M.Akers. 1982.Influence of nutrition on mammary development in pre- and post-pubertal heifers. J. Dairy ScL 65:793. Tucker, H. A. 1%. Influence of number of rmckling young on nucleic acid content of Ihe lactating rat mammary

Effects of increased dietary energy and protein during late gestation on mammary development in gilts.

Thirty-two gilts were used to evaluate the effects of increased dietary energy and CP during late gestation on mammary development. On d 75 of gestati...
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