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Effect of Plane of Nutrition on Growth and Mammary Gland Development in Holstein Heifers
Effect of Plane of Nutrition on Growth and Mammary Gland Development in Holstein Heifers K. STELWAGEN and D. G. GRIEVE Department of Animal and POUltry Science University of Guelph Ontario, Canada N1G 2W1 ABSTRACT
Forty-one Holstein and 6 Holstein crossbred heifers, 6 to 8 mo of age, were used to determine the effect of plane of nutrition on growth and mammogenesis prior to and during puberty. Animals were fed to gain 611 g (low), 737 g (medium), and 903 g (high) by a diet of cracked com and chopped alfalfa-grass hay. Mammary biopsies were carried out in vivo to determine if they provide acceptable infonnation on mammary composition (based upon morphometric evaluation) in comparison with dissected glands. Results indicated that NRC (1978) recommendations for average daily gain of Holstein heifers between 6 and 16 mo may be too generous. At puberty (first estrus), age and wither height decreased linearly with increasing plane of nutrition, whereas body weight and hip height were not affected by plane of nutrition. Five heifers were slaughtered at the beginning and 18 at the conclusion of the trial. Increasing plane of nutrition resulted in fatter mammary glands with decreased concentration of DNA, whereas total mammary DNA did not differ among treatment groups. In this study, morphometric evaluation of mammary tissue obtained through biopsies did not yield useful infonnation in comparison to chemical analysis of dissected glands. (Key words: mammogenesis, plane of nutrition, growth) INTRODUCTION
Normal age at frrst parturition in large dairy breeds is between 2 and 3 yr (25, 28); however,
Received April 10, 1989. Accepted April 19. 1990.
1990 1 Dairy Sci 73:2333-2341
heifers can calve at 2 yr of age or less (11, 25, 29). Early calving reduces the unproductive period of the dairy heifer, which implies that less feed is required to raise it to productive age. Because 40 to 70% of the total costs of raising replacement heifers can be attributed to feed costs (1), a reduction would have a positive impact on fann cash flow. To accomplish the objective of early parturition, heifers should be bred at an early age. Feeding heifers a high plane of nutrition, allowing for rapid growth after weaning, can lower the age at which sexual maturity occurs (2, 25, 29). However, Gardner et al. (8) and Little and Kay (18) observed lower milk production from heifers raised on a high plane of nutrition than that from their counterparts raised on a more moderate feeding regimen. A recent study by Gardner et al. (9) seems to contradict these earlier results; however, results in this study may have been confounded by differences in nutrient composition of the control and treatment diets. Depressed milk production may be caused by incomplete mammary development in heifers raised on a high plane of nutrition (11). Most reliable infonnation on mammary gland development has been obtained by dissecting glands of slaughtered animals. Such experiments are costly, particularly if many animals are involved. Furthennore, studying mammary development on the same animal during successive intervals becomes impossible. For these reasons, in vivo techniques for mammary gland evaluation are more desirable. The objective of the study was 1) to investigate growth and mammary development in Holstein heifers before and during puberty, and 2) to discover whether or not mammary biopsies carried out on live animals provided acceptable information on mammary composition (based upon morphometric evaluation) in comparison with chemical analysis of dissected glands from slaughtered animals.
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STELWAGEN AND GRIEVE
MATERIALS AND METHODS
TABLE 1. Nubient composition of trace-mineral salt, alfalfa grass hay, and cracked com.
Animal Management Composition
The experiment was designed as a randomized complete block design (RCBD) with four blocks and involved 47 dairy heifers, which were divided into a production (P) and slaughter (S) group. The P group comprised 18 female Canadian Holstein calves (block 1, 9 calves of approximately 8 mo of age; block 2, 9 6-mo-old calves); and block 3, 6 female New Zealand X Canadian Holstein crossbred calves of 6 to 8 mo of age. The S group consisted of 23 6- to 7-mo-old Canadian Holstein heifers, of which 5 heifers were randomly chosen to be slaughtered (stunned with captive bolt) at the beginning of the experiment. The remaining 18 S heifers (block 4) were slaughtered after 279 d on trial. Animals were housed in individual stalls at the University of Guelph Dairy Research Station and were removed from the experiment immediately after the second biopsy had been performed on each animal within a block, which was 230 d (block 1), 347 d (block 2 and 3), and 279 d (block 4) after the beginning of the experiment, Le., when the animals were about 16 mo of age. During the entire experiment, all animals were subjected to artificiallighting daily from 0500 to 1800 h. Heifers in block 4 were also exposed to daylight during the first 183 d (June to December) on trial. Animals were fed a diet containing cracked com, chopped (2.5 cm) alfalfa grass hay, and trace-mineral salt (Table 1) and had access to fresh water at all times. To feed each animal the same hay to com ratio at the same age and to allow for realistic intakes, the hay to com ratio was changed four times. From 6 to 7 mo of age, this ratio was 70:30, from 8 to 9 mo 65: 35, from 10 to 12 mo 60:40, and from 13 mo to the end of trial 55:45. The trace-mineral mix was fed at .25% of daily OM intake. Based upon these feedstuffs and NRC (20) recommendations for growing heifers, feeding amounts were established to allow for an average daily gain (AOO) of 600 g (L, low treatment), 750 g (M, medium treatment), and tOOO g (H, high treatment). Heifers were randomly allotted to one of the three feeding regimens. Feed intake and orts were recorded for each animal. Diets were adjusted biweekly according to age and every 4 wk according to age and Iournal of Dairy Science Vol. 73,
No.9, 1990
DM. % Cp,! % ADp 1 % ME, {,2 MI/kg NaCl, % Zinc, ppm Iron, ppm
Manganese, ppm Copper, ppm Iodine, ppm Cobalt, ppm
Mineral salt
Hay
Com
91.5 15.6 43.6
87.1 9.4 3.5 14.3
8.0 96.5 4000 1600 1200 330
70 40
IMeasured in DM. 2caIculated from ADP (details in text).
weight gain during the previous 4-wk period. Com and hay were sampled weekly, frozen, and composited in 4-wk intervals. Subsamples were analyzed for DM, CP, and ADF according to the procedures of AOAC (4). Metabolizable energy (ME) was calculated from the percentage of ADF in DM by means of equations from the Ontario Ministry of Agriculture and Food: com: %1DN = 92.22 - (1.535x%ADF); mixed hay: %1DN = 92.62 - (.9093x%ADF), DE (Mcallkg) = .4409x%1DN, and ME (Mcal/kg) = .45 + (1.0IxDE). Live weight and body height at the withers and hip of each animal were measured on two consecutive days at the beginning and end of the trial and at 28-d intervals during the trial. Mammary Biopsies
All experimental animals, except for those slaughtered at the beginning of the trial, were subjected to mammary biopsies to obtain tissue samples for morphometric evaluation. The first biopsy (B1) was carried out when animals were confirmed to have estrous cycles (i.e., attained puberty). Confirmation was based upon blood plasma progesterone concentrations (6) 0btained from weekly jugular vein blood samples. Progesterone concentrations greater than 1 ng/ ml were considered to be indicative of a ftmctional corpus luteum (22, 25). A second biopsy (B2) was performed 2 mo after Bl. The S heifers underwent a third biopsy (B3) 3 d before being slaughtered, except for six animals
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MAMMOGENESIS AND PLANE OF NUTRITION
in which B3 coincided with B2. All biopsies were performed during the luteal phase of the estrous cycle. Biopsies were taken approximately 3 cm above the right rear teat (B 1 and B3) and left rear teat (B2). After preparation of the operative area and application of local anesthesia, a small incision (l to 1.5 cm) was made and a disposable biopsy needle (fru-cut biopsy needle, Travenol Laboratories, Inc., Deerfield, IL) (l5.2-cm cannula and 20-mm specimen notch) was inserted to remove a tissue sample. Tissues were immediately washed in saline and ftxed in Bouin's fluid. After 45 min, excessive fIxative was removed with 70% ethanol. The tissue was stained in Ehrlich's hematoxylin, eosin, and phloxine; embedded in paraffin; and cut into 6-1J.IIl slices. Slices were examined under a light microscope (100)<) equipped with a grid having 344 visible intersections. Adipose, remaining connective tissue, epithelial cell area, and ductolar-luminal area were quantified by counting intersections overlying each of the cell types. Cell types at the top and bottom of each of two fields per biopsy were counted. Chemical Analysis of Mammary Tissue
The S group heifers were slaughtered during the luteal phase of the estrous cycle. Immediately after exsanguination, mammary glands were detached from the body cavity and separated along the median suspensory ligament. The right half was subsequently trimmed from skin and teats and weighed to obtain trimmed half gland weight (TrGIWt) and frozen in liquid nitrogen to be stored at -ISoC until chemical analysis. All analyses were performed on ground lyophilized tissue. Dry matter was determined by drying for 2 h at 135°C and fat according AOAC (4). Twenty-gram samples of lyophilized tissue were subjected to a 5-h fat extraction followed by a 3-h extraction with anhydrous ether (4), and the remaining tissue was dried overnight in a fume hood yielding dried fat-free tissue (DFFf). Crude protein (4) and DNA content were determined according to methods of Martin et al. (19) in aliquots of finely ground DFFf. Relative Growth Coefficients
Relative growth coefficients for DNA and DFFf were calculated to estimate the rate of
increase of these parameters in relation to increases in metabolic live weight. Calculations were similar to those outlined for heifers (26) and lambs (14). Statistical Analysis
Growth and biopsy data were analyzed using the model: Yijk = J1 + T i + Bj + (TB)ij + &jk and for mammary composition data: Yij = J1 + Ti + E;.J' where Yij(k) = adjusted value for observations ij(k) of the dependent variable; J1 = true mean; Ti = treatment L, M, or H; Bj = block 1,2,3, or 4; (TB)ij = treatment by block interaction; and E;.j(k) = residual error of observation ij(k). Biopsy data were expressed as a percentage of the total number of counts, and results from Bland B2 were analyzed as two separate data sets. Prior to analysis of variance, biopsy data were transformed, using the In (x + 1) transformation (27) to stabilize the variance. Analyses were performed using the general linear models procedure of SAS (24). Results were considered significant at ~.05. When a significant treatment effect occurred in the absence of a significant treatment by block interaction, the treatment effect was partitioned into linear and nonlinear components. RESULTS AND DISCUSSION
One animal on the H treatment died during the ftrst half of the experiment from causes unrelated to the experiment, and data were not included in the analyses. Significant differences in DM intake (Table 2) among treatment groups were expected, because differences in ADG could only be obtained by feeding the animals different quantities of the same diet in order to prevent any potentially confounding effects of different protein to energy ratios. Recent work with rats (21) suggested that different dietary protein and energy intakes may influence mammogenesis. Actual ADG for heifers on the L or M treatments was in agreement with proposed ADG for these animals (Table 2). However, despite significantly higher intakes, H heifers failed to attain the target growth rate. Nevertheless, actual ADO (903 g) of H heifers in this experiments can be qualified as high for study of the effects of rapid rearing on mammary gland development (1). Journal of Dairy Science Vol. 73,
No.9, 1990
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STELWAGEN AND GRIEVE
TABLE 2. Average daily DM intake and proposed and actual ADO for slaughter and production group heifers. Treatment Variable n DM Intake, kg/d Actual ADO, g Proposed ADO, g Difference ADO, g
lL
= Low,
M
= medium,
14 4.6 611 600 +11 H
= high
M
H
SEM2
Lin3
14 5.7 737 750 -13
13 7.5 903 1000
.1 32.0
• •
..JT1
IIeatmenl.
2SEM averaged over treatments.
~in = Linear treatment effect. ·P<.Ol.
Average age at the beginning and conclusion of the experiment as well as initial and final body weight and height measured at the withers and hip are given in Table 3. Final body weight increased in a linear way with increasing plane of nutrition. No treatment effect was noted on wither height or hip height at the conclusion of the trial. Final body weight and wither height of heifers in this trial are in agreement with body growth data for Holstein heifers of similar age (12) and with heights and weights of 16-mo-old Holstein heifers derived from growth charts used by the Ontario Ministry of Agriculture and Food (3). This indicates that the low plane of nutrition was sufficient for skeletal and body development in Holstein heifers between 6 and 16 mo of age. Therefore, NRC (20) recommendations for AOO of heifers of large dairy breeds (700 to 800 g) may be too generous. At the beginning of the trial, on the basis of blood plasma progesterone, five animals had apparently already attained puberty (i.e., frrst estrus). The average age, body weight, and height of the remaining animals at the time they attained puberty are shown in Table 4. Results from this study support earlier findings that age at puberty declines with increasing feed intake (2, 17, 23), whereas body weight at puberty was not significantly affected by feeding (17, 23). Wither height decreased linearly with increasing plane of nutrition, which is in agreement with observations made by Amir et al. (2) and Larsen et al. (17). Why hip height was unaffected by feeding level is unknown. Attainment of puberty seems to be determined by body weight rather than chronological age and Journal of Dairy Science Vol. 73,
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can be manipulated by the feeding regimen. Mammary biopsy data for epithelial cell area, ductular-Iuminal area, adipose cell area, and remaining connective tissue cell area were expressed as mean percentage of the total area identified of each of four fields per biopsy slide. Transformed (In {x + I}) means of B1 and B2 are reported in Table 5. Within each biopsy (Bl or B2), no significant effect due to plane of nutrition was noted for any of the parameters measured (fable 5). Neither did any statistical evidence emerge of changes in the area of different tissue types between the time heifers attained puberty (B 1) and 2 mo later (B2). Chemical analyses of dissected mammary glands from S group heifers are shown in Table 6. Data of the initial slaughter group serve as reference data. Trimmed gland weight (frGIWt) increased linearly with increasing plane of nutrition. Fat and DM also increased linearly, explaining the increases in TrGIWt, because DFFT and CP in DFFT did not significantly differ between feeding regimens. Deoxyribonucleic acid is often used to estimate mammary cell numbers (15). In this study, total mammary DNA was measured instead of DNA in parenchyma that was visually separated from extra parenchymal tissue (22, 25). Due to the way mammary parenchyma branches into the surrounding fat pad, extraparenchymal tissue can never be completely separated visually from parenchymal tissue. Either true parenchyma will be removed with the extraparenchymal adipose tissue or extraparenchymal adipose tissue may unavoidably be included in parenchymal tissue, thus diluting
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MAMMOGENESIS AND PLANE OF NUTRITION
TABLE 3. Average initial and final age, body weight, wither' height (WHT), and hip height (HH'I) for slaughter and production group animals. Treatment
L1
Variable No. cows
SEM
M
14
SEM
H
14
SEM
13
Initial age, d Final age, d
185 477
11 5
194 487
11 5
185 473
12 5
Initial BW, kg Final BW. kg
199.1 378.7
13.2 7.1
204.0 420.6
13.2 7.1
187.8 447.2
13.7 7.3
Initial WHT, em Final WHT, em
106.0 127.6
1.9 .6
105.6 127.9
1.9 .6
104.1 127.7
2.0 .6
NS
Initial HHT, em Final HHT, em
112.0 130.5
2.0 .6
111.5 131.1
2.0 .6
110.6 131.5
2.1 .6
NS
= =
•
=
IL Low, M medium, H = high treatment. 2Lin Linear treatment effect.
·P<.OOL
parenchymal DNA. Total mammary DNA (Table 6) was not affected by feeding. We expected that animals on the M and H planes would have had less mammary DNA, indicating a lower potential for future milk production. However, the inclusion of extraparenchymal adipose cell DNA may explain the lack of effect of plane of nutrition on total mammary DNA. Heifers receiving the M and H plane of nutrition had 57 and 129% more mammary fat than heifers on the low plane (Table 6); a larger proportion of the total mammary DNA probably consisted of nonparenchymal DNA, since increases in adipose tissue in young developing animals can, at least in part, be ascribed to increases in adipose cell number
(5, 13). Thus, although total mammary DNA did not differ among treatment groups, the amount of adipose tissue significantly decreased with decreasing plane of nutrition, indicating indirectly a larger proportion of parenchymal DNA for animals on the lower planes of nutrition. Mammary DNA expressed per unit of TrGIWt or DFFT declined with increasing plane of nutrition, suggesting also a potential adverse effect of feeding on mammogenesis. Although these results are in agreement with similar adverse dietary effects on mammary DNA observed in heifers (22, 25) and lambs (14), the current data represent total mammary DNA, i.e., parenchymal and nonparenchymal DNA.
TABLE 4. Average age. body weight, wither height (WH1), and hip height (HH'I) of slaughter' and production group heifers at attainment of puberty. Treatment Variable
LI
SEM
M
SEM
H
SEM
Lin2
n Age, d BW,kg WHT. em HHT, em
11 365 284.9 121.5 125.4
13 12.1 1.1 1.0
12 313 283.7 118.0 122.7
12 11.6 1.0 1.0
13 305 298.0 117.6 123.0
12 11.2 1.0 1.0
• NS ••
NS
= Low, M = medium, H = high treatment. 2Lin = Linear treatment effect. lL
·P<.05. ··P<.01. Journal of Dairy Science Vol. 73,
No.9. 1990
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STELWAGEN AND GRIEVE
TABLE 5. Tnmsformed (In [x + 1)) means of percentage epithelial. adipose, remaining connective tissue area, and ductuIar-luminal space obtained through mammary biopsies (Bl and B2). Treatment SEM2
r.m3
2.34 2.48 +.14
.24 .31 .41 4
NS NS NS
3.53 3.67 +.14
3.50 3.10 -.40
.22 .21 .304
NS NS NS
3.79 3.86 +.07
3.80 3.62 -.18
3.78 3.75 -.03
.12 .14 .204
NS NS NS
.73 .71
.92 .71 -21
.90
.94
.18 .18 .25 4
NS NS NS
Variable
Biopsy
L1
M
H
No. heifers
Bl B2
14 13
14 14
13 13
Epithelium
Bl B2 B2-81
2.10 2.35 +.25
2.36 2.32 -.04
Adipose
BI B2 B2-Bl
3.37 3.31 -.06
Connective
Bl B2 B2-Bl
Ductular-Iuminal
Bl B2 B2-81
-.02
+.04
lL = Low, M = medium, and H = high treatment. 2sEM averaged over treabncnts. 3r.in = Linear treabnent effect.
4standard
errors of difference averaged ovee treabnents.
TABLE 6. Chemical analysis of mammary gland tissue of slaughter group heifers expressed as total amounts, per unit bimmed half gland weight (TrGlWt). and dried fat-free tissue
Initial slaughter group
SEM
L1
M
H
SBM
Lin2
5 630.0 403.5 351.3 52.2 45.4 540.0 96.5
154.6 91.0 76.5 14.5 12.5 190.0 10.3
6 1106.7 783.3 703.2 80.0 71.4 1050.9 68.9
6 1583.7 1208.7 1096.1 112.6 97.3 1216.4 75.9
6 2136.7 1673.9 1552.3 121.6 106.6 1207.7 90.9
127.7 107.7 96.6 21.1 18.0 197.8 6.5
•• •••
DNA,§ mg
66.4 58.8 7.7 6.7 74.8
2.7 3.2 .8 .6 9.9
69.6 62.1 7.5 6.7 103.1
76.0 69.4 6.6 5.7 735
78.4 72.8 5.7 5.0 56.4
2.0 2.4 .9 .8 10.9
Pee g, DFFf DNA, mg
9.6
1.3
13.6
11.7
10.0
1.1
Variable n TrGIWt, g DM, g Fat, g DFFf, g
cp 3
DNA,~ IDJ a>3:DNA
Pee 100 g TrGIWt DM, g Fat. g DFFf. g
cp 3
lL
Treatment
= Low, M = medium, H = high treatment. = Linear treabnent effect.
2Lin
3Measured in DFPT. ·P<.05. ·"P<.OI. ·"P<.OOL
Journal of Dairy Science Vol. 73,
No.9. 1990
•••
NS NS NS
•
•• •• NS NS
••
•
MAMMOGENESIS AND PLANE OF NUTRITION
7.2 b=2.86
7.0
.... ~
-S'"
6.8
«:
6.6
z
b=2.50
0
I
I:: 0
2
6.4 6.2
~ 6.0
'" 0
...J
5.8 5.6 3.5
3.6
3.7
3.8
3.9
4.0
4.1
4.2
Log [live weight (kg)] .67
Figure 1. Increase in total mammary DNA, relative to metabolic live weight, between \be beginning (initial slaughter group) and conclusion (slaughter group) of \be experiment for heifers on \be low ( - ) , medium (--), and high (....) treatments.
Mammary parenchyma increased at a substantially faster rate than did metabolic weight in heifers between 3 and 9 mo (26) and in lambs between 4 and 20 wk of age (14). Furthennore, animals that received the lowest feeding regimen had the highest relative growth coefficients (14). The design of the current experiment did not allow calculation of separate relative growth coefficients pre- and postpuberty; however, we assumed that the differences in mammary growth that occurred during the allometric mammary growth phase were sustained during the period following puberty. Mammary growth returns to an isometric rate shortly after puberty has been reached (26) and continues at this rate until the onset of gestation. Coefficients calculated for the entire experimental period (Figure 1) revealed that total DNA in DFFf increased most rapidly per unit increase in metabolic body weight in heifers on the L diet Although no differences in total mammary DNA (Table 6) were observed at conclusion of the trial, final body weight differed significantly resulting in different mammary growth rates relative to body weight. A p·eriod of allometric mammary growth commences at 2 to 3 mo of age and tenninates around puberty (i.e., first estrus; 26). Heifers
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raised on different planes of nutrition attained puberty at approximately the same body weight (Table 4) (17, 23). Therefore, heifers on the lower planes of nutrition would be expected to contain more mammary DNA (which may ultimately result in an increased production capacity) at the end of the allometric growth phase than would animals fed higher planes, because the increase in mammary DNA per unit increase in body weight was greatest for heifers on the low plane of nutrition. All relative growth coefficients for mammary parenchyma reported in the literature are based on DNA analysis. In the present study, coefficients were also calculated for DFFf. These coefficients (L, 2.01; M, 2.18; H, 2.36) appeared to be opposite from those of total DNA in DFFr (Figure 1) and, as a result, seem to contradict the hypothesis of adverse dietary effects on mammary cell numbers in animals on high planes of nutrition. However, an increased mammary DNA concentration concurrent with a decrease in cell size (protein:DNA; Table 6) in heifers on the lower treatments may indicate a more densely organized cell matrix, which may be due to an increased amount of smaller parenchyma or secretory cells. The current data do not provide conclusive evidence to support this hypothesis, and fwther re~h (e.g., assessment of treatment differences in epithelial cell numbers per unit epithelium, mitotic characteristics and parenchymal cell size) is required. The prepubertal period during which mammogenesis may be adversely affected by plane of nutrition has not been adequately defined. Foldager and Sejrsen (7) suggested that this period is from 90 to 300 kg of live body weight in large dairy breeds. Although initial body weight of the heifers in the current experiment (Table 3) was within this range (approximately 200 kg) and actual AOO (Table 2) were within the range where plane of nutrition may be expected to affect mammogenesis (7), the allometric period may have started before the animals entered the experiment. Further research to define more precisely the timing of the initiation of allometric mammary growth will be essential. Results from the third biopsy (B3) were compared with chemical analysis of the same, but dissected, glands. A suitable biopsy sample could not be obtained from one animal, even 10umal of Dairy Science Vol. 73,
No.9, 1990
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STELWAGEN AND GRIEVE
TABLE 7. Correlations between mammary gland composition parameters obtained Ihrough biopsy and chemical analysis on the same, but dissected, glands. Chemical analysis per kg TrGIW~ B3
DPFT,I g
CP, g
DNA, mg
Fat, g
Epithelial area, % Adipose area, % Connective tissue area, % Ductular-luminal space, %
.51· .06 .09 .24
.53· .06 .06 26
.42 .12 -.11 .30
-28 -.04 -.02 .30
IDFTT
= Dried fat free tissue.
2nGIWt = Trimmed gland weight.
·P<.10.
though several attempts were made. From two other animals, biopsy slides could not be quantified due to excessive staining. All data are proportional and were transformed to a natural logarithmic scale before comparisons were made (fable 7). Correlations (fable 7) were low and nonsignificant and in some cases unexpected. A significant negative relationship (r = -.65) between epithelial cell area and DM intake in approximately ll-mo-old heifers (10) was not substantiated in the present study (B 1; r = -.24, P<.14, n = 41), with a substantially large number of animals. Mammary biopsy is a relatively fast and simple method of obtaining qualitative information about mammary gland composition in live animals. However, biopsy results of this study correlated poorly to chemical analysis (Table 7), so the usefulness of mammary biopsy in obtaining reliable data may be limited. Knight (16) hypothesized that the biopsy technique may give more accurate results in mature animals, which have more homogeneous mammary tissue. In summary, although plane of nutrition did not affect total mammary DNA, mammary glands contained significantly more adipose tissue in animals fed high planes of nutrition, indicating indirectly an adverse dietary effect on mammogenesis in these animals. In addition, mammary DNA increased at a greater rate relative to body growth in heifers on the low plane of nutrition. Furtbennore, morphometric evaluation of mammary tissue, obtained through mammary biopsies, did not yield satisfactory results in heifers between 6 and 16 mo of age. 10urnal of Dairy Science Vol. 73,
No.9, 1990
ACKNOWLEDGMENTS
Financial assistance was provided by the Ontario Ministry of Agriculture and Food, Agriculture Canada, and the Natural Sciences and Engineering Research COlUlCil of Canada. The authors thank George Werchola for carrying out the progesterone radioimmunoassays. REFERENCES 1 Amir, S., and 1. Kali. 1974. Influence of plane of nutrition of the dairy heifer on growth and performance after calving. Dairy Sci. Handbook 7:183. 2 Amir, S., 1. Kali, and R Vulcani. 1967. Influence of growth rate on reproduction and lactation in dairy cattle. Growth and developmenl of mammals. Proc. 14th Easter School Agric. Sci., Univ. NottiDgbam. G. A. Lodge and G. E. lamming, ed. Butterworth, London, Engl. 3 Anonymous. 1982. Calf care and raising yonng stock. W. D. Hoard's and Son Co., Fort Atkinson, WI. 4 Association of Official Analytical Chemists. 1980. Official methods of analysis of the Association of Official Chemists. 13th ed. Association of Official Analytical Chemists, Arlington, VA. 5 Cleary, M. P., M.R.C. Greenwood, and 1. A. Brasel. 1977. A multifaetor analysis of growth in the rat epididymal fat pad. 1. Nutr. 107:1969. 6 DeBoer, G., R.I. Etches, and 1. S. Walton. 1980. A solid phase radioimmunoassay for progesterone in bovine plasma. Can. 1. Anim. Sci. 60:783. 7Foldager, I., and K. Sejrsen. 1987. Mammary gland development and milk production in dairy cows in relation to feeding and hormone manipulation during rearing. Research in dairy cattle: Danish status and perspectives. Del kgl.danske Landhusholdningsselskab,
Copenhagen, DK. 8 Gardner, R W., 1. D. Schuh, and L. G. Vargus. 1977. Accelerated growth and early breeding in Holstein heifen. 1. Dairy Sci. 60:1941. 9 Gardner, R W., L. W. Smith, and R. L. PD. 1988. Feeding and management of dairy heifers few optimal lifetime productivity. 1. Dairy Sci. 71:996. 10 Grieve, D. G., P. 1. Visser, and B. W. McBride. 1986. Effect of forage source and feeding level on growth and
MAMMOGENESIS AND PLANE OF NUTRlTION
mammary development of Holstein calves. J. Dairy Sci. 69(Suppl. 1):132. (Abstr.) 11 Harrison, R D., I. P. Reynolds, and W. Little. 1983. A quantitative analysis of mammary glands of dairy heifers reared at different rates of live weight gain. J. Dairy Res. 50:405. 12 Heinrichs, A. J., and G. L. Hargrove. 1987. Standards of weight and height for Holstein heifers. J. Dairy Sci. 70: 653. I3 Hirsch, J., and P. W. Han. 1969. Cellularity of rat tissue: effects of growth, starvation, and obesity. J. Lipid Res. 10:77. 14 Johnson, I. D., and I. C. Hart. 1985. Pre-pubertal mammogenesis in sheep. I. The effects of nutrition on growth and mammary development in female lambs. Anim. Prod. 41:323. 15 Kirkham, W. R., and C. W. Turner. 1953. Nucleic acids ofthemammarygIaodofrats.Proc.Soc.Exp.BioI.Med. 83:123. 16 Knight, C. H., 1984. Mammary growth and development strategies of animals and investigators. Symp. Zool. Soc. Lond. 51:197. 17 Larsen, J. B., J. Foldager, and K. Sejrsen. 1975. A comparison between different feeding intensities using various concentrate/roughage/grass rations for rearing dairy heifers to calve at an early age. 1be early calving of heifers and its impact on beef production. J. C. Taylor. ed. Comm. Eur. Commun., Brussels, Belgium. 18 Little, W., and R M. Kay. 1979. The effects of rapid rearing and early calving on the subsequent performance of dairy heifers. Anim. Prod. 29:131. 19 Martin, R F., D. C. Donahue, and L. R. Finch. 1972. New analytical procedure for the estimation of DNA with p-nitrophenylhydrazine. Anal. Biochem. 47:562.
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20 National Research Council. 1978. Nutrient requirements of domestic animals, No. 13. Nutrient requirements of dairy cattle, 5th rev. ed. Natl. Acad. Sci., Washington, DC. 21 Park, C. S., Y. J. Choi, and R. L. Harrold. 1987. Protein energy interrelationships in development and function of mammary glands in rats. Nutr. Rep. lot. 36:1251. 22 Petitclerc, D., L. T. Chapin, and H. A. Tucker. 1984. Carcass composition and mammary development to photoperiod and plane of nutrition in Holstein heifers. J. Anim. Sci. 50:913. 23 Pritchard, D. E., H. D. Hafs, H. A. Tucker, L. J. Boyd, R. W. Purchas, and J. T. Hubert. 1972. Growth, mammary, reproductive, and pituitary hormone characteristics of Holstein heifers. fed extra grain and melengestrol acetale. J. Dairy Sci. 55:995. 24 SAS~ User's Guide: Statistics, Version 5 ed. 1985. SAS Institute, Inc., Cary, NC. 25 Sejrsen, K., J. T. Hubert, H. A. Tucker, and R. M. Akers. 1982. Influence of nutrition on mammary development in pre- and postpubertal heifers. J. Dairy Sci. 65:793. 26 Sinha, Y. N., and Tucker, H. A. 1969. Mammary development and pituitary prolactin level of heifers from birth through puberty and during estrous cycle. J. Dairy Sci. 52:507. 27 Snedecor, G. W. and W. G. Cochran. 1980. Statistical methods. 7th ed. The Iowa State University Press, Ames. 28 Swanson, E. W., 1960. Effect of rapid growth with fattening of dairy heifers on their lactational ability. J. Dairy Sci. 43:377. 29 Swanson, E. W. 1975. Future research on problems of increasing meat production by early calving. The early calving of heifers and impact on beef production. J. C. Tayler, eeL Comm. Eur. Commun., Brussels. Belgium.
Journal of Dairy Science Vol. 73,
No.9, 1990
Forty-one Holstein and 6 Holstein crossbred heifers, 6 to 8 mo of age, were used to determine the effect of plane of nutrition on growth and mammogenesis prior to and during puberty. Animals were fed to gain 611 g (low), 737 g (medium), and 903 g (high) by a diet of cracked com and chopped alfalfa-grass hay. Mammary biopsies were carried out in vivo to determine if they provide acceptable infonnation on mammary composition (based upon morphometric evaluation) in comparison with dissected glands. Results indicated that NRC (1978) recommendations for average daily gain of Holstein heifers between 6 and 16 mo may be too generous. At puberty (first estrus), age and wither height decreased linearly with increasing plane of nutrition, whereas body weight and hip height were not affected by plane of nutrition. Five heifers were slaughtered at the beginning and 18 at the conclusion of the trial. Increasing plane of nutrition resulted in fatter mammary glands with decreased concentration of DNA, whereas total mammary DNA did not differ among treatment groups. In this study, morphometric evaluation of mammary tissue obtained through biopsies did not yield useful infonnation in comparison to chemical analysis of dissected glands. (Key words: mammogenesis, plane of nutrition, growth) INTRODUCTION
Normal age at frrst parturition in large dairy breeds is between 2 and 3 yr (25, 28); however,
Received April 10, 1989. Accepted April 19. 1990.
1990 1 Dairy Sci 73:2333-2341
heifers can calve at 2 yr of age or less (11, 25, 29). Early calving reduces the unproductive period of the dairy heifer, which implies that less feed is required to raise it to productive age. Because 40 to 70% of the total costs of raising replacement heifers can be attributed to feed costs (1), a reduction would have a positive impact on fann cash flow. To accomplish the objective of early parturition, heifers should be bred at an early age. Feeding heifers a high plane of nutrition, allowing for rapid growth after weaning, can lower the age at which sexual maturity occurs (2, 25, 29). However, Gardner et al. (8) and Little and Kay (18) observed lower milk production from heifers raised on a high plane of nutrition than that from their counterparts raised on a more moderate feeding regimen. A recent study by Gardner et al. (9) seems to contradict these earlier results; however, results in this study may have been confounded by differences in nutrient composition of the control and treatment diets. Depressed milk production may be caused by incomplete mammary development in heifers raised on a high plane of nutrition (11). Most reliable infonnation on mammary gland development has been obtained by dissecting glands of slaughtered animals. Such experiments are costly, particularly if many animals are involved. Furthennore, studying mammary development on the same animal during successive intervals becomes impossible. For these reasons, in vivo techniques for mammary gland evaluation are more desirable. The objective of the study was 1) to investigate growth and mammary development in Holstein heifers before and during puberty, and 2) to discover whether or not mammary biopsies carried out on live animals provided acceptable information on mammary composition (based upon morphometric evaluation) in comparison with chemical analysis of dissected glands from slaughtered animals.
2333
2334
STELWAGEN AND GRIEVE
MATERIALS AND METHODS
TABLE 1. Nubient composition of trace-mineral salt, alfalfa grass hay, and cracked com.
Animal Management Composition
The experiment was designed as a randomized complete block design (RCBD) with four blocks and involved 47 dairy heifers, which were divided into a production (P) and slaughter (S) group. The P group comprised 18 female Canadian Holstein calves (block 1, 9 calves of approximately 8 mo of age; block 2, 9 6-mo-old calves); and block 3, 6 female New Zealand X Canadian Holstein crossbred calves of 6 to 8 mo of age. The S group consisted of 23 6- to 7-mo-old Canadian Holstein heifers, of which 5 heifers were randomly chosen to be slaughtered (stunned with captive bolt) at the beginning of the experiment. The remaining 18 S heifers (block 4) were slaughtered after 279 d on trial. Animals were housed in individual stalls at the University of Guelph Dairy Research Station and were removed from the experiment immediately after the second biopsy had been performed on each animal within a block, which was 230 d (block 1), 347 d (block 2 and 3), and 279 d (block 4) after the beginning of the experiment, Le., when the animals were about 16 mo of age. During the entire experiment, all animals were subjected to artificiallighting daily from 0500 to 1800 h. Heifers in block 4 were also exposed to daylight during the first 183 d (June to December) on trial. Animals were fed a diet containing cracked com, chopped (2.5 cm) alfalfa grass hay, and trace-mineral salt (Table 1) and had access to fresh water at all times. To feed each animal the same hay to com ratio at the same age and to allow for realistic intakes, the hay to com ratio was changed four times. From 6 to 7 mo of age, this ratio was 70:30, from 8 to 9 mo 65: 35, from 10 to 12 mo 60:40, and from 13 mo to the end of trial 55:45. The trace-mineral mix was fed at .25% of daily OM intake. Based upon these feedstuffs and NRC (20) recommendations for growing heifers, feeding amounts were established to allow for an average daily gain (AOO) of 600 g (L, low treatment), 750 g (M, medium treatment), and tOOO g (H, high treatment). Heifers were randomly allotted to one of the three feeding regimens. Feed intake and orts were recorded for each animal. Diets were adjusted biweekly according to age and every 4 wk according to age and Iournal of Dairy Science Vol. 73,
No.9, 1990
DM. % Cp,! % ADp 1 % ME, {,2 MI/kg NaCl, % Zinc, ppm Iron, ppm
Manganese, ppm Copper, ppm Iodine, ppm Cobalt, ppm
Mineral salt
Hay
Com
91.5 15.6 43.6
87.1 9.4 3.5 14.3
8.0 96.5 4000 1600 1200 330
70 40
IMeasured in DM. 2caIculated from ADP (details in text).
weight gain during the previous 4-wk period. Com and hay were sampled weekly, frozen, and composited in 4-wk intervals. Subsamples were analyzed for DM, CP, and ADF according to the procedures of AOAC (4). Metabolizable energy (ME) was calculated from the percentage of ADF in DM by means of equations from the Ontario Ministry of Agriculture and Food: com: %1DN = 92.22 - (1.535x%ADF); mixed hay: %1DN = 92.62 - (.9093x%ADF), DE (Mcallkg) = .4409x%1DN, and ME (Mcal/kg) = .45 + (1.0IxDE). Live weight and body height at the withers and hip of each animal were measured on two consecutive days at the beginning and end of the trial and at 28-d intervals during the trial. Mammary Biopsies
All experimental animals, except for those slaughtered at the beginning of the trial, were subjected to mammary biopsies to obtain tissue samples for morphometric evaluation. The first biopsy (B1) was carried out when animals were confirmed to have estrous cycles (i.e., attained puberty). Confirmation was based upon blood plasma progesterone concentrations (6) 0btained from weekly jugular vein blood samples. Progesterone concentrations greater than 1 ng/ ml were considered to be indicative of a ftmctional corpus luteum (22, 25). A second biopsy (B2) was performed 2 mo after Bl. The S heifers underwent a third biopsy (B3) 3 d before being slaughtered, except for six animals
2335
MAMMOGENESIS AND PLANE OF NUTRITION
in which B3 coincided with B2. All biopsies were performed during the luteal phase of the estrous cycle. Biopsies were taken approximately 3 cm above the right rear teat (B 1 and B3) and left rear teat (B2). After preparation of the operative area and application of local anesthesia, a small incision (l to 1.5 cm) was made and a disposable biopsy needle (fru-cut biopsy needle, Travenol Laboratories, Inc., Deerfield, IL) (l5.2-cm cannula and 20-mm specimen notch) was inserted to remove a tissue sample. Tissues were immediately washed in saline and ftxed in Bouin's fluid. After 45 min, excessive fIxative was removed with 70% ethanol. The tissue was stained in Ehrlich's hematoxylin, eosin, and phloxine; embedded in paraffin; and cut into 6-1J.IIl slices. Slices were examined under a light microscope (100)<) equipped with a grid having 344 visible intersections. Adipose, remaining connective tissue, epithelial cell area, and ductolar-luminal area were quantified by counting intersections overlying each of the cell types. Cell types at the top and bottom of each of two fields per biopsy were counted. Chemical Analysis of Mammary Tissue
The S group heifers were slaughtered during the luteal phase of the estrous cycle. Immediately after exsanguination, mammary glands were detached from the body cavity and separated along the median suspensory ligament. The right half was subsequently trimmed from skin and teats and weighed to obtain trimmed half gland weight (TrGIWt) and frozen in liquid nitrogen to be stored at -ISoC until chemical analysis. All analyses were performed on ground lyophilized tissue. Dry matter was determined by drying for 2 h at 135°C and fat according AOAC (4). Twenty-gram samples of lyophilized tissue were subjected to a 5-h fat extraction followed by a 3-h extraction with anhydrous ether (4), and the remaining tissue was dried overnight in a fume hood yielding dried fat-free tissue (DFFf). Crude protein (4) and DNA content were determined according to methods of Martin et al. (19) in aliquots of finely ground DFFf. Relative Growth Coefficients
Relative growth coefficients for DNA and DFFf were calculated to estimate the rate of
increase of these parameters in relation to increases in metabolic live weight. Calculations were similar to those outlined for heifers (26) and lambs (14). Statistical Analysis
Growth and biopsy data were analyzed using the model: Yijk = J1 + T i + Bj + (TB)ij + &jk and for mammary composition data: Yij = J1 + Ti + E;.J' where Yij(k) = adjusted value for observations ij(k) of the dependent variable; J1 = true mean; Ti = treatment L, M, or H; Bj = block 1,2,3, or 4; (TB)ij = treatment by block interaction; and E;.j(k) = residual error of observation ij(k). Biopsy data were expressed as a percentage of the total number of counts, and results from Bland B2 were analyzed as two separate data sets. Prior to analysis of variance, biopsy data were transformed, using the In (x + 1) transformation (27) to stabilize the variance. Analyses were performed using the general linear models procedure of SAS (24). Results were considered significant at ~.05. When a significant treatment effect occurred in the absence of a significant treatment by block interaction, the treatment effect was partitioned into linear and nonlinear components. RESULTS AND DISCUSSION
One animal on the H treatment died during the ftrst half of the experiment from causes unrelated to the experiment, and data were not included in the analyses. Significant differences in DM intake (Table 2) among treatment groups were expected, because differences in ADG could only be obtained by feeding the animals different quantities of the same diet in order to prevent any potentially confounding effects of different protein to energy ratios. Recent work with rats (21) suggested that different dietary protein and energy intakes may influence mammogenesis. Actual ADG for heifers on the L or M treatments was in agreement with proposed ADG for these animals (Table 2). However, despite significantly higher intakes, H heifers failed to attain the target growth rate. Nevertheless, actual ADO (903 g) of H heifers in this experiments can be qualified as high for study of the effects of rapid rearing on mammary gland development (1). Journal of Dairy Science Vol. 73,
No.9, 1990
2336
STELWAGEN AND GRIEVE
TABLE 2. Average daily DM intake and proposed and actual ADO for slaughter and production group heifers. Treatment Variable n DM Intake, kg/d Actual ADO, g Proposed ADO, g Difference ADO, g
lL
= Low,
M
= medium,
14 4.6 611 600 +11 H
= high
M
H
SEM2
Lin3
14 5.7 737 750 -13
13 7.5 903 1000
.1 32.0
• •
..JT1
IIeatmenl.
2SEM averaged over treatments.
~in = Linear treatment effect. ·P<.Ol.
Average age at the beginning and conclusion of the experiment as well as initial and final body weight and height measured at the withers and hip are given in Table 3. Final body weight increased in a linear way with increasing plane of nutrition. No treatment effect was noted on wither height or hip height at the conclusion of the trial. Final body weight and wither height of heifers in this trial are in agreement with body growth data for Holstein heifers of similar age (12) and with heights and weights of 16-mo-old Holstein heifers derived from growth charts used by the Ontario Ministry of Agriculture and Food (3). This indicates that the low plane of nutrition was sufficient for skeletal and body development in Holstein heifers between 6 and 16 mo of age. Therefore, NRC (20) recommendations for AOO of heifers of large dairy breeds (700 to 800 g) may be too generous. At the beginning of the trial, on the basis of blood plasma progesterone, five animals had apparently already attained puberty (i.e., frrst estrus). The average age, body weight, and height of the remaining animals at the time they attained puberty are shown in Table 4. Results from this study support earlier findings that age at puberty declines with increasing feed intake (2, 17, 23), whereas body weight at puberty was not significantly affected by feeding (17, 23). Wither height decreased linearly with increasing plane of nutrition, which is in agreement with observations made by Amir et al. (2) and Larsen et al. (17). Why hip height was unaffected by feeding level is unknown. Attainment of puberty seems to be determined by body weight rather than chronological age and Journal of Dairy Science Vol. 73,
No.9, 1990
can be manipulated by the feeding regimen. Mammary biopsy data for epithelial cell area, ductular-Iuminal area, adipose cell area, and remaining connective tissue cell area were expressed as mean percentage of the total area identified of each of four fields per biopsy slide. Transformed (In {x + I}) means of B1 and B2 are reported in Table 5. Within each biopsy (Bl or B2), no significant effect due to plane of nutrition was noted for any of the parameters measured (fable 5). Neither did any statistical evidence emerge of changes in the area of different tissue types between the time heifers attained puberty (B 1) and 2 mo later (B2). Chemical analyses of dissected mammary glands from S group heifers are shown in Table 6. Data of the initial slaughter group serve as reference data. Trimmed gland weight (frGIWt) increased linearly with increasing plane of nutrition. Fat and DM also increased linearly, explaining the increases in TrGIWt, because DFFT and CP in DFFT did not significantly differ between feeding regimens. Deoxyribonucleic acid is often used to estimate mammary cell numbers (15). In this study, total mammary DNA was measured instead of DNA in parenchyma that was visually separated from extra parenchymal tissue (22, 25). Due to the way mammary parenchyma branches into the surrounding fat pad, extraparenchymal tissue can never be completely separated visually from parenchymal tissue. Either true parenchyma will be removed with the extraparenchymal adipose tissue or extraparenchymal adipose tissue may unavoidably be included in parenchymal tissue, thus diluting
2337
MAMMOGENESIS AND PLANE OF NUTRITION
TABLE 3. Average initial and final age, body weight, wither' height (WHT), and hip height (HH'I) for slaughter and production group animals. Treatment
L1
Variable No. cows
SEM
M
14
SEM
H
14
SEM
13
Initial age, d Final age, d
185 477
11 5
194 487
11 5
185 473
12 5
Initial BW, kg Final BW. kg
199.1 378.7
13.2 7.1
204.0 420.6
13.2 7.1
187.8 447.2
13.7 7.3
Initial WHT, em Final WHT, em
106.0 127.6
1.9 .6
105.6 127.9
1.9 .6
104.1 127.7
2.0 .6
NS
Initial HHT, em Final HHT, em
112.0 130.5
2.0 .6
111.5 131.1
2.0 .6
110.6 131.5
2.1 .6
NS
= =
•
=
IL Low, M medium, H = high treatment. 2Lin Linear treatment effect.
·P<.OOL
parenchymal DNA. Total mammary DNA (Table 6) was not affected by feeding. We expected that animals on the M and H planes would have had less mammary DNA, indicating a lower potential for future milk production. However, the inclusion of extraparenchymal adipose cell DNA may explain the lack of effect of plane of nutrition on total mammary DNA. Heifers receiving the M and H plane of nutrition had 57 and 129% more mammary fat than heifers on the low plane (Table 6); a larger proportion of the total mammary DNA probably consisted of nonparenchymal DNA, since increases in adipose tissue in young developing animals can, at least in part, be ascribed to increases in adipose cell number
(5, 13). Thus, although total mammary DNA did not differ among treatment groups, the amount of adipose tissue significantly decreased with decreasing plane of nutrition, indicating indirectly a larger proportion of parenchymal DNA for animals on the lower planes of nutrition. Mammary DNA expressed per unit of TrGIWt or DFFT declined with increasing plane of nutrition, suggesting also a potential adverse effect of feeding on mammogenesis. Although these results are in agreement with similar adverse dietary effects on mammary DNA observed in heifers (22, 25) and lambs (14), the current data represent total mammary DNA, i.e., parenchymal and nonparenchymal DNA.
TABLE 4. Average age. body weight, wither height (WH1), and hip height (HH'I) of slaughter' and production group heifers at attainment of puberty. Treatment Variable
LI
SEM
M
SEM
H
SEM
Lin2
n Age, d BW,kg WHT. em HHT, em
11 365 284.9 121.5 125.4
13 12.1 1.1 1.0
12 313 283.7 118.0 122.7
12 11.6 1.0 1.0
13 305 298.0 117.6 123.0
12 11.2 1.0 1.0
• NS ••
NS
= Low, M = medium, H = high treatment. 2Lin = Linear treatment effect. lL
·P<.05. ··P<.01. Journal of Dairy Science Vol. 73,
No.9. 1990
2338
STELWAGEN AND GRIEVE
TABLE 5. Tnmsformed (In [x + 1)) means of percentage epithelial. adipose, remaining connective tissue area, and ductuIar-luminal space obtained through mammary biopsies (Bl and B2). Treatment SEM2
r.m3
2.34 2.48 +.14
.24 .31 .41 4
NS NS NS
3.53 3.67 +.14
3.50 3.10 -.40
.22 .21 .304
NS NS NS
3.79 3.86 +.07
3.80 3.62 -.18
3.78 3.75 -.03
.12 .14 .204
NS NS NS
.73 .71
.92 .71 -21
.90
.94
.18 .18 .25 4
NS NS NS
Variable
Biopsy
L1
M
H
No. heifers
Bl B2
14 13
14 14
13 13
Epithelium
Bl B2 B2-81
2.10 2.35 +.25
2.36 2.32 -.04
Adipose
BI B2 B2-Bl
3.37 3.31 -.06
Connective
Bl B2 B2-Bl
Ductular-Iuminal
Bl B2 B2-81
-.02
+.04
lL = Low, M = medium, and H = high treatment. 2sEM averaged over treabncnts. 3r.in = Linear treabnent effect.
4standard
errors of difference averaged ovee treabnents.
TABLE 6. Chemical analysis of mammary gland tissue of slaughter group heifers expressed as total amounts, per unit bimmed half gland weight (TrGlWt). and dried fat-free tissue
Initial slaughter group
SEM
L1
M
H
SBM
Lin2
5 630.0 403.5 351.3 52.2 45.4 540.0 96.5
154.6 91.0 76.5 14.5 12.5 190.0 10.3
6 1106.7 783.3 703.2 80.0 71.4 1050.9 68.9
6 1583.7 1208.7 1096.1 112.6 97.3 1216.4 75.9
6 2136.7 1673.9 1552.3 121.6 106.6 1207.7 90.9
127.7 107.7 96.6 21.1 18.0 197.8 6.5
•• •••
DNA,§ mg
66.4 58.8 7.7 6.7 74.8
2.7 3.2 .8 .6 9.9
69.6 62.1 7.5 6.7 103.1
76.0 69.4 6.6 5.7 735
78.4 72.8 5.7 5.0 56.4
2.0 2.4 .9 .8 10.9
Pee g, DFFf DNA, mg
9.6
1.3
13.6
11.7
10.0
1.1
Variable n TrGIWt, g DM, g Fat, g DFFf, g
cp 3
DNA,~ IDJ a>3:DNA
Pee 100 g TrGIWt DM, g Fat. g DFFf. g
cp 3
lL
Treatment
= Low, M = medium, H = high treatment. = Linear treabnent effect.
2Lin
3Measured in DFPT. ·P<.05. ·"P<.OI. ·"P<.OOL
Journal of Dairy Science Vol. 73,
No.9. 1990
•••
NS NS NS
•
•• •• NS NS
••
•
MAMMOGENESIS AND PLANE OF NUTRITION
7.2 b=2.86
7.0
.... ~
-S'"
6.8
«:
6.6
z
b=2.50
0
I
I:: 0
2
6.4 6.2
~ 6.0
'" 0
...J
5.8 5.6 3.5
3.6
3.7
3.8
3.9
4.0
4.1
4.2
Log [live weight (kg)] .67
Figure 1. Increase in total mammary DNA, relative to metabolic live weight, between \be beginning (initial slaughter group) and conclusion (slaughter group) of \be experiment for heifers on \be low ( - ) , medium (--), and high (....) treatments.
Mammary parenchyma increased at a substantially faster rate than did metabolic weight in heifers between 3 and 9 mo (26) and in lambs between 4 and 20 wk of age (14). Furthennore, animals that received the lowest feeding regimen had the highest relative growth coefficients (14). The design of the current experiment did not allow calculation of separate relative growth coefficients pre- and postpuberty; however, we assumed that the differences in mammary growth that occurred during the allometric mammary growth phase were sustained during the period following puberty. Mammary growth returns to an isometric rate shortly after puberty has been reached (26) and continues at this rate until the onset of gestation. Coefficients calculated for the entire experimental period (Figure 1) revealed that total DNA in DFFf increased most rapidly per unit increase in metabolic body weight in heifers on the L diet Although no differences in total mammary DNA (Table 6) were observed at conclusion of the trial, final body weight differed significantly resulting in different mammary growth rates relative to body weight. A p·eriod of allometric mammary growth commences at 2 to 3 mo of age and tenninates around puberty (i.e., first estrus; 26). Heifers
2339
raised on different planes of nutrition attained puberty at approximately the same body weight (Table 4) (17, 23). Therefore, heifers on the lower planes of nutrition would be expected to contain more mammary DNA (which may ultimately result in an increased production capacity) at the end of the allometric growth phase than would animals fed higher planes, because the increase in mammary DNA per unit increase in body weight was greatest for heifers on the low plane of nutrition. All relative growth coefficients for mammary parenchyma reported in the literature are based on DNA analysis. In the present study, coefficients were also calculated for DFFf. These coefficients (L, 2.01; M, 2.18; H, 2.36) appeared to be opposite from those of total DNA in DFFr (Figure 1) and, as a result, seem to contradict the hypothesis of adverse dietary effects on mammary cell numbers in animals on high planes of nutrition. However, an increased mammary DNA concentration concurrent with a decrease in cell size (protein:DNA; Table 6) in heifers on the lower treatments may indicate a more densely organized cell matrix, which may be due to an increased amount of smaller parenchyma or secretory cells. The current data do not provide conclusive evidence to support this hypothesis, and fwther re~h (e.g., assessment of treatment differences in epithelial cell numbers per unit epithelium, mitotic characteristics and parenchymal cell size) is required. The prepubertal period during which mammogenesis may be adversely affected by plane of nutrition has not been adequately defined. Foldager and Sejrsen (7) suggested that this period is from 90 to 300 kg of live body weight in large dairy breeds. Although initial body weight of the heifers in the current experiment (Table 3) was within this range (approximately 200 kg) and actual AOO (Table 2) were within the range where plane of nutrition may be expected to affect mammogenesis (7), the allometric period may have started before the animals entered the experiment. Further research to define more precisely the timing of the initiation of allometric mammary growth will be essential. Results from the third biopsy (B3) were compared with chemical analysis of the same, but dissected, glands. A suitable biopsy sample could not be obtained from one animal, even 10umal of Dairy Science Vol. 73,
No.9, 1990
2340
STELWAGEN AND GRIEVE
TABLE 7. Correlations between mammary gland composition parameters obtained Ihrough biopsy and chemical analysis on the same, but dissected, glands. Chemical analysis per kg TrGIW~ B3
DPFT,I g
CP, g
DNA, mg
Fat, g
Epithelial area, % Adipose area, % Connective tissue area, % Ductular-luminal space, %
.51· .06 .09 .24
.53· .06 .06 26
.42 .12 -.11 .30
-28 -.04 -.02 .30
IDFTT
= Dried fat free tissue.
2nGIWt = Trimmed gland weight.
·P<.10.
though several attempts were made. From two other animals, biopsy slides could not be quantified due to excessive staining. All data are proportional and were transformed to a natural logarithmic scale before comparisons were made (fable 7). Correlations (fable 7) were low and nonsignificant and in some cases unexpected. A significant negative relationship (r = -.65) between epithelial cell area and DM intake in approximately ll-mo-old heifers (10) was not substantiated in the present study (B 1; r = -.24, P<.14, n = 41), with a substantially large number of animals. Mammary biopsy is a relatively fast and simple method of obtaining qualitative information about mammary gland composition in live animals. However, biopsy results of this study correlated poorly to chemical analysis (Table 7), so the usefulness of mammary biopsy in obtaining reliable data may be limited. Knight (16) hypothesized that the biopsy technique may give more accurate results in mature animals, which have more homogeneous mammary tissue. In summary, although plane of nutrition did not affect total mammary DNA, mammary glands contained significantly more adipose tissue in animals fed high planes of nutrition, indicating indirectly an adverse dietary effect on mammogenesis in these animals. In addition, mammary DNA increased at a greater rate relative to body growth in heifers on the low plane of nutrition. Furtbennore, morphometric evaluation of mammary tissue, obtained through mammary biopsies, did not yield satisfactory results in heifers between 6 and 16 mo of age. 10urnal of Dairy Science Vol. 73,
No.9, 1990
ACKNOWLEDGMENTS
Financial assistance was provided by the Ontario Ministry of Agriculture and Food, Agriculture Canada, and the Natural Sciences and Engineering Research COlUlCil of Canada. The authors thank George Werchola for carrying out the progesterone radioimmunoassays. REFERENCES 1 Amir, S., and 1. Kali. 1974. Influence of plane of nutrition of the dairy heifer on growth and performance after calving. Dairy Sci. Handbook 7:183. 2 Amir, S., 1. Kali, and R Vulcani. 1967. Influence of growth rate on reproduction and lactation in dairy cattle. Growth and developmenl of mammals. Proc. 14th Easter School Agric. Sci., Univ. NottiDgbam. G. A. Lodge and G. E. lamming, ed. Butterworth, London, Engl. 3 Anonymous. 1982. Calf care and raising yonng stock. W. D. Hoard's and Son Co., Fort Atkinson, WI. 4 Association of Official Analytical Chemists. 1980. Official methods of analysis of the Association of Official Chemists. 13th ed. Association of Official Analytical Chemists, Arlington, VA. 5 Cleary, M. P., M.R.C. Greenwood, and 1. A. Brasel. 1977. A multifaetor analysis of growth in the rat epididymal fat pad. 1. Nutr. 107:1969. 6 DeBoer, G., R.I. Etches, and 1. S. Walton. 1980. A solid phase radioimmunoassay for progesterone in bovine plasma. Can. 1. Anim. Sci. 60:783. 7Foldager, I., and K. Sejrsen. 1987. Mammary gland development and milk production in dairy cows in relation to feeding and hormone manipulation during rearing. Research in dairy cattle: Danish status and perspectives. Del kgl.danske Landhusholdningsselskab,
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Journal of Dairy Science Vol. 73,
No.9, 1990