Colostral Immunoglobulin Absorption Linearly Related to Concentration for Calves1

Colostral Immunoglobulin Absorption Linearly Related to Concentration for Calves 1 G. H. STOTT and A. F E L L A H Department of Animal Sciences University of Arizona Tucson 85721

liters, had less influence on immunoglobulin absorbed than did concentration.

ABSTRACT

This experiment shows colostrat immunoglobulin concentration is a major factor in the rate of immunoglobulin absorption and the amount of absorption when it is fed to newborn calves. First-milking colostrum was collected from two cows and blended with fresh whole milk in immunoglobulin concentrations ranging from 7.5 to 123.8 mg/ml for immunoglobulin G, . 38 to 5.53 mg/ml for immunoglobulin A, and .46 to 11.19 mg/ml for immunoglobulin M. Six batches of colostrum were prepared separately with each having a different range of immunoglobulin concentration. Calves were separated from their dams at birth and fed either 1 or 2 liters of the prepared colostrum at the appropriate concentration. Feeding was repeated after 12 h. One-hundred and twenty HolsteinFriesian calves were fed in six blocks, a block for each freshly prepared colostrum, of 20 animals each. Blood samples were taken at prefeeding and at 12 and 24 h postpartum. The three isotypes in serum and colostrum were quantitated by single radial immunodiffusion procedure. Immunogtobulin G and A concentrations in serum of the calf at 24 h after feeding colostrum had a positive linear relationship with immunoglobulin G and A concentrations in the colostrum fed, whereas immunoglobulin M concentrations in the serum had a quadratic response. When compared on equal mass, the amount of colostrum fed, 1 or 2

INTRODUCTION

Received March 22, 1982. I Journal Paper 1433 of the Arizona Agricultural Experiment Station. 1983 J Dairy Sci 66:1319--1328

Passive immunization of the bovine neonate depends on absorption of antibodies from colostrum. Calves deprived of colostrum or calves absorbing inadequate amounts of colostraI immunoglobulins (Ig) are susceptible to the bacterial invasion and predisposed to septicemia, enteritis, and enterictoxemia (6, 8, 12, 31). Low Ig concentrations in blood serum have been associated with high morbidity and mortality rates in calves (1, 18, 21, 22, 30, 32). Low concentrations after colostrum feeding usually are explained by 1) insufficient amount of colostrum consumed (4, 16, 23, 36), 2) low colostral Ig concentration (14, 20, 24), 3) late feeding of colostrum (3, 28, 30, 34), 4) variation in birth weight (15), 5) genetic ability to absorb Ig (8, 13, 16), and 6) influences of environmental temperature (38). The interaction of these factors in affecting serum Ig concentration has been studied in several experiments. However, the influence of colostral Ig concentration on Ig absorption b y calves has not been defined clearly. A number of authors (4, 13, 15, 21, 22) concluded that serum Ig concentration in the calf after colostral feeding was primarily dependent on colostral Ig mass. Others noted a lack of significant correlation between colostral and serum Ig concentration but they did not determine the amount suckled (2, 13, 22, 27). Most studies were not designed to examine the influence of colosl:ral Ig concentrations per se. In some experiments, colostral Ig concentrations vcere determined but limited to narrow ranges (4, 36) or confounded with quantities fed at different ages (15, 16). In practice, calves commonly are fed on volume with little or no consideration of Ig concentration or its value. Our experiment was designed to supply more accurate information on the relationship

1319

1320

STOTT AND FELLAH

of colostral Ig concentration to the Ig by the calf. Confounding and the amount of colostrum fed and treatments were replicated evaluation.

absorption of factors of age were avoided, for statistical

MATERIALS AND METHODS

To satisfy objectives of this experiment, we planned to test absorption of colostral Ig by newborn calves at two volumes and five concentrations for each colostrum used. The two volumes, 1 and 2 liters, are the amounts generally recommended for feeding calves. The five concentrations were to cover a wide range that represented high, low, and intermediate concentrations that could be expected in colostrum fed to calves at their first feeding. The chosen concentrations were based on various reports (10) and numerous colostrum analyses in our laboratory from pooled colostral samples and individual cows (3 3). Experimental Design

To facilitate the rapid use of fresh colostrum, without a delay in time for full analysis, a hydrometer was used for rapid estimation of colostral Ig concentration. First-milking colostrum from 2 cows, carefully selected for volume and concentration, was pooled, and five dilutions were made with fresh milk to produce a range of Ig concentration. Dilutions then were divided into portions for feeding treatments for one block (20 calves). Portions were refrigerated until fed. Feeding treatments for each block consisted of the five concentrations, each fed at two volumes, 1 and 2 liters. Each treatment was replicated twice in each block. One hundred-twenty Holstein-Friesian calves were assigned randomly to 1 of 10 treatments within blocks. The general linear model was used to identify and isolate effects of absorption of colostral Ig from: 1) colostral Ig concentration, 2) volume fed, 3) interaction, volume, and concentration (mass), and 4) other differences in colostrum from different cows (between blocks). Collecting of Calves

Calves were separated from their dams immediately after birth to prevent suckling. Each calf was assigned randomly to a treatment within the ongoing block until the block was Journal of Dairy Science Vol. 66, No. 6, 1983

completed. The assigned treatments were fed within 1 h after birth and again 12 h later. Collecting fresh colostrum, feeding, and bleeding newborn calves were generally completed within 2 to 3 days from the start of each block. Parturient cows, newborn calves, and facilities were made available through the cooperation of a large Holstein-Friesian commercial dairy farm (2600 milking cows). Usually 8 to 20 cows freshened each day. Collecting of Blood Samples

Blood samples were taken from each experimental calf prior to the first feeding (0 h) to determine the Ig concentration in the newborn sera. Samples also were taken at 12 h to compare the rate of Ig absorption and 24 h to quantitate the total Ig concentration absorbed (34). Serum was separated by centrifugation within 24 h after collection and stored at - 2 0 ° C until analyzed. Samples of colostrum from each treatment also were stored at - 2 0 ° C until analyzed. Single Radial Immunodiffusion Analysis (sRID)

Whole colostrum and blood serum IgG, IgA, and IgM were quantitated by a modification of sRID procedures (7, 19). Whole colostrum was used in sRID analysis to assure maximum accuracy (10). Polyethlene glycol 4000 (.5 wt/ vol) was added to the agarose (1% wt/vol) to enhance precipitin rings. Immunoglobulin G plates were allowed to incubate 12 h at 4°C, whereas IgM and IgA plates were incubated 24 h at 25°C. All plates were incubated in humidifier chambers to prevent dessication. Antisera to the bovine IgG, IgA, and IgM were produced in rabbits by methods similar to those of (5). Antisera were rendered monospecific by immunoabsorption and were characterized by double diffusion (Ouchterlony) and immunoelectrophoresis. Standards for immunodiffusion analysis were quantitated by Lowry protein determination with purified bovine gammaglobulin. Standard serum was diluted serially with Ig-free serum to generate a standard curve. A four parameter logistic transformation (11) compiled by Kuehl (17) for competitive ligand assays and modified for sRID fitted all standard curves. Daily controls were within two standard deviations.

IMMUNOGLOBULIN ABSORPTION IN CALVES

13 21

Statistical Analysis

Absorption of Colostral IgG

Experimental calves between blocks received colostrum with different ranges of Ig concentration (Table 1). Therefore, the effect of concentrations within each combination of volume of concentration x block was summarized by regressing concentrations on responses for the calves in that particular treatment group of volume x block. Differences between serum Ig concentrations from one group of volume X block (regression line) to another were statistically analyzed by comparing regression lines (25). The equality of blocks, equality of volumes, and equality of blocks and volumes also were analyzed by this method. Data were analyzed in part by a statistical computer package (26).

A positive linear relationship best fit IgG as in Figure 1. Absorption of colostral IgG by experimental calves, as expressed by their serum concentration at 24 h after feeding was plotted from pooled data. The slope of the regression line for 2 liters (.26) indicates the greater amount of colostral Ig absorption compared to 1 liter (.17) at the same concentration fed. Coefficients of determination (r 2) for the two volumes are comparable. For colostral Ig absorption of the two volumes fed at the same concentration, efficiency of absorption also, may be considered based on mass (volume x concentration), that is, the number of IgG molecules ingested by the calf when fed i liter versus 2 liters at the same concentration. Lines in Figure 1 compare equal IgG mass absorption at the two volumes at low and high colostral concentrations. At low concentrations (10 to 20 mg/ml), 2 liters of colostrum fed at 10 mg/ml results in approximately the same absorption as 1 liter at 20 mg/ml, both volumes having the same IgG mass. However, at higher concentrations the relationship changes. One liter at 100 mg/ml, having the same mass as 2 liters at 50 mg/ml, has a greater absorption (18 mg/ml versus 15 mg/mI).

R ESU LTS

Regression analysis of data from the experiment, showing relationship of colostral Ig concentrations (Table 1) fed experimental calves to absorption of Ig as measured by Ig concentration in their serum, was plotted for each isotype (IgG, IgA, and IgM) within blocks and as pooled data for each isotope. Multiple regression methodology was used to determine the best fit absorption pattern.

TABLE 1. Experimental colostrum immunoglobulin concentrations (mg/ml), by treatments, blocks, and isotypes. Treatments IgG

IgA

lgM

T1 T2 T3 T4 T5

Blocks B1

B2

B3

B4

B5

B6

123.8 83.6

83.6 59.0 42.9 23.7 11.6

83.6 54.4 42.9

91.8 83.6 76.4

16.6

46.4

7.5

36.8

101.0 83.6 59.0 39.7 31.7

85.8 71.4 43.7 32.5 19.9

50.2

27.4 15.5

T1 T2 T3 T4 T5

5.53 3.45 2.43 1.04 .59

3.28 2.59 1.40 .87 .62

3.64 2.93 2.59 1.40 .38

T1 T2 T3 T4 T5

10.54 5.57 3.78 2.68 1.31

7.04 4.47 3.40 2.25 1.10

8.01 5.34 4.22 1.84 .46

3.83 3.09 2.75 1.55 1.04

3.83 3.45 3.09 1.55

7.33 6.77

9.05 7.05

5.89 4.58 3.64

5.59 4.02 2.10

1.04

4.42 3.64 2.28 1.68 1.13 11.19 9.38 5.34 4.22 2.49

Journal of Dairy Science Vol. 66, No. 6, 1983

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STOTT AND FELLAH

Each regression line in Figure 1 is based on 30 colostral samples of concentrations ranging from 7.5 to 123.8 mg/ml (Table 1, IgG) with each concentration fed to two calves at the same volume. Thus, each regression line has the same colostral samples fed but at different volumes to different calves, 60 calves for each. The IgG absorption based on the serum IgG concentration of the 120 calves ranged from 1.96 to 39.7 mg/ml. Regression lines for each block are shown in Figure 2 for 2 liters fed, in Figure 3 for 1 liter. Although the regression lines for blocks in each graph show slopes that appear nearly alike (positive linear), they are different (P<.01) within each volume fed and between volumes fed. Because each block represents colostrum from different cows, we observed variances of absorption between colostrum. Coefficients of determination (r 2) are not much higher in individual blocks than the pooled data, but the high r 2 in each block verifies the consistently close relationship of colostral IgG concentration with absorption of IgG by recipient calves.

Absorption of Colostral IgA

A positive linear regression related to concentration of IgA (Table 1) in the colostrum, also best fit absorption of IgA by the experimental calves at 24 h postcolostral feeding. Pooled data for the two volumes, from all six blocks, are in Figure 4. The slope of the regression for calves fed 2 liters (.24) illustrates greater absorption than when 1 liter was fed (.13) at the same concentration. Similar to IgG, the same colostral mass (volume × concentration), but at different concentrations, had variable influences on efficiency of IgA absorption. This is demonstrated in Figure 4 by the vertical and horizontal lines drawn from the points on the two regression lines representing equal masses at different concentrations. At low colostral IgA concentrations (1 to 2 mg/ml) feeding different volumes with equal mass resuits in similar IgA absorption. However, at higher concentrations the relationship changes.

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COLOSTRAL IgG CONCENTRATION(mg/ml) Figure 1. Twenty-four hour serum IgG concentration in calves related to colostral IgG concentration fed.

Journal of Dairy Science VoL 66, No. 6, 1983

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Figure 2. Relationship between colostrum and serum lgG concentration at 24 h postpartum for each block (B) of the neonatal calves fed 2 liters of colostrum.

IMMUNOGLOBULIN

ABSORPTION

One liter at 6 mg/ml has a 12.5% greater absorption than 2 liters at 3 mg/ml. Figures 5 and 6 show regression lines within blocks for absorption of IgA at the two volumes. The positive linear regression line for each block within each volume is different from the other blocks (P<.01), and each shows relatively high r 2 but lower than those in the same blocks for lgG.

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Absorption of Colostral IgM

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COLOSTRAL IgG CONCENTRATION(mg/ml) F i g u r e 3. R e l a t i o n s h i p b e t w e e n c o l o s t r u m a n d serum lgG concentration at 24 h postpartum for each b l o c k (B) o f t h e n e o n a t a l calves f e d 1 liter o f c o l o s trum.

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COLOSTRAL I g A CONCENTRATION

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F i g u r e 5. R e l a t i o n s h i p b e t w e e n c o l o s t r u m a n d s e r u m I g A c o n c e n t r a t i o n at 2 4 h p o s t p a r t u m f o r e a c h b l o c k (B) o f t h e n e o n a t a l calves f e d 2 liters o f c o l o s trum. J o u r n a l o f D a i r y S c i e n c e Vol. 6 6 , N o . 6, 1 9 8 3

1324

STOTT AND FELLAH [

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I 2.0

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IgA CONCENTRATION (mg/ml)

Figure 6. Relationship between colostrum and serum IgA concentration at 24 h postpartum for each block (B) of the neonatal calves fed 1 liter of colostrum,

creased serum IgM concentration at a decreased rate. Similar to IgG and IgA, absorption of IgM differed between volumes of colostrum fed ( P < . 0 1 ) . Calves receiving 2 liters of c o l o s t r u m a b s o r b e d m o r e t h a n t h o s e receiving 1 liter at t h e same c o n c e n t r a t i o n b u t were m u c h less efficient w h e n t o t a l m o l e c u l a r mass of IgM was considered. T h e i n f l u e n c e o f mass a n d c o n c e n t r a t i o n o n a b s o r p t i o n is d e m o n s t r a t e d w i t h vertical a n d h o r i z o n t a l lines in Figure 7. Calves receiving a r a t i o n low in IgM c o n c e n t r a t i o n w i t h t h e t w o v o l u m e s o f e q u a l mass (1 m g / m l at 2 liters versus 2 m g / m l at 1 liter) h a d similar IgM a b s o r p t i o n . This r e l a t i o n s h i p c h a n g e d as c o l o s t r u m s o f h i g h e r c o n c e n t r a t i o n s were fed. F e e d i n g at I liter at 10 m g / m l had a 30% greater a b s o r p t i o n t h a n 2 liters at 5 m g / m l (equal mass). Regression analysis o n IgM d a t a w i t h i n b l o c k s for calves receiving 2 liters (Figure 8) p r o d u c e d a positive linear r e l a t i o n s h i p b e t w e e n colostral a n d s e r u m IgM c o n c e n t r a t i o n f o r f o u r blocks only. The other two showed a quadratic Journal of Dairy Science Vol. 66, No. 6, 1983

( b l o c k 4) and a c u b i c r e s p o n s e ( b l o c k 5). T h e r 2 w h e r e 2 liters was fed are c o m p a r a t i v e l y high in all b l o c k s including t h o s e w i t h q u a d r a t i c a n d c u b i c responses. D a t a f r o m all b l o c k s fed 1 liter show linear a b s o r p t i o n r e s p o n s e to increased colostral c o n c e n t r a t i o n (Figure 9) w i t h o n e e x c e p t i o n , b l o c k 6, w h i c h s h o w s a q u a d r a t i c response. T h e b l o c k r 2 f o r l - l i t e r feedings is n o t as great as for 2 liters a n d is m o r e c o m p a r a b l e to t h e p o o l e d r 2.

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IMMUNO GLOBULIN ABSORPTION IN CALVES r BI-r2=.56

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of Absorption

Serum lg concentrations at 12 h after feeding colostrum (Figures 10, 11, and 12) for both 1 and 2 liters represent a high portion (IgG, liter 67%, 2 liters 70%; IgA, 1 liter 85%, 2 liters 85%; lgM, 1 liter 75%, 2 liters 90%) of the total amounts absorbed by 24 h, indicating the rapid intake and transport by the intestinal absorptive cells of the colostral Ig immediately after initial colostrum feeding. Regression lines in the figures for 12-h absorption are based on calculations for each concentration of colostrum fed (Table 1) and represent interactions of the amount fed (1 and 2 liters) and concentration of Ig in the colostrum. In all regressions, except for IgM at 2 liters, the amount absorbed by 12 h for each class has a positive linear relationship with concentration fed. With the 12-h interval as a time factor, the rate of Ig absorption for the first 12 h after initial feeding is a positive linear response related to concentration of IgG and IgA in the colostrum fed and also for IgM when the initial feeding is limited to 1 liter. When 2 liters are fed, IgM is slightly quadratic at high concentrations (Figure 12). In short, the higher the con-

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2: liters y = - 2 4 + . 2 2 X (IZh) r2=,74 P<.0i I liter y = , 0 2 + . 1 2 x (f2 h) r2=,66 P<.01

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COLOSTRAL IgA CONCENTRATION (mg/ml)

Figure 11. Relationship of colostral IgA concentration to rate of absorption based on m e a s u r e m e n t s of calf serum IgA concentration at 12 and 24 h postfeeding. Journal o f Dairy Science Vol. 66, No. 6, 1983

1326

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STOTT AND FELLAH

2liters y = .12+ .31X - , O I X 2 ~ (J2 h) r2= .55 P < .01 t I l i t e r v= 8 + 16X J " ( 2 h) " r 2 = 59 P < 0 1 / '

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Figure 12. Relationship of colostral lgM concentration to rate of absorption based on measurements of calf serum IgM concentration at 12 and 24 h postfeeding.

centration of colostral Ig fed, the more rapidly the calf absorbs Ig immediately after being fed. DISCUSSION

The primary objective of this experiment was to evaluate the role of colostral Ig concentration on absorption of Ig in the neonatal calf. We had measured the effect of calf age (hours postpartum) at the time of initial feeding of colostrum on absorption rate (35), amount of Ig absorbed (36), and influence on closure (34) from birth to 24 h postpartum. Interacting with age, three volumes (.5, 1, and 2 liters) of pooled colostrum were fed. The age of the calf at first colostral feeding and the volume fed had major roles in determining rate of absorption, time of closure, and amount of Ig absorbed. Now age at first feeding, volume fed, and maternal source of colostrum were constant whereas concentration of colostral Ig (30 concentrations) varied from 7.5 to 123.8 mg/ml for IgG, .38 to 5.53 mg/ml for IgA, and .46 to 11.19 mg/ml for IgM (Table 1). The replicated feeding of each concentration of two volumes within blocks (same colostrum) shows that concentration of Ig in colostrum is a major factor in determining rate of Ig absorption and amount absorbed before cessation of absorption occurs near 24 h postpartum. For the two isotypes IgG and IgA, the relationship of colostral Ig concentration to absorption, as indicated by Ig concentration in the experimental calf serum, is linear. This is Journal of Dairy Science Vol. 66, No. 6, 1983

demonstrated in Figures 1 and 4 for the pooled data for all blocks. That the relationship is linear (Figures 2, 3, 5, and 6) within blocks (different colostrums) with even greater r 2 further verifies linearity of the relationship of colostral Ig concentration to absorption in the calf. For IgM, the data indicate a quadratic relationship at high volumes and concentrations. This could be due to the chance distribution of the small numbers of observations (5) within volumes x blocks. Because four of the six blocks in Figure 8 and five of the six blocks in Figure 9 showed a linear response, samples from blocks that were not linear were reassayed (sRID) as well as the colostrum fed in the particular blocks. The repeated analysis verified the first analysis. The distribution question is not resolved. It is worth considering, however, that the quadratic response of IgM at high volumes and with high concentrations has been reported (36, 37) and suggests that in some way the absorption efficiency of the large molecules is altered when they are available in the gut in excessive amounts, either by volume, or concentration, or both, and particularly when colostrum feeding is delayed (36). If true, from a practical standpoint is suggests that feeding excessive volumes of colostrum (2 liters or more) at the initial feeding or later, defeats the purpose of gaining a high absorption of IgM in the neonate calf. F o r all three isotypes, there are high r 2 within blocks, indicating that when calves are fed at an early age, absorption of colostral lg is dependent upon colostral Ig concentration, and the amount absorbed can be estimated by the colostral concentration where age and volume are constant. The phenomenon is of practical importance in establishing reliable passive immunity in calves, because a simple, rapid method for determining colostral Ig concentration has been developed for field use (9). This experiment also was designed to show the simple interaction of colostrum volume and colostrum Ig concentration on absorption of colostral Ig. Two volumes were fed, one double the other (2 liters and 1 liter), for a purpose. Doubling the volume at any concentration would double the mass fed (concentration x volume) and could be compared with an equivalent mass with half the volume but double the concentration. At medium and high lg con-

IMMUNOGLOBULIN ABSORPTION IN CALVES c e n t r a t i o n s e q u i v a l e n t mass o f l - l i t e r v o l u m e is absorbed much more efficiently than equal mass at 2 liters. H o w e v e r , at low c o n c e n t r a tions, e q u i v a l e n t mass at 2 liters a n d 1 liter h a s practically e q u i v a l e n t a b s o r p t i o n . S o m e investig a t i o n s have placed m u c h e m p h a s i s o n mass w i t h limited a t t e n t i o n to c o n c e n t r a t i o n (4, 13, 15, 21, 22). F r o m o u r data, b o t h v o l u m e a n d c o n c e n t r a t i o n are i m p o r t a n t . E a c h n e e d s to b e e v a l u a t e d as a s e p a r a t e e n t i t y , b e c a u s e t h e v o l u m e of c o l o s t r u m t h a t can b e fed at initial feeding is limited. Also, as s h o w n , a b s o r p t i o n o f colostral lg f r o m equal mass b u t d i f f e r e n t Ig c o n c e n t r a t i o n varies w i t h c o n c e n t r a t i o n . Some authors advocate feeding large v o l u m e s of c o l o s t r u m (15, 20). However, if t h e Ig c o n c e n t r a t i o n of t h e c o l o s t r u m is low, i n a d e q u a t e Ig a b s o r p t i o n o c c u r s regardless o f t h e v o l u m e , as in Figure 1. Calves ingesting 2 or 3 liters will n o t a b s o r b e n o u g h Ig to e q u a t e a s t a t u s a b o v e h y p o g a m m a g l o b u l i n a e m i a if t h e c o n c e n t r a t i o n is b e l o w 2 0 m g / m l . P e n h a l e et al. (29, 30), s t u d y i n g t h e Ig c o n c e n t r a t i o n in calf s e r u m in w h i c h s p o n t a n e o u s disease occurred, f o u n d t h a t surviving calves had m e a n s of 7.5 m g / m t of lgG, .8 m g / m l IgM, a n d .2 m g / m l IgA; b e l o w these c o n t e n t s was considered h y p o g a m m a g l o b u l i n a e m i c . It is r e a s o n a b l e t h a t t h e Ig c o n c e n t r a t i o n in c o l o s t r u m used to t r a n s f e r m a t e r n a l i m m u n i t y t o t h e o f f s p r i n g is of p r i m a r y i m p o r t a n c e in assuring a desirable passive i m m u n i t y . C o n c e n t r a t i o n o f Ig in c o l o s t r u m , age at first feeding, a n d v o l u m e o f c o l o s t r u m f e d ( w i t h i n limits) are p r o b a b l y t h e m a j o r f a c t o r s a f f e c t i n g colostral Ig absorption. ACKNOWLEDGMENTS

T h e a u t h o r s are g r a t e f u l to S h a m r o c k Dairies, Inc., T u c s o n , AZ, for a n i m a l s a n d facilities t h a t were supplied g e n e r o u s l y f o r this p r o j e c t . T h e f i n a n c i a l assistance of t h e U n i t e d D a i r y m e n of A r i z o n a also is a c k n o w l e d g e d gratefully. REFERENCES

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