Research in Veterinary Science 1992, 53, 219-222

Effect of colostrum intake on serum of calves

-lactalbumin concentrations in

C. G. PROSSER, S. J. EICHLER, V. C. FARR, S. R. DAVIS, Dairying Research Corporation,

Ruakura Agricultural Centre, Private Bag, Hamilton, New Zealand

Seven Friesian calves were fed colostrum for four days beginning within 24 hours of birth, and milk thereafter. The concentration of ~-lactalbumin in serum was measured by specific radioimmunoassay and compared to IgG assayed by electroimmunodiffusion. Serum concentrations of ~-lactalbumin peaked at 387 + 85 ng ml-1 within eight hours of initial intake of colostrum, declining to 12 + 3 ng mi-1 by day 6. IgG rose steadily to 17 mg ml-1 by 48 hours of birth and remained relatively constant thereafter. The temporal pattern of c~-lactalbumin in serum following colostrum intake confirms previous studies suggesting reduced absorption of colostral proteins between 24 and 36 hours. The presence of variable amounts of c~-lactalbumin in serum even after 17 days, however, indicates limited transfer of milk-derived proteins across the gut at this time. The data further show that cessation of maximal gut transfer does not relate to molecular weight of transferred protein.

WHILE conducting experiments assessing functionality of mammary epithelial cells grown in culture it was found that commercial sources of fetal calf serum and serum from newborn calves differ in that the latter possesses high concentrations of cMactalbumin. The presence of a-lactalbumin in calf serum is a novel observation of unknown physiological significance, presumably relating to the intake of colostrum by the calf. Other colostral proteins are known to be transferred intact across the gut of the neonate, including y-globulins (Bangham et al 1958, Brandon and Lascelle~s 1971, Husband et al 1972, Bush and Staley 1980) and 7-glutamyl transferase (Braun et al 1982). These proteins, which are much larger than o~-lactalbumin, are only transferred during the first 24 to 36 hours of birth (Kruse 1970, Husband et al 1972, Braun et al

1982). The present study was designed to follow the temporal pattern of oMactalbumin in calf serum to determine whether the timing of gut closure varied according to the relative size of the protein. Materials and methods

Animals Seven Friesian calves (30 to 42 kg liveweight at birth) were fed colostrum for four days beginning within 24 hours of birth. For four of these calves, colostrum was fed twice daily (3.0 litres per feed) at approximately 08.00 and 16.00. The other three calves received 6 litres of colostrum once a day. After the fourth day each calf received 6 litres of milk per day. Colostrum was pooled from several recently calved animals. Blood was taken by jugular venipuncture from each calf at birth and thereafter twice daily before feeding colostrum until four days, then daily for a further three days and then every second day until day 17 following birth. The blood was allowed to clot at room temperature and serum harvested for subsequent analysis of c~-lactalbumin and IgG.

Radioimmunoassay for ~-lactalbumin The radioimmunoassay (RIA)for o~-lactalbumin utilised rabbit anti-bovine (x-lactalbumin (donated by M. Grigor, University of Otago, New Zealand) at a dilution of 1:18,000 in 50 mM sodium phosphate buffer, pH 7-5, containing 0.25 per cent (w/v) bovine serum albumin. Standard c~-lactalbumin was diluted in the same buffer to give 0.16 to 100 ng ml-~. Samples of serum and colostrum were diluted 1:10 and 1:100,000, respectively. 219

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C G. Prosser, S. J. Eichler, V. C. Farr, S. R. Davis

Samples (500 gl) of standards and unknowns were incubated with antiserum (100 gl) and [1251]_ labelled o~-lactalbumin (approximately 22,000 cpm 100 gl-1) overnight at room temperature. c~-Lactalbumin was iodinated using iodogen (Pierce, Rockford, Illinois) according to the method of Salacinski et al (1981). The o~-lactalbumin used for iodination and as standard was purchased from Sigma (St Louis, Missouri) and repurified on a phenyl-sepharose column (Lindahl and Vogel 1984). Following the overnight incubation, diluted goat anti-rabbit I g G and normal rabbit serum were added with 3.0 per cent (w/v) PEG 6000. Incubation continued for two hours at room temperature, then bound radioactivity was precipitated by centrifugation. The supernatant was decanted and precipitated radioactivity measured with a 5I(B 1261 Multigamma gamma-counter. All samples were run in the one assay. Electroimmunodiffusion o f lgG The procedure of electroimmunodiffusion (EID) for measurement of concentrations of IgG in serum was based on the method of Laurell (1966) as applied to IgG by Weeke (1968) and Grub (1970). Standard bovine IgG (Sigma), was diluted to give 0-25 to 1 mg mkl and serum was diluted 1:20. Agarose (1 per cent w/v) containing 0.8 to 1-5 per cent final concentration rabbit anti-bovine IgG (Sigma) was spread on a 100 x 100 mm glass plate. The buffer used for dilution of serum, standards and 1 per cent agarose was 0.08 M Tris, 0-02 M tricine buffer, p H 8.5, with 0,5 m M calcium lactate. For assay, standards and samples were diluted 1:3 with 2 M potassium cyanate, heated to 45°C for 45 to 60 minutes then 3 ~1 applied to wells punched out of the agarose plates. Plates were electrophoresed for three hours at 8°C at 200 volts. Gels were dried in an oven, stained with Coomassie blue R250 and the area under the immunoprecipitation arc for standards and unknowns measured.

lysozyme in the assay. Only [3-1actoglobulin xcaused any significant displacement of radiolabelled ~x-lactalbumin (equivalent to l ng mkl czlactalbumin at 100 ng ml-1 [3-1actoglobulin). This was later found to relate to contamination of [3lactoglobulin preparation with minor amounts of a-lactalbumin (data not shown). Addition of standard a-lactalbumin to bovine serum yielded 100 per cent recovery and dilutions of serum gave displacement curves parallel to standard (zlactalbumin. The lower limit of detectability of the assay was 0.2 ng ml-1. The concentration of o~-lactalburnin and IgG in serum of calves ingesting 6 litres of colostrum is shown in Fig 1. Although four of the seven calves received colostrum in two lots of 3 litres each and the others as one lot of 6 litres, the concentrations of o~-lactalbumin or IgG did not differ substantially between treatments. Therefore data for all seven animals were combined. No cz-lactalbumin was detected in sera from newborn calves before the first intake of colostrum. However, within eight hours of the initial feed, concentrations had risen to 387 + 85 ng ml 1 which represented the highest level attained. Thereafter concentrations declined rapidly, reaching lowest levels approximately six days after birth. Significant amounts of o~-lactalbumin were present in sera collected up to 17 days after birth. Concentrations of IgG in serum showed a different pattern to o~-lactalbumin, being detectable even in pre-colostral sera (Fig 1). As with o~-lactalbumin, concentrations rose rapidly following the initial intake of colostrum, but unlike o~-lactalbumin no true peak could be discerned. Rather, concentrations plateaued at approximately 17 mg mkX within the first 48 hours of birth. 5OO 7T_ E 400

The RIA of a-lactalbumin in serum was based on that described by Akers et al (1986). The specificity of the antiserum was tested by including various amounts (up to 100,000 ng m1-1) of purified casein, [3-1actoglobulin, lactoferrin or

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FIG 1 : Concentrations of c~-Iactalbumin (0) and IgG (O) in serum from colostrum fed calves. Results are mean _+SEM of seven calves receiving 6 litres of colostrum for four days beginning within 24 hours of birth (time O) and 6 litres of milk thereafter

~-Lactalbumin in calf serum

Discussion Akers et al (1986) was one of the first to address the question of the level of ~-lactalbumin in bovine serum, but reportedly found no detectable a-lactalbumin in serum from calves. However, the serum was obtained from animals which were three to nine months old and presumably fed a forage/grain diet. In the present report it has been shown that sera from newly born calves contain significant amounts of c~-lactalbumin, but only following intake of colostrum, implying transfer of this whey protein across the gut without degradation. The temporal pattern for c~-lactalbumin in serum of colostrum fed calves more closely resembles that ofy-glutamyl transferase activity (Braun et al 1982) than IgG (Husband et al 1972, Logan et al 1973), with an initial rapid rise, but declining very quickly after reaching maximum in the first 24 hours of birth. McEwan et al (1968) have shown that plasma volume of calves increases nearly 50 per cent during the first few days of life and Logan et al (1973) reported that most of this expansion takes place within several hours of birth. Thus part of the initial decline in serum concentrations of o~-lactalbumin and y-glutamyl transferase may be an effect of dilution. The decline in y-glutamyl transferase activity in colostrum occurring during the first week post partum (Braun et al 1982) may also contribute to the continuing fall in activity in serum of calves by reducing intake of this protein. However, this would not be the case for the continuing decline in o~-lactalbumin because its concentration in colostrum and milk fed to the calves was constant over the period of the present study. This decline in c~-lactalbumin concentrations in calves is consistent with previous studies suggesting reduced absorption ofimmunoglobulin from colostrum between 24 and 36 hours (Kruse 1970, Husband et al 1972). The similarity between the pattern of absorption of y-glutamyl transferase and a-lactalbumin which vary approximately fivefold in weight (80 kDa versus 15 kDa; Baumrucker 1980) confirms the lack of selectivity of the gut of the young to absorb any particular protein (Bangham et al 1958, Brandon and Lascelles 1971). The present data extend these findings, indicating the timing of cessation of maximal transfer of milk-derived proteins is not dependent on the relative size of the protein either.

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The presence of variable amounts of c~-lactalbumin in serum up to 17 days post partum, also reported for y-glutamyl transferase, indicates limited transfer of milk-derived proteins across the gut at this time. In this instance ~-lactalbumin may be a more reliable indicator of the degree of gut permeability to milk proteins because of its uniqueness to milk, whereas y-glutamyl transferase is also synthesised by the calf (Braun et al 1978). Thus ~-lactalbumin, similar to IgG and y-glutamyl transferase, may be used as a reliable indicator ofcolostrum intake in calves. The presence of significant quantities of ~-lactalbumin in newborn calf serum, however, precludes its use in tissue culture studies where measurement of cellular output of cMactalbumin is required. It is of further interest that, unlike IgG or y-glutamyl transferase, no o~-lactalbumin was detectable before colostrum intake. This is consistent with the unique origin of this protein, being derived solely from milk whey. The absence of detectable o~-lactalbumin in serum from either fetal or unfed newborn calves also means there is little placental transfer of c~-lactalbumin present in maternal circulation during pregnancy (Akers et al 1986). This contrasts to the situation in the human where significant amounts of c~-lactalbumin were detected in cord blood and serum of non-fed infants less than one day old (Jokobsson et al 1986).

Acknowledgements We thank S. Burke for the care of calves, Associate Professor M. Grigor, University of Otago, for antisera to bovine a-lactalbumin and R. McLaren for assistance with determination of serum levels of IgG.

References AKERS, R. M., McFADDEN, T. B., BEAL, W. E., GUIDRY, A. J. & FARRELL, H. M. (1986) Radioimmmloassay for measurement of bovine c~-lactalbumin in serum, milk and tissue culture media. Journal of Dairy Research 53, 419-429 BANGHAM, D. R., INGRAM, P. L., ROY, J. H. B., SHILLAM, K. W. G. & TERRY, R. J. (1958) The absorption of 13q-Iabelled serum and colostral proteins from the gut of the young calf. Proceedings of the Royal Society of London (Series B) 149, 184-191 BAUMRUCKER, C. R. (1980) Purification and identification of yglutamyl transpeptidase of milk membranes. Journal of Dairy Science 63, 49-54 BRANDON, M. R. & LASCELLES, A. K. (1971) Relative efficiency of absorption of IgG l, IgG 2, IgA and IgM in the newborn calf.

Australian Journal of Experimental Biological and Medical Science 49, 629-633

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C. G. Prosser, S. J. Eichler, V. C. Farr, S. R. Davis

BRAUN, J. P., RICO, A. G., BENARD, P., THOUVENOT, J. P. & BONNEFIS, M. J. (1978) Blood and tissue distribution of gamma glutamyl transferase in calves. Journal of Dairy Science 61, 596-599 BRAUN, J. P., TAINTURIER, D., LAUGIER, C., BENARD, P., THOUVENOT, J. P. & RICO, A. G. (1982) Early variations of blood plasma gamma-glutamyl transferase in newborn calves - a test of eolostrum intake. Journal of Dairy Science 65, 2178-2181 BUSH, L. J. & STALEY, T. E. (1980) Absorption of colostral irnmunoglobulins in newborn calves. Journal of Dairy Science 63, 672-677 GRUB, A. (1970) Quantification ofimmmaoglobinG by electrophoresis in agarose gel containing antibodies. Scandinavian Journal of Laboratory Investigation 26, 249-255 HUSBAND, A. J., BRANDON, M. R. & LASCELLES, A. K. (1972) Absorption and endogenous production ofimmtmoglobins in calves. Journal of Experiemental Biological and Medical Sciences 50, 491498 JOKOBSSON, I., LINDBERG, T., LOTHE, L., AXELSSON, I. & BENEDIKTSSON, B. (1986) Human c~-lactalbumin as a marker of macromolecular absorption. Gut 27, 1029-1034 KRUSE, V. (1970) Absorption of immunoglobin from colostrum in newborn calves. Animal Production 12, 627-638 LAURELL, C.-B. (1966) Quantitative estimation of proteins by elec-

trophoresis in agarose gel containing antibodies. Analytical

Biochemistry 15, 45-52 LINDAHL, L. & VOGEL, H. J. (1984) Metal-ion-dependent hydrophobic-interaction chromatography of c~-lactalbumins. Analytical Biochemistry 140, 394-402 LOGAN, E. F., PENHALE, W. J. & JONES, R. A. (t973) Changes in the serum immunoglobin levels of colostrum-fed calves during the first 12 weeks post partum. Research in Veterinary Science 14, 394-397 MeEWAN, A. D., FISHER, E. W. & SELMAN, I. E. (1968) The effect of colostrum on the volume and composition of the plasma of calves. Research in Veterinary Science 9, 284-286 SALACINSKI, P. R. P., McLEAN, C., SYKES, J. E. C., CLEMENTJONES, V. V. & LOWRY, P. J. (1981) Iodination of proteins, glycoproteins and peptides using a solid-phase oxidising agent, 1,3,4,6-tetrachloro-3c~,60~-diphenyl glycoluril (lodogen). Analytical Biochemistry 117, 136-146 WEEKE, B. (1968) Carbamylated human immunoglobins tested by electrophoresisin agarose and antibody containing agarose. Journal of Laboratory Investigation 21, 351-354

Received November 14, 1991 Accepted February 7, 1992