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Serum Lactoferrin Concentrations In Calves Fed Fresh Or Frozen Colostrum
Nicole M. Holloway, MS*
Jeffrey Lakritz, DVM, PhD*
Jeff W. Tyler, DVM, MPVM, PhD*
Steven L. Carlson, DVM†
*Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, Missouri.
†Private practice, Visalia, CA.
The enclosed research was supported in part by USDA Formula Funds and USDA National Research Initiative Competitive Grants Program (Agreement No. 2001–35204–10799). Additional support was provided by the Minority Biomedical Researchers Training Initiative, and the MU/NIH Science Education Partnership Awards, University of Missouri-Columbia and the University of Missouri Chancellor’s Gus T. Ridgel Fellowship for Underrepresented Minority Americans.
WORDS: Calves, colostrum,
Experiments were conducted to determine whether serum lactoferrin concentrations in neonatal calves are affected by frozen storage of colostrum. Calves were randomly assigned to receive fresh (n = 13) or frozen (n = 10) colostrum. Significant differences in serum lactoferrin concentrations were seen among fresh and frozen colostrum-fed calves on days 4 and 7. There were significantly increased serum lactoferrin concentrations relative to day 0 concentrations on days 4, 6, and 7 in calves receiving frozen colostrum and on day 6 in calves receiving fresh colostrum. Serum lactoferrin concentrations are not decreased in calves receiving frozen colostrum relative to calves receiving fresh colostrum.
Colostrum composition varies with several factors, including individual, breed, parity, prepartum diet, length of dry period, and time interval postpartum.1–3 The timely ingestion of colostrum is an important determinant of calf health.2,3 Colostral immunoglobulin is actively absorbed by the calf intestinal epithelium, providing a source of protective antibodies. The absorption of colostral immunoglobulins is critically important because transplacental transfer of immunoglobulins is negligible in livestock, and calves are born essentially agammaglobulinemic. Although the role of immunoglobulins has been well described, colostrum contains other factors whose role in the neonatal immune response is less well described. One such factor is lactoferrin.
Lactoferrin is a protein with multiple functions.4,5 Lactoferrin is present in colostrum, mammary secretions, tears, seminal fluid, synovial fluid, plasma, and neutrophil granules. Lactoferrin is an iron transport and storage glycoprotein.4,6 In the presence of bicarbonate, lactoferrin sequesters iron, making it unavailable to support bacterial growth.7 Lactoferrin binds the lipid A portion of endotoxin, preventing lipopolysaccharide activation of macrophages and decreasing cytokine production.5 Thus, lactoferrin inhibits bacterial colonization of the gastrointestinal tract.
The purpose of this study was to determine the effect of frozen colostrum storage on serum lactoferrin concentrations in neonatal calves. Frozen storage is frequently recommended as a method both to preserve colostrum and as a mechanism to limit the spread of viral pathogens.
Materials and Methods
Sample collection took place at two privately owned dairies in California’s San Joaquin Valley. Twenty-three Holstein calves were randomly assigned to one of two treatment groups, fresh (group 1, n = 13) or frozen/thawed (group 2, n = 10) colostrums. Calves were isolated from dams immediately after birth. A 10-mL sample was obtained from each aliquot and frozen (–20˚C) for later determinations of colostral lactoferrin. Fresh colostrum was stored at refrigerator temperatures until used. Frozen colostrum was stored for 24 hours in a –20˚C freezer. Four L of colostrum were administered via oroesophageal intubation at 3 hours postpartum, allowing frozen colostrum adequate time to thaw in an ambient temperature water bath (approximately 25˚C).
Serum samples were collected from each calf before the ingestion of colostrum (day 0) and at 2, 4, 6, and 7 days postpartum. The precolostral status of calves included in the study was confirmed by the presence of serum immunoglobulin G concentrations less than 200 mg/dL at the time of the precolostral sampling. Serum samples were stored at –20˚C until laboratory determinations of serum lactoferrin concentration.
Colostral lactoferrin concentrations were determined using an enzyme-linked immunosorbent assay (ELISA).8 Briefly, plates were first coated with (1 ng/well) of goat antibovine lactoferrin (Bethyl Laboratories, Montgomery, TX) at 22˚C. The plates were then washed and blocked with 3% bovine serum albumin (BSA; Sigma Chemical Company, St. Louis, MO). The plates were washed and 1:1000 dilutions of colostrum were placed into wells. After incubation, the plates were washed. Goat antibovine lactoferrin HRP-conjugate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added to each of the sample wells.
After incubation with the secondary antibody, the plates were washed, and 100 nl of 2,2’-azino-di (3-ethyl-benzthiazoline-6- sulfonate) (ABTS; Sigma Chemical Company) solution was added to each well and allowed to incubate. Plates were analyzed by examining the intensity of color change at 405 nm using an automated plate reader. Sample concentration was determined by comparing their absorbance values to those of the standard curve. Standards were prepared by diluting known concentrations of lactoferrin (500 ng/mL, 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31.25 ng/mL, 15.625 ng/mL, and 7.8 ng/mL). Serum lactoferrin concentrations were determined using a similar ELISA assay. Serum samples were diluted 1:100. Colostrum and serum immunoglobulin G concentrations were determined using a radial immunodiffusion assay.3,9
A two-way analysis of variance (ANOVA) for repeated measures was performed, comparing serum lactoferrin concentrations for the treatment groups at successive serum collection times. In this analysis, time was considered a repeating independent variable. The null hypothesis, that serum lactoferrin concentrations do not differ between treatment groups at subsequent collection times, was rejected with P values < .05.
Colostral lactoferrin concentrations did not differ among fresh (630 ± 128 ng/mL) and frozen colostrum (672 ± 98 ng/mL). Day 2 serum lactoferrin concentrations did not differ significantly from precolostral (day 0) concentrations in calves fed either fresh or frozen colostrum. In calves fed fresh colostrum, day 6 serum lactoferrin concentrations were significantly greater than day 0 concentrations, and in calves fed frozen colostrum, day 4, 6, and 7 serum lactoferrin concentrations were significantly increased relative to day 0 concentrations.. A statistically significant difference was present among mean serum lactoferrin concentrations on days 4 and 7 and on day 2. No other comparison among treatment groups or sampling times differed significantly. Complete results are provided in Tables 1–3.
Passive transfer of colostral immunoglobulin is a phenomenon that has been described in great detail. Passive transfer is a nonselective process in which proteins within the gastrointestinal lumen are entrapped within pinocytotic vesicles that are then transported across the cytoplasm of the villous epithelial cells and released into the extracellular fluid compartment. Although the transfer of immunoglobulins is most often studied and has been best described, other proteins are transported by this mechanism. Studies have shown the transfer of gamma-glutamyl transferase, alkaline phosphatase, and aspartate aminotransferase activities in lambs, calves, and puppies after the ingestion of colostrum.10–14 These studies substantiate that the passive transfer process is relatively nonselective and that nonimmunoglobulin proteins can and will be transferred in this fashion.
Although it is tempting to attribute the observed increases in serum lactoferrin concentrations to passive transfer of colostral lactoferrin, this hypothesis is not supported by the present study. In both experiments, day 2 serum lactoferrin concentrations did not differ significantly from precolostral (day 0) concentrations in calves fed either fresh or frozen colostrum. In experiment 2, calves fed fresh colostrum showed day 6 serum lactoferrin concentrations that were significantly greater than day 0 concentrations, and calves fed frozen colostrum had day 4, 6, and 7 serum lactoferrin concentrations that were significantly higher than day 0 concentrations. Previous studies have shown that closure of the gastrointestinal epithelium to protein macromolecules occurs by 24 hours of age in calves.15,16 The lack of a significant increase in serum lactoferrin concentration on day 2 does not support the hypothesis that increases in serum lactoferrin concentration observed on days 4, 6, and 7 were caused by passive transfer of colostral lactoferrin.
Perhaps the easiest way to place the ability of the calf to absorb colostral lactoferrin in perspective is to compare the absorption of colostral lactoferrin with that of colostral immunoglobulin. Colostrum from Holstein cows will have approximately 75 g/L immunoglobulin G.1,3 A serum immunoglobulin G concentration of 1500 mg/dL (15 g/L) is typical of calves that are fed an appropriate volume of colostrum in a timely manner.1,2,17 Consequently, a 5:1 ratio of colostrum to serum immunoglobulin G concentrations is the expected norm. This contrasts sharply with the absorption of colostral lactoferrin. If we compare colostral lactoferrin concentrations with day 2 calf serum lactoferrin concentrations, the analogous ratio is greater than 100 (672 ?g/mL to 6 ng/mL). This observation suggests that colostral lactoferrin is absorbed less efficiently than is colostral immunoglobulin. Although we have no evidence supporting a link between colostral and calf serum lactoferrin concentrations, suprapharmacologic colostral lactoferrin may result in higher serum concentrations.
It appears that feeding frozen colostrum will cause consistent and significant increases in serum lactoferrin concentrations. Significant differences were noted between the 2 groups on days 4 and 7. We are left with the conclusion that the administration of a previously frozen colostrum source will result in increased calf serum lactoferrin concentrations.
Either administration of frozen colostrum up-regulates lactoferrin production or a normally present mechanism that down-regulates lactoferrin production is removed by the freezing process. Studies that measure serum concentrations of acute phase proteins, cytokines, and inflammatory mediators in calves fed fresh and frozen colostrum may help clarify these hypotheses.
An important point that deserves passing mention is the dearth of information regarding the effect of frozen colostrum on calf health. Although this practice is often advocated, studies documenting that immunoglobulins in frozen colostrum are absorbed were only published in the past year.9 Furthermore, studies of reasonable statistical power that compare the health of calves fed fresh and frozen colostrum have never been reported.
The authors thank Dr. James Cullor and the staff of the University of California-Davis Veterinary Medicine Teaching and Research Center, Tulare, California for assistance and the use of their facilities.
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Table 1. Mean Serum Lactoferrin Concentrations in Calves of Varying Ages After Administration of Fresh Colostrum in Experiment 2*
Day 0 2.5 – – – –
Day 2 6.1 0.95 – – –
Day 4 12.0 0.32 0.76 – –
Day 6 17.1 0.03 0.19 0.85 –
Day 7 13.6 0.18 0.55 0.99 0.96
*Reported P values represent the probability that daily means do not differ.
Table 2. Mean Serum Lactoferrin Concentrations in Calves of Varying Ages After Administration of Frozen Colostrum in Experiment 2
Day 0 2.1 – – – –
Day 2 6.2 0.95 – – –
Day 4 33.4 < 0.01 < 0.01 – –
Day 6 21.9 0.01 0.05 0.27 –
Day 7 29.0 < 0.01 < 0.01 0.94 0.73
*Reported P values represent the probability that daily means do not differ.
Table 3. Serum Lactoferrin Concentrations at 0, 2, 4, 6 and 7 Days of Age in Calves Receiving Fresh or Frozen Colostrum in Experiment 2
Calves Receiving Calves Receiving
Fresh Colostrum Frozen Colostrum
Lactoferrin Lactoferrin P
0 2.5 2.1 0.95
2 6.1 6.2 0.98
4 12.0 33.4 < 0.01
6 17.1 21.9 0.41
7 13.6 29.0 0.01
*Reported P values represent the probability that daily means do not differ.
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