Lactoferrin supplementation of the neonatal calf has no impact on immunoglobulin G absorption and intestinal development in the first days of life¹

Abstract

The objectives of this study were to determine if newborn calves receiving supplemental lactoferrin (LF) had improved IgG uptake and if supplemental LF enhanced intestinal development through estimation of xylose uptake. Twenty-four newborn Holstein bull calves were randomly assigned to 1 of 2 treatments: 0 or 1 g/d of supplemental LF. Calves were fed pooled maternal colostrum from 9 cows in 2 feedings: at birth and 12 h later. Calves consumed in excess of 200 g of IgG. Blood samples were taken before colostrum feeding (0 h) and at 12, 18, and 24 h after birth. Blood samples were analyzed for IgG concentration. On d 2 of life, calves were fed milk replacer with the added LF and 0.5 g/kg of BW xylose to determine if supplemental LF affected intestinal development. Blood was sampled at 0, 0.5, 1, 2, 3, 4, 6, 8, and 12 h after the xylose dose. All calves attained passive transfer and supplemental LF did not affect IgG uptake (P ≥ 0.36) or apparent efficiency of absorption of IgG (P = 0.49). Lactoferrin did not enhance rate of absorption at any time point (P ≥ 0.36). There were no differences in xylose (P = 0.28) or glucose (P = 0.27) area under the curve values in calves supplemented with either 0 or 1 g/d LF. Lactoferrin did not enhance IgG uptake during the first 24 h or intestinal development in calves on the second day of life.

INTRODUCTION

Lactoferrin (LF) is an iron binding glycoprotein found in whey and endocrine excretions (Steijns and Hooijdonk, 2000) and is found in high concentrations in colostrum (1–2 mg/mL; Molenaar et al., 1996). Colostral LF may play a role in calf health because of its involvement in intestine and immune development in humans and mice (Shah, 2000; Zhang et al., 2001). Feeding 1 or 10 g/d LF to preweaned calves fed milk replacer (MR) improved grain intake and ADG (Joslin et al., 2002). Feeding 1, 2, or 3 g/d LF increased ADG before weaning (Robblee et al., 2003) and 1 g/d LF reduced fecal scores compared with calves fed no supplemental LF. Fecal scores are a measure of diarrhea (1 being firm and 4 being liquid). Calves are born agammaglobulinemic and are dependent on passive transfer of immunoglobulin to acquire functional immunity. Over 40% of calves in the United States have failure of passive transfer (serum IgG concentrations < 10 g/L; NAHMS, 2008). Colostral LF is absorbed intact on d 1 of life along with other macromolecules (Talukder et al., 2002). Supplemental LF may have a positive impact on IgG absorption in the neonatal calf. Prgomet et al. (2007) supplemented colostrum (transition milk) on d 3 of life with 1.5 to 1.6 g/d LF and observed increased serum IgG concentration on d 6 and 2 wk postpartum compared with calves not fed supplemental LF. Shea et al. (2009) reported no benefit of LF on IgG uptake in calves fed colostrum replacer. Lactoferrin supplementation has been shown to increase intestinal development in chicks (Humphrey et al., 2002) and mice (Zhang et al., 2001). The null hypotheses of this study were that LF would not increase IgG or xylose uptake. Therefore, the objectives were to further investigate the effect of adding supplemental LF to pooled maternal colostrum on IgG uptake and intestinal development.

MATERIAL AND METHODS

Calves and Treatments

Twenty-four Holstein bulls (45.8 ± 6.2 kg BW) were assigned at birth to a randomized complete block design. Initial BW was taken within the first 12 h of life using a platform scale (Salter Housewares USA Inc., Oakbrook, IL). Calves were removed from their dams at birth and not allowed to suckle. Navels were dipped in a tincture of iodine and calves were placed in individual pens bedded with kiln-dried sawdust. This study was approved by the Institutional Animal Care and Use Committee at the University of New Hampshire (number 040304).

Colostrum (at least 2 L) was collected from 9 cows after parturition and frozen (–20°C) in 2-L plastic bottles. After colostrum harvest, colostrum quality was tested using a colostrometer. The concentration of IgG was recorded for each cow and recorded on each 2-L bottle. Frozen colostrum was thawed by allowing the plastic bottles to stand at room temperature until thawed and pooled to provide a minimum IgG concentration of 50 g/L based on colostrometer values at harvest. At time of feeding, frozen pooled colostrum was thawed in warm water and LF was added to each according to treatment. Two liters of colostrum were fed within 4 h of birth (±3 h) and the second feeding (2 L) was 8 h later with or without 1.0 g of supplemental LF (0.5 g LF/feeding) according to treatment. The dose of 1.0 g/d LF was chosen due to the beneficial effects shown in 2 studies conducted in our laboratory (Joslin et al., 2002; Robblee et al., 2003). Iron saturation of the supplemental LF was 13.2 g/100 g (Immucell Corp., Westbrook, ME). This is a measure of the amount of Fe bound to the LF molecule. Lactoferrin functions by binding available Fe.

According to colostrometer estimations, pooled colostrum concentrations were similar. However, on analysis using radial immunodiffusion (Global Beta Health, Nevada, IA), colostrum IgG concentration was 55.5 g/L for the 0 g supplemental LF treatment and 53.25 g/L for the LF treatment. Therefore, total IgG fed was 222 g (0 g supplemental LF) and 213 g (1 g supplemental LF). Colostrum was fed via nipple bottle. After the 2 feedings of colostrum, calves were fed a milk protein–based MR (Blue Seal Feeds, Kent Nutrition Group, Muscatine, IA) twice daily (0700 and 1700 h) with or without supplemental LF (calves that received LF in colostrum also received LF in MR). Milk replacer was sampled from each bag used (250 g) and analyzed for CP by Kjeldahl analysis (AOAC, 1979), fatty acid content was determined by saponification in KOH in ethyl alcohol (Oser, 1965), and Ca, P, Mg and Fe (method 985.01; AOAC, 1990). The MR was dried for 6 h at 60°C in a vacuum oven for DM determination. Analyses of MR (DM basis) were 28.5% CP, 18.4% fatty acids, 0.92% Ca, 0.94% P, and 78 mg/kg Fe. Dry matter of the MR was 97%. Milk replacer was fed to provide 0.2 Mcal ME/kg BW^0.75 (Tikofsky et al., 2001) and reconstituted to 15% DM.

Immunoglobulin G

Samples of colostrum were stored at –20°C for later analysis of IgG and LF concentration by radial immunodiffusion (Cardiotech Services, Inc., Louisville, KY). Blood samples were obtained from the jugular vein into an evacuated tube (Monoject; Sherwood Medical Industries Inc., St. Louis, MO) for serum IgG concentration. The first sample (referred to as 0 h) was taken immediately before the first feeding of colostrum. Additional blood samples were obtained at 12, 18, and 24 h after the initial sample. Blood samples were stored for at least 1 h after collection to allow for clotting. Serum was harvested from blood via centrifugation at 3,300 × g at 25°C for 20 min and stored at –20°C until analyzed for IgG. Apparent efficiency of absorption at 12, 18, and 24 h of age was estimated using the equation [serum IgG (g/L) × BW (kg) × 0.09/IgG intake (g)] × 100 (Quigley and Drewry, 1998). Rate of absorption was calculated by subtracting the previous serum sample IgG concentration from the current serum sample IgG concentration and dividing the difference by the time in hours between samples.

Xylose Challenge

On d 2 of life, D-xylose was mixed (0.5 g/kg of BW) in MR and fed during the morning feeding. Blood samples were taken at 0.5, 1, 2, 3, 4, 6, 8, and 12 h after feeding. Samples were obtained from the jugular vein into an evacuated tube containing tripotassium EDTA (Monoject). Calves had free access to water but were not fed their second MR feeding until sampling was completed. Plasma was harvested from blood via centrifugation at 3,300 × g at 25°C for 20 min. Two aliquots of plasma were stored at –20°C and later analyzed for d-xylose and glucose concentrations. d-Xylose was analyzed as described by Merritt and Duelly (1983). Glucose was analyzed using a glucose oxidase–based kit (Wako Chemicals USA, Inc., Richmond, VA).

Area under the Curve

Area under the curve (AUC) for serum or plasma samples was calculated using the trapezoidal rule (Phillips and Taylor, 1973). This analysis calculates the area of each trapezoid with hour being the independent variable and concentration of IgG (g/L), xylose (mmol/L), or glucose (mg/dL) being the dependent variable. Units for IgG AUC are (grams per liter) ∙ hour, units for xylose AUC are (millimoles per liter) ∙ hour, and units for glucose AUC are (milligrams per deciliter) ∙ hour.

Statistical Analysis

The main effect was dietary supplementation of LF (0 or 1 g/d). Analysis of variance was analyzed using the MIXED procedure of SAS (version 9.3; SAS Inst. Inc., Cary, NC). Significance was determined at P ≤ 0.05. The mixed effects model was

in which Y_i is the dependent continuous variable, µ is the overall mean, B_i is the random effect of block (i = 1, …, 12), L_j is the random effect of LF source (j = 0, 1), H_k is the effect of hour (12, 18, and 24 for IgG and 0.5, 1, 2, 3, 4, 6, 8, and 12 for xylose and glucose measurements), and E_ijk is the random residual ∼N(0, σ²).

Calf was considered random. Degrees of freedom were calculated using the Kenward–Roger option of the MIXED procedure of SAS. The lowest Bayesian information criteria based on the following tests of covariance structure were used: autoregressive type 1, compound symmetry, unstructured, and Toeplitz.

RESULTS AND DISCUSSION

Birth weight, colostrum and IgG intake, serum concentration, AUC, and absorption rates over the first day of life of calves fed supplemental LF are in Table 1. The amount of LF derived from colostrum consumed by each calf was 4.3 g for calves not supplemented with LF and 4.6 g/d for calves supplemented with LF. Therefore, treated calves receiving supplemental LF consumed 1.3 g more than the control calves. Pooling of colostrum was based on IgG concentration of the colostrum and not on LF content. Calves on the treatment colostrum plus supplemental LF consumed 5.6 g of LF (colostrum LF + 1 g supplemental LF). It is unlikely that this small difference (0.3 g) would elicit any effects.

Table 1.

Birth weight, colostrum and IgG intake, serum IgG concentration, and absorption rates at various times over the first day of life and area under the curve (AUC) of calves fed supplemental lactoferrin. Xylose and glucose area under the curve on d 2 of life of calves fed supplemental lactoferrin

	Treatment1
Item	0 g lactoferrin	1 g lactoferrin	SE2	P-value
BW, kg	46.3	45.2	–	–
Colostrum fed, L	4.0	4.0	–	–
Total IgG fed, g	222	213	–	–
Serum IgG, g/L
12 h	14.6	15.0	0.78	0.36
18 h	16.9	18.0	1.16	0.42
24 h	15.8	16.5	1.00	0.61
Serum IgG, AUC, (g/L)·h	289	305	17.7	0.54
IgG absorption rate, g/h
0– 12 h	1.20	1.30	0.06	0.36
12–18 h	0.40	0.44	0.18	0.89
18–24 h	–0.15	–0.29	0.11	0.38
AEA,3 %	30.0	32.0	1.92	0.49
Xylose, AUC, (mmol/L)·h	23.8	25.7	1.18	0.28
Glucose, AUC, (mg/dL)·h	870	904	21	0.27

¹Treatment = 0 g/d supplemental lactoferrin or 1 g/d supplemental lactoferrin.

²SE is the standard error of least squares means.

³AEA = apparent efficiency of absorption {[serum IgG (g/L) × BW × 0.09/IgG intake] × 100; Quigley and Drewry, 1998}.

All calves obtained passive transfer (≥10 g IgG/L serum) by 12 h and IgG concentration remained elevated at 18 and 24 h. No IgG was detected in any calf at 0 h. Area under the curve for IgG was 305 and 289 (g/L)∙ h for calves supplemented with 1 g of LF or no LF, respectively (P = 0.54). Rate of absorption was greatest during the first 12 h but did not differ between treatments (P = 0.36) and decreased to less than 0.5 g/h between 12 and 18 h after birth. Immunoglobulin G absorption rate was negative between 18 and 24 h postpartum. These data are similar to Cabral et al. (2012), using colostrum replacer, who observed reduced IgG uptake between 18 and 24 h. Apparent efficiency of absorption for IgG was similar between treatments, supporting the IgG concentration and AUC and rate of absorption data. Lactoferrin did not enhance IgG uptake in this study. These results are similar to that of Dawes et al. (2004), who fed 1 g LF/kg of colostrum. Shea et al. (2009) fed 0, 0.5, 1, or 2 g/d LF to calves offered a reconstituted lacteal-based colostrum replacer. Shea et al. (2009) observed a quadratic effect for apparent efficiency of absorption, where calves receiving 0.5 and 1 g LF had lower values than calves fed 0 or 2 g/d LF. They observed a similar response for xylose AUC. It is unlikely that the results in the current study are due to the amount of IgG provided, as it was similar to the amount provided in the colostrum replacer fed by Shea et al. (2009).

Prgomet et al. (2007) fed LF from 3 d of age until slaughter at d 61. In that study, they observed increased circulating IgG and increased proliferation of Peyer's patches in the ileum in calves receiving 1.5 to 1.6 g/d LF (Prgomet et al., 2007). They hypothesized that LF acted as an antigen and an immune modulatory compound resulting in local production of both IgG and IgA. However, they fed LF after the colostrum feeding phase (first 24 h of life). Joslin et al. (2002) observed increased preweaning ADG and starter intake and greater heart girth gains in calves fed either 1 or 10 g of supplemental LF. These researchers added LF to colostrum on d 1 and then added it to MR until weaning by d 35. In the study of Robblee et al. (2003), calves were supplemented with 0, 1, 2, or 3 g/d LF beginning on d 3 and were weaned between d 26 and 29. In their study, Robblee et al. (2003) observed lower preweaning fecal scores and fewer days medicated for calves consuming 1 g/d supplemental LF. They also observed a positive linear effect, with increasing supplementation of LF resulting in increased ADG and feed efficiency. Therefore, it is likely that the results observed during the MR feeding period were due to increased local production of IgG and IgA as suggested by Prgomet et al. (2007). This improved immunity could have resulted in improved growth due to healthier calves.

Area under the curve for xylose was similar between LF and control calves (25.7 vs. 23.8 [mmol/L] · h, respectively; Table 1). Glucose AUC was also similar between LF and control calves (904 vs. 870 [mg/dL] ∙ h, respectively; Table 1). Xylose absorption is used as an indirect method of estimating intestinal epithelium size and function (Hammon and Blum, 1997), and these data indicate that supplemental LF did not enhance intestinal development or function.

The amount of LF used in this study was based on the beneficial results obtained by supplementing MR-fed calves with 1 g/d LF (Joslin et al., 2002; Robblee et al., 2003). However, Cowles et al. (2006) and English et al. (2007) observed no benefit to calves fed supplemental LF added to either MR or whole milk, respectively. Cowles et al. (2006) fed LF with an accelerated MR feeding program and observed no benefit in calves being fed up to 1,300 g of MR powder/d. This could have been due to an increased rate of passage through the gastrointestinal tract decreasing the opportunity for LF to stimulate any observable growth or health response. English et al. (2007) fed 3.8 L of whole milk and observed no benefits of either 0.5 or 1.0 g supplemental LF. They attributed the lack of response to LF due to lack of stress and healthy calves. Prgomet et al. (2007) began feeding LF on d 3 of life and saw a benefit to calves during the preweaning period. In other species, LF supplementation was shown to increase intestinal development (Zhang et al., 2001; Humphrey et al., 2002). Zhang et al. (2001) fed milk from transgenic mice with an LF concentration of 12 mg/mL to pups for 10 d. This concentration of LF was over 10 times greater than the concentration of LF used in the current study. Humphrey et al. (2002) observed greater villous heights, improved nutrient absorption, and greater feed efficiency in chicks fed 0.125 g LF/kg diet in combination with lysozyme. There was no effect on intestinal development when LF was fed alone. Lactoferrin is absorbed during the colostrum feeding phase (first 24 h; Talukder et al., 2002; Dawes et al., 2004), suggesting that in calves, absorbed LF is not responsible for intestinal development or functionality. Lactoferrin supplemented during the first 24 h of life would likely be absorbed by the enterocytes in the small intestine. However, after gut closure (>24 h of age), LF may have stimulated the local production of IgG and IgA, resulting in the positive results observed by others (Joslin et al., 2002; Robblee et al., 2003; Prgomet et al., 2007). These data suggest that calves supplemented with 1 g/d LF does not improve IgG absorption on d 1 of life, which concurs with Dawes et al. (2004) and Shea et al. (2009), or enhance intestinal development during the first 2 d of life. Based on the results of this study and that of Shea et al. (2009), LF supplementation was not beneficial on d 1 of life.

Footnotes

1

Partial funding was provided by the New Hampshire Agricultural Experiment Station. This is Scientific Contribution Number 2583 of the New Hampshire Agricultural Experiment Station. This work was supported by the USDA National Institute of Food and Agriculture (NC–1042; accession number 1001283) and The George Walker Milk Fund.

© American Society of Animal Science 2016