immunoglobu-A meta-analysis of the effects of colostrum heat treatment on colostral viscosity, immunoglobulin G concentration, and the transfer of passive immunity in newborn dairy calves

Newborn ruminants depend on colostrum intake immediately after birth to obtain immunoglobulins for effective transfer of passive immunity (TPI). As colostrum may also be a vehicle of infectious agents, heat treatment of raw colostrum is a practice aimed at eliminating or reducing its pathogen load. Despite the usefulness of heat treatment in preventing the transmission of infectious colostrum-borne diseases, heat treatment of colostrum may have some side effects. A systematic review and meta-analysis were conducted to summarize the effects of colostrum heat treatment on colostral viscosity and IgG concentration, and serum IgG concentration as a proxy for TPI in newborn calves fed raw versus heat-treated colostrum. Moderators were studied to identify sources of heterogeneity. Literature databases were searched for peer-reviewed articles published between 1946 and 2022. A Master of Science thesis was also included. Five, 21, and 19 original publications were quantitatively evaluated in 3 separate meta-analyses, based on predefined selection criteria. Two-level and 3-level random-effects meta-analysis revealed a significant overall effect of heat treatment on colostral viscosity and IgG concentration, and serum IgG concentration in newborns. Heat-treated colostrum had significantly higher viscosity (21.0 cP, 95% CI: 3.8 to 38.2) and lower IgG concentration (−7.4 g/L, 95% CI: −11.1 to −3.7) compared with raw colostrum. Overall, newborn calves fed heat-treated colostrum had higher serum IgG concentrations (2.8 g/L, 95% CI: 1.4 to 4.0) 24–48 h after birth than those fed with raw colostrum. Particularly, this positive effect on the serum IgG concentrations was seen when colostrum was heat-treated at ≤60°C (2.9 g/L, 95% CI: 0.9 to 4.2) and when the standard low-temperature low-time (LTLT) method was used for heat treatment (2.6 g/L, 95% CI: 0.1 to 5.1). Colostrum treated at >60–63.5°C tended to have higher viscosity (275.6 cP, 95% CI: −37.9 to 589.3) and had lower IgG concentration (−21.7 g/L, 95% CI: −27.3 to −16.1). Calves fed colostrum treated at this temperature range had significantly lower serum IgG (−4.2 g/L, 95% CI: −7.9 to −0.4) compared with those fed raw colostrum. Heat treatment of colostrum at 72–76°C was not associated with a significant increase in colostral viscosity (6.3 cP, 95% CI: −324.3 to 336.9) nor a reduction in IgG colostral concentration (−13.1 g/L, 95% CI: −26.5 to 0.2), but calves fed colostrum treated at this temperature range had a significant reduction in serum IgG (−11.3 g/L, 95% CI: −17.1 to −5.4). Feeding newborn calves with colostrum heat-treated at ≤60°C by the standard LTLT method, particularly within 2 h after birth, resulted in increased serum IgG concentration at 24–48 h of age. Importantly, delaying feeding of heat-treated colostrum to newborns beyond 2 h of age resulted in no significant difference in IgG serum levels compared with feeding raw colostrum, highlighting the importance of early administration of heat-treated co-lostrum to favor TPI. On-farm colostrum heat treating should achieve an equilibrium between pathogen elimination and the preservation of colostral immunoglobulins while minimizing undesired increases in viscosity. The beneficial effects of colostrum heat treatment on TPI can be negligible if colostrum feeding is not performed within 2 h after birth.


INTRODUCTION
Ruminants have a 5-layered synepitheliochorial placenta that prevents the transmission of immunoglobu-lins from the dam to the fetus during the intrauterine life.Therefore, neonates are born agammaglobulinemic or hypogammaglobulinemic (Wooding, 1992).This makes newborns almost entirely dependent on colostrum intake after birth to provide the immunoglobulins necessary to achieve adequate transfer of passive immunity (TPI).
Early ingestion and absorption of adequate amounts of colostral immunoglobulins are essential for establishing immunity until the newborn's own immune system is completely functional (Davis and Drackley, 1998).The main determinants for an adequate TPI include the quality and volume of colostrum fed, and the time elapsed between birth and colostral ingestion (Heinrichs and Elizondo-Salazar, 2009).There is no clear indication of the volume of colostrum required for newborn calves, though it is suggested that approximately 8-12% of the body weight (e.g., 3-4.5 L in a 37.5 kg calf) should be fed within 2 h and no later than 6-12 h after birth; afterward, the efficiency of intestinal antibody absorption declines progressively and is negligible after ~24-36 h of age (Besser et al., 1985;Weaver et al., 2000;Patel et al., 2014;Godden et al., 2019).Quality colostrum should contain >50 g/L of IgG (Lombard et al., 2020), which is ≥22° Brix by digital refractometry (Quigley et al., 2013), with bacterial counts <100,000 cfu/mL (McGuirk and Collins, 2004).
The traditional definition of failure of TPI (FTPI) in newborns involves a serum IgG concentration of <10 g/L measured at 24-48 h of age (Weaver et al., 2000;McGuirk and Collins, 2004).However, this historical threshold has recently been questioned, and a higher (15 g/L) minimum serum IgG concentration has been proposed (Urie et al., 2018;Godden et al., 2019).Animals experiencing FTPI have a reduced ability to fight diseases, an increased risk of morbimortality, and reduced milk production (Faber et al., 2005).
The colostrum may be a vehicle of infectious agents in neonates.Numerous pathogens including Mycobacterium avium ssp.paratuberculosis, Mycobacterium bovis, Mycoplasma bovis, Salmonella enterica, Escherichia coli, Listeria monocytogenes, Coxiella burnetii, Brucella abortus, and bovine leukemia virus, to list some, are colostrum-borne (Catlin and Sheehan, 1986;Okolo, 1992;Streeter et al., 1995;Rowe and East, 1997;Godden et al., 2006;Tutusaus et al., 2013).Pathogen contamination of colostrum is of concern as a source of infectious diseases and because of possible interference with adequate TPI.Many bacteria can alter the composition of colostrum, especially protease-producing bacteria, which negatively affects their protein fraction, reducing colostrum quality (Godden et al., 2006).
Heat treatment of fresh colostrum is one of the most frequently used approaches to reduce microbial bur-den.Although the heat treatment of colostrum is an evident tool to prevent pathogen transmission, it may have some side effects including the degradation of immunoglobulins, the alteration of colostral fluidity and the induction of unacceptable feeding characteristics that challenge colostrum consumption (e.g., increased viscosity).The drawbacks associated with heat-treated colostrum can interfere with adequate TPI.Qualitative studies have highlighted these aspects (Elizondo-Salazar and Heinrichs, 2008); however, integrated quantitative field evaluations are scarce.A recent study investigated the effects of heat-treated colostrum, with particular emphasis on the temperature used for heat treatment, on colostral IgG concentration and serum IgG and serum total protein concentrations as proxies of TPI in calves (Malik et al., 2022).The study reported an overall effect size that indicated decreases in colostrum IgG by −7.67 g/L (95% CI: −10.3 to −5.0) and nonsignificant increase in serum IgG by 1.37 g/L (95% CI: −0.1 to 2.8).
To evaluate the effects of heat treatment of colostrum on these and other variables of practical application in dairy farms, such as colostrum viscosity (not assessed by Malik et al., 2022), we conducted a similar literature search and meta-analyses.Unlike Malik et al. (2022), we explored potential moderators such as the age of calves (hours after birth) at colostrum feeding, the calves' sex and breed, and the method of colostrum feeding on the serum IgG concentration of newborn calves fed raw versus heat-treated colostrum as an indicator of TPI.Although there is an overlap in the literature reviewed in both studies, ours includes more references and trials as we searched for literature in a broader spectrum of databases with no language restriction, and applied different inclusion criteria.Our study reaffirms the overall conclusions reached by Malik et al. (2022), while deepening the analysis and complementing the information with new results worth discussing.Of note, as opposed to the borderline nonsignificant effect found by Malik et al. (2022), we found a significant overall increase in serum IgG in calves fed heat-treated colostrum.

MATERIALS AND METHODS
A systematic literature review and meta-analysis were conducted to summarize and combine the results of multiple studies on the effects of heat treatment on bovine colostrum viscosity and IgG concentration, and serum IgG concentration as indicator of TPI in newborn calves that consumed raw or heat-treated colostrum.No human or animal subjects were used, so this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board.

Literature Search and Study Selection
The PRISMA guidelines (Moher et al., 2009) were followed in this study (Figure 1).The search strategy was developed for exploring the effects of heat treatment on colostrum viscosity and IgG concentration, and the effect of consuming heat-treated and fresh non-heattreated raw colostrum on the serum IgG concentration of newborn calves.Electronic databases including CAB Abstracts, PubMed, Google Scholar, Web of Science, Medline, Scopus, and Science Direct (the last 4 not consulted by Malik et al., 2022), were used to screen for literature using the following search terms: "pasteurization" or "heat treatment" or "heat process" and "colostrum" or "colostral" and "cattle" or "cow" or "bovine" or "ruminant" and "immunoglobulins" or "antibodies" or "transfer of passive immunity."No language or study design restrictions were applied.The reference lists cited in relevant studies were checked for further related publications.The search retrieved publications from January 1946 to January 2022.Duplicate publications were identified using reference management software (Mendeley, London, United Kingdom) and discarded.Two authors (AR and MF) independently read and selected eligible publications; discrepancies in eligibility were further discussed with a third author (FG) until a consensus was reached.

Inclusion Criteria
Published research work fulfilling the following criteria were included: (a) fresh, whole (nonskimmed) colostrum from primiparous or multiparous cows was used after heat treatment, (b) colostrum heat treatment was performed at a standardized temperature during a standardized period of time, (c) raw colostrum was defined as colostrum that had not undergone any sort of heat treatment, (d) heat-treated and non-heat-treated aliquots from the same colostrum sample were subjected to evaluation, (e) IgG evaluation was conducted instead of analysis of whole immunoglobulin content of colostrum, (f) IgG quantification was performed by using radial immunodiffusion (RID), ELISA, or turbidimetric immunoassay (TIA), (g) newborn calves were assigned to treatments randomly or systematically, (h) newborn calves were not allowed to suckle colostrum from their dams and were allocated to places with no access to dams, (i) colostrum was artificially fed, (j) serum IgG was quantified in newborns 24-48 h after birth, (k) the studies represented original (primary) research (reviews were not included).Authors of publications that did not state all the information required for our study were contacted by email to request missing data.

Extraction of Data and Meta-Analyses
The studies were first screened by title and abstract, and no relevant publications were eliminated.The full texts of the remaining publications were then checked against the inclusion criteria.Data systematically obtained from the publications that met the inclusion criteria were as follows: first author identity, year of publication, year and country where the study was conducted, study title, journal name, study design, time, and temperature applied for the heat treatment of colostrum; method of newborn assignment to treatments; newborn breed; hours elapsed between birth and colostrum intake; hours after birth when the serologic assessment was performed; method for IgG quantification in colostrum and serum; mean IgG colostral concentration; mean colostral viscosity; and mean IgG serum concentration.Data regarding colostrum or serum IgG concentrations reported in different units (mg/mL, mg/dL) were converted to grams per liter (g/L).Different units of viscosity [log 10 (cP), Pascal-second (Pa•s), log 10 (Pa•s) 2 ] were converted and expressed in centipoize (cP).When the standard deviation (SD) of the mean was not reported, it was derived from other measures of dispersion, that is, from the standard error (SE) of the mean as SD = SE × √n, or from mean values and 95% CI.
The colostral viscosity and IgG concentration, and the serum IgG concentration in newborns (dependent variables) were considered as the effect size.The mean difference (MD; outcome for treatment group-outcome for control group, Borenstein et al., 2021) was used to estimate the pooled effect size for each outcome variable.Random-effect meta-analyses were conducted to evaluate the effect of heat treatment of colostrum on the 3 variables, the 95% CI, and the statistical significance using the DerSimonian and Laird method (DerSimonian and Laird, 1986).Because some studies used different combinations of temperature and time for heat treating colostrum or different tests to measure IgG, leading to multiple effects in each study, 3-level meta-analytic random-effects models were additionally conducted as described by Assink and Wibbelink (2016) and Harrer et al. (2021).We assumed that trial/ experiment effect sizes (level 2) were nested within studies/publications (level 3; Cheung, 2014;Assink and Wibbelink, 2016).The variation (I 2 ) amount at each level was calculated using the 'var.comp'function.An ANOVA using ANOVA function was run, and Akaike and Bayesian information criterion values (AIC and BIC, respectively) were compared for model selection (Harrer et al., 2021).Heterogeneity among studies was first explored by Cochran's Q statistic (X 2 ), and then quantified using Higgins' I 2 statistic (Borenstein et al.,  (Moher et al., 2009) flow diagram describing the study design process for the systematic review and meta-analyses of the effect of colostrum heat treatment on IgG concentration and viscosity, and the effect of feeding heat-treated colostrum on the serum IgG concentration in newborn calves.2021).Results from the meta-analyses with corresponding 95% confidence intervals (CI) are summarized in independent forest plots (Figures 2, 3, and 4).A set of analyses was run to examine the robustness of the pooled results by identifying outliers and influential cases.Different influence diagnostics were calculated to detect the studies that most influenced the overall estimate of the meta-analysis and assess if this influence distorted the pooled effect (Viechtbauer and Cheung, 2010).Influence diagnostic plots were created, including a Baujat plot and the leave-one-out analyses (ordered by heterogeneity and by effect size; Baujat et al., 2002;Viechtbauer and Cheung, 2010;O'Connor et al., 2014).The studies were quality assessed for bias and graded as high, low or unclear risk across the following domains, when corresponded: bias arising from the randomization process, bias due to deviations from intended intervention, bias due to missing outcome data, bias in the measurement of the outcome, and bias in the selection of the reported results (RevMan version 5.3.5;The Cochrane Collaboration, The Nordic Cochrane Centre, Copenhagen, Denmark).
Publication bias was visually assessed using funnel plots constructed by plotting the specific effect (MD) against a measure of its precision (SE) using "funnel" function in R. Symmetrical distribution of points (trials) in the shape of an inverted funnel is expected in the absence of publication bias.The studies with the least variable effect sizes can be found at the top of the plot, while smaller, less precise publications are located at the bottom of the funnel (Palmer et al., 2008).The asymmetry was further evaluated by Egger's linear regression analysis, with P < 0.05 set as the level of significance (Egger et al., 1997).If statistical significance was evidenced, Duval and Tweedie's trimand-fill analysis was applied to adjust the outcome by imputing results from the assumed missing trials.The contours of statistical significance in contour-enhanced funnel plots enable the evaluation of whether the areas where studies exist correspond to areas of statistical significance (e. g., <0.01, <0.05) and whether the areas where studies are possibly missing relate to areas of low significance (e. g., >0.05-0.1;Peters et al., 2008).If apparently missing studies are in areas of higher statistical significance, then asymmetry is less likely triggered by publication bias (i.e., variable study quality).Contour-enhanced funnel plots are shown in Supplemental Figure S1 (https: / / data .mendeley.com/datasets/ hc9kmswy5z/ 1).
Three-level moderator models were fit to assess putative moderators of the overall effect (Assink and Wibbelink, 2016).The heat treatment method, temperature, and time of heating were the moderators included for colostrum viscosity.The heat treatment method was classified into standard and alternative low-temperature long-time heat treatment (LTLT) [the standard LTLT is usually performed at 60-63°C for 30 min, and a longer-time alternative to the stan-   dard LTLT proposed elsewhere is performed at 55-63°C for 60-120 min (Godden, 2007)], and high-temperature short-time heat treatment (HTST) performed at 72-76°C for 0.25 min (15 s).The moderators evaluated for colostrum IgG concentration included heat treatment method, temperature, and time of heating as separate variables, and IgG quantification techniques (RID, ELISA, or TIA).The moderators analyzed for serum IgG concentration comprised: heat treatment method, temperature, and time of heat treatment as separate variables; IgG quantification method; calf breed; calf sex; method of colostrum administration; timing of colostrum administration (hours after birth); volume (L) of ingested colostrum; and time gap (hours) between birth and blood sampling for serum IgG quantification.The statistical analyses and plotting were done using version 4.0.3 of R (R Studio Team, 2015), using the packages dplyr (Hadley et al., 2019), meta (Balduzzi et al., 2019), metafor (Viechtbauer, 2010), dmetar (Harrer et al., 2021), esc (Lüdecke, 2019), and tidyverse (Wickham et al., 2019).
The number of publications excluded and reasons for exclusion are shown in Figure 1.These investigations were performed in the United States (n = 3), Egypt (n = 1), and Germany (n = 1).Three studies reported multiple trials, resulting in the inclusion of 12 trials.Globally, these studies evaluated 265 raw colostrum samples and an equal number of heat-treated colostrum samples (median = 25 and interquartile range (IQR) = 13.5, each).Heat treatment was performed at 60°C for 30, 60 and 120 min, at 63°C for 30 and 120 min, at 63.5°C for 30 min, and at 72°C for 0.25 min (15 s), for which the studies employed the standard LTLT method (n = 5), alternative LTLT (n = 6), and HTST (n = 1).
Because nesting trial effect sizes in studies did not improve the model, a 2-level model was finally used.Heat treatment increased the viscosity of colostrum by an average of 21.0 cP [95% CI: 3.8 to 38.2, P = 0.03, unadjusted MD 117.2 cP (95% CI: 64.3 to 170.1)] compared with the viscosity measured in raw colostrum, after outliers and influential removal, and correction by Duval and Tweedie's trim-and-fill analysis (with 2 added studies; Duval and Tweedie, 2000).Outliers and influential studies contributing to the heterogeneity or influencing the overall effect size were identified and excluded [McMartin et al., 2006 (2 trials); Elizondo-Salazar et al., 2010 (1 trial);Hassan et al., 2020 (1 trial)].Because these influential trials have a very high effect, their removal resulted in a reduction of the overall effect, but with no substantial I 2 reduction.There was asymmetry in the contour-enhanced funnel plot for the viscosity of colostrum; however, Eggers' test was not significant (coefficient = 1.4,P = 0.188).Despite this, publication bias remained a possibility because the statistical power of Egger's test to reveal bias is likely reduced with low number of studies.The overall effect of heat treatment on the colostral viscosity tended to be moderated only by the temperature used for heat treatment (P = 0.057).The MD of heat treatment done at different temperatures was 5.2 cP at ≤60°C (95% CI: −15.5 to 26.0), 275.6 cP at 60-63.5°C (95% CI: −37.9 to 589.3), and 6.3 cP at 72°C (95% CI: −324.3 to 336.9).
Bovine colostrum naturally has a wide range of thicknesses, and its viscosity is often only visually estimated using qualitative scales (e. g., watery, liquid, and thick; Hassan et al., 2020).We did not find published information regarding the normal quantitative viscosity range for colostrum.Given this information gap, it is rational to consider the viscosity values reported here for raw colostrum samples, ranging from 19.6 to 93.33 cP, as a ballpark figure of the physiological viscosity of colostrum.Only studies analyzing colostrum samples from Holstein cattle met the inclusion criteria (Supplemental Table S1; https: / / data .mendeley.com/datasets/ p25d2ry44v/ 1); thus, this value does not necessarily reflect the normal colostral viscosity in other breeds.Thickened colostrum consistency is undesirable and makes the colostrum unsuitable for consumption.A significant change in viscosity would turn colostrum into a pudding-like material with unacceptable consistency, which could not be easily fed to calves through a nipple bottle or an esophageal tube, the 2 most widely used methods for colostrum feeding.
Changes in colostrum thickness could be correlated with the concentration of total solids; specifically, increments in viscosity could be attributed to interactions between denatured whey proteins (Singh and Singh, 1980).Denatured β-LG molecules can form aggregates with other proteins such as casein micelles or α-LA (Felipe et al., 1997;Needs et al., 2000;Patel et al., 2006).The denatured protein aggregates occupy a higher phase volume, leading to increased viscosity (Damodaran, 2008).No substantial correlations were found between IgG reduction and viscosity increase (Foster et al., 2016); therefore, denaturation of colostral IgG may not have significantly contributed to the viscosity increment observed in the heat-treated colostrum.Because most of the studies included in the meta-analysis merely evaluated the IgG content and no other colostral components, possible reasons for the increase in viscosity observed in heat-treated colostrum need to be further researched.
Aside from the IgG content, some less-studied hypotheses are worth to be mentioned.Because batch heat treatment, the method used by most of the included studies, is a closed system where colostrum is heated and held in an enclosed vessel, little water evaporation can be assumed.However, the possibility of some concentration effect due to water evaporation should not be disregarded.We are unaware of research comparing open and closed heat-treatment systems on the effect of water evaporation and colostrum viscosity.The exact mechanism(s) by which heat treatment increases colostral viscosity remain to be elucidated; this information is needed to better understand how to avoid undesired increases in colostrum viscosity at the farm level.
Mixing thickened colostrum with warm water can mitigate the complications during calf feeding (Foster et al., 2016).However, this would dilute the IgG content and negatively affect TPI.Increased viscosity may also detrimentally affect palatability of colostrum.Additionally, thick colostrum cannot be easily cleaned or washed from instruments used for artificial feeding; removing organic matter from such instruments is key before their disinfection.
The pooled effect size based on the 3-level metaanalytic model was −7.4 g/L [95% CI: −11.1 to −3.7, P = 0.0001, unadjusted MD −7.7 g/L (95% CI: −11.9 to −3.4)] after the exclusion of influential and outliers trials [Elizondo-Salazar et al., 2010 (2 trials); Hesami et al., 2020 (1 trial)], that substantially contributed to heterogeneity and distorted the pooled effect estimate.The post-heat-treatment reduction in colostral immunoglobulins, specifically IgG, may be due to the loss of the primary 3-dimensional functional structure of the glycoproteins (Law and Leaver, 1999).This denaturation may initially occur as a reversible unfolding of the native structure, affecting the globular configuration, which can progress to irreversible changes and aggregation with other whey proteins or casein via hydrophobic interactions or disulfide bonds (Law and Leaver, 1999;Indyk et al., 2008).Each IgG molecule contains 2 antigen-binding fragments (Fab) at one end, known as the variable region, and a constant fragment (Fc) at the other end (Hurley and Theil, 2011).Unfolding of the protein structure induced by heat can affect the integrity of both the Fab and Fc regions (Indyk et al., 2008).However, structural alterations are mainly seen in the Fab fragments; thus, altering the ability of IgG to bind antigens (Roterman et al., 1994;Jasion and Burnett, 2015).
The contour-enhanced funnel plot for IgG concentration in colostrum was symmetric and was consistent with the results of Eggers' test (coefficient = −1.4,95% CI: −11.9 to 9.0, P = 0.792).No studies were added when the trim-and-fill procedure was done, and the quality assessment of the studies revealed that most studies were at low or unclear risk of bias.The subgroup analyses in the 3-level model revealed that only the temperature used for heat treatment of colostrum was significantly associated with colostrum IgG concentration (P < 0.0001).Moderators' analysis showed that the MD in the studies that heat-treated colostrum at ≤60°C was −3.6 g/L (95% CI: −7.3 to 0.1).The MD was −21.7 g/L (95% CI: −27.3 to −16.1) in the primary studies that conducted heat treatment between 60 and 63°C, and −13.1 g/L (95% CI: −26.5 to 0.2) in the only study that heat-treated colostrum at 72°C (Table 1).
We found that heat treatment of colostrum decreased the IgG content in a very similar magnitude to that reported by Malik et al., 2022 (−7.67 g/L).This slight variation could be attributed in part to the different models used.Malik et al., 2022 conducted a randomeffects model, while a multilevel random-effects metaanalysis provided a better fit to our meta-analytic data set.In addition, we included studies that used fresh colostrum samples as the control group, whereas Malik

Table 1 (Continued).
Moderator analysis of the effect of colostral heat treatment on colostrum viscosity (cP), IgG concentration (g/L), and serum IgG concentration (g/L) in newborn calves 1 et al. ( 2022) analyzed studies using fresh or frozen colostrum samples, underestimating the effect of freezingthawing on colostral IgG concentration.
The adjusted MD of IgG serum concentration based on the 3-level meta-analytic model between calves fed heat-treated colostrum or raw colostrum was 2.8 g/L [95% CI: 1.4 to 4.0, P = 0.04, unadjusted MD: 0.76 g/L (95% CI: −1.4 to 2.9)], after removing influential and outlier trials (Godden et al., 2003;Kryzer et al., 2015;Salazar-Acosta and Elizondo-Salazar, 2019) and trim-and-fill correction for missing data (with 8 added studies).Despite heat treatment significantly decreasing the IgG concentration of colostrum, feeding heat-treated colostrum improves TPI, as assessed by serum IgG concentration at 24-48 h of age, possibly indicating enhanced intestinal absorption of IgG.A higher IgG serum concentration in newborns fed heattreated colostrum can result from a greater apparent efficiency of IgG absorption from the total mass of IgG in the small intestine.This indicates the benefit of the administration of heat-treated colostrum in terms of TPI.Although these results seem contradictory, some possible explanations can be found.The mechanisms involved in epithelial cell absorption of IgG and bacterial internalization are shared and bacteria and IgG compete for common nonspecific receptors on neonatal enterocytes (Corley et al., 1977;Baintner, 2007).The viable bacteria present in raw colostrum can prevent IgG adhesion and interfere with its intestinal absorption into the systemic circulation.Another hypothesis suggests that the physical binding of microbes to freely transportable IgG reduces its availability for absorption (James and Polan, 1978;James et al., 1981;Staley and Bush, 1985).The heat treatment of colostrum decreases its bacterial load, reducing the microbial population that reaches the intestine and compete for receptors or bind to immunoglobulins.Consequently, heat treatment leads to an increase in the amount of IgG available for absorption (James and Polan, 1978;James et al., 1981;Staley and Bush, 1985;Johnson et al., 2007).It is worth mentioning that some studies have reported mean serum IgG concentrations below the traditional threshold for FTPI in calves (<10 g/L) receiving either raw or heat-treated colostrum (Lakritz et al., 2000;Tyler et al., 2000;Gelsinger et al., 2015a;Salazar-Acosta and Elizondo-Salazar, 2019;Supplemental Table S3; https: / / data .mendeley.com/datasets/ yprnr9w62w/ 1).This reinforces that feeding low-quality colostrum (e. g., 20.1-22.5 g/L of IgG, Salazar-Acosta and Elizondo-Salazar, 2019) can lead to FTPI, regardless of prompt administration after birth.Heat treatment will not improve poor quality colostrum.
Competition between IgG and nonimmunoglobulin proteins for intestinal absorption has also been reported (Quigley et al., 1998).The addition of large macromolecules such as bovine serum albumin to bovine colostrum reduced the efficiency of IgG absorption.This is consistent with the saturable capacity of the intestinal transport mechanism of macromolecules (Besser and Osborn, 1993).Another hypothesis states that heat treatment can denature proteins that compete with immunoglobulins, allowing greater intestinal absorption of IgG (Elizondo-Salazar and Heinrichs, 2009a,b).It is worth mentioning the role of colostral bioactive proteins, such as the epidermal growth factor involved in the cessation of macromolecular absorption (gut closure) in newborn animals (Xu et al., 2000).These bioactive proteins are also heat sensitive and can be denatured and aggregated by heat treatment (Gingerich and McPhillips, 2005), altering the ability of colostrum to promote gastrointestinal maturation.This could additionally contribute to a more effective TPI among calves fed heat-treated colostrum (Ballou and Liang, 2015).
Most of the trials were at low or unclear risk of bias across most domains; however, the contour-enhanced funnel plot for serum IgG concentration in calves and Egger's test suggested publication bias (coefficient = −1.1,95% CI: −2.1 to −0.1, P = 0.03).Duval and Tweedie's trim-and-fill analysis suggested that studies were missing and had to be imputed for publication bias correction (Duval and Tweedie, 2000).When assessing moderators of the overall effect, the subgroup analyses in the 3-level model provided some evidence that the magnitude of the intervention effect varied with the heat treatment method (P = 0.001), the temperature of heat treatment (P = 0.0001), and time of colostral consumption (P = 0.03; Table 1).The beneficial effect of heat-treating colostrum on serum IgG concentrations was greater in calves fed colostrum heat treated at standard LTLT, lower temperatures, and those fed soon after birth (Table 1).
The HTST heat treatment of colostrum was associated with a significant reduction in serum IgG by −9.2 g/L (95% CI: −15.2 to −3.2), while a significant increase in serum IgG by 2.6 g/L (95% CI: 0.1 to 5.1, P = 0.03) for standard LTLT was observed.The MD of alternative LTLT was 0.2 g/L (95% CI: −1.9 to 2.5) and trended to be lower than standard LTLT (P = 0.06).The mean effect of heat treatment done at ≤60°C was 2.9 g/L (95% CI: 0.9 to 4.2, P = 0.002).The heat treatment conducted at >60-63.5°C or 76°C showed MD of −4.2 g/L (95% CI: −7.9 to −0.4) and −11.3 g/L (95% CI: −17.1 to −5.4), respectively.These effects were significantly lower than the mean effect of heat-treating colostrum at ≤60°C (P = 0.001 and P = 0.0001, respectively).Moderator analysis showed that delaying feeding of heat-treated colostrum after 2 h of age resulted in no difference in IgG serum levels compared with feeding raw colostrum at ≤2 h of age.Newborn calves aged ≤2 h fed heat-treated colostrum had significantly higher serum IgG levels than calves fed raw colostrum (MD 3.1 g/L; 95% CI: 0.6 to 5.5, P = 0.02).The MD declined and differences were nonsignificant when calves were fed between 2 to 6 h of age (MD −1.7 g/L, 95% CI: −4.9 to 1.5, P = 0.292), or later than 6 h of age (MD −3.1 g/L, 95% CI: −8.6 to 2.5, P = 0.263).The age at the first colostrum feeding is one of the most critical factors determining the efficiency of IgG absorption (Davis and Drackley, 1998).The apparent efficiency of IgG absorption consistently declines as the age increases from birth to 12 h, which is the inflection point when this efficiency drops sharply (Osaka et al., 2014).
The current meta-analysis reinforced the previously described inverse exponential relationship between IgG serum concentration and time of colostrum feeding (Matte et al., 1982).Time and resources invested in heating colostrum can be worthless in terms of IgG concentration in calves' serum (TPI) if colostrum administration is delayed.Our work suggests that among the management factors that have critical roles in TPI, the age at first colostrum administration might have a greater influence than the volume of colostrum fed.The hypothesis that ensuring access to high-quality colostrum soon after birth might be a stronger determinant of TPI than the volume of colostrum provided can be proposed; however, this should be further investigated in specifically designed trials.
Our work validated the conclusions of Malik et al. (2022) by finding the same pattern of overall effects, but more precise estimates of the true effects were obtained by identifying and quantitatively assessing more published studies and trials what would explain the slightly different magnitudes we found.We included peer-reviewed studies published late in 2021 and an MS thesis from the University of Alberta, Canada (Kent-Dennis, 2014), and because we did not restrict our literature search by language, we also found publications in Spanish that were not included in the study by Malik et al. (2022).Arguably, the inclusion of an MS thesis, which is considered a non-peer-reviewed publication, could be seen as a limitation of our study.We found an overall adjusted effect size that showed significant decreases in colostrum IgG by −7.4 g/L (−7.67 g/L reported by Malik et al., 2022) and a significant increase in serum IgG by 2.8 g/L (nonsignificant increase by 1.37 g/L reported by Malik et al., 2022).We applied different inclusion criteria for serum IgG in calves.Because serum IgG measured at 24 to 48 h of age has long been used as the gold standard measure of TPI (Lombard et al., 2020), publications with later blood sampling were excluded.This criterion determined the exclusion of 3 studies, 2 of which were large-size studies (n = 533 in Godden et al., 2012a andn = 309 in Teixeira et al., 2013).The elimination of these great weight studies would also explain the variation in the overall effect size.In contrast to what Malik et al. (2022) described for serum IgG in calves (nonsignificant decrease by −3.40 g/L for heat treatments >60°C), the effect size found in our study indicated a significant reduction in serum IgG in calves fed colostrum treated at higher temperature (−4.2 g/L for 60-63.5°Cand −11.3 g/L for 76°C) compared with those fed raw colostrum.
Appropriate colostrum management practices are crucial for successful rearing of newborn dairy calves (Godden et al., 2019).Adequate TPI is linked to protection against infections in pre-weaned animals (Fur-man-Fratczak et al., 2011).Calves with FTPI are prone to infectious diseases (Lofstedt et al., 1999;Berchtold and Constable, 2009), have enhanced morbidity and mortality risks, and reduced productive performance (Robison et al., 1988;Ahmad et al., 2000;Faber et al., 2005;Nowak and Poindron, 2006).Heat treatment of colostrum is becoming a frequent practice (Gelsinger and Heinrichs, 2017) and can have both beneficial and side effects on colostrum quality, physical properties, pathogen inactivation, and TPI to newborn calves.The current meta-analyses support the effect of heat treatment on increasing colostral viscosity and reducing its IgG concentration.Despite this, newborn calves fed heat-treated colostrum ≤2 h of age had an overall enhanced serum IgG concentration at 24 to 48 h of age (TPI), suggesting a higher efficiency of intestinal absorption of IgG.Feeding heat-treated colostrum after 2 h of age does not seem to have a significant effect on the TPI compared with feeding raw colostrum.On-farm heat treatment of colostrum should achieve an equilibrium between preserving colostral immunoglobulins and preventing significant increases in viscosity while reaching adequate levels of pathogen inactivation.

Figure 1 .
Figure 1.PRISMA(Moher et al., 2009) flow diagram describing the study design process for the systematic review and meta-analyses of the effect of colostrum heat treatment on IgG concentration and viscosity, and the effect of feeding heat-treated colostrum on the serum IgG concentration in newborn calves.
Figure 2. Forest plot for the meta-analysis of the effect of colostrum heat treatment on viscosity (cP) from the 5 original studies that met the inclusion criteria in the systematic review.The length of the horizontal line embodies the 95% CI for the effect size from each experiment, and the center of the square is the point estimate from that survey.MD = mean difference; I 2 = I-squared statistic; τ 2 = variance of the model.References: McMartin et al. (2006), Elizondo-Salazar and Heinrichs (2009a), Elizondo-Salazar et al. (2010), Abd El-Fattah et al. (2014), Hassan et al. (2020).
Rabaza et al.: HEAT-TREATED COLOSTRUM AND PASSIVE IMMUNITY

Table 1 .
Rabaza et al.:HEAT-TREATED COLOSTRUM AND PASSIVE IMMUNITY Moderator analysis of the effect of colostral heat treatment on colostrum viscosity (cP), IgG concentration (g/L), and serum IgG concentration (g/L) in newborn calves 1 Item