High bacterial counts in colostrum can impact calf health both directly and by negatively impacting immunoglobulin absorption [11, 12, 35]. Fresh/raw colostrum fed to calves should contain less than 100,000 cfu/mL total bacterial count and less than 10,000 cfu/mL total coliform count [4, 5]. However, these levels are often exceeded, for example, an observational study in which 827 colostrum samples from 67 farms in 12 states were tested, reported almost 43% of samples had total plate counts greater than 100,000 cfu/mL and 17% of samples had greater than 1 million cfu/mL [36]. Strategies to reduce bacterial counts in colostrum are indicated. The current study lends support to the use of acidification of bovine colostrum to improve colostrum quality through bacterial reduction. Acid is already being used to manipulate whole milk or milk replacer for calves [27,28,29,30,31,32,33] and was chosen here partly because of its increasing popularity. Moreover, it is a relatively simple and inexpensive technique accessible to most producers. Acidification has already been shown to reduce bacterial load including some specific pathogens [34, 37,38,39]. However, it has some important drawbacks including danger of handling, palatability of end-product (viscosity and taste), and potential damage to colostrum components, specifically immunoglobulins. Nevertheless, acidification offers an additional advantage to some other methods of colostrum treatment in that it may permit prolonged storage without the need for freezing or refrigeration. However, extended shelf-life would only be of value if it could be demonstrated that acid decreased bacterial load, without negatively affecting levels, absorption, and neutralizing capability of colostral immunoglobulins. Research into the effects of feeding acidified colostrum to newborn Holstein dairy calves is limited, particularly in terms of functional outcomes. This study showed that acidification of colostrum reduced bacterial load, potentially including pathogens (total coliforms), without negatively affecting immunoglobulins or their ability to neutralize selected pathogens after feeding to newborn calves.
Handling of acid should be done with care and eye and skin protective measures should always be used. United States Food and Drug Administration (FDA) regulations state that formic acid and formate salts from all added sources (if multiple sources of formic acid and its salts are used in combination) cannot exceed 1.2% of complete feed [40]. Although there are currently no guidelines for whole milk or colostrum, formic acid levels in all AC samples in this trial were ≤ 0.45%, i.e., approximately one third or lower than the established guidelines for livestock feeds. Throughout the study’s 6-month sample collection period, no ill-effects were noted in any calves that received AC and all study animals went on to be productive members of the participating herd. Moreover, this herd has years of experience feeding ad libitum acidified milk to calves and has never recorded any detrimental effects, including no impacts on intake such as those described in some studies (see below). Observations made during the current trial indicated that three factors chiefly contributed to successful colostrum acidification. First of all, it was important to chill the colostrum prior to addition of acid. Adding acid to colostrum with a temperature at or above 15–16 °C (60 °F) resulted in coagulation and increased viscosity, changes which make calves refuse to drink and essentially preclude esophageal tube feeding. Secondly, expect to use slightly more acid when acidifying colostrum compared to acidification of milk. For example, compared to the same volume of whole raw milk, we used 15 mL more (40–45 vs 25–30 mL) of the 10% acid solution per 4 L of colostrum to reach the desired pH (4.0 to 4.5). Finally, the colostrum must be vigorously stirred while slowly adding the acid solution. The use of a drill attachment designed for mixing paint worked well to rapidly mix the acid and colostrum thereby preventing coagulation.
The method we used, resulted in colostrum with a low bacterial concentration, that was stable at room temperature for days (unpublished observations and a currently funded research project), and acceptable to calves via bottle or esophageal tube feeding. The main drawback found with formic acid acidified colostrum was the effect on palatability, which is likely encountered with all acids. We have observed calves to occasionally reject suckling or take longer to consume the desired amount of acidified colostrum or milk when bottle-fed. Moreover, acidification with formic acid to pH levels used in the current study i.e., between 4.0 and 4.5, has been reported to limit voluntary intake of milk replacer by approximately 1 L/d [31]. Other studies have shown that some calves permitted to feed ad libitum, reject colostrum [28] or milk replacer acidified to pH below 4.5 [29] or exhibit fragmented feeding patterns [32]. In common with our own observations, these findings indicate that low pH can alter the palatability of milk or colostrum fed to calves. However, at least in the case of colostrum, that decrease in palatability and risk of rejection can be overcome by esophageal tube feeding which is not hindered in any way. In the current study, to ensure complete intake of 4 L, colostrum (MC and AC) was administered to all calves by esophageal feeder. Although acidification of milk replacer has been fairly widely used in feeding of dairy calves [27,28,29,30,31,32,33] there is a paucity of information on what impact if any acidification might have on the gastrointestinal microbiome of young calves. There is evidence that calves fed acidified milk replacer ad libitum have lower abomasal and fecal pH than calves fed restricted amounts of nonacidified milk replacer [41], raising the concern that lower gastric pH might negatively impact digestive function, or cause gastrointestinal discomfort. However, a study designed to evaluate feeding and other behaviors potentially indicative of GI discomfort with free-access feeding of acidified milk, found that apart from a possible effect on palatability, despite which there was no feed avoidance in the first week of life, no behavioral impacts were observed [32]. Interestingly, a recent study at our institution has shown that feeding calves acidified colostrum increased abundance of Faecalibacterium in the first week of life (Hennessy et al. unpublished data, June 2022), and Faecalibacterium is associated with decreased diarrhea and better calf growth [42, 43], suggesting that acidified colostrum might actually enhance GI function. To be considered excellent quality, colostrum should have a low bacterial (including pathogens) load, but the process of harvesting colostrum could result in bacterial contamination. Furthermore, cattle suffering from mammary gland infections at the time of calving could also contribute additional bacteria to the collected colostrum. In addition to bacterial pathogens, colostrum can also be a channel of transfer for certain viruses [44]. The results of this study showed what might well be a clinically meaningful reduction in total bacteria and more specifically a significant reduction in total coliforms. Reduction of pathogens is a critical component of colostrum management because they are known to bind to free immunoglobulin in the gut lumen or block immunoglobulin uptake and transport across intestinal epithelial cells, thereby interfering with absorption [5, 10]. Decreasing bacteria to remove potential pathogens is permissive to enhanced passive transfer [5, 10, 12], and subsequent promotion of calf health. From a microbial standpoint, the colostrum on our well-managed study farm was of excellent quality from the outset, with ranges for total bacterial and total coliform counts (1,000–62,300 and 0–1,700 cfu/mL, respectively) being well below published targets for untreated colostrum. Nonetheless, levels of bacteria and specifically coliforms were, reduced by acidification suggesting that routine use of acid might be a valuable tool in bacterial management. However, work with more heavily contaminated colostrum is necessary to definitively demonstrate this.
Estimation of the effect of acidification on immunoglobulin levels and on their immunoactivity both in colostrum and in serum after absorbtion were essential parts of this study. For measurement of IgG concentrations, we chose to use commercially available turbidometric immunoassay (TIA) kits. Available for bovine colostrum and bovine serum,Footnote 1 advantages of which include accessibility, portability, simplicity, and cost-effectiveness. Although designed for point-of-care use, in this study all IgG concentrations were quantitatively determined spectrophotometrically in our clinical laboratory. Compared to the reference method of radial immunodiffusion (RID) and a point-of-care IgG ELISA, performance of the same manufacturer’s equine TIA test kit for measurement of immunoglobulins was good in quantifying equine serum IgG [45]. It might therefore be reasonable to expect that this methodology would perform just as well for measurement of bovine IgG. However, studies on comparative performance of RID and ELISA [46], ELISA and TIA [47], RID and Brix [48], RID, TIA and Brix [49], RID, Brix and colostrometer [50], RID and refractometer [51] have been somewhat inconsistent, with some studies showing relatively good comparative results [48, 49] while others report poor or contradictory correlations between tests and/or medium i.e., colostrum vs serum [46, 47, 50, 51] For example, Quigley et al. reported correlations between Brix and both TIA and RID, but TIA and RID, while correlated, were not consistent throughout the full concentration range of samples tested [49]. By contrast, Gelsinger et al. reported weak correlations between ELISA and RID results in plasma and unheated colostrum, such that IgG concentrations were significantly lower in all sample types when measured by ELISA, consequently they did not recommend direct comparison of ELISA and RID [46]. Although Schneider et al. found that colostrum IgG concentrations measured using the same test kit as that employed in the current study to be correlated with ELISA, the TIA values were on average 66.4% lower than those measured by ELISA and did not recommend use of TIA [47]. However, given the inconsistency of results across studies it is difficult to be definitive about the accuracy of the assays. When we compared correlations between colostrometer, Brix refractometer and TIA for IgG colostral concentrations, both colostrometer and Brix values were substantively and statistically correlated with TIA.
Other studies report Brix refractometry to be among the most consistent of methods available for measurement of colostral IgG [48, 49, 52]. Our data indicated that a Brix reading of 22.2% was equivalent to a colostral IgG concentration of 50 g/L measured by TIA, a value which is very similar to that reported for other studies [48, 49, 52]. By contrast, despite the apparent correlation, the colostrometer cutoff of 86.8 g/L did not closely match the TIA measurement. Moreover, examination of the scatter plots (not shown) revealed that for colostrometer readings only 5 samples were within 5 g/L of the fitted line for TIA IgG concentrations and 16 samples differed from the TIA measurement by ≥ 15 g/L. By contrast, 15 of the Brix readings were within 1% of the TIA fitted line and only 8 differed by more than 2% (i.e., approximately 5 g/L based on the 50 g/L cutoff being 22.2% Brix). In hindsight, it would have been better to have included Brix refractometry for assessment of serum IgG concentration, but we nevertheless feel that our use of TIA for both colostrum and serum is reasonable. In serum, total protein measured by refractometry and TIA were correlated and the total protein cutoffs of 5 and 5.5 g/dL often considered as gold standards for good and excellent passive transfer, respectively [4, 5, 53], generally matched the calculated equivalents for serum IgG concentration (8.1 and 11.6 g/L) measured by TIA. There was only one mismatch where an AC calf’s total protein of 5.2 g/dL did not match the serum IgG concentration of 3.1 g/L. Serum IgG measurements on subsequent days were also low indicating that the low level in this calf was a real effect. It is unclear why the total protein on day 3 was higher than might be expected based on the IgG concentration but could be related to the subjective nature of refractometry.
Colostrum quality has for the most part been judged by immunoglobulin concentration and bacterial load. The use of standard pathogen reducing techniques (heat treatment or true pasteurization, ultraviolet light irradiation, and pressure) are, under certain conditions, known to induce denaturing of immunoglobulins leading to reduced passive transfer and compromised serum immunoglobulin levels in calves [22,23,24,25]. Even maternal antibodies that do reach the blood of calves may have decreased immunoactivity. The ability of absorbed, undamaged immunoglobulin to neutralize pathogens is a key component in disease prevention leading to healthier calves. It has been reported that free-access feeding of acidified milk replacer results in improved growth and overall health of both dairy and veal calves [31]. Here we show that formic acid acidification of colostrum decreased bacterial load and was neutral in terms of its effect on immunoglobulin absorption measured as serum total protein and IgG concentrations, and AEA. More importantly, the neutralizing capabilities of the absorbed immunoglobulins against both viruses (IBRV, and BVDV Types 1 and 2) and bacteria (serovars of Leptospira spp) were not different between MC and AC groups suggesting that the immunoglobulins remained functionally intact after acidification. For IBRV the target titer for protection against the virus is ≥ 1:8 [54, 55], whereas those for BVDV types 1 and 2 are ≥ 1:128 [55, 56]. On day 3 all calves in this study had titers that exceeded the target levels for protection against said viruses.
Information gained from this project could benefit the production animal through supplementing existing management strategies directed at maintaining animal health and well-being. Additional studies to investigate the combined effect of feeding acidified colostrum followed by the provision of acidified whole milk or milk replacer on health in dairy youngstock and their subsequent productivity could further elucidate the benefits of acidification to the dairy industry. Future work is planned to investigate another potential benefit of acidification in the preservation of colostrum with reduced bacterial load without the need for freezing or refrigeration.
