Temporal kinetics of bovine mammary IgG secretion into colostrum and transition milk

Abstract

Neonatal calf survival and health is predominantly dependent on sufficient consumption of immunoglobulin G (IgG) and the resulting transfer of passive immunity (TPI). In this study, we investigate the potential for continued IgG secretion and temporal kinetics of mammary IgG output in sequential milkings performed at 0, 4, 16, 28, 40, and 52 hr postcalving in Holstein dairy cows. For colostrum (0 hr), we also scrutinize the relationships between IgG concentration, volume, refractometer readings (˚Bx values, Brix) and concentration of sugars (lactose and glucose). Mammary transcripts postpartum (0 hr) indicated that active IgG secretion continues beyond the first milking (colostrum; n = 4 to 5). IgG measurements at the different timepoints indicated that colostrum represents only 25.1% of the total IgG produced across the 6 sequential milking timepoints, with a substantial 48.9% being secreted into transition milk over the next 3 timepoints (4-, 6-, and 28-hr) combined. The differences on the basis of IgG concentrations across 0-, 4-, and 16-hr milking timepoints were not statistically significant (P = 0.1522; n = 9). For colostrum, volume remained highly variable, even with induced let-down prior to milking (n = 27). Nonetheless, colostrum IgG secretion was significantly co-regulated with volume (R2 = 0.915; P < 0.001; n = 18), an association that was stronger than that measured for lactose (R2 = 0.803; P < 0.001; n = 18) and glucose (R2 = 0.467; P = 0.002; n = 17). Comparing colostrum ˚Bx values to absolute IgG concentrations showed no correlation (R2 = 0.127; P = 0.07; n = 27); biochemical separation of colostrum components indicated that both proteins and nonprotein solutes could affect ˚Bx values (P < 0.0001 for both; n = 5). This suggests that ˚Bx values do not reasonably indicate IgG concentration to serve as a measure of "colostrum quality." Additionally, our finding that early transition milk (4-, 6-, and 28-hr) can contribute substantially more IgG than colostrum forces a rethink of existing feeding paradigms and means to maximize TPI in calves. Collectively, our results reveal the remarkable value of early transition milk and caveats to colostrum assessments that could advance application in enhancing neonatal calf health.

Keywords: calf; dairy; immunoglobulin; mammary; nutrition.

Figure 1.

Transport system that incorporates IgG into colostrum is actively transcribed in the mammary gland postpartum. (A) Site of mammary gland biopsy at the midpoint of the right hind quarter of cows. Biopsies were collected after the first/colostrum milking, within 1 to 2 hr after parturition. Step of passing the needle guide, prior to the core biopsy procedure is shown. (B) Physiological expression of the FcRN heterodimer components, FcGRT and B2M, is significantly higher at day 0 compared with baseline levels at days 40 to 50 of lactation (*P < 0.05; ***P < 0.001). This is indicative of an active process extending to the period of transition milk secretion. As a control, expression of the IgA and IgM transporter, PIGR, was not different between day 0 and days 40 to 50 of lactation.

Figure 2.

Volume of first milking/colostrum within 2 hr postpartum is highly variable. (A) First milking colostrum volume data from Holstein dairy cows (n = 130) was collected and sorted based on cow parity. When primiparous and multiparous groups were compared, there was no difference in colostrum volume produced. Within the multiparous group, comparing colostrum volume produced across the 5 different lactations, also did not show a significant difference in volume produced. The full range of colostrum volumes was between 0.1 and 22 L as recorded across all animals/groups. (B) First milking after oxytocin for inducing complete let-down also showed significant variability in volume between individual animals (range = 0.75 to 15.36 L), indicating differences to mammary secretion eliminating environmental factors that might have affected milking yield.

Figure 3.

Refractive index measurements (Bx values) does not directly correlate to IgG concentrations and is affected by both protein and nonprotein solute levels. (A) Standard curve generated using purified IgG detecting the heavy chain (IgG-H) in fluorescent Western blots for quantitating IgG concentration in colostrum (n = 3). Quantitative measurements of band intensity profiles are presented as RFU/relative fluorescence units. Representative standard blot with defined concentrations of purified bovine IgG is shown. (B) Representative blot with sample-specific variable dilutions as used to quantify colostrum (COL) IgG concentrations is shown with calculated values. (C) Concurrent measurements of Bx values and IgG concentration in 27 colostrum samples indicating the lack of a strong relationship between these 2 dimensions. (D) Absence of a significant correlation between IgG concentration and Bx values in the range of the 27 colostrum samples tested. (E) Removal of lipids/fat globules (CLR/coarse lipid removed), from colostrum had no effect on Bx values. Protein precipitated (PPT) CLR colostrum showed a significant reduction in Bx values (different letters indicate P < 0.0001). (F) Removal of nonprotein solutes (<6 kDa) from CLR colostrum by dialysis (DL) also caused substantial reduction in Bx values (different letters indicate P < 0.0001). Dialysis was validated by the significant decrease to osmolality (*P < 0.05), and confirming the absence of protein loss during this process.

Figure 4.

Time course of IgG secretion by the mammary gland reveals substantially greater amounts secreted in transition milk. (A) Representative Western blot showing colostrum (COL) and transition milk IgG levels in periodic postpartum milking (1, 4, 16, 28, 40, and 52 hr). (B) Graph showing changes to IgG concentration and colostrum/transition milk volume during the periodic post-partum milking (n = 9). The threshold line (at 50 g/L) indicates minimum concentration recommended for feeding by industry standards. Data show that colostrum/milk IgG secretion in the mammary gland continues beyond the first milking (colostrum), with a gradual decline over the sampling period in transition milk. IgG concentrations were not significantly different (ns) between <1-, 4-, and 16-hr milking. Increases to volume was not significantly different between <1-, 4-, and 16-hr milking, but progressively increased at the 28-, 40-, and 52-hr milkings. (C) Graph showing total postpartum mammary IgG secretion (cumulative values) across the different timepoints indicating the overall mammary output over time. Notable are the increased combined IgG output over 4- and 16-hr milking that are timepoints without significant changes to volume. Overall, there remains a linear increase in total mammary IgG released over time (R² = 0.7076; P = 0.0004). Of the total mammary IgG released, 25% is contained in colostrum and 32.3% is contained in the 4- and 16-hr transition milk. (D) Comparing total IgG produced in the first milking compared with transition milk combined (4 to 52 hr) indicated that an ~3-fold significantly higher levels of total IgG is secreted as part of transition milk (****P < 0.00001).

Figure 5.

IgG secretion and colostrum volume are coregulated, with a stronger association than other secreted components such as glucose and lactose. (A,B) IgG concentration and Bx values were not correlated to colostrum volume. Measurements for IgG concentration and Bx values remained within a constant range even with the highly variable range in colostrum volume (0.75 to 11 L; n = 18). (C, D) Total IgG secreted (and total protein secreted) was in strong correlation to colostrum volume (P < 0.001), indicating that IgG secretion and acquisition of colostrum fluid volume are significantly associated. (D, E) Total glucose and total lactose secreted also showed a significant association with colostrum volume (P = 0.002 and P < 0.001, respectively), but the relationships appeared weaker than that observed for total IgG towards higher volumes.