Final report for GNE19-220
This project addressed the problem of a lack of knowledge on the timing of gut closure in calves at the intestine level. To the best of our knowledge, there has not been a study directly examining the kinetics of IgG enterocyte absorption machinery expression in the neonatal small intestine, nor abomasa fluid pH over time- both of which are important to known the digestive environment IgG must pass through to be absorbed intact and functional. To address this, we 1) directly measured the spatio-temporal gene expression profile of FCGRT, the IgG receptor, in the small intestine of the neonatal Holstein calf at 0h, 12, 24, 48 hours and 7 days of age and 2) measured the abomasa fluid pH at the same timepoints. Together, this data allows us to map the timeframe for IgG absorption/gut closure in the neonate. Our research approach was to collect intestine samples from Holstein dairy calves at the specified time points (0, 12, 24, 48 hours and 7 days of age) for RNA extraction and RT-qPCR, and to record abomasum fluid pH measurements at the same timepoints- and to analyze changes over time from 0h (birth) to 7 days of age. The animal project is complete, and qPCR (gene expression) and IHC (IgG protein) lab work are also complete. We concluded that FCGRT expression profile indicates diverse roles in enteric immune homeostasis, with IgG absorption occurring in a reduced capacity out to 48 hours of life at least. Recent literature suggests IgG in colostrum is stable even in acidic pH, meaning that IgG absorption can indeed occur despite a drop in abomasa lumen pH. We have communicated our findings to scientists, consultants and producers at Cornell CNC 2020 conference, and ADSA 2021 conference. We are working with Cornell PRO-Dairy to further communicate findings with farmers for adoption of practices (feed calves out to 48 hours at least).
As intestinal immunoglobulin absorption machinery expression is the determining factor for immunoglobulin absorption and thus TPI, the window for neonatal gut closure needs to be completely re-evaluated and based directly on the expression of the intestinal immunoglobulin IgG absorption machinery. We recently published a paper (Schalich et al., 2021) on postpartum mammary IgG secretion kinetics (0-52 hours after parturition), and found that there is no significant difference in IgG concentration 1-16 hours postpartum, and that surprisingly only 25% of IgG is secreted into colostrum (first milking, 1 hour postpartum) while the remaining 75% is secreted into transition milk (next subsequent 5 milkings out to 52 hours). In consideration of the co-evolution of cow-calf physiology, and the lack of direct and substantial evidence for mainstream thought that enterocyte IgG absorption in the neonatal gut ceases at 24 hours, we believe that current colostrum feeding practices up to only 24 hours of life effectively truncate the physiological period of time in which immunoglobulin absorption and enteric protection can occur in the calf intestine.
For this project, the following objectives allowed us to address this gap in our knowledge, and to apply new information learned to help dairy producers with colostrum management: 1. Precisely and quantitatively determined the full window of time that a neonatal calf intestine can absorb colostrum immunoglobulins. We used quantitative polymerase chain reaction (qPCR) to directly measure immunoglobulin receptor expression (mRNA) across the full length of the calf intestine (duodenum, jejunum and ileum). Samples will be collected within 0 hours- 7 days after birth, with calves randomly assigned to one of five different sample collection time point groups: 0, 12, 24, 48 hours and 7 days after birth. Concurrently, we examined for abomasal function (onset of pH and protein degrading enzymes) to determine the period of permissive conditions for IgG uptake. 2. We developed a colostrum feeding protocol founded on calf intestinal physiology for maximum immunoglobulin absorption.
The purpose of this project was 1) to reduce the waste of colostrum and transition milk and ensure more is fed to calves for transfer of passive immunity (TPI) and sustained enteric protection in the neonatal period 2) in achieving point #1, to reduce the overuse of antibiotics in calves (calves that have failure of TPI or do not get sustained enteric IgG protection from drinking transition milk may fall ill and need antibiotics to recover). If met these objectives will be economically helpful to dairy farmers, and environmentally beneficial to all to reduce our risk of antimicrobial resistance, which is a major threat to One Health.
This project was conducted from September 2019 - June 2021 (calf sampling, lab work, analysis, communication to scientific audiences).
All protocols in this study were performed according to approved IACUC protocols. This study was performed at Walnut Ridge Dairy (LLC) in Lansing, NY. Holstein calves were enrolled on a rolling basis at the time of birth. At birth, calves were immediately collected from the dairy maternity ward and were relocated to a separate pen reserved for study calves. Calves were randomly enrolled into one study group (0, 12, 24, 48 hours, or 7 day; n=3-5 calves/group). These timepoints were chosen to span the complete timeframe for passive transfer of immunity (PTI) in the calf, with 0 hours representing the initiation of PTI (known capacity for IgG absorption) and day 7 gut closure. At the time of sample collection, calves were euthanized by non-penetrating captive bolt and exsanguination of the jugular vein. Following euthanasia, the abomasum and intestines were exposed by a ventral abdominal midline incision, then removed and separated from one another. The small intestine was first placed into a bucket containing ice cold saline solution (0.9 % NaCl) before being unwound and placed onto ice for sampling of 1 x 1 cm intestine pieces every 100 cm along the length of the duodenum (Toofanian et al., 1974). Upon collection, each sample was quickly rinsed with ice cold 1X phosphate buffered saline (PBS), placed into 1.5 mL tubes, and snap frozen in liquid nitrogen. All tissue samples were transported in liquid nitrogen back to the lab, where they were stored at -80 C until further processing. Total RNA was extracted from tissue samples in a two-step protocol using TRIzol® (Life Technologies) with the following modification: samples were completely homogenized in 1 mL of TRIzol reagent per sample, with immediate 1:10 dilution in TRIzol. RNA concentration was measured in a NanoDrop ND-100 Spectrophotometer (Wilmington, DE, USA). Reverse transcription of 2.0 μg of total RNA was performed using MultiscribeTM reverse transcriptase (ThermoFisher Scientific) with random hexamer primers.
Expression of specific genes were analyzed by quantitative PCR (qPCR) using SYBR-green detection method with intron-spanning primers. Gene expression of FCGRT in the small intestine duodenum, jejunum and ileum were FCGRT was analyzed after normalization to the expression of housekeeping gene hydroxymethylbilane synthase (HMBS). Abomasa fluid pH was measured with an Inlab® Easy Combination pH meter Electrodes (METTLER TOLEDO), which was calibrated before each use. Briefly, an incision was made, the abomasum opened, and the pH probe inserted into abomasum fluid surrounding the clot. After stabilizing, the pH was recorded. Relative quantifications of fold change were performed after normalization to housekeeping genes by comparing Ct values from individual samples by the 2-∆∆Ct method (Livak & Schmittgen, 2001). For all gene expression assays, fold change was calculated relative to 0 hours (0 Hr) which served as the baseline timepoint. For statistical analyses, Prism 9 (GraphPad) was used. Gene expression data across the different timepoints were compared using one-way ANOVA and post-hoc Tukey’s test and p < 0.05 was considered significant. In the results, data are represented as mean ± standard error of the mean (SEM). In all figures independent data points are displayed when possible.
We have collected all intestine samples and done RNA extraction, and have completed the gene expression analysis (qPCR) and pH measurements. Our findings suggest that IgG absorption can occur at a reduced capacity out to 48 hours, while mammary secretion kinetics suggest sustained enteric protection during the entire neonatal period- a crucial factor for neonate immune health that has not been well studied or communicated to dairy producers. We found that the IgG receptor, FCGRT, expression profile indicates diverse roles in enteric immune protection (see below) and that elevated levels of a novel IgG absorption marker remains elevated/does not change 12-48 hours in the calf gut, suggesting sustained IgG absorption out to at least 48 hours of life. pH is highest at 0H and declines to hold steady 12h-7 days of life, indicating an acidic environment IgG must traverse for absorption. The PI (Kasey Schalich) presented this research at Cornell Nutrition Conference 2020 and will be presenting this research as a 12 minute talk at ADSA 2021. We anticipate that we will publish 1-2 papers from this research. We have written 2 articles for Cornell PRO-Dairy that translates our findings on gut physiology into recommended colostrum and transition milk feeding programs for calves, which are in the process of being published hopefully this year. Graphs for all FCGRT gene expression and abomasum fluid pH are linked below.
Key findings are: 1) IgG absorption can occur at a reduced capacity out to ~48 hours in neonatal calves. 2) Feeding IgG past the absorptive period could still be beneficial to confer enteric support to the gut.
Based on our findings, we recommended feeding calves colostrum/transition milk out to at least 48 hours of life for better success of TPI, enteric protection, and reduced need to administer antibiotics to calves. If implicated, we think that these practices could improve calf health and return potential gains (ADG, lactation performance) to producers due to the other benefits of feeding colostrum/TM to calves.
Education & Outreach Activities and Participation Summary
I did one 5 min graduate student research presentation at the Cornell Nutrition Conference in October 2020, to share our preliminary findings, and will be giving a 12 minute talk on our research at ADSA 2021. We wrote 2 articles for dairy producers on our findings, which we are in the process of publishing with Hoard's Dairyman. In this way, we aim to share our research findings with other scientists and producers.
It will hopefully lead to less colostrum waste, more calves being fed colostrum and transition milk, and reduced antibiotic use on farms. This will be beneficial for farms economically, and for the environment and human health due to decreased risk of antibiotic resistance.
Our view on sustainable agriculture did not change significantly, although we have become more aware of the labor challenges on U.S. dairy farms. We are surprised to learn about how transition milk is generally not fed to calves. I (Kasey) am pursuing a career in academia as a professor in an animal science department, and the research data and experience generated from this project has greatly increased my interest and enthusiasm for a career in research that can use basic science to improve the sustainability of animal agriculture.
The research method of directly sampling the small intestine, and testing the current consensus on the kinetics of enterocyte IgG absorption in the neonatal gut,were key to this project's success, as was the funding which made this entire study possible. We believe that great applications for producers can come from pursuing basic science on animal physiology, as historically has been the case. Our future endeavors will likely take this proven approach, hoping that funding will allow us to do so. We learned a great deal about gut physiology and hope to use our knowledge to advance the science and collaborate with others interested in the topic, and that ultimately this research improves animal health and human health, as recognized by the One Health initiative.