Progress report for GNE20-242
The development of morbidities including diarrhea, septicemia, and respiratory disease
impairs growth and increases mortality in dairy calves. Calf morbidity and mortality are also
sources of economic loss for the dairy industry. Effective colostrum management is one
approach to enhance passive immunity but the benefit is short-lived. Indeed, the calf is most
likely to develop a morbidity pre-weaning because of underdeveloped innate and adaptive
immune systems. This “gap in immunity” predisposes calves to infection and morbidity, which
are commonly managed by antibiotic administration. However, antibiotic mismanagement and
resistance are societal concerns. Non-antibiotic therapies are needed to improve calf health and
sustainable dairy production. We propose to investigate dietary lysophospholipid
supplementation to enhance innate and adaptive immunity in pre-weaned calves. In non-
ruminants, the lysophospholipid lysophosphatidylcholine (LPC) activates neutrophils and
peripheral blood mononuclear cells to protect against endotoxemia. Therefore, we aim to
determine whether dietary LPC supplementation enhances innate and adaptive immune function
(objective 1) and growth (objective 2) in pre-weaned Holstein calves. Our approach will involve
feeding pre-weaned calves milk replacer unsupplemented or supplemented with LPC. Calves
will be intravenously challenged by bacteria-derived endotoxin to study infection. We will
leverage our experience studying bovine lipid biology and an integrated commercial partnership
to fast-track new milk replacer product development for on-farm utilization. Our outreach plan
involves the dissemination of knowledge and practice through peer-review publications,
conference proceedings, and community extension. The project outcome is the development of a
dietary approach to elevate immune function in pre-weaned calves to protect against morbidity.
- Our first objective is to test the hypothesis that dietary lysophospholipid supplementation enhances innate and adaptive immune function in pre-weaned Holstein calves.
- Our second objective is to test the hypothesis that dietary lysophospholipid supplementation enhances growth performance in pre-weaned Holstein calves.
The purpose of this project is to identify a dietary approach to enhance immune function in pre-weaned calves. This purpose aligns with our long-term goal, which is to identify non-antibiotic therapies to prevent morbidity and mortality in dairy calves. One approach to prevent pathogen infection is to feed colostrum immediately after birth. However, the approach is partially inadequate because immune transfer is influenced by timing of delivery, immunoglobulin concentrations, and volume of colostrum fed, which are due to differences in management, season, and cow. Colostrum feeding is a form of passive immunity and benefit to calf is short-lived. The calf must then rely on innate (fast-acting and broadly effective) and adaptive (slow-acting but highly specific) immunity; however, these systems are naive at birth and won’t mature until after weaning. Indeed, wk 2 through wk 5 of life is referred to as a “gap in immunity” in calves. This is why pre-weaned calves are susceptible to pathogen infection and morbidities including diarrhea, septicemia, and bovine respiratory disease. These morbidities predispose calves to mortality, which may be as extreme as 7.8% on dairy farms . Similar observations are observed in beef herds . Even though a calf may survive a morbidity occurrence, deleterious effects on growth, milk production, and reproduction may occur later in life. For example, pneumonia and umbilical infection reduce average daily gain in calves . Heifers that develop diarrhea early in life are more likely to calve later in life  and produce less milk during their future lactation . Approaches that enhance innate and adaptive immune responses to pathogen infection are likely to prevent morbidity, mortality, and compromised growth and lactation performance later in life.
A common approach to treat morbidity and prevent mortality is the use of antibiotics. However, the extensive use and potential mismanagement of antibiotics is a concern for consumers. In addition, the development of antibiotic resistant bacteria has emerged as a major problem for livestock industries. The development of a dietary therapy that bolsters immune function in pre-weaned calves has potential to limit antibiotic use on farms. We have centered our attention on the dietary nutrient and lysophospholipid called lysophosphatidylcholine (LPC). In rodents, LPC is a potent immunomodulator that enhances innate and adaptive immunity. This includes elevated neutrophil bactericidal activity, blocking endotoxin-induced inflammation, increased T cell and macrophage interferon-γ (IFNγ) secretion, and increased antibody production [6-9]. LPC therapy also protects against bacterial infection and sepsis to prevent mortality in rodents . Therefore, we will test the hypothesis that feeding a milk replacer enriched in LPC enhances innate and adaptive immune function in pre-weaned calves subject to pathogen infection. If effective, the potential long-term impact on dairy farms includes improved calf health, lower antibiotic usage, and an enhanced productive lifespan of livestock. Such outcomes are likely to reduce economic and environmental costs associated with veterinary treatment and raising heifer replacements but also improve the consumer’s acceptability of farm management practices. These achievements would help farmers achieve a sustainable operation.
Pre-weaned Holstein calves (~2 to 5 wks of life) have underdeveloped innate and adaptive immune systems. This “gap in immunity” predisposes calves to early-life morbidities including diarrhea and septicemia. Lysophospholipid therapy enhances innate and adaptive immune function in non-ruminants to protect against endotoxemic shock and prevent mortality. Therefore, feeding pre-weaned Holstein calves milk replacer enriched in lysophospholipids is a potential means to bolster immune function and protect against endotoxin. In support, Brianna Tate has cultured neonatal calf neutrophils with 18:0-LPC and observed neutrophil activation (Figure 1).
In compliance with the Cornell Institutional Animal Care and Use Committee (protocol #2018-0110), twenty-newborn Holstein heifer calves will be obtained from the Cornell University Dairy Research Center (Harford, NY) and housed in the Large Animal Research and Teaching Unit (Ithaca, NY). Inclusion criteria will include no twins, birth weights greater than 34.5 kg, high ease of calving, and complete consumption of colostrum. Starting on d 2 of life, calves will be fed a 26% crude protein, 20% fat milk replacer on a DM basis at 1.75% of body weight (BW) provided as two equal meals per day. The custom milk replacer blend provided by Milk Specialties Global Animal Nutrition will contain a basal amount of soy lecithin (~1% of dry matter) but not contain lysolecithin (i.e., LPC), or lasalocid, essential oils or mannan-oligosaccharides because of their potential to influence microbial ecology of the gastrointestinal tract and host immunity. Calves will be provided ad libitum access to a 22% crude protein starter pellet and water. Following a 2-wk acclimation period (first 2 wk of life), calves will be blocked by birth weight and average daily gain (kg/d) and randomly assigned to either: control (unsupplemented; n = 10) or supplemented (275 mg of soy lysolecithin enriched with LPC/kg of BW/d; n = 10) diets. The previously described milk replacer blend will be utilized for both treatment groups; however, lysolecithin will be mixed into the milk replacer prior to each feeding. The dietary treatments will be provided for 28 d, reduced by half during weaning transition, and terminated post-weaning (Figure 2). For objective #1, we realize that dietary LPC therapy may only be proven effective in calves that experience infection. Therefore, our experimental approach will include two endotoxin challenges to mimic acute infections. First, calves will be intravenously challenged with 2.5 μg/kg of E. coli O111:B4 lipopolysaccharide (i.e., endotoxin) on d 3 of the experiment (“early” exposure). In support, evidence in rodents suggests that 48 h of LPC therapy is sufficient to protect against a bacterial infection . Calves will be challenged with endotoxin again on d 28 (“late” exposure). Jugular catheters will be inserted 96 h prior to each endotoxin challenge and removed immediately following each test. Calves will be weaned starting at d 42 of age by restricting milk replacer to 0.875% of BW daily (dry matter basis; half of prior intake) fed only in the evening until d 49 of age at which point the study will conclude. Calves that receive antibiotic therapy or die prematurely because of morbidity will be replaced and excluded from the sample analyses but defined in all presentations.
Record and sample collection:
Feed samples will be collected weekly, composited monthly and stored at -20ᵒC. Feed intakes and general health observations will be recorded daily. Rectal temperatures and respiration rates will be recorded daily and every 30 min during the endotoxin challenge (until h 8 post administration). Signs of dehydration and fecal scores will be monitored. Body weights, and hip and wither heights will be recorded twice weekly. Blood will be sampled daily before morning feeding from d 1 through 7, then weekly until d 35. Additional samples will be taken post endotoxin administration (i.e., 0, 1, 2, 3, 4, 6, and 8 h, relative to injection) and post-weaning (i.e., 0, 1, 2, and 3 d, relative to start of starter only feeding). Whole blood will be utilized to study neutrophil activation the day of collection (d 3 and 28). Separated plasma and serum will be stored at -80ᵒC for future analyses.
For both objectives, we will utilize liquid chromatography and mass spectrometry to quantify plasma lysophospholipids using targeted lipidomics within the Cornell University Metabolomics Facility. This will include measuring changes in plasma total and 16:0-, 18:0-, 18:1-, 18:2-LPC. For objective 1, white blood cell counts and differentials (i.e., neutrophils, lymphocytes, monocytes, eosinophils, and basophils) will be analyzed within the Cornell Animal Health Diagnostic Center. To evaluate changes in innate immunity, we will measure neutrophil activation by flow cytometry using the PHAGOBURST™ assay (Celonic Group, Germany) to quantify oxidative burst activity. We will also measure the ability of neutrophils to kill E. coli by counting the number of colony forming units propagated on Luria broth agar plates after co-incubation with neutrophils isolated from unsupplemented and supplemented calves. Circulating concentrations of tumor necrosis factor-α (TNFα) and serum amyloid A (acute phase response protein) will be quantified by bovine ELISA (basal and endotoxin challenge samples). Serum immunoglobulin concentrations including IgM and IgG will be measured by ELISA to investigate adaptive immunity. Lastly, circulating IFNγ concentrations will be measured by ELISA to better understanding the innate and adaptive immune response.
Treatment, time (day or hour), and their interactions will be examined as fixed effects. The individual calf will be an independent variable and evaluated as a random effect nested within treatment. For objective 1, analyzed dependent variables will include LPC, white blood cell counts, neutrophil oxidative burst and E. coli killing, TNFα, IgM, IgG, and IFNγ. For objective 2, dependent variables will include dry matter and energy intakes, rectal temperatures and respiration rates, BW, hip and wither heights, gain to feed ratio, and average daily gain. Data will be analyzed using the SAS® software (version 9.4; SAS Institute Inc., Cary). First, normality will be tested using the Univariate Procedure and residual plots. Data will then be evaluated using the MIXED procedure of SAS with repeated measures or by evaluating the least square means through the GLM Procedure using Scheffé’s method. Pearson’s correlation performed using the CORR procedure to characterize associations between dependent variables.
Dietary lysophospholipid supplementation is expected to increase circulating LPC concentrations including 16:0-, 18:0- and 18:1-LPC. For objective 1, dietary lysophospholipid supplementation is expected to enhance neutrophil oxidative burst activity and E. coli killing. These results would suggest LPC therapy will enable neutrophils to exhibit an increase in bactericidal activity and be more effectively fight pathogen infection. Endotoxin administration will increase rectal temperatures and respiration rates as well as circulating levels of serum amyloid A and TNFα. Because of LPC’s inhibitory effect on pro-inflammatory precursors (i.e., phospholipase A2), we expect that LPC feeding will decrease circulating TNFα and serum amyloid A in response to endotoxin administration . TNFα is a pro-inflammatory cytokine that is secreted by macrophages and monocytes in order to fight infection; serum amyloid A is an acute phase protein that provokes chemotaxis. We also anticipate that feeding LPC will increase circulating INFγ, IgM, and IgG. These outcomes would suggest that LPC feeding will help the calf develop an antibody-mediated immune response that will neutralize pathogens and protect against subsequent infections. This finding may be mediated by LPC interacting with surface receptors (i.e. G2A) of macrophages and other phagocytic/antigen-presenting cells to enhance their bactericidal and phagocytic capabilities. In turn, these cells will stimulate B cells to produce antibodies [7, 19]. For objective 2, improved immunity will likely support increases in growth performance. It is expected that we will observe increases in BW, gain to feed ratio, and average daily gain with LPC treatment. We postulate that this will be due to maintenance of energy intake as well as increased utilization of nutrients (e.g., glucose) that would otherwise be utilized for immune cell activation and metabolism.
1. The presence of lecithin (e.g., phosphatidylcholine) in the milk replacer is needed to form a fat emulsion. We recognize that dietary phosphatidylcholine could be digested to form LPC for intestinal absorption. To control for this uncertainty, all calves will be fed a low amount of crude lecithin (~1% of ration dry matter; containing triglyceride and mixed phospholipids), which is expected to provide ~2 g of phosphatidylcholine to all calves fed 1 kg of milk replacer powder.
2. We have chosen to work with Milk Specialties Global Animal Nutrition because they can secure deoiled (triglyceride removed) and fractioned (LPC purified) lysolecithin. We expect the LPC composition of the supplement to be >75%. However, we recognize that the supplement will likely include other types of lysophospholipids including lysophosphatidic acid and lysophosphatidylethanalomine. While lysophosphatidic acid has been shown to have immunomodulatory effects  we do not expect that LPE will influence study outcomes. We will quantify the concentrations of these lysophospholipids in the supplement and plasma samples and correlate these plasma lysophospholipid concentrations with parameters of immune function in order to determine whether there is a correlation. Pure LPC (100%) is not economical to use as a livestock ration ingredient and the reason why it was avoided in this proposal.
3. Our dose of lysolecithin (LPC) was carefully considered. In rodents, 20 or 40 mg of LPC (16:0 or 18:0)/kg of BW/d protects against sepsis when delivered as a subcutaneous injection . This would equate to 1.2 or 2.4 g of pure LPC per d in a 60 kg calf. We opted for 275 mg of lysolecithin enriched in LPC/kg of BW/d. This equates to 16.5 g of lysolecithin for a 60 kg calf (~1.5% of ration DM). This is expected to provide a minimum of 12.4 g of LPC for a 60 kg calf, which is ~5 times as much as Yan et al.  but chosen because lysolecithin is subject to gastrointestinal degradation and absorption is expected to be less. We suspect that bioavailability of our dietary LPC will be ~50-75% (LPC delivery would still exceed Yan et al. . Moreover, our dose is expected to negligibly influence milk replacer product cost if product development moves forward. Please note that our other support will evaluate the effectiveness of subcutaneous LPC at 20 and 40 mg of LPC/kg of BW/d for comparison.
4. Our use of a “low-dose” lipopolysaccharide injection is expected to cause a short-term infection response. Calves are fully expected to recover in 24 to 48 h. Therefore, effects on growth may be negated by quick recovery time and thus difficult to observe treatment effects. In the event that we do not detect treatment effects on growth, we will conduct secondary statistical analyses using linear regression to relate circulating LPC concentrations in early life (wk 1 of experiment) with average daily gain for the trial duration. Future research using larger study populations would be needed to determine whether dietary LPC supplementation prevents morbidity and mortality, and long-term outcomes on growth trajectory, age at first calving, and milk production.
Progress Report Update:
Due to complications that arose as a result of the COVID-19 pandemic, the timeline of the anticipated project will be partially delayed. The six-week project is expected to be conducted during the Summer of 2021. Laboratory and sample analyses (Phase 2) will be expedited in order to allow the remaining projected timetable to progress as expected (i.e. results presented at both the Cornell Nutrition Conference and ADSA). A revised timetable for Phase 2 and 3 will be provided below.
Phase 2, Summer 2021 through Fall 2021:
June: Enroll calves in experiment and begin sample collection
June – August: Manage calf experiment and collect samples
August: Complete sample collection and summarize field data including statistical analysis
August – October: Complete laboratory sample analyses
October – December: Complete statistical analyses and prepare manuscripts; present available data at Cornell Nutrition Conference
Phase 3, Presentation of Findings, Spring through Summer 2022:
Spring/Summer 2022: Submit manuscripts for peer-review to Journal of Dairy Science; present
findings at ADSA; publish findings in Cornell PRO-DAIRY e-editorial
Spring/Summer 2022 Present findings at ADSA Annual Meeting
During the Fall of 2020, further laboratory experiments were carried out in order to determine the optimal LPC species to be administered to calves during the approaching in vivo trial. Trypan blue assays carried out on neutrophils treated with and without three different LPC species (16:0, 18:0, and 18:1) showed that after PMA stimulation and a subsequent one-hour incubation with LPC, although reactive oxygen species production was increased as a result of LPC treatment, viability was significantly compromised. This was especially the case with saturated LPC species 16:0 and 18:0. In order to mitigate this effect and to more closely recapitulate physiological conditions, we added 50mM of bovine serum albumin to 100mM of each respective LPC and used this treatment cocktail for all future assays. This was found to salvage viability in PMA-stimulated neutrophils while also maintaining previously observed elevated levels of reactive oxygen species production in comparison to non-LPC treated stimulated neutrophils. Saturated 18:0 LPC was found to have the greatest difference in hydrogen peroxide production between LPC and non-LPC treated neutrophils whilst also maintaining cell viability. As a result, we decided to move forward with this LPC species for our treatments in our in vivo trial.
Education & Outreach Activities and Participation Summary
Nutritional therapies that enhance calf immune health are needed to prevent morbidity, mortality, antibiotic use, and economic loss. It is imperative that dairy producers, researchers, nutritionists, and veterinarians receive the most up-to-date and relevant information regarding the development of novel therapeutics to bolster calf health. Considering the diversity of our target audience, multiple methods of communication are deemed necessary. First, findings will be presented at the American Dairy Science Association (ADSA) Annual Meeting, the Cornell Nutrition Conference, and Cornell Advanced Dairy Nutrition and Management Shortcourse. Second, we will submit a minimum of two publications to the Journal of Dairy Science for peer-review. Third, we will highlight practical applications within a PRO-DAIRY e-Leader newsletter or The Manager magazine which is circulated to ~12,000 farms across the eastern United States. Through these methods, we will be able to reach and educate a broad audience.