Determine Whether Dietary Lysophospholipid Supplementation Enhances Immunity in Holstein Dairy Calves

Grant Figure 2- Experimental Design Grant Figure 1 - LPC 

Experimental Approach

Rationale:

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).

Experimental design:

In compliance with the Cornell Institutional Animal Care and Use Committee (protocol #2018-0110), twenty-newborn Holstein heifer calves were obtained from the Cornell University Dairy Research Center (Harford, NY) and housed in the Large Animal Research and Teaching Unit (Ithaca, NY). Inclusion criteria included 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 were 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 contained a basal amount of soy lecithin (~1% of dry matter) but did 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 were 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 were 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 was utilized for both treatment groups; however, lysolecithin will be mixed into the milk replacer prior to each feeding. The dietary treatments were provided for 28 d, reduced by half during weaning transition, and terminated post-weaning (Figure 2). For objective #1, we realized that dietary LPC therapy may only be proven effective in calves that experience infection. Therefore, our initial experimental approach was include two endotoxin challenges to mimic acute infections: first, calves would 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) and again on d 28 (“late” exposure).  Unfortunately, due to IACUC restrictions put in place we were no longer able to carry out the endotoxin challenge. Calves were 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 received antibiotic therapy or died prematurely because of morbidity were replaced and excluded from the sample analyses but defined in all presentations.

Record and sample collection:

Feed samples were collected weekly, composited monthly and stored at -20ᵒC. Feed intakes and general health observations were recorded daily. Rectal temperatures and respiration rates were recorded daily and every 30 min during the endotoxin challenge (until h 8 post administration). Signs of dehydration and fecal scores were monitored. Body weights, and hip and wither heights were recorded twice weekly. Blood were sampled daily before morning feeding from d 1 through 7, then weekly until d 35. Additional samples were taken post-weaning (i.e., 0, 1, 2, and 3 d, relative to start of starter only feeding). Whole blood was utilized to study neutrophil activation the day of collection (d 3 and 28). Separated plasma and serum were stored at -80ᵒC for future analyses.

Sample analyses:

For both objectives, we utilized liquid chromatography and mass spectrometry to quantify plasma lysophospholipids using targeted lipidomics within the Cornell University Metabolomics Facility. This included 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) were analyzed within the Cornell Animal Health Diagnostic Center. To evaluate changes in innate immunity, we measured neutrophil activation by flow cytometry using the PHAGOBURST™ assay (Celonic Group, Germany) to quantify oxidative burst activity. We also measured 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) were quantified by bovine ELISA (basal and endotoxin challenge samples). Serum immunoglobulin concentrations including IgM and IgG were measured by ELISA to investigate adaptive immunity. Lastly, circulating IFNγ concentrations were measured by ELISA to better understanding the innate and adaptive immune response.

Statistical analyses:

Treatment, time (day or hour), and their interactions were examined as fixed effects. The individual calf was an independent variable and evaluated as a random effect nested within treatment. For objective 1, analyzed dependent variables included LPC, white blood cell counts, neutrophil oxidative burst and E. coli killing, TNFα, IgM, IgG, and IFNγ. For objective 2, dependent variables included dry matter and energy intakes, rectal temperatures and respiration rates, BW, hip and wither heights, gain to feed ratio, and average daily gain. Data was analyzed using the SAS® software (version 9.4; SAS Institute Inc., Cary). First, normality was tested using the Univariate Procedure and residual plots. Data was then 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 was performed using the CORR procedure to characterize associations between dependent variables.

Expected results:

Dietary lysophospholipid supplementation was expected to increase circulating LPC concentrations including 16:0-, 18:0- and 18:1-LPC. For objective 1, dietary lysophospholipid supplementation was expected to enhance neutrophil oxidative burst activity and E. coli killing. Due to LPC’s inhibitory effect on pro-inflammatory precursors (i.e., phospholipase A2), we expected that LPC feeding would decrease circulating TNFα and serum amyloid A [18]. 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 anticipated that feeding LPC will increase circulating INFγ, IgM, and IgG. These outcomes would suggest that LPC feeding would assist in the development of the calf's antibody-mediated immune response that would neutralize pathogens and protect against subsequent infections. We deduced that 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 would likely support increases in growth performance. It was expected that we would observe increases in BW, gain to feed ratio, and average daily gain with LPC treatment. We postulated 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.

Potential pitfalls:

1. The presence of lecithin (e.g., phosphatidylcholine) in the milk replacer is needed to form a fat emulsion. We recognized that dietary phosphatidylcholine could be digested to form LPC for intestinal absorption. To control for this uncertainty, all calves were 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 [20] 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 [7]. 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. [7] but chosen because lysolecithin is subject to gastrointestinal degradation and absorption is expected to be less. We suspected that bioavailability of our dietary LPC will be ~50-75% (LPC delivery would still exceed Yan et al. [7]. Moreover, our dose is expected to negligibly influence milk replacer product cost if product development moves forward. Please note that our other support evaluated 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 was expected to cause a short-term infection response. Calves were fully expected to recover in 24 to 48 h. Unfortunately, during a pilot LPS challenge calf losses occurred that resulted in the removal of the endotoxin challenge from our study, in accordance with IACUC recommendations. 

2020 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).

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.

2021 Update:

In May of 2021, pilot LPS challenges were conducted in order to confirm that the determined concentration of LPS to be administered to the calves would result in the expected physiological effects that would indicate that the calf was indeed immunologically challenged (fever, increased respiration rate, etc.) Unfortunately, complications arose in the calves at the concentration of LPS administered intravenously and, as a result, delays and adaptations needed to be made in order to secure approval from our university IACUC committee. Therefore, the intravenous LPS challenge was replaced with an ex vivo LPS challenge that was conducted on whole-blood samples collected from the jugular vein of calves. Restrictions on the number of calves that could be procured from our provider also necessitated the enrollment of calves in 5 blocks instead of all at once, as previously planned. The project was conducted from late July 2021 until mid-October 2021. Laboratory and sample analyses are ongoing and are expected to finish, with the data analyzed, by the end of February. The remaining projected timetable is expected to progress as expected (i.e. results presented at ADSA and published in JDS).