Subcutaneous lysophosphatidylcholine administration promotes a febrile and immune response in Holstein heifer calves

Lysophosphatidylcholine (LPC) is immunomodulatory in non-ruminants; however, the actions of LPC on immunity in cattle are undefined. Our objective was to study the effects of LPC administration on measures of immunity, liver health, and growth in calves. Forty-six healthy Holstein heifer calves (age 7 ± 3 d) were randomly assigned to 1 of 4 treatments (n = 10 to 11 calves/treatment): a milk replacer diet unsupplemented with lecithin in the absence (CON) or presence of subcutaneous (s.c.) administered mixed (mLPC; 69% LPC-16:0, 25% LPC-18:0, 6% other) or pure (pLPC; 99% LPC-18:0) LPC, or a milk replacer diet supplemented with 3% lecithin enriched in lysophospholipids containing LPC in the absence of s.c. administered LPC (LYSO) for 5 wk. Calves received 5 subcutaneous (s.c.) injections of vehicle (10 mL of phosphate-buffered saline containing 20 mg of bovine serum albumin/mL; CON and LYSO) or vehicle containing mLPC or pLPC to provide 10 mg of total LPC/kg of body weight [BW]/ injection every 12 h during wk 2 of life. Calves were fed a milk replacer containing 27% crude protein (CP) and 24% fat at 1.75% of BW per d (dry matter basis) until wk 6 of life (start of weaning). Starter grain and water were provided ad libitum. Body measurements were recorded weekly and clinical observations were recorded daily. Blood samples were collected weekly before morning feeding and at 0, 5 and 10 h, relative to the final s.c. injection of vehicle or LPC. Data were analyzed using a mixed model with repeated measures including fixed effects of treatment, time, and their interaction. A Dunnett’s test was used to compare treatments to CON. Peak rectal temperatures were higher in mLPC or pLPC, relative to CON. Plasma LPC concentrations were greater in mLPC and LYSO calves 5 h and 10 h post-final injection, relative to CON. Calves receiving mLPC and pLPC also had higher circulating serum amyloid A concentrations, relative to CON. Calves receiving mLPC had greater serum aspartate aminotransferase, γ-glutamyltransferase, and glutamate dehydrogenase concentrations, relative to CON. Calves provided mLPC experienced lower average daily gain (ADG) post-weaning, relative to CON. The LYSO treatment did not modify rectal temperatures, ADG, or measures of liver health, relative to CON. We conclude that LPC administered as s.c. injections induced an acute febrile response, modified measures of liver and immune function, and impaired growth in calves.


INTRODUCTION
The immune system of the dairy calf is classified as "naïve" during the first few weeks of life (Chase et al., 2008a).This is due in part to the physiology of the ruminant placenta that prevents exchange of immunoglobulins between the dam and fetus in utero.As a consequence, dairy calves are agammaglobulinemic at birth and key effector mechanisms of their adaptive and innate immune cells are underdeveloped (Chase et al., 2008b;Lombard et al., 2020).For example, neutrophils are "first responder" innate immune cells that traffic to an infiltrating pathogen and neutralize it using bactericidal mechanisms (i.e., the oxidative burst and phagocytosis).However, neutrophils have a reduced phagocytic ability in neonatal dairy calves (Menge et al., 1998).This is likely due to reduced expression of key receptors necessary for successful recognition and phagocytosis of bacteria, such as the fragment crystallizable (Fc) receptors, in comparison to neutrophils isolated from older animals (Zwahlen et al., 1992;Barrington and Parish, 2001;Chase et al., 2008b).Calves also have reduced antimicrobial peptide (i.e., myeloperoxidase) production (Zwahlen et al., 1992)  memory B and T cells, and lower antibody production) (Chase et al., 2008a).
The calf is reliant on passive immunity acquired from consuming colostral secretions from the dam, which include immunoglobulins and cytokines.The industry standard to bolster calf immune protection is to feed quality colostrum shortly after birth.However, failure of passive transfer (FPT), or insufficient concentrations of circulating colostrum-derived antibodies, is a prevalent issue with regard to colostrum management.Failure of passive transfer can occur as often as in 20% of calves born on dairy operations and the ramifications of this can be perceived well into adulthood (e.g., reduced milk production) (Raboisson et al., 2016).Due to the high prevalence of FPT, it is imperative that alternative interventions are implemented that bolster calf immunity.Although antibiotics are one tool for disease prevention, their use can potentially result in the emergence of antibiotic resistant strains of bacteria and are becoming progressively discouraged by veterinarians and consumers alike (Langford et al., 2003;Berge et al., 2005;Walker et al., 2012).Therefore, the development of efficacious, non-antibiotic therapies that effectively boost immune function and mitigate disease in calves is essential.
Lysophosphatidylcholine (LPC) has been scrutinized as a potential immunomodulator and corollary for disease outcomes in non-ruminants (Hong and Song, 2008).The lysophospholipid (LPC) is derived from the enzymatic activity of phospholipase A 2 (PLA 2 ) on phosphatidylcholine (PC) in circulation or by the transfer of fatty acids to cholesterol via lecithincholesterol acyltransferase (LCAT) (Law et al., 2019).Circulating concentrations of LPC are significantly lower in patients with sepsis as compared with healthy individuals (Drobnik et al., 2003), and specific types of LPC have been suggested to be potential markers for disease severity and mortality in septic patients (Drobnik et al., 2003;Park et al., 2014).In a murine model of sepsis, mice that received stearoyl-LPC (i.e., LPC-18:0) subcutaneously, either before or after cecal ligation puncture, had a dose-dependent increased rate of survivorship than mice that received only vehicle (Yan et al., 2004).Lysophosphatidylcholine was found to increase the oxidative burst response of cultured murine neutrophils in a dose-dependent manner (Yan et al., 2004).Moreover, phagocytic activity is enhanced following stearoyl-LPC treatment in murine neutrophils (Quan et al., 2016), and LPC enhances antibody production and interferon-γ secretion from isolated human peripheral blood mononuclear cells (Huang et al., 1999).These findings highlight the extensive immunomodulatory capabilities of LPC at both the systemic and cellular level.
In an exploratory manner, we have previously established that saturated LPC (e.g., LPC-18:0) enhances the oxidative burst, potentiates the ability of endotoxin to promote tumor necrosis factor-α (TNFα) and interleukin-6 secretion, and accelerates the killing of Escherichia coli in neutrophils isolated from the neonatal calf (Tate et al., In Review).These data suggested that LPC activates bovine neutrophils; however, the effects of LPC in the neonatal dairy calf had not been previously evaluated.Therefore, the objective of this study was to determine the effects of LPC on parameters of growth as well as markers of inflammation and immunity in dairy calves.To test our objective, we carried out a longitudinal study to test the effects of either subcutaneous (s.c.) administration of LPC or dietary lysolecithin enriched in LPC in Holstein calves.We hypothesized that the provision of LPC would modify measures of immune and metabolic health to influence growth in healthy calves.

Experimental design and treatments
All experimental procedures were approved by the Cornell University Institutional Animal Care and Use Committee (protocol #2018-0110).Forty-six heifer calves (4.2 ± 0.8 d of age [mean ± SD] and 38 ± 3.91 kg of BW) were acquired from Lincoln Dairy (Auburn, NY).Calves received 4 L colostrum within 2 h of birth followed by 2 additional liters 6 h later (±2 h).Due to the limited number of calves available each week, calves were acquired and enrolled in 5 blocks and completely randomized within block to study the effects of LPC.Following transport, calves were housed in individual pens bedded with sawdust in the Cornell University Block Barn (Ithaca, NY).Following 7 d of acclimation, calves were assigned to one of 4 treatment groups: a milk replacer diet unsupplemented with lecithin in the absence (CON; n = 11) or presence of s.c.administered mixed (mLPC; 69% LPC-16:0, 25% LPC-18:0, 6% other; n = 11) or pure (pLPC; 99% LPC-18:0; n = 11) LPC, or a milk replacer diet supplemented with 3% lecithin enriched in lysophospholipids containing LPC (6% total LPC) in the absence of s.c.administered LPC (LYSO; n = 10) for a 5-wk experimental period.Exclusion criteria for calf enrollment included adequate hydration as assessed by hematocrit, a serum Brix value ≥8.3 (Deelen et al., 2014), and visual confirmation of sound clinical health by assessing mobility, fecal scores, and respiration rates.Figure 1 illustrates the treatment and sampling timeline.During acclimation, calves were fed a common milk replacer (24% CP and 17% fat) at 1.75% of BW on a DM basis twice daily at 0700 and 1600 h.Feeding amounts were adjusted weekly based on the body weights measured at the start of each week.At the start of the experimental period, the provision of milk replacer unsupplemented or supplemented with lecithin was initiated (Table 1).The prepared lecithin-supplemented milk replacer was purchased directly from Milk Specialties Global Animal Nutrition (Eden Prairie, MN).During wk 2 of the experimental period, calves received a single s.c.injection of either vehicle (phosphate buffered saline [PBS] with 20 mg of bovine serum albumin [BSA]/mL; CON and LYSO) or LPC in vehicle (10 mg of total LPC/kg of BW; mLPC and pLPC) every 12 h over the course of 48 h beginning at 0700 h on d 1 and terminating at 0700 h on d 3 of the experimental period (5 total injections).The concentration of LPC given as well as the intervals at which it was administered were based on experimental methods described by Yan et al. (2004).To facilitate weaning, milk replacer intake was reduced by half beginning wk 6 and terminated by wk 7 of the experimental period.Starter grain and water were provided ad libitum for the duration of the study.

Treatment preparation and administration
L-α-lysophosphatidylcholine derived from egg yolk (cat.#: 830071; 69% LPC-16:0, 25% LPC-18:0, and 6% other) and stearoyl-LPC (>99% LPC-18:0; cat.#: 855775) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL) and Cayman Chemical Company (Ann Arbor, MI), respectively.Treatments were prepared by dissolving the necessary amount of LPC (10 mg of total LPC/kg of BW/injection) and BSA (200 mg/injection) for 5 injections (plus 5% extra) into 52.5 mL of PBS.Treatments were sonicated 3 times at 5-s intervals and then heated on a hot plate with intermittent gentle swirling until clear.Control injections containing vehicle were prepared in a similar manner with the omission of LPC.Single-dose treatments were then aliquoted into sterile 50-mL conical tubes and frozen at −20°C until use.Immediately before administration, injections were prepared by thawing aliquots in a warm water bath until clear and drawn into 12-mL syringes using 18-gauge needles.Injections were administered s.c.near the scapula.

Sample and data collection
Milk replacer and starter grain intakes were recorded daily.Samples of each milk replacer, in addition to starter grain, were collected weekly, composited by month, and stored at −20°C.Clinical assessments were performed daily at morning feedings (0700 h).During these assessments, rectal temperatures, respiration rates, and fecal scores were recorded for the duration of the study.In addition, rectal temperatures were recorded at h 0, 5, and 10, relative to each s.c.injection of either vehicle or LPC beginning at the first s.c.injection during wk 2 of the experimental period.Daily respiration rates were determined by counting flank movements for a 15-s duration, then multiplied by 4 to obtain movements per minute.Body weights were recorded weekly.
Blood samples (10 mL) were collected once weekly before morning feeding and at h 0, 5, and 10, relative to the final s.c.injection of vehicle or LPC during the experimental period (Figure 1).Blood was collected via jugular venipuncture into evacuated blood tubes containing potassium EDTA as an anticoagulant when blood was collected for plasma or whole blood collection.Plasma and serum samples were separated using centrifugation (3,400 × g for 20 min).Separated plasma or serum was samples were initially stored at −20°C and then transferred to −80°C for long-term storage within 2 wk of collection.
Serum samples were submitted to the University of Missouri Veterinary Medical Diagnostic Laboratory for  c. administered mixed (mLPC; 69% LPC-16:0, 25% LPC-18:0, 6% other; n = 11) or pure (pLPC; 99% LPC-18:0; n = 11) LPC, or a milk replacer diet supplemented with 3% lecithin enriched in lysophospholipids containing LPC in the absence of s.c.administered LPC (LYSO; n = 10) for a 5-wk experimental period.Calves were fed twice daily (0700 and 1400 h) a milk replacer containing 27% crude protein, 24% fat at 1.75% of BW per d (dry matter basis) until wk 6 of life (start of weaning).At wk 6 of life, milk replacer intake was reduced by half and terminated by wk 7. Starter (22% CP) and water were provided ad libitum throughout study.Body measurements were recorded weekly and intakes and clinical observations were recorded daily.Blood samples were collected weekly as well as 0, 5, and 10 h following the final injection.

Calculations and statistical analyses
Average daily gains were calculated by dividing the differences in weight between each week by 7 d.Feed to gain ratios (F:G) were computed by dividing ADG by overall DMI within week.Statistical analyses were carried out using the mixed model procedure of SAS (v9.4,SAS Institute Inc., Cary, NC, USA).The model included the random effects of calf, block, and baseline measurements, and the fixed effects of treatment, time (i.e., h, d, wk), and their interaction.The preweaning period is defined as wk 1 to wk 5 of the experimental period.The post-weaning period is defined as wk 6 to wk 7 of the study.A partitioned analysis of least squares means comparison within time points was utilized to analyze treatment differences by time.The covariance structures used to test fit statistics included variance components, compound symmetry, autoregressive one, and unstructured.Smaller fit values (BIC) were always selected.The model was used to evaluate body measurements, clinical parameters, and plasma or serum metabolites or proteins.Normality of the residuals were checked with normal probability and box plots and homogeneity of variances with plots of residuals versus predicted values in order ensure no violation of model assumptions.Studentized residual values >3.0 or < −3.0 were considered outliers and removed from the analysis (typically 1 per response variable).A Dunnett's multiple comparisons test was performed between treatments (i.e., mLPC, pLPC, and LYSO) and reference control (i.e., CON).Main effects and interactions were declared significant at P ≤ 0.05 and trending toward significance at 0.05 > P ≤ 0.10.Results are expressed as least squares means ± SEM, unless otherwise noted.

RESULTS
Three calves developed serious complications during the study to warrant veterinary intervention during wk 3 of the experimental period.One LYSO calf and one mLPC calf developed pneumonia.One mLPC calf developed vasculitis and skin lesions at the site of injection.These calves received veterinary treatment (i.e., flunixin meglumine); however, they were euthanized by captive bolt within 1 wk of initial diagnosis.For these calves, all available data collected were utilized in our statistical analyses.

Effects of LPC administration on rectal temperatures, respiration rates, and fecal scores
Changes in daily and hourly rectal temperatures, respiration rates, and fecal scores in response to LPC administration are shown in Table 3 and Figure 2.For changes in rectal temperatures, we observed a significant effect of treatment, treatment × day, and treatment × hour (P < 0.05, Figure 2A and 2B).Calves receiving mLPC or pLPC treatment experienced increases in mean rectal temperatures beginning on d 2 of injection, relative to CON (P < 0.05).Rectal temperatures were greater for mLPC and pLPC calves at h 5 and 10 from d 2 to d 4 following the initial LPC injection, relative to CON (P < 0.001).The rectal temperatures of mLPC and pLPC calves remained elevated until d 4 following the initial s.c.injection, relative to CON (P < 0.001).Over the duration of the pre-weaning period, rectal temperatures were highest for mLPC calves (treatment × week, P < 0.01).Calves provided the LYSO treatment did not experience a change in rectal temperatures in the short term or long-term following the initiation of treatment, relative to CON.We also did not observe an effect of treatment for respiration rates or fecal scores.

Effects of LPC administration on dry matter intake and growth
Changes in DMI and growth parameters in response to LPC administration are provided in Table 4.During the pre-weaning period (wk 1 to 5 of the study), no significant effects of treatment were observed for DMI, BW, ADG, heart girth, mid girth, flank girth, hip height, hip width, body length, or feed: gain.During the post-weaning period (wk 6 to 7 of the study), mLPC calves had significantly lower BW (P < 0.05) and ADG (P < 0.001), relative to CON calves.We also observed a significant treatment × week effect for post-weaning and overall heart girth.Specifically, mLPC calves had significantly lower heart girth measurements at wk 8, relative to CON calves (P < 0.05).Additionally, mLPC calves had significantly lower overall DMI, relative to CON calves (P < 0.05).No significant differences in pre-weaning, post-weaning, or overall BW, ADG, heart girth, mid girth, flank girth, hip height, hip width, body length, DMI, and feed: gain were detected for pLPC and LYSO calves, relative to CON calves.

Effects of LPC administration on plasma glucose, total fatty acid, and insulin concentrations
Changes in plasma glucose, total fatty acids, and insulin concentrations in response to LPC administration are provided in Table 5. Treatment did not modify plasma glucose, total fatty acid, or insulin concentrations.There was a tendency for mLPC calves to have lower plasma glucose concentrations during the postweaning period, relative to CON calves (P = 0.08).

Effects of LPC administration on circulating markers of immune and liver health
Changes in circulating concentrations of immune and liver health markers in response to LPC administration are provided in Table 6.We observed significant effects of treatment, hour, and treatment × hour for albumin and total protein concentrations (P < 0.01).For all calves, serum albumin, total protein, globulin, total bilirubin, AST, GGT, and cortisol concentrations decreased at 10 h post final injection, relative to 0 h (P < 0.01).In addition, at h 10 serum concentrations of AST significantly decreased from 0 h values (P < 0.001) while GLDH tended to decrease with time (P = 0.082).Serum albumin and total protein concentrations were significantly reduced in mLPC and pLPC calves, relative to CON calves (P < 0.001).Serum globulin concentrations tended to be modified by treatment (P = 0.06).Specifically, serum globulin concentrations were lowest for calves that received the mLPC treatment.Relative to CON calves, at 0 h, mLPC calves had significantly higher serum concentrations of cortisol (P < 0.05).Aspartate transaminase concentrations were significantly greater in mLPC (P < 0.001) and pLPC (P < 0.05) calves, relative to CON calves.Calves receiving mLPC tended to have higher serum concentrations of total (P = 0.076) and direct (P = 0.052) bilirubin at 10 h, relative to CON calves.Serum GLDH was significantly higher in mLPC calves at h 0 and 10, relative to CON calves (P < 0.05).No significant differences were detected between LYSO and CON calves in concentrations of serum markers of liver health during the experimental period.Plasma concentrations of TNFα and serum IgG were not found to be modified by LPC administration.A significant effect of treatment was observed for plasma SAA concentrations (P < 0.05; Figure 3).Specifically, mLPC and pLPC calves had greater circulating SAA concentrations, relative to CON (P < 0.05).Plasma SAA concentrations were not different between LYSO and CON.

Effects of LPC on calf white blood cell profiles
Changes in acute and long-term white blood cell profiles in response to LPC administration are displayed in Table 7. Significant time effects were observed for segmented neutrophils, monocytes, eosinophils and basophils: the absolute counts of segmented neutrophils significantly decreased (P < 0.001), while absolute counts of monocytes, eosinophils, and basophils significantly increased over time (P < 0.05).Relative percentages of segmented neutrophils significantly decreased over time (P < 0.001), while relative percentages of lymphocytes, eosinophils, and basophils significantly increased over time (P < 0.05).Treatment tended to modify WBC (P = 0.073).Specifically, WBC were greatest in calves provided mLPC and lowest in CON calves.Additionally, while relative percentage of segmented neutrophils was not significantly higher in this group, absolute numbers of segmented neutrophils were significantly higher in mLPC calves than CON calves (P < 0.05).No significant treatment or treatment × week effects were detected in segmented neutrophil, monocyte, lymphocyte, eosinophil or basophil profiles at any time point among treatment groups.

DISCUSSION
This longitudinal study sought to examine the effects of acute s.c. and long-term dietary LPC administration It should be considered that the differential immunological responses to antigenic compounds encountered within the gut mucosa versus those introduced and encountered parenterally may explain the discrepancy between calves fed LPC and those provided s.c.LPC.The gastrointestinal immune system develops tolerance to dietary antigens to blunt potential inflammatory responses that could disrupt commensal microorganisms (Yokanovich et al., 2021), a phenomenon known as oral tolerance.This is driven in part by gut CD103 + dendritic cells found within the gastrointestinal tract that capture antigens and present them to naïve T cells, causing them to differentiate into T regulatory cells that are more tolerogenic and anti-inflammatory, thus preventing aberrant inflammatory responses to these antigens when encountered again (Tordesillas and Berin, 2018).This response is distinct from the largely inflammatory immune cascades that are induced and carried out by innate immune cells when an antigen is introduced directly into circulation (Marshall et al., 2018).Indeed, one study found that antigen-specific proliferation of transgenic T cells was induced more efficiently in the spleens of mice injected with low doses of antigen, but that oral administration of a 1,000-fold higher antigen dose failed to induce de novo T cell proliferation of antigen-specific T cells in peripheral lymphoid organs (Worbs et al., 2006).It is possible that the dampened physiological responses to the ingested LPC we observed may be due to induced oral tolerance as opposed to modifications in its structure during digestion.
Plasma concentrations of total and individual species of LPC (LPC-16:0, −18:0, and −18:1) were found to be significantly elevated in the hours following LPC administration in both mLPC calves and LYSO calves.The focus on circulating LPC composition has merit because the acyl chain length and saturation of LPC has been shown to modulate its effector mechanisms.For example, Yan et al. (2004) documented that s.c.administered LPC-18:0 improved survival in mice with experimentally-induced sepsis while LPC-16:0 and LPC-18:1 did not have this effect.It has also been documented that circulating concentrations of LPC-16:0, −18:0, −18:1, and −18:2 are all reduced in human septic patients (Drobnik et al., 2003).We have also demonstrated reductions in plasma LPC-18:0, −16:0, and −18:1 concentrations in dairy cattle following lipopolysaccharide exposure (Javaid et al., 2022).Therefore, we considered that marked increases in total circulating LPC in mLPC and LYSO calves may potentially protect against an immunogenic challenge, though additional experiments elucidating the effects of LPC administration within the context of a pathogenic challenge would be necessary to draw such conclusions.The pre-weaning period is defined as wk 1 to wk 5 of the experimental period.The post-weaning period is defined as wk 6 to wk 7 of the study.
Calves receiving mixed-LPC showed a spike in plasma LPC concentrations at 5 h post the final s.c.injection, followed by a decline at 10 h.These data may indicate the clearance of circulating LPC by uptake or degradation via the action of enzymes such as LPC acyltransferase (LPCAT) and PLA 2 over time (Law et al., 2019).Conversely, although pLPC calves received s.c.injection of pure stearoyl-LPC at the same times concentrations as their mLPC counterparts, LPC species were not found to be significantly elevated within these animals.In circulation, LPC is short-lived, being rapidly converted into different metabolites within lipoproteins or enzymatically modified (Law et al., 2019).It has been previously suggested that the catalytic efficiency of LPC may be determined by its acyl chain's binding affinity (Liu et al., 2008), therefore perhaps the mixed-LPC product's predominately LPC-16:0 composition coupled with the incorporation of unsaturated LPC may influence the action of LPC-degrading enzymes such as PLA 2 and LPCAT, which have been demonstrated to show preferential activity for different LPC based on their acyl chain position, saturation, and carbon length (Kazachkov et al., 2008;Mouchlis et al., 2019).It is possible that the purified LPC-18:0 that was administered to the pLPC calves was more quickly degraded relative to the egg yolk-derived mLPC administered to mLPC calves, resulting in plasma LPC concentrations returning to baseline levels before or circa the 5 h sampling time point.In the case of the lecithin supplemented calves (i.e., LYSO), their continuous consumption of the lysolipid-enriched milk replacer over the course of the experimental period likely contributed to their consistently elevated LPC concentrations in circulation, especially at the post-prandial 10 h time point.
We discovered that acute s.c.administration of mLPC or pLPC induced a febrile response and modified physiological parameters of liver, immune, and metabolic health in calves.Liver markers of health and functionality were significantly modified as a result of s.c.LPC.The significant decrease in serum albumin and total protein concentrations is indicative of an acute inflammatory response.Albumin metabolism has been demonstrated to become attenuated due to decreased hepatic protein synthesis (Liao et al., 1986).Previous studies have shown a similar drop in albumin during instances of systemic inflammation (Jacobsen et al., 2004;Joshi et al., 2018).Interestingly, while both mLPC and pLPC calves displayed elevated concentrations of AST relative to CON calves, these concentrations were higher in mLPC calves than in pLPC calves.Additionally, mLPC calves had significantly lower concentrations of serum globulin at h 10 post final s.c.injection and significantly higher concentrations of cor-
tisol at h 0, relative to CON calves.These findings were not observed for pLPC calves.These findings suggest that s.c.injections of mLPC potentially induced more severe liver injury than pLPC.This is underpinned by the finding that mLPC calves, but not pLPC calves, had significantly higher concentrations of GLDH at 0 and 10 h, relative to the final injection.Calves that received mLPC also tended to have higher concentrations of total and direct bilirubin at h 10.Because the liver is an important site of LPC metabolism, changes in its functional integrity could have profound impact on the production and catabolism of other lipid species and intermediates.It could be considered that the observed changes in markers of liver health could be a result of enhanced LPC clearance from circulation or modulation of lipoprotein metabolism to mitigate the significant increases in circulating LPC, creating an increased physiological demand on the liver.Measurements of the expression of key liver enzymes involved in LPC and lipoprotein metabolism such as LPCAT or hepatic lipases are needed to determine whether this is indeed the case.Labeling of the administered LPC and quantifying its levels within the liver could also determine whether the LPC is indeed localizing here and carrying out its effector mechanisms.
Significant increases in rectal temperatures observed in mLPC and pLPC calves during the treatment period were accompanied by significant increases in plasma SAA concentrations.Serum amyloid A is a potent marker for inflammation and an increase in its circulating concentrations suggests a systemic inflammatory response to s.c.LPC.Interestingly, this response was observed in the absence of changes in TNFα concentrations.Typically, inflammatory cytokines such as TNFα stimulate the production of SAA, and, consequently, SAA and TNFα concentrations are observed to increase in tandem during instances of systemic inflammation and disease (Lassen et al., 2015).Our findings suggest that, following s.c.LPC administration, SAA production and secretion is potentially upregulated independent of mechanisms that would also stimulate TNFα production.Indeed, there are many other cytokines that can induce an acute phase response in the liver independent of TNFα (i.e., IL-1β) (Ehlting et al., 2021).However, it could also be speculated that the frequency of our sampling time points was inadequate to capture transient changes in TNFα that may have occurred in response to LPC treatment.Alternatively, the lack of a TNFα response may also be in part explained by the physiological effect of continuously     elevated body temperatures on TNFα production.It has been previously reported that recurrent exposure to febrile temperatures (40°C) can reduce TNFα production in macrophages (Ensor et al., 1995).In fact, this phenomenon has been demonstrated across multiple studies and has been attributed to reduced stability of TNFα mRNA and premature deactivation of TNFα transcription (Fouqueray et al., 1992;Snyder et al., 1992;Ensor et al., 1994).
The observed changes in markers of inflammation and liver function in our mLPC and pLPC calves may be attributed to the effector mechanisms of activated immune cells in the liver.These changes could be indicative of a concerted inflammatory response in the liver in response to LPC treatment, during which the organ switches protein synthesis toward the production of acute phase proteins (Ehlting et al., 2021).Additionally, liver damage can be induced by neutrophil migration to, and activation within, the liver during instances of inflammation (Gujral et al., 2004).Prior studies have also established that LPC can enhance neutrophil bactericidal mechanisms such as reactive oxygen species (ROS) production and E. coli killing (Yan et al., 2004;Hong et al., 2010).It has been shown that phosphatidylcholine and LPC can be recognized by pattern recognition receptors of the innate immune system such as TLR2 (Liu et al., 2020).Therefore, LPC interactions with these receptors can potentially amplify an innate immune response by activating pattern recognition receptors on innate immune cells.While this can be beneficial for the swift resolution of an infection, rampant inflammation can damage surrounding cells and tissue.Therefore, s.c.LPC could have potentiated neutrophil responses and, in turn, induced tissue damage in the liver as a result.Due to an observed acute febrile response in pLPC calves in the absence of increased plasma LPC, it is likely that the LPC administered was rapidly cleared from circulation and that the transient increase in LPC in this treatment group may have primed neutrophil activity to a lesser extent, thus explaining why changes in liver markers were still observed in these animals, though to a slightly lesser degree than seen in mLPC calves.Interestingly, elevated plasma LPC in LYSO calves did not correlate with increased SAA and liver markers, suggesting that dietary consumption may attenuate LPC's effects on immune cells and, consequently, its impact on liver health in these animals.Further experimentation determining the role of WBC on parameters of liver function during the acute phase response in dairy calves and how s.c. and dietary LPC may differentially modulate this activity is also needed.
Significant reductions in parameters of growth (e.g., ADG) were observed post-weaning in calves given mLPC.These changes could be attributed to their concurrent reduction in DMI.Due to the fact that the most significant differences in growth were detected in the post-weaning period, it is unlikely that these changes are directly due to treatment-related changes in circulating LPC but more so due to the long-term effects that treatment had on immune or metabolic function.While circulating insulin and total fatty acid concentrations were not found to be significantly affected by LPC treatment, plasma glucose in mLPC calves did show a tendency to be lower than the other treatment groups during the post-weaning period.This outcome is likely attributed to lower DMI in these mLPC calves.Although lower glucose supply could limit growth, we were unable to evaluate glucose partitioning in the present study.In mLPC calves, WBC counts were significantly higher during the post-weaning period relative to CON calves, with a higher relative percentage of segmented neutrophils.It is possible that LPC could be causing increases in white blood cell populations and/ or activity.Prior work in our lab has demonstrated that saturated LPC-16:0 and LPC-18:0 is able to enhance the bactericidal mechanisms (i.e., production of the ROS hydrogen peroxide) in neutrophils harvested from dairy calf and stimulated ex vivo (Tate et al., In Review).Immune cells are known consumers of glucose, especially in instances of immune activation where they upregulate their glucose uptake to accommodate increased intracellular metabolic activity (Wolowczuk et al., 2008).Increased numbers of WBCs create an increased requirement for glucose, directing these stores away from growth (Maratou et al., 2007;Schuster et al., 2007).Indeed, increased immune activation during systemic inflammation or disease negatively impacts growth in animals (Hiss and Sauerwein, 2003;Sauerwein et al., 2013).
While this study was novel in scope in terms of determining the immunomodulatory effects of LPC within the dairy calf, certain limitations of the study are present.First, due to the gap in knowledge regarding LPC as an exogenously supplied immunomodulator in dairy calves, optimal concentrations for treatment could not be determined.Therefore, LPC treatment doses had to be extrapolated from murine studies (Yan et al., 2004), creating a potential source of uncertainty regarding whether the chosen dosages were sufficient, inadequate, or in excess to achieve a beneficial immune response without compromised growth.We also recognize that a further limitation of our study was that we did not test an unsaturated pure form of s.c.LPC to determine whether the treatment effects we observed were modulated by the degree of saturation of the LPC molecule.Alternative forms of LPC confer differential biological effects; for example, LPC-DHA has documented antiinflammatory properties.Lastly, we were unable to test the effects of LPC in the presence of an immune challenge.Our own work has shown that LPC administered in tandem with a pathogenic stimulus such as LPS potentiates antimicrobial responses in bovine neutrophils (i.e., proinflammatory cytokine production; Tate et al., In Review).Future studies characterizing the relationship between LPC status, immune function, and pathogen exposure are needed.

CONCLUSION
Our experimental findings reveal a complex story highlighting the effects of LPC on the febrile and acute phase responses, immune cell profile, measures of liver health, and growth in Holstein heifer calves.Five serial s.c.injections of LPC every 12 h of the course of 3 d were found to induce an acute febrile response and marked increases in measures of liver inflammation.In the case of mLPC calves, acute LPC therapy also had long-term effects, demonstrated by significant reductions in post-weaning BW and ADG as well as overall DMI.Changes in growth, inflammation, and immune and liver health were found to not be modified by LYSO treatment.Experiments further optimizing and refining the conditions under which LPC can be administered to dairy calves to confer continuous immune protection and improve immune responses while negating any adverse outcomes that may result from an overactivated and unregulated immune response are needed.
quantification of liver health markers (i.e., albumin, globulin, total protein, total bilirubin, direct bilirubin, aspartate aminotransferase [AST], glutamate dehydrogenase [GLDH], gamma-glutamyl transferase [GGT]) as well as cortisol.Serum samples were also submitted to the Cornell University Animal Health Diagnostic Center (Ithaca, NY) for IgG quantification via radial immunodiffusion kits.Plasma samples were submitted to the Johns Hopkins University School of Medicine for quantification of LPC using liquid chromatography and tandem mass spectrometry (LC-MS/MS).Whole blood samples collected into potassium EDTA tubes were immediately transported on ice to the Cornell University Animal Health Diagnostic Center for white blood cell count and profile analyses via automated hemogram.
Figure 2. Effects of s.c.vehicle (CON & LYSO) or LPC (mLPC and pLPC) on mean (A & B) and maximum rectal temperatures (C) taken over the course of the 3 d experimental period as well as 24 h post-final injection.Calves were randomly assigned to 1 of 4 treatments: a milk replacer diet unsupplemented with lecithin in the absence (CON; n = 11) or presence of s.c.administered mixed (mLPC; 69% LPC-16:0, 25% LPC-18:0, 6% other; n = 11) or pure (pLPC; 99% LPC-18:0; n = 11) LPC, or a milk replacer diet supplemented with 3% lecithin enriched in lysophospholipids containing LPC in the absence of s.c.administered LPC (LYSO; n = 10) for a 5-wk experimental period.Rectal temperatures were taken immediately before (0700 h) and 5 h after (1200 h) the first injection and immediately before the second injection (1700 h) of LPC or vehicle every day during the experimental period (d 1 to 3) and 24 h after the final injection (d 4 of wk 2).Data are presented as LSM ± maximum SEM *, P < 0.05; **, P < 0.01; ***, P < 0.001, relative to CON.

2
Blood was collected immediately prior (0 h) and 10 h following the final of 5 serial s.c.injections of either LPC or vehicle.

Table 1 . Nutrient composition (% of DM unless otherwise noted) of experimental milk replacer and starter (mean ± SD) fed to calves 1
1During the experimental period, calves were fed twice daily (0700 h and 1700 h) either an unsupplemented control milk replacer or a milk replacer containing 3% lysolecithin enriched in lysophospholipids including LPC at 1.75% of BW per d on a dry matter basis until wk 6 of life (start of weaning).At wk 6 of life, milk replacer intake was reduced by half and terminated by wk 7. Starter and water were provided ad libitum throughout study.CP = crude protein, NDF = neutral detergent fiber, ADF = acid detergent fiber, TDN = total digestible nutrients, EE = ether extract.
Tate et al.: LYSOPHOSPHATIDYLCHOLINE AND CALF HEALTH

Table 2 .
Tate et al.: LYSOPHOSPHATIDYLCHOLINE AND CALF HEALTH Changes in plasma lysophosphatidylcholine concentrations relative to the final injection of s.c.vehicle or LPC in pre-weaned Holstein heifer calves parameters of growth, metabolism, and immunity in clinically healthy dairy calves.Our findings highlight the differential effects of dietary and s.c.LPC on physiological parameters in neonatal dairy calves.It is important to note the difference in how our treatments delivered LPC into bovine circulation.Whereas s.c.LPC with albumin directly increased the presence of LPC in circulation, the consumption of dietary LPC might have triggered its metabolism in the enterocyte or incorporation with chylomicrons (Komoda et al., 2009).It is not entirely clear how LPC bound to albumin or incorporated into chylomicrons unique modify immune function and animal performance; however, our data may suggest that the digestive and postabsorptive modifications of LPC damped the bioactive properties of LPC. on

Table 4 .
Changes in plasma body measurements and dry matter intake in pre-weaned Holstein heifer calves receiving injections of s.c.vehicle or LPC

Table 5 .
Changes in plasma glucose, total fatty acid, and insulin in pre-weaned Holstein heifer calves receiving injections of s.c.vehicle or LPC

Table 6 .
Tate et al.: LYSOPHOSPHATIDYLCHOLINE AND CALF HEALTH Changes in concentrations of serum markers of liver health relative to the final injection of s.c.vehicle or LPC in pre-weaned Holstein heifer calves with 3% lecithin enriched in lysophospholipids containing LPC in the absence of s.c.administered LPC (LYSO; n = 10) for a 5-wk experimental period.Data are presented as LSM ± SEM *, P < 0.05; **, P < 0.01; ***, P < 0.001, relative to CON.

Table 7 .
Changes in the white blood cell profiles of pre-weaned Holstein heifer calves receiving injections of s.c.vehicle or LPC