Supplementation with N -carbamoylglutamate during the transition period improves the function of neutrophils and reduces inflammation and oxidative stress in dairy cows

The aim of this study was to investigate the effects of N -carbamoylglutamate (NCG) supplementation during the transition period on the functions of blood polymorphonuclear neutrophils (PMN), inflammation, and oxidative stress in dairy cows. Thirty multiparous Chinese Holstein dairy cows at wk 4 before parturition were blocked into 2 groups by parity, body weight, and milk yield of previous lactation, and randomly allocated to 2 dietary treatments of basal diet supplemented without (control, n = 15) or with 20 g/d per cow of NCG (NCG, n = 15). The supplementation was carried out from d −21 to 21 relative to calving. Health incidents (mastitis, retained placenta, and lameness) were recorded, and blood samples were collected at d −21, −7, 0 (the calving date), 7, and 21 relative to parturition and analyzed for variables related to inflammation and oxidative stress. In addition, whole blood was collected at d 7 to isolate PMN and used for analysis of the expression of functional genes and from d −21 to 21 for determination of weekly hematological parameters. The number of lymphocytes was greater at d 7 in the blood of NCG cows. The plasma level of malondialdehyde was lower in the NCG group, and blood reactive oxygen species were lower at d 7, whereas total antioxidant capacity tended to be greater in the NCG group and glutathione peroxidase tended to be higher at d 21 in cows fed NCG, suggesting that NCG supplementation improved antioxidation in cows. In addition, the concentration of serum amyloid A was lower in NCG-fed animals during the postpartum stage. Blood concentrations of IL6 and tumor necrosis factor-α were lower and tended to be lower in NCG-fed animals at d 7, respectively. Meanwhile, the concentrations of IL6 tended to be lower in NCG-fed animals at d 21. Furthermore, the expression of S100A9 and MMP9 in the PMN was lower and tended to be lower, respectively, whereas the expression of ITGB2 , XBP1 tended to be higher and expression of CLEC6A was higher in NCG-fed cows. Overall, our results indicated that supplementation with NCG during the transition period showed the beneficial effects on animal health, by improving PMN functions and alleviating inflammation status and oxidative stress in dairy cows.


INTRODUCTION
The transition period is a critical stage in dairy cows, during which the risk of diseases increases (Ingvartsen et al., 2003).It has been reported that 30 to 50% of dairy cows experience health disorders, and approximately 75% of infectious diseases and metabolic disorders in dairy cows occur during this stage (Ingvartsen, 2006;LeBlanc et al., 2006).This is partly attributed to the negative energy balance that leads to excessive inclusion of fatty acids resulting from adipose mobilization in the leukocyte membrane, which is known to play a role in impairing immune functions and dysfunctional inflammation in transition period (Sordillo and Raphael, 2013).Thus, the impaired functions of PMN were considered to be one of the risk factors contributing to increased susceptibility to disease of transition dairy cows (Meglia et al., 2005).The PMN are the first line of defense against invading pathogens, especially those causing inflammation such as mastitis (Paape et al., 2003).Additionally, inflammatory responses to metabolic disorders are among the key causes of oxidative stress during the transition stage in dairy cows (Hayajneh, 2014).Transition cows undergo substantial metabolic and physiological changes as they shift from nonlactating to lactating, which often leads to the progressive development of oxidative stress (Bernabucci et al., 2005;Castillo et al., 2005).Increasing evidence has shown that high oxidative stress impairs milk production (Li et al., 2016) and plays an important Supplementation with N-carbamoylglutamate during the transition period improves the function of neutrophils and reduces inflammation and oxidative stress in dairy cows F. F. Gu, 1 L. Y. Jiang, 1 D. M. Wang, 1 F. Q. Zhao, 2 and J. X.Liu* role in several pathological conditions (Lykkesfeldt and Svendsen, 2007;Sordillo and Aitken, 2009).Therefore, transition cows are characterized by high oxidative stress and inflammation, which may be alleviated by improving PMN functions (Bionaz et al., 2007;Trevisi et al., 2012).
Multiple nutritional approaches have been employed to improve the health status of transition cows via modification of PMN functions.Improving the absorption of microminerals such as copper, zinc, and manganese has also been shown to be beneficial to PMN, facilitating better functions (Dietz et al., 2017).Supplementing Met led to positive effects on phagocytosis of blood PMN (Osorio et al., 2013;Li et al., 2016).Therefore, nutritional strategies are effective ways to improve the functions of PMN.Arginine is another essential and functional AA for dairy cows (NRC, 2001) and plays an important role in immune function (Wu and Meininger, 2002;Ding et al., 2019).Savoini et al. (1984) found that defensins in bovine PMN are rich in cysteine and Arg (38-42 residues) and display bactericidal activity against many gram-positive, gram-negative, and anaerobic bacteria as well as fungi and viruses.Recently, Garcia et al. (2016) showed that Arg has the highest utilization by PMN in early lactating dairy cows.These findings in cows were consistent with those in human PMN (Li et al., 2007;Wijnands et al., 2015).
N-Carbamoylglutamate (NCG) is a structural analog of N-acetylglutamate and is an Arg enhancer in ruminants (Chacher et al., 2014).Supplementation with NCG resulted in greater milk production and higher blood Arg concentrations in early lactating cows (Gu et al., 2021) and in high-yield mid-lactation cows (Chacher et al., 2014) indicating a beneficial role of NCG in improving animal performance.However, little information is available on whether and how NCG regulates animal health such as alleviating inflammation and oxidative stress in dairy cows, especially during the transition period.Therefore, the objective of the present study was to investigate the effects of NCG on PMN functions, inflammation, and oxidative stress by analyzing the expression profile of functional genes in PMN and biomarkers of inflammation and oxidative stress in perinatal dairy cows fed with or without NCG.

Animals and Experimental Design
The experimental procedures used in this study were approved by the Animal Care Committee of Zhejiang University (Hangzhou, China) and were conducted in accordance with the university's guidelines for animal research.
The animal experimental design was described in our companion study (Gu et al., 2021).In brief, 30 multiparous Chinese Holstein dairy cows at d 28 before parturition with similar BW (657 kg, SD = 58) were assigned to 15 blocks according to parity and 305-d milk yield in previous lactation (8,692 kg, SD = 607).The milk yield of these dairy cows was selected as the target parameter for a power test, and the calculated power was 0.9184 when n = 15 at each group.The first 7 d were the adaptation period.The cows were then randomly allocated into 2 groups that were fed a basal diet without (CON group) or with 20 g/d NCG (Beijing Animore Sci.& Tech.Co. Ltd.; NCG group).The average day of prepartum period was 19 d (SD = 1.15) and 18.8 d (SD = 2.63) in the CON and NCG group, respectively.The experiment ended at d 21 after calving.Two cows were removed due to severe clinical mastitis in the CON group, and 2 cows were removed from NCG group: a cow that suffered from severe mastitis and a cow that delivered more than 7 d earlier than expected calving day.Throughout the whole trial period, cows were housed in a barn with individual tie-stalls and had free access to water.The TMR was offered at 0600, 1400, and 1900 h during the day.The pure NCG was added once per day at 1400 h by scattering it on the top of TMR and then mixed with the part of TMR for individual cows.Feeds were offered ad libitum to yield approximately 5% orts.Collection and analysis of TMR samples were performed as described previously (Gu et al., 2021).The ingredients of the diets are shown in Supplemental Table S1 (https: / / doi .org/ 10 .6084/m9 .figshare.19358747.v1).

Disease Incidence and Blood Sampling
During the postpartum stage, the incidence of some diseases, including mastitis, retained placenta, and lameness, was recorded according to the diagnosis by veterinarians.Mastitis was identified based on the somatic cell number (>200,000/mL) in milk.The criteria for determining the retained placenta were to see whether the placenta was shed naturally within 12 h after delivery.Lameness included abscess, pododermatitis circumscripta (ulcer), interdigital skin hyperplasia (corns), interdigital phlegmon (foot rot), digital dermatitis, and white line diseases.The incidence of several diseases that often occur during the transition period was shown in Supplemental Table S2 (https: / / doi .org/ 10 .6084/ m9 .figshare .19358747 .v1).
Blood samples were collected into tubes containing an anticoagulant (heparin lithium) from the coccygeal vein of cows at d −21, −7, 0 (the calving date), 7, and 21 relative to the calving date.The samples were centrifuged at 3,000 × g for 15 min at 4°C to collect plasma and then frozen at −20°C until subsequent analysis.The reactive oxygen species (ROS) was detected immediately after plasma collection.In addition, whole blood was collected weekly into 2-mL EDTA evacuated tubes from d −21 to 21 relative to calving at 3 h after the morning feeding.Subsequently, the blood samples were sent to an animal hospital (Nargis Animal Hospital, Hangzhou, China) within 2 h relative to the collected time for analysis of blood variables, including the number of total white blood cells and the numbers and percentages of lymphocytes, monocytes, and PMN, using an automatic blood cell analyzer (B2600, Mandray).The plasma levels of superoxide dismutase, glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and total antioxidant capacity (T-AOC) were analyzed using an Auto Analyzer 7020 instrument (Hitachi High-Technologies Corporation) with commercial colorimetric kits (Ningbo Medical System Biotechnology Co. Ltd.).The plasma concentrations of glutathione peroxidase, IL-6, IL-10, IL-4, tumor necrosis factor-α (TNF-α), amyloid A (SAA), and haptoglobin were analyzed using commercially available ELISA kits specific for the bovine species (Jiangsu Enzyme Biotechnology Co. Ltd.).

Isolation of PMN and Gene Expression Analysis
The PMN were isolated from whole blood samples using a method modified from Zhou et al. (2015).In brief, blood samples (~20 mL) were collected at approximately 0700 h on d 7 postpartum from the coccygeal vein into two 10-mL evacuated tubes containing acid citrate dextrose.The ratio of blood to acid citrate dextrose was 4:1.After blood collection, the tubes were placed on ice until isolation within 30 min.The collected samples were slowly poured into a 15-mL centrifuge tube (6-7 mL per tube) to collect PMN cells.An equal volume of PBS was added to each tube and then centrifuged at 792 × g for 15 min at 4°C.The plasma, buffy coat, and approximately one-third of the red cells were removed, and 5 mL of lysate was added to completely lyse the remaining red blood cells.Then, 5 mL of osmolarity recovery solution was added and kept stable for 3 min followed by centrifugation at 397 × g for 10 min at 4°C.Subsequently, the precipitate was added to 5 mL of PBS and centrifuged at 309 × g for 5 min at 4°C.After washing with PBS, PMN were collected with 1 mL of TRIzol and stored at −80°C until further analysis.Last, 13 PMN samples were collected in each group.
Total RNA was extracted from the PMN with TRIzol reagent according to the manufacturer's procedures (catalog no.RN03, Aidlab Inc.).The concentration and purity of the total RNA were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.).The purity was calculated by absorbance ratio of 260/280 measured and the cutoff was 1.80.Finally, and the sample purity averaged 1.85 (SD = 0.01).In addition, the Agilent 2100 Bioanalyzer and RNA 6000 Nano LabChip kit (Agilent Technologies) was applied to detected the RNA integrity.Samples had an average RNA integrity number of 7.98 (SD = 0.34), and the average concentration of RNA was 132 ng/µL (SD = 16.5).Total RNA of PMN was reverse transcribed to cDNA using the PrimeScript 1st Strand cDNA Synthesis Kit (catalog no.FSQ-101, Toyobo).Quantitative real-time PCR was performed using the 2 × SYBR Premix Ex Taq kit (code no.PC5902, Aidlab Inc.) and the Applied Biosystems 7500 Analyzer.The PCR conditions were set as follows: 1 cycle at 95°C for 2 min, 40 cycles of 95°C for 15 s, and 60°C for 34 s, followed by a melting curve program (60 to 95°C).

Statistical Analysis
Kolmogorov-Smirnov test was used to evaluate the normal distribution, and Levene test was used to evaluate the homogeneity of variance using SPSS software (version 20.0,IBM Corp.); the outliers were evaluated by Data Processing System (version 15.1) with Grubbs method.The animal disease incidence was analyzed with the chi-squared test using SPSS (version 20.0).
Other data were analyzed using SAS software (version 9.0, SAS Institute Inc.) with the Spatial Power for repeated measures with mixed models.For the analysis of blood cells, plasma inflammation, and oxidative stress parameters, the data at −21 d (before NCG addition) relative to parturition were included as a covariate and were analyzed by considering day, treatment, and the interaction of treatment × day as fixed effects and block and cow as random effects.The effect of day was included as a repeated measure.Cows within diets were subjected to repeated measurements.The mRNA expression of PMN was analyzed by considering treatment as a fixed effect and block and cow as random effects.The means were separated using the PDIFF option in the LSMEANS treatment.The experimental results were reported as least squares means.Significance was declared at P ≤ 0.05, and 0.05 < P ≤ 0.10 was considered a trend.

Hematological Traits and Disease Incidence
The hematological data are shown in Table 1.Compared with the CON group, the mastitis incidence tended to be lower in the NCG group (P = 0.06).The quantity of lymphocytes in the blood (P = 0.04) was higher in the NCG-fed cows than in the control cows throughout the experiment.A significant interaction effect was observed (P = 0.04) between treatment and days in the number of monocytes, with a higher number of blood monocytes in NCG cows than in CON cows at d 7 (P < 0.05).
The incidence of several diseases that often occur during the transition period is shown in Supplemental Table S2 (https: / / doi .org/ 10 .6084/m9 .figshare.19358747.v1).Compared with the CON group, the mastitis incidence tended to be lower in the NCG group (P = 0.06), but no significant difference was observed in retained placenta and lameness between 2 groups.

Inflammation and Oxidative Stress
The results for the inflammatory response and oxidative stress are presented in Table 2.No treatment effects were found for inflammatory variables, but significant interactions between treatment and days were observed for SAA (P = 0.03), and the tendency were observed for TNF-α (P = 0.08) and IL6 (P = 0.07).The blood SAA concentration was lower in cows fed NCG than in control cows at d 0 (P < 0.05) and d 21 (P < 0.05) and tended to be lower at d 7 (P = 0.07; Figure 1).The concentrations of IL6 decreased at d 7 (P < 0.05) and tended to decrease at d 21 (P = 0.06) with NCG addition, whereas the concentration of TNF-α tended to be lower at d 7 in the NCG group (P = 0.07).
The plasma concentrations of MDA was lower (P < 0.01) in the NCG group, and the T-AOC (P = 0.08) tended to be higher in the NCG cows.No treatment effect but significant interactions between treatment and days were observed for ROS (P = 0.04), and the tendency was observed for GSH-Px (P = 0.06; Table 2).Compared with the CON cows, the ROS content was lower at d 7 in the NCG cows (P < 0.05) and the concentrations of GSH-Px tended to be higher in NCG-fed cows than in CON cows at d 21 (P = 0.10; Figure 1).

Gene Expression of PMN
The expression of the functional genes in PMN is presented in Table 3.The expression of genes encoding Arg and glucose transporters (SLC6A14, SLC7A1, SLC7A6, SLC7A7, SLC7A9, and SLC2A1) and genes involved in cell apoptosis and survival (FAS, CASP9, CASP2, and BAK1) was not affected by NCG supplementation.The mRNA levels of S100A9 (P = 0.05) and MMP9 (P = 0.10) tended to be lower in NCG cows than in control cows.In contrast, the expression of ITGB2 (P = 0.08) and XBPP1 (P = 0.06) tended to be higher and CLEC6A (P = 0.03) was higher in NCG cows.The sample size was 15, 15, 15, and 13 at d −7, 0, 7, and 21 in the CON group, respectively. 2 The sample size was 14, 14, 13, and 13 at d −7, 0, 7, and 21 in the NCG group, respectively.

DISCUSSION
The postpartum dairy cattle often suffer from oxidative stress and inflammation, resulting in multiple diseases (Bionaz et al., 2007;Trevisi et al., 2012).Additionally, the impaired PMN functions and a reduced lymphocyte proliferation rate contribute to the increased susceptibility to disease in transition dairy cows (Meglia et al., 2005).In our previous study, the plasma Arg and nitric oxide (NO) levels were greater in animals fed NCG, contributing to improved lactation performance during the transition period (Gu et al., 2021).However, it is still unclear whether NCG can improve health status by alleviating the inflammation and oxidative stress of transition cows.The present study provided evidence that NCG supplementation plays a role in alleviating postpartum inflammation and oxidative stress and has the potential to enhance PMN functions and increase lymphocyte numbers during the transition stage.
The perinatal inflammatory response is characterized by increased production of positive acute phase proteins, including haptoglobin and SAA (Bertoni et al., 2008).Although no difference in haptoglobin was observed between cows in the CON and NCG groups in our study, the lower (at d 0 and 21) and trend lower (at d 7) concentration of SAA indicated reduced inflammation in NCG-fed cows.The postpartum changed trends of the concentrations of IL6 and TNF-α were consistent with the changes of SAA.These results suggest that the benefits of NCG addition are the reduction of inflammation and improvement of animal health.Numerous studies have reported that supplementation with Arg can reduce inflammation (Fan et al., 2009;Zhao et al., 2018).Inflammation is often accompanied by oxidative stress (Hayajneh, 2014).One of the major sources of oxidative stress is lipid peroxidation, especially during the perinatal period when body fat is often mobilized to compensate energy requirement for postpartum dairy cows (Sordillo and Raphael, 2013).The significantly lower concentrations of MDA in the NCG group indicate that these cows faced less stress caused by lipid peroxidation.Although other biomarkers of oxidative stress did not present the significant differences between 2 groups, the changed trends of the concentration ROS and GSH-PX indicate the lower oxidative stress in NCG cows especially at d 7 when the oxidative stress is often the highest during the postpartum stage (Guan et al., 2020).Integrated with these results, we speculated that the oxidative stress was alleviated with NCG addition during the transition period.Xiao et al. (2016) found that dietary supplementation with 0.1% NCG and 1% Arg was effective in enhancing antioxidant status and maintaining the morphological structure of the jejunum under oxidative stress in rats.Cao et al. (2016) reported that NCG and Arg supplementation could improve the T-AOC in the liver and plasma of rats and alleviate the plasma MDA concentration, and 0.1% NCG was more effective than 1% Arg.In terms of ruminants, Zhang et al. (2018) found that NCG and Arg supplementation reduced MDA levels and increased GSH activity in the intestine of lambs.Arginine is considered to be in short supply under oxidative stress or inflammation (Wu et al., 2009).In our previous study, the plasma Arg concentration was higher in NCG-fed cows (Gu et al., 2021).Therefore, the beneficial effects of alleviating  inflammation and oxidative stress with NCG addition may be largely attributed to the increased Arg concentration and the important role of Arg in immunological functions in the body, especially in PMN (Li et al., 2007).
The PMN are the primary line of defense against invading pathogens such as bacteria (Wright et al., 2010).Arginine is an important component of defense in bovine PMN (Savoini et al., 1984) and maintains PMN activities (Heys et al., 1997).Synthesis of NO is an essential mechanism against viruses and bacteria for PMN in mammals (Bronte and Zanovello, 2005).Garcia et al. (2016) reported that Arg has the greatest utilization in the PMN of dairy cows in early lactation.Although the number of PMN was not affected by NCG addition in the present study, the expression of some functional genes in PMN changed when NCG was added, which may affect the functionality of PMN.The ITGB2 is a key gene related to migration and adhesion of PMN (Heiser et al., 2018), and cross-linking of surface ITGB2 proteins leads to PMN degranulation (Hussen et al., 2016).Therefore, the tendency to higher expression of ITGB2 may facilitate PMN migration to the site of infection and reduce inflammation (Crookenden et al., 2016).The CLEC6A gene encodes a pattern recognition receptor that regulates the inflammatory response.Thomas and Schroder (2013) suggested that PMN pattern recognition receptor-mediated signaling is important for orchestrating innate and adaptive immunity via cytokine and chemokine production and antimicrobial release.Thus, higher expression of CLEC6A in NCG-fed cows in this study is likely to benefit cows, and enhanced CLEC6A signaling may induce various inflammatory cytokines, chemokines, and type I interferons, consequently triggering an array of antimicrobial immune responses (Kumar et al., 2011;Heiser et al., 2018).The XBP1 is a key gene involved in toll-like receptors that plays an important role in the antimicrobial function of PMN (Crookenden et al., 2016).The S100A9 gene encodes a calcium-binding protein that regulates oxidative metabolism of PMN and activates NADPH oxidase (Bréchard et al., 2013) via membrane translocation (Schenten et al., 2010(Schenten et al., , 2011)).The lower expression of S100A9 and the tendency to reduced MMP9 expression in NCG-fed cows in this study might indicate lower inflammation and oxidative stress, which is consistent with the changes in SAA and ROS levels in the blood of these animals.Moreover, PMN egress from the bone marrow when the body experiences systemic inflammation and increased immature PMN in circulation (Van Merris et al., 2002).On the calving day, increased MMP9 expression is associated with increased immature PMN levels in the blood (Crookenden et al., 2016).Therefore, the tenden-cy to lower expressing of MMP9 in PMN in NCG-fed cows in this study might indicate more mature PMN in their circulation, which is beneficial for neutralizing invading pathogens.In summary, NCG supplementation may improve PMN functionality by improving their chemotactic, phagocytic and antimicrobial functions.However, current study only detected the PMN changes at the transcriptional level.Further studies are warranted to validate the effects of NCG on the PMN functions in future.
Finally, the numbers of lymphocytes in both groups of cows in this study were within the normal range for cattle (Kulberg et al., 2002), but cows with NCG supplementation had a higher number of lymphocytes, indicating that these cows may have better immunity.This may be attributed to the higher plasma concentrations of Arg and NO in the NCG cows (Gu et al., 2021).The NO is a metabolite of Arg and has been recognized to play an important role in both innate and acquired immunity capacities (Bogdan et al., 2000).Some stud- ies have shown that Arg is required for lymphocyte development (Field et al., 2000;Calder and Yaqoob, 2004).Thus, the higher numbers of lymphocytes in the blood of cows fed NCG may indicate better immunity in these cows, contributing to reduced postpartum inflammation and oxidative stress (Figure 2).

CONCLUSIONS
Feeding NCG to cows starting in the prepartum stage showed the beneficial effects on animal health, reflective of the reduction of inflammation and oxidative stress.These findings may be attributed to the improved immunity that is characterized by the increased numbers of lymphocytes in the blood and the potentially improved functions of PMN.Our study provided evidence that the use of NCG supplementation tended to improve immunological function and reduce oxidative stress and inflammation in dairy cows during the transition stage.
Gu et al.: N-CARBAMOYLGLUTAMATE AND THE HEALTH OF TRANSITION COWS

Figure 2 .
Figure 2. Diagram of the working hypothesis on how N-carbamoylglutamate (NCG) supplementation improves animal health in dairy cows during the transition period.In the diagram, genes with a solid background are upregulated in NCG-supplemented cows, and genes with a hollow background are downregulated.NO = nitrite oxide.

Table 1 .
Gu et al.: N-CARBAMOYLGLUTAMATE AND THE HEALTH OF TRANSITION COWS Whole blood white cell profiles in dairy cows supplemented without (CON) or with N-carbamoylglutamate (NCG) during the transition period

Table 2 .
Gu et al.: N-CARBAMOYLGLUTAMATE AND THE HEALTH OF TRANSITION COWS Inflammatory responses and oxidative stress in plasma of dairy cows supplemented without (CON) or with N-carbamoylglutamate (NCG) during the transition period

Table 3 .
Gu et al.: N-CARBAMOYLGLUTAMATE AND THE HEALTH OF TRANSITION COWS mRNA abundance of functional genes in PMN isolated from blood at postpartum 7 d of dairy cows supplemented without (CON) or with N-carbamoylglutamate (NCG) during the transition period