High-concentrate feeding upregulates the expression of inflammation-related genes in the ruminal epithelium of dairy cattle
© The Author(s). 2016
Received: 25 August 2015
Accepted: 12 July 2016
Published: 29 July 2016
The objective of this study was to characterize the mRNA expression profile related to rumen epithelial inflammation through the in vivo and in vitro experiments. In the in vivo experiment, rumen papillae were collected from four dairy cows adapted to either a 40 % (LC) or 70 % (HC) concentrate feeds for microarray analysis.
Results showed that 245 differentially expressed genes (DEGs) were detected in the cows fed the HC relative to the LC diet. The DEGs were first annotated, and results revealed that the expression of inflammation-related genes, including IL-1β, IL-2, IL-22, CCL19, CCL8, CX3CR1, CXCL6, INHBE, LEPR, PRL, and TNFRSF9 found in the cytokine-cytokine receptor pathway were up-regulated in the HC-fed cows, indicating local inflammation in the rumen epithelium was triggered. The expression of IL-1β, IL-2, and IL-6 was further validated by qRT-PCR. To demonstrate whether there were relationships between cytokine mRNA expression and ruminal factors (pH and LPS), the isolated ruminal epithelial cells were cultured in vitro. Results showed that the mRNA expression of IL-1β, IL-2, IL-6, and IL-8 increased after the LPS treatment, while low-pH treatment elevated the mRNA expression of TNF-α, suggesting that low-pH coupled with higher levels of LPS in rumen of cows fed the HC may be mainly responsible for the triggered local ruminal inflammation.
Our results indicate that ruminal local inflammation response might be triggered during HC feeding, and these findings also enhance the knowledge of rumen epithelial adaptation to HC at the molecular level.
Feeding ruminants diets rich in fermentable carbohydrates alters ruminal microbiota composition and metabolism, resulting in the accumulation of volatile fatty acids (VFAs) and leading to a dramatic depression of the rumen pH [1, 2]. Long-term high-concentrate feeding likely causes the rumen metabolic disorder commonly termed subacute ruminal acidosis (SARA), which is defined as the daily duration time of ruminal pH remains below 5.6 or 5.8 [3, 4]. SARA has been shown to depress ruminal diet digestibility, feed intake, milk production, and milk fat and has become a significant economic issue in the dairy industry [3, 5].
Dairy cattle health can be compromised by grain-induced SARA since it has been associated with rumenitis, metabolic acidosis, lameness and hepatic abscesses [3, 5]. Many previous reports revealed that grain-induced SARA increased the levels of acute phase proteins such as haptoglobin (Hp) and serum amyloid A (SAA), which indicate a systemic inflammatory response in peripheral blood [6, 7]. This systemic inflammation is considered to be related with the diet-induced massive disruption of rumen epithelial barrier function  and increased permeability of the rumen epithelium , which aids microbes and immunogenic compounds to transmigrate into portal circulation , and further induces systemic inflammation. The principle mechanisms responsible for disrupting the barrier function of the rumen epithelium during SARA are not clear. A study that used isolated rumen and colon tissue from steers demonstrated that LPS and decreased pH may act synergistically to disrupt the epithelial barrier function and then breach the epithelial barrier . Once the epithelium has been breached, the cells of mucosa-associated lymphoid tissue, which are found throughout the digestive mucosa and consist of clusters of white blood cells including innate lymphoid cells and mast cells [11, 12], respond by triggering local inflammation of rumen epithelium and altering cytokine production [11, 13]. Currently the structural transformation of the rumen epithelium during grain-induced SARA has been characterized [8, 14], but inflammation of rumen and the molecular mechanisms that trigger these events are not very clear.
In this study, we hypothesized that a local inflammatory response in the rumen epithelium occurred when cows were fed a high-concentrate diet, and therefore, mRNA expression of genes involved in inflammation would be up-regulated. The first objective of the this study was to characterize the mRNA expression of genes involved in inflammation in the rumen papillae from dairy cows fed high-concentrate diets using bovine genome microarray. The second objective of this study was to explore the relationship between expression of genes related to cytokines and ruminal factors.
Animals, experimental design, and diets
The experimental procedures and animal care conditions were approved by the Experimental Animal Welfare Ethics Committee of Nanjing Agricultural University.
This study was part of a larger experiment investigating the impact of subacute ruminal acidosis adaptation on rumen health in dairy cattle. A detailed description of the experimental design has been previously described . Briefly, four ruminally cannulated Holstein cattle (average body weight, 460 ± 16.4 kg; 84 ± 25 d in milk at the beginning of the trial) were used in a 2 × 2 crossover design trial with 2 cows in each square and 2 periods. All animals were fed a low-concentrate diet (30 % concentrate feed, DM basis) for 3 weeks before the start of this experiment. The dietary treatments were a low-concentrate diet (LC; 40 % concentrate feed, DM basis) and a high-concentrate diet (HC; 70 % concentrate feed, DM basis) (Additional file 1: Table S1). Each experimental period was 21 d, with the first 11 d used for diet adaptation and 10 d of measurements. For the HC group, the dietary concentrate level was stepped up (by approximately 15 percentage units/d compared with the LC diet) during the first 2 d. Throughout the experimental period, the cows received a total mixed ration (TMR), ad libitum to about 5 % orts. Cows were housed in tie stalls and had free access to water.
Blood and rumen papillae samples
Blood samples were taken from the coccygeal vein at 0 and 4 h following the morning feeding on d 12, 17, and 21 of each experimental period. Blood hematological analysis was carried out using an automated haematological analyser (Sysmex K-1000D; Sysmex, Tokyo, Japan), and blood chemistry was measured by Vitros 250 Chemistry System (Ortho Clinical Diagnostics, Markham, Canada).
Rumen papillae samples were collected at 4 h after the morning feeding on d 21 of each experimental period. The ventral sac of the rumen was chosen for collected site . The rumen papillae were excised (approximately 150 mg) from the rumen, washed 20 times in ice-cold PBS, and then frozen immediately in liquid nitrogen until the RNA was isolated.
Total RNA preparation
Total RNA was isolated from about 100 mg of frozen rumen tissues using TRIzol (Takara Bio, Otsu, Japan) following the manufacturer’s instructions. Qualified total RNA was further purified with the RNeasy mini kit (Qiagen, Hilden, Germany) and RNase-Free DNase Set (Qiagen, Hilden, Germany). The RNA concentration was determined using a NanoDrop spectrophotometer ND-1000UV-Vis (Thermo Fisher Scientific, Madison, WI). The RNA quality was routinely checked by measuring the optical density (260/280 and 260/230 ration). The total RNA from the rumen papillae was amplified and labelled with the Low Input Quick Amp Labeling Kit, One-Color (Agilent technologies, Santa Clara, CA), and the labelled cRNA was purified with RNeasy mini kit (Qiagen, Hilden, Germany).
Microarray hybridizations and data analysis
The hybridization of the cRNA was performed with Agilent Whole Bovine Genome Oligo (4 × 44 K) Microarrays using the Gene Expression Hybridization Kit (Agilent Technologies) according to the manufacturer’s protocols. After washing in staining dishes, hybridized slides were scanned with the Agilent Microarray Scanner (Agilent Technologies) with the default setting. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies).
Isolation and culturing of ruminal epithelial cells
Ruminal epithelial cells were isolated using the modified serial tryptic digestion procedure as described previously . Rumen papillae were taken from the ventral blind sac of healthy lactating Holstein cows (n = 3). The cows were fed grass hay. During isolation, each fraction was examined under a phase contrast microscope. The fractions that contained increasing numbers of cells from the stratum basal and the stratum spinosum of the rumen epithelium were pooled. The collected cells were seeded into 25-mL cell culture flasks and cultured with DMEM (Corning Cellgro, Manassas, VA) supplemented with 10 % fetal bovine serum (Corning Cellgro, Manassas, VA) and 1 % antibiotic/antimycotic solution at 38 °C under 5 % CO2 for 24 h. Thereafter, the cells were allocated for the following treatments: (1) pH 7.4; (2) pH 5.5; (3) pH 7.4 + 10 μg/mL LPS; (4) pH 5.5 + 10 μg/mL LPS. After incubation for 24 h, the cells were collected and stored at - 80 °C for further analysis. All reagents used were purchased from Sigma (St. Louis, MO) unless indicated otherwise.
Quantitative RT- PCR and data analysis
Primers for real-time quantitative PCR
Primer sequence (5′ → 3′)
Data for blood chemistry and hematology parameters were analysed by using the general linear model (GLM) procedure in SPSS Version 18 (SPSS, Chicago, IL, USA), according to the model shown below: yijkmn = μ + Si + C(S)ij + Pk + Tm + (ST)im + eijkmn, where μ is the overall mean, Si is the diet treatment (i = 1–2), C(S)ij is the random effect of cow j (j = 1–4) nested within the diet i, Pk is the fixed effect of period k (k = 1–2), Tm is the fixed effect of day m (m = 1–3), (ST)im is the fixed effect of the diet by day interaction, and eijkmn is the random residual error. Significance was declared at P < 0.05.
Statistical calculations for the microarray data were conducted by GLM procedure in SPSS Version 18 (SPSS, Chicago, IL, USA), according to the model shown below: yijk = μ + Di + Pj + (DP)ij + eijk, where μ is the overall mean, Di is the diet treatment (i = 1–2), Pj is the fixed effect of period j (j = 1–2), (DP)ij is the interaction between treatment and experimental period, and eijk is the random residual error. Significance was declared at P < 0.05, and a tendency was considered to exist at 0.05 ≤ P < 0.10. Differentially expressed genes (DEGs) were defined by genes whose fold-change in expression (the LC group vs. the HC group) was equal to or higher than 1.5-fold and P < 0.05. DEGs and heat-map analysis were performed by SAS software from SHANGHAI BIOTECHNOLOGY CORPORATION eBioService (http://www.ebioservice.com). For better understanding of DEGs, overrepresentation analysis of Gene Ontology (GO) terms and pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was performed. Correlation analysis was performed using GraphPad Prism 5 software (Graphpad Software, San Diego, CA).
Data obtained from the in vitro cell culture experiment were analyzed by GLM procedure of SPSS Version 18 (SPSS, Chicago, IL, USA) to determine the effects of LPS and pH on the mRNA expression of cytokines, according to the model shown below: yijk = μ + Li + Pj + (LP)ij + eijk, where μ is the overall mean, Li is the fixed effect of LPS (i = 1–2 for dosage), Pj is the fixed effect of pH (j = 1–2), (LP)ij is the fixed effect of the LPS treatment × pH, and eijk is the random residual error. Differences with P < 0.05 were considered significant.
Ruminal pH, SCFA and LPS
The results presented here must be interpreted in light of the overall effects of HC feeding on rumen fermentation in the experimental cattle reported in Mao’s prior study . Briefly, the ruminal pH of the HC-fed cattle was lower (P < 0.001) than that of the LC-fed cattle, and the duration of time for which the ruminal pH was less than 5.8 was about 5.1 h after the first feeding in the HC group. The HC feeding increased (P < 0.001 to P = 0.018) the concentrations of propionate (24.32 mmol/L vs. 20.39 mmol/L), butyrate (14.39 mmol/L vs. 11.28 mmol/L), valerate (7.27 mmol/L vs. 4.81 mmol/L), isovalerate (2.64 mmol/L vs. 2.08 mmol/L), total volatile fatty acid (124.85 mmol/L vs. 111.23 mmol/L), lactic acid (0.24 mmol/L vs. 0.18 mmol/L) and LPS (26,266.88 EU/mL vs. 14,741.13 EU/mL) compared with the LC group.
Blood chemistry and hematology parameters
Mean values for blood chemistry and hematology for cows fed two different diets
Diet x Day
White blood cells, 1012/L
Total protein, g/L
Urea nitrogen, mmol/L
Low density lipoprotein, mmol/L
High density lipoprotein, mmol/L
Annotation of DEGs using Gene Ontology
Enriched GO terms within molecular function, cellular component and biological process
Enrichment test p-value
auxiliary transport protein activity
channel regulator activity
cyclase regulator activity
extracellular region part
establishment of localization in cell
regulation of localization
regulation of multicellular organismal process
immune system process
negative regulation of sequestering of calcium ion
regulation of homeostatic process
negative regulation of developmental process
response to other organism
maintenance of location
multicellular organismal process
establishment of localization
regulation of biological quality
Assignments of DEGs based on KEGG
Functions of inflammatory cytokines in response to ruminal environment variations
Validation of microarray gene expressions (IL-1β, IL-2 and IL-6) using Realtime-PCR method
qRT-PCR relative expression
1.15 ± 0.164
2.40 ± 0.159
P < 0.05
P < 0.05
0.80 ± 0.142
1.58 ± 0.135
P < 0.05
P < 0.05
1.31 ± 0.010
0.66 ± 0.068
P < 0.05
P < 0.05
The relationship between the ruminal pH, LPS levels, and cytokines mRNA expression level
Effects of pH and LPS on selected cytokines’ mRNA expression in cultured ruminal epithelial cells
The relative expression of cytokines after low pH and lipopolysaccharides treatments in vitro
LPS, 10 μg/mL
LPS, 10 μg/mL
pH x LPS
The overall goal of this study was to characterize the profile of gene expression especially target genes associated with inflammation after SARA challenge in vivo, and to ascertain to find out how environmental factors in rumen affect the mRNA expression of these cytokines in vitro. Therefore, defining SARA was imperative. The ruminal pH, SCFA, and LPS data have been reported previously by our team , and the duration time for ruminal pH below 5.8 was about 5.1 h in the HC group during the diurnal period, and the a higher LPS level was observed in the HC group, suggesting that SARA was successfully induced in cows fed the HC diet. Many studies have confirmed that the grain based-SARA challenge can cause translocation of lipopolysaccharide and triggers systemic inflammation leading to an increase in acute phase proteins in blood [7, 24, 25]. In the present study, we cannot absolutely certify the occurrence of systemic inflammation in cows fed HC diet in the absence of detecting acute phase proteins or inflammatory cytokines in blood, as our research centred on local inflammation of the rumen epithelium. However, the increase in the serum white blood cells, lymphocytes, and albumin and the decrease in total protein and globulin in HC-fed cows, indicating that the inflammatory responses may be activated and the synthesis ability of globulin and albumin in liver were affected by HC feeding [26, 27].
In the present study, the analyses of GO categories revealed that most changes centralized in biological processes. In this category, our study showed that GO terms transport, establishment of localization, and cellular localization were enriched by the HC feeding, and these terms were defined as the process or result of the movement of a cell, substance or cellular entity, such as a protein complex or organelle, to a specific location. Considerable evidence has accumulated describing the changes in ruminal epithelial nutrient and ion transport (VFA, glucose, Na+), as well as the gene expression of transporters regulating these processes during the adaptation of HC diets . Moreover, HC feeding also induced accelerated differentiation and cellular migration in the stratum basale, spinosum and granulosum layers of the rumen epithelium . It is well established that rumen fermentation profile altered dramatically in response to highly fermentable diets; therefore the transports of these substances and cellular migrations may be driven or stimulated by ruminal fermentation products, and the exact mechanism warrants further exploration. In addition, our study also showed that GO term immune system process and its child terms leukocyte homeostasis and immune response were enriched by the different diet treatments. Indeed, several studies have confirmed that immune system is involved in the adaptation of the rumen epithelium to HC diets, and these immune responses are thought to be initiated from the stimulation of ruminal microbiota or toxin compounds [29, 30]. Correspondingly, the present study demonstrated that the level of some harmful or pro-inflammatory compounds such as LPS, biogenic amines, ethanolamine and glutaric acid in rumen were elevated in the cows fed HC diet (data not published). It is noticeable that a certain degree of immune response may be the self-protective behavior which prevents the further ruminal epithelial injury, and thus, future studies should evaluate the relationship between the degree of immune response and the severity of ruminal acidosis, as well as the process of local inflammation in the rumen epithelium.
To date, relatively little information is available about the global effects of HC feeding on the molecular functions and pathways of the ruminal epithelium. However, a recent study revealed that the alteration of multiple pathways including calcium signaling pathway, gap junction, and Jak-STAT signaling pathway contributes to the changes in ruminal fermentation induced by HC diets . Consistent with the above report, calcium signaling pathway and Jak-STAT signaling pathway were both found enriched in the present study. Enriched calcium pathway in the present study also supported and confirmed the previously observed physiological change that HC feeding clearly altered calcium transport , and this alteration in the rumen epithelium may be associated with the stimulation of accumulated VFA in the rumen . Thus, some calcium-dependent events occurred in the rumen epithelium, such as cytokine release and signal transduction process may be affected when cows were fed HC diets. Jak-STAT pathway is one of a handful of pleiotropic cascades used to transduce a multitude of signals for development and homeostasis in mammals . Jak-STAT signaling pathway was reported to be closely related with the activation of inflammation, and the activate JAK and STAT proteins usually played a positive role in inflammation progresses . In the present study, even though the key genes encoded JAK kinases (JAK1, JAK2, JAK3 and Tyk2) and STATs were not changed, enriched Jak-STAT signaling pathway may to some extent reflect that the functional homeostasis of rumen epithelium were impaired after HC feeding.
In the present study, aside from the changes in Calcium signaling pathway and Jak-STAT signaling pathway, inflammation related pathways including Cytokine-cytokine receptor interaction and Chemokine signaling pathway were also enriched by the different diet treatments. Inflammation is a complex set of synergistic actions of inflammatory mediators (such as cytokines, chemokines, and nitric oxide) and immune cells (such as macrophages, lymphocytes, and monocytes), as the first response of the immune system to infection or tissue injury [36, 37]. Although some review articles reported HC feeding may lead to rumenitis [3, 38] local inflammation in the rumen epithelium, there is still a lack of understanding of how inflammatory-related genes respond to HC diet at the molecular level. Our study revealed that the expression of genes found in cytokine-cytokine receptor pathway, including IL-1β, IL-2, IL-22, CCL19, CCL8, CX3CR1, CXCL6, INHBE, LEPR, and PRL, were up-regulated in HC-fed cows; only IL-6 and IL-15RA were down-regulated. It was well known that IL-1β, IL-2 and IL-22 have the potential to stimulate pro-inflammatory responses, and mediated inflammatory diseases . Therefore, the higher expression of IL-1β, IL-2 and IL-22 mRNA in the rumen epithelium indicates that local inflammation might occur in the rumen epithelium of the HC group. Chemokines are chemotactic cytokines that can participate in regulating inflammatory leukocyte recruitment, lymphocyte recruitment and homing . Chemokines were categorized as inflammatory or homeostatic chemokine according to the functional role. Results from previous studies indicated that bovine mammary epithelial cells can express CXCL6 (also called GCP2) and CCL8 (also called MCP2) in response to certain bacterial cell components during the simulation course of mastitis in vitro [41, 42]. In addition, CX3CR1, the receptor of chemokine CX3CL1, was also classified as an inflammatory chemokine . In the present study, up-regulated genes (CCL19, CCL8, and CX3CR1) were observed in HC-fed cows, further indicating that local inflammation in the rumen epithelium might be triggered.
Inflammation may be the interaction result of multiple pathways or biological processes. In this study, the GO term immune system process was enriched in the HC group, and most affected inflammatory cytokines such as IL-1β, IL-2 and IL-6 were also in the category immune system process, indicating that the immune system activation might be involved in the adaptation of the rumen epithelium to the HC diet. Of the affected pro-inflammatory cytokines, there is a paucity of information on the expression of IL-1β in ruminants fed HC diet. However, IL-1β has been shown to increase the permeability of the intestinal tight junctions of Caco-2 cells . If IL-1β acts similarly in the rumen epithelium, the interleukin may play a role in reducing the barrier function of the rumen epithelium in cattle fed the HC diet. Previous studies showed that inducers of IL-1β expression in the gut include hyperosmolarity, T and B cells, LPS, and other interleukin proteins [44, 45] indicating that these factors may have played a role in IL-1β up-regulation in cattle fed high grain diets. This is also consistent with our in vitro results that LPS treatment significantly increased the expression of IL-1β. The role of IL-2 in the inflammatory process is complex and involves pro-inflammatory as well as regulatory aspects, and dysregulation seem to contribute to various immune system-related diseases in humans [46, 47]. A previous study showed that the aberrant production of IL-2 can disrupt primary human T lymphocytes function by leading to the loss of T cell anergy and induction of autoimmunity . Thus, it is reasonable to postulate that an increase in the expression of IL-2 during HC feeding might have a negative impact on the innate immune response in the rumen epithelium. IL-6 is a multifunctional, pleiotropic cytokine involved in regulating immune responses, acute-phase responses and inflammation [49, 50]. Previous studies revealed that IL-6 is involved in tissue repair and cytoprotection in the human gut, and preventing IL-6 production could favor wound healing and remodelling following injury [39, 50, 51]. In this study, the true cause of the observed effects in the down-regulation of IL-6 mRNA in the HC group is unclear. Nevertheless, since the reduction of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α can reduce the length and severity of the immune response , it follows that decreasing the expression of IL-6 during HC feeding can have beneficial effects.
To validate the results of the in vivo experiment and get a better understanding of the inflammation response during HC feeding, the rumen factors (LPS and low pH) which are most associated with SARA were selected for the in vitro experiment. In vitro, the mRNA expression of tested pro-inflammatory mediators IL-1β, IL-2, and IL-8 was up-regulated after LPS was stimulated. These results further strengthened the well-documented theory of the pro-inflammation ability of LPS, and demonstrated that the higher levels of LPS in rumen fluid during HC feeding could contribute to local inflammation in the rumen epithelium. Our in vitro results also showed that the expression of IL-6 mRNA was increased by the LPS challenge, and this is not consistent with the down-regulation results observed in vivo in the present study. These differences indicate that IL-6 mRNA expression might be affected by factors other than LPS. Indeed, recent progress in the understanding of the role butyrate plays in inflammation confirmed that n-butyrate can down-regulate the level of IL-6 during LPS-induced inflammation response but not affect the level of TNF-α . In this study, a higher concentration of butyrate was observed in the HC group compared with the LC group. Therefore, the stimulation effect of LPS on the production of IL-6 might be concealed by the inhibition effect of butyrate, and this might finally result in the lower mRNA expression of IL-6 observed in the in vivo tests.
Although it has been well known that feeding dairy cows diets containing high proportions of grain is associated with a rapid decline in ruminal pH, and low ruminal pH can lead to rumenitis and eventually to ruminal parakeratosis, erosion, and ulceration of the rumen epithelium , there is little information on the effects of extracellular pH on rumen immune function. Clinical studies of organic acidosis in humans revealed clinical acidaemias are usually accompanied by immune deficiency, including a decrease in white cell numbers, globulins, and mitogenic responses, a diminution of the inflammatory response and delayed phagocytosis . In this study, our findings revealed that low-pH treatment significantly elevated the mRNA expression of gene TNF-α compared with the high pH group. These results suggest that lower pH may contribute to local inflammation of the rumen epithelium by up-regulating certain genes. However, the mechanism underlying the up-regulation of TNF-α expression induced by lower pH during HC feeding is unclear, and more research is needed to evaluate the effects of extracellular pH on the immunity function of the rumen epithelium.
Our findings revealed that the biological functional changes in the rumen epithelium were mainly focused on biological processes in dairy cattle fed a high-concentrate diet, and the local inflammation response was triggered during HC feeding in the present study. The lower pH and high level of LPS in rumen during HC feeding may play an important role in the process of local inflammation in the rumen epithelium, and their pro-inflammatory effects may weaken the permeability barrier of the rumen epithelium. Future research is required to explore the mechanism of rumen epithelium inflammation as well as the role of lower ruminal pH in the damage to the rumen tissue of dairy cattle during SARA.
DEGs, differentially expressed genes; HC, high-concentrate; Hp, haptoglobin; LC, low-concentrate; SAA, serum amyloid A; SARA, subacute ruminal acidosis; TMR, a total mixed ration; VFA, volatile fatty acids
The authors thank Dongsheng Wang for the collection of biopsy samples.
This study was carried out with the support of the National Basic Research Program of China (2011CB100801). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
The datasets generated during the current study are available in the Gene Expression Omnibus (GEO) repository. The accession number is GSE82272 (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc = GSE82272).
The authors’ contributions are as follows: RYZ carried out the majority of the animal studies including animal care, RNA isolation and real-time PCR; RYZ and SYM carried out data interpretation and manuscript preparation. SYM and WYZ were responsible for the conception of the project and the oversight of the experiment. All authors read and approved this manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The experimental procedures and animal care conditions were approved by the Experimental Animal Welfare Ethics Committee of Nanjing Agricultural University.
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