Dynamics of oxidative stress and immune responses in neonatal calves during diarrhea

Oxidative stress is the imbalanced redox status be-tween oxidant production and their scavengers leading to intestinal physiological dysfunction. However, the role of systemic and local oxidative status during neo-natal calf diarrhea is not known. This study assessed systemic (serum) and local (fecal) oxidative status when calves either naturally developed diarrhea or naturally recovered. Healthy calves were enrolled in the study at d 18 of age, and their health status was monitored from the enrollment. Based on their enteric health status on d 21 and 28, calves were grouped as continuous diarrhea from d 21 to 28 (n = 14), diarrhea at d 21 but recovered at d 28 (DH group, n = 19), healthy at d 21 but developed diarrhea at d 28 (HD group, n = 15), and healthy throughout the study (HH group, n = 16). Serum and fecal samples were collected at d 21 and 28 from all calves in the morning 2 h after feeding. Dynamics of oxidative stress indicators including reactive oxygen species (ROS), malondialdehyde (MDA), H 2 O 2 , 8-hydroxy-2 ′ -deoxyguanosine (8-OHDG), glutathione peroxidase, superoxide dismutase, catalase (CAT), and total antioxidant capacity and inflammatory indicators TNF-α, IL-1β, IL-4, IL-6, IL-10, and IFN-γ were evaluated using serum samples. In addition, fecal oxidative stress indicators ROS and MDA were measured. Serum ROS, MDA, 8-OHDG, as well as fecal ROS and MDA, were higher, whereas serum CAT and H 2 O 2 were lower in diarrheic calves than those of healthy calves. Serum ROS, MDA, and 8OHDG and fecal ROS and MDA increased in the HD group from d 21 to 28 as they developed diarrhea. In contrast, all these oxidative stress markers decreased in the DH group from d 21 to 28 as they recovered. However, serum H 2 O 2 had an opposite changing trend, which became lower in the HD group and higher in the DH group at d 28. In conclusion, both systemic and local oxidative stress markers and cytokine profiles altered as calves moved from being healthy to having diarrhea or vice versa. Serum ROS, MDA, and 8-OHDG can be used to develop biomarkers to screen calves prone to enteric infections during the preweaning period.


INTRODUCTION
The redox state is the fine balance between oxidants (pro-oxidants) and antioxidants in a biological system (Tauler Riera et al., 2012).Oxidative stress is defined as an imbalance between oxidants and antioxidants, leading to a disruption of redox signaling and control or molecular damage (Rahal et al., 2014;Sies, 2018).Oxidants mainly refers to reactive oxygen species (ROS) including hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (OH − ) and superoxide anions (O 2 − ; Schieber and Chandel, 2014), which are produced by the cell activities (e.g., the aerobic metabolism; Das and Roychoudhury, 2014;Villalpando-Rodriguez and Gibson, 2021).They can actively react with biomolecules, such as DNA, lipid and protein (Juan et al., 2021).The overaccumulation of ROS can cause oxidative stress that damage the redox homeostasis, which is harmful to cells and damage biomolecules to produce oxidative products such as malonaldehyde (MDA) and 8-hydroxy-2′-deoxyguanosine (8-OHDG).There are host antioxidant systems, including enzymatic antioxidants and nonenzymatic antioxidants.Superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), and peroxiredoxin are the most common antioxidant enzymes, while glutathione, inositol, cysteine, vitamins, and minerals are nonenzymatic antioxidants (Guan and Lan, 2018).Oxidative stress has been involved in intestinal diseases (Kim et al., 2012), as such the control and prevention of oxidative stress has been a research focus recently (Pizzino et al., 2017;Luca et al., 2019).
Diarrhea in neonatal calves can cause more than 50% of mortality, calf malnutrition, digestive dysfunction, and reduced growth and production performance, which results in significant economic losses (Heinrichs et al., 2005;Windeyer et al., 2014).Neonatal calf diarrhea is a multifactorial syndrome, usually caused by pathogenic infection mainly including bacteria, virus, and nonpathogenic factors such as malnutrition and environmental changes (Beheshtipour and Raeeszadeh, 2020;Morrison, 2020).Studies have shown that the oxidative stress occurs when animals suffer from diarrhea in rodent models (Sammari et al., 2021;Zeeshan et al., 2021).Meanwhile, suppressing oxidative stress can potentially be beneficial to alleviate diarrhea in rodents (Song et al., 2011).However, the conclusion on the oxidative stress and diarrhea can be controversial as it has been also reported that oxidative stress is not affected by the nonmalnutrition diarrhea (Granot et al., 2001).
To date, the direct relationship between oxidative stress occurrence and calf diarrhea is not well defined.Moreover, the link between intestinal and systemic oxidative stress indicators has not been understood.Redox status has important role in priming immune responses.This study hypothesized that redox and inflammatory status would be altered as calves developed diarrhea or recovered.Therefore, we evaluated the serum (ROS, MDA, 8-OHDG, and antioxidant enzymes) and fecal (ROS and MDA) oxidative stress markers as well as serum cytokine profiles during wk 3 of life (d 21-28) of dairy calves.

Animals and Sample Collection
The study was conducted during September to December in Chifeng, Inner Mongolia, China.The experi-mental calves (n = 94) were separated from their dams immediately after birth and housed in individual calf hutches.The overall feeding management followed the standard operational practice of the commercial farm.Briefly, the calves received 2 L of pasteurized colostrum (IgG >55 g/L) via esophageal tube immediately after birth and 2 L in the next 6 h.Calves received whole milk during the first 6 d (2, 2.5, and 3 L twice for d 1, 2-3, and 4-6, respectively), followed by milk replacer (3, 3.5, and 4 L twice for d 7-14, 15-21, and 22-28, respectively).Calves had access to starter and water ad libitum from d 1.The nutrient composition is shown in Table 1.The animal study was conducted following the Institutional Animal Care and Use Committee of the Institute of Animal Sciences at the Chinese Academy of Agricultural Sciences (protocol no.IAS2022-90).
Healthy male Holstein calves (n = 94) with similar BW were enrolled in the study at d 18 and their health status were monitored until d 28 (Figure 1).Fecal scores were recorded daily: 0 (normal), 1 (soft), 2 (runny), and 3 (watery), and a fecal score ≥2 for 2 consecutive days was considered as diarrhea (Chae et al., 2021).Study calves (n = 64) were set into healthy and diarrhea status based on their fecal scores on d 21 and 28.Based on the dynamics of diarrhea between d 21 and 28, calves were grouped into 4 categories: (1) continuous diarrhea from d 21 to 28 (DD; n = 14), (2) diarrhea at d 21 but recovered at d 28 (DH; n = 19), (3) healthy at d 21 but had diarrhea at d 28 (HD; n = 15), and (4) healthy during study period from d 21 to 28 (HH; n = 16), respectively (Figure 2).Thirty calves were eliminated due to their eligible health dynamics such as inconsistent diarrhea.
On d 21 and 28, sampling (blood and feces) and weighing were performed.Calves were kept on a standup position during the sampling process to minimize stress due to handling.A 10-mL blood sample was collected 2 h (±0.5 h) after feeding milk in the morning through jugular venipuncture.Serum was separated within 30 min after sampling through centrifugation at 3,000 × g for 20 min, stored in liquid nitrogen, and then transferred to −80°C until further analysis.Fe- cal samples were collected via rectal palpitation in the morning 2 h (±0.5 h) after feeding milk, snap freeze in liquid nitrogen, and then transferred to −80°C.

Serum Oxidative Stress and Inflammation Indicator Measurement
Serum ROS was measured using serum ROS Detection Kit (BBoxiProbe O13, BestBio Shanghai, China).Briefly, a 100-μL serum sample was added into 96-well plate (black with clear bottom) and mixed well with a 1-μL probe O13.After incubating for 30 min at 37°C in the dark, the fluorescence intensity was measured using a fluorescence microplate reader at excitation/emission = 535/610 nm.Serum MDA was measured using the MDA assay kit (thiobarbituric acid method; Nanjing Jian Cheng Bioengineering Institute, Nanjing, China).Serum H 2 O 2 , CAT, GPX, and SOD were measured by commercial kits (Nanjing Jian Cheng Bioengineering Institute, Nanjing, China).The concentration of 8-OHDG was measured using a commercial ELISA kit (Nanjing Jian Cheng Bioengineering Institute, Nanjing, China).Total antioxidant capacity (T-AOC) was measured with total antioxidant capacity assay kit (FRAP method; Nanjing Jian Cheng Bioengineering Institute, Nanjing, China).The concentrations of serum IL-1β, IL-4, IL-6, IL-10, TNF-α, and IFN-γ were determined using ELISA method with Cusabio bovine ELISA kits (Cusabio Biotech Co. Ltd., Wuhan, Hubei, China).The calves in the DH group had diarrhea on d 20 and 21, then gradually recovered and became all healthy on d 28.The calves in the HD group were all healthy before d 22, then gradually suffered from diarrhea, and all had diarrhea on d 27 and 28.The calves were healthy the whole time of the experiment.

Fecal Oxidative Stress Indicator Measurement
Fecal samples (1 g of dry weight) were mixed with PBS buffer (10 mL) and then disrupted in bead mill machine (Retsch, MM400) at 4°C.After centrifugation, the supernatant was used to measure the concentration of ROS and MDA.The fecal ROS was measured similar to serum ROS described above.The MDA concentration was measured through HPLC according to methods described by Briviba et al. (2004).Briefly, MDA in the fecal samples was reacted with thiobarbituric acid.Then the products were analyzed by HPLC on a 4 × 250 mm Vydac RP C18 column and tested by fluorescence detector with a 515-nm excitation and 550-nm emission wavelengths.The MDA levels in the fecal samples were calculated in relative to a standard (1 mM MDA, Nanjing Jian Cheng Bioengineering Institute, Nanjing, China).

Data Analysis
All data were checked for normality and outliers the UNIVARIATE procedure of SAS (version 9.4, SAS Institute Inc., Cary, NC).The data were analyzed using a repeated measurement design in R using nlme package and following statistical model.
where Y ijk is the response variable, μ is the overall mean, τ i is the effect of the group, β k is the effect of age, (τ β) ik is the interaction effect between τ i and β k , j indexes the animal, and ε jk(i) ~N (0,  ij ) is a random error.The error notation, ε jk(i) , shows that the animal-age combination is nested in the treatment.Compound symmetry correlation structure (corCompSymm in nlme::gls) was used based on Akaike and Bayesian information criteria (Pinheiro et al., 2017).The model imposes that the variances and covariances of the residuals are equal but different from 0. The matrix  ij is The model included group and age as fixed effects, interaction effects between group and age, and individual calf as a random effect.If the interaction effects were significant, between-group differences at each age were tested at α = 0.05.If not, the group effect or age effect was tested.The BW and ADG of different groups and all the indices between different health status (diarrhea or healthy) of the calves were analyzed using a oneway ANOVA.Significance level was set at P < 0.05.Pearson correlation analyses were performed, with r = 0 referring to no correlation and r = −1/+1 referring to a negative or positive correlation.If R 2 = 1, all of the data points fall perfectly on the regression line.If R 2 = 0, the estimated regression line is perfectly horizontal.Post hoc had been tested in G-power (version 3.1) based on the mean of primary indicators (ROS and MDA) with all power values >0.75.

Growth Performance of Calves
Calf BW at birth (P = 0.435), d 21 (P = 0.392), and d 28 (P = 0.421) did not differ among different groups.However, the average daily gain of DD group tended to be lower than that of HH group (P = 0.075, Table 2) during the experimental period (from d 21 to 28).3).The concentration of CAT was lower (P < 0.05), whereas T-AOC was tended to be lower (P = 0.071) on d 28 compared with d 21 (A), regardless of the calf group (Table 3).The concentration of SOD was higher (P < 0.05) on d 28 compared with d 21, regardless of the calf group.The concentration of CAT and T-AOC was tended to be lower in DD compared with other groups (Table 3).

Fecal Oxidative Stress Markers
Concentrations of fecal ROS and MDA were significantly affected by the interaction of calf group and age (G×A) and health status (H; Table 4, Figure 4).In DD and HH calves, fecal ROS and MDA levels were stabilized during the experimental period (d 21 to 28).In contrast, when calves recovered from diarrhea (DH), the concentration of fecal MDA and ROS decreased significantly (P < 0.05) from d 21 to 28.Both fecal oxidative stress markers tended to be increased (MDA: P = 0.061; ROS: P = 0.067) when calves developed diarrhea (HD).Fecal ROS and MDA levels were significantly higher in calves with diarrhea compared with the healthy calves (P < 0.05).

Cytokine Measurement
Concentrations of cytokines (IL-6, IL-4, IL-10, TNF-α, and IFN-γ) were affected by group G, A, and      H (Table 5).TNF-α and IL-10 were higher in calves with diarrhea compared with the healthy calves (P < 0.05), regardless of group.When the effect of group and calf age was evaluated, the concentration of IL-1β, TNF-α and IL-10 was affected by the interaction effect of G×A (Table 5, Figure 5).The concentration of TNF-α decreased significantly (P < 0.01) as calves recovered from diarrhea (DH), whereas increased (P < 0.01) as calves got diarrhea from healthy (HD).IL-10 decreased significantly in DD, DH, and HH group (P < 0.05), whereas it was stable in HD group (P = 0.978).IL-1β increased significantly in DH, HD, and HH group (P < 0.05), whereas it was stable in DD group (P = 0.998).The concentration of IL6 was higher in HD group compared with that in HH group (P < 0.05).Almost all the indices were affected by age (A) except TNF-α (P = 0.431).IL-1β and IL-6 increased significantly (P < 0.01) whereas IL-4, IL-10 and IFN-γ decreased significantly as the calves grew from d 21 to 28 (P < 0.01).

Associations Between Serum Oxidative Stress Indicators, Immune Factors, and Fecal Indicators
Further correlation analysis revealed weak positive correlations between serum MDA and ROS, serum ROS and fecal ROS, as well as fecal MDA and serum MDA (P < 0.001, R = 0.40, 0.34, and 0.43, respectively, Figure 6).Fecal MDA and ROS had a strong positive relationship (P < 0.001, R = 0.58).In addition, the correlation analysis showed that the TNF-α and serum ROS had a positive relationship (P < 0.001, R = 0.48), whereas no correlation was found between serum ROS and IL-10 (P = 0.478, R = 0.10, Figure 7).

DISCUSSION
The present study investigated the oxidative stress and cytokine responses when the enteric health status of neonatal dairy calves was either stable (remained healthy-HH or remained diarrheic-DD) or changing (develop diarrhea -HD or recovered from diarrhea -DH) during the third week of life.We reported that serum oxidative stress markers ROS, MDA, and 8-OHDG increased as calves developed diarrhea but decreased as calves recovered from diarrhea.This suggests that dynamics of serum ROS, MDA, and 8-OHDG could be linked to enteric health status of the neonatal calves.Moreover, serum ROS increased while CAT and total antioxidant ability decreased when calves developed diarrhea.Similar to the present study, Akyüz and Kükürt (2021) have reported an increase in the total oxidant and a decrease in the antioxidant ability in calves with diarrhea.The ROS are important compounds in activating physiological and immunological responses (Shadel and Horvath, 2015;Aviello and Knaus, 2017).The increase of ROS sometimes is necessary for the body to respond to a physiological change such as cellular proliferation, immune response (Vaccaro et al., 2020).Therefore, the increase at the ROS level when calves developed diarrhea suggest that alteration in cellular functions as well as regulation of inflammatory responses in these calves during diarrhea.Determining MDA and 8-OHDG to identify lipid peroxidation and DNA damage is an essential to assess oxidative stress (Gaweł et al., 2004;Rahal et al., 2014;Cherian et al., 2019;Dai et al., 2019).The phospholipid membranes of enterocytes are prone to oxidative damage caused by ROS (Farag and Alagawany, 2018;Yu et al., 2020).Groups with different lowercase letters (a and b) showed significant differences at the same age (P < 0.05).The symbol * above the lines refers to a significant difference (P < 0.05) as the calves grew in one group.As a result, enterocytes produce MDA (Farag and Alagawany, 2018;Yu et al., 2020).Studies have reported increased serum MDA level in calves infected with coccidiosis or pathogenic bacteria (Ramadan et al., 2021;Aydin et al., 2022).Higher MDA content has been reported in erythrocytes of calves with acute diarrhea (Ranjan et al., 2006).In addition to MDA, 8-OHDG indicates DNA damage activated due to increased ROS (Valavanidis et al., 2009).Increased serum MDA and 8-OHDG content in diarrheic calves in the present study confirmed that oxidative stress leads to enterocyte damage during diarrhea.This is the first study to report dynamics of oxidative stress markers in serum as animals change their health status either from healthy to diarrhea or vice versa.
In addition to serum (systemic) oxidative stress, the present study also explored the dynamics of oxidative stress markers in feces as a mean to study the intestinal oxidative status.To date, little studies focus on the fecal oxidative stress markers.Studies in other animals model such as sheep and rodents have reported that the intestinal oxidative stress when animals suffer from diarrhea according to intestinal epithelial indices (Cheng et al., 2021;Sammari et al., 2021).In contrast, knowledge on fecal oxidative stress will generate noninvasive approaches to assess intestinal oxidative status, which has been shown to associate with fecal microbial communities (Million et al., 2016;Million and Raoult, 2018).The fecal oxidative stress markers (ROS and MDA) displayed a pattern similar to that inserum (systemic), suggesting that serum oxidative status can be used as an indicator to study the dynamics of enteric health during neonatal period.
Superoxide dismutase, GPX, and CAT are considered as the first-line defense of antioxidant systems, which can be constantly produced by the host (Ighodaro and Akinloye, 2018).Studies have reported a decrease in serum GPX in calves infected with coccidiosis or pathogenic bacteria (Akyüz and Kükürt, 2021;Ramadan et al., 2021;Aydin et al., 2022).However, the present results were not consistent with previous studies showing the serum antioxidant enzymes decrease as animals suffer from diarrhea in mice or humans (O Al-Gazally, 2006;Koriem et al., 2019).Although we found GPX was higher in DD group than that in HH group and CAT content and T-AOC tended to be lower in in DD group, SOD, GPX and CAT as well as T-AOC were not affected by health dynamics of calves as calves developed diarrhea or recovered from diarrhea.Therefore, it is unclear whether the calves had low antioxidant ability following with diarrhea occurrence.Saleh et al. have reported that SOD and T-AOC content increased during mild infection of dermatophyte while decreased during severe infection, but MDA continuously increased no matter the severity of infection in a sheep model (Saleh et al., 2022).We did not sort the calves based on diarrhea severity, types and causes, which might lead to the inconsistent results.
H 2 O 2 , as the most common ROS, has been reported for a long time to be an important intracellular messenger involved in many cell activities (Rhee, 1999).Therefore, we speculated that serum H 2 O 2 would increase as calves develop diarrhea.However, serum H 2 O 2 decreased when calves developed diarrhea while increased as calves recovered from diarrhea, which showed an opposite trend compared with other ROS.The sources of serum H 2 O 2 are excretion of immune cells that migrate through endothelium (Forman et al., 2016).Although the blood immune cells in high-ROS environment tends to produce more H 2 O 2 (Cooper et al., 2002;Mohanty et al., 2014) and H 2 O 2 can transfer more freely through membranes than other ROS (Chanin et al., 2020).,Although the blood immune cells in high-ROS environment tends to produce more H 2 O 2 (Cooper et al., 2002;Mohanty et al., 2014) and H 2 O 2 can transfer more freely through membranes than other ROS (Chanin et al., 2020).Although the blood immune cells in high-ROS environment tends to produce more H 2 O 2 (Cooper et al., 2002;Mohanty et al., 2014) and H 2 O 2 can transfer more freely through membranes than other ROS (Chanin et al., 2020).,Although the blood immune cells in high-ROS environment tends to produce more H 2 O 2 (Cooper et al., 2002;Mohanty et al., 2014) and H 2 O 2 can transfer more freely through membranes than other ROS (Chanin et al., 2020).Because it was speculated that intestinal H 2 O 2 concentration decreased in calves with diarrhea.However, it has been reported that increased ROS transfer to leaky gut to trigger immune response, and intestinal epithelial cells produce more H 2 O 2 to respond to chronic inflammation and dysbiosis (Aviello and Knaus, 2017;Burgueño et al., 2019).
Regulation of inflammation plays a vital role in maintaining homeostasis during enteric infections.Although proinflammatory cytokines control infectious pathogens, anti-inflammatory cytokines activate regulatory responses to control unnecessary inflammation and promote recovery.The anti-inflammatory cytokine IL-10 and the proinflammatory cytokine TNF-α were significantly higher in calves with diarrhea and decreased as calves recovered, which is consistent with the previous studies in calves (Steen et al., 2020;Chuang et al., 2022).Although IL-10 tends to inhibit the production of TNF-α (Tatiya-aphiradee et al., 2019), IL-10 and TNF-α usually show a positive correlation in the immune regulation (Azim et al., 1999;Machado et al., 2014;Wang et al., 2021;Zhang et al., 2021), suggesting an activation of both inflammatory and regulatory responses as calves developed diarrhea.The linkage between oxidative stress and inflammation has been reported in much research, which plays an important role in maintaining homeostasis (Lei et al., 2015).The ROS production after TNF-α stimulation plays an important role in cell death (Kim et al., 2010).It has been reported that TNF-α regulation depends on ROS stimuli and TNF-α can also trigger ROS production (Jin et al., 2008;Babu et al., 2015).Ling et al. have reported that serum ROS and TNF-α in LPS stimulation of calf whole blood were higher simultaneously, suggesting the interaction of oxidative stress and inflammation (Ling et al., 2018).The positive correlation of serum ROS and TNF-α in the present study confirmed the association between oxidative stress markers and inflammatory response during an infection.If ROS, as the main downstream mediators, can be regulated well, it might be possible to regulate any controlled inflammation, which is crucial to maintain intestinal homeostasis during an infection.
In the present study, we did not test these calves for pathogens nor we evaluated other clinical symptoms to determine if the calves suffered from infectious diarrhea.We considered this to be one of the limitations.However, both infectious and noninfectious diarrhea can lead to gut dysbiosis due to the changes in the gut microbial communities (Li et al., 2021b).The present study provided a general connection between diarrhea and oxidative stress.An important limitation of the study is that the causative relationship cannot be distinguished.It is speculated that the oxidative stress can be part of the causes that lead to calf diarrhea.Both infectious and noninfectious diarrhea have been reported to change the inflammatory cytokine levels in calves (Cho and Yoon, 2014;Li et al., 2021a).Healthy calves maintain intestinal barrier integrity and protect gut from pathogen invasion (Chase, 2018).After an infection or other stimuli disturbing barrier integrity, immune cells can be recruited to the damaged area that can produce ROS (Aviello and Knaus, 2017).Although the overaccumulation of ROS induces the leaky gut, altered barrier functions lead to the gut dysfunction resulted in diarrhea (Mankertz and Schulzke, 2007).The intestinal epithelium has been gradually repaired as ROS are scavenged to the normal level (Jung et al., 2020).This can potentially explain the oxidative stress occurrence as calves had diarrhea whereas it disappeared as calves recovered in the present study.However, the systematic pathogenesis of oxidative stress in calf diarrhea has not been studied well.Wu et al. reported that infection-induced intestinal oxidative stress can stimulate immune response in distant organs of body (Wu et al., 2012).This has been reflected in the present study to show a positive correlation of serum and fecal indicators, which indicate the interconnection of the host and lumen redox status.

CONCLUSIONS
We revealed that oxidative stress levels (both systemic and local) change as calves move from being healthy to having diarrhea or vice versa.Serum ROS, MDA, and 8-OHDG could be used as diagnostic biomarkers to screen calves based on enteric health status, especially if they are prone to developing enteric infections.Further studies need to be conducted to find the contribution of gut microbiota, diet, and host to intestinal and systemic redox status in neonatal calves, especially when the susceptibility to enteric infections is high.

FuFigure 1 .
Figure 1.Experimental design.A total of 64 calves were selected from 94 neonatal calves reared on a commercial farm.Based on the fecal score records, calves that suffered from diarrhea from 21 to 28 d of age were put into the DD group (n = 15).Calves that suffered diarrhea at 21 d of age but recovered at 28 d of age were put into the DH group (n = 19).Calves that stayed healthy at 21 d of age but developed diarrhea at 28 d of age were put into HD group (n = 14).Calves that stayed healthy during the experimental periods were put into the HH group (n = 16).The serum and fecal samples were collected from all the experimental calves on d 21 and 28.

Figure 2 .
Figure 2. Diarrhea incidence of each treatment during the experimental period (%).All calves were healthy at the beginning on d 18.All calves in the DD group had diarrhea on d 20 that lasted to d 28.The calves in the DH group had diarrhea on d 20 and 21, then gradually recovered and became all healthy on d 28.The calves in the HD group were all healthy before d 22, then gradually suffered from diarrhea, and all had diarrhea on d 27 and 28.The calves were healthy the whole time of the experiment.

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et al.: OXIDATIVE STRESS IN CALVES WITH DIARRHEA

2P
-value of treatment effects: G = effects of groups (DD, DH, HD, and DD); A = effects of ages (d 21 vs. d 28); H = effects of health status (healthy vs. diarrhea); G × A = interaction between group and age.

Figure 3 .
Figure 3.The effect of the dynamics of calf health (group by age interaction effect) on serum oxidative stress (mean ± SE). (A) Reactive oxygen species (ROS), (B) H 2 O 2 , (C) malondialdehyde (MDA), and (D) 8-hydroxy-2′-deoxyguanosine (8-OHDG).DD group: the calves had diarrhea all the time from d 21 to 28.DH group: the calves had diarrhea on d 21 and recovered on d 28.HD group: the calves were healthy on d 21 and had diarrhea on d 28.HH group: the calves were healthy the whole time from d 21 to 28.Groups with different lowercase letters (a and b) showed significant differences at the same age (P < 0.05).The symbols * and † above the lines refer to a significant difference (P < 0.05) and trend (0.05 < P < 0.1), respectively.A.U. = arbitrary unit.

Figure 4 .
Figure 4.The effect of the dynamics of calf health (group by age interaction effect) on fecal indicators of redox status (mean ± SE). (A) Reactive oxygen species (ROS), (B) malondialdehyde (MDA).DD group: the calves had diarrhea all the time from d 21 to 28.DH group: the calves had diarrhea on d 21 and recovered on d 28.HD group: the calves were healthy on d 21 and had diarrhea on d 28.HH group: the calves were healthy the whole time from d 21 to 28.Groups with different lowercase letters (a and b) showed significant differences at the same age (P < 0.05).The symbols * and † above the lines refer to a significant difference (P < 0.05) and changing trend (0.05 < P < 0.1), respectively, as the calves grew in one group.A.U. = arbitrary unit.

FuFigure 5 .
Figure 5.The effect of the dynamics of calf health (group by age interaction effect) on serum immune factors (mean ± SE). (A) IL-1β.(B) TNF-α.(C) IL-10.DD group: the calves had diarrhea all the time from d 21 to 28.DH group: the calves had diarrhea on d 21 and recovered on d 28.HD group: the calves were healthy on d 21 and had diarrhea on d 28.HH group: the calves were healthy the whole time from d 21 to 28.Groups with different lowercase letters (a and b) showed significant differences at the same age (P < 0.05).The symbol * above the lines refers to a significant difference (P < 0.05) as the calves grew in one group.

FuFigure 6 .
Figure 6.The correlation of systematic and intestinal indicators of redox status.S_MDA and F_MDA refer to serum and fecal malondialdehyde, respectively.S_ROS and F_ROS refer to serum and fecal reactive oxygen species, respectively.All of the calves at each age (n = 128) were involved.Shaded areas represent the 95% confidence interval around the line of best fit.A.U. = arbitrary unit.

Figure 7 .
Figure 7.The correlation of indicators of oxidative stress and inflammatory cytokines.S_ROS refers to serum reactive oxygen species.Randomly selected calves at each age (n = 100) were involved.Shaded areas represent the 95% confidence interval around the line of best fit.A.U. = arbitrary unit.

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et al.: OXIDATIVE STRESS IN CALVES WITH DIARRHEA

Table 1 .
Fu et al.: OXIDATIVE STRESS IN CALVES WITH DIARRHEA Composition of diets of calves

Table 2 .
The BW and ADG of calves in different groups 1 1DD group: the calves had diarrhea all the time from d 21 to 28.DH group: the calves had diarrhea on d 21 and recovered on d 28.HD group: the calves were healthy on d 21 and had diarrhea on d 28.HH group: calves stayed healthy from d 21 to 28.2The ADG from d 21 to 28 during the experimental periods.).Calves in HH group showed a decreasing trend (P = 0.08) for serum 8-OHDG from d 21 to 28.Concentration of CAT and SOD was affected by calf age (A), whereas that of CAT and T-AOC was tended to be affected by calf group (G, Table

Table 3 .
The serum indicator of redox status affected by groups (G), ages (A), and health status (H) calves had diarrhea all the time from d 21 to 28.DH group: the calves had diarrhea on d 21 and recovered on d 28.HD group: the calves were healthy on d 21 and had diarrhea on d 28.HH group: calves stayed healthy from d 21 to 28.
P < 0.05: significant differences among groups within a row, regardless of calf age. 1 DD group: the

Table 4 .
The fecal indicators of intestinal oxidative stress affected by groups (G), ages (A), and health status (H) calves had diarrhea all the time from d 21 to 28.DH group: the calves had diarrhea on d 21 and recovered on d 28.HD group: the calves were healthy on d 21 and had diarrhea on d 28.HH group: calves stayed healthy from d 21 to 28.
P < 0.05: significant differences among groups within a row, regardless of calf age. 1 DD group: the calves had diarrhea all the time from d 21 to 28.DH group: the calves had diarrhea on d 21 and recovered on d 28.HD group: the calves were healthy on d 21 and had diarrhea on d 28.HH group: calves stayed healthy from d 21 to 28. 2 P-value of treatment effects: G = effects of groups (DD, DH, HD, and DD); A = effects of ages (d 21 vs. d 28), H = effects of health status (healthy vs. diarrhea); G × A = interaction between group and age. 3 MDA = malondialdehyde; ROS = reactive oxygen species; A.U. = arbitrary unit.Table 5.The serum immune factors (pg/mL) affected by groups (G), ages (A), and health status (H) P < 0.05: significant differences among groups within a row, regardless of calf age. 1 DD group: the