Dietary Sodium Butyrate Supplementation Reduces High-Concentrate

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Dietary Sodium Butyrate Supplementation Reduces High-Concentrate Diet Feeding-Induced Apoptosis in Mammary Cells in Dairy Goats Guangjun Chang, Jinyu Yan, Nana Ma, Xinxin Liu, Hongyu Dai, Muhammad Shaid Bilal, and Xiangzhen Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05882 • Publication Date (Web): 15 Feb 2018 Downloaded from http://pubs.acs.org on February 19, 2018

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Journal of Agricultural and Food Chemistry

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Dietary Sodium Butyrate Supplementation Reduces High-Concentrate Diet

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Feeding-Induced Apoptosis in Mammary Cells in Dairy Goats

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Guangjun Chang#, Jinyu Yan#, Nana Ma, Xinxin Liu, Hongyu Dai, Muhammad

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Shaid Bilal, Xiangzhen Shen*

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College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, P. R. China

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# Equal contributors

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*Corresponding author:

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Xiangzhen Shen

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Department of Veterinary Clinical Science

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College of Veterinary Medicine, Nanjing Agricultural University

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Nanjing, 210095, P. R. China

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Phone: +86 25 84395505

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Fax：+86 25 84398669

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E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Eighteen lactating goats (38.86 ± 2.06 kg) were randomly allocated to three

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groups. One group was fed a low-concentrate (LC) diet (forage:concentrate = 6:4, LC),

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while the other two groups were fed a high-concentrate (HC) diet (forage:concentrate

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= 4:6, HC) or an HC diet supplemented with sodium butyrate (BHC) for 20 weeks.

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Samples of ruminal fluid, milk, hepatic blood plasma, and mammary gland tissue

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were prepared for the experimental analysis. The LPS concentration, caspase-3 and -8

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enzymatic activity, caspase-3 and -8 mRNA expression, and NF-κB (p65),

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phosphorylated-p65, bax, cytochrome C, and caspase-3 protein expression were

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higher in the HC group than those in the LC group; however, the levels of these

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parameters were lower in the BHC group than those in the HC group. Moreover, bcl-2

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mRNA and protein expression was higher in the BHC group than that in the HC or LC

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groups, and no significant difference was observed between the HC and LC groups.

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Thus, feeding lactating goats an HC diet induces apoptosis in mammary cells, and

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supplementing the diet with sodium butyrate reduces the concentrations of LPS and

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proinflammatory cytokines, subsequently attenuating the activation of NF-κB and

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caspase-3 and eventually inhibiting apoptosis in mammary cells.

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Keywords: High concentrate, Sodium butyrate, Mammary cell apoptosis, NF-κB,

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Dairy goats

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Introduction

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In the dairy-farming industry, dairy cows and goats are often fed

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high-concentrate diets to increase milk production. However, this dietary intake is

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associated with subacute ruminal acidosis (SARA) in ruminants. SARA occurs when

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the ruminal pH values are below 5.5-5.8 for more than 3 h daily.1 One study has

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shown that SARA is successfully induced in dairy cows fed a total mixed rations

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(TMR) containing 20% finely ground wheat.2 A decrease in the milk fat percentage is

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observed in cows suffering from SARA.3 Moreover, SARA induced by the long-term

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feeding of a high-concentrate diet can cause the accumulation of lipopolysaccharide

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(LPS), which is released by gram-negative bacteria, in the gastrointestinal tract of

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ruminants.4 A recent study also suggested that the expression levels of chaperone

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proteins in ruminal epithelial cells are decreased during SARA, indicating that these

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cells are less protected against potentially harmful toxic compounds in the rumen and

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are more vulnerable to cellular damage; hence, impairment of the ruminal barrier

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function is promoted.5 Thus, the accumulated LPS can be translocated from the

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gastrointestinal tract into the circulatory system.6

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LPS can interact with toll-like receptor 4 (TLR4) on the cell surface and evoke an

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NF-κB-dependent signaling cascade via a series of intermediate reactions.7 NF-κB

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plays an important role in cellular apoptosis by regulating the transcription of

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apoptosis-related genes.8 According to Ken K et al., LPS can disrupt the milk-blood

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barrier in mice by damaging the mammary alveolar tight junctions.9 According to Sun Y,

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LPS can induce apoptosis in mammary cells in both dairy cows and mice.10, 11



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Sodium butyrate has been shown to stimulate ruminal and intestinal

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development in newborn calves.12-14 According to P. Guilloteau et al., sodium butyrate

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supplementation enhances the gastrointestinal tract defense system in young calves by

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increasing the levels of the heat shock proteins HSP27 and HSP70.15 Dietary sodium

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butyrate also increases the intestinal expression of occludin and decreases the

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permeability of the small intestine.16 Moreover, sodium butyrate plays an important

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role in anti-inflammatory activity in ruminants.17

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We hypothesized that a high-concentrate diet could induce apoptosis in

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mammary cells and that the addition of sodium butyrate could mitigate the induction of

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apoptosis. Therefore, the objective of this study was to investigate the effect of sodium

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butyrate supplementation on mammary cell apoptosis in dairy goats fed a

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high-concentrate diet.

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MATERIALS AND METHODS

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Chemicals. Dietary sodium butyrate was purchased from Dongying Degao

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Biotechnology, Ji nan, China. Heparin sodium injection (1.25×106 IU/mL) was

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obtained from Shenzhen Sendi Biotechnology Co.Ltd. Ultrapure water was prepared

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from a Milli-Q system (Bedford, MA, United States). Analytical grade phenol,

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isopropanol and chloroform were acquired from Shanghai LingfengChemical ，

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Shanghai, China. And other reagents used in present study were of analytical grade.

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Ethics Statement. All animal experiments were reviewed and approved by the

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Institutional Animal Care and Use Committee of Nanjing Agricultural University. The

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experimental procedures strictly complied with the “Guidelines for Experimental


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Animals” of the Ministry of Science and Technology (2006, Beijing, China).

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Animals and Experimental Design. Eighteen multiparous, lactating Chinese

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Saanen goats (average body weight 38.86 ± 2.06 kg, aged 2-3 years, 4-6 weeks

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postpartum, and average milk yield 1.84 ± 0.72 kg/day) fitted with ruminal fistulas

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and hepatic vein catheters were randomly allocated to three groups; each group

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included 6 goats that were individually raised in stalls at a standard animal feeding

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house at Nanjing Agricultural University (Nanjing, China). One group was fed a

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low-concentrate (LC) diet (forage:concentrate = 6:4) as a control, the second group

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received a high-concentrate (HC) diet (forage:concentrate = 4:6), and the third group

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was fed an HC diet supplemented with sodium butyrate (BHC). Before the initiation

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of formal experiment, all goats were administrated the LC diet for one month adaption

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period to ensure the similarity of the rumen fermentation. During the adaption period,

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rumen fistulas were installed at first week, and hepatic vein catheters at third week.

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After surgical recovery and adaption period, the goats (average dry matter intake

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(DMI) 1.96 kg/day) exhibited an averaged milk yield of 1.22 ± 0.55 kg/day. All goats

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were fed at 8:00 A.M. and 6:00 P.M., the forage and concentrate dietary components

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were fed separately, and the goats had free access to fresh water throughout the

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experimental period. Each feed quantity was weighed as described in Table 1. Table 2

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presents the ingredients and nutritional composition of the diets. The experiment

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duration was 20 weeks. Hepatic vein catheters flushed with sterilized heparin saline

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(150 IU/mL) three times daily at 8 h intervals.

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Ruminal Fluid Sampling and Analysis. Approximately 10 mL of ruminal fluid



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were collected at 0 h (15 min before feeding), 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h after the

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morning feeding for three consecutive days during the 20th week. All samples were

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filtered through two layers of cheesecloth, and 1 mL aliquots of the ruminal fluid

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samples were used to measure the pH using a pH-meter (Sartorius, Germany). The

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remaining fluid from each sample was stored at -20°C until further analysis.

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Milk Sampling and Analysis. Milking was performed after each feeding, and the

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milk yield of each goat was measured and recorded daily from the 2nd week to the 20th

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week. A 50-mL aliquot of milk was preserved with potassium dichromate and stored

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at 4°C to determine the concentrations of milk fat and protein using a FossoMatic

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5000 analyzer (FOSS Electric A/S, Denmark).

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Blood Sampling and Analysis. Blood samples were collected from the hepatic

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vein into 5-mL heparin vacuum tubes 4 h after the morning feeding on the final three

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days of the twentieth week. The plasma was separated by centrifuging the blood

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samples at 3000×g for 15 min, collected into pyrogen-free glass tubes, and

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subsequently stored at -20°C to determine the LPS and proinflammatory cytokine

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levels.

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LPS was detected using a Chromogenic Endpoint Tachypleus Amebocyte Lysate

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Assay Kit (cat. CE64406; Chinese Horseshoe Crab Reagent Manufactory Co., Ltd;

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Xiamen, China) with a minimum detection limit of 0.01 EU/mL in the plasma.

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Radioimmunoassay kits were used to determine the concentrations of the

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proinflammatory cytokines IL-1β (cat. C09DJB, Beijing North Institute of Biological

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Technology, Beijing, China), IL-6 (cat. C12DJB, Beijing North Institute of Biological


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Technology, Beijing, China) and TNF-α (cat. C06PJB, Beijing North Institute of

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Biological Technology, Beijing, China).

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RNA Extraction, cDNA Synthesis and Real-time PCR. After collecting the

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ruminal fluid and blood samples, all goats were anesthetized and slaughtered by diet

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group in the following order: LC, HC and BHC. The mammary tissue was

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immediately collected, transferred to liquid nitrogen and stored at -80°C until RNA

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extraction. Total RNA was extracted using an RNAiso Plus Kit according to the

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manufacturer’s instructions (cat. 9108, Takara, Dalian, China), and the concentration

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and quality of the RNA were assessed using a NanoDrop ND-1000 Spectrophotometer

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(Thermo Fisher Scientific Inc., Waltham, USA). Then, 400 ng of total RNA was used

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to synthesize cDNA using the PrimeScript RT Master Mix Kit (cat. RR036A, Takara,

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Dalian, China). Real-time PCR was conducted using a SYBR Premix Ex TaqTM Kit

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(cat. DRR420A, Takara, Dalian, China) on an ABI 7300 Real-Time PCR System

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(Applied Biosystems, Foster City, CA, USA). The PCR protocol included a

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denaturation step at 95°C for 15 s, followed by 40 cycles at 95°C for 5 s and 60°C for

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31 s. The mRNA expression levels of the candidate genes were normalized to the

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mRNA level of the housekeeping gene glyceraldehyde phosphate dehydrogenase

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(GAPDH), and the data were analyzed using the 2-△△Ct method. The primers used to

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amplify the candidate genes were synthesized by Generay Company (Shanghai, China)

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and are listed in Table 3.

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Caspase-3 and -8 Activity Analysis. In total, 100 mg of frozen minced mammary

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tissue were prepared for the total protein extraction and homogenized using a Dounce



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homogenizer after adding 1 mL ice-cold RIPA lysis buffer (cat. SN338; Sunshine

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Biotechnology Co., Ltd; Shanghai, China). The supernatant was collected after

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centrifugation at 12,000 rpm for 15 min at 4°C. The protein concentration was

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measured using a PierceTM BCA Protein Assay Kit (cat. 23225, Thermo Fisher, USA).

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The protein was diluted to the same final concentration for all samples. The enzyme

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activity of caspase-3 (cat. KGA203, KeyGEN Biotech. Co. Ltd, Nanjing, China) and

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caspase-8 (cat. KGA303, KeyGEN Biotech. Co. Ltd, Nanjing, China) was determined

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using caspase activity assay kits according to the manufacturer’s instructions.

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Western Blotting. The procedure performed for the extraction from the mammary

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gland is the same as that described in the above paragraph. After the total protein

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preparation, 50 µg of total protein from each sample was loaded onto 10%

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SDS-PAGE gels to separate the proteins. After the separation, the proteins were

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transferred onto nitrocellulose (NC) membranes (Bio Trace, Pall Co., Washington,

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USA). The membranes were blocked with a buffer containing either 10% skim milk

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or 10% BSA (to detect the phosphorylated proteins) dissolved in Tris-buffered saline

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with Tween (TBST) for 2 h at room temperature. Subsequently, the NC membranes

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were washed and incubated with a primary antibody diluted in TBST at 4°C overnight.

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The primary antibodies were diluted at the following ratios: NF-κB p65 (1:300; cat.

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sc-293072, Santa Cruz Biotechnology, USA), phosphorylated NF-κB p65 (1:300; cat.

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sc-33020, Santa Cruz Biotechnology, USA), bax (1:500; cat. AB026, Beyotime,

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Shanghai, China), bcl-2 (1:1000; cat. AB112, Beyotime, Shanghai, China),

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cytochrome C (1:200; cat. AC908, Beyotime, Shanghai, China), caspase-3 (1:1000;


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cat. AC030, Beyotime, Shanghai, China), cleaved caspase-3 (1:1000; cat. Ac033,

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Beyotime, Shanghai, China) and β-actin (1:10000; cat. LM240D, BioWorld, USA),

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which was used as a reference protein. Then, the membranes were washed and

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incubated with the corresponding secondary antibodies for 2 h at room temperature.

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The following secondary antibodies were used: goat anti-rabbit (1:5000, cat. sc-2004,

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Santa Cruz Biotechnology, USA) was used for p65, phosphorylated p65, bcl-2,

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caspase-3 and cleaved caspase-3, and goat anti-mouse (1:10000; cat. SN133,

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SunShineBio, China) was used for β-actin, bax and cytochrome C. Finally, the

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membranes were treated with an enhanced chemiluminescence (ECL) detecting kit

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(cat. E411-04, Vazyme Biotech. Co., Ltd; Nanjing, China). The signals were recorded

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using the LAS4000 imaging system (, GE Healthcare Bio-Sciences AB, Uppsala,

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Sweden), and the results were analyzed using Quantity One (Bio-Rad Laboratories,

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Inc., Hercules, CA, United States).

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Statistical Analysis. The ruminal pH value, milk yield, milk fat concentration, milk

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protein concentration, milk fat/milk protein ratio, and milk lactose concentration were

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analyzed by performing repeated measures analysis of variance using the SAS

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software MIXED procedure (SAS version 9.4, SAS Institute Inc.). Diet and time were

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considered fixed factors, and the individual goat was considered a random factor.

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Time, diet and goat were considered repeated measures variables, and compound

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symmetry (CS) was used as a type of covariance. The LPS concentration, enzyme

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activity, and mRNA and protein expression were analyzed by performing an ANOVA

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using SAS. The differences were considered significant at P < 0.05.



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RESULTS

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Rumen pH. The rumen pH value in the BHC group was between that in the HC

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group and that in the LC group at all tested time points. Additionally, in the HC group,

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a pH value below 5.8 was observed for 4 h. The BHC group had a significantly higher

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pH value than the HC group for 2 h after the morning feeding (P < 0.05, Figure 1).

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Furthermore, the pH value in the HC group was lower than that in the BHC group 8 h

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after the morning feeding, which lasted for 2 h (P < 0.05, Figure 1). The pH value in

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the BHC group was lower than that in the LC group after the morning feeding, which

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lasted for 6 h (P < 0.05, Figure 1).

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Milk Yield and Milk Composition. The BHC group exhibited a significantly

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higher (P < 0.05, Table 4) milk yield than the LC and HC groups throughout the

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experiment. From week 7 to week 19, the milk fat percentage in the LC and BHC

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groups was higher (P < 0.05, Table 4) than that in the HC group. From week 2 to

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week 15, the BHC group exhibited a significantly higher (P < 0.05, Table 4) protein

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percentage than the HC group. From week 7 to week 19, the lactose percentage in the

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HC and BHC groups was higher (P < 0.05, Table 4) than that in the LC group.

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LPS Concentration in Hepatic Venous Plasma. As shown in Figure 2, the LPS

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concentration in the HC and BHC groups was higher (P < 0.01) than that in the LC

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group, whereas that in the BHC group was lower (P < 0.05) than that in the HC group.

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Proinflammatory Cytokines in Hepatic Venous Plasma. The concentrations of

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proinflammatory cytokines (IL-1β, IL-6 and TNF-α) were higher (P < 0.05, Table 5)

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in the HC group than those in the LC and BHC groups. A significant difference was


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observed in the IL-6 level (P < 0.05, Table 5), but no significant difference in the

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IL-1β and TNF-α levels was observed between the LC and BHC groups.

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Gene Expression in Mammary Tissue. The mRNA expression of caspase-3 in the

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mammary gland tissue was higher (P < 0.05, Figure 3) in the HC and BHC groups

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than that in the LC group but was lower (P < 0.05, Figure 3) in the BHC group than

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that in the HC group. The mRNA expression of caspase-8 in the HC and BHC groups

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was higher (P < 0.05, Figure 3) than that in the LC group, but no significant

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difference was observed between the HC and BHC groups. In addition, the mRNA

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expression of bcl-2 in the BHC group was higher (P < 0.01, Figure 3) than that in the

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HC and LC groups.

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Caspase Activity in Mammary Tissue. The enzymatic activity of caspase-3 in the

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mammary tissue in the HC group was higher (P < 0.01, Figure 4) than that in the LC

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and BHC groups. Compared to the LC diet, the HC and BHC diets increased (P

Dietary Sodium Butyrate Supplementation Reduces High-Concentrate

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