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Dietary addition of Humulus scandens improved the intestinal barrier in rabbits

Dietary addition of Humulus scandens improved the intestinal barrier in rabbits JOURNAL OF APPLIED ANIMAL RESEARCH 2023, VOL. 51, NO. 1, 62–68 https://doi.org/10.1080/09712119.2022.2154215 a,b a,b a,b a,b a,b a,b Gongyan Liu , Lihong Hao , Cheng Wang , Yin Zhang , Haitao Sun and Hongmei Hu Institute of Animal Husbandry and Veterinary, Shandong Academy of Agricultural Sciences, Shandong Key Laboratory of Animal Disease Control and Breeding, Jinan, People’s Republic of China; Key Laboratory of Livestock and Poultry Multi-omics of MARA, Jinan, People’s Republic of China ABSTRACT ARTICLE HISTORY Received 19 April 2022 This study was conducted to investigate the effect of Humulus scandens (HS) in diet on the production Accepted 28 November 2022 performance and intestinal barrier of rabbits. One hundred and sixty Laiwu black rabbits at 35 days of age with body weight of 692.5 ± 48.3 g were divided into four groups (five replicates per group and KEYWORDS eight rabbits per replicate): fed on a basal diet free of HS (control), 4%, 8% or 12% HS powder Humulus scandens; intestinal supplement. The results showed that the dietary addition of HS decreased the ADFI, F/G, diarrhoea barrier; rabbits; production ratio and mortality ratio. Besides, the final body weight and ADG (P < .05) increased with the level of performance HS. Rabbits in 12% HS group had a higher sIgA and IgG concentration in serum and ileum than control, while lower TNFα, IFN-γ and IL-6 concentration (P < .05). Compared with the control, the HS supplementation groups for 12% level could decrease the mRNA expression of ZO-1, JAM3 and mucin1 (P < .05). In conclusion, dietary supplement of HS modulates immune responses and enhances intestinal barrier, meanwhile inhibits the synthesis of cytokine. Besides, our experiment offers positive evidence in improving rabbit health of HS as rabbit feed resource. 1. Introduction it is reported that flavonoids play a strong immune role by inhibiting the proliferation of immune cells and the pro- The disease of digestive tract is the most important disease in duction of pro-inflammatory cytokines (Rios 2010). HS is rabbits, accounting for 70% (Carabaño et al. 2008). The mor- wild on the roadside in the field, which adapts to various tality of the weaned rabbits up to 60% due to epizootic environmental conditions and has high yield (Urziceanu rabbit enteropathy (Fann et al. 2001; Carabaño et al. 2008). et al. 2022). In addition, because of its strong vitality and Rabbit diarrhoea is a big problem in present rabbit pro- harm to the growth of crops, it is often regarded as a malig- duction, especially in the weaning stage (Martens and Van nant weed, so far it has not been systematically developed Herck 2000). Weaned rabbits had thinner intestinal walls, and utilized, resulting in serious waste of resources (Zhou incomplete intestinal barrier, frequent diarrhoea or gastroin- and Feng 2020). However, there was little information avail- testinal flatulence (Fann et al. 2001). Antibiotics have been able about HS use as feedstuff in rabbit production and widely used to control mortality, increase breeding costs, special its effect on improving intestinal barriers. The affect product quality and reduce the economic benefits of present experiment was conducted to investigate the effect many farms. For this reason, some herbs and plant extracts of HS on the performance and intestinal barrier of rabbits, to substitute antibiotic have been found and studied by to identify applicative effects HS as a feed resource in rabbit many researchers. Humulus scandens (HS) is widely used in production. China to treat dysentery and chronic colitis as one traditional Chinese medicine, and contains polysaccharides, flavonoids, volatile oil and other chemical components (Chen et al. 2. Materials and methods 2012). Li et al. (2008) had reported that HS was rich in flavo- 2.1. Production of HS powder noids and there have been three kinds (vitexin, luteolin and cosmosiin) of flavonoids separated from HS. Flavonoids are Harvest the HS from countryside in Shandong Province in the a kind of common polyphenols in the plant world. They autumn mature stage, and naturally drying and pulverizing. have biological activities such as anti-inflammatory, anti- oxidant and anti-cancer (Korkina and Afanas’ev 1997; Neuhou- 2.2. Animals, diet and feed chemical composition ser 2005; Talhouk et al. 2007; Zhang et al. 2021; Nath et al. 2022). Li et al. (2022) flavonoids could inhibit alcohol- One hundred and sixty Laiwu black rabbits (male–female rate induced hepatocyte injury, which might be attributed to alle- of 1:1) at 35 days of age weight of 692.5 ± 48.3 g were divided viating oxidative stress and mitigating inflammatory response into four groups (five replicates per group and eight rabbits by activating Nrf2 and inhibiting NF-κB pathways. In addition, per replicate): f fed on a basal diet with 0 (control), 4%, 8% CONTACT Hongmei Hu huhongmeipatty@163.com; Haitao Sun wwww8888@163.com Institute of Animal Husbandry and Veterinary, Shandong Academy of Agricultural Sciences, Shandong Key Laboratory of Animal Disease Control and Breeding, Jinan 250100, People’s Republic of China © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, dis- tribution, and reproduction in any medium, provided the original work is properly cited. JOURNAL OF APPLIED ANIMAL RESEARCH 63 Table 1. Chemical composition of Humulus scandens powder (air-dry basis, %). histological measurements. The other intestinal segments Items Humulus scandens powder were collected by cryopreservation tube, rapidly frozen in Dry matter (DM) 91.60 liquid nitrogen and stored at −80°C until analysis. Crude protein (CP) 16.00 Ether extract (EE) 0.90 Neutral detergent fibre (NDF) 47.40 Acid detergent fibre (ADF) 36.90 Aid detergent lignin (ADL) 9.20 2.4. Determination of indicators and methods Ash 16.50 Calcium 1.93 2.4.1. Feed chemical analysis Phosphorus 0.35 The chemical composition of the SH and diets was analysed fol- Polysaccharide (mg/g) 19.53 Total flavonoids (mg/g) 6.86 lowing the procedures of AOAC International (2005). The dry matter was determined by oven drying at 105°C (procedure 934.01), and the ether extract was determined by extraction or 12% HS powder, and the chemical of HS were listed in with petroleum ether in a Soxtec 1043 apparatus (FOSS Table 1. The basal diets were formulated according to the Tecator AB, Hoganas, Sweden; procedure 920.39). The protein values for growing from De Blas and Mateos (2020) and the content was determined using a Kjeltec Auto 1030 Analyser ingredients and composition of diet are listed in Table 2. (procedure 954.01). The ash content was determined according Besides, the diet pelleted by the use of pressure and the to procedure 920.153. Analysis of fibre components was per- diameter of the pellets was 4 mm. The experiment lasted formed according to Van Soest et al. (1991). The calcium and for nine weeks which included one week adaptation period phosphorus contents were determined by inductively and eight weeks experimental period. During the rearing coupled plasma atomic emission spectrometry (ICP-EMS; period, the rabbits were individually housed cage and had Optima 8000, Perkin Elmer, U.S.A.). free access to feed and water. The content of polysaccharides in HS was detected by phenol-sulphuric acid method. Preparation of standard 2.3. Sample collection and preparation glucose solution: accurately weigh 20 mg of anhydrous glucose dried at 105°C to constant weight, put it in a 250 mL At the end of the trial (98 days), 40 rabbits (10 rabbits per volumetric flask, fix the volume and shake well for use. group, male and female had half each, and the average body Drawing the standard curve: accurately absorb 0, 0.2, 0.4, 0.6, weight of the two rabbits equals to the average body weight 0.8, 1.0 and 1.2 mL of glucose standard solution, put them in of each replicate) were sacrificed by cervical dislocation. a test tube with a stopper, add water to 2 mL respectively, About 4 cm segment of the middle ileum was collected and and shake well, then add 1.0 mL of 5% phenol solution, divided into two segments. One intestinal segments were quickly add concentrated H SO 5 mL, shake well and leave it 2 4 flushed gently with phosphate-buffered saline (PBS, pH 7.4) at room temperature for 30 min until it is cooled. Then, use and then placed in 10% fresh, chilled formalin solution for an ultraviolet spectrophotometer at 490 nm wavelength. Determination of polysaccharide content in the sample: the Table 2. Composition and nutrient levels of basal diets (air-dry basis, %). absorbance value is determined according to the above stan- Levels of Humulus scandens dard method, and the content is calculated according to the Items 0 (Control) 4 8 12 standard curve. Raw material composition The content of flavonoids in HS was detected by colorime- Corn 15.0 15.0 15.0 15.0 try. Accurately weigh 1 g of raw materials, add 50% ethanol sol- Soybean meal 10.0 10.0 10.0 10.0 ution according to the ratio of material to liquid (1:20–1:40, g/ Wheat bran 20.0 20.0 20.0 20.0 Corn germ meal 15.0 15.0 15.0 15.0 ml), perform ultrasonic treatment for 2 h, centrifuge at 3000 r/ Soybean straw powder 7.0 7.0 7.0 7.0 min for 20 min and collect supernatant for determination. Peanut seedling powder 30.0 26.0 22.0 18.0 Taking rutin as standard, and mix 1.5 mL of extract with Humulus scandens 0.0 4.0 8.0 12.0 Premix 3.0 3.0 3.0 3.0 1.5 mL of 2% AlCl -methanol solution (containing 5% acetic Total 100.0 100.0 100.0 100.0 acid), stand for 10 min, and measure the absorbance at Nutrient levels 430 nm. With rutin as the standard sample, a standard curve Digestible energy (DE, MJ/kg) 10.14 10.21 10.26 10.29 Crude protein (CP) 15.10 15.60 16.30 16.70 was established in the range of 10–500 μg/mL. Ether extract (EE) 2.00 2.10 2.20 2.50 Neutral detergent fibre (NDF) 56.80 56.70 52.50 50.00 Acid detergent fibre (ADF) 43.30 43.00 41.40 40.60 Aid detergent lignin (ADL) 5.77 5.80 5.41 5.32 2.4.2. Growth performance Ash 11.60 11.20 11.80 11.10 Calcium 1.27 1.14 1.26 1.19 Body weights of each replicate rabbits were measured before Phosphorus 0.62 0.67 0.75 0.68 feeding at the beginning and end of the trial, and the ADG Premix provided the following per kg of diets: vitamin A 10,000 IU, vitamin D was calculated from 42 to 98 days of age. The ADFI was calcu- 2000 IU, vitamin E 50 mg, vitamin K 2.5 mg, thiamine 5 mg, riboflavin lated according to total feed intake divided by the total number 10 mg, nicotinic acid 20 mg, pantothenic acid 50 mg, folic acid 2.5 mg, vitamin B 1 mg, choline chloride 400 mg, iron 100 mg, zinc 50 mg, copper of experimental days in each replicate. The F/G was then calcu- 40 mg, manganese 30 mg, iodine 0.5 mg, selenium 0.05 mg, calcium hydrogen lated as the feed intake/weight gain. The number of rabbits phosphate 15 g, sodium chloride 5 g, lysine 1.5 g, methionine 1.5 g, the rest is with diarrhoea or dead was recorded daily throughout the miscellaneous meal carrier complement. Digestible energy was calculated, whereas the others were measured values. study. 64 G. LIU ET AL. Table 3. Gene-specific primers used for the analysis of rabbit gene expression. 2.4.3. Slaughter performance ′ ′ Gene name GeneBank Primer sequences (5 –3 ) Product size, bp As described by Blasco and Ouhayoun (1996), the slaughter GAPDH NM_001082253 GTCAAGGCTGAGAACGGGAA 129 procedure and carcass analysis were performed. Twelve hours GAAGACGCCAGTGGATTCCA before slaughter, rabbits were fasted and weighed, which claudin1 NM_001089316 AAAGATGCGGATGGCTGTCA 187 was pre-slaughter body weight. After bleeding, the pelts, AAGGCAGAGAGAAGCAGCAG Occludin XM_008262318 TCGTTTGAGCAGCAGCAGTA 161 paws and full gastrointestinal tract were removed, and the ACTCCCTGATCCAGTCCTCC commercial carcass was weighed (preserving the head, ZO1 XM_017348357 AGAGGATTTGTCAGCCCAGC 179 trachea, oesophagus, thoracic organs, liver, kidney and perire- CTCGCGGTTCACTTTTTGCA JAM3 XM_008248362 GAAGTCCTGGGGCAGACTTC 158 nal fat). The semi-clean carcass weight was commercial CTGCAGACAGGGGTTACTGG carcass weight removing the head at the first cervical vertebra, mucin1 XM_017345830 CACACCATCTTCGGTTCCCA 197 removing the trachea and oesophagus, retaining the liver, ACTTGCTGCTTGGATCCTCC kidney and perirenal fat. The full-clean carcass weight is the GAPDH = glyceraldehyde 3-phosphate dehydrogenase; ZO1 = zonula occluden protein 1; JAM3 = junctional adhesion molecules 3. semi-clean carcass weight removing the heart, liver, kidney and perirenal fat. At the same time, the thymus, spleen, heart, liver and kidney are weighed. At the same time, the com- (Liu et al. 2017). The PCR primers used in this study (Table 3) mercial slaughter ratio, semi-clean slaughter ratio, full-clean were designed using a Premier 5.0 Software and synthesized slaughter ratio, thymus ratio, spleen ratio, heart ratio, liver by Ruibo Biological Engineering Co., Ltd. (Qingdao, China). ratio and kidney ratio were calculated by dividing the live The relative mRNA expression levels were calculated using weight before slaughter. −△△Ct the arithmetic formula 2 (Livak and Schmittgen 2001). 2.4.4. Morphological structure of small intestine The ileum tissue samples were cut longitudinally at the mesen- 2.5. Statistical analysis teric attachment and immediately fixed in 10% neutral forma- All of the data collected were subjected to one-way ANOVA lin. The samples were then dehydrated in graded alcohols, analysis with the Statistical Analysis Systems statistical software cleared with xylene and embedded in paraffin. The serial micro- package (Version 8e, SAS Institute, Cary, NC), and the primary tome sections (5 µm thick) were stained with haematoxylin and effect of HS treatments on the responses among the various eosin stain. In each slide, 10 well-oriented villi and crypts were groups, and Duncan’s test was used for multiple comparisons. analysed at a low magnification (40×) with a light microscope Besides, the data are shown as the means and root mean errors (using Image-Pro Plus 6.0 software, U.S.A.). (RMSE), P < .05 was statistically significant. 2.4.5. Immunoglobulins and cytokines levels The ileum samples were disrupted and homogenized in PBS 3. Results buffer (1 g samples: 9 ml PBS). After centrifuging with a speed of 5000 g for 10 min, the supernatant was isolated. The 3.1. Growth performance IgG, IgM and sIgA concentrations were measured with an The growth performance of the experimental rabbits is pre- immunoturbidimetry using Enzyme-Linked Immunosorbent sented in Table 4. Dietary HS supplemental level had significant Assay kits (CEA544Rb, CEA543Rb, SEA641Rb, respectively; effect on ADFI, ADG, F/G, diarrhoea ratio and mortality ratio (P Cloud-Clone Corp, Wuhan, China). The IgG, IgM and sIgA in < .05) of meat rabbits, with the increase of addition level, the supernatant could combine the IgG, IgM and sIgA antibody feed intake, F/G, diarrhoea ratio and mortality ratio decreased, in 0.02 mol/L phosphate buffer substrate turnover was moni- and the FBW and ADG increased. tored spectrophotometrically at 450 nm with an automated reader (BioTek, Winooski, Vt.). The levels of IgA, TNFα, IFN-γ and IL-6 were determined using Enzyme-Linked Immunosor- 3.2. Slaughter performance bent Assay kits (CSB-E06946Rb, CSB-E06998Rb, CSB- E06925Rb, CSB-E06903Rb, respectively; CUSABIO, Qingdao, Dietary HS supplemental level had significant effect on pre- China). After 1 h of incubation at 37°C, samples were slaughter body weight, commercial carcass weight, semi- removed and the plates were washed with a PBS buffer. After clean carcass weight, full-clean carcass weight and kidney that, 100 μL horseradish peroxidase, and 100 μL of tetramethyl- weight (P < .05) of experimental rabbits, the pre-slaughter benzidine substrate was added to each well. The mixture was body weight, commercial carcass weight, semi-clean carcass incubated in the dark for 30 min at room temperature. weight, full-clean carcass weight in the higher group (8% and Optical density value at 450 nm was measured by a Bio-Rad 12% group) were higher than those of the control group. ELISA reader (Bio-Rad, Richmond, CA). Besides, the kidney weight of the experimental group (4%, 8% and 12% group) with HS was significantly higher than 2.4.6. RNA isolation and analysis those of the control group. Furthermore, HS supplemental of Total RNA Extraction Reagent Kit (RNAiso Plus, Takara, Code maternal diets had no influence on the heart weight, lung No. 9108/9109) was used to ileum RNA Isolation, and weight, liver weight or the commercial slaughter ratio, semi- qRT-PCR by SYBR Premix Ex Taq™ II (Tli RNaseH Plus, Takara, clean carcass ratio, full-clean carcass ratio, heart index, lung Code No. RR820A) was performed as described previously index, liver index (P > .05; Table 5). JOURNAL OF APPLIED ANIMAL RESEARCH 65 Table 4. Effects of Humulus scandens supplementation on growth performance and feed intake in rabbits. Levels of Humulus scandens,% Items 0 (Control) 4 8 12 RMSE P-value IBW, g 876.5 886.8 887.0 892.7 57.2615 .9755 b b a a FBW, g 2313.0 2367.8 2495.3 2567.5 84.2817 .0008 c bc ab a ADG, g/d 25.65 26.45 28.72 29.91 1.9991 .0136 ab a b b ADFI, g/d 100.54 101.80 98.91 98.91 2.3075 .0201 a ab bc c F/G 4.13 3.96 3.71 3.40 0.2699 .0032 1 a ab b b Diarrhoea ratio , % 12.50 7.50 5.00 5.00 0.2245 .0209 2 a ab b b Mortality ratio , % 7.50 5.00 2.50 2.50 0.2058 .0490 Diarrhea ratio (%) = 100 × (number of rabbits with diarrhoea × average diarrhoea days)/(number of experimental rabbits × experimental days). Mortality (%) = 100 × (number of rabbits with death/number of experimental rabbits). a,b,c Different superscript letters means different (p < .005). RMSE, root mean square error; IBW, initial body weight; ADFI, average daily feed intake; ADG, average daily gain; F/G, feed to gain ratio. Table 5. Effects of Humulus scandens supplementation on carcass traits of rabbits. Levels of Humulus scandens,% Items 0 (Control) 4 8 12 RMSE P-value b ab a a Pre-slaughter body weight, g 2259.6 2379.0 2473.0 2535.0 114.7642 .0089 c bc ab a Commercial carcass weight, g 1254.9 1331.5 1396.7 1488.0 82.7896 .0029 c bc ab a Semi-clean carcass weight, g 1109.5 1186.4 1247.6 1336.0 79.5357 .0027 c bc ab a Full-clean carcass weight, g 1023.0 1091.0 1145.6 1234.2 72.8901 .0024 Heart weight, g 5.93 5.97 6.49 6.20 0.3882 .1253 Lung weight, g 11.52 10.94 13.26 12.10 1.6660 .1932 Liver weight, g 65.33 71.22 75.33 78.65 8.2528 .1040 b a a a Kidney weight, g 12.09 14.16 14.58 14.42 1.0303 .0049 Commercial slaughter ratio, % 55.55 55.97 56.50 58.65 1.9648 .1003 Semi-clean slaughter ratio, % 49.12 49.85 50.46 52.65 1.9214 .0544 Full-clean slaughter ratio, % 45.31 45.86 46.35 48.64 1.9357 .0700 Heart ratio, g/kg 2.62 2.52 2.56 2.51 0.1762 .7443 Lung ratio, g/kg 5.10 4.61 5.24 4.90 0.7468 .5877 Liver ratio, g/kg 28.92 29.81 29.71 31.72 2.2454 .2770 Kidney ratio, g/kg 5.10 4.61 5.24 4.90 0.7468 .5877 RMSE, root mean square error. intestinal was decreased by HS supplementation level 3.3. Immune organ development increased. Compared with the control, the HS supplementation As shown in Table 6, dietary HS supplemental level had no sig- in the diet did not alter the level of IgM (P > .05) in serum. nificant effect on thymus weight, spleen weight or thymus ratio, spleen ratio (P > .05) of experimental rabbits. 3.6. Gene expression of physical barrier 3.4. Morphological structure of small intestine Agarose gel map for RNA quality was seen in Figure 1. The HS supplementation in the diet did not alter the mRNA expression The HS supplementation in the diet did not alter the villus of claudin1 and occludin (P > .05) in the small intestine (P > .05, height, crypt depth, villus width, mucosal layer thickness and Figure 2). Compared with the control, the HS supplementation muscle layer thickness (P > .05) of the small intestine (Table 7). groups for 12% level could decrease the mRNA expression of ZO-1, JAM3 and mucin1 (P < .05). 3.5. Immune active compounds Table 7. Effects of Humulus scandens supplementation on intestinal morphology The HS supplementation in the diet could improve the titre of of rabbits. the IgG, IgA and sIgA (P < .05) in serum and intestinal (Table 8). Levels of Humulus scandens,% However, the titre of the TNF-α, IFN-γ and IL-6 in serum and 0 P- Items (Control) 4 8 12 RMSE value Table 6. Effects of Humulus scandens supplementation on immune organ Villus height, 865.30 900.61 803.88 771.05 180.1309 .3533 development of rabbits. µm Crypt depth, 147.45 169.63 179.81 167.53 35.6639 .5539 Levels of Humulus scandens,% µm Items 0 (Control) 4 8 12 RMSE P-value Villus width, µm 137.48 147.83 157.65 162.46 22.7420 .3466 Thymus weight, g 4.55 4.72 4.82 4.80 0.8512 .9529 Mucosal layer 1017.60 1143.90 1134.90 1228.70 199.0111 .4396 Thymus ratio, g/kg 1.91 1.94 2.01 1.99 0.3742 .9665 thickness, µm Spleen weight, g 1.16 1.28 1.09 1.05 0.2061 .3272 Muscle layer 104.29 117.45 106.16 124.46 22.8826 .4764 Spleen ratio, g/kg 0.51 0.54 0.43 0.42 0.0841 .0918 thickness, µm RMSE, root mean square error. RMSE, root mean square error. 66 G. LIU ET AL. Table 8. Effects of Humulus scandens supplementation on immune active main factor responsible for a reduction in digestibility of raw compounds of rabbits. feed and feedstuffs (Gidenne and Perez 1994). The cell wall lig- Levels of Humulus scandens,% nification is lower in higher HS level (12%group) than that in control groups (Table 2). Thus, the lower fibre level should Items (Control) 4 8 12 RMSE P-value explain the higher energy and protein in diets containing HS Immune active compounds in serum b a a a and the subsequent reduction in ADFI. Carcass weight is IgG titre, μg/mL 7.53 8.01 8.25 8.32 0.4612 .0080 IgM titre, ng/mL 645.31 659.96 671.89 692.10 41.6420 .1695 closely related to live weight before slaughter, the results of c b ab a IgA titre, ng/mL 114.20 121.13 127.57 130.29 6.4129 .0001 slaughter ratio for rabbits showing no or few effects of fibre c bc a ab sIgA titre, ng/mL 159.90 177.37 209.08 190.05 24.9001 .0041 sources inclusion, which was agree with the previous studies TNF-α titre, ng/mL 3.28 3.00 3.17 2.99 0.4218 .4701 a a a b IFN-γ titre, pg/mL 362.07 352.68 328.26 237.77 66.8278 .0033 (Dal Bosco et al. 2012; Margüenda et al. 2012). Guo et al. a b b b IL-6 titre, pg/mL 327.41 263.98 270.62 269.45 38.4523 .0079 (2008) reported that the mixture (50:50) of HS and alfalfa consti- Immune active compounds in intestinal b a a a tutes an alternative source of fibre for fattening rabbits and pre- IgG titre, μg/g 14.10 23.80 21.68 25.54 6.4128 .0071 c b a a IgM titre, ng/g 21.82 34.31 43.25 50.71 8.0591 <.0001 vents diarrhoea effectively. In this study, we firstly investigate the b a a ab IgA titre, ng/g 3.42 13.74 12.94 10.01 7.1810 .0311 addition effect of HS in rabbit production. After detecting the b a a a sIgA titre, ng/g 222.26 354.17 320.14 311.71 68.4362 .0046 a a a b indicators of performance, intestinal morphology and intestinal TNF-α titre, ng/g 22.83 22.78 22.56 14.66 4.5753 .0023 a a a b IFN-γ titre, ng/g 73.48 70.05 68.57 59.64 7.4278 .0064 barrier, the present study showed a positive effect of HS as a ab ab b IL-6 titre, ng/g 1.74 1.47 1.38 1.27 0.3309 .0499 rabbit feed resource. RMSE, root mean square error. The intestinal mucosal barrier is mainly composed of epi- thelial cells, tight junctions between adjacent enterocytes, and critical components of the mucosal immune system (Turner 4. Discussion 2009). It acts as a selective barrier that allows the absorption As we all know, HS often grows wild in ditches, wastelands, ruins and excretion of substances required for the homogenate and forest edges, which adapts to various environmental con- state of the body and prevents the passage of various pathogens ditions and can be harvested in large quantities in the autumn or their products. Under normal circumstances, epithelial cells mature stage. HS has been used to treat inflammatory diseases allow only a small amount of intact antigens to enter the in China. Luteolin is one of the flavonoids isolated from HS, it mucosa, where they interact with the mucosal immune system is a common flavonoid, which exists in many herbal medicines. to reduce the inflammatory response. At the same time, intesti- Plants rich in luteolin have been used in traditional Chinese nal pathogens can activate immune cells and initiate the inflam- medicine to treat inflammatory disorders diseases (Ziyan et al. matory response required to eliminate infection (Chadwick et al. 2007; Lin et al. 2008). The main constituent of HS is fibre 2002). Under the condition of inflammatory bowel disease, the (Table 1). In our study showed that in rabbits of similar initial excessive penetration of antigen through the epithelial layer body weight (Table 3), the inclusion of HS in the diet decreased may lead to inappropriate immune stimulation and chronic gas- the ADFI and F/G in rabbits between 42 and 98 days of age. This trointestinal inflammation (Baumgart and Carding 2007). In result may be due to the difference in energy level in the four addition, epithelial cells dilute, flush and bind harmful sub- diets by different level with HS powder. Accordingly, the levels stances by secreting liquid and mucus and sIgA into the of fibre and fibre proportions in diet decreased when dietary lumen, so as to play an important physiological defence role HS levels increased (Table 2). The degree of lignification is the (Shang et al. 2008). sIgA is a major mucosal immune effector Figure 1. Agarose gel map for RNA quality. A, B, C and D represent Humulus scandens were supplemented at 0, 4%, 8% and 12%, 1–6 represent different samples of this group. JOURNAL OF APPLIED ANIMAL RESEARCH 67 Figure 2. Effect of Humulus scandens on mRNA expression of physical barrier gene in ileum of rabbits. Values are presented as the means ± std. error (n = 6). * indicates P < .05, ** indicates P < .001, *** indicates P < .0001. A, B, C and D represent Humulus scandens were supplemented at 0, 4%, 8% and 12%. molecule, which provides an important first line of defence RT–PCR analysis of the small intestine showed that the HS against pathogens (Woof and Kerr 2006). In line with the pre- addition affects the gene expression of ZO-1, indicating that vious studies, rabbits after HS treatment increased the intestinal there may be improvement of the intestinal barrier mechanism. sIgA concentration. IgG is another important immunoglobulin, which can promote immune cells to phagocytize pathogens 5. Conclusion and neutralize bacterial toxins. In this study, the addition of HS to the diet increased the secretion of intestinal immunoglobulin Dietary supplement of Humulus scandens modulated immune in rabbits, which suggests HS treatment could increase the responses via increasing the secretion of IgA and IgG and ability of immune cells to swallow pathogens and toxins. improved intestinal barrier might by regulating ZO1, JAM3 Cytokines play an important role in the immune system and and mucin1 genes expression, meanwhile inhibit the synthesis are also potential targets of immune regulation (Hill and Sarvet- of cytokine. These maybe cause low death rate of weaned nick 2002). Some studies indicate that some herbs and plant rabbits, so our experiment offered a positive evidence of extracts inhibit the synthesis of pro-inflammatory cytokines Humulus scandens as rabbit feed resource. such as TNF-α and other cytokines related to inflammatory pro- cesses such as IFN-γ and IL6 (Pérez-Köhler et al. 2015). In our Acknowledgement study, dietary addition of 12% HS decreased significantly the TNF-α, IFN-γ and IL6 concentration in ileum, which imply that The author thanks the staff of Shandong Zhengyu Rabbit Industry Co., Ltd. for their excellent support during this experiment. the HS also had an inhibition in synthesis of cytokines, it is con- sistent with Aziz et al. (2018), the luteolin in HS could regulate pro-inflammatory mediators by reducing the synthesis of a Disclosure statement variety of pro-inflammatory cytokines (Aziz et al. 2018). No potential conflict of interest was reported by the author(s). Tight junctions are assembled from at least three transmem- brane proteins, including occludin, claudin and junction adhesion molecule (JAM), which are anchored to the cytoskele- Funding ton through peripheral membrane proteins, such as zonula This work was supported by Earmarked Fund for Modern Agro-industry occluden protein 1 (ZO1), ZO2 and ZO3 (Bazzoni et al. 2000). Technology Research System (CARS-43-G-7); Shandong Province Modern Mucin is a major glycoprotein expressed on the surface of Agricultural Industry Technology System (SDAIT-21); Scientific and Techno- breast epithelial cells and plays an important role in maintain- logical Problems Project Unveiled by Shandong Academy of Agricultural ing mucus protective layer and resisting inflammation. The Sciences (SHJB2021-43). main determinant of intestinal barrier function is intercellular tight junction (Martìn-Padura et al. 1998). These tight junctions Ethics statement proteins interact with other intracellular plaque ZO1, ZO2 and ZO3 proteins in turn anchor the transmembrane proteins to The experimental procedures were approved by the Shandong the actin cytoskeleton (Gonzalez-Mariscal et al. 2003). The Academy of Agricultural Sciences Animal Care and Use Com- association of tight junctions proteins with the perijunctional mittee (SAAS-2020-03) and were conducted in accordance actin cytoskeleton ring is vital for maintaining the tight junc- with the Guidelines for Experimental Animals established by tions structure and function (Gonzalez-Mariscal et al. 2003). the Ministry of Science and Technology (Beijing, China). 68 G. LIU ET AL. Liu GY, Wu ZY, Zhu YL, Liu L, Li FC. 2017.Effects of dietary vitamin B 6 on the ORCID skeletal muscle protein metabolism of growing rabbits. Anim Prod Sci. Gongyan Liu http://orcid.org/0000-0003-1478-7759 57:2007–2015. Hongmei Hu http://orcid.org/0000-0001-7064-4037 Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C (T)) method. Methods. 25:402–408. 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Fruscella P, Panzeri C, Stoppacciaro A, Ruco L, Villa A, et al. 1998. 2000. Interaction of junctional adhesion molecule with the tight junction Junctional adhesion molecule, a novel member of the immunoglobulin components ZO-1, cingulin, and occludin. J Biol Chem. 275:20520–20526. superfamily that distributes at intercellular junctions and modulates Blasco A, Ouhayoun J. 1996. Harmonization of criteria and terminology in monocyte transmigration. J Cell Biol. 142:11–127. rabbit meat research. World Rabbit Sci. 4:93–99. Nath R, Das C, Kityania S, Nath D, Das S, Choudhury MD, Patra JK, Talukdar Carabaño R, Badiola I, Chamorro S, García J, GarcíaRuiz AI, García-Rebollar P, AD. 2022. Natural flavonoids in the prevention and treatment of lung Gómez-Conde MS, Gutiérrez I, Nicodemus N, Villamide MJ, et al. 2008. cancer: A pharmacological aspect. Comb Chem High T Scr. doi:10. Review. New trends in rabbit feeding: influence of nutrition on intestinal 2174/1386207325666220701121537. health. 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Chem Biodivers. doi:10.1002/cbdv.202200471. inflammatory activity of luteolin in experimental animal models. Lin Y, Shi R, Wang X, Shen HM. 2008. Luteolin, a flavonoid with potential for Planta Med. 73:221–226. cancer prevention and therapy. Curr Cancer Drug Targets. 8:634–646. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Animal Research Taylor & Francis

Dietary addition of Humulus scandens improved the intestinal barrier in rabbits

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Abstract

JOURNAL OF APPLIED ANIMAL RESEARCH 2023, VOL. 51, NO. 1, 62–68 https://doi.org/10.1080/09712119.2022.2154215 a,b a,b a,b a,b a,b a,b Gongyan Liu , Lihong Hao , Cheng Wang , Yin Zhang , Haitao Sun and Hongmei Hu Institute of Animal Husbandry and Veterinary, Shandong Academy of Agricultural Sciences, Shandong Key Laboratory of Animal Disease Control and Breeding, Jinan, People’s Republic of China; Key Laboratory of Livestock and Poultry Multi-omics of MARA, Jinan, People’s Republic of China ABSTRACT ARTICLE HISTORY Received 19 April 2022 This study was conducted to investigate the effect of Humulus scandens (HS) in diet on the production Accepted 28 November 2022 performance and intestinal barrier of rabbits. One hundred and sixty Laiwu black rabbits at 35 days of age with body weight of 692.5 ± 48.3 g were divided into four groups (five replicates per group and KEYWORDS eight rabbits per replicate): fed on a basal diet free of HS (control), 4%, 8% or 12% HS powder Humulus scandens; intestinal supplement. The results showed that the dietary addition of HS decreased the ADFI, F/G, diarrhoea barrier; rabbits; production ratio and mortality ratio. Besides, the final body weight and ADG (P < .05) increased with the level of performance HS. Rabbits in 12% HS group had a higher sIgA and IgG concentration in serum and ileum than control, while lower TNFα, IFN-γ and IL-6 concentration (P < .05). Compared with the control, the HS supplementation groups for 12% level could decrease the mRNA expression of ZO-1, JAM3 and mucin1 (P < .05). In conclusion, dietary supplement of HS modulates immune responses and enhances intestinal barrier, meanwhile inhibits the synthesis of cytokine. Besides, our experiment offers positive evidence in improving rabbit health of HS as rabbit feed resource. 1. Introduction it is reported that flavonoids play a strong immune role by inhibiting the proliferation of immune cells and the pro- The disease of digestive tract is the most important disease in duction of pro-inflammatory cytokines (Rios 2010). HS is rabbits, accounting for 70% (Carabaño et al. 2008). The mor- wild on the roadside in the field, which adapts to various tality of the weaned rabbits up to 60% due to epizootic environmental conditions and has high yield (Urziceanu rabbit enteropathy (Fann et al. 2001; Carabaño et al. 2008). et al. 2022). In addition, because of its strong vitality and Rabbit diarrhoea is a big problem in present rabbit pro- harm to the growth of crops, it is often regarded as a malig- duction, especially in the weaning stage (Martens and Van nant weed, so far it has not been systematically developed Herck 2000). Weaned rabbits had thinner intestinal walls, and utilized, resulting in serious waste of resources (Zhou incomplete intestinal barrier, frequent diarrhoea or gastroin- and Feng 2020). However, there was little information avail- testinal flatulence (Fann et al. 2001). Antibiotics have been able about HS use as feedstuff in rabbit production and widely used to control mortality, increase breeding costs, special its effect on improving intestinal barriers. The affect product quality and reduce the economic benefits of present experiment was conducted to investigate the effect many farms. For this reason, some herbs and plant extracts of HS on the performance and intestinal barrier of rabbits, to substitute antibiotic have been found and studied by to identify applicative effects HS as a feed resource in rabbit many researchers. Humulus scandens (HS) is widely used in production. China to treat dysentery and chronic colitis as one traditional Chinese medicine, and contains polysaccharides, flavonoids, volatile oil and other chemical components (Chen et al. 2. Materials and methods 2012). Li et al. (2008) had reported that HS was rich in flavo- 2.1. Production of HS powder noids and there have been three kinds (vitexin, luteolin and cosmosiin) of flavonoids separated from HS. Flavonoids are Harvest the HS from countryside in Shandong Province in the a kind of common polyphenols in the plant world. They autumn mature stage, and naturally drying and pulverizing. have biological activities such as anti-inflammatory, anti- oxidant and anti-cancer (Korkina and Afanas’ev 1997; Neuhou- 2.2. Animals, diet and feed chemical composition ser 2005; Talhouk et al. 2007; Zhang et al. 2021; Nath et al. 2022). Li et al. (2022) flavonoids could inhibit alcohol- One hundred and sixty Laiwu black rabbits (male–female rate induced hepatocyte injury, which might be attributed to alle- of 1:1) at 35 days of age weight of 692.5 ± 48.3 g were divided viating oxidative stress and mitigating inflammatory response into four groups (five replicates per group and eight rabbits by activating Nrf2 and inhibiting NF-κB pathways. In addition, per replicate): f fed on a basal diet with 0 (control), 4%, 8% CONTACT Hongmei Hu huhongmeipatty@163.com; Haitao Sun wwww8888@163.com Institute of Animal Husbandry and Veterinary, Shandong Academy of Agricultural Sciences, Shandong Key Laboratory of Animal Disease Control and Breeding, Jinan 250100, People’s Republic of China © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, dis- tribution, and reproduction in any medium, provided the original work is properly cited. JOURNAL OF APPLIED ANIMAL RESEARCH 63 Table 1. Chemical composition of Humulus scandens powder (air-dry basis, %). histological measurements. The other intestinal segments Items Humulus scandens powder were collected by cryopreservation tube, rapidly frozen in Dry matter (DM) 91.60 liquid nitrogen and stored at −80°C until analysis. Crude protein (CP) 16.00 Ether extract (EE) 0.90 Neutral detergent fibre (NDF) 47.40 Acid detergent fibre (ADF) 36.90 Aid detergent lignin (ADL) 9.20 2.4. Determination of indicators and methods Ash 16.50 Calcium 1.93 2.4.1. Feed chemical analysis Phosphorus 0.35 The chemical composition of the SH and diets was analysed fol- Polysaccharide (mg/g) 19.53 Total flavonoids (mg/g) 6.86 lowing the procedures of AOAC International (2005). The dry matter was determined by oven drying at 105°C (procedure 934.01), and the ether extract was determined by extraction or 12% HS powder, and the chemical of HS were listed in with petroleum ether in a Soxtec 1043 apparatus (FOSS Table 1. The basal diets were formulated according to the Tecator AB, Hoganas, Sweden; procedure 920.39). The protein values for growing from De Blas and Mateos (2020) and the content was determined using a Kjeltec Auto 1030 Analyser ingredients and composition of diet are listed in Table 2. (procedure 954.01). The ash content was determined according Besides, the diet pelleted by the use of pressure and the to procedure 920.153. Analysis of fibre components was per- diameter of the pellets was 4 mm. The experiment lasted formed according to Van Soest et al. (1991). The calcium and for nine weeks which included one week adaptation period phosphorus contents were determined by inductively and eight weeks experimental period. During the rearing coupled plasma atomic emission spectrometry (ICP-EMS; period, the rabbits were individually housed cage and had Optima 8000, Perkin Elmer, U.S.A.). free access to feed and water. The content of polysaccharides in HS was detected by phenol-sulphuric acid method. Preparation of standard 2.3. Sample collection and preparation glucose solution: accurately weigh 20 mg of anhydrous glucose dried at 105°C to constant weight, put it in a 250 mL At the end of the trial (98 days), 40 rabbits (10 rabbits per volumetric flask, fix the volume and shake well for use. group, male and female had half each, and the average body Drawing the standard curve: accurately absorb 0, 0.2, 0.4, 0.6, weight of the two rabbits equals to the average body weight 0.8, 1.0 and 1.2 mL of glucose standard solution, put them in of each replicate) were sacrificed by cervical dislocation. a test tube with a stopper, add water to 2 mL respectively, About 4 cm segment of the middle ileum was collected and and shake well, then add 1.0 mL of 5% phenol solution, divided into two segments. One intestinal segments were quickly add concentrated H SO 5 mL, shake well and leave it 2 4 flushed gently with phosphate-buffered saline (PBS, pH 7.4) at room temperature for 30 min until it is cooled. Then, use and then placed in 10% fresh, chilled formalin solution for an ultraviolet spectrophotometer at 490 nm wavelength. Determination of polysaccharide content in the sample: the Table 2. Composition and nutrient levels of basal diets (air-dry basis, %). absorbance value is determined according to the above stan- Levels of Humulus scandens dard method, and the content is calculated according to the Items 0 (Control) 4 8 12 standard curve. Raw material composition The content of flavonoids in HS was detected by colorime- Corn 15.0 15.0 15.0 15.0 try. Accurately weigh 1 g of raw materials, add 50% ethanol sol- Soybean meal 10.0 10.0 10.0 10.0 ution according to the ratio of material to liquid (1:20–1:40, g/ Wheat bran 20.0 20.0 20.0 20.0 Corn germ meal 15.0 15.0 15.0 15.0 ml), perform ultrasonic treatment for 2 h, centrifuge at 3000 r/ Soybean straw powder 7.0 7.0 7.0 7.0 min for 20 min and collect supernatant for determination. Peanut seedling powder 30.0 26.0 22.0 18.0 Taking rutin as standard, and mix 1.5 mL of extract with Humulus scandens 0.0 4.0 8.0 12.0 Premix 3.0 3.0 3.0 3.0 1.5 mL of 2% AlCl -methanol solution (containing 5% acetic Total 100.0 100.0 100.0 100.0 acid), stand for 10 min, and measure the absorbance at Nutrient levels 430 nm. With rutin as the standard sample, a standard curve Digestible energy (DE, MJ/kg) 10.14 10.21 10.26 10.29 Crude protein (CP) 15.10 15.60 16.30 16.70 was established in the range of 10–500 μg/mL. Ether extract (EE) 2.00 2.10 2.20 2.50 Neutral detergent fibre (NDF) 56.80 56.70 52.50 50.00 Acid detergent fibre (ADF) 43.30 43.00 41.40 40.60 Aid detergent lignin (ADL) 5.77 5.80 5.41 5.32 2.4.2. Growth performance Ash 11.60 11.20 11.80 11.10 Calcium 1.27 1.14 1.26 1.19 Body weights of each replicate rabbits were measured before Phosphorus 0.62 0.67 0.75 0.68 feeding at the beginning and end of the trial, and the ADG Premix provided the following per kg of diets: vitamin A 10,000 IU, vitamin D was calculated from 42 to 98 days of age. The ADFI was calcu- 2000 IU, vitamin E 50 mg, vitamin K 2.5 mg, thiamine 5 mg, riboflavin lated according to total feed intake divided by the total number 10 mg, nicotinic acid 20 mg, pantothenic acid 50 mg, folic acid 2.5 mg, vitamin B 1 mg, choline chloride 400 mg, iron 100 mg, zinc 50 mg, copper of experimental days in each replicate. The F/G was then calcu- 40 mg, manganese 30 mg, iodine 0.5 mg, selenium 0.05 mg, calcium hydrogen lated as the feed intake/weight gain. The number of rabbits phosphate 15 g, sodium chloride 5 g, lysine 1.5 g, methionine 1.5 g, the rest is with diarrhoea or dead was recorded daily throughout the miscellaneous meal carrier complement. Digestible energy was calculated, whereas the others were measured values. study. 64 G. LIU ET AL. Table 3. Gene-specific primers used for the analysis of rabbit gene expression. 2.4.3. Slaughter performance ′ ′ Gene name GeneBank Primer sequences (5 –3 ) Product size, bp As described by Blasco and Ouhayoun (1996), the slaughter GAPDH NM_001082253 GTCAAGGCTGAGAACGGGAA 129 procedure and carcass analysis were performed. Twelve hours GAAGACGCCAGTGGATTCCA before slaughter, rabbits were fasted and weighed, which claudin1 NM_001089316 AAAGATGCGGATGGCTGTCA 187 was pre-slaughter body weight. After bleeding, the pelts, AAGGCAGAGAGAAGCAGCAG Occludin XM_008262318 TCGTTTGAGCAGCAGCAGTA 161 paws and full gastrointestinal tract were removed, and the ACTCCCTGATCCAGTCCTCC commercial carcass was weighed (preserving the head, ZO1 XM_017348357 AGAGGATTTGTCAGCCCAGC 179 trachea, oesophagus, thoracic organs, liver, kidney and perire- CTCGCGGTTCACTTTTTGCA JAM3 XM_008248362 GAAGTCCTGGGGCAGACTTC 158 nal fat). The semi-clean carcass weight was commercial CTGCAGACAGGGGTTACTGG carcass weight removing the head at the first cervical vertebra, mucin1 XM_017345830 CACACCATCTTCGGTTCCCA 197 removing the trachea and oesophagus, retaining the liver, ACTTGCTGCTTGGATCCTCC kidney and perirenal fat. The full-clean carcass weight is the GAPDH = glyceraldehyde 3-phosphate dehydrogenase; ZO1 = zonula occluden protein 1; JAM3 = junctional adhesion molecules 3. semi-clean carcass weight removing the heart, liver, kidney and perirenal fat. At the same time, the thymus, spleen, heart, liver and kidney are weighed. At the same time, the com- (Liu et al. 2017). The PCR primers used in this study (Table 3) mercial slaughter ratio, semi-clean slaughter ratio, full-clean were designed using a Premier 5.0 Software and synthesized slaughter ratio, thymus ratio, spleen ratio, heart ratio, liver by Ruibo Biological Engineering Co., Ltd. (Qingdao, China). ratio and kidney ratio were calculated by dividing the live The relative mRNA expression levels were calculated using weight before slaughter. −△△Ct the arithmetic formula 2 (Livak and Schmittgen 2001). 2.4.4. Morphological structure of small intestine The ileum tissue samples were cut longitudinally at the mesen- 2.5. Statistical analysis teric attachment and immediately fixed in 10% neutral forma- All of the data collected were subjected to one-way ANOVA lin. The samples were then dehydrated in graded alcohols, analysis with the Statistical Analysis Systems statistical software cleared with xylene and embedded in paraffin. The serial micro- package (Version 8e, SAS Institute, Cary, NC), and the primary tome sections (5 µm thick) were stained with haematoxylin and effect of HS treatments on the responses among the various eosin stain. In each slide, 10 well-oriented villi and crypts were groups, and Duncan’s test was used for multiple comparisons. analysed at a low magnification (40×) with a light microscope Besides, the data are shown as the means and root mean errors (using Image-Pro Plus 6.0 software, U.S.A.). (RMSE), P < .05 was statistically significant. 2.4.5. Immunoglobulins and cytokines levels The ileum samples were disrupted and homogenized in PBS 3. Results buffer (1 g samples: 9 ml PBS). After centrifuging with a speed of 5000 g for 10 min, the supernatant was isolated. The 3.1. Growth performance IgG, IgM and sIgA concentrations were measured with an The growth performance of the experimental rabbits is pre- immunoturbidimetry using Enzyme-Linked Immunosorbent sented in Table 4. Dietary HS supplemental level had significant Assay kits (CEA544Rb, CEA543Rb, SEA641Rb, respectively; effect on ADFI, ADG, F/G, diarrhoea ratio and mortality ratio (P Cloud-Clone Corp, Wuhan, China). The IgG, IgM and sIgA in < .05) of meat rabbits, with the increase of addition level, the supernatant could combine the IgG, IgM and sIgA antibody feed intake, F/G, diarrhoea ratio and mortality ratio decreased, in 0.02 mol/L phosphate buffer substrate turnover was moni- and the FBW and ADG increased. tored spectrophotometrically at 450 nm with an automated reader (BioTek, Winooski, Vt.). The levels of IgA, TNFα, IFN-γ and IL-6 were determined using Enzyme-Linked Immunosor- 3.2. Slaughter performance bent Assay kits (CSB-E06946Rb, CSB-E06998Rb, CSB- E06925Rb, CSB-E06903Rb, respectively; CUSABIO, Qingdao, Dietary HS supplemental level had significant effect on pre- China). After 1 h of incubation at 37°C, samples were slaughter body weight, commercial carcass weight, semi- removed and the plates were washed with a PBS buffer. After clean carcass weight, full-clean carcass weight and kidney that, 100 μL horseradish peroxidase, and 100 μL of tetramethyl- weight (P < .05) of experimental rabbits, the pre-slaughter benzidine substrate was added to each well. The mixture was body weight, commercial carcass weight, semi-clean carcass incubated in the dark for 30 min at room temperature. weight, full-clean carcass weight in the higher group (8% and Optical density value at 450 nm was measured by a Bio-Rad 12% group) were higher than those of the control group. ELISA reader (Bio-Rad, Richmond, CA). Besides, the kidney weight of the experimental group (4%, 8% and 12% group) with HS was significantly higher than 2.4.6. RNA isolation and analysis those of the control group. Furthermore, HS supplemental of Total RNA Extraction Reagent Kit (RNAiso Plus, Takara, Code maternal diets had no influence on the heart weight, lung No. 9108/9109) was used to ileum RNA Isolation, and weight, liver weight or the commercial slaughter ratio, semi- qRT-PCR by SYBR Premix Ex Taq™ II (Tli RNaseH Plus, Takara, clean carcass ratio, full-clean carcass ratio, heart index, lung Code No. RR820A) was performed as described previously index, liver index (P > .05; Table 5). JOURNAL OF APPLIED ANIMAL RESEARCH 65 Table 4. Effects of Humulus scandens supplementation on growth performance and feed intake in rabbits. Levels of Humulus scandens,% Items 0 (Control) 4 8 12 RMSE P-value IBW, g 876.5 886.8 887.0 892.7 57.2615 .9755 b b a a FBW, g 2313.0 2367.8 2495.3 2567.5 84.2817 .0008 c bc ab a ADG, g/d 25.65 26.45 28.72 29.91 1.9991 .0136 ab a b b ADFI, g/d 100.54 101.80 98.91 98.91 2.3075 .0201 a ab bc c F/G 4.13 3.96 3.71 3.40 0.2699 .0032 1 a ab b b Diarrhoea ratio , % 12.50 7.50 5.00 5.00 0.2245 .0209 2 a ab b b Mortality ratio , % 7.50 5.00 2.50 2.50 0.2058 .0490 Diarrhea ratio (%) = 100 × (number of rabbits with diarrhoea × average diarrhoea days)/(number of experimental rabbits × experimental days). Mortality (%) = 100 × (number of rabbits with death/number of experimental rabbits). a,b,c Different superscript letters means different (p < .005). RMSE, root mean square error; IBW, initial body weight; ADFI, average daily feed intake; ADG, average daily gain; F/G, feed to gain ratio. Table 5. Effects of Humulus scandens supplementation on carcass traits of rabbits. Levels of Humulus scandens,% Items 0 (Control) 4 8 12 RMSE P-value b ab a a Pre-slaughter body weight, g 2259.6 2379.0 2473.0 2535.0 114.7642 .0089 c bc ab a Commercial carcass weight, g 1254.9 1331.5 1396.7 1488.0 82.7896 .0029 c bc ab a Semi-clean carcass weight, g 1109.5 1186.4 1247.6 1336.0 79.5357 .0027 c bc ab a Full-clean carcass weight, g 1023.0 1091.0 1145.6 1234.2 72.8901 .0024 Heart weight, g 5.93 5.97 6.49 6.20 0.3882 .1253 Lung weight, g 11.52 10.94 13.26 12.10 1.6660 .1932 Liver weight, g 65.33 71.22 75.33 78.65 8.2528 .1040 b a a a Kidney weight, g 12.09 14.16 14.58 14.42 1.0303 .0049 Commercial slaughter ratio, % 55.55 55.97 56.50 58.65 1.9648 .1003 Semi-clean slaughter ratio, % 49.12 49.85 50.46 52.65 1.9214 .0544 Full-clean slaughter ratio, % 45.31 45.86 46.35 48.64 1.9357 .0700 Heart ratio, g/kg 2.62 2.52 2.56 2.51 0.1762 .7443 Lung ratio, g/kg 5.10 4.61 5.24 4.90 0.7468 .5877 Liver ratio, g/kg 28.92 29.81 29.71 31.72 2.2454 .2770 Kidney ratio, g/kg 5.10 4.61 5.24 4.90 0.7468 .5877 RMSE, root mean square error. intestinal was decreased by HS supplementation level 3.3. Immune organ development increased. Compared with the control, the HS supplementation As shown in Table 6, dietary HS supplemental level had no sig- in the diet did not alter the level of IgM (P > .05) in serum. nificant effect on thymus weight, spleen weight or thymus ratio, spleen ratio (P > .05) of experimental rabbits. 3.6. Gene expression of physical barrier 3.4. Morphological structure of small intestine Agarose gel map for RNA quality was seen in Figure 1. The HS supplementation in the diet did not alter the mRNA expression The HS supplementation in the diet did not alter the villus of claudin1 and occludin (P > .05) in the small intestine (P > .05, height, crypt depth, villus width, mucosal layer thickness and Figure 2). Compared with the control, the HS supplementation muscle layer thickness (P > .05) of the small intestine (Table 7). groups for 12% level could decrease the mRNA expression of ZO-1, JAM3 and mucin1 (P < .05). 3.5. Immune active compounds Table 7. Effects of Humulus scandens supplementation on intestinal morphology The HS supplementation in the diet could improve the titre of of rabbits. the IgG, IgA and sIgA (P < .05) in serum and intestinal (Table 8). Levels of Humulus scandens,% However, the titre of the TNF-α, IFN-γ and IL-6 in serum and 0 P- Items (Control) 4 8 12 RMSE value Table 6. Effects of Humulus scandens supplementation on immune organ Villus height, 865.30 900.61 803.88 771.05 180.1309 .3533 development of rabbits. µm Crypt depth, 147.45 169.63 179.81 167.53 35.6639 .5539 Levels of Humulus scandens,% µm Items 0 (Control) 4 8 12 RMSE P-value Villus width, µm 137.48 147.83 157.65 162.46 22.7420 .3466 Thymus weight, g 4.55 4.72 4.82 4.80 0.8512 .9529 Mucosal layer 1017.60 1143.90 1134.90 1228.70 199.0111 .4396 Thymus ratio, g/kg 1.91 1.94 2.01 1.99 0.3742 .9665 thickness, µm Spleen weight, g 1.16 1.28 1.09 1.05 0.2061 .3272 Muscle layer 104.29 117.45 106.16 124.46 22.8826 .4764 Spleen ratio, g/kg 0.51 0.54 0.43 0.42 0.0841 .0918 thickness, µm RMSE, root mean square error. RMSE, root mean square error. 66 G. LIU ET AL. Table 8. Effects of Humulus scandens supplementation on immune active main factor responsible for a reduction in digestibility of raw compounds of rabbits. feed and feedstuffs (Gidenne and Perez 1994). The cell wall lig- Levels of Humulus scandens,% nification is lower in higher HS level (12%group) than that in control groups (Table 2). Thus, the lower fibre level should Items (Control) 4 8 12 RMSE P-value explain the higher energy and protein in diets containing HS Immune active compounds in serum b a a a and the subsequent reduction in ADFI. Carcass weight is IgG titre, μg/mL 7.53 8.01 8.25 8.32 0.4612 .0080 IgM titre, ng/mL 645.31 659.96 671.89 692.10 41.6420 .1695 closely related to live weight before slaughter, the results of c b ab a IgA titre, ng/mL 114.20 121.13 127.57 130.29 6.4129 .0001 slaughter ratio for rabbits showing no or few effects of fibre c bc a ab sIgA titre, ng/mL 159.90 177.37 209.08 190.05 24.9001 .0041 sources inclusion, which was agree with the previous studies TNF-α titre, ng/mL 3.28 3.00 3.17 2.99 0.4218 .4701 a a a b IFN-γ titre, pg/mL 362.07 352.68 328.26 237.77 66.8278 .0033 (Dal Bosco et al. 2012; Margüenda et al. 2012). Guo et al. a b b b IL-6 titre, pg/mL 327.41 263.98 270.62 269.45 38.4523 .0079 (2008) reported that the mixture (50:50) of HS and alfalfa consti- Immune active compounds in intestinal b a a a tutes an alternative source of fibre for fattening rabbits and pre- IgG titre, μg/g 14.10 23.80 21.68 25.54 6.4128 .0071 c b a a IgM titre, ng/g 21.82 34.31 43.25 50.71 8.0591 <.0001 vents diarrhoea effectively. In this study, we firstly investigate the b a a ab IgA titre, ng/g 3.42 13.74 12.94 10.01 7.1810 .0311 addition effect of HS in rabbit production. After detecting the b a a a sIgA titre, ng/g 222.26 354.17 320.14 311.71 68.4362 .0046 a a a b indicators of performance, intestinal morphology and intestinal TNF-α titre, ng/g 22.83 22.78 22.56 14.66 4.5753 .0023 a a a b IFN-γ titre, ng/g 73.48 70.05 68.57 59.64 7.4278 .0064 barrier, the present study showed a positive effect of HS as a ab ab b IL-6 titre, ng/g 1.74 1.47 1.38 1.27 0.3309 .0499 rabbit feed resource. RMSE, root mean square error. The intestinal mucosal barrier is mainly composed of epi- thelial cells, tight junctions between adjacent enterocytes, and critical components of the mucosal immune system (Turner 4. Discussion 2009). It acts as a selective barrier that allows the absorption As we all know, HS often grows wild in ditches, wastelands, ruins and excretion of substances required for the homogenate and forest edges, which adapts to various environmental con- state of the body and prevents the passage of various pathogens ditions and can be harvested in large quantities in the autumn or their products. Under normal circumstances, epithelial cells mature stage. HS has been used to treat inflammatory diseases allow only a small amount of intact antigens to enter the in China. Luteolin is one of the flavonoids isolated from HS, it mucosa, where they interact with the mucosal immune system is a common flavonoid, which exists in many herbal medicines. to reduce the inflammatory response. At the same time, intesti- Plants rich in luteolin have been used in traditional Chinese nal pathogens can activate immune cells and initiate the inflam- medicine to treat inflammatory disorders diseases (Ziyan et al. matory response required to eliminate infection (Chadwick et al. 2007; Lin et al. 2008). The main constituent of HS is fibre 2002). Under the condition of inflammatory bowel disease, the (Table 1). In our study showed that in rabbits of similar initial excessive penetration of antigen through the epithelial layer body weight (Table 3), the inclusion of HS in the diet decreased may lead to inappropriate immune stimulation and chronic gas- the ADFI and F/G in rabbits between 42 and 98 days of age. This trointestinal inflammation (Baumgart and Carding 2007). In result may be due to the difference in energy level in the four addition, epithelial cells dilute, flush and bind harmful sub- diets by different level with HS powder. Accordingly, the levels stances by secreting liquid and mucus and sIgA into the of fibre and fibre proportions in diet decreased when dietary lumen, so as to play an important physiological defence role HS levels increased (Table 2). The degree of lignification is the (Shang et al. 2008). sIgA is a major mucosal immune effector Figure 1. Agarose gel map for RNA quality. A, B, C and D represent Humulus scandens were supplemented at 0, 4%, 8% and 12%, 1–6 represent different samples of this group. JOURNAL OF APPLIED ANIMAL RESEARCH 67 Figure 2. Effect of Humulus scandens on mRNA expression of physical barrier gene in ileum of rabbits. Values are presented as the means ± std. error (n = 6). * indicates P < .05, ** indicates P < .001, *** indicates P < .0001. A, B, C and D represent Humulus scandens were supplemented at 0, 4%, 8% and 12%. molecule, which provides an important first line of defence RT–PCR analysis of the small intestine showed that the HS against pathogens (Woof and Kerr 2006). In line with the pre- addition affects the gene expression of ZO-1, indicating that vious studies, rabbits after HS treatment increased the intestinal there may be improvement of the intestinal barrier mechanism. sIgA concentration. IgG is another important immunoglobulin, which can promote immune cells to phagocytize pathogens 5. Conclusion and neutralize bacterial toxins. In this study, the addition of HS to the diet increased the secretion of intestinal immunoglobulin Dietary supplement of Humulus scandens modulated immune in rabbits, which suggests HS treatment could increase the responses via increasing the secretion of IgA and IgG and ability of immune cells to swallow pathogens and toxins. improved intestinal barrier might by regulating ZO1, JAM3 Cytokines play an important role in the immune system and and mucin1 genes expression, meanwhile inhibit the synthesis are also potential targets of immune regulation (Hill and Sarvet- of cytokine. These maybe cause low death rate of weaned nick 2002). Some studies indicate that some herbs and plant rabbits, so our experiment offered a positive evidence of extracts inhibit the synthesis of pro-inflammatory cytokines Humulus scandens as rabbit feed resource. such as TNF-α and other cytokines related to inflammatory pro- cesses such as IFN-γ and IL6 (Pérez-Köhler et al. 2015). In our Acknowledgement study, dietary addition of 12% HS decreased significantly the TNF-α, IFN-γ and IL6 concentration in ileum, which imply that The author thanks the staff of Shandong Zhengyu Rabbit Industry Co., Ltd. for their excellent support during this experiment. the HS also had an inhibition in synthesis of cytokines, it is con- sistent with Aziz et al. (2018), the luteolin in HS could regulate pro-inflammatory mediators by reducing the synthesis of a Disclosure statement variety of pro-inflammatory cytokines (Aziz et al. 2018). No potential conflict of interest was reported by the author(s). Tight junctions are assembled from at least three transmem- brane proteins, including occludin, claudin and junction adhesion molecule (JAM), which are anchored to the cytoskele- Funding ton through peripheral membrane proteins, such as zonula This work was supported by Earmarked Fund for Modern Agro-industry occluden protein 1 (ZO1), ZO2 and ZO3 (Bazzoni et al. 2000). Technology Research System (CARS-43-G-7); Shandong Province Modern Mucin is a major glycoprotein expressed on the surface of Agricultural Industry Technology System (SDAIT-21); Scientific and Techno- breast epithelial cells and plays an important role in maintain- logical Problems Project Unveiled by Shandong Academy of Agricultural ing mucus protective layer and resisting inflammation. The Sciences (SHJB2021-43). main determinant of intestinal barrier function is intercellular tight junction (Martìn-Padura et al. 1998). These tight junctions Ethics statement proteins interact with other intracellular plaque ZO1, ZO2 and ZO3 proteins in turn anchor the transmembrane proteins to The experimental procedures were approved by the Shandong the actin cytoskeleton (Gonzalez-Mariscal et al. 2003). The Academy of Agricultural Sciences Animal Care and Use Com- association of tight junctions proteins with the perijunctional mittee (SAAS-2020-03) and were conducted in accordance actin cytoskeleton ring is vital for maintaining the tight junc- with the Guidelines for Experimental Animals established by tions structure and function (Gonzalez-Mariscal et al. 2003). the Ministry of Science and Technology (Beijing, China). 68 G. LIU ET AL. Liu GY, Wu ZY, Zhu YL, Liu L, Li FC. 2017.Effects of dietary vitamin B 6 on the ORCID skeletal muscle protein metabolism of growing rabbits. Anim Prod Sci. Gongyan Liu http://orcid.org/0000-0003-1478-7759 57:2007–2015. Hongmei Hu http://orcid.org/0000-0001-7064-4037 Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C (T)) method. Methods. 25:402–408. 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Journal

Journal of Applied Animal ResearchTaylor & Francis

Published: Dec 31, 2023

Keywords: Humulus scandens; intestinal barrier; rabbits; production performance

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