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Effect of the dietary supplementation with extracts of chestnut wood and grape pomace on performance and jejunum response in female and male broiler chickens at different ages

Effect of the dietary supplementation with extracts of chestnut wood and grape pomace on... Background: Recently, interest in the use of herbs and phytogenic compounds has grown because of their potential role in the production and health of livestock animals. Among these compounds, several tannins have been tested in poultry, but those from chestnut wood and grape-industry byproducts have attracted remarkable interest. Thus, the present study aimed to gain further insights into the mechanisms involved in the response to the dietary supplemen- tation with extracts of chestnut wood or grape pomace. To this purpose, 864 broiler chickens were fed a control diet (C) or the same diet supplemented 0.2% chestnut wood (CN) extract or 0.2% grape pomace (GP) extract from hatch- ing until commercial slaughtering (at 45 days of age) to assess their effects on performance, meat quality, jejunum immune response and whole-transcriptome profiling in both sexes at different ages (15 and 35 d). Results: Final live weight and daily weight gain significantly increased (P < 0.01) in chickens fed GP diets compared to CN and C diets. The villi height was lower in chickens fed the CN diet than in those fed the C diet (P < 0.001); moreover, a lower density of CD45 cells was observed in chickens fed the CN diet (P < 0.05) compared to those fed the C and GP diets. Genes involved in either pro- or anti-inflammatory response pathways, and antimicrobial and antioxidant responses were affected by GP and CN diets. There was no effect of the dietary treatment on meat quality. Regarding sex, in addition to a lower growth performance, females showed a lower occurrence of wooden breast (16.7% vs. 55.6%; P < 0.001) and a higher occurrence of spaghetti meat (48.6% vs. 4.17%; P < 0.001) in pectoralis major muscles after slaughtering than those in males. Based on the results of whole-transcriptome profiling, a significant activation of some molecular pathways related to immunity was observed in males compared with those of females. Conclusions: The GP supplementation improved chicken performance and promoted immune responses in the intestinal mucosa; moreover, age and sex were associated with the most relevant transcriptional changes. Keywords: Growth, Immunohistochemistry, Meat quality, Tannins, Transcriptome Background *Correspondence: angela.trocino@unipd.it During the last decade, interest in the use of herbs and phytogenic compounds has grown because of their Department of Comparative Biomedicine and Food Science (BCA), University of Padova, Viale dell’Università 16, 35020 Padova, Legnaro, Italy potential role in the production and health of livestock Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 2 of 17 animals. Among these compounds, plant tannins are examined the effect of dietary treatments on jejunum polyphenolic compounds that exhibit antioxidant activ- morphology, inflammatory patterns, and whole-tran - ity, which positively affect the composition of the gut scriptome profiling. microbial flora, stimulate the animal immune system and possess antibacterial properties [1–3]. Additionally, at the Methods intestinal level polyphenols downregulate the expression Experimental facilities of various proinflammatory cytokines, such as interleu - This study was conducted at the Experimental Farm of kin (IL)-1β, IL-4, IL-6, IL-10, tumor necrosis factor alpha the University of Padova (Legnaro, Padova, Italy) in a (TNF-α), and interferon gamma [4]. poultry house equipped with a cooling system, forced In the poultry industry, several tannins have been ventilation, radiant heating, and controlled light systems. tested, but those from chestnut wood  (CN) and grape- Thirty-six wire-net pens (2.5 m × 2.4 m; 6 m ) were used, industry byproducts have attracted remarkable inter- each equipped with five nipple drinkers and a circular est. In  vitro studies have shown the antimicrobial and feeder for manual distribution of feed. Each pen had a antiparasitic effects of CN tannins [5, 6]. Moreover, in concrete floor covered with wood shaving litter (depth broiler chickens [7], CN extract increased jejunal mRNA 5 cm, 2.5 kg/m ). A total of 24 h of light was provided levels of some growth-related antioxidant genes, such during the first 2 d after the arrival of the chicks. Sub - as epidermal growth factor and its receptor, and heat sequently, the hours of light were progressively reduced shock protein 70. However, results of in  vivo trials lack until an 18L:6D photoperiod was achieved, which was consistency in their effects on poultry performance; posi - then maintained from 13 days of age onward. tive effects have been reported by some authors [8, 9], whereas others [10, 11] did not find any improvement. Animals, experimental groups and in vivo recordings Regarding byproducts of the grape-industry, supplemen- A total of 864 broiler chicks (1 day old; 432 males and 432 tations with grape pomace (GP) concentrate and grape females; Ross 308, Aviagen) were delivered by a commer- seed extracts have been shown to affect the gut morphol - cial truck, in compliance with Council Regulation (EC) ogy and increase the microflora biodiversity in broilers, No 1/2005 to the experimental facilities. All chicks were in addition to improving growth performance [12]. vaccinated against Marek’s disease, infectious bronchitis, Additionally, tannins from both CN and grape-indus- and Newcastle disease at the hatchery. They were ran - try byproducts exhibit antioxidant effects when sup - domly allocated in six experimental groups according to plemented in diets for broiler chickens [13, 14]. The an experimental arrangement based on two sexes × three antioxidant properties of tannins contained in GP have dietary treatments (control diet, CN diet, and GP diet), been observed in both the liver and breast meat of broiler with 6 pens per experimental group and 24 birds per pen chickens either after slaughter [14] or during storage [15]. (total of 36 pens). Owing to their antioxidant properties, tannins can allevi- Chicks were individually weighed on the day of ate oxidative stress that occurs during the onset of breast their arrival, identified by a plastic band at the leg, and myopathies [16, 17]. weighed once per week to measure live weight and any Interestingly, the effects of tannins could vary during difference among treatments at different ages, besides animal development [10, 18], particularly with age and promptly identifying any health problem. The chick - sex. Modern hybrids are highly precocious, and large ens were fed ad  libitum. The pen feed consumption was sex-differences are observed from the very first days after measured daily through a computerized weighing system hatching. Regarding age, animals fed a diet supplemented connected to all feeders. with a GP concentrate showed differences in perfor - The end of the growth trial was set at 44 d and birds mance and gut morphology at the end of an experimen- were slaughtered at 45 days of age. tal trial, suggesting a time-dependent cumulative effect of tannins [19]. Humer et  al. [20] reported remarkable Diets and feeding plans differences in intestinal histometric and microbial traits Three commercial diets were formulated as the control between male and female broilers fed with phytogenic (C) diets to be fed during three periods, i.e., from arrival additives. to 15 d; from 15 to 29 d; and from 29 d until slaughter- u Th s, the present study aimed to gain further insights ing (Table  1). These C diets, produced in a mash form, into the mechanisms involved in the response of broiler were supplemented with 0.2% CN extracts (CN diets) or chickens of both sexes and different ages to different 0.2% GP extract (GP diets). The CN extract (Saviotan tannin extracts (i.e., CN and GP) supplemented in diets Feed, 57 mg/g phenolic compounds, GAE equivalent fed from hatching until slaughtering (45 days of age). In Folin-Ciocalteu) was produced by Sedepan (Radicofani, addition to chicken performance and meat quality, we SI, Italy). The GP extract (77 mg/g phenolic compounds, P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 3 of 17 Table 1 Ingredients and chemical composition measured in the two steps (starting with 1 kg) and mixed by hand in a lab (if not specified otherwise) of the control (C) diets. These diets box prior mixing them with other 47 kg of mash diet in were supplemented with 0.2% chestnut wood (CN) extracts or the electric concrete mixer. 0.2% grape pomace (GP) extracts to obtain the CN and GP diets, Diets were analyzed to determine their dry matter respectively content, crude protein, and crude fiber using AOAC Period of administration 1-15 d 15-29 d 29 d [22] methods. The ether extract was analyzed after acid to slaughtering hydrolysis [23]. Ingredients Corn meal, % 56.60 59.25 63.15 Sampling of jejunum tissues Soybean meal (CP 48%), % 34.50 30.00 24.60 At 15 and 35 days post hatching, 36 chickens per period Toasted full-fat soybean meal, % 3.00 5.00 7.00 (1 chick per pen with mean BW) were selected and Animal fat, % 2.50 2.50 2.50 euthanized with C O asphyxiation, prior to jejunum Dicalcium phosphate, % 1.00 0.50 0.25 tissue collection. One sample of approximately 2 cm Calcium carbonate, % 0.92 1.33 1.30 was taken from the jejunum, at the midpoint between Sodium chloride, % 0.26 0.27 0.27 the end of the duodenal loop and the location of the L-lysine base, liquid (50%), % 0.25 0.24 0.16 Meckel’s diverticulum [24], and washed in phosphate- Methionine hydroxy analogue, % 0.31 0.28 0.23 a buffered saline (PBS). Sections (approximately 1-cm Vitamin-mineral premix , % 0.30 0.30 0.30 thick) were fixed in paraformaldehyde in PBS (0.1 6-phytase (EC 3.1.3.26), % 0.20 0.20 0.20 mol/L, pH 7.4), dehydrated, embedded in paraffin at L-Threonine, % 0.11 0.08 0.04 b the laboratory, and later submitted to the histological Coccidiostat , % 0.05 0.05 0.00 analyses and immunohistochemistry as detailed in the Chemical composition following section. Dry matter, % 89.2 89.2 89.3 Immediately before fixing, small jejunum sections Crude protein, % 22.2 20.8 20.2 were collected under RNase-free conditions and stored Ether extract, % 5.4 5.5 6.4 in RNAlater reagent (Applied Biosystems, Foster City, Crude fiber, % 1.3 1.2 1.8 CA, USA) for transcriptomic analyses. In the laboratory, Ash, % 5.7 5.5 6.6 RNA-seq samples were stored at 4 °C overnight and then Starch, % 41.1 43.3 40.2 transferred to −80 ºC until further processing. Calcium , % 0.81 0.80 0.71 Phosphorous , % 0.58 0.47 0.42 Digestible phosphorous , % 0.34 0.24 0.18 Histological analyses and immunohistochemistry Digestible lysine , % 1.32 1.23 1.05 Two serial 4-μm sections per jejunum sample were Digestible methionine + cysteine , % 0.91 0.85 0.76 obtained using a microtome and stained with hematoxy- Digestible threonine , % 0.83 0.76 0.66 lin/eosin for morphometric evaluation and Alcian blue Apparent metabolizable energy , 2982 3045 3087 (pH 2.5)-PAS method for quantitative analysis of goblet kcal/kg cells. Moreover, two further serial sections were used Premix provided per kg of feed: vit. A, 10,000 IU; vit. D , 3500 IU; vit. E acetate, + + for CD3 and C D45 immunohistochemical analyses. 90 mg; vit. K , 6 mg; Biotin, 0.38 mg; Thiamine, 3.75 mg; Riboflavin, 8 mg; vit. B , 3 6 5.75 mg; vit. B , 0.1 mg; Niacin, 70 mg; Pantothenic acid, 17.5 mg; Folic acid, The villi length and crypt depth were collected by a slide 2.25 mg; Fe, 45 mg; Cu, 10 mg; Mn, 70 mg; Zn, 65 mg; Se, 0.25 mg scanner (D-Sight, A. Menarini Diagnostics, Firenze, Italy) Sodium Monensin, 100 mg/kg feed and measured using image analysis software (DP-soft, Values calculated according to FEDNA [21] Olympus Optical, Co., Hamburg, Germany), according to the procedure described by Hampson [25]. The goblet GAE equivalent Folin-Ciocalteu) was produced by Tamp- cells positive for Alcian blue (pH 2.5)-PAS staining were ieri Financial Group (Faenza, RA, Italy). counted using NIH ImageJ software [26] on 10 different The supplementation level of extracts was chosen villi per animal along 300 μm of the villus surface. Immu- based on commercial standards and previous results nohistochemical analyses to identify CD3 intraepithelial [8]. The inclusion of CN and GP extracts in the diets T-cells and CD45 intraepithelial leukocytes in broiler was performed at the experimental farm by thoroughly jejunal mucosa were performed following the procedure mixing the C diets with the dried extracts using an elec- described by Röhe et  al. [27]. Intraepithelial leukocytes tric concrete mixer (Suncoo 4/5HP Concrete mixer, 140 were counted in the epithelium using a reference rectan- L, 600 W, 2800 r/min; SUNCOO, China). A total of 3 kg gle with the short side at 100 μm and expressed as the + + 2 of mash diet were progressively added with extracts in density of CD45 and CD3 cells (cells/10,000 μm ). Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 4 of 17 RNA-seq library preparation and sequencing took approximately 3 h. Ready-to-cook carcasses were Total RNA from the 72 chickens slaughtered at 15 and recovered after 2 h of refrigeration at 2°C and indi- 35 d was extracted using the RNAeasy Mini Kit (Qiagen, vidually weighed to measure the slaughter dressing Hilden, Germany), following the manufacturer’s instruc- percentage. tions; total RNA concentration was then determined A total of 144 carcasses (four per pen), previously using a Qubit RNA BR (Broad-Range) kit in a Qubit 2.0 selected on the basis of the final live weight as corre - Fluorometer (Life Technologies, Carlsbad, CA, USA). sponding to the mean BW within a pen, were subjected RNA quality was assessed using a 2100 Bioanalyzer (Agi- to gross examination to evaluate the occurrence in pec- lent Technologies, Waldbronn, Germany). All samples toralis major muscles (presence or absence) of white had an RNA integrity number > 7. striping (WS) (either moderate or severe) [33]; wooden Equal amounts of RNA from three different chickens of breast (WB) (firm upon palpation, prominent ridge like the same sex, fed with the same diet and slaughtered at bulge on caudal area of fillet, clear viscous fluid cover the same age (15 or 35 d) were pooled; a total of 24 RNA and/or petechial multifocal lesions on the fillet surface) pools were obtained (i.e., two pools/replicates per sex [34]; and spaghetti meat (SM) (exhibiting an overall for each of the three diets and per two slaughtering age). impaired integrity and tendency toward separation of Notably, the RNA pooling is a common practice among the muscle fiber bundles especially within the cranial gene expression studies, and it is well justified based on part of the fillet) [35]. The 144 carcasses were stored at statistical and practical considerations [28]. Twenty-four 2  °C before meat quality analyses. Twenty-four hours tagged RNA-seq libraries were prepared using the Illu- after slaughter, carcasses were dissected for the main mina TruSeq Stranded mRNA kit and sequenced on an cuts (breast, wings, thighs, and drumsticks). Pectora- Illumina NovaSeq 6000 instrument at the NGS Sequenc- lis major muscles were separated from the breasts for ing Core (Padova, Italy) following a 100-bp paired-end meat quality analyses [36]. The pH values of the pectora - approach. lis major muscles were measured in triplicates on their ventral side with a pH meter (Basic 20, Crison Instru- ments Sa, Carpi, Italy) equipped with a specific elec - RNA-seq reads processing and mapping trode (cat. 5232, Crison Instruments Sa, Carpi, Italy). Initial quality control was performed using FastQC soft- The L*a*b* color indexes were measured in triplicate on ware version 0.11.9 [29]. Read trimming and adapter the ventral side of the same muscles covered by a trans- removal were performed using Trimmomatic (version parent plastic film, using a Minolta CM-508 C spectro - 0.39) with default parameters [30]. Reads shorter than photometer (Minolta Corp., Ramsey, NJ, USA) [37]. 36 bp were excluded from the analysis. Residual ribo- After measuring the pH and color indexes, one meat somal RNAs (rRNAs) were removed through the local portion (8 cm × 4 cm × 3 cm) was separated from the sequence alignment tool SortMeRNA 2.1 [31] against dif- cranial side of the pectoralis major muscle, parallel to ferent databases (Rfam 5.8S; Rfam 5S; Silva 16S archaeal, the direction of the muscle fibers, and stored under vac - bacterial; Silva 18S eukaryote; Silva 23S archaeal, bacte- uum in plastic bags at -18 °C until meat analyses. Thaw - rial; Silva 28S eukaryote). Reads trimmed and cleaned as ing and cooking losses were measured in this cut [36]. described above were then mapped against the chicken After thawing, the meat portion was placed in a plastic Ensembl reference genome (GCA_000002315.5) using bag and cooked in a water bath for 45 min until an inter- the STAR aligner and following the two-pass map- nal temperature of 80  °C was achieved. After 40 min of ping mode [32]. The maximum number of allowed mis - cooling, another meat portion (4 cm × 2 cm × 1 cm) matches and the maximum number of loci to which the was separated to assess the maximum shear force using reads could map were set to 8 and 10, respectively. Read an LS5 dynamometer (Lloyd Instruments Ltd, Bognor counts per sample at the gene level were extracted by set- Regis, UK) using the Allo-Kramer (10 blades) probe ting the GeneCounts quantification while running STAR. (load cell: 500 kg; distance between the blades: 5 mm; thickness: 2 mm; cutting speed: 250 mm/min) [37]. Commercial slaughtering and carcass and meat quality The pectoralis major muscles of the remaining 72 recordings carcasses (2 chickens per pen, 12 per experimen- At 45 days of age, all remaining chickens were slaugh- tal group) were dissected and subsequently stored at tered in a commercial slaughterhouse. The chickens -20  °C to assess the meat oxidation level of thiobarbi- were weighed individually before crating. Loading turic acid reactive substances (TBARs) [38] using spec- took approximately 1 h, transport from the experimen- trophotometric measurements (Jasco Mod. 7800 UV/ tal facilities to the commercial slaughterhouse took VIS) at 532 nm. The results were expressed as μg of approximately 15 min, and lairage before slaughtering malondialdehyde/kg. P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 5 of 17 Statistical analysis The input was prepared as follows: the raw P values (pval) Individual data of live weights, daily growth, and carcass obtained through the pairwise DE analysis were used and meat traits were subjected to analysis of variance to rank the list of genes by significance. When multiple (ANOVA) with diets (C, CN, and GP) and sex as main genes with the same gene name were detected, only the factors of variability and their interactions, and the pens most significant gene (based on pval) was retained. The as a random effect, using the PROC MIXED procedure pval were replaced by 1-pval or -(1-pval) when a gene in SAS [39]. Being live weight of male and female chicks was over- or under-expressed, respectively. The analysis significantly different at the first hatching day (Table  2) it was performed using the gseKEGG function in the Clus- was included in the model as a covariate for live weights terProfiler package [41]. and daily growth. Pen data of feed intake and feed con- version were subjected to ANOVA, with diet and sex as Results main factors of variability, and their interactions, using Growth performance the PROC GLM procedure [39]. Individual data related to Live weight at 15 d did not differ among groups (513 g on + + jejunum morphology, goblet cells, and C D3 and C D45 average), whereas live weight at 29 d was higher in broil- cell densities were analyzed using the PROC GLM proce- ers fed the GP diet than in those fed the CN diets (P < dure, with diet, age, and sex as the main effects and their 0.01), and this difference was confirmed at the end of the interactions. The Chi-square test was used to test differ - study in comparison with those fed the C and CN diets ences in mortality according to diet and the rate of myo- (45 d; P < 0.01) Table  2. Therefore, the daily weight gain pathies according to diet and sex. Adjusted means were during the study was higher in broilers fed the GP diet compared using Bonferroni’s t-test. Differences between than in those fed the other two diets (P < 0.01), without the means with P ≤ 0.05 were considered statistically differences in feed intake and feed conversion. significant. Males were heavier than females from the first day to For whole-transcriptome profiling, a pairwise differ - the end of the study (P < 0.001), resulting in a higher daily ential expression (DE) analysis was performed using the weight gain (+22%) and feed intake (+17%), without dif- likelihood ratio test implemented in EdgeR [40] to com- ferences in feed conversion (1.59) (Table 2). A significant pare mRNA profiles between experimental groups. A interaction was observed between dietary treatment and false discovery rate (FDR) of ≤ 0.05 and a fold change sex at 45 d (P < 0.05); males fed the GP diet were heavier (FC) of ≥ 1.5 were used as thresholds of significance than those fed C and CN diets, whereas females fed C between age and sex. Regarding changes due to diets, an and GP diets were heavier than those fed the CN diet. FC threshold of 2 was selected to mitigate possible false Losses were low (3.4%, 27 chickens) and due to mortal- positives due to the limited number of replicates per ity (1.1%) and lameness (2.3%) (Table 2). experimental group (i.e., two per diet, per age, per sex). Overall, four sex/age-specific datasets (each consisting Slaughter results, meat quality, and myopathy rate of six samples) were analyzed separately, i.e., females at Table  3 shows that the carcass weight (with feet) was 15 d; females at 35 d; males at 15 d; and males at 35 d. higher in chickens fed the GP diet than in those fed the A functional interpretation of significant differentially other two diets (P < 0.05). No diet-related effects on myo - expressed genes (DEGs) was obtained through a Gene pathy occurrence (Table 3) or meat quality (Table 4) were Ontology over-representation test, performed using recorded. the ClusterProfiler package in the R environment [41]. Regarding the effect of sex, males had heavier carcasses Only KEGG pathways were considered, using the func- (+17%, P < 0.001) and a higher proportion of leg weight tion enrichKEGG. Ensembl gene identifiers were used (thighs + drumsticks) (+0.7; P < 0.05) but a lower breast to establish a list of significantly upregulated and down - yield (-1.7%; P < 0.001) and pectoralis major muscle pro- regulated genes and a “background” (i.e., the whole set of portion (-0.6%; P < 0.001) than those of females (Table 3). expressed genes). The ClusterProfiler package was used Females showed a lower meat pH (P < 0.01) (Table  4), to produce plots representing enriched terms (P ≤ 0.05). lower WB rate (P < 0.001), and higher SM rate (P < 0.001) A pre-ranked KEGG Gene Set Enrichment Analy- than those of males but had similar WS rates to those of sis (GSEA) [42] was performed to investigate whether males (Table 3). gene sets defined a priori showed a statistically signifi - cant enrichment at either end of the ranking. A statisti- Jejunum morphology and immuno-histochemical analyses cally significant enrichment value (Benjamini–Hochberg On average of the two slaughtering times of 15 and 35 adjusted P value ≤ 0.05) indicates that the biological d, the villi height was lower in chickens fed the CN diet activity (e.g., the biomolecular pathway) characterized than in those fed the C diet (P < 0.001); moreover, chick- by the gene set is correlated with the supplied ranking. ens fed the CN diet had a lower density of CD45 cells (P Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 6 of 17 Table 2 Growth performance (LS means) and mortality of broiler chickens until slaughter Diet (D) Sex (S) P value MSE Items C CN GP Females Males D S D×S Chickens, n 258 252 255 383 382 Pens, n 12 12 12 18 18 Live weight, g Initial (1 d) 44.3 43.9 44.5 43.7 44.9 0.15 <0.001 0.16 3.45 15 d 512 510 518 498 529 0.23 <0.001 0.12 50.7 ab a b 29 d 1689 1669 1703 1559 1815 <0.01 <0.001 0.12 124 2 a a b Final (44 d) 3099 3087 3146 2816 3406 <0.01 <0.001 0.02 217 Whole trial (1-44 d) 2 a a b Daily weight gain , g/d 69.4 69.1 70.5 63.0 76.4 <0.01 <0.001 0.02 4.94 Daily feed intake, g/d 111 110 112 102 119 0.16 <0.001 0.70 2.47 Feed conversion 1.59 1.59 1.59 1.59 1.59 0.85 0.91 0.26 0.36 Losses , % 2.27 4.55 3.41 3.28 3.54 0.33 0.70 0.70 - MSE root mean square error. C, control diet. CN, control diet supplemented with 0.2% chestnut wood extracts. GP, control diet supplemented with 0.2% grape pomace extracts Individual data: live weight and daily growth rate. Pen data: feed intake and feed conversion Interaction Diet × Sex, Final live weight: P = 0.02: 2827 g, 2789 g and 2822 g in females fed C, CN and GP diets; 3372 g, 3381 g and 3473 g in males fed C, CN, and GP diets, respectively. Daily weight gain: P = 0.02: 63.2 g/d, 62.4 g/d and 63.1 g/d in females fed C, CN, and GP diets, 75.6 g/d, 75.8 g/d and 77.9 g/d in males fed C, CN, GP diets, respectively. Dead and lame chickens a,b Values with different superscript letters significantly differ (P < 0.05) Table 3 Slaughter results, carcass traits (LS means) and myopathy rates in chickens slaughtered at 45 days of age  Items Diet (D) Sex (S) P value MSE C CN GP Females Males D S D×S Chickens , n 258 252 255 383 382 Pens, n 12 12 12 18 18 2 a a b Cold carcasses , g 2352 2342 2392 2122 2602 0.03 <0.001 <0.01 173.06 Dressing out percentage, % 76.6 76.8 76.8 76.4 77.1 0.45 <0.001 0.51 1.48 Chickens, n 48 48 48 72 72 Cold carcasses (CC), g 2274 2243 2306 2063 2486 0.32 <0.001 0.10 143.58 Dressing percentage, % 73.3 73.0 73.0 73.3 73.0 0.48 0.18 0.92 1.32 Breast yield , % CC 39.3 38.8 39.5 40.0 38.3 0.16 <0.001 0.56 1.76 P. major, % CC 12.2 12.1 13.4 12.5 11.9 0.33 <0.001 0.37 0.85 Wings, % CC 9.8 10.0 9.9 9.9 9.9 0.06 0.44 0.65 0.51 Legs (thighs+drumsticks), % CC 30.1 30.1 29.8 29.6 30.3 0.67 0.04 0.43 2.02 Myopathy rates at P. major White striping, % 72.9 60.4 79.2 66.7 75.0 0.12 0.26 - - Wooden breast, % 41.7 25.0 41.7 16.7 55.6 0.11 <0.001 - - Spaghetti meat, % 29.5 25.5 25.0 48.6 4.2 0.83 <0.001 - - MSE root mean square error. C, control diet. CN, control diet supplemented with 0.2% chestnut wood extracts. GP, control diet supplemented with 0.2% grape pomace extracts 1 2 Carcasses with feet. Interaction Diet × Sex, Cold carcasses: P < 0.01: 2126 g, 2103 g, 2126 g in females fed C, CN and GP diets, respectively; 2571 g, 2580 g, and 2652 3 4 g in males fed CN and GP diets. Without feet. With bone a,b Values with different superscript letters significantly differ (P < 0.05) P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 7 of 17 Table 4 Rheological traits and lipid oxidation status ( TBARs) of the pectoralis major muscle in chickens slaughtered at 45 days of age  Items Diet (D) Sex (S) P-value MSE C CN GP Females Males D S D×S P. major, n 48 48 48 72 72 pH 5.99 5.99 5.95 5.94 6.02 0.56 <0.01 0.99 0.16 L* 50.4 50.2 49.7 49.9 50.1 0.66 0.63 0.10 2.40 a* -0.04 -0.07 -0.15 -0.03 -0.15 0.56 0.23 0.20 0.54 b* 10.4 10.3 10.2 10.5 10.1 0.70 0.09 0.65 1.38 P. major, n 24 24 24 36 36 Cooking losses, % 12.0 11.7 11.8 11.4 12.3 0.99 0.65 0.92 12.4 Shear force, kg/g 4.17 4.06 4.30 4.17 4.18 0.39 0.96 0.72 0.83 TBARs, mg MDA/kg 0.083 0.080 0.075 0.078 0.080 0.38 0.74 0.22 0.021 MSE root mean square error. C, control diet. CN, control diet supplemented with 0.2% chestnut wood extracts. GP, control diet supplemented with 0.2% grape pomace extracts < 0.05) than those chickens fed the GP diet (Table 5). Die- mapped to the chicken reference genome (Table S3). The tary treatment did not affect the density of goblet cells. multidimensional scaling plot provided unsupervised As the age increased from 15 to 35 d, villi height (P < clustering of the samples (Fig.  1). The first dimension 0.01), crypt depth (P < 0.05), and density of both C D3 (x-axis) clearly separates females from males, whereas and CD45 cells (P < 0.001) increased (Table 5), whereas the second dimension (y-axis) separates samples by age. the density of goblet cells decreased (P = 0.001). The biological variability within dietary treatments was Regarding sex, villi height was lower in females than in low, as demonstrated by the clusters formed when MDS males (P < 0.01), whereas the densities of C D45 cells (P distances between expression profiles of all the replicates = 0.01) and goblet cells (P = 0.06) were higher (Table 5). were plotted. Jejunum samples showed largely different + + As for villi height, CD3 and C D45 cells, significant transcriptional profiles between sexes and between ages, interactions were recorded between sex and age (Table whereas compared with the C diet, the CN and GP diets S1), and between sex and diet (Table S2). had a moderate impact on jejunum gene expression. Whole transcriptome analysis A total of 725,278,263 raw reads were obtained. Raw Illu- Dietary effects on jejunum whole transcriptome mina sequencing data were deposited in GenBank under Transcriptional profiles of same-sex and same-age the BioProject accession number PRJNA666129. All chickens fed CN and GP diets were compared to those samples passed the quality control measures. After trim- of chickens fed the C diet, thus identifying significant ming and rRNA removal, an average of approximately 30 DEGs, that are shown in Table  6 and in the supplemen- million reads per sample was retained, with ~94% reads tary Tables S4 and S5. + + Table 5 Jejunum morphometry, number of goblet cells and densities of CD45 and CD3 cells at 15 and 35 days of age  Items Diet (D) Age (A) Sex (S) P-value MSE 1 2 C CN GP 15 35 F M D A S D×A A×S D×S D×A×S Broilers, n 24 24 24 36 36 36 36 b a ab Villi height, μm 1033 934 954 866 1082 936 1011 <0.001 <0.01 <0.01 0.44 0.02 0.03 0.13 111 Crypt depth, μm 145 139 144 138 147 140 145 0.45 0.04 0.23 0.70 0.29 0.79 0.31 18,5 Villi / Crypt ratio 7.49 7.06 6.96 6.67 7.67 7.06 7.28 <0.001 0.16 0.37 0.70 0.37 0.20 0.68 1,00 Goblet cells, n/300 μm 21.02 21.67 22.25 22.84 20.45 22.20 21.09 0.24 0.001 0.06 0.34 0.12 0.43 0.27 2.46 + 2 CD3 cells, n/10,000 μm 2242 2219 2297 2069 2436 2223 2283 0.16 <0.001 0.09 0.23 <0.001 <0.001 0.46 459 + 2 ab a b CD45 cells, n/10,000 μm 2879 2793 2925 2437 3295 2915 2816 0.02 <0.001 0.01 0.18 <0.001 0.01 0.45 537 MSE root mean square error. C, control diet. CN, control diet supplemented with 0.2% chestnut wood extracts. GP, control diet supplemented with 0.2% grape pomace extracts 1 2 Averages of traits according to Age × Sex are provided in Table S1. Averages of traits according to Diet × Sex are provided in Table S2. a,b Values with different superscript letters significantly differ (P < 0.05) Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 8 of 17 Fig. 1 Multiple dimensional scaling (MDS) plot of distances between expression profiles (as log fold change, logFC) of RNA-seq libraries (24 samples) of the jejunum of female (F) and male (M) broiler chickens fed control diet (C diet), diet added with 0.2% chestnut extracts (CN diet), and diet added with 0.2% grape pomace extracts (GP diet) at 15 d (libraries C_15d, CN_15d, GP_15d) and 35 days of age (libraries C_35d, CN_35d, GP_35d) [1F, 2F, 1M, 2M stand for the two replicates for females and the two replicates for males per age] At 15 d, when comparing females fed the CN diet At 35 d, 30 DEGs were detected when comparing with those fed the C diet, 15 DEGs were identified. females fed the CN and C diets. Some genes involved in Cytochrome P450 1A1 (CYP1A1; logFC: 1.71), CYP1A2 immunity and inflammation, such as complement C1q (logFC: 1.47) and STEAP4 Metalloreductase (STEAP4; C chain (logFC: 1.07), major histocompatibility complex logFC: 4.65) were upregulated. Guanylate-binding pro- class I polypeptide-related sequence A (MICA; logFC: tein 1-like (GBP1-like; logFC: -8.43), butyrophilin sub- 1.72), extracellular fatty acid-binding protein (logFC: family 1 member A1-like (BTN1A1-like; logFC: -1.69), 1.32), and bradykinin receptor B1 (BDKRB1; logFC: 1.26), and leukocyte immunoglobulin-like receptor subfamily were upregulated. Likewise, the glutathione S-transferase A member 2 (logFC: -4.12) were downregulated. Regard- class-alpha (GSTA) gene was upregulated (logFC: 1.25). ing the effects of the GP diet compared with those of the In females at 35 d, compared with the C diet the GP C diet in females at 15 d, avian beta-defensin 9 and 10 diet upregulated 197 genes. Among the genes involved (AvBD9 and AvBD10, respectively) and class I histocom- in inflammatory processes, the following were upregu - patibility antigen, F10 alpha chain-like (HA1F-like) were lated: nephroblastoma overexpressed (NOV; logFC: remarkably upregulated (logFC of 7.18, 9.23, and 2.43, 1.06), netrin 1 (logFC: 1.05), C1q and tumor necrosis respectively). Both GBP1-like and BTN1A1-like mRNA factor related protein 4 (C1QTNF4; logFC: 1.69), and levels were lowered (logFC: -6.09 and -1.45). Gap junc- BDKRB1 (logFC: 1.09). Additional upregulated genes tion protein beta 1 and pyruvate dehydrogenase kinase 4 that play a role in the regulation of immune defense were among the most significantly downregulated genes. were the junctional adhesion molecule 2 (logFC: 1.04), P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 9 of 17 Table 6 Number of significantly upregulated and downregulated protein that protects the intestinal mucosa from inflam - differentially expressed genes (Fold change ≥ 2; False Discovery mation. The gene most significantly induced by the GP Rate ≤ 0.05) in the jejunum of male or female broiler chickens fed diet was glutathione peroxidase 4 (logFC: 1.26), whereas the experimental diets at 15 or 35 days of age the suppressor of cytokine signaling 3 (logFC: -1.04) and IL-22 (logFC: -2.80) were downregulated. Comparison Number of Number of Total DEGs At 35 d, CN diet in males significantly induced HA1F- up-regulated down-regulated like (logFC: 1.46) and MICA mRNA levels (logFC: 1.41), genes genes while lysozyme-g-like (logFC: -2.47) and NOV (logFC: Female chickens -1.89) were downregulated. at 15 d On comparing chickens at 35 d and those at 15 d (Table CN diet vs. C diet 7 8 15 S6), 324 DEGs were identified (FDR ≤ 0.05; FC ≥ 1.5). GP diet vs. C diet 14 18 32 The top 10 upregulated and downregulated genes are Male chickens at shown in Table 7. Only two KEGG pathways were statis- 15 d tically enriched by the 213 genes upregulated after 15 d, CN diet vs. C diet 7 7 14 namely, the “peroxisome proliferator-activated receptor GP diet vs. C diet 21 10 31 (PPAR) signaling pathway” and the “neuroactive ligand– Female chickens receptor interaction pathway” (Table S7). However, a at 35 d higher number of KEGG pathways (i.e., 14) were sig- CN diet vs. C diet 18 12 30 nificantly enriched by the upregulated genes in chickens GP diet vs. C diet 197 74 271 at 35 d (Fig.  2 and Table S7); some of them were related Male chickens at to PPAR signaling, xenobiotic metabolism, glutathione 35 d metabolism, steroid hormone synthesis, IgA production, CN diet vs. C diet 8 8 16 and amino acid metabolism. GP diet vs. C diet 2 4 6 In 15-d chickens, the GSEA (Table S7)  pointed out DEGs differentially expressed genes. C, control diet. CN, control diet supplemented with 0.2% chestnut wood extracts. GP, control diet the activation of 18 KEGG pathways related to cell–cell supplemented with 0.2% grape pomace extracts junctions (i.e., tight junction, focal adhesion, and adher- ent junction), cytoskeleton, and transforming growth factor-β (TGF-β). In 35-d chickens, the GSEA highlighted C-X-C motif chemokine ligand 12 (CXCL12; logFC: the activation of 33 KEGG pathways; the most significant 1.08), and T-cell surface glycoprotein CD8 alpha chain- ones include gene sets related to xenobiotic metabolism, like (logFC: 1.74). Moreover, the mRNA levels of hemo- antioxidant response (i.e., glutathione metabolism), pro- globin beta, subunits A, A1, and AD (HBBA, HBA1, and tein processing (i.e., proteasome, protein export, and HBAD), as well as GSTA, were increased (logFC: 2.25, ribosome), and amino acid metabolism. The top-35 sig - 1.85, 2.27, and 1.18, respectively). Finally, as for upregu- nificant KEGG pathways activated in 35-d chickens com - lated genes, glucagon like peptide 1 receptor (GLP1R; pared to 15-d chickens are reported in Fig. 3. logFC: 1.02), and solute carrier family 2, facilitated glu- On comparing females and males, 246 significant DEGs cose transporter member 4-like (GLUT4-like; logFC: were identified; among these, 47 and 199 genes were 1.17) play a role in nutrients intestinal absorption. upregulated in females and males, respectively. The top Among the downregulated genes, some are involved 10 upregulated and downregulated genes are listed in in vitamin and protein absorption, such as scavenger Table  8. The KEGG enrichment analysis of upregulated receptor class B member 1 (logFC: -2.00) and beta-car- genes in males showed that only the “ribosome biogen- otene oxygenase 1 (logFC: -1.55). Further downregu- esis in eukaryotes” pathway was significantly enriched. In lated genes are involved in protein and carbohydrate contrast, GSEA identified a total of 19 KEGG pathways metabolism, such as carboxypeptidase O (logFC: -1.80), that were significantly enriched, 9 in females and 13 in glutamic-pyruvic transaminase 2 (logFC: -1.27), and males (Fig.  4; Table S7). In females, a considerable num- lactase (logFC: -1.10). Additionally, the expression of ber of activated gene sets was related to cell cycle regula- AvBD9 and AvBD10 was downregulated (logFC: -7.59 tion (e.g., DNA replication and mismatch repair). and -6.21, respectively). At 15 d, compared with the C diet, the CN diet signifi - cantly upregulated STEAP4 (logFC: 3.31), CYP1A (logFC: Discussion 1.89), and HA1F-like (logFC: 1.61) genes in males. Among In animal feeding, tannins can have either beneficial or the seven downregulated genes, ectonucleotide pyroph- detrimental effects depending on their chemical struc - osphatase/phosphodiesterase 7 (logFC: 1.66) encodes a ture and dosage, in addition to other individual (e.g., Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 10 of 17 Table 7 Top 10 upregulated ( ) and downregulated ( ) differentially expressed genes (DEGs) in broiler chickens at 35 d compared with those at 15 d. For each DEG, Ensembl gene ID, log fold change (logFC), and False Discovery Rate (FDR), as reported in edgeR output and the Ensembl gene description, are provided Fig. 2 Over-represented KEGG pathways among the upregulated genes in broiler chickens at 35 d compared with those in broiler chickens at 15 d. The number of differentially expressed genes (DEGs) belonging to each enriched KEGG pathway is reported in the x axes. Color gradient represents the P-value significance, as specified in the legend. P-values were adjusted using the Benjamini–Hochberg method P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 11 of 17 Fig. 3 Results of the Gene Set Enrichment Analysis (GSEA): top-35 significantly enriched KEGG pathways in broiler chickens at 35 d compared with those in broiler chickens at 15 d. Pathways showing an enrichment value > 0 are activated in 35-d broiler chickens, whereas those with an enrichment value < 0 are activated in 15-d broiler chickens. Color gradient represents the P-value significance, as specified in the legend. The P-values were adjusted using the Benjamini–Hochberg method animal species, age, sex, physiological state) and feeding The different nature of tannins of CN and GP and factors [43]. Standardization of conditions (in terms of their effects at the gut level can partly explain our quantity and quality of the different polyphenolic frac - results, even if different effects are likely to expected tions) is even more difficult when using extracts from depending on the animal age and gut tract [46]. The natural sources. Thus, in the present trial, we compared CN mainly contains hydrolysable tannins, which usu- two products of a similar nature (byproducts containing ally possess a relatively low molecular weight and high different quantities of polyphenols of different types) in bioavailability [8, 9, 47]. Thus, CN hydrolysable tannins the same form (powder) at the same moderate supple- can be early hydrolyzed and absorbed in the first gastro mentation level. intestinal tract, as observed in rats [48], exerting both We observed that the GP diet improved growth per- cytotoxic or cytoprotective effects on jejunal mucosa formance compared with the other dietary treatments, [46, 48, 49]. On the other hand, GP condensed tannins whereas CN diet reduced the villi height compared to can remain active along the whole gut and can be trans- the control diet and the density of CD45 cells compared formed by gut microbiota in other bioactive metabolites to the GP diet. The highest growth performance meas - which can further affect gut mucosa and microbiota ured in broilers fed GP diet might be in part supported composition [46, 50]. by an increased intestinal absorption of nutrients. In the Schiavone et  al. [8] showed dose-dependent effects of present study, this is suggested by the upregulation in dietary supplementation with CN tannins, i.e., null at the females of genes such as the receptor of glucagon-like lowest inclusion rate (1.5 g CN wood extract/kg), positive peptide (GLP1R), that promotes efficient nutrient assimi - at the intermediate level (2.0 g/kg) in terms of final live lation [44], and GLUT4, a glucose transporter whose weight and feed intake, and negative at the highest rate expression can be induced by tannins [45]. (2.5 g/kg), which reduced live weight, compared with the Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 12 of 17 Table 8 Top 10 upregulated ( ) and downregulated ( ) differentially expressed genes (DEGs) in females compared with those in males. For each DEG, Ensembl gene ID, log fold change (logFC), and False Discovery Rate (FDR), as reported in edgeR output and the Ensembl gene description, are provided Fig. 4 Results of the Gene Set Enrichment Analysis (GSEA): enriched KEGG pathways in female broiler chickens compared with those in male broiler chickens. Pathways showing an enrichment value > 0 are activated in females, whereas those with an enrichment value < 0 are activated in males. Color gradient represents the P-value significance, as specified in the legend. The P-values were adjusted using the Benjamini–Hochberg method P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 13 of 17 C diet (from 14 to 56 days of age). Similarly, Jamroz et al. [61]. Indeed, genes involved in either pro- or anti- [10] did not observe effects on performance with low CN inflammatory response pathways and antimicrobial tannin inclusion rates (0.25 and 0.50 g of sweet CN tan- responses were affected by GP and CN diets, besides nin/kg), whereas the highest dose (1.0 g/kg) decreased genes contributing to the antioxidant responses. Nev- the live weight at 41 d and caused alterations in the intes- ertheless, the majority of these genes, especially those tinal wall morphology and a decreased proliferation rate related to immune functions, were significantly regu - in the mother-cell zone. These findings are consistent lated by the GP supplementation consistently with the with the decreased villi length observed in our study in higher CD45 density in the jejunum of broilers fed chickens fed the CN diet. In contrast, CN tannin supple- GP diet compared to those fed CN diet. Finally, the mentation increased the average daily weight gain and improved immune competences observed in broilers jejunum villus height under a heat-stress challenge [51] fed GP diet might have positively impacted on their and reduced the proliferation of Clostridium perfringens growth performance compared to the other dietary and the severity of gut damage in necrotic enteritis [52]. treatments. Similar to CN, condensed tannins of GP byproducts In the present study, the majority of changes in the (extracted from seeds, skin, and stems as byproducts of chicken jejunum transcriptome was observed in chick- winemaking) can reduce diet digestibility and negatively ens of the same sex but at different ages. In fact, in 15-d affect the growth performance of monogastric animals females, the GP and CN diets downregulated genes [53]. In poultry, during the first period (from hatching involved in the inflammatory response [62] and pro - until 21 days of age), the increase in GP supplementa- tection against microbes and viruses, such as GBP1- tion (from 2.5 to 5.0 g of GP seed extract/kg) decreased like [63] a robust marker of inflammation. In 15-d male both animal performance and diet digestibility [54]. Some and female broilers, the CN diet upregulated STEAP4, authors reported negative effects on performance with which plays a role in the response to chronic inflamma - high inclusion rates of grape-based extracts [12], which tion in colon cancer [64], the suppression or inhibition also showed a negative effect on villus length. In contrast, of cytokine production and signaling (IL-6 and TNF-α- 6% dietary inclusion of a GP concentrate did not modify induced NF-κB signaling [65]), the response to nutri- performance or diet digestibility but showed antioxidant ents, oxidative stress, fatty acid metabolism, and glucose potential that was as effective as vitamin E in diet, excreta, metabolism [66]. In 15-d chickens of both sexes, CN ileal content, and breast muscle [15]. Other authors evi- supplementation modulated the expression of CYP1A, denced that the dietary supplementation of grape byprod- an important detoxifying monooxygenase that can be ucts increased activity of total superoxide dismutase and induced by natural polyphenols [67] as demonstrated in decreased the content of malondialdehyde in plasma [19] the gut of pigs fed CN extracts [68]. and leg meat [55], whereas this was not observed in the In 35-d females, the GP diet upregulated C1QTNF4 present study when measuring meat TBARs. (involved in the regulation of the inflammatory networks In our study, we neither observed any effect of CN or and in feed intake suppression in mice) [69] and CXCL12 GP supplementation on myopathy rate, which is consist- (a constitutive and inflammatory chemokine of the intes - ent with the findings of previous studies that tested vari - tinal immune system) [70]. Conversely, in 35-d females, ous antioxidants for this purpose [56–58]. Nevertheless, the GP diet downregulated beta-defensins (AvBD9, oxidative stress, localized hypoxia, increased intracellular AvBD10), which possess modest antimicrobial properties calcium, and the presence of muscle fiber-type switching and display a wide range of immunomodulatory activi- are pathways responsible for the occurrence of WB and ties, such as modulation of pro- and anti-inflammatory WS [16, 59, 60]; hence, the administration of antioxidant responses, promotion of wound healing [71]. Addition- substances, such as GP or CN extracts, could be expected ally, in 35-d females, both GP and CN diets induced the to affect myopathy occurrence. expression of GSTA, primarily involved in the defense In the present study, the increased mucosal immune against oxidative stress. The GP diet also induced three responses with the GP diet (i.e. the higher density of hemoglobin subunits (i.e., HBBA, HBAD, and HBA1), intraepithelial leukocytes, especially CD45 ) compared which are upregulated in the presence of oxidative stress to the CN diet could be attributed to the tannin nature and are believed to alleviate it [72]. and availability along the intestinal tract as discussed Notably, most of the differences in favor of GP supple - above (condensed compared to hydrolysable tannins in mentation regarding the overall transcriptomic response GP compared to CN). On the other hand, when com- appeared in the second period of growth. Overall, this is paring different dietary supplementation doses of tan - consistent with the results of Farahat et  al. [73] and Yang nic acid, effects on broiler chicken immunity changed et al. [19], who observed a time-dependent cumulative effect from positive to negative in a dose-dependent manner of dietary supplementation with grape seed extracts and Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 14 of 17 pomace concentrate. However, according to some authors interaction,” and “regulation of actin cytoskeleton” gene [8, 53], the dietary supplementation with tannins (from grape sets). On the other hand, differences in growth rates and seed and CN extracts) was more effective in younger broilers final live weight between sexes are also associated to a than in older ones, likely based on their effects on pathogenic different occurrence of myopathies whereas the results microorganisms as well as on commensal microbiota [74, 75]. of the present study confirmed that SM occurred more In the present study, the most significant age-dependent often in females than in males, whereas WB was more transcriptional variations (i.e., top 10 upregulated DEGs) frequent in males than in females [81, 82]. were related to immunoglobulin functions, for which the KEGG pathway “intestinal immune network for IgA pro- Conclusions duction” was significantly enriched with the increase in age. The dietary supplementation of GP extracts can be ben - Overall, this age-dependent improvement of immune com- eficial to broilers because it increases growth performance petencies, together with the increased densities of jejunum and the final live weight of animals. In the absence of any + + CD3 and CD45 cells we observed in older chickens, was specific challenges, it still improved the jejunum morphol - expected. Additionally, four of the top 10 upregulated DEGs ogy and the overall immune response. The addition of CN encoded GSTs, which suggests that glutathione-dependent extracts affected the jejunum morphology, although growth detoxifying capability significantly increases with chicken traits remained unaffected. Overall, the chicken jejunum development. This was further strengthened by the enrich - transcriptome was scarcely affected by extracts, although ment of the KEGG pathways “glutathione metabolism” and in 35-d females fed the GP diet, some positive effects on “metabolism of xenobiotics by cytochrome P450,” both nutrient absorption, immune and antioxidant responses including several differentially regulated GST s. were observed. Regarding age and sex, further molecular The downregulation of three hemoglobin subunits (i.e., investigations are required in view of the interactions with HBBA, HBAD, and HBA1) in 35-d chickens compared dietary additives, as observed in the present study, and sub- with 15-d chickens could be related to an imbalance sequent effect on chicken health and performance. between the muscular development of birds and the vas- cularization/blood supply and might reflect the possible muscle suffering that leads to muscle fiber degeneration Abbreviations ANOVA: Analysis of variance; AvBD10: Avian beta-defensin 10; AvBD9: Avian and myopathies with the increase in age [76, 77]. beta-defensin 9; BDKRB1: Bradykinin receptor B1; BTN1A1: Butyrophilin Expected differences in performance between males subfamily 1 member A1; C: Control; C1QTNF4: C1q and tumor necrosis factor and females were confirmed and found to be associ - related protein 4; CN: Chestnut wood; GP: Grape pomace; CXCL12: C-X-C motif chemokine ligand 12; CYP1A1: Cytochrome P450 1A1; DE: Differential ated with differences in the jejunum transcriptome. Top expression; DEGs: Differentially expressed genes; FC: Fold change; FDR: False upregulated genes in females are involved in ubiquitina- discovery rate; GBP1: Guanylate-binding protein 1; GSEA: Gene Set Enrich- tion, a post-translational mechanism for protein deg- ment Analysis; GSTA: Glutathione S-transferase class-alpha; HA1F-like: F10 alpha chain-like; HBA1: Hemoglobin beta subunit A1; HBAD: Hemoglobin beta radation via the proteasome, ensuring the structural subunit AD; HBBA: Hemoglobin beta subunit A; IL: Interleukin; MICA: major integrity control and/or protein turnover rate. However, histocompatibility complex class I polypeptide-related sequence A; NOV: these transcriptional variations seem related to DNA Nephroblastoma overexpressed; PAS: Periodic Acid-Schiff; PBS: Phosphate- buffered saline; PPAR: Peroxisome proliferator-activated receptor; pval: P val- repair pathways and cell fate decisions, rather than pro- ues; rRNAs: Ribosomal RNAs; SM: Spaghetti meat; STEAP4: Metalloreductase; teolytic processes. This hypothesis is consistent with a TBARs: Thiobarbituric acid reactive substances; TGF-β: Transforming growth non-degrading role of protein ubiquitination [78], and factor-β; TNF-α: Tumor necrosis factor Alpha; WB: Wooden breast; WS: White striping. supported by the enriched KEGG pathways reported in females (e.g., “mismatch repair,” “cell cycle”). Supplementary Information Overall, the gene expression data showed that DNA The online version contains supplementary material available at https:// doi. repair processes are likely to be differentially regulated in org/ 10. 1186/ s40104- 022- 00736-w. male and female jejunum. Notably, sex differences in the control of cell cycle and DNA repair have been reported Additional file 1. RNA-seq libraries sequenced and transcriptional in mammals [79, 80]. Upregulated DEGs playing a role in changes. Averages of traits for jejunum morphology and immuno-histo- chemical analyses according to interactions between sex and age and the mitochondrial respiratory chain and ATP production between sex and diet are provided in Table S1 and Table S2, respectively. were also reported, e.g., cytochrome c oxidase subunit 7C Details on sequenced RNA-seq libraries are provided in Table S3. Compari- and NADH:ubiquinone oxidoreductase subunit S4. This sons among transcriptional profiles of same-sex and same-age chickens fed CN and GP diets compared to those of chickens fed the C diet, thus is most likely linked to the different growth rate between identifying significant DEGs, are shown in supplementary Tables S4, S5 sexes (higher in males), which is confirmed by the sig - and S6. Annotated genes significantly regulated in more than one com- nificant activation in males of genes involved in tissue parison are listed in Tables S5. Gender-related and sex-related transcrip- tional changes are reported in Tables S5 and S6. Functional enrichment morphogenesis and maintenance of cell and tissue struc- analysis is shown in Tables S7. ture and function (e.g. “focal adhesion,” “ECM-receptor P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 15 of 17 4. Li H, Li Z, Wei Z, Liu T, Zou X, Liao Y, et al. Long-term effects of oral tea Acknowledgments polyphenols and Lactobacillus brevis M8 on biochemical parameters, The PhD grant (PhD Course in Animal and Food Science of the University of digestive enzymes, and cytokines expression in broilers. J Zhejiang Padova) of Giulio Pillan is funded by Unismart and OFFICINE FACCO & C. Spa Univ Sci B. 2015;16:1019–26. (year 2020). The authors wish to thank Giovanni Caporale for his assistance in 5. Lupini C, Cecchinato M, Scagliarini A, Graziani R, Catelli E. In vitro antiviral preparing the samples for histological analyses. activity of chestnut and quebracho woods extracts against avian reovirus and metapneumovirus. Res Vet Sci. 2009;87:482–7. Authors’ contributions 6. Brus M, Gradišnik L, Trapečar M, Škorjanc D, Frangež R. Beneficial effects of AT, MD, and GX conceived and designed the experiment. AP, MB, FB and GP water-soluble chestnut (Castanea sativa Mill.) tannin extract on chicken performed the trial, collected the experimental data. MP, AT, MG, FB, and MG small intestinal epithelial cell culture. Poult Sci. 2018;97:1271–82. collected and prepared samples for histological and whole transcriptome 7. Xiong Y, Dong S, Zhao X, Guo KJ, Gasco L, Zoccarato I. Gene expres- analyses. AP, CB, and MB performed histological analyses. MP, MG, and MD sions and metabolomic research on the effects of polyphenols from the performed whole transcriptome analysis and related statistical analyses. AT involucres of Castanea mollissima Blume on heat-stressed broilers chicks. and AP performed the statistical analyses. MP, AT, GX, and MD analysed and Poult Sci. 2016;95:1869–80. interpreted the data. AT and AP wrote the first draft of the manuscript. All 8. Schiavone A, Guo K, Tassone S, Gasco L, Hernandez E, Denti R, et al. authors critically reviewed the manuscript for intellectual content and gave Eec ff ts of a natural extract of chestnut wood on digestibility, per - final approval of the version to be published. formance traits, and nitrogen balance of broiler chicks. Poult Sci. 2008;87:521–7. Funding 9. Huang Q, Liu X, Zhao G, Hu T, Wang Y. Potential and challenges of tannins The project was funded by the University of Padova (BIRD Ateneo di Padova; as an alternative to in-feed antibiotics for farm animal production. Anim CUP: C24I20000260005). Nutr. 2018;4:137–50. 10. Jamroz D, Wiliczkiewicz A, Skorupińska J, Orda J, Kuryszko J, Tschirch H. Availability of data and materials Eec ff t of sweet chestnut tannin (SCT ) on the performance, microbial Raw Illumina sequencing data have been deposited in GenBank (SRA) under status of intestine and histological characteristics of intestine wall in the SRA accessions SRR12728661-SRR12728684, and they will be published chickens. Br Poult Sci. 2009;50:687–99. after manuscript acceptance. During reviewing process, BioProject’s metadata 11. Rezar V, Salobir J. Eec ff ts of tannin-rich sweet chestnut (Castanea sativa are available at the following link: mill.) wood extract supplementation on nutrient utilisation and excreta https:// datav iew. ncbi. nlm. nih. gov/ object/ PRJNA 666129? revie wer= 398l4 cbdnf dry matter content in broiler chickens. Europ Poult Sci. 2014;78:42. nmu1m ris3g 20ol1c https:// doi. org/ 10. 1399/ eps. 2014. 42. The other datasets analysed in the current study are available from the cor- 12. Viveros A, Chamorro S, Pizarro M, Arija I, Centeno C, Brenes A. Eec ff ts of responding author upon reasonable request. dietary polyphenol-rich grape products on intestinal microflora and gut morphology in broiler chicks. Poult Sci. 2011;90:566–78. Declarations 13. Wang ML, Suo X, Gu JH, Zhang WW, Fang Q, Wang X. Influence of grape seed proanthocyanidin extract in broiler chickens: effect on chicken coc- Ethics approval and consent to participate cidiosis and antioxidant status. Poult Sci. 2008;87:2273–80. The study was approved by the Ethical Committee for Animal Experimenta- 14. Starčević K, Krstulović L, Brozić D, Maurić M, Stojević Z, Mikulec Ž, et al. tion (Organismo Preposto al Benessere Animale) of the University of Padova Production performance, meat composition and oxidative susceptibil- (project 92/2019 - prot. nr. 469509). All animals were handled according to ity in broiler chicken fed with different phenolic compounds. J Sci Food the principles of EU Directive 2010/63/EU regarding the protection of animals Agric. 2015;95:1172–8. used for experimental and other scientific purposes. The researchers involved 15. Brenes A, Viveros A, Goñi I, Centeno C, Sáyago-Ayerdy SG, Arija I, in animal handling were either animal specialists (PhD or MSc in Animal Sci- et al. Eec ff t of grape pomace concentrate and vitamin e on digest - ences) and/or veterinary practitioners. ibility of polyphenols and antioxidant activity in chickens. Poult Sci. 2008;87:307–16. Consent for publication 16. Abasht B, Mutryn MF, Michalek RD, Lee WR. Oxidative stress and meta- Not applicable bolic perturbations in wooden breast disorder in chickens. PloS One. 2016;11:e0153750. Competing interests 17. Soglia F, Laghi L, Canonico L, Cavani C, Petracci M. Functional property The authors declare that they have no competing interests. issues in broiler breast meat related to emerging muscle abnormalities. Food Res Int. 2016;89:1071–6. 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Dietary supplementation with sodium butyrate: growth, gut response at different ages, and meat quality of female and male broiler chickens. Ital J Anim Sci. 2020;20:1135–46. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Animal Science and Biotechnology Springer Journals

Effect of the dietary supplementation with extracts of chestnut wood and grape pomace on performance and jejunum response in female and male broiler chickens at different ages

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Abstract

Background: Recently, interest in the use of herbs and phytogenic compounds has grown because of their potential role in the production and health of livestock animals. Among these compounds, several tannins have been tested in poultry, but those from chestnut wood and grape-industry byproducts have attracted remarkable interest. Thus, the present study aimed to gain further insights into the mechanisms involved in the response to the dietary supplemen- tation with extracts of chestnut wood or grape pomace. To this purpose, 864 broiler chickens were fed a control diet (C) or the same diet supplemented 0.2% chestnut wood (CN) extract or 0.2% grape pomace (GP) extract from hatch- ing until commercial slaughtering (at 45 days of age) to assess their effects on performance, meat quality, jejunum immune response and whole-transcriptome profiling in both sexes at different ages (15 and 35 d). Results: Final live weight and daily weight gain significantly increased (P < 0.01) in chickens fed GP diets compared to CN and C diets. The villi height was lower in chickens fed the CN diet than in those fed the C diet (P < 0.001); moreover, a lower density of CD45 cells was observed in chickens fed the CN diet (P < 0.05) compared to those fed the C and GP diets. Genes involved in either pro- or anti-inflammatory response pathways, and antimicrobial and antioxidant responses were affected by GP and CN diets. There was no effect of the dietary treatment on meat quality. Regarding sex, in addition to a lower growth performance, females showed a lower occurrence of wooden breast (16.7% vs. 55.6%; P < 0.001) and a higher occurrence of spaghetti meat (48.6% vs. 4.17%; P < 0.001) in pectoralis major muscles after slaughtering than those in males. Based on the results of whole-transcriptome profiling, a significant activation of some molecular pathways related to immunity was observed in males compared with those of females. Conclusions: The GP supplementation improved chicken performance and promoted immune responses in the intestinal mucosa; moreover, age and sex were associated with the most relevant transcriptional changes. Keywords: Growth, Immunohistochemistry, Meat quality, Tannins, Transcriptome Background *Correspondence: angela.trocino@unipd.it During the last decade, interest in the use of herbs and phytogenic compounds has grown because of their Department of Comparative Biomedicine and Food Science (BCA), University of Padova, Viale dell’Università 16, 35020 Padova, Legnaro, Italy potential role in the production and health of livestock Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 2 of 17 animals. Among these compounds, plant tannins are examined the effect of dietary treatments on jejunum polyphenolic compounds that exhibit antioxidant activ- morphology, inflammatory patterns, and whole-tran - ity, which positively affect the composition of the gut scriptome profiling. microbial flora, stimulate the animal immune system and possess antibacterial properties [1–3]. Additionally, at the Methods intestinal level polyphenols downregulate the expression Experimental facilities of various proinflammatory cytokines, such as interleu - This study was conducted at the Experimental Farm of kin (IL)-1β, IL-4, IL-6, IL-10, tumor necrosis factor alpha the University of Padova (Legnaro, Padova, Italy) in a (TNF-α), and interferon gamma [4]. poultry house equipped with a cooling system, forced In the poultry industry, several tannins have been ventilation, radiant heating, and controlled light systems. tested, but those from chestnut wood  (CN) and grape- Thirty-six wire-net pens (2.5 m × 2.4 m; 6 m ) were used, industry byproducts have attracted remarkable inter- each equipped with five nipple drinkers and a circular est. In  vitro studies have shown the antimicrobial and feeder for manual distribution of feed. Each pen had a antiparasitic effects of CN tannins [5, 6]. Moreover, in concrete floor covered with wood shaving litter (depth broiler chickens [7], CN extract increased jejunal mRNA 5 cm, 2.5 kg/m ). A total of 24 h of light was provided levels of some growth-related antioxidant genes, such during the first 2 d after the arrival of the chicks. Sub - as epidermal growth factor and its receptor, and heat sequently, the hours of light were progressively reduced shock protein 70. However, results of in  vivo trials lack until an 18L:6D photoperiod was achieved, which was consistency in their effects on poultry performance; posi - then maintained from 13 days of age onward. tive effects have been reported by some authors [8, 9], whereas others [10, 11] did not find any improvement. Animals, experimental groups and in vivo recordings Regarding byproducts of the grape-industry, supplemen- A total of 864 broiler chicks (1 day old; 432 males and 432 tations with grape pomace (GP) concentrate and grape females; Ross 308, Aviagen) were delivered by a commer- seed extracts have been shown to affect the gut morphol - cial truck, in compliance with Council Regulation (EC) ogy and increase the microflora biodiversity in broilers, No 1/2005 to the experimental facilities. All chicks were in addition to improving growth performance [12]. vaccinated against Marek’s disease, infectious bronchitis, Additionally, tannins from both CN and grape-indus- and Newcastle disease at the hatchery. They were ran - try byproducts exhibit antioxidant effects when sup - domly allocated in six experimental groups according to plemented in diets for broiler chickens [13, 14]. The an experimental arrangement based on two sexes × three antioxidant properties of tannins contained in GP have dietary treatments (control diet, CN diet, and GP diet), been observed in both the liver and breast meat of broiler with 6 pens per experimental group and 24 birds per pen chickens either after slaughter [14] or during storage [15]. (total of 36 pens). Owing to their antioxidant properties, tannins can allevi- Chicks were individually weighed on the day of ate oxidative stress that occurs during the onset of breast their arrival, identified by a plastic band at the leg, and myopathies [16, 17]. weighed once per week to measure live weight and any Interestingly, the effects of tannins could vary during difference among treatments at different ages, besides animal development [10, 18], particularly with age and promptly identifying any health problem. The chick - sex. Modern hybrids are highly precocious, and large ens were fed ad  libitum. The pen feed consumption was sex-differences are observed from the very first days after measured daily through a computerized weighing system hatching. Regarding age, animals fed a diet supplemented connected to all feeders. with a GP concentrate showed differences in perfor - The end of the growth trial was set at 44 d and birds mance and gut morphology at the end of an experimen- were slaughtered at 45 days of age. tal trial, suggesting a time-dependent cumulative effect of tannins [19]. Humer et  al. [20] reported remarkable Diets and feeding plans differences in intestinal histometric and microbial traits Three commercial diets were formulated as the control between male and female broilers fed with phytogenic (C) diets to be fed during three periods, i.e., from arrival additives. to 15 d; from 15 to 29 d; and from 29 d until slaughter- u Th s, the present study aimed to gain further insights ing (Table  1). These C diets, produced in a mash form, into the mechanisms involved in the response of broiler were supplemented with 0.2% CN extracts (CN diets) or chickens of both sexes and different ages to different 0.2% GP extract (GP diets). The CN extract (Saviotan tannin extracts (i.e., CN and GP) supplemented in diets Feed, 57 mg/g phenolic compounds, GAE equivalent fed from hatching until slaughtering (45 days of age). In Folin-Ciocalteu) was produced by Sedepan (Radicofani, addition to chicken performance and meat quality, we SI, Italy). The GP extract (77 mg/g phenolic compounds, P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 3 of 17 Table 1 Ingredients and chemical composition measured in the two steps (starting with 1 kg) and mixed by hand in a lab (if not specified otherwise) of the control (C) diets. These diets box prior mixing them with other 47 kg of mash diet in were supplemented with 0.2% chestnut wood (CN) extracts or the electric concrete mixer. 0.2% grape pomace (GP) extracts to obtain the CN and GP diets, Diets were analyzed to determine their dry matter respectively content, crude protein, and crude fiber using AOAC Period of administration 1-15 d 15-29 d 29 d [22] methods. The ether extract was analyzed after acid to slaughtering hydrolysis [23]. Ingredients Corn meal, % 56.60 59.25 63.15 Sampling of jejunum tissues Soybean meal (CP 48%), % 34.50 30.00 24.60 At 15 and 35 days post hatching, 36 chickens per period Toasted full-fat soybean meal, % 3.00 5.00 7.00 (1 chick per pen with mean BW) were selected and Animal fat, % 2.50 2.50 2.50 euthanized with C O asphyxiation, prior to jejunum Dicalcium phosphate, % 1.00 0.50 0.25 tissue collection. One sample of approximately 2 cm Calcium carbonate, % 0.92 1.33 1.30 was taken from the jejunum, at the midpoint between Sodium chloride, % 0.26 0.27 0.27 the end of the duodenal loop and the location of the L-lysine base, liquid (50%), % 0.25 0.24 0.16 Meckel’s diverticulum [24], and washed in phosphate- Methionine hydroxy analogue, % 0.31 0.28 0.23 a buffered saline (PBS). Sections (approximately 1-cm Vitamin-mineral premix , % 0.30 0.30 0.30 thick) were fixed in paraformaldehyde in PBS (0.1 6-phytase (EC 3.1.3.26), % 0.20 0.20 0.20 mol/L, pH 7.4), dehydrated, embedded in paraffin at L-Threonine, % 0.11 0.08 0.04 b the laboratory, and later submitted to the histological Coccidiostat , % 0.05 0.05 0.00 analyses and immunohistochemistry as detailed in the Chemical composition following section. Dry matter, % 89.2 89.2 89.3 Immediately before fixing, small jejunum sections Crude protein, % 22.2 20.8 20.2 were collected under RNase-free conditions and stored Ether extract, % 5.4 5.5 6.4 in RNAlater reagent (Applied Biosystems, Foster City, Crude fiber, % 1.3 1.2 1.8 CA, USA) for transcriptomic analyses. In the laboratory, Ash, % 5.7 5.5 6.6 RNA-seq samples were stored at 4 °C overnight and then Starch, % 41.1 43.3 40.2 transferred to −80 ºC until further processing. Calcium , % 0.81 0.80 0.71 Phosphorous , % 0.58 0.47 0.42 Digestible phosphorous , % 0.34 0.24 0.18 Histological analyses and immunohistochemistry Digestible lysine , % 1.32 1.23 1.05 Two serial 4-μm sections per jejunum sample were Digestible methionine + cysteine , % 0.91 0.85 0.76 obtained using a microtome and stained with hematoxy- Digestible threonine , % 0.83 0.76 0.66 lin/eosin for morphometric evaluation and Alcian blue Apparent metabolizable energy , 2982 3045 3087 (pH 2.5)-PAS method for quantitative analysis of goblet kcal/kg cells. Moreover, two further serial sections were used Premix provided per kg of feed: vit. A, 10,000 IU; vit. D , 3500 IU; vit. E acetate, + + for CD3 and C D45 immunohistochemical analyses. 90 mg; vit. K , 6 mg; Biotin, 0.38 mg; Thiamine, 3.75 mg; Riboflavin, 8 mg; vit. B , 3 6 5.75 mg; vit. B , 0.1 mg; Niacin, 70 mg; Pantothenic acid, 17.5 mg; Folic acid, The villi length and crypt depth were collected by a slide 2.25 mg; Fe, 45 mg; Cu, 10 mg; Mn, 70 mg; Zn, 65 mg; Se, 0.25 mg scanner (D-Sight, A. Menarini Diagnostics, Firenze, Italy) Sodium Monensin, 100 mg/kg feed and measured using image analysis software (DP-soft, Values calculated according to FEDNA [21] Olympus Optical, Co., Hamburg, Germany), according to the procedure described by Hampson [25]. The goblet GAE equivalent Folin-Ciocalteu) was produced by Tamp- cells positive for Alcian blue (pH 2.5)-PAS staining were ieri Financial Group (Faenza, RA, Italy). counted using NIH ImageJ software [26] on 10 different The supplementation level of extracts was chosen villi per animal along 300 μm of the villus surface. Immu- based on commercial standards and previous results nohistochemical analyses to identify CD3 intraepithelial [8]. The inclusion of CN and GP extracts in the diets T-cells and CD45 intraepithelial leukocytes in broiler was performed at the experimental farm by thoroughly jejunal mucosa were performed following the procedure mixing the C diets with the dried extracts using an elec- described by Röhe et  al. [27]. Intraepithelial leukocytes tric concrete mixer (Suncoo 4/5HP Concrete mixer, 140 were counted in the epithelium using a reference rectan- L, 600 W, 2800 r/min; SUNCOO, China). A total of 3 kg gle with the short side at 100 μm and expressed as the + + 2 of mash diet were progressively added with extracts in density of CD45 and CD3 cells (cells/10,000 μm ). Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 4 of 17 RNA-seq library preparation and sequencing took approximately 3 h. Ready-to-cook carcasses were Total RNA from the 72 chickens slaughtered at 15 and recovered after 2 h of refrigeration at 2°C and indi- 35 d was extracted using the RNAeasy Mini Kit (Qiagen, vidually weighed to measure the slaughter dressing Hilden, Germany), following the manufacturer’s instruc- percentage. tions; total RNA concentration was then determined A total of 144 carcasses (four per pen), previously using a Qubit RNA BR (Broad-Range) kit in a Qubit 2.0 selected on the basis of the final live weight as corre - Fluorometer (Life Technologies, Carlsbad, CA, USA). sponding to the mean BW within a pen, were subjected RNA quality was assessed using a 2100 Bioanalyzer (Agi- to gross examination to evaluate the occurrence in pec- lent Technologies, Waldbronn, Germany). All samples toralis major muscles (presence or absence) of white had an RNA integrity number > 7. striping (WS) (either moderate or severe) [33]; wooden Equal amounts of RNA from three different chickens of breast (WB) (firm upon palpation, prominent ridge like the same sex, fed with the same diet and slaughtered at bulge on caudal area of fillet, clear viscous fluid cover the same age (15 or 35 d) were pooled; a total of 24 RNA and/or petechial multifocal lesions on the fillet surface) pools were obtained (i.e., two pools/replicates per sex [34]; and spaghetti meat (SM) (exhibiting an overall for each of the three diets and per two slaughtering age). impaired integrity and tendency toward separation of Notably, the RNA pooling is a common practice among the muscle fiber bundles especially within the cranial gene expression studies, and it is well justified based on part of the fillet) [35]. The 144 carcasses were stored at statistical and practical considerations [28]. Twenty-four 2  °C before meat quality analyses. Twenty-four hours tagged RNA-seq libraries were prepared using the Illu- after slaughter, carcasses were dissected for the main mina TruSeq Stranded mRNA kit and sequenced on an cuts (breast, wings, thighs, and drumsticks). Pectora- Illumina NovaSeq 6000 instrument at the NGS Sequenc- lis major muscles were separated from the breasts for ing Core (Padova, Italy) following a 100-bp paired-end meat quality analyses [36]. The pH values of the pectora - approach. lis major muscles were measured in triplicates on their ventral side with a pH meter (Basic 20, Crison Instru- ments Sa, Carpi, Italy) equipped with a specific elec - RNA-seq reads processing and mapping trode (cat. 5232, Crison Instruments Sa, Carpi, Italy). Initial quality control was performed using FastQC soft- The L*a*b* color indexes were measured in triplicate on ware version 0.11.9 [29]. Read trimming and adapter the ventral side of the same muscles covered by a trans- removal were performed using Trimmomatic (version parent plastic film, using a Minolta CM-508 C spectro - 0.39) with default parameters [30]. Reads shorter than photometer (Minolta Corp., Ramsey, NJ, USA) [37]. 36 bp were excluded from the analysis. Residual ribo- After measuring the pH and color indexes, one meat somal RNAs (rRNAs) were removed through the local portion (8 cm × 4 cm × 3 cm) was separated from the sequence alignment tool SortMeRNA 2.1 [31] against dif- cranial side of the pectoralis major muscle, parallel to ferent databases (Rfam 5.8S; Rfam 5S; Silva 16S archaeal, the direction of the muscle fibers, and stored under vac - bacterial; Silva 18S eukaryote; Silva 23S archaeal, bacte- uum in plastic bags at -18 °C until meat analyses. Thaw - rial; Silva 28S eukaryote). Reads trimmed and cleaned as ing and cooking losses were measured in this cut [36]. described above were then mapped against the chicken After thawing, the meat portion was placed in a plastic Ensembl reference genome (GCA_000002315.5) using bag and cooked in a water bath for 45 min until an inter- the STAR aligner and following the two-pass map- nal temperature of 80  °C was achieved. After 40 min of ping mode [32]. The maximum number of allowed mis - cooling, another meat portion (4 cm × 2 cm × 1 cm) matches and the maximum number of loci to which the was separated to assess the maximum shear force using reads could map were set to 8 and 10, respectively. Read an LS5 dynamometer (Lloyd Instruments Ltd, Bognor counts per sample at the gene level were extracted by set- Regis, UK) using the Allo-Kramer (10 blades) probe ting the GeneCounts quantification while running STAR. (load cell: 500 kg; distance between the blades: 5 mm; thickness: 2 mm; cutting speed: 250 mm/min) [37]. Commercial slaughtering and carcass and meat quality The pectoralis major muscles of the remaining 72 recordings carcasses (2 chickens per pen, 12 per experimen- At 45 days of age, all remaining chickens were slaugh- tal group) were dissected and subsequently stored at tered in a commercial slaughterhouse. The chickens -20  °C to assess the meat oxidation level of thiobarbi- were weighed individually before crating. Loading turic acid reactive substances (TBARs) [38] using spec- took approximately 1 h, transport from the experimen- trophotometric measurements (Jasco Mod. 7800 UV/ tal facilities to the commercial slaughterhouse took VIS) at 532 nm. The results were expressed as μg of approximately 15 min, and lairage before slaughtering malondialdehyde/kg. P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 5 of 17 Statistical analysis The input was prepared as follows: the raw P values (pval) Individual data of live weights, daily growth, and carcass obtained through the pairwise DE analysis were used and meat traits were subjected to analysis of variance to rank the list of genes by significance. When multiple (ANOVA) with diets (C, CN, and GP) and sex as main genes with the same gene name were detected, only the factors of variability and their interactions, and the pens most significant gene (based on pval) was retained. The as a random effect, using the PROC MIXED procedure pval were replaced by 1-pval or -(1-pval) when a gene in SAS [39]. Being live weight of male and female chicks was over- or under-expressed, respectively. The analysis significantly different at the first hatching day (Table  2) it was performed using the gseKEGG function in the Clus- was included in the model as a covariate for live weights terProfiler package [41]. and daily growth. Pen data of feed intake and feed con- version were subjected to ANOVA, with diet and sex as Results main factors of variability, and their interactions, using Growth performance the PROC GLM procedure [39]. Individual data related to Live weight at 15 d did not differ among groups (513 g on + + jejunum morphology, goblet cells, and C D3 and C D45 average), whereas live weight at 29 d was higher in broil- cell densities were analyzed using the PROC GLM proce- ers fed the GP diet than in those fed the CN diets (P < dure, with diet, age, and sex as the main effects and their 0.01), and this difference was confirmed at the end of the interactions. The Chi-square test was used to test differ - study in comparison with those fed the C and CN diets ences in mortality according to diet and the rate of myo- (45 d; P < 0.01) Table  2. Therefore, the daily weight gain pathies according to diet and sex. Adjusted means were during the study was higher in broilers fed the GP diet compared using Bonferroni’s t-test. Differences between than in those fed the other two diets (P < 0.01), without the means with P ≤ 0.05 were considered statistically differences in feed intake and feed conversion. significant. Males were heavier than females from the first day to For whole-transcriptome profiling, a pairwise differ - the end of the study (P < 0.001), resulting in a higher daily ential expression (DE) analysis was performed using the weight gain (+22%) and feed intake (+17%), without dif- likelihood ratio test implemented in EdgeR [40] to com- ferences in feed conversion (1.59) (Table 2). A significant pare mRNA profiles between experimental groups. A interaction was observed between dietary treatment and false discovery rate (FDR) of ≤ 0.05 and a fold change sex at 45 d (P < 0.05); males fed the GP diet were heavier (FC) of ≥ 1.5 were used as thresholds of significance than those fed C and CN diets, whereas females fed C between age and sex. Regarding changes due to diets, an and GP diets were heavier than those fed the CN diet. FC threshold of 2 was selected to mitigate possible false Losses were low (3.4%, 27 chickens) and due to mortal- positives due to the limited number of replicates per ity (1.1%) and lameness (2.3%) (Table 2). experimental group (i.e., two per diet, per age, per sex). Overall, four sex/age-specific datasets (each consisting Slaughter results, meat quality, and myopathy rate of six samples) were analyzed separately, i.e., females at Table  3 shows that the carcass weight (with feet) was 15 d; females at 35 d; males at 15 d; and males at 35 d. higher in chickens fed the GP diet than in those fed the A functional interpretation of significant differentially other two diets (P < 0.05). No diet-related effects on myo - expressed genes (DEGs) was obtained through a Gene pathy occurrence (Table 3) or meat quality (Table 4) were Ontology over-representation test, performed using recorded. the ClusterProfiler package in the R environment [41]. Regarding the effect of sex, males had heavier carcasses Only KEGG pathways were considered, using the func- (+17%, P < 0.001) and a higher proportion of leg weight tion enrichKEGG. Ensembl gene identifiers were used (thighs + drumsticks) (+0.7; P < 0.05) but a lower breast to establish a list of significantly upregulated and down - yield (-1.7%; P < 0.001) and pectoralis major muscle pro- regulated genes and a “background” (i.e., the whole set of portion (-0.6%; P < 0.001) than those of females (Table 3). expressed genes). The ClusterProfiler package was used Females showed a lower meat pH (P < 0.01) (Table  4), to produce plots representing enriched terms (P ≤ 0.05). lower WB rate (P < 0.001), and higher SM rate (P < 0.001) A pre-ranked KEGG Gene Set Enrichment Analy- than those of males but had similar WS rates to those of sis (GSEA) [42] was performed to investigate whether males (Table 3). gene sets defined a priori showed a statistically signifi - cant enrichment at either end of the ranking. A statisti- Jejunum morphology and immuno-histochemical analyses cally significant enrichment value (Benjamini–Hochberg On average of the two slaughtering times of 15 and 35 adjusted P value ≤ 0.05) indicates that the biological d, the villi height was lower in chickens fed the CN diet activity (e.g., the biomolecular pathway) characterized than in those fed the C diet (P < 0.001); moreover, chick- by the gene set is correlated with the supplied ranking. ens fed the CN diet had a lower density of CD45 cells (P Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 6 of 17 Table 2 Growth performance (LS means) and mortality of broiler chickens until slaughter Diet (D) Sex (S) P value MSE Items C CN GP Females Males D S D×S Chickens, n 258 252 255 383 382 Pens, n 12 12 12 18 18 Live weight, g Initial (1 d) 44.3 43.9 44.5 43.7 44.9 0.15 <0.001 0.16 3.45 15 d 512 510 518 498 529 0.23 <0.001 0.12 50.7 ab a b 29 d 1689 1669 1703 1559 1815 <0.01 <0.001 0.12 124 2 a a b Final (44 d) 3099 3087 3146 2816 3406 <0.01 <0.001 0.02 217 Whole trial (1-44 d) 2 a a b Daily weight gain , g/d 69.4 69.1 70.5 63.0 76.4 <0.01 <0.001 0.02 4.94 Daily feed intake, g/d 111 110 112 102 119 0.16 <0.001 0.70 2.47 Feed conversion 1.59 1.59 1.59 1.59 1.59 0.85 0.91 0.26 0.36 Losses , % 2.27 4.55 3.41 3.28 3.54 0.33 0.70 0.70 - MSE root mean square error. C, control diet. CN, control diet supplemented with 0.2% chestnut wood extracts. GP, control diet supplemented with 0.2% grape pomace extracts Individual data: live weight and daily growth rate. Pen data: feed intake and feed conversion Interaction Diet × Sex, Final live weight: P = 0.02: 2827 g, 2789 g and 2822 g in females fed C, CN and GP diets; 3372 g, 3381 g and 3473 g in males fed C, CN, and GP diets, respectively. Daily weight gain: P = 0.02: 63.2 g/d, 62.4 g/d and 63.1 g/d in females fed C, CN, and GP diets, 75.6 g/d, 75.8 g/d and 77.9 g/d in males fed C, CN, GP diets, respectively. Dead and lame chickens a,b Values with different superscript letters significantly differ (P < 0.05) Table 3 Slaughter results, carcass traits (LS means) and myopathy rates in chickens slaughtered at 45 days of age  Items Diet (D) Sex (S) P value MSE C CN GP Females Males D S D×S Chickens , n 258 252 255 383 382 Pens, n 12 12 12 18 18 2 a a b Cold carcasses , g 2352 2342 2392 2122 2602 0.03 <0.001 <0.01 173.06 Dressing out percentage, % 76.6 76.8 76.8 76.4 77.1 0.45 <0.001 0.51 1.48 Chickens, n 48 48 48 72 72 Cold carcasses (CC), g 2274 2243 2306 2063 2486 0.32 <0.001 0.10 143.58 Dressing percentage, % 73.3 73.0 73.0 73.3 73.0 0.48 0.18 0.92 1.32 Breast yield , % CC 39.3 38.8 39.5 40.0 38.3 0.16 <0.001 0.56 1.76 P. major, % CC 12.2 12.1 13.4 12.5 11.9 0.33 <0.001 0.37 0.85 Wings, % CC 9.8 10.0 9.9 9.9 9.9 0.06 0.44 0.65 0.51 Legs (thighs+drumsticks), % CC 30.1 30.1 29.8 29.6 30.3 0.67 0.04 0.43 2.02 Myopathy rates at P. major White striping, % 72.9 60.4 79.2 66.7 75.0 0.12 0.26 - - Wooden breast, % 41.7 25.0 41.7 16.7 55.6 0.11 <0.001 - - Spaghetti meat, % 29.5 25.5 25.0 48.6 4.2 0.83 <0.001 - - MSE root mean square error. C, control diet. CN, control diet supplemented with 0.2% chestnut wood extracts. GP, control diet supplemented with 0.2% grape pomace extracts 1 2 Carcasses with feet. Interaction Diet × Sex, Cold carcasses: P < 0.01: 2126 g, 2103 g, 2126 g in females fed C, CN and GP diets, respectively; 2571 g, 2580 g, and 2652 3 4 g in males fed CN and GP diets. Without feet. With bone a,b Values with different superscript letters significantly differ (P < 0.05) P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 7 of 17 Table 4 Rheological traits and lipid oxidation status ( TBARs) of the pectoralis major muscle in chickens slaughtered at 45 days of age  Items Diet (D) Sex (S) P-value MSE C CN GP Females Males D S D×S P. major, n 48 48 48 72 72 pH 5.99 5.99 5.95 5.94 6.02 0.56 <0.01 0.99 0.16 L* 50.4 50.2 49.7 49.9 50.1 0.66 0.63 0.10 2.40 a* -0.04 -0.07 -0.15 -0.03 -0.15 0.56 0.23 0.20 0.54 b* 10.4 10.3 10.2 10.5 10.1 0.70 0.09 0.65 1.38 P. major, n 24 24 24 36 36 Cooking losses, % 12.0 11.7 11.8 11.4 12.3 0.99 0.65 0.92 12.4 Shear force, kg/g 4.17 4.06 4.30 4.17 4.18 0.39 0.96 0.72 0.83 TBARs, mg MDA/kg 0.083 0.080 0.075 0.078 0.080 0.38 0.74 0.22 0.021 MSE root mean square error. C, control diet. CN, control diet supplemented with 0.2% chestnut wood extracts. GP, control diet supplemented with 0.2% grape pomace extracts < 0.05) than those chickens fed the GP diet (Table 5). Die- mapped to the chicken reference genome (Table S3). The tary treatment did not affect the density of goblet cells. multidimensional scaling plot provided unsupervised As the age increased from 15 to 35 d, villi height (P < clustering of the samples (Fig.  1). The first dimension 0.01), crypt depth (P < 0.05), and density of both C D3 (x-axis) clearly separates females from males, whereas and CD45 cells (P < 0.001) increased (Table 5), whereas the second dimension (y-axis) separates samples by age. the density of goblet cells decreased (P = 0.001). The biological variability within dietary treatments was Regarding sex, villi height was lower in females than in low, as demonstrated by the clusters formed when MDS males (P < 0.01), whereas the densities of C D45 cells (P distances between expression profiles of all the replicates = 0.01) and goblet cells (P = 0.06) were higher (Table 5). were plotted. Jejunum samples showed largely different + + As for villi height, CD3 and C D45 cells, significant transcriptional profiles between sexes and between ages, interactions were recorded between sex and age (Table whereas compared with the C diet, the CN and GP diets S1), and between sex and diet (Table S2). had a moderate impact on jejunum gene expression. Whole transcriptome analysis A total of 725,278,263 raw reads were obtained. Raw Illu- Dietary effects on jejunum whole transcriptome mina sequencing data were deposited in GenBank under Transcriptional profiles of same-sex and same-age the BioProject accession number PRJNA666129. All chickens fed CN and GP diets were compared to those samples passed the quality control measures. After trim- of chickens fed the C diet, thus identifying significant ming and rRNA removal, an average of approximately 30 DEGs, that are shown in Table  6 and in the supplemen- million reads per sample was retained, with ~94% reads tary Tables S4 and S5. + + Table 5 Jejunum morphometry, number of goblet cells and densities of CD45 and CD3 cells at 15 and 35 days of age  Items Diet (D) Age (A) Sex (S) P-value MSE 1 2 C CN GP 15 35 F M D A S D×A A×S D×S D×A×S Broilers, n 24 24 24 36 36 36 36 b a ab Villi height, μm 1033 934 954 866 1082 936 1011 <0.001 <0.01 <0.01 0.44 0.02 0.03 0.13 111 Crypt depth, μm 145 139 144 138 147 140 145 0.45 0.04 0.23 0.70 0.29 0.79 0.31 18,5 Villi / Crypt ratio 7.49 7.06 6.96 6.67 7.67 7.06 7.28 <0.001 0.16 0.37 0.70 0.37 0.20 0.68 1,00 Goblet cells, n/300 μm 21.02 21.67 22.25 22.84 20.45 22.20 21.09 0.24 0.001 0.06 0.34 0.12 0.43 0.27 2.46 + 2 CD3 cells, n/10,000 μm 2242 2219 2297 2069 2436 2223 2283 0.16 <0.001 0.09 0.23 <0.001 <0.001 0.46 459 + 2 ab a b CD45 cells, n/10,000 μm 2879 2793 2925 2437 3295 2915 2816 0.02 <0.001 0.01 0.18 <0.001 0.01 0.45 537 MSE root mean square error. C, control diet. CN, control diet supplemented with 0.2% chestnut wood extracts. GP, control diet supplemented with 0.2% grape pomace extracts 1 2 Averages of traits according to Age × Sex are provided in Table S1. Averages of traits according to Diet × Sex are provided in Table S2. a,b Values with different superscript letters significantly differ (P < 0.05) Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 8 of 17 Fig. 1 Multiple dimensional scaling (MDS) plot of distances between expression profiles (as log fold change, logFC) of RNA-seq libraries (24 samples) of the jejunum of female (F) and male (M) broiler chickens fed control diet (C diet), diet added with 0.2% chestnut extracts (CN diet), and diet added with 0.2% grape pomace extracts (GP diet) at 15 d (libraries C_15d, CN_15d, GP_15d) and 35 days of age (libraries C_35d, CN_35d, GP_35d) [1F, 2F, 1M, 2M stand for the two replicates for females and the two replicates for males per age] At 15 d, when comparing females fed the CN diet At 35 d, 30 DEGs were detected when comparing with those fed the C diet, 15 DEGs were identified. females fed the CN and C diets. Some genes involved in Cytochrome P450 1A1 (CYP1A1; logFC: 1.71), CYP1A2 immunity and inflammation, such as complement C1q (logFC: 1.47) and STEAP4 Metalloreductase (STEAP4; C chain (logFC: 1.07), major histocompatibility complex logFC: 4.65) were upregulated. Guanylate-binding pro- class I polypeptide-related sequence A (MICA; logFC: tein 1-like (GBP1-like; logFC: -8.43), butyrophilin sub- 1.72), extracellular fatty acid-binding protein (logFC: family 1 member A1-like (BTN1A1-like; logFC: -1.69), 1.32), and bradykinin receptor B1 (BDKRB1; logFC: 1.26), and leukocyte immunoglobulin-like receptor subfamily were upregulated. Likewise, the glutathione S-transferase A member 2 (logFC: -4.12) were downregulated. Regard- class-alpha (GSTA) gene was upregulated (logFC: 1.25). ing the effects of the GP diet compared with those of the In females at 35 d, compared with the C diet the GP C diet in females at 15 d, avian beta-defensin 9 and 10 diet upregulated 197 genes. Among the genes involved (AvBD9 and AvBD10, respectively) and class I histocom- in inflammatory processes, the following were upregu - patibility antigen, F10 alpha chain-like (HA1F-like) were lated: nephroblastoma overexpressed (NOV; logFC: remarkably upregulated (logFC of 7.18, 9.23, and 2.43, 1.06), netrin 1 (logFC: 1.05), C1q and tumor necrosis respectively). Both GBP1-like and BTN1A1-like mRNA factor related protein 4 (C1QTNF4; logFC: 1.69), and levels were lowered (logFC: -6.09 and -1.45). Gap junc- BDKRB1 (logFC: 1.09). Additional upregulated genes tion protein beta 1 and pyruvate dehydrogenase kinase 4 that play a role in the regulation of immune defense were among the most significantly downregulated genes. were the junctional adhesion molecule 2 (logFC: 1.04), P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 9 of 17 Table 6 Number of significantly upregulated and downregulated protein that protects the intestinal mucosa from inflam - differentially expressed genes (Fold change ≥ 2; False Discovery mation. The gene most significantly induced by the GP Rate ≤ 0.05) in the jejunum of male or female broiler chickens fed diet was glutathione peroxidase 4 (logFC: 1.26), whereas the experimental diets at 15 or 35 days of age the suppressor of cytokine signaling 3 (logFC: -1.04) and IL-22 (logFC: -2.80) were downregulated. Comparison Number of Number of Total DEGs At 35 d, CN diet in males significantly induced HA1F- up-regulated down-regulated like (logFC: 1.46) and MICA mRNA levels (logFC: 1.41), genes genes while lysozyme-g-like (logFC: -2.47) and NOV (logFC: Female chickens -1.89) were downregulated. at 15 d On comparing chickens at 35 d and those at 15 d (Table CN diet vs. C diet 7 8 15 S6), 324 DEGs were identified (FDR ≤ 0.05; FC ≥ 1.5). GP diet vs. C diet 14 18 32 The top 10 upregulated and downregulated genes are Male chickens at shown in Table 7. Only two KEGG pathways were statis- 15 d tically enriched by the 213 genes upregulated after 15 d, CN diet vs. C diet 7 7 14 namely, the “peroxisome proliferator-activated receptor GP diet vs. C diet 21 10 31 (PPAR) signaling pathway” and the “neuroactive ligand– Female chickens receptor interaction pathway” (Table S7). However, a at 35 d higher number of KEGG pathways (i.e., 14) were sig- CN diet vs. C diet 18 12 30 nificantly enriched by the upregulated genes in chickens GP diet vs. C diet 197 74 271 at 35 d (Fig.  2 and Table S7); some of them were related Male chickens at to PPAR signaling, xenobiotic metabolism, glutathione 35 d metabolism, steroid hormone synthesis, IgA production, CN diet vs. C diet 8 8 16 and amino acid metabolism. GP diet vs. C diet 2 4 6 In 15-d chickens, the GSEA (Table S7)  pointed out DEGs differentially expressed genes. C, control diet. CN, control diet supplemented with 0.2% chestnut wood extracts. GP, control diet the activation of 18 KEGG pathways related to cell–cell supplemented with 0.2% grape pomace extracts junctions (i.e., tight junction, focal adhesion, and adher- ent junction), cytoskeleton, and transforming growth factor-β (TGF-β). In 35-d chickens, the GSEA highlighted C-X-C motif chemokine ligand 12 (CXCL12; logFC: the activation of 33 KEGG pathways; the most significant 1.08), and T-cell surface glycoprotein CD8 alpha chain- ones include gene sets related to xenobiotic metabolism, like (logFC: 1.74). Moreover, the mRNA levels of hemo- antioxidant response (i.e., glutathione metabolism), pro- globin beta, subunits A, A1, and AD (HBBA, HBA1, and tein processing (i.e., proteasome, protein export, and HBAD), as well as GSTA, were increased (logFC: 2.25, ribosome), and amino acid metabolism. The top-35 sig - 1.85, 2.27, and 1.18, respectively). Finally, as for upregu- nificant KEGG pathways activated in 35-d chickens com - lated genes, glucagon like peptide 1 receptor (GLP1R; pared to 15-d chickens are reported in Fig. 3. logFC: 1.02), and solute carrier family 2, facilitated glu- On comparing females and males, 246 significant DEGs cose transporter member 4-like (GLUT4-like; logFC: were identified; among these, 47 and 199 genes were 1.17) play a role in nutrients intestinal absorption. upregulated in females and males, respectively. The top Among the downregulated genes, some are involved 10 upregulated and downregulated genes are listed in in vitamin and protein absorption, such as scavenger Table  8. The KEGG enrichment analysis of upregulated receptor class B member 1 (logFC: -2.00) and beta-car- genes in males showed that only the “ribosome biogen- otene oxygenase 1 (logFC: -1.55). Further downregu- esis in eukaryotes” pathway was significantly enriched. In lated genes are involved in protein and carbohydrate contrast, GSEA identified a total of 19 KEGG pathways metabolism, such as carboxypeptidase O (logFC: -1.80), that were significantly enriched, 9 in females and 13 in glutamic-pyruvic transaminase 2 (logFC: -1.27), and males (Fig.  4; Table S7). In females, a considerable num- lactase (logFC: -1.10). Additionally, the expression of ber of activated gene sets was related to cell cycle regula- AvBD9 and AvBD10 was downregulated (logFC: -7.59 tion (e.g., DNA replication and mismatch repair). and -6.21, respectively). At 15 d, compared with the C diet, the CN diet signifi - cantly upregulated STEAP4 (logFC: 3.31), CYP1A (logFC: Discussion 1.89), and HA1F-like (logFC: 1.61) genes in males. Among In animal feeding, tannins can have either beneficial or the seven downregulated genes, ectonucleotide pyroph- detrimental effects depending on their chemical struc - osphatase/phosphodiesterase 7 (logFC: 1.66) encodes a ture and dosage, in addition to other individual (e.g., Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 10 of 17 Table 7 Top 10 upregulated ( ) and downregulated ( ) differentially expressed genes (DEGs) in broiler chickens at 35 d compared with those at 15 d. For each DEG, Ensembl gene ID, log fold change (logFC), and False Discovery Rate (FDR), as reported in edgeR output and the Ensembl gene description, are provided Fig. 2 Over-represented KEGG pathways among the upregulated genes in broiler chickens at 35 d compared with those in broiler chickens at 15 d. The number of differentially expressed genes (DEGs) belonging to each enriched KEGG pathway is reported in the x axes. Color gradient represents the P-value significance, as specified in the legend. P-values were adjusted using the Benjamini–Hochberg method P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 11 of 17 Fig. 3 Results of the Gene Set Enrichment Analysis (GSEA): top-35 significantly enriched KEGG pathways in broiler chickens at 35 d compared with those in broiler chickens at 15 d. Pathways showing an enrichment value > 0 are activated in 35-d broiler chickens, whereas those with an enrichment value < 0 are activated in 15-d broiler chickens. Color gradient represents the P-value significance, as specified in the legend. The P-values were adjusted using the Benjamini–Hochberg method animal species, age, sex, physiological state) and feeding The different nature of tannins of CN and GP and factors [43]. Standardization of conditions (in terms of their effects at the gut level can partly explain our quantity and quality of the different polyphenolic frac - results, even if different effects are likely to expected tions) is even more difficult when using extracts from depending on the animal age and gut tract [46]. The natural sources. Thus, in the present trial, we compared CN mainly contains hydrolysable tannins, which usu- two products of a similar nature (byproducts containing ally possess a relatively low molecular weight and high different quantities of polyphenols of different types) in bioavailability [8, 9, 47]. Thus, CN hydrolysable tannins the same form (powder) at the same moderate supple- can be early hydrolyzed and absorbed in the first gastro mentation level. intestinal tract, as observed in rats [48], exerting both We observed that the GP diet improved growth per- cytotoxic or cytoprotective effects on jejunal mucosa formance compared with the other dietary treatments, [46, 48, 49]. On the other hand, GP condensed tannins whereas CN diet reduced the villi height compared to can remain active along the whole gut and can be trans- the control diet and the density of CD45 cells compared formed by gut microbiota in other bioactive metabolites to the GP diet. The highest growth performance meas - which can further affect gut mucosa and microbiota ured in broilers fed GP diet might be in part supported composition [46, 50]. by an increased intestinal absorption of nutrients. In the Schiavone et  al. [8] showed dose-dependent effects of present study, this is suggested by the upregulation in dietary supplementation with CN tannins, i.e., null at the females of genes such as the receptor of glucagon-like lowest inclusion rate (1.5 g CN wood extract/kg), positive peptide (GLP1R), that promotes efficient nutrient assimi - at the intermediate level (2.0 g/kg) in terms of final live lation [44], and GLUT4, a glucose transporter whose weight and feed intake, and negative at the highest rate expression can be induced by tannins [45]. (2.5 g/kg), which reduced live weight, compared with the Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 12 of 17 Table 8 Top 10 upregulated ( ) and downregulated ( ) differentially expressed genes (DEGs) in females compared with those in males. For each DEG, Ensembl gene ID, log fold change (logFC), and False Discovery Rate (FDR), as reported in edgeR output and the Ensembl gene description, are provided Fig. 4 Results of the Gene Set Enrichment Analysis (GSEA): enriched KEGG pathways in female broiler chickens compared with those in male broiler chickens. Pathways showing an enrichment value > 0 are activated in females, whereas those with an enrichment value < 0 are activated in males. Color gradient represents the P-value significance, as specified in the legend. The P-values were adjusted using the Benjamini–Hochberg method P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 13 of 17 C diet (from 14 to 56 days of age). Similarly, Jamroz et al. [61]. Indeed, genes involved in either pro- or anti- [10] did not observe effects on performance with low CN inflammatory response pathways and antimicrobial tannin inclusion rates (0.25 and 0.50 g of sweet CN tan- responses were affected by GP and CN diets, besides nin/kg), whereas the highest dose (1.0 g/kg) decreased genes contributing to the antioxidant responses. Nev- the live weight at 41 d and caused alterations in the intes- ertheless, the majority of these genes, especially those tinal wall morphology and a decreased proliferation rate related to immune functions, were significantly regu - in the mother-cell zone. These findings are consistent lated by the GP supplementation consistently with the with the decreased villi length observed in our study in higher CD45 density in the jejunum of broilers fed chickens fed the CN diet. In contrast, CN tannin supple- GP diet compared to those fed CN diet. Finally, the mentation increased the average daily weight gain and improved immune competences observed in broilers jejunum villus height under a heat-stress challenge [51] fed GP diet might have positively impacted on their and reduced the proliferation of Clostridium perfringens growth performance compared to the other dietary and the severity of gut damage in necrotic enteritis [52]. treatments. Similar to CN, condensed tannins of GP byproducts In the present study, the majority of changes in the (extracted from seeds, skin, and stems as byproducts of chicken jejunum transcriptome was observed in chick- winemaking) can reduce diet digestibility and negatively ens of the same sex but at different ages. In fact, in 15-d affect the growth performance of monogastric animals females, the GP and CN diets downregulated genes [53]. In poultry, during the first period (from hatching involved in the inflammatory response [62] and pro - until 21 days of age), the increase in GP supplementa- tection against microbes and viruses, such as GBP1- tion (from 2.5 to 5.0 g of GP seed extract/kg) decreased like [63] a robust marker of inflammation. In 15-d male both animal performance and diet digestibility [54]. Some and female broilers, the CN diet upregulated STEAP4, authors reported negative effects on performance with which plays a role in the response to chronic inflamma - high inclusion rates of grape-based extracts [12], which tion in colon cancer [64], the suppression or inhibition also showed a negative effect on villus length. In contrast, of cytokine production and signaling (IL-6 and TNF-α- 6% dietary inclusion of a GP concentrate did not modify induced NF-κB signaling [65]), the response to nutri- performance or diet digestibility but showed antioxidant ents, oxidative stress, fatty acid metabolism, and glucose potential that was as effective as vitamin E in diet, excreta, metabolism [66]. In 15-d chickens of both sexes, CN ileal content, and breast muscle [15]. Other authors evi- supplementation modulated the expression of CYP1A, denced that the dietary supplementation of grape byprod- an important detoxifying monooxygenase that can be ucts increased activity of total superoxide dismutase and induced by natural polyphenols [67] as demonstrated in decreased the content of malondialdehyde in plasma [19] the gut of pigs fed CN extracts [68]. and leg meat [55], whereas this was not observed in the In 35-d females, the GP diet upregulated C1QTNF4 present study when measuring meat TBARs. (involved in the regulation of the inflammatory networks In our study, we neither observed any effect of CN or and in feed intake suppression in mice) [69] and CXCL12 GP supplementation on myopathy rate, which is consist- (a constitutive and inflammatory chemokine of the intes - ent with the findings of previous studies that tested vari - tinal immune system) [70]. Conversely, in 35-d females, ous antioxidants for this purpose [56–58]. Nevertheless, the GP diet downregulated beta-defensins (AvBD9, oxidative stress, localized hypoxia, increased intracellular AvBD10), which possess modest antimicrobial properties calcium, and the presence of muscle fiber-type switching and display a wide range of immunomodulatory activi- are pathways responsible for the occurrence of WB and ties, such as modulation of pro- and anti-inflammatory WS [16, 59, 60]; hence, the administration of antioxidant responses, promotion of wound healing [71]. Addition- substances, such as GP or CN extracts, could be expected ally, in 35-d females, both GP and CN diets induced the to affect myopathy occurrence. expression of GSTA, primarily involved in the defense In the present study, the increased mucosal immune against oxidative stress. The GP diet also induced three responses with the GP diet (i.e. the higher density of hemoglobin subunits (i.e., HBBA, HBAD, and HBA1), intraepithelial leukocytes, especially CD45 ) compared which are upregulated in the presence of oxidative stress to the CN diet could be attributed to the tannin nature and are believed to alleviate it [72]. and availability along the intestinal tract as discussed Notably, most of the differences in favor of GP supple - above (condensed compared to hydrolysable tannins in mentation regarding the overall transcriptomic response GP compared to CN). On the other hand, when com- appeared in the second period of growth. Overall, this is paring different dietary supplementation doses of tan - consistent with the results of Farahat et  al. [73] and Yang nic acid, effects on broiler chicken immunity changed et al. [19], who observed a time-dependent cumulative effect from positive to negative in a dose-dependent manner of dietary supplementation with grape seed extracts and Pascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 14 of 17 pomace concentrate. However, according to some authors interaction,” and “regulation of actin cytoskeleton” gene [8, 53], the dietary supplementation with tannins (from grape sets). On the other hand, differences in growth rates and seed and CN extracts) was more effective in younger broilers final live weight between sexes are also associated to a than in older ones, likely based on their effects on pathogenic different occurrence of myopathies whereas the results microorganisms as well as on commensal microbiota [74, 75]. of the present study confirmed that SM occurred more In the present study, the most significant age-dependent often in females than in males, whereas WB was more transcriptional variations (i.e., top 10 upregulated DEGs) frequent in males than in females [81, 82]. were related to immunoglobulin functions, for which the KEGG pathway “intestinal immune network for IgA pro- Conclusions duction” was significantly enriched with the increase in age. The dietary supplementation of GP extracts can be ben - Overall, this age-dependent improvement of immune com- eficial to broilers because it increases growth performance petencies, together with the increased densities of jejunum and the final live weight of animals. In the absence of any + + CD3 and CD45 cells we observed in older chickens, was specific challenges, it still improved the jejunum morphol - expected. Additionally, four of the top 10 upregulated DEGs ogy and the overall immune response. The addition of CN encoded GSTs, which suggests that glutathione-dependent extracts affected the jejunum morphology, although growth detoxifying capability significantly increases with chicken traits remained unaffected. Overall, the chicken jejunum development. This was further strengthened by the enrich - transcriptome was scarcely affected by extracts, although ment of the KEGG pathways “glutathione metabolism” and in 35-d females fed the GP diet, some positive effects on “metabolism of xenobiotics by cytochrome P450,” both nutrient absorption, immune and antioxidant responses including several differentially regulated GST s. were observed. Regarding age and sex, further molecular The downregulation of three hemoglobin subunits (i.e., investigations are required in view of the interactions with HBBA, HBAD, and HBA1) in 35-d chickens compared dietary additives, as observed in the present study, and sub- with 15-d chickens could be related to an imbalance sequent effect on chicken health and performance. between the muscular development of birds and the vas- cularization/blood supply and might reflect the possible muscle suffering that leads to muscle fiber degeneration Abbreviations ANOVA: Analysis of variance; AvBD10: Avian beta-defensin 10; AvBD9: Avian and myopathies with the increase in age [76, 77]. beta-defensin 9; BDKRB1: Bradykinin receptor B1; BTN1A1: Butyrophilin Expected differences in performance between males subfamily 1 member A1; C: Control; C1QTNF4: C1q and tumor necrosis factor and females were confirmed and found to be associ - related protein 4; CN: Chestnut wood; GP: Grape pomace; CXCL12: C-X-C motif chemokine ligand 12; CYP1A1: Cytochrome P450 1A1; DE: Differential ated with differences in the jejunum transcriptome. Top expression; DEGs: Differentially expressed genes; FC: Fold change; FDR: False upregulated genes in females are involved in ubiquitina- discovery rate; GBP1: Guanylate-binding protein 1; GSEA: Gene Set Enrich- tion, a post-translational mechanism for protein deg- ment Analysis; GSTA: Glutathione S-transferase class-alpha; HA1F-like: F10 alpha chain-like; HBA1: Hemoglobin beta subunit A1; HBAD: Hemoglobin beta radation via the proteasome, ensuring the structural subunit AD; HBBA: Hemoglobin beta subunit A; IL: Interleukin; MICA: major integrity control and/or protein turnover rate. However, histocompatibility complex class I polypeptide-related sequence A; NOV: these transcriptional variations seem related to DNA Nephroblastoma overexpressed; PAS: Periodic Acid-Schiff; PBS: Phosphate- buffered saline; PPAR: Peroxisome proliferator-activated receptor; pval: P val- repair pathways and cell fate decisions, rather than pro- ues; rRNAs: Ribosomal RNAs; SM: Spaghetti meat; STEAP4: Metalloreductase; teolytic processes. This hypothesis is consistent with a TBARs: Thiobarbituric acid reactive substances; TGF-β: Transforming growth non-degrading role of protein ubiquitination [78], and factor-β; TNF-α: Tumor necrosis factor Alpha; WB: Wooden breast; WS: White striping. supported by the enriched KEGG pathways reported in females (e.g., “mismatch repair,” “cell cycle”). Supplementary Information Overall, the gene expression data showed that DNA The online version contains supplementary material available at https:// doi. repair processes are likely to be differentially regulated in org/ 10. 1186/ s40104- 022- 00736-w. male and female jejunum. Notably, sex differences in the control of cell cycle and DNA repair have been reported Additional file 1. RNA-seq libraries sequenced and transcriptional in mammals [79, 80]. Upregulated DEGs playing a role in changes. Averages of traits for jejunum morphology and immuno-histo- chemical analyses according to interactions between sex and age and the mitochondrial respiratory chain and ATP production between sex and diet are provided in Table S1 and Table S2, respectively. were also reported, e.g., cytochrome c oxidase subunit 7C Details on sequenced RNA-seq libraries are provided in Table S3. Compari- and NADH:ubiquinone oxidoreductase subunit S4. This sons among transcriptional profiles of same-sex and same-age chickens fed CN and GP diets compared to those of chickens fed the C diet, thus is most likely linked to the different growth rate between identifying significant DEGs, are shown in supplementary Tables S4, S5 sexes (higher in males), which is confirmed by the sig - and S6. Annotated genes significantly regulated in more than one com- nificant activation in males of genes involved in tissue parison are listed in Tables S5. Gender-related and sex-related transcrip- tional changes are reported in Tables S5 and S6. Functional enrichment morphogenesis and maintenance of cell and tissue struc- analysis is shown in Tables S7. ture and function (e.g. “focal adhesion,” “ECM-receptor P ascual et al. Journal of Animal Science and Biotechnology (2022) 13:102 Page 15 of 17 4. Li H, Li Z, Wei Z, Liu T, Zou X, Liao Y, et al. Long-term effects of oral tea Acknowledgments polyphenols and Lactobacillus brevis M8 on biochemical parameters, The PhD grant (PhD Course in Animal and Food Science of the University of digestive enzymes, and cytokines expression in broilers. J Zhejiang Padova) of Giulio Pillan is funded by Unismart and OFFICINE FACCO & C. Spa Univ Sci B. 2015;16:1019–26. (year 2020). The authors wish to thank Giovanni Caporale for his assistance in 5. Lupini C, Cecchinato M, Scagliarini A, Graziani R, Catelli E. In vitro antiviral preparing the samples for histological analyses. activity of chestnut and quebracho woods extracts against avian reovirus and metapneumovirus. Res Vet Sci. 2009;87:482–7. Authors’ contributions 6. Brus M, Gradišnik L, Trapečar M, Škorjanc D, Frangež R. Beneficial effects of AT, MD, and GX conceived and designed the experiment. AP, MB, FB and GP water-soluble chestnut (Castanea sativa Mill.) tannin extract on chicken performed the trial, collected the experimental data. MP, AT, MG, FB, and MG small intestinal epithelial cell culture. Poult Sci. 2018;97:1271–82. collected and prepared samples for histological and whole transcriptome 7. Xiong Y, Dong S, Zhao X, Guo KJ, Gasco L, Zoccarato I. Gene expres- analyses. AP, CB, and MB performed histological analyses. MP, MG, and MD sions and metabolomic research on the effects of polyphenols from the performed whole transcriptome analysis and related statistical analyses. AT involucres of Castanea mollissima Blume on heat-stressed broilers chicks. and AP performed the statistical analyses. MP, AT, GX, and MD analysed and Poult Sci. 2016;95:1869–80. interpreted the data. AT and AP wrote the first draft of the manuscript. All 8. Schiavone A, Guo K, Tassone S, Gasco L, Hernandez E, Denti R, et al. authors critically reviewed the manuscript for intellectual content and gave Eec ff ts of a natural extract of chestnut wood on digestibility, per - final approval of the version to be published. formance traits, and nitrogen balance of broiler chicks. Poult Sci. 2008;87:521–7. Funding 9. Huang Q, Liu X, Zhao G, Hu T, Wang Y. Potential and challenges of tannins The project was funded by the University of Padova (BIRD Ateneo di Padova; as an alternative to in-feed antibiotics for farm animal production. Anim CUP: C24I20000260005). Nutr. 2018;4:137–50. 10. Jamroz D, Wiliczkiewicz A, Skorupińska J, Orda J, Kuryszko J, Tschirch H. Availability of data and materials Eec ff t of sweet chestnut tannin (SCT ) on the performance, microbial Raw Illumina sequencing data have been deposited in GenBank (SRA) under status of intestine and histological characteristics of intestine wall in the SRA accessions SRR12728661-SRR12728684, and they will be published chickens. Br Poult Sci. 2009;50:687–99. after manuscript acceptance. During reviewing process, BioProject’s metadata 11. Rezar V, Salobir J. Eec ff ts of tannin-rich sweet chestnut (Castanea sativa are available at the following link: mill.) wood extract supplementation on nutrient utilisation and excreta https:// datav iew. ncbi. nlm. nih. gov/ object/ PRJNA 666129? revie wer= 398l4 cbdnf dry matter content in broiler chickens. Europ Poult Sci. 2014;78:42. nmu1m ris3g 20ol1c https:// doi. org/ 10. 1399/ eps. 2014. 42. The other datasets analysed in the current study are available from the cor- 12. Viveros A, Chamorro S, Pizarro M, Arija I, Centeno C, Brenes A. Eec ff ts of responding author upon reasonable request. dietary polyphenol-rich grape products on intestinal microflora and gut morphology in broiler chicks. Poult Sci. 2011;90:566–78. Declarations 13. Wang ML, Suo X, Gu JH, Zhang WW, Fang Q, Wang X. Influence of grape seed proanthocyanidin extract in broiler chickens: effect on chicken coc- Ethics approval and consent to participate cidiosis and antioxidant status. Poult Sci. 2008;87:2273–80. The study was approved by the Ethical Committee for Animal Experimenta- 14. Starčević K, Krstulović L, Brozić D, Maurić M, Stojević Z, Mikulec Ž, et al. tion (Organismo Preposto al Benessere Animale) of the University of Padova Production performance, meat composition and oxidative susceptibil- (project 92/2019 - prot. nr. 469509). All animals were handled according to ity in broiler chicken fed with different phenolic compounds. J Sci Food the principles of EU Directive 2010/63/EU regarding the protection of animals Agric. 2015;95:1172–8. used for experimental and other scientific purposes. The researchers involved 15. Brenes A, Viveros A, Goñi I, Centeno C, Sáyago-Ayerdy SG, Arija I, in animal handling were either animal specialists (PhD or MSc in Animal Sci- et al. Eec ff t of grape pomace concentrate and vitamin e on digest - ences) and/or veterinary practitioners. ibility of polyphenols and antioxidant activity in chickens. Poult Sci. 2008;87:307–16. Consent for publication 16. Abasht B, Mutryn MF, Michalek RD, Lee WR. Oxidative stress and meta- Not applicable bolic perturbations in wooden breast disorder in chickens. PloS One. 2016;11:e0153750. Competing interests 17. Soglia F, Laghi L, Canonico L, Cavani C, Petracci M. Functional property The authors declare that they have no competing interests. issues in broiler breast meat related to emerging muscle abnormalities. Food Res Int. 2016;89:1071–6. 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Dietary supplementation with sodium butyrate: growth, gut response at different ages, and meat quality of female and male broiler chickens. Ital J Anim Sci. 2020;20:1135–46. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

Journal

Journal of Animal Science and BiotechnologySpringer Journals

Published: Aug 17, 2022

Keywords: Growth; Immunohistochemistry; Meat quality; Tannins; Transcriptome

References