Table_3_A phytobiotic extract, in an aqueous or in a cyclodextrin encapsulated form, added in diet affects meat oxidation, cellular responses and intestinal morphometry and microbiota of broilers.xlsx

The present trial examined the effects of diet supplementation with an extract including Greek oregano, garlic, rock samphire, and camelina, administered either in aqueous form or encapsulated in cyclodextrin, on broiler chickens. The duration of the trial was 35 days. Mixed broiler chicks (Ross-308, 120 individuals, 1 day old) were randomly allocated to one of three groups, each with four replicates. Control group A (CONTROL) was fed a basal diet consisting of maize and soybean. The diet of the AQORGCC and CDORGCC groups was further supplemented with aqueous and cyclodextrin-encapsulated herbal extracts, respectively. Levels of lipid and protein oxidation were determined in breast and thigh meat samples. Furthermore, to address cellular stress and signaling responses, the expression patterns of heat shock proteins (Hsp60, Hsp70, and Hsp90), mitogen-activated protein kinases (P38 and P44/42 MAPKs), and apoptotic-related proteins (Bcl-2/Bad ratio) were investigated in breast and thigh tissues using Western blot analysis. The intestinal morphometry of the duodenum, jejunum, and ileum was also assessed. To investigate ileal and cecal bacterial community diversity, 16S rRNA gene high-throughput amplicon sequencing on the V3–V4 hypervariable region was performed. The results showed that the herbal extract in cyclodextrin delayed meat lipid oxidation. According to the protein expression patterns, the formulated diets elicited tissue-specific cellular responses. Compared with the CONTROL group, dietary supplementation with the encapsulated form resulted in significant Hsp induction and MAPK activation, whereas, in the group whose diet was supplemented with the aqueous form, the expression of most of the examined proteins decreased or was maintained at a constant level. Villus height and lamina propria width were mostly affected by the aqueous herbal extract, whereas the number of goblet cells remained unchanged among the groups. Firmicutes, Proteobacteria, and Bacteroidota were the major phyla in mean relative abundance in all diets in both cecal and ileal samples. Alpha-diversity indices highlighted higher species richness and diversity in the cecum than in the ileum, as well as in chicks treated with the aqueous extract of the herbal mixture, but only in the cecum. Cecal beta-diversity differed between the cyclodextrin and the CONTROL groups, while ileal beta-diversity varied only between the aqueous-treated group and the CONTROL group. In conclusion, the dietary mixtures of herbal extracts (particularly those encapsulated in cyclodextrin) improved protein and lipid oxidation and increased the number of beneficial lactic acid-producing bacteria in the cecum, whereas the aqueous herbal extract mostly affected bacterial activity in the proximal part of the chicken intestine. Similarly, intestinal morphometry in the duodenum, jejunum, and ileum was mostly affected by the aqueous herbal extract, which seems to inhibit proteins associated with stress signaling in meat.

本试验探究了以希腊牛至、大蒜、海蓬子及亚麻荠提取物为成分的日粮添加剂，分别以水溶液形式或环糊精包被形式给药，对肉用仔鸡的影响。试验周期为35天。将120只1日龄罗斯308（Ross-308）混合肉用雏鸡随机分为3组，每组设4个重复单元。对照组A（CONTROL）饲喂以玉米和大豆为基础的基础日粮；AQORGCC组与CDORGCC组日粮分别添加水溶液形式与环糊精包被形式的植物提取物。
对胸肌与腿肌样本的脂质与蛋白质氧化水平进行测定。此外，为探究细胞应激与信号通路响应，采用蛋白质印迹法（Western blot）检测胸肌和腿肌组织中热休克蛋白（Hsp60、Hsp70、Hsp90）、丝裂原活化蛋白激酶（P38、P44/42 MAPKs）及凋亡相关蛋白（Bcl-2/Bad比值）的表达模式。同时评估十二指肠、空肠及回肠的肠道形态学参数。为探究回肠与盲肠的细菌群落多样性，针对V3-V4高变区开展16S rRNA基因高通量扩增子测序。
试验结果显示，环糊精包被的植物提取物可延缓肌肉脂质氧化进程。蛋白表达模式分析表明，不同配方日粮可引发组织特异性的细胞响应。与对照组相比，饲喂包被提取物日粮的试验组出现显著的热休克蛋白诱导与丝裂原活化蛋白激酶激活效应；而饲喂水溶液提取物日粮的试验组，多数检测蛋白的表达水平出现下调或维持恒定。水溶液形式的植物提取物对肠绒毛高度与固有层宽度影响最为显著，而各组的杯状细胞数量无显著差异。所有日粮处理的盲肠与回肠样本中，厚壁菌门（Firmicutes）、变形菌门（Proteobacteria）与拟杆菌门（Bacteroidota）均为平均相对丰度最高的菌门。α多样性指数（Alpha-diversity）显示，盲肠内的物种丰富度与多样性均高于回肠；同时，经植物混合物水溶液处理的雏鸡仅在盲肠中表现出更高的物种丰富度与多样性。盲肠的β多样性（Beta-diversity）在环糊精包被组与对照组间存在显著差异，而回肠的β多样性仅在水溶液处理组与对照组间存在差异。
综上，日粮添加植物提取物混合物（尤其是环糊精包被形式）可改善肌肉的蛋白质与脂质氧化状态，并增加盲肠内有益产乳酸细菌的数量；而水溶液形式的植物提取物主要影响鸡肠道近端区域的细菌活性。同理，十二指肠、空肠与回肠的肠道形态学参数主要受水溶液植物提取物影响，该提取物似乎可抑制肌肉中与应激信号通路相关的蛋白表达。