One hundred fourty-four bulls (164.8 ± 5.91 kg BW and 135 ± 7.2 d of age) were randomly allocated to one of 8 pens and assigned to control (C) or citrus flavonoid (BF) treatments (Citrus aurantium, 0.4 kg per ton of concentrate of Bioflavex CA, > 20% naringin; BF). Each pen had one drinker, one separate five-space straw feeder, and one separate three-space feeder where mash concentrate containing mostly corn, barley, DDG and wheat was offered. Concentrate intake was recorded daily, whilst BW and animal behavior were recorded fortnightly. Animals were slaughtered after 168 d of study (12 periods of 14 d), and HCW and carcass quality were recorded, and rumen papillae samples were collected. Final BW (437.9 ± 1.85 kg), HCW (238.7 ± 2.02 kg), and concentrate intake (7.1 ± 0.13 kg/d) were not affected by treatment. Concentrate feed conversion ratio (kg of concentrate/ kg of BW) tended (P < 0.10) to be lesser in BF than in C bulls (5.11 vs. 5.36 ± 0.108 kg/kg). Percentage of animals eating concentrate during visual scan was greater (P < 0.01) in BF compared with C bulls (10.02 % vs. 7.97 % ± 0.512). Oral non-nutritive behaviors, agonistic interactions (fighting, butting, and chasing) and sexual behaviors (flehmen, attempted and complete mounts) were greater (P < 0.01) in C than in BF bulls. In the rumen epithelium, gene expression of bitter taste receptor 7, bitter taste receptor 16, bitter taste receptor 38 and bitter taste receptor 39 was greater (P < 0.05) in C compared with BF bulls, as well as was gene expression of free fatty acid receptor 2, pancreatic polypeptide receptor 1, cholecystokinin receptor 4, cytokine IL-25, Toll-like receptor-4 and β-defensin1. In conclusion, supplementation with flavonoids extracted from Citrus aurantium in bulls fed high-concentrate diets tends to improve efficiency, and reduces oral non-nutritive behaviors, agonistic interactions and sexual behavior. Moreover, flavonoid supplementation modifies the expression of genes in the rumen epithelium that could be related with eating and animal behavior regulation.

Feed Science and Technology © Elsevier after peer review. To access the final edited and published work see https://doi.org/10.1016/j.anifeedsci.2018.08.010 FLAVONOIDS AND RUMEN HEALTH AND PERFORMANCE 1 2 Effects of flavonoids extracted from Citrus aurantium on performance, eating and 3 animal behavior, rumen health, and carcass quality in Holstein bulls fed high-4 concentrate diets. 5 6 MABSTRACT 22The effects of flavonoids extracted from Citrus aurantium (Bioflavex® CA) on eating 23 pattern, performance, carcass quality, and rumen wall health of Holstein bulls fed on a 24 single feeder were studied. One hundred ninety-eight bulls (195.3 ± 19.6 kg of body 25 weight and 149 ± 6.8 d of age) were used in a complete block randomized design. 26 Groups of animals with the same mean and coefficient of variation of body weight 27(replicates) were randomly allocated in 1 of 6 pens (20 animals per pen), and each 28 pen was assigned to one of 6 pens and assigned to a Control (C) diet or to a diet 29 supplemented with flavonoids (Bioflavex® CA, Interquim S.L., Spain) (BF, 0.4 kg 30 per ton of concentrate of Bioflavex® CA) in two consecutive fattening cycles. 31Concentrate intake was recorded daily, and BW fortnightly. Animal behavior was 32 monitored by visual scan procedure every fourteen days. Animals were slaughtered after 33 168 d of study, hot carcass weight and carcass quality were recorded, and internal 34 rumen wall was examined. Concentrate intake was higher (P < 0.05) in C than in BF 35 bulls; however, ADG and concentrate efficiency were not affected by treatments. The 36 final BW tended (P = 0.06) to be higher in C than in BF bulls, but this difference 37 disappeared for carcass weight. In the finishing phase, the proportion of meal size 38 values above 750 g was higher (P < 0.05) in C compared with BF bulls. Throughout the 39 study exhibited more displacements and fighting than C bulls, whilst C bulls performed 40 more (P < 0.05) oral behaviors. During the finishing phase, sexual behaviors such as 41 flehmen and complete mounts were higher (P < 0.01 and P < 0.05, respectively) in C 42 bulls as well, and C bulls tended (P = 0.10) to perform more attempted mounts 43 compared with BF bulls. In the slaughterhouse, color of rumen wall tended (P = 0.06) 44 to be lighter for BF compared with C bulls, and presence of baldness areas in the rumen 45 was lesser (P = 0.01) in BF animals. In conclusion, when bulls were supplemented with 46 3 Bioflavex® CA, feed intake was reduced. Flavonoids supplementation increased time 47 eating straw, reduced agonistic behaviors throughout the study and sexual interactions 48 during the finishing phase, potentially improving animal welfare. Rumen wall 49 parameters analyzed were indicative of a better rumen health in BF than in C bulls, 50 which maybe due to the reduction of large meal sizes. 51

Flavonoid supplementation may modify the behavior and rumen inflammatory response of fattening bulls, and this could be related to the concentrate presentation (mash or pellet) form. In the present study, 150 Holstein bulls (183.0 ± 7.53 kg BW and 137 ± 1.8 d of age) were randomly allocated to one of eight pens and assigned to control (C) or (BF) (Citrus aurantium, Bioflavex CA, HealthTech Bio Actives, Spain, 0.4 kg per ton of concentrate of Bioflavex CA, 20% naringin). Concentrate (pellet) intake was recorded daily, and BW and animal behavior fortnightly. Animals were slaughtered after 168 d of study, and ruminal epithelium samples were collected for gene expression analyses. Treatment did not affect animal performance; however, BF supplementation reduced agonistic interactions and oral non-nutritive behaviors and increased the time devoted to eating concentrate and ruminating activity (p < 0.05). The gene expression of some genes in the rumen epithelium was greater or tended to be greater in BF than C bulls (bitter taste receptor 16, cytokine IL-25, β-defensin; p < 0.10; pancreatic polypeptide receptor 1 and tumor necrosis factor alpha; p < 0.05). In conclusion, flavonoid supplementation modifies the expression of genes in the rumen epithelium that could be related to inflammation and animal behavior modulation.

One hundred and forty-six bulls (178.2 ± 6.64 kg BW and 146.0 ± 0.60 d of age) were randomly allocated to one of eight pens and assigned to control (C) or citrus flavonoid (BF) treatments (Citrus aurantium, Bioflavex CA, HTBA, S.L.U., Spain, 0.4 kg per ton of Bioflavex CA). At the finishing phase, the dietary fat content of the concentrate was increased (58 to 84 g/kg DM). Concentrate intake was recorded daily, and BW and animal behavior by visual scan, fortnightly. After 168 d, bulls were slaughtered, carcass data were recorded, and rumen and duodenum epithelium samples were collected. Performance data were not affected by treatment, except for the growing phase where concentrate intake (p < 0.05) was lesser in the BF compared with the C bulls. Agonistic and sexual behaviors were more frequent (p < 0.01) in the C than in the BF bulls. In the rumen epithelium, in contrast to duodenum, gene expression of some bitter taste receptors (7, 16, 39) and other genes related to behavior and inflammation was higher (p < 0.05) in the BF compared with the C bulls. Supplementing citrus flavonoids in high-fat finishing diets to Holstein bulls reduces growing concentrate consumption and improves animal welfare.