Effects of dietary supplementation with purple garlic powder and oregano essential oil on intestinal health in post-weaning piglets from commercial farms.

This work studied the effects of the inclusion of Purple Garlic Powder (PGP) and Oregano Essential Oil (OEO) in the feed, at different doses and combinations, on intestinal health and the growth performance of 140 and 3000 piglets, respectively, weaned at 21 days of age. Seven dietary treatments were used: a negative control group (basal diet), a positive control group with ZnO (3000 mg/Kg of feed), two groups with OEO at 0.4% and 1.2% respectively, two groups with PGP at 0.4% and 2% respectively and one group with OEO at 1.2% combined with PGP at 2%. Only the positive control group received ZnO in the diet. Each group of piglets received the treatment for seven weeks, from weaning, and were later sacrificed to obtain jejunum and ileum samples for counting of goblet cells, intraepithelial lymphocytes, and IgA-producing cells. The growth performance were measured at the beginning and at the end of the seven weeks. In jejunum and ileum, the number of goblet cells increased in the groups with ZnO, PGP 2%, OEO 1.2% and PGP 2% + OEO 1.2%, presenting significant differences with the rest of the groups. The results obtained for the intraepithelial lymphocyte count were in line with those obtained for the count of goblet cells. Regarding IgA-producing cells, the groups that showed significantly favourable results in the jejunum and ileum were OEO 1.2%, PGP 2% and their combination, but the groups that showed the most similar means to ZnO were the OEO 0.4% and the PGP 0.4%. Regarding the growth performance, PGP 2%, OEO 1.2% and their combination had similar results to ZnO. The intestinal health of piglets could be improved, without harming the growth performance, by means of the supplementation of PGP 2%, OEO 1.2% and PGP 2% + OEO 1.2% offering a natural alternative to the use of ZnO.

Oregano Essential Oil and Purple Garlic Powder Effects on Intestinal Health, Microbiota Indicators and Antimicrobial Resistance as Feed Additives in Weaning Piglets.

Finding alternatives to zinc oxide is a pressing issue for the pig production sector. We studied the impact of the bioactive components degradation of oregano essential oil (OEO) and purple garlic powder (PGP) during storage in silos, their effect on the morphometry of the jejunum and ileum and the cecal microbiota as intestinal health indicators in piglets during the post-weaning period. We also monitored antimicrobial resistance in the commensal indicator E. coli. Histological parameters and intestinal microbiota were measured in 140 piglets weaned at 21 days of age. Seven dietary treatments were used: a negative control group (basal diet), a positive control group with ZnO (3000 mg/kg of food), two groups with OEO at 0.4% and 1.2%, respectively, two groups with PGP 0.4% and 2%, respectively, and a group with OEO with 1.2% combined with PGP with 2%. Each group of piglets received the treatment for seven weeks, from weaning, before samples were taken. Antibiotic resistance profiles were measured in 81 E. coli strains. On this occasion, only the control groups, ZnO, OEO 1.2%, PGP 2% and OEO 1.2% + PGP 2% were used, and the samples were obtained from the cecal content. A progressive loss of the bioactive components of OEO and PGP was observed during the 34 days of storage (p < 0.05). PGP 2%, OEO 1.2% and their combination showed results similar to ZnO (p > 0.05), or superior in the study of intestinal morphometry and the values of E. coli and Lactobacillus. All categories showed high levels of resistance. Only the strains isolated from the OEO 1.2% group did not show resistance to colistin and presented the lowest resistance values. In general, high doses of the additives studied showed the best results, obtaining levels like or higher than those offered by ZnO.

Antimicrobial Resistance of Campylobacter jejuni, Escherichia coli and Enterococcus faecalis Commensal Isolates from Laying Hen Farms in Spain.

Antimicrobial resistance (AMR) is a global threat for human and animal health. Few studies have been carried out in laying hens. We evaluated the antimicrobial susceptibility of commensal Campylobacter jejuni, Escherichia coli, and Enterococcus faecalis isolates in Spanish laying hens in 2018. The Minimum Inhibitory Concentration (MIC) was used to identify any AMR of the studied isolates by means of a broth microdilution method. C. jejuni was highly resistant to the B category antimicrobials, and 52% of the isolates were susceptible to all the antimicrobials tested. E. coli showed medium and high percentages of resistance to the B and A antibiotic categories, respectively, and 33.33% of the isolates were susceptible to all antimicrobials. The E. faecalis resistance to A category antimicrobials was variable, and 4.62% of the isolates were susceptible to all antimicrobials. In our work, novel data on AMR in laying hen commensal isolates in Spain is provided, and the AMR levels differ from those reported for poultry in the EU. A high resistance to key drugs for human medicine was found, representing a public health risk.

Resistance patterns to C and D antibiotic categories for veterinary use of Campylobacter spp., Escherichia coli and Enterococcus spp. commensal isolates from laying hen farms in Spain during 2018.

Antimicrobial Resistance (AMR) is a global threat for human and animal health. Few studies have been carried out on laying hens. The aim of this work was to evaluate the antimicrobial susceptibility of commensal Campylobacter spp., E. coli, and Enterococcus spp. isolates in Spanish laying hens in 2018. Samples were collected from 39 laying hen farms. The microorganisms of interest were isolated and confirmed by PCR. The Minimum Inhibitory Concentration (MIC) to antimicrobials of C and D categories were determined. 195 E. coli, 195 Enterococcus spp. and 25 Campylobacter spp. isolates were obtained. E. coli isolates showed high resistance to D category antimicrobials (sulfamethoxazole 76.41 %, tetracycline 62.05 %, trimethoprim 50.77 %, ampicillin 30.77 %) and lower resistance to C category (azithromycin 30.26 %, gentamicin 12.31 %, chloramphenicol 4.62 %). A 10.26 % of E. coli isolates were susceptible to all antimicrobials tested, Multi Drug Resistance (MDR) to 3 antimicrobial families was found in 23.08 % of the isolates and 13.85 % were MDR to 4 families, being Erythromycin-Sulfamethoxazole-Tetracycline the most common resistance profile (10.77 %). Enterococcus spp. showed very high resistance to D category tetracycline (78.47 %) and C category erythromycin (76.42 %). The 11.79 % of Enterococcus spp. isolates were susceptible to all antimicrobials and 53.33 % were resistant to 2 families, being Erythromycin-Tetracycline the most common AMR profile (51.79 %). Regarding Campylobacter spp., resistance to tetracycline (48 %) was higher than resistance to C category antimicrobials (erythromycin 28 %, streptomycin 24 %, gentamicin 16 %). There was a 52 % sensitivity to all tested antimicrobials and 24 % showed MDR to aminoglycosides, macrolides and tetracyclines (Gentamicin-Streptomycin-Erythromycin-Tetracycline MDR profile). Novel data on AMR in laying hen commensal isolates in Spain was provided. High resistance to several antimicrobials was found, especially to key drugs for the treatment of zoonosis, which represents a public health risk. Better surveillance and careful regulation of antimicrobial use is required in laying hen production.

Effects of Dietary Supplementation of Garlic and Oregano Essential Oil on Biomarkers of Oxidative Status, Stress and Inflammation in Postweaning Piglets.

The effects of two different concentrations of micro capsuled oregano essential oil (OEO) and purple garlic powder on biomarkers of oxidative status, stress, and inflammation, as well as on average daily gain (ADG) and feed conversion ratio (FCR), were evaluated in piglets during the postweaning period. The trial was carried out with 300 crossbred pigs of 21 days of age fed with different concentrations of OEO and purple garlic powder and ZnO. Saliva and serum samples were taken to evaluate a panel of biomarkers of oxidative status, stress, and inflammation. OEO and garlic powder at 0.4% did not produce significant changes in C-reactive protein (CRP) and cortisol and yielded higher levels of the antioxidant biomarker CUPRAC in serum than higher doses (p < 0.01); they yielded a better ADG than the control and ZnO diets. OEO and garlic powder at higher concentrations than 0.4% showed higher concentrations of CRP (p < 0.05). Overall, doses of OEO and garlic powder at 0.4% did not lead to inflammation, stress, or negative changes in oxidative biomarkers in piglets during the postweaning period and gave better productive performance than the control and ZnO diets. High doses of OEO and garlic powder were ineffective and could negatively affect the animals. Therefore, our results highlight the importance of the dose used when OEO or garlic are supplemented to piglets.

The Combined Effect of Mediterranean Shrubland Pasture and the Dietary Administration of Sage By-Products on the Antioxidant Status of Segureña Ewes and Lambs.

The objective of this study was to determine if the combined effect of ewe grazing and the incorporation of sage by-products in the lamb feed enhances the Segureña ewe and lamb antioxidant status. For that, the endogenous (related to blood antioxidant enzymes) and exogenous (plasma antioxidant activity) antioxidant systems of ewes and lambs were studied at the physiological stages of lactation, after weaning and at the end of the fattening period. Major results indicated that grazing improved the enzymatic antioxidant defense of ewes during the stressful period of lamb weaning, and also, the antioxidant status of the ewe plasma at both physiological stages: lactation and after lamb weaning. With regards to the lambs, ewe grazing stimulated the antioxidant enzymatic defense during lactation, but not the antioxidant capacity of the plasma. At the end of the fattening period, the inclusion of sage by-products in the lamb diet (10% w/w) enhanced both the enzymatic cascade and antioxidant capacity of the lamb plasma. The antioxidant capacity of the lamb meat was also studied and the benefits of sage were detected in samples from the deltoideus muscle. Thus, Mediterranean grazing stimulated the antioxidant defense in ewes, while the inclusion of sage by-products in the lamb diets enhanced the antioxidant status of both blood and meat.

Relevance of the carnosic acid/carnosol ratio for the level of rosemary diterpene transfer and for improving lamb meat antioxidant status.

The aim of the present work was to evaluate whether the relation between the concentrations of the two major diterpenes present in two typified rosemary extracts affects their levels of deposition and antioxidant capacity in different lamb tissues. The composition of the extracts expressed as percentage of weight/weight was 1:1 (14-16)% and 2:1 (25-11)% (carnosic acid-carnosol), respectively. Thirty weaned lambs were assigned randomly to three homogeneous groups. One group was fed a basal diet as a control and the diets of the other two were enriched with rosemary extracts 1:1 and 2:1, respectively. HPLC-ESI-MS/TOF identified a metabolite (C19H22O3) described for the first time in lamb tissues, along with carnosol, carnosic acid, rosmanol and carnosol-p-quinone. The results obtained corroborate the importance of the presence of carnosol in the dietary administration of rosemary extract as a way of improving the stability of the diterpene fraction during feed manufacturing and the level of deposition and antioxidant efficacy of diterpenes after ruminal fermentation.

Isoflavone supplementation reduces DNA oxidative damage and increases O-ß-N-acetyl-D-glucosaminidase activity in healthy women.

Phenolic compounds are believed to boost the human antioxidant defense system and health; therefore, the aim of this research was to investigate the hypothesis that soy isoflavones (IFs) provide antioxidant protection in healthy women by evaluating DNA resistance to oxidative damage and O-ß-N-acetyl-D-glucosaminidase (OGA) activity. An IF supplement (80 mg/d) was given to 9 postmenopausal women and 13 young women for 6 months and then stopped up to the 14th month. The women were allowed to consume their normal diet. Blood samples were collected at the beginning of the study after 2, 4, and 6 months and then at the 8th and 14th months. Plasma concentrations of genistein and daidzein, total antioxidant capacity, plasma vitamin status, markers of oxidative stress (red blood cell membrane fluidity, activity of the red blood cell cytosolic enzyme OGA and lymphocyte DNA susceptibility to oxidative stress), and serum lipid profile were analyzed. Analysis of variance for repeated measures was used for statistical analysis. Plasma concentrations of IFs rose significantly during the supplementation period, and plasma total antioxidant capacity increased in young women; membrane fluidity and OGA activity increased, and DNA oxidative damage decreased (P < .05) at 4 months, then returned to the basal level. There was a significant inverse correlation between DNA damage and plasma IF concentrations (P < .01). The results indicated a positive effect of IF supplementation on oxidative stress in women, thus suggesting that the healthful action ascribed to soy consumption may be partially related to the antioxidant potential of IFs.

In vitro and in vivo effect of IFNalpha on B16F10 melanoma in two models: subcutaneous (C57BL6J mice) and lung metastasis (Swiss mice).

Interferon alpha tends to be the only agent used to treat melanoma. The objective of this study was to assess the effect of interferon alpha on the growth of the B16F10 melanoma, both in vitro and in vivo. We study the in vitro effect of interferon alpha (250,000, 500,000 and 1,000,000 IU/ml) on the B16F10 melanoma cell line (at 24, 48 and 72 h) and the in vivo effect in a subcutaneous (1x10(6) cells; 300,000 IU) and a pulmonary metastatic model (5x10(5) cells/lateral vein of the tail; 300,000, 600,000 and 1,200,000 IU). Necropsy included a morphological and immunohistochemical study (subcutaneous model), while the number of superficial lung metastases, implantation percentage and growth and invasion indices were calculated in the latter model. In vitro, interferon alpha decreased cell survival in a time- and dose-dependent manner; 250,000 IU/ml: 77% (24h), 80% (48 h) and 92% (72 h); 500,000 IU/ml: 62% (24h), 32% (48 h), 20% (72 h); 1,000,000 IU/ml: 41% (24h), 16% (48 h), 10% (72 h). In the subcutaneous model, it reduced tumor weights (77.74%) and cell proliferation (70.8%), and increased necrotic areas (8%) and inflammatory infiltrates (34.46%). Metastatic model: 300,000 IU reduced pleural nodules by 38.79%, implantation by 59.42%, growth by 43.48%, invasion by 25.06%; the corresponding figures for 600,000 and 1,200,000 IU were 38.79, 59.42, 43.48, 25.06%, and 65.55, 84.98, 56.52, 66.19%, respectively. Interferon alpha inhibited cell proliferation in all the models and had immunomodulatory (subcutaneous model) and antimetastatic (pulmonary metastatic model) effects in vivo.

Efectos del IFN a y la diosmina en el melanoma metastásico pulmonar murino / Effects of IFN a and diosmin on metastasic murine-lung melanoma

Antecedentes: El melanoma metastatiza en aproximadamente un tercio de los pacientes, causando una caída de la supervivencia hasta el 1-2% a los dos años. El único tratamiento eficaz es el interferón alfa (IFN a) a dosis altas, que resulta muy tóxico. Por ello se buscan antitumorales menos tóxicos, entre los que destacan los flavonoides. Nuestro objetivo ha sido estudiar el tratamiento del melanoma metastásico pulmonar combinando IFN a y diosmina en un modelo murino. Material y Métodos: Utilizamos 60 ratones Swiss inoculados con células (5 x 105) de la línea de melanoma murino B16F10, tratados durante 11 días antes y 21 días después de la inoculación: Grupo I: etanol + PBS; Grupo II: etanol + IFN a (600.000 UI); Grupo III: etanol + IFN a (1.200.000 UI); Grupo IV: diosmina + PBS; Grupo V: diosmina + IFN a (600.000 UI); Grupo VI: diosmina + IFN a (1.200.000 UI). Tras el tratamiento, los animales fueron sacrificados y realizamos el contaje macroscópico de los nódulos metastásicos subpleurales. Resultados: Encontramos diferencias significativas entre el grupo control y los tratados (p<0,001), produciéndose la mayor reducción del número de metástasis subpleurales respecto al control en el grupo III (79,74%; p<0,001), seguido del grupo V (77,38%; p<0,001), del VI (72,33%; p<0,001), del IV (61,4%; p<0,001) y del II (59,59%; p<0,001). Conclusiones: La combinación de diosmina con la dosis menor de IFN a mostró la potenciación de la actividad antimetastásica de ambos compuestos, resultando igual de eficaz que la dosis más elevada de IFNa de forma individualizada

Background: Melanoma metastasizes in approximately one third of patients, causing a drop in survival of 1-2% at two years. The only effective treatment is Interferon alpha (IFN a) at elevated doses, which is highly toxic. Thus, less toxic antitumoral agents are being sought, among which flavonoids are to be highlighted. Our aim was to study the combined treatment of metastasic lung melanoma with IFN a and diosmin in a murine model. Material & Methods: 60 Swiss mice inoculated with cells (5 x 105) from the B16F10 murine melanoma cell line, treated over 11 days prior and 21 days following inoculation: Group I: ethanol + PBS; Group II: ethanol + IFN a (600,000 IU); Group III: ethanol + IFN a (1,200,000 IU); Group IV: diosmin + PBS; Group V: diosmin + IFN a (600,000 IU); Group VI: diosmin + IFN a (1,200,000 IU). Following treatment, animals were sacrificed and a macroscopic count of subpleural metastasic nodules was performed. Results: We found significant differences between the control and the treated groups (p<0.001), there being a greater drop in the number of subpleural metastasis in group III with respect to the control (79.74%; p<0.001), followed by group V (77.38%; p<0.001), VI (72.33%; p<0.001), IV (61.4%; p<0.001) and II (59.59%; p<0.001). Conclusions: The combination of diosmin with the lower dose of IFN a showed a strengthening of the anti-metastasic action of both compounds, being equally as effective as the highest dose of IFN a on its own

Treatment of metastatic melanoma B16F10 by the flavonoids tangeretin, rutin, and diosmin.

Melanoma is one of the most frequently metastasizing malignant neoplasias. This study examines an experimental model of pulmonary metastasis and the B16F10 cell subline, highly metastatic in the lung. Antimetastatic effects of the flavonoids tangeretin, rutin, and diosmin were analyzed, and at the same time an analysis of the metastatic activity of ethanol was performed, considered to be necessary because it is used as a vehicle for administering the flavonoids. Lentini's model, which complements the macroscopic evaluation of nodule numbers by using a stereoscopic microscope and image analysis at the microscopic level, was used. The greatest reduction in the number of metastatic nodules (52%) was obtained with diosmin; similarly, the percentages of implantation, growth index, and invasion index (79.40, 67.44, and 45.23%, respectively), were all compared with those of the ethanol group, considered to be an effective control group. Rutin- and tangeretin-treated groups also showed reductions of the same index compared with the ethanol group. It would seem that structural factors would better explain these results and the antimetastatic activity of each flavonoid and the respective metabolites.

[Experimental model for treating pulmonary metastatic melanoma using grape-seed extract, red wine and ethanol]. / Modelo experimental de tratamiento con extracto de semillas de uva, vino tinto y etanol del melanoma metastásico pulmonar.

INTRODUCTION: Melanoma is one of the neoplasias that most frequently metastasize, especially in the lung, where represents a challenge in oncology since current treatment is ineffective, and mortality is high. MATERIAL AND METHODS: Swiss mice (n = 52) were inoculated with 0.5 x 106 B16F10 cell lines and, later, given an oral administration of grape-seed extract, red wine or ethanol. Metastatic nodules on the lung surface were counted and, after processing for microscopy, five sections were selected for image analysis and the invasion index was calculated. RESULTS: Macroscopic analysis showed that grape-seed extract and red wine reduced the number of metastatic nodules by 26.07 and 20.81%, respectively, compared with a control group treated with ethanol. Microscopically, the reduction in the invasion index was 31.65 for grape-seed extract and 17.57% for red wine. CONCLUSION: Ethanol administration significantly increased pulmonary metastasis while grape-seed extract and red wine led to their reduction.

Modelo experimental de tratamiento con extracto de semillas de uva, vino tinto y etanol del melanoma metastásico pulmonar / Experimental model for treating pulmonary metastatic melanoma using grape-seed extract, red wine and ethanol

Introducción. El melanoma es una de las neoplasias malignas que metastatiza con mayor frecuencia, preferentemente en pulmón, representando un reto para la oncología por la ineficacia del tratamiento y la alta tasa de mortalidad. Material y métodos. Inoculamos a 52 ratones Swiss 0,5X106 melanocitos B16F10, a los que administramos oralmente extracto de semilla de uva, vino tinto y etanol. Realizamos el recuento de los nódulos metastásicos de la superficie pulmonar y tras el procesado de los pulmones seleccionamos 5 secciones para su estudio microscópico mediante análisis de imagen, calculando el índice de invasión. Resultados. En el estudio macroscópico el extracto de semillas de uva y el vino tinto originaron una reducción de nódulos metastásicos del 26,07% y del 20,81% respecto al grupo tratado con etanol. Microscópicamente la reducción del índice de invasión fue del 31,65% en el grupo tratado con extracto de semillas de uva y del 17,57% para el del vino. Conclusión. El tratamiento con etanol originó un aumento significativo de las metástasis pulmonares, mientras que el extracto de semillas de uva y el vino tinto las redujeron

Introduction. Melanoma is one of the neoplasias that most frequently metastasize, especially in the lung, where represents a challenge in oncology since current treatment is ineffective, and mortality is high. Material and methods. Swiss mice (n = 52) were inoculated with 0.5 x 106 B16F10 cell lines and, later, given an oral administration of grape-seed extract, red wine or ethanol. Metastatic nodules on the lung surface were counted and, after processing for microscopy, five sections were selected for image analysis and the invasion index was calculated. Results. Macroscopic analysis showed that grape-seed extract and red wine reduced the number of metastatic nodules by 26.07 and 20.81%, respectively, compared with a control group treated with ethanol. Microscopically, the reduction in the invasion index was 31.65 for grape-seed extract and 17.57% for red wine. Conclusion. Ethanol administration significantly increased pulmonary metastasis while grape-seed extract and red wine led to their reduction

Melanoma metastásico pulmonar Efectos del etanol y de flavonoides / Pulmonary metastatic melanoma. Effects of ethanol and flavonoids

Antecedentes: El Melanoma es el responsable del 75 por ciento de las muertes por cáncer de piel, fundamentalmente por las metástasis, preferentemente pulmonares. La dificultad del tratamiento estriba en las interacciones entre las células tumorales y los mecanismos homeostáticos que usurpan. Métodos: Hemos establecido un modelo experimental de metástasis pulmonares mediante la inoculación intravenosa de células B16F10 a ratones Swiss, a los que administramos oralmente apigenina, diosmina y rutina, y el etanol como disolvente, durante once días antes y veintiuno después de la inoculación, procediéndose al sacrificio de los animales y al recuento de los nódulos en toda la superficie pulmonar mediante lupa estereoscópica. Resultados: La inoculación de 0,5 x 106 células de B16F10 origina un modelo metastásico pulmonar cuantificable macroscópicamente, destacando en el tratamiento el grupo del etanol (II) con el mayor número de nódulos pulmonares y diferencias significativas respecto a los restantes grupos (330ñ86 nódulos, con un aumento del 87 por ciento respecto al control (I)). Ninguno de los grupos de los flavonoides mostró diferencias significativas respecto al control ni entre sí, solo el grupo tratado con Diosmina (V) presentaba menor número de nódulos que el control (I) y diferencias significativas con el tratado con etanol (II) con una reducción del 52 por ciento de los nódulos observados. Conclusión: El grupo tratado con etanol casi duplicaba las metástasis pulmonares del control. Todos los tratados con flavonoides presentaban reducción del número de metástasis respecto del etanol, siendo el de diosmina el de mayor reducción (52 por ciento) (AU)