Development and Validation of a UPLC-ESI(-)-MS/MS Methodology for the Simultaneous Quantification of Hesperidin, Naringin, and their Aglycones in Chicken Tissue Samples.

BACKGROUND: The dietary supplementation of livestock with antioxidants to improve the meat quality represents an active research area of high commercial impact. In order to investigate the optimal dosing, analytical methodologies need to be developed in various tissues to evaluate which concentration does remain in the tissue. OBJECTIVE: We aimed to develop and validate a sensitive and specific methodology for the simultaneous quantitative determination of hesperidin, naringin, hesperetin, and naringenin in chicken tissue samples employing ultra-performance LC-tandem MS. METHODS: Lipid extraction using cold chloroform was performed followed by protein precipitation by cold acetone. Chromatography was performed on a C18 column using a ternary gradient of water, acetonitrile, and isopropanol-acetonitrile-acetone (58+40+2, v/v) as the mobile phase. Detection was performed by electrospray ionization in negative ion mode with the selected reaction monitoring technique. RESULTS: Calibration plots exhibited good linearity (r2 > 0.99) over the concentration range from 0.125 to 25 µg/g tissue for the four analytes, and the lower LOQ for the four analytes was 0.125 µg/g tissue. The repeatability as percent relative SD and precision as percent accuracy were <20 and >80%, respectively. CONCLUSIONS: The developed methodology was applied for the quantitative determination of hesperidin, naringin, hesperetin, and naringenin in tissue samples after dietary supplementation with 1.5 g/kg hesperidin and 1.5 g/kg naringin in Ross 308 broiler chickens. HIGHLIGHTS: This is the first methodology to access naringin, naringenin, hesperidin, and hesperetin in chicken tissue. It involved simple sample preparation, and the mass spectrometry based detection ensures high specificity and sensitivity.

Elemental Metabolomics: Modulation of Egg Metallome with Flavonoids, an Exploratory Study.

The basic principles of elemental metabolomics were applied to investigate whether alteration of egg metallome could be achieved after two flavonoids addition, namely hesperidin and naringin in diets of laying hens. A total of 72 hens were divided into six groups: Control (C) (basal diet), E1 (750 mg hesperidin/kg diet), E2 (1500 mg hesperidin/kg diet), N1 (750 mg naringin/kg diet), N2 (1500 mg naringin/kg diet), and VE (200 mg vitamin E/kg diet). The same diet was provided to birds of all treatments, with the exception of added supplements. The diets had the same vitamin and mineral premix; thus, all birds received the same number of elements because no differences on feed intake existed. The egg elemental profile consisted of As, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, Pb, Sb, Se, Sr, V, Zn, and was determined using ICP-MS. Flavonoid supplementation altered the elemental profile. Most notably, in both albumen and yolk, hesperidin increased Ni, Pb, and Sr concentration while it decreased that of Co and Sb. Naringin increased Cd, Cr, Cu, Ni, and V and lowered the concentration of Co and Sb in both yolk and albumen. Vitamin E supplementation, in comparison to the control, decreased Co in both albumen and yolk and also raised Sb in albumen. Flavonoid presence led to the differences in deposition of certain trace minerals in egg compared to that of hens fed a basal diet or a diet with vitamin E supplementation.

Antioxidant status, meat oxidative stability and quality characteristics of lambs fed with hesperidin, naringin or α-tocopheryl acetate supplemented diets.

BACKGROUND: Dietary supplementation of sheep with bioactive compounds such as citrus flavonoids appears as a great alternative for improving meat antioxidant capacity and could also have beneficial effects on growth performance, carcass traits and meat quality characteristics. An experiment was therefore conducted to examine the effects of hesperidin or naringin dietary supplementation on lambs' growth performance, antioxidant status and meat quality characteristics. Forty-four male 3-month-old Chios lambs were randomly assigned to four groups: C, fed with a basal diet, or H or N or VE offered the same diet further supplemented with hesperidin at 2500 mg or naringin at 2500 mg or α-tocopheryl acetate (vitamin E) at 200 mg kg-1 feed respectively. At the end of the experiment (day 35), samples of longissimus thoracis muscle were collected for meat quality evaluation. RESULTS: Both flavonoids and vitamin E dietary supplementation reduced plasma malondialdehyde (MDA) levels on day 35 (P < 0.05). No significant differences were observed in performance traits and meat quality characteristics among the experimental groups (P > 0.05). Hesperidin and naringin also reduced MDA levels in meat stored at 4 °C for up to 8 days (P < 0.05), although to a lesser extent than α-tocopheryl acetate. CONCLUSION: Incorporation of flavonoids in sheep diets resulted in an improvement of plasma and meat antioxidant capacity. © 2018 Society of Chemical Industry.

UHPLC-HRMS-based tissue untargeted metabolomics study of naringin and hesperidin after dietary supplementation in chickens.

To date numerous metabolomic studies have been performed in order to characterize nutritional intervention studies. The aim of the current study was to present a comprehensive pipeline for characterizing the metabolic changes that occur in chickens tissues in response to naringin and hesperidin dietary supplementation. Forty-nine chickens were randomly divided into 3 groups: the first one fed with diet supplemented with naringin, the second with hesperidin whereas the control group was fed by commercial basal diet. After 30 days of administration chicken muscle samples were analyzed by UHPLC-HRMS whereas data were analyzed by the proposed pipeline. Three significant variables were detected to discriminate the control from the group after naringin administration and thirteen variables after hesperidin supplementation. Furthermore, a more detailed pipeline (encompassing multiple internal standards, internal validation of the clustering, extended statistical significance scores and multiple identification procedures) has been proposed aiming towards a more accurate untargeted analysis.

NMR Metabolomics Investigates the Influence of Flavonoid-Enriched Rations on Chicken Plasma.

The use of flavonoids as dietary supplements is well established, mainly due to their intense antioxidant and anti-inflammatory properties. In the present study, hesperidin, naringin, and vitamin E were used as additives at different concentrations in poultry rations in order to achieve meat of improved quality. NMR metabolomics was applied to chicken blood serum samples to discern whether and how the enriched rations affected the animals' metabolic profile. Variations in the metabolic patterns according to sustenance consumption were traced by orthogonal projections to latent structures discriminant analysis (OPLS-DA) models and were attributed to specific metabolites by using S-line plots. In particular, serum samples from chickens fed with vitamin E displayed higher concentrations of glycine and succinic acid compared to control samples, which were mainly characterized by betaine, formic acid, and lipoproteins. Samples from chickens fed with hesperidin were characterized by increased levels of lactic acid, citric acid, creatine, carnosine, creatinine, phosphocreatine, anserine, glucose, and alanine compared to control samples. Lastly, naringin samples exhibited increased levels of citric and acetic acids. Results verify the scalability of NMR metabolomics to highlight metabolite variations among chicken serum samples in relation to food rations.

The Influence of Naringin or Hesperidin Dietary Supplementation on Broiler Meat Quality and Oxidative Stability.

An experiment was conducted to examine the effects of supplementing broiler feed with hesperidin or naringin, on growth performance, carcass characteristics, breast meat quality and the oxidative stability of breast and thigh meat. Two hundred and forty 1-day-old Ross 308 broiler chickens were randomly assigned to 6 groups. One of the groups served as a control (C) and was given commercial basal diets, whereas the other five groups were given the same diets further supplemented with naringin at 0.75 g/kg (N1), naringin at 1.5 g/kg (N2), hesperidin at 0.75 g/kg (E1), hesperidin at 1.5 g/kg (E2) and a-tocopheryl acetate at 0.2 g/kg (E). At 42 days of age, 10 chickens per treatment group were slaughtered for meat quality and oxidative stability assessment. No significant differences were observed among groups in final body weight, carcass weight and internal organs weights (P>0.05) apart from liver that decreased linearly with increased levels of naringin (P-linear<0.05). Regarding the breast meat quality parameters, only redness (a*) value was higher in E1 and N1 group compared to VE group (P<0.05), while all the others i.e. shear values (N/mm2), pH24, cooking loss (%) and L* and b* color parameters were not significantly different among groups (P>0.05). Measurement of lipid oxidation values showed that after hesperidin and naringin dietary supplementation, malondialdehyde values decreased in tissue samples in a dose depended manner (P-linear<0.05). In conclusion, hesperidin and naringin, positively influence meat antioxidative properties without negative implications on growth performance and meat quality characteristics in poultry, thus appearing as important additives for both the consumer and the industry.

Effects of gestational maternal undernutrition on growth, carcass composition and meat quality of rabbit offspring.

An experiment was conducted in order to evaluate the effects of gestational undernutrition of rabbit does on growth, carcass composition and meat quality of the offsprings. Thirty primiparous non lactating rabbit does were artificially inseminated and randomly divided in three treatment groups: Control (C; fed to 100% of maintenance requirements throughout gestation, n = 10), early undernourished (EU; fed to 50% of maintenance requirements during days 7-19 of gestation, n = 10) and late undernourished (LU; fed to 50% of maintenance requirements during days 20-27 of gestation, n = 10). During the 4th week of the gestation period, LU does significantly lost weight compared to C and EU groups (P<0.05). At kindling, C does produced litters with higher proportions of stillborn kits (P<0.05) while the total litter size (alive and stillborn kits) was not different among groups (10.7, 12.8 and 12.7 kits in C, EU and LU groups, respectively). Kit birth weight tended to be lower in the LU group. During fattening, body weight and feed intake were not different among offsprings of the three experimental groups. Moreover, the maternal undernutrition did not have any impact on carcass composition of the offsprings in terms of carcass parts and internal organs weights as well as meat quality of L. lumborum muscle (pH24, colour, water holding capacity and shear values) at slaughter (70 days of age). Therefore, it can be concluded that the gestational undernutrition of the mother does not have detrimental effects on the productive and quality traits of the offsprings.

Severe maternal undernutrition and post-weaning behavior of rabbits.

The objective of the present experiment was to investigate the implications of severe maternal undernutrition on the post-weaning behavior of rabbits. Thirty two does were randomly assigned to four groups: the control group (C) that was fed 100% of the recommended energy maintenance requirements throughout pregnancy and lactation and the U1, U2 and U3 groups that were fed 50% of the recommended energy maintenance requirements between the 6th and the 19th day of pregnancy, between the 20th and the 27th day of pregnancy and between the 3rd and the 10th day of lactation, respectively. At the age of 50 and 65 days, behavior of rabbits in cages was recorded and rabbits were further subjected to an open-field test; a paradigm used as an indicator of fear and emotional distress. Significant differences were found between the U2 and the other experimental groups for the duration of eating and drinking, and duration of locomotory and investigatory behaviors (P<0.05). At the age of 65 days, duration of comfort behaviors was also lower in the U2 compared to the other groups (P<0.05). On the other hand, duration of resting appeared to be the highest in the group of rabbits born from undernourished does between the 20th and the 27th day of pregnancy (P<0.05). Duration of resting was significantly increased, although frequency and duration of eating and drinking were significantly decreased at the age of 65 days (P<0.05). Male rabbits had greater values for locomotory and investigatory behaviors, and duration of eating and drinking compared to the females (P<0.01). A decreased activity of rabbits during the light period was observed (as expected), whereas an increase in duration of locomotory and investigatory behaviors was observed during the first four and the last 4h of the light and dark periods, respectively (P<0.05). Rabbits born from the U1 and U2 group of does displayed increased rates of latency to leave the start position compared to the other groups during the implementation of the open-field test (P<0.05). No other significant differences in the exhibition of the examined behaviors were found between the treatment groups, with the exception of 'moving forelegs' activity that had the lowest value in the U3 group of rabbits. It is concluded that severe maternal undernutrition (50% of the recommended maintenance requirements) especially between the 20th and the 27th day of pregnancy has significant effects on the exhibition of rabbits' post-weaning behavior, since the offspring from the U2 undernourished does were significantly less active compared to the other experimental groups.

Effects of dietary hesperidin supplementation on lamb performance and meat characteristics.

An experiment was conducted to examine the effects of supplementing feed with hesperidin or α-tocopheryl acetate on lambs' growth performance, carcass and meat characteristics. Sixteen male lambs were randomly assigned to four groups. One of the groups served as control and was given a basal diet, whereas the other three groups were given the same diet further supplemented with hesperidin at 1500 mg/kg, or hesperidin at 3000 mg/kg, or α-tocopheryl acetate at 200 mg/kg. At the end of the experiment, lambs were fasted, weighed and slaughtered. After overnight chilling, samples of Longissimus thoracis muscle were taken and were used for meat quality evaluation. No significant differences were observed in final body weight, body weight gain and organ weights among the four groups. pH, color, water-holding capacity, shear force values and intramuscular fat concentration of Longissimus thoracis muscle were also not significantly influenced by the dietary treatments. Measurement of lipid oxidation values showed that hesperidin supplementation positively influenced meat antioxidant properties during refrigerated storage.

Feeding preferences in lambs influenced by prenatal flavour exposure.

The aim of the present study is to highlight and evaluate the role of flavour prenatal exposure in postnatal feeding preferences in sheep after weaning and until the early stages of puberty. 16 lambs were selected from two groups of ewes; the first group was fed with a control diet, consisted of concentrated feed and alfalfa hay and the second group with the same diet, with the only difference that the concentrate was supplemented with oregano essential oil (1 ml/kg), during the period of pregnancy (50th-130th day). Lambs were later individually subjected to feeding preference tests, at the age of 3, 4.5, 6, 7.5 months old. Each test lasted 25 min and it was a free choice situation between 3 different test feeds supplemented with eucalyptus or orange or oregano essential oil (1 ml/kg). Lambs born to oregano-treated ewes ate higher amounts of the oregano supplemented test feed during all feeding preference tests compared to lambs from the control group (P<0.01). Rates for occurrences and duration of eating were also greater in lambs born to oregano-treated ewes than the offspring of the control ewe group (P<0.01). On the other hand, animals without a flavour exposure precedent did not exhibit an evident strong preference for a specific test feed, although orange supplemented feed tended to be more preferable compared to the other offered feeds. Average total intake of lambs were not different between groups during all feeding preference tests at 3, 41/2, 6 and 71/2 months old. As it is concluded, prenatal exposure to oregano essential oil via maternal ingestion drastically influences lamb feeding preferences till adulthood.

Effect of breed and age on sexual behaviour of rams.

The objective of this study was to highlight the problems that arise during the reproduction between thin-tailed rams and fat-tailed ewes. At the same time, particular emphasis laid on the influence of sheep breed, sheep age, time after ram introduction and day of the ewe estrus cycle on ram and ewe sexual behaviour. Rams were subjected to sexual performance tests by being individually exposed to 12 ewes for 3 h daily, 19 consecutive days. The 16 rams of the experiment were separated according to their age (9 and 21 months old) and breed (Chios and Karagouniki), and the 96 ewes of Chios fat-tailed breed, were divided by age (9 and 21 months old). The main characteristics of courtship behaviour, like sniffing, nudging, flehmen response and following were recorded and studied in detail. Mature Chios rams, which were the only one with previous experience of Chios ewes, exhibited higher rates of sexual interest per ewe than the other rams (P < 0.05). On the other hand, rams sniffed and nudged more young than mature ewes (P < 0.05), probably due to the fact that young ewes did not express intense symptoms of estrus. Young rams exhibited substandard sexual interest towards mature ewes, when they first came in contact with them (P < 0.05). In general, Karagouniki thin-tailed rams exhibited reduced rates of mating behaviour when they courted with Chios fat-tailed ewes in comparison with Chios rams (P < 0.05). Moreover, as the time after ram introduction passed, the frequency and duration of sexual behaviour components decreased (P < 0.001). Finally, the effect of the day of the experiment was only significant in the case of sniffing, which increased during the first 2 days and then declined and stabilized (P < 0.01). As it was demonstrated, ram age and ram breed played a fundamental role in the exhibition of sexual interest elements.