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Absorption and deposition of xanthophylls in broilers challenged with three dosages of Eimeria acervulina oocysts a

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X. Hernández-Velasco , H.D. Chapman , C.M. Owens , V.A. Kuttappan , B. Fuentec

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Martínez , A. Menconi , J.D. Latorre , G. Kallapura , L.R. Bielke , T. Rathinam , B.M. b

Hargis & G. Tellez

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Departamento de Medicina y Zootecnia de Aves, Facultad de Medicina Veterinaria y Zootecnia (FMVZ), Universidad Nacional Autonóma de México (UNAM), D. F., 04510 México City, México b

Department of Poultry Science, University of Arkansas, Fayetteville, AR 7270, USA

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Centro de Enseñanza, Investigación y Extensión en Producción Avícola, FMVZ, UNAM, D.F., 13209 México City, México Accepted author version posted online: 10 Apr 2014.Published online: 24 Apr 2014.

To cite this article: X. Hernández-Velasco, H.D. Chapman, C.M. Owens, V.A. Kuttappan, B. Fuente-Martínez, A. Menconi, J.D. Latorre, G. Kallapura, L.R. Bielke, T. Rathinam, B.M. Hargis & G. Tellez (2014) Absorption and deposition of xanthophylls in broilers challenged with three dosages of Eimeria acervulina oocysts, British Poultry Science, 55:2, 167-173, DOI: 10.1080/00071668.2013.879095 To link to this article: http://dx.doi.org/10.1080/00071668.2013.879095

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British Poultry Science, 2014 Vol. 55, No. 2, 167–173, http://dx.doi.org/10.1080/00071668.2013.879095

Absorption and deposition of xanthophylls in broilers challenged with three dosages of Eimeria acervulina oocysts X. HERNÁNDEZ-VELASCO, H.D. CHAPMAN1, C.M. OWENS1, V.A. KUTTAPPAN1, B. FUENTE-MARTÍNEZ2, A. MENCONI1, J.D. LATORRE1, G. KALLAPURA1, L.R. BIELKE1, T. RATHINAM1, B.M. HARGIS1, AND G. TELLEZ1

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Departamento de Medicina y Zootecnia de Aves, Facultad de Medicina Veterinaria y Zootecnia (FMVZ), Universidad Nacional Autonóma de México (UNAM), D. F., 04510 México City, México, 1Department of Poultry Science, University of Arkansas, Fayetteville, AR 7270, USA, and 2Centro de Enseñanza, Investigación y Extensión en Producción Avícola, FMVZ, UNAM, D.F., 13209 México City, México

Abstract 1. An experiment was designed to evaluate the effect of different doses of oocysts of Eimeria acervulina on intestinal absorption and skin deposition of xanthophylls (XAs) in broilers. 2. A total of 192 broiler chickens were randomly assigned to 4 groups: an uninfected control group and three groups inoculated with either 1 × 102, 1 × 104 or 1 × 105 sporulated oocysts of E. acervulina by gavaging at 21 d. There were 4 replicate pens (2 male and 2 female) per group. 3. Plasma xanthophyll (PX) and skin yellowness (SY) were measured in live birds weekly. At 42 d of age, SY was measured in the breast and abdomen after chilling and in the breast 24 h post-processing on refrigerated carcasses. 4. In general, in all challenged treatments, and for the duration of the study, the average PX decreased by 0.02 μg/ml (R2 = 61.6%) for every 1000 inoculated oocysts, whereas PX increased by 1.26 μg/ml/d in uninfected birds. 5. The average SY in live birds from 21 to 42 d of age decreased by 0.019 b*/every 1000 oocysts administered, while SY of uninfected controls increased by 0.57 b*/d. It was also noted that in all treatments females had a greater SY (6.17 b*) than males for the duration of the study. The SY of the breast and abdomen was correlated (r = 0.76) in chilled carcasses. Breast SY in 24 h refrigerated carcasses was greater in the control group and for female birds. 6. Oocyst excretion was different between inoculated treatments only on 7 d post-inoculation (PI). Coccidia lesion scores in the duodenum averaged 1+ in infected birds and 2+ in birds given the highest oocyst dose.

INTRODUCTION Despite increasing quantities of further-processed chicken being produced commercially, a large portion of the poultry meat market continues to be whole carcasses, of which pigmented skin (skin yellowness (SY)) is an important component (Sirri et al., 2010). The consumer preference for yellow colouration in broiler skin in various countries, such as China, Peru, Italy and Mexico, depends upon cultural and historical factors, as well as the local

availability of crops capable of providing appropriate pigmentation (Heffner et al., 1964; Fletcher, 1999). Carotenoids are synthesised by microorganisms and plants, and accumulated in many biological tissues throughout the food chain. Dietary carotenoids, particularly the xanthophylls (XAs, also referred to as dihydroxycarotenoids) lutein and zeaxanthin, are the main compounds of interest in Marigold flower (Tagetes erecta) extract that causes a yellow colouration in chicken skin and fat (Hadden et al., 1999; Kotake-Nara and Nagao, 2011).

Correspondence to: G. Tellez, Department of Poultry Science, University of Arkansas, POSC O-114, Fayetteville, AR 72701, USA. E-mail: [email protected] Accepted for publication 5 November 2013.

This work was authored as part of the Contributor’s official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105, no copyright protection is available for such works under U.S. Law.

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Skin colour in poultry is mainly attributed to the ingestion of dietary XA and its deposition on the skin. Colouration has been associated with many factors such as the health status of the bird and freshness of the carcass, and is considered to impart an improved flavour to poultry meat (Breithaupt, 2007; Liu et al., 2008). Due to these issues, in several Latin American countries some companies differentiate their chicken meat brand by the degree of skin pigmentation. Commercial competition and shorter grow-out periods require the use of high concentrations of dietary pigment, which may represent 8–10% of the final cost of production (Castañeda et al., 2005; Muñoz-Díaz et al., 2012). Additionally, due to the antioxidant properties of XA, recent findings have attributed further advantages (reduction of UV-induced damage, risk for ocular diseases and atherosclerosis) to XA inclusion in animal feeds destined for human consumption (Baker and Günther, 2004; Kijlstra et al., 2012). There is a high likelihood, therefore, that pigment will continue to be included in diets for commercial broilers. The deposition of XA in poultry skin is affected by conditions which could impair the digestion and absorption of dietary XA, such as coccidiosis, an intestinal disease caused by protozoan parasites of the genus Eimeria. Further investigation of the effects of coccidia upon pigmentation in broilers is therefore desirable. Avian coccidiosis may cause body weight (BW) loss, a reduction in feed conversion ratio (FCR) and poor digestion due to physical damage and physiological changes induced by the invasive effect of the parasite upon the enterocyte (McDougald and Fitz-Coy, 2008; Assis et al., 2010). The severity of these effects varies depending upon the Eimeria species involved, magnitude of the infective dose, immune status of the bird and other factors. Plasma xanthophyll (PX) concentrations have proven to be sensitive indicators of the severity of Eimeria infections (Conway et al., 1993; Fetterer and Allen, 2000) compared to other criteria such as live weight, macroscopic lesion scoring and the number of oocysts shed in faeces (Conway et al., 1990). Among the Eimeria species that commonly affect broiler chickens, Eimeria tenella causes a moderate decrease in PX due to XA contained in the blood being lost in the caeca (Ruff and Fuller, 1975; Allen, 1989). However, E. acervulina and E. maxima parasitise the upper and mid-intestine, respectively, and can cause significant reductions in XA (Ruff and Fuller, 1975; Sharma and Fernando, 1975; Ogbuokiri and Edgar, 1986; Tyczkowski and Hamilton, 1991). These two species are responsible for malabsorption of nutrients caused by sloughing and shortening of the intestinal villi and affect the upper intestine (duodenum and jejunum), where most pigment absorption occurs (Littlefield et al., 1972; Tyczkowski and Hamilton, 1986; Allen, 1989).

Although skin pigmentation in some countries has a significant economic impact, and is well known to be affected by Eimeria spp., there are few published studies in which E. acervulina has been evaluated for its effect on XA absorption or SY. Moreover, it is not known how skin pigmentation is affected by the gender of birds challenged with this species. In this study, we investigate the effect of E. acervulina on the absorption and deposition of XA in chicken skin when Aztec Marigold pigment is added to the diet.

MATERIALS AND METHODS Experimental birds A total of 192 1-d-old broiler chickens (96 males and 96 females) were separated by sex, individually neck-tagged and randomly allocated to 16 floor pens with new pine shavings as litter in an environmentally controlled room at the University of Arkansas Poultry Health Laboratory. All animal handling procedures were in compliance with the Institutional Animal Care and Use Committee of the University of Arkansas. Feed The feed was a maize and soya based diet that met NRC requirements (NRC, 1994). Salinomycin (60 mg/kg; Alpharma Inc., Bridgewater, NJ, USA) was added to starter and finisher diets of uninfected control birds throughout the duration of the experiment. Additionally, these birds were medicated with amprolium (Huvepharma, Peachtree City, GA, USA) in the drinking water for two 3-d periods (29–31 d and 36–38 d) to ensure no cross-contamination with coccidia. The birds that were to be inoculated with E. acervulina received salinomycin in the starter feed from 1 to 14 d of age. All birds received 74 mg/kg of XA from Aztec Marigold flowers (T. erecta; Florafil HP Powder 40 g/kg which contain 90.7% of translutein, 5% of trans-zeaxanthin 0.4% of cis-lutein 0.4% of criptoxanthin and 0.4% of esters; Vepinsa S.A. de C.V. Los Mochis, Sinaloa, México) in the finisher feed (21–42 d of age). BW was recorded at 21 and 42 d. Feed intake was recorded weekly, and BW gain and FCR were calculated for the period of 21–42 d. Chicks had ad libitum access to feed and water throughout the trial. Parasite A strain of E. acervulina (US-AR-05-01) isolated from a broiler farm in northwest Arkansas and maintained at the parasitology laboratory of the Center of Excellence for Poultry Science, University of Arkansas, was used in the present

ABSORPTION AND DEPOSITION OF XANTHOPHYLLS

study. All birds from groups 2, 3 and 4 were individually challenged at 21 d of age with 1 ml of 1 × 102, 1 × 104 and 1 × 105 sporulated oocysts of E. acervulina, respectively, via oral gavage. Birds in group 1 (uninfected controls) received 1 ml of sterile distilled water.

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PXs and skin colour On a weekly basis, 2 ml of blood was collected from the wing veins of three birds per pen and mixed with 1 mg of EDTA/ml (Sigma Chemical Co., St. Louis, MO, USA) in sample tubes. The tubes were covered to protect them from light, immediately placed in ice and centrifuged at 466 × g/10 min to obtain plasma. XA concentration in plasma was determined by spectrophotometry (Spectronic 20D + , Thermo Spectronic, Rochester, NY, USA) as previously described (Allen 1987) and expressed as µg/ml. On d 21, 28, 35 and 42, SY (yellowness b* value) in 48, 48, 40 and 32 live birds, respectively per treatment (2 replicates of males and 2 of females), was measured on the right apterial latero-pectoral area using the CIE (1978) system colour profile and a CR300 Minolta colorimeter (CR-300, Konica Minolta, Ramsey, NJ, USA). All birds were processed when 42 d of age using an inline slaughter system as described by Kuttappan et al. (2013), placed on ice and refrigerated at 2°C. SY measurement was taken from the breast and abdominal areas of all carcasses immediately after slaughter and from the breast 24 h post-slaughter. Oocyst counts Fresh droppings were collected from 5 birds per pen at 14 and 21 d of age to confirm the absence of oocysts of Eimeria spp. prior to infection. Thereafter, quantification of oocysts in faecal samples from all pens was conducted weekly using the McMaster technique (Hodgson, 1970; Long et al., 1976).

1 × 104 and 1 × 105) and gender (male, female). For the main effects, if statistical significance was detected (P < 0.05), means were separated by Tukey’s multiple comparison procedure. SY and PX concentration were fitted to a multiple regression model where the independent variables were the time of pigment consumption and the size of the infective oocyst dose. The coefficients zero and one were used as dummy variables for males and females, respectively, to indicate the presence or absence of a categorical variable. A correlation matrix was built for SY in the breast and abdominal areas (Neter et al., 1996; Kuehl, 1999). Oocyst numbers in the faeces were analysed using the Kruskal–Wallis test. The treatment median difference was analysed with the Mann–Whitney U-test using the Statistical Program Social Science software (SPSS).

RESULTS No difference was detected in BW gain, feed consumption or FCR on d 42 at the end of the study (Table 1). However, in uninfected birds the PX level increased by 1.26 µg/ml for every day of pigment consumption. However, the average PX in all challenged treatments and for the duration of the study was reduced by 0.02 µg/ml (R2 = 61.6%; P < 0.01) for every 1000 oocysts inoculated (Figure 1). This response is expressed by the equation: Y = 1 .105 –0.02 (number of oocysts × 103) + 1.26 (d PI) – 0.04 (d PI) 2 + 5.15 (sex). There was a significant linear response in pigment deposition in the skin of male and female live birds for every day of pigment consumption Table 1. Cumulative feed consumption, FCR, and body weight gain from 21 to 42 d of age in chickens fed 74 mg/kg of XAs from Aztec Marigold flowers and infected with Eimeria acervulina

Lesion scoring Two birds per pen were killed on d 7 and d 14 postinoculation (PI) according to approved guidelines (American Veterinary Medical Association, 2013). Macroscopic intestinal lesions were recorded according to the method described by Johnson and Reid (1970). Statistical analysis Feed intake, BW gain, FCR, SY in live birds, chilled and 24-h refrigerated carcasses were measured and analysed by ANOVA utilising a 4 × 2 completely randomised factorial design. The factors included the inoculated oocyst dose (0, 1 × 102,

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Dose of oocysts None 1 × 102 1 × 104 1 × 105 Sex Males Females SEM ANOVA (P-value) Dose of oocysts Sex Dose of oocysts × sex

Cumulative feed consumption (g)

FCR

Weight gain (g)

3658a 3554a 3608a 3627a

1.871a 1.851a 1.926a 1.930a

1956a 1921a 1873a 1880a

3660a 3564b 58

1.886b 1.902a 0.043

1941a 1874b 33

0.40 (NS)