Effect of in ovo injection of raffinose on growth performance and gut health parameters of broiler chicken

Effect of in ovo injection of raffinose on growth performance and gut health parameters of broiler chicken J. D. Berrocoso, R. Kida, A. K. Singh, Y. S. Kim, and R. Jha1 Department of Human Nutrition, Food and Animal Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI 96822, USA

Key words: growth performance, in ovo injection, ileum mucosa morphology, prebiotic, immunology 2017 Poultry Science 00:1–8 http://dx.doi.org/10.3382/ps/pew430

INTRODUCTION

vorably alter the colonic microflora. Bednarczyk et al. (2011) reported a prebiotic effect of in ovo injection with raffinose family oligosaccharides (RFOs) on growth performance and concluded that in ovo injection of raffinose could replace antibiotic growth promoters as a non-antimicrobial enhancer additive. However, the available information on the effects of in ovo injection of prebiotic on gut health and immune response is scarce and needs to be elucidated. The beneficial effects of probiotics or prebiotics are considerably dependent on the interaction with the innate immune system and possible modulation of adaptive immunity (K¨ ohler et al., 2003). Cytokines also have important functions in the activation and regulation of other cells and tissues when any antigen invades in the body of broilers (Wigley and Kaiser, 2003). Although several beneficial effects of prebiotics are reported on the avian immune system, the effect of RFO inoculation on the avian immune system is still unclear. This study investigated how RFO inoculation alters the expression levels of marker genes of immune cells or cytokines. The hypothesis of this study was that in ovo injection of RFOs as a prebiotic effect in the air sac at 12 d of incubation of chicken eggs could support the development of ileum mucosa morphology and stimulate the immune

The injection of nutrients in ovo may provide the poultry industry with an alternative method to increase the weight of newly hatched chicks (Ohta et al., 1999). Studies have shown that the in ovo feeding of exogenous substance into fertile eggs such as inulin and lactobacillus (Pruszynska-Oszmalek et al., 2015), organic minerals (Oliveira et al., 2015), vitamins (Nowaczewski et al., 2012), carbohydrates (Uni et al., 2005), and amino acids (Bhanja et al., 2004), enhances growth performance, bone mineralization, and development of the digestive tract and increases body weight and nutritional status of the hatchling. The use of prebiotics is an approach that has been widely studied in chickens. Most of the researches conducted in vivo show that these substances reduce enteric diseases and also enhance their productivity. Oligosaccharides such as raffinose are considered to be prebiotic compounds because they are not hydrolyzed in the upper gastrointestinal tract and are able to fa C 2016 Poultry Science Association Inc. Received July 22, 2016. Accepted October 26, 2016. 1 Corresponding author: [email protected]

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d 21, the relative weight of the proventriculus, drumstick, breast, and gizzard was not affected (P > 0.05) by RFO. On hatch d, the villus height increased linearly (P < 0.01) with an increasing dose of RFO. Also, an increasing dose of RFO increased the villus height and villus height:crypt depth ratio (P < 0.05) but did not affect the crypt depth on d 21. The expression levels of CD3 and chB6, which are T cell and B cell marker genes, respectively, were significantly enhanced by high dose RFO (4.5 mg). In conclusion, although an increasing dose of RFO in ovo injection did not significantly influence growth performance or slaughter yield of broilers, RFO has the potential of enhancing ileum mucosa morphology and improving immunity in the small intestine, which are indicators of improved gut health.

ABSTRACT The effects of in ovo injection of raffinose (RFO) as a prebiotic on growth performance, relative weight of proventriculus, gizzard, drumstick and breast muscles, and ileum mucosa morphology were examined in Cobb 500 broilers. A total of 240 fertilized eggs were divided into 4 groups: a non-injected with intact shell and 3 levels of RFO solution (1.5, 3.0, and 4.5 mg in 0.2 mL of an aqueous diluents). The RFO solution was injected into the air sac on d 12 of incubation. In total 144 birds were fed a standard diet and management and sacrificed at d 21 post hatch for collection of samples. Total RNA was extracted from the small intestine, and RT-qPCR was performed to quantify mRNA levels of marker genes of immune cells. Injection of RFO had no significant effect (P > 0.05) on d one body weight of chicks. On

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system. The objective of this experiment was to evaluate the impact of in ovo injection with 3 levels of RFO as a prebiotic on growth performance, gut health, and immune system development of broiler chicks at 21 d post hatch.

MATERIALS AND METHODS All animal care procedures were approved by the Institutional Animal Care and Use Committee of University of Hawaii.

Fertile eggs from a breeder flock at 34 wk of age (Cobb 500) were obtained from a local hatchery (Asagi Hatchery Inc., Honolulu, HI). Upon arrival, the eggs were individually weighed, numbered, and incubated at 37.5◦ C and 60% relative humidity in an incubator (GQF incubator, Savannah, GA) in the Animal Nutrition Laboratory of University of Hawaii at Manoa (Honolulu, HI). Initially, the 240 eggs were equally and randomly distributed with 60 eggs assigned to each of 4 pre-specified treatment groups on each of 3 replicate tray levels (20 eggs per treatment in each tray level). Eggs were incubated under standard commercial conditions. At the 12th d of incubation, eggs were candled, and those unfertilized or with dead embryos were discarded. After hatching, 144 chicks were moved to the Small Animal Facility of the University of Hawaii at Manoa (Honolulu, HI) and randomly allocated to 4 treatment groups.

Item Ingredient, % Corn SBM Soya oil Limestone Di-cal phosphate Methionine Threonine NaCl Phytase Vitamin and mineral premix1 Calculated composition, % Dry matter Total ash Metabolizable energy (kcal/kg) Crude protein Crude fiber Lysine Methionine Threonine Methionine + Cysteine Ca Total P Na Cl Analyzed composition, % Dry matter Total ash Crude protein Gross energy (kcal/kg)

Inclusion level 64.22 29.93 0.50 1.09 0.65 0.22 0.04 0.34 0.01 1.00 88.12 4.23 2971.00 21.13 2.64 1.08 0.54 0.79 0.89 0.84 0.61 0.32 0.26 90.15 90.15 4.84 20.63 4002.12

1 Providing the following (per kg of diet): vitamin A (trans-retinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 3,000 IU; vitamin E (all-rac-tocopherolacetate), 30 mg; vitamin B1 , 2 mg; vitamin B2 , 8 mg; vitamin B6 , 4 mg; vitamin B12 (cyanocobalamin), 0.025 mg; vitamin K3 (bisulphatemenadione complex), 3mg; choline (choline chloride), 250 mg; nicotinic acid, 60 mg; pantothenic acid (D-calcium pantothenate), 15 mg; folic acid, 1.5 mg; beta´ıne anhydrous, 80 mg; Dbiotin, 0.15 mg; zinc (ZnO), 80 mg; manganese (MnO), 70 mg iron (FeCO3 ), 60 mg; copper (CuSO4 ·5H2 O), 8 mg; iodine (KI), 2 mg; selenium (Na2 SeO3 ), 0.2 mg.

In Ovo Injection In ovo injection of prebiotic solution was carried out on the 12th d of incubation into the air sac in a biosafety cabinet (37.0 ± 0.3◦ C and a relative humidity of ≥75%). Briefly, the eggs were disinfected with 70% ethanol and then a punch hole (perforating the shell) was done at the blunt end of the egg with an 18-gauge needle. Each group was injected with suitable in ovo RFO treatment using a pipette along with the pipette tip fitted with a 21-gauge needle that was inserted into the air sac, which was identified and marked after candling. The chicks were divided into 4 groups: a non-injected group (no injection) and 3 levels of RFO solution (1.5, 3.0, and 4.5 mg in 0.2 mL of a commercial diluent) injection. After in ovo injection, the hole was sealed with sterile paraffin, and eggs were returned to the incubator. All eggs remained outside the incubator for the same amount of time (approximately 15 min) during the injection period to avoid any variability.

Growth Performance and Relative Weight of the Organs At hatch, the number of live hatched chicks and unhatched chicks were counted to determine hatchability of fertile eggs. Unhatched eggs were opened to determine the cause of death. The day-old chicks (n = 144) were weighed individually, wing tagged, and placed randomly into one of 24 floor pens (6 birds per pen), making 6 replicates of each treatment. Birds in all the floor pens were raised under a standard commercial broiler rearing environment (temperature, humidity, and light). The temperature in the first wk was maintained at 35◦ C and gradually decreased to 28o C by the end of the third week. All birds were fed with a commercial corn-soybean meal-based pellet diet during the 21-day post-hatch trial period (Table 1). The diet met or exceeded the nutritional requirements of broiler chickens (standard guidelines of breeder) and birds had

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Experimental Design and Egg Incubation

Table 1. Ingredient composition and nutrient content of diet fed to the broilers in the study (as-fed basis; % unless otherwise indicated).

IN OVO INJECTION OF RAFFINOSE IN BROILER

unrestricted access to feed and water at all times. Body weight and feed consumption of the birds were measured by pen at 7, 14, and 21 d of age, and ADG, ADFI, and gain:feed ratio (G:F) were calculated from these data by period and cumulatively. Feed wastage was recorded daily and the feed consumption was adjusted for wastage and bird mortality. On d 21, 6 birds per treatment were randomly chosen for the determination of organ weights and were dissected after euthanizing with CO2 gas. The weight of breast muscle, drumsticks, gizzard, and proventriculus were recorded, and the relative weight (% of live body weight) was calculated.

At d 20 of incubation, 24 eggs (6 eggs per treatment) were opened and embryos were killed by cervical dislocation and separated from residual yolk. In addition, at hatch d (6 birds per treatment) and 21 daof age (6 birds per treatment) were randomly selected and euthanized by CO2 inhalation. A section of approximately 1 cm of small intestine (0.5 cm anterior and posterior to the Meckel’s diverticulum) was collected. The sample mucosal morphology was conducted as described by Xu et al. (2003). Segments 10 cm proximal to the ileocecal junction were taken for ileum histology study. A total of 6 intact, well-oriented crypt-villus units were selected in triplicate (18 measurements for each sample). An upright light microscope (Zeiss Axioskop II; Carl Zeiss Microscopy, New York City, NY) was used for histological analysis. Villus height was measured from the tip of the villi to the villus crypt junction, and crypt depth was determined as the depth of the invagination between adjacent villi. Measurement of villus height and crypt depth was performed using image processing and analysis system of the software, AxioVision, specialized for the microscope. The objective magnification was used at 20X, 10X, and 5X at incubation, hatch, and 21 d post hatch for ileum histological slides for better measurement.

RNA Extraction, Reverse Transcription and Real-Time Quantitative PCR A section of approximately 1 cm of small intestine (0.5 cm anterior and posterior to the Meckel’s diverticulum) of 6 birds per treatment was collected. The collected tissue was soaked in RNAlater solution immediately in order to prevent degradation of RNA and stored at −80◦ C until total RNA isolation. For RNA isolation, the tissue was removed from RNAlater solution to a micro-tube. Approximately 50 mg of the tissue was homogenized directly in TRIzol RNA isolation reagent (Invitrogen, Waltham, MA). Total RNA was extracted from collected tissues using 1 mL of TRIzol per sample according to the manufacturer’s instruction.

After RNA extraction, RNA concentration was measured with NanoPhotometer (IMPLEN, M¨ unchen, Germany). QuantiTect Reverse Transcription Kit (QIAGEN, Hilden, Germany) was used for reverse transcription, and 500 ng of total RNA was used as a template. First, RNA solution was treated with gDNA wipeout buffer to break remaining genome DNA, and was incubated for 2 min at 42◦ C, then placed immediately on ice. The genome DNA wiped-out RNA solution was reverse transcribed using Quantiscript Reverse Transcriptase. The solution was incubated for 15 min at 42◦ C, and incubated for 3 min at 95◦ C to inactivate Quantiscript Reverse Transcriptase, then the cDNA sample solution was stored at −20◦ C. Amplification and detection were conducted using Applied Biosystems 7300 Real-Time PCR System and R Fast SYBR Green Master Mix (QIAGEN, Hilden, Germany). The synthesized cDNA was used as the template for RT-qPCR. Thermal cycling parameters consisted of an initial one cycle of hot-start at 95◦ C for 20 s and 40 cycles of denaturation at 95◦ C for 15 s, and annealing and extension at 60◦ C for 30 seconds. The Ct value was determined, and the abundance of gene transcripts was analyzed using the ΔΔCt method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene (Kida et al., 2016). The CD56 gene marker for NK-cells, TLR4 gene marker for macrophage, IL-10 gene marker for antiinflammatory cytokines, and IL-1b gene marker for proinflammatory cytokines were amplified to see their relative abundance, which can explain the status of innate immunity and any ongoing inflammation. In order to access the influence of the treatment on adaptive immune response, the CD3 and chB6 gene markers of T-cell and B-cell were amplified using the respective primers. The oligonucleotide primers used are shown in Table 5.

Statistical Analysis Treatment sums of squares for the effects of RFO injection on initial weight of broiler chicks, growth performance, and jejunum mucosa morphology were partitioned into linear (L) and quadratic (Q) effects using the MIXED procedure of SAS (SAS V9.2, SAS Institute Inc., Cary, NC) when the fixed effects were significant. The level of significance was fixed at P < 0.05. The contrast coefficients for the polynomial contrast were acquired from the orthogonal polynomial coefficients table for 4 levels of a factor. Differences among gene expressions of applied treatments were examined by unpaired t-tests command using the TTEST Procedure of SAS (SAS V9.2, SAS Institute Inc., Cary, NC), and the significance was declared at P < 0.05. Data on gene expressions were log-transformed to provide an approximation of a normal distribution before analysis, and the results were expressed as the mean ± standard error (SE).

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Ileum Mucosa Histology

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and villus height:crypt depth ratio (P < 0.05), but did not affect the crypt depth at d 21 post hatch.

RESULTS Effects of RFO Injection on Growth Performance and Relative Organs Weight

Effect of RFO Injection on Adaptive Immune Cells The expression level of CD3 in the small intestine of broilers was significantly up-regulated by 4.5 mg RFO injection (Figure 2A). The RFO injection of 3.0 mg tended to increase the expression level of CD3 (P = 0.051). The expression level of chB6 was significantly enhanced in broilers treated with 3.0 mg and 4.5 mg RFO injection (Figure 2A).

Effects of RFO Injection on Ileum Mucosa Morphology of Chickens

Effect of RFO Injection on Innate Immune Cells and Cytokines

Injection of RFO affected the ileum mucosa morphology of the chickens differentially depending on stages of growth (Table 4). At 20 da of incubation and on d of hatch, linear (P < 0.001) and quadratic (P < 0.001) effects were observed for villus height and villus height:crypt ratio with an increasing dose of RFO. Crypt depth was not affected by injection of RFO at 20 d of incubation; however, at hatch, crypt depth tended to increase linearly with injection of RFO (P = 0.051). At d 21 of age, villus height increased linearly (P < 0.01) with an increasing dose of RFO. Also, an increasing dose of RFO increased the villus height

CD56 or Neural Cell Adhesion Molecule 1 (NCAM1) is expressed on the surface of neurons, glia, skeletal muscle, and natural killer (NK) cells. .CD14 and Toll-like receptor 4 (TLR4) are widely utilized as marker genes of macrophage. Interleukin-1β (IL-1β ) and IL-10 are known as a pro-inflammatory cytokine and an anti-inflammatory cytokine, respectively. No significant difference was observed among the expression levels of CD56 in RFO-injected broilers (Figure 2B). The expression level of CD14 was lower than the detection limit (data not shown). TLR4 was detected but no effect of RFO injection was seen

Table 2. Effects of RFO in ovo injection on growth performance of broilers.

Initial BW, g d 0 to 7 ADG1 , g ADFI2 , g G:F3 d 7 to 14 ADG, g ADFI, g G:F d 14 to 21 ADG, g ADFI, g G:F d 0 to 21 ADG, g ADFI, g G:F

Non-injected

1.5 mg RFO

3.0 mg RFO

4.5 mg RFO

SEM (n = 6)

P-value

45.4

45.4

45.2

46.2

0.12

0.896

15.1 13.3 1.138

15.4 14.2 1.079

16.6 15.1 1.104

16.8 15.8 1.061

0.6 1.3 0.08

0.635 0.487 0.684

36.3 59.1 0.615

36 60.6 0.594

37 60.6 0.609

36 59.3 0.607

0.81 2.67 0.02

0.478 0.287 0.642

56.9 110.3 0.516

53.9 115 0.469

55.3 115.4 0.479

57.3 113.5 0.505

0.89 0.5 0.02

0.698 0.458 0.112

36.1 60.9 0.593

35.1 63.3 0.554

36.3 63.7 0.57

36.7 62.9 0.584

0.52 2.2 0.05

0.125 0.984 0.654

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Average daily gain. Average daily feed intake. 3 Gain: feed ratio. 2

Table 3. Effects of RFO in ovo injection on relative weight (% of BW) of breast muscle, drumsticks, gizzard, and proventriculus.

Breast, % Drumsticks, % Gizzard, % Proventriculus, %

Non-injected

1.5 mg RFO

3.0 mg RFO

4.5 mg RFO

SEM (n = 6)

P-value

18.03 9.22 1.54 0.51

18.26 9.14 1.55 0.45

18.54 9.21 1.54 0.51

18.14 9.25 1.78 0.6

0.715 0.021 0.099 0.073

0.156 0.548 0.072 0.736

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Injection of RFO had no significant (P > 0.05) effect on d one body weight of chicks. From zero to 21 d of age, injection of RFO did not affect the resultant growth performance of the chicks (Table 2). The effects of injection of RFO on the relative weights of digestive organs are summarized in Table 3. On d 21, the relative weight of the proventriculus, drumstick, breast muscle, and gizzard were not affected (P > 0.05) by RFO.

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IN OVO INJECTION OF RAFFINOSE IN BROILER Table 4. Effects of RFO in ovo injection on ileum mucosa morphology of broiler chickens. Non-injected 20 d inc VH1 CD2 VH/CD3 Birth d VH CD VH/CD 21 d of age VH CD VH/CD

1.5 mg RFO

3.0 mg RFO

4.5 mg RFO

SEM (n = 6)

Linear effects

Quadratic effects

P-value

102 14 7.20

99 15 6.53

96 14 7.00

134 15 8.94

3.93 0.58 0.34

< 0.0001 0.7300 0.0003

< 0.0001 0.8364 < 0.0001

< 0.0001 0.2249 < 0.0001

124 22 5.65

110 22 4.92

141 36 3.95

164 40 4.12

8.93 2.3 0.31

0.0002 < 0.0001 < 0.0001

0.0446 0.4468 0.0563

0.0003 < 0.0001 < 0.0001

914 234 3.91

998 193 5.16

1096 205 5.36

1164 230 5.06

32.65 17.23 0.33

< 0.0001 0.9874 0.0001

0.8051 0.0586 0.2168

< 0.0001 0.2822 0.0004

Significant differences were considered at P < 0.05. 1 Villus height (μ m). 2 Crypt depth (μ m). 3 Villus height to crypt depth ratio.

Gene

Primer

Immune cell: related genes: CD3 Forward Reverse CD45 Forward Reverse CD56 (NCAM1) Forward Reverse chB6 (Bu-1) Forward Reverse TLR4 Forward Reverse Cytokine: IL-1β Forward Reverse IL-10 Forward Reverse Reference: GAPDH Forward Reverse

Genbank Accession number

Sequence

Amplicon Size (bp)

5 -GGACGCTCCCACCATATCAG-3 5 -TGTCCATCATTCCGCTCACC-3 5 -TATTCTTGGTGTTCTTGATTGTTGTG-3 5 -CTGCTACAAGGCTGATGACTTCA-3 5 -GTTCATGAGCAGAGGGTGCT-3 5 -ACATGGCCTGGATGATGCAA-3 5 -TACTTTGTCGGCCGAGTGTC-3 5 -AGTCTGCAGTTCCATTGGGG-3 5 -AGTCTGAAATTGCTGAGCTCAAAT-3 5 -GCGACGTTAAGCCATGGAAG-3

NM 205512

180

NM 204417

120

NM 001242604

196

NM 205182

197

NM 001030693

190

5 -CGCTTCATCTTCTACCGCCT-3 5 -GATGTTGACCTGGTCGGGTT-3 5 -TGTCACCGCTTCTTCACCTG-3 5 -CTCCCCCATGGCTTTGTAGA-3

NM 204524

144

NM 001004414

105

5 -AGCTTACTGGAATGGCTTTCCG-3 5 -ATCAGCAGCAGCCTTCACTACC-3

NM 204305

122

(Figure 2B). The treatment with RFO injection did not affect either IL-1β or IL-10 mRNA level in any dose of RFO (Figure 2B).

DISCUSSION Effects of RFO Injection on Growth Performance and Relative Organs Weight In ovo administration of RFO had no effect on the relative weight of the proventriculus, breast muscle, or drumsticks. Similarly, Pilarski et al. (2005) reported that in ovo injection of oligosaccharides at a dose of 1.763 mg/egg did not have a significant effect on weights of the carcass or breast muscle of broiler chickens at 42 d of age. Interestingly, the injection of 4.5 mg of RFO increased the relative weight of the gizzard. In poultry, the main function of the gizzard is to grind and digest larger feed particles, implying that the increase in gizzard weight can enhance normal storage and physical digestion of solid feed. This can potentially lead to

the improvement of performance in the broiler chickens. However, in the current study, the growth performance post hatch was not affected by RFO injection.

Effects of RFO in ovo Injection on Ileum Mucosa Morphology of Chickens In chickens, the first d after hatch is a critical period for development of the mucosa because a major change occurs in the source of nutrients as the yolk is replaced by an exogenous diet (Noy and Sklan, 1998). Intestine, as the main interface between an organism and its nutritional environment, plays a vital role in the development and growth of a newborn animal (Noy and Sklan, 1998). The growth of the chickens is dependent on the digestion and absorption of nutrients, which is a direct result of the morphological and functional development of the small intestine. Villus height, crypt depth, and villus length/crypt depth ratio are good indicators for functional capacity of the intestine (Fasina and Olowo, 2013). It has been proposed that

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Table 5. Nucleotide sequences of primers used in real-time qPCR analyses.

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Effect of RFO Injection on Adaptive Immune cells In this study, the injection of 4.5 mg of RFO upregulated the gene expression of CD3 of the small intestine and enhanced that of chB6 (Figure 2A). CD3 is a membrane protein expressed in T cells at all stages of development. It is commonly used as a T cell marker (Bernot and Auffray, 1991). The chB6, also known as Bu-1, was originally recognized by using alloantisera a and b between inbred lines of chickens. The marker is expressed on early and mature B cells, except plasma cells (Igy´art´ o et al., 2008). Gut associated lymphoid tissue (GALT) plays a significant role in the avian immune system, as this part is exposed to a variety of non-self external materials including pathogenic microbes (Gallego et al., 1995). Therefore, the development of GALT has the potential to enhance avian gut health. Various types of lymphocytes localize in GALT, and they contribute to the maintenance of intestinal health. This result suggests that the number of T cells and B cells was increased by adequate concentration of RFO, which can lead to development of immunity in the small intestine. Madej and Bednarczyk (2016) studied the effect of in ovo-delivered prebiotics and synbiotics on the composition of T cells and B cells in gutassociated lymphoid tissue, and they utilized inulin or Bi2 tos as a prebiotic and Lactococcus lactis subsp. lactis IBB SL1 or Lactococcus lactis subsp. cremoris IBB SC1 as a probiotic. Although the number of CD3 expressed

Figure 1. Effects of RFO in ovo injection on ileum mucosa morphology of chickens at 21 d of hatch. Each character on the figure means the following treatments: A: Non-injected, B: 1.5 mg RFO, C: 3.0 mg RFO, D: 4.5 mg RFO. Typical images of ileum mucosa morphology are shown. Images were captured with light microscopy at 50 × magnification. Scale bar indicates 500 μ m.

cells was increased by some symbiotic, there was no significant effect on population of CD3 or chB6 expressing cells in only prebiotics-treated birds. Considering our result that 4.5 mg RFO supplementation enhanced the gene expression of B cells and T cells and increased the length of the villus (Table 4 and Figure 1), RFO is likely more appropriate as a prebiotic for in ovo injection.

Effect of RFO Injection on Innate Immune cells and Cytokines It has been reported that prebiotics improve the innate immune system in many species (Schley and Field, 2002; Hardy et al., 2013). Ibuki et al. (2011) reported in their in vitro study that macrophages of 1,4 mannobiose-treated chicken showed increased Salmonella-killing activity. However, much less work has been done to investigate effects of in ovo injection of prebiotics or probiotics on the innate immune system. In the innate immune system, a variety of leukocytes work for maintenance of avian health, including phagocytes such as macrophage, neutrophil, and dendritic and NK cells. Macrophage is the most efficient phagocyte, which engulfs and decomposes pathogens and then produces cytokines that activate other immune cells. NK cells destroy pathogen-infected cells (Juul-Madsen et al., 2006). Cytokines play important roles in the avian immune system; proinflammatory cytokines, such as IL-1β , increase immune cell migration to the infected site and activate them; antiinflamattory cytokines, on the other hand, suppress immune cell activity (Shanmugasundaram and Selvaraj, 2012). The up-regulation of IL-1β can cause inflammatory symptoms and harm avian gut health. On the other hand, the enhancement of IL-10 has the possibility to alleviate inflammation and contribute to improvement of gut

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a deeper crypt is indicative of a faster tissue turnover and, perhaps, a higher demand for new tissue. Furthermore, it has been reported that a high intestinal villus is associated with a well-differentiated intestinal mucosa with high digestive and absorptive capabilities (Jeurissen et al., 2002). For example, Tako et al. (2004) evaluated the effects of in ovo feeding of carbohydrates and β -hydroxy-β -methylbutyrate on the development of the chicken intestine, and the authors observed that the administration of exogenous nutrients into the amnion increased the size of the villi and, thus, led to an increase in the intestinal capacity to digest disaccharides. Since the late-term embryo naturally consumes the amniotic fluids, it is possible that insertion of a nutrient solution into the embryonic amniotic fluid may enhance development of the digestive system. Injecting of RFO into the air sac of chicken embryos seems to be able to increase intestinal villus length and width, potentially increasing digestive and absorptive capacity. However, in the current study, the improvement in the development of the ileum mucosa, as was measured by villus length and villus height/crypt ratio, had no effect in improving growth performance from zero to 21 d post hatch, indicating that the measure of intestinal morphology such as villus length and villus height/crypt ratio does not necessarily translate into improved growth performance.

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health (Chichlowski et al., 2007). An immune response is a result of the balance among these active and inhibitory cytokines. The CD56 (NCAM1) and TLR4 are marker genes as NK cell and macrophage, respectively. RFO injection had no effect on the mRNA level of CD56 or TLR4 (Figure 2B). Moreover, the expression level of neither IL-1β nor IL-10 was affected by RFO injection. The birds in this study were kept in a sanitary environment, so it is likely that they had not had a negative effect of infection of pathogens and the innate immune system did not need to be improved. In conclusion, although an increasing dose of RFO in ovo injection did not significantly influence growth performance or slaughter yield of broilers, it enhanced ileum mucosa morphology and improved immune response indicators in the small intestine, which are indicators of improved gut health. Thus, raffinose injection in ovo can be a potential early nutrition programming strategy to improve gut health of broiler chickens.

ACKNOWLEDGEMENTS This work was supported by the USDA, Agricultural Research Service, Agreement no. 58-5320-3-022, entitled, “Regionally Grown Feedstock,” managed by the College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI. Additional support for the microscopy core at the University of Hawaii was provided by a Research Center for Minority Institutions grant from the National Institute on Minority Health and Health Disparities (G12MD007601).

REFERENCES Bednarczyk, M., M. Urbanowski, P. Gulewicz, K. Kasperczyk, G. Maiorano, and T. Szwaczkowski. 2011. Field and in vitro study on prebiotic effect of raffinose family oligosaccharides in chickens. Bull Vet. Inst. Pulawy. 55:465–469. Bernot, A., and C. Auffray. 1991. Primary structure and ontogeny of an avian CD3 transcript. Proc. Natl. Acad. Sci. 88:2550–2554.

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Figure 2. Effects of RFO injection on immunity of small intestine of broilers. Total RNA was extracted from the small intestine of broilers at 21 d of age. The expression of each gene was examined using RT-qPCR and expressed as ratio to GAPDH, with the level being set to 1 in broilers treated without RFO in each gene. A: Effect of RFO injection on marker genes of adaptive immune cells. B: Effect of RFO injection on innate immune cells and cytokines. Data shown are the mean ± SE (n = 6). # indicates P = 0.051 and ∗ indicates P < 0.05. Statistical analysis was conducted in comparison to non-injected. RFO = raffinose (mg/0.2 mL).

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BERROCOSO ET AL. Madej, J. P., and M. Bednarczyk. 2016. Effect of in ovo-delivered prebiotics and synbiotics on the morphology and specific immune cell composition in the gut-associated lymphoid tissue. Poult. Sci. 95:19–29. Noy, Y., and D. Sklan. 1998. Yolk utilisation in the newly hatched poult. Br. Poult. Sci. 39:446–451. Nowaczewski, S., H. Kontecka, and S. Krystianiak. 2012. Effect of in ovo injection of vitamin C during incubation on hatchability of chickens and ducks. Folia Biol. (Prague). 60:93–97. Ohta, Y., N. Tsushima, K. Koide, M. T. Kidd, and T. Ishibashi. 1999. Effect of amino acid injection in broiler breeder eggs on embryonic growth and hatchability of chicks. Poult. Sci. 78:1493– 1498. Oliveira, T. F. B., A. G. Bertechini, R. M. Bricka, E. J. Kim, P. D. Gerard, and E. D. Peebles. 2015. Effects of in ovo injection of organic zinc, manganese, and copper on the hatchability and bone parameters of broiler hatchlings. Poult. Sci. 94:2488– 2494. Pruszynska-Oszmalek, E., P. A. Kolodziejski, K. Stadnicka, M. Sassek, D. Chalupka, B. Kuston, L. Nogowski, P. Mackowiak, G. Maiorano, J. Jankowski, and M. Bednarczyk. 2015. In ovo injection of prebiotics and synbiotics affects the digestive potency of the pancreas in growing chickens. Poult. Sci. 94:1909–1916. Pilarski, R., M. Bednarczyk, M. Lisowski, A. Rutkowski, Z. Bernacki, M. Wardenska, and K. Gulewicz. 2005. Assessment of the effect of galactosides injected during embryogenesis on selected chicken traits. Folia Biol. 53:13–20. Shanmugasundaram, R., and R. Selvaraj. 2012. Effect of killed whole yeast cell prebiotic supplementation on broiler performance and intestinal immune cell parameters. Poult. Sci. 91:107–111. Schley, P. D., and C. J. Field. 2002. The immune-enhancing effects of dietary fibres and prebiotics. Br. J. Nutr. 87:S221–S230. Tako, E., P. R. Ferket, and Z. Uni. 2004. Effects of in ovo feeding of carbohydrates and β -Hydroxy-β - Methylbutyrate on the development of chicken intestine. Poult. Sci. 83:2023–2028. Uni, Z., P. Ferket, E. Tako, and O. Kedar. 2005. In ovo feeding improves energy status of late-term chicken embryos. Poult. Sci. 84:764–770. Xu, Z. R., C. H. Hu, M. S. Xia, X. A. Zhan, and M. Q. Wang. 2003. Effects of dietary fructooligosaccharide on digestive enzyme activities, intestinal microflora and morphology of male broilers. Poult. Sci. 82:1030–1036. Wigley, P, and P. Kaiser. 2003. Avian cytokines in health and disease. Rev. Bras. Cienc. Avic. 5:1–14.

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Bhanja, S. K.1, A. B. Mandal, and T. K. Goswami. 2004. Effect of in ovo injection of amino acids on growth, immune response, development of digestive organs and carcass yields of broiler. Indian J. Poult. Sci. 39:212–218. Chichlowski, M., J. Croom, B. W. McBride, L. Daniel, G Davis, and M. D. Koci 2007. Direct-fed microbial PrimaLac and salinomycin modulate whole-body and intestinal oxygen consumption and intestinal mucosal cytokine production in the broiler chick. Poult. Sci. 86:1100–1108. Fasina, Y. O., and Y. L. Olowo. 2013. Effect of commercial yeastbased product (Maxigen) on intestinal villi morphology and growth performance of broiler chickens. International J. Poult. Sci. 12:09–14. Gallego, M., E. D. Cacho, and J. A. Bascuas. 1995. Antigenbinding cells in the Cecal tonsils and Peyer’s patches of the chicken after bovine serum albumin administration. Poult. Sci. 74: 472–479. Hardy, H., J. Harris, E. Lyon, J Beal, and A. D. Foey. 2013. Probiotics, prebiotics and immunomodulation of gut mucosal defenses: Homeostasis and immunopathology. Nutrients. 5:1869–1912. Ibuki, T., K. Imada, T. Minamino, T. Kato, T. Miyata, and K. Namba. 2011. Common architecture of the flagellar type III protein export apparatus and F- and V-type ATPases. Nat. Struct. Mol. Biol. 18:277–282. Igy´art´ o, B. Z., N. Nagy, A. Magyar, and I Ol´ ah. 2008. Identification of the avian B-cell-specific Bu-1 alloantigen by a novel monoclonal antibody. Poult. Sci. 87:351–355. Jeurissen, S. H. M., F. Lewis, J. D. Van der Klis, Z. Mroz, J. M. J. Rebel, and A. A. ter Huurne. 2002. Parameters and techniques to determine intestinal health of poultry as constituted by immunity, integrity and functionality. Curr. Issues Intest. Microbiol. 3:1–14. Juul-Madsen, H. R., T. S. Dalgaard, C. M. Rontved, K. H. Jensen, and N. Bumstead. 2006. Immune response to a killed infectious bursal disease virus vaccine in inbred chicken lines with different major histocompatibility complex haplotypes. Poult. Sci. 85:986– 998. Juul-Madsen, H., B. Viertlboeck, L. Smith, and T. G¨ obel. 2008. Avian innate immune responses. Avian Immunol. 4:129–158. Kida, R., H. Yoshida, M. Murakami, M. Shirai, O. Hashimoto, T. Kawada, T. Matsui, and M. Funaba. 2016. Direct action of capsaicin in brown adipogenesis and activation of brown adipocytes. Cell Biochem. Funct. 34:34–41. K¨ ohler, H., B. A. McCormick, and W. A. Walker. 2003. Bacterialenterocyte crosstalk: Cellular mechanisms in health and disease. J. Pediatr. Gastroenterol. Nutr. 36:175–185.