The Impact of Organic and Inorganic Selenium on the Immune System of Growing Broilers Submitted to Immune Stimulation and Heat Stress

One to 42-d-old (432) female broilers were fed different levels of inorganic selenium (ISe) and organic selenium (OSe), according to the following treatments: (1) 0.3 mg ISe; (2) 0.3 mg ISe + 0.2 mg OSe; (3) 0.5 mg ISe and (4) 0.3 mg OSe/ kg of feed. All birds were vaccinated against infectious bursal disease (IBD) at 19d of age and three birds/replicate (R) were inoculated with sheep red blood cells (SRBC) at 32d. Three other birds/R received Freund’s adjuvant at 37d and avian tuberculin (AT) in the wattle at 47d of age. All birds were submitted to heat stress after 21d. Performance parameters, bursa and spleen weights, lymphocyte bursa depletion, antibody (Ab) production against IBD and SRBC, hematocrit, leukocytes, heterophil/lymphocyte ratio (H/L), and cellular reaction to AT were evaluated. The contrast analysis showed that OSe has improved feed intake (FI) between day 1 and 42 (p<0.10). Birds fed ISe presented worse H/L ratio (p<0.10), but higher Ab titers against IBD (p<0.04) and SRBC (p<0.05) than birds fed OSe, but OSe supplemented birds showed lower lymphocyte depletion scores in the bursa. The higher FI promoted by OSe may be beneficial when rearing broilers in hot weather. The use of ISe induced higher humoral immune response.


INTRODUCTION
Nutrient requirements for performance are well established in broilers, but not for efficient immune response.The additive effect of selenium (Se) and vitamin E deficiencies has been traditionally associated with muscular dystrophy and exudative diathesis in poultry due to their antioxidant action and capacity to protect the cell membrane (Finch & Turner, 1996;Edens, 2001;Payne & Southern, 2005).Selenium is involved in antibody production, and stimulates phagocytosis and chemotaxis of macrophages and neutrophils, depending on the pathogen and on the levels of vitamin E in the diet.It is an essential component of enzyme glutathione peroxidase (GPx), and plays a major role against diseases (Kidd, 2004).It was shown that, in E. tenella infections, Se was related to lower broiler mortality and less cecal lesions due to its capacity of protecting leukocytes against normal phagocytosis at the site of infection (Colnago et al.,1984).Selenium also acts in the metabolism of Cys and Met, and in the synthesis of thyroid hormones (Dahlke et al., 2005).In humans and animals, Se deficiency has been associated with liver necrosis, poor feathering, and cancer (Edens, 2001).Selenium is found in forages and grains, and its availability varies according to location and climate (Finch & Turner, 1996).Corn-soybean meal diets are usually supplemented with Se, and feeding recommendations for broilers range from 0.15 (NRC, 1994) to 0.33 mg/kg (Rostagno, 2005).Inorganic sources of selenium (ISe), particularly sodium selenite (Na 2 SeO 3 ) were added to broilers feeds until recently, when organic Se sources became commercially available.When da Silva ICM 1b Ribeiro AML 1ab  Canal CW 2ab Trevizan L 1a Macagnan M 2b Gonçalves TA 1c Hlavac NRC 3c de Almeida LL 2 Pereira RA 4 selenium is chelated to amino acids (Se-AA), such as methionine-associated Se (Se-Met), it is better absorbed and more available (Surai, 2000;Edens & Gowdy 2004) than ISe, and is considered an "organic" source (OSe).Although ISe can be used for the biosynthesis of selenoproteins, it is incorporated into body proteins only in the form of Se-AA because Met and Se-Met become analogs when sulfur is replaced by Se in the molecule, and therefore, are not differentiated by the genetic code that regulates this incorporation.Se-Met can also be used to synthesize body proteins (Daniels, 1996;Shcrauzer, 2000).Hydrogen selenide (H 2 Se), a byproduct of Na 2 SeO 3 (ISe) metabolism, is produced before Se is incorporated into body proteins, producing oxygen-reactive compounds in a fast recycling and diffusion process between the plasma and red blood cells.Se-AA sources are also converted into H 2 Se, but their use in the synthesis of proteins other than body proteins reduces the damage from pro-oxidative effects (Leng et al., 2003).
The present experiment studied the effects of dietary Se level and source on the immune status of broilers challenged by heat stress (HS) and a vaccine against Infectious Bursal Disease by evaluating humoral and cell-mediated immune responses.

Birds and treatments
In this experiment, 432 one-d-old female Ross 308 chicks were used.Birds were housed in 36 metal cages, in a climate-controlled room, and received 24 hours/day of light throughout the experiment.Bird performance was measured from 1 to 42 d.The experiment was divided in a starter (1 to 21 d) and a grower phase (22 to 42 d).However, in order to evaluate wattle-cell reaction (reaction to avian tuberculin), some birds were maintained until day 48.
The experimental diets (Table 1) were based on a basal diet, which was formulated using the Brazilian Tables for Poultry and Swine (Rostagno, 2005) and to which Se levels and sources (inorganic or organic) were added to make up four treatments: 0.3 mg/kg ISe, 0.3 mg/kg ISe + 0.2 mg/kg OSe, 0.5 mg/kg ISe, or 0.3 mg/ kg OSe.The inorganic selenium source contained 45% sodium selenite, and the organic selenium source was Sel-Plex ® , a yeast-based commercial product containing at least 50% Se-Met (Edens, 2001).Both sodium selenite and Sel-Plex ® were diluted in rice hulls, mixed into 3kg of the basal feed, and then added to the final feed batches.Vitamin E levels in the basal diet were 40 mg/kg (starter phase) and 25 mg/kg (grower phase).The commercial mineral premix used did not contain Se.Each of the four treatments had nine replicates (cages) of 12 birds each.Birds were fed the experimental diets and water ad libitum from 1 day of age.During the grower phase, birds were submitted daily to cyclic heat stress (CHS), consisting of 12 hours at 24ºC, three hours of a gradual increase from 24 up to 30-31ºC, six hours between 30 and 31ºC, and three hours of gradual decrease, from 30-31 down to 24ºC.Relative air humidity was kept at 74.1% ± 10.3%.All methods used in this experiment followed the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999).

Immune stimulation
All birds were submitted to the following regime of immune stimulation: 1) vaccines administered at the hatchery, on day 1, against Marek's Disease, Fowl Pox and Infectious Bronchitis; 2) vaccination against Infectious Bursal Disease (IBD) at 19d of age; 3) i.v.inoculation of 10% SRBC at 32d of age; and 4) i.m. inoculation of complete Freund's adjuvant at 37d of age and avian tuberculin at 47d of age.
The vaccine against IBD (Winterfield strain, CEVAC IBD-L ® , a live, freeze-dried vaccine) was added to the drinking water supplied to all birds, according to the manufacturer's instructions.Complete Freund's adjuvant (containing 4 mg/mL of killed Mycobacterium avium) was administered i.m at 0.5mL in three birds/ replicate in order to sensitize them to a subsequent intradermal administration of 0.01mL of avian tuberculin in the wattle to measure cellular immunity.

Measurements
Birds were weekly weighed and their feed intake was measured to calculate feed conversion ratio (FCR), considering cage as replicate.At 19, 29, 35, and 42d, blood samples were collected from three birds per replicate for IBD antibody determination (not the same birds were collected at all ages).On day 42, blood samples from other three birds per replicate were collected for SRBC and hematological profile analysis, measuring hematocrit, total leukocytes and their subtypes: lymphocytes, heterophils, monocytes, eosinophils, basophils, and heterophil:lymphocyte (H/L) ratio.On the same day, three birds per replicate were euthanized by cervical dislocation, weighed, and had their spleens and bursas collected and weighed.The bursas were also measured using a bursometer, and then identified and fixed in 10% formalin.The remaining birds in each replicated were reared until day 47, when three birds/replicate that had been previously inoculated with adjuvant (but not SRBC -these birds were not sampled for other measurements) were inoculated with avian tuberculin in one wattle and euthanized by electrocution 24 hours later.Inoculated and non-inoculated wattles from each bird were removed and weighed.
The immunological parameters measured were humoral immunity (ELISA for IBD and hemagglutination-HA for SRBC).The optical densities found in ELISA tests for IBD were converted into antibody titers by using a formula recommended by the kit manufacturer (IDEXX Corporation ® ).SRBC titers were determined according to a protocol adapted from Bartlett & Smith (2003).Cellular immunity was measured by avian tuberculin reaction, using the weight difference between inoculated vs. non-inoculated wattles in the same bird.
Lymphocyte depletion was detected in collected bursas stored in 10% buffered formalin, cut and then dehydrated, clarified, and embedded in paraffin.The tissue blocks were cut into 0.5 µm-thick sections and stained with hematoxylin-eosin.Lesions were examined under optical microscopy and classified according to Muskett's score, in a scale from one to five (Muskett, 1979).

Statistical analysis
A completely randomized experimental design was applied with four treatments of nine replicates per treatment, with 12 birds per replicate.Experimental data were submitted to analysis of variance (ANOVA) after their normality was verified, using SAS (2001).When significant F values (p<0.10) were obtained, means were compared by the LS-means test.SRBC antibody titers were submitted to square-root transformation.Orthogonal contrasts were also applied to compare the treatments containing inorganic vs. organic selenium (0.3 and 0.5 mg/kg ISe vs. 0.3 mg/kg OSe) as to their effects on performance, antibody titers, quantitative and qualitative blood analyses (hematological profile), absolute and relative spleen and bursa weights, bursal lymphocyte depletion, and weight difference between inoculated and non-inoculated wattles.Except for performance (nine replicates/treatment), statistical analyses were conducted on the results obtained from 27 birds/treatment (three birds per replicate).The Chi-Square Test (χ 2 ) was also used to analyze the results for bursal lymphocyte depletion.

RESULTS AND DISCUSSION
No significant differences in mortality were found among treatments throughout the experiment.The performance results are shown in Table 2. ANOVA did not detect any significant effect of selenium levels and sources on performance, but the contrast analysis of mean values revealed that the use of OSe improved feed intake (FI) between days 1 and 21 (p<0.09)and 21 and 42 (p<0.10).However, the highest observed FI did not affect body weight gain (BWG) or feed conversion ratio (FCR) (p>0.10).The diets were formulated using a single basal diet, thereby reducing any potential errors in feed mixing, which supports any possible differences detected.
These results partially disagree with those observed by Ribeiro et al. (2008), who observed that birds submitted to CHS and supplemented with 0.3 ppm OSe + 0.3 ppm ISe, in addition to vitamin E and C and organic Zn, presented lower FI and better FCR.In the study of Moreira et al. (2001), zero to 1.35 mg/kg OSe and ISe were tested and birds fed 1.05 mg/kg from 1 to 21 d presented higher FI, with no effect of Se source.Those authors mentioned that that response was unexpected, and was not observed in the period from 22 to 42 d.On the other hand, Dahlke et al. (2005) did not find any influence of Se levels or sources on performance responses, including FI, of broilers submitted to different temperatures.Yoon et al. (2007), comparing ISe levels from zero to 0.3 mg/kg and two sources of OSe, did not find any effect of Se levels or sources on performance.However, Se retention in the body was higher with OSe and also more efficient as compared to ISe as the birds aged and as the levels of Se in the diet were reduced.Those authors suggested that differences in the bioavailability of OSe sources based on the results of Se in the blood and the activity of GPx, may explain that result.Similarly, Payne & Southern (2005) also did not find any differences in performance, carcass traits, or GPx activity in broilers fed 0 or 0.3 mg/kg of Se, regardless of the source; however, Se retention in the muscle was higher when OSe was fed.Edens (2001) showed that the combined use of both Se sources did not improve body weight as compared to OSe alone, whereas FCR enhanced with OSe+ISe as compared to ISe alone.
The birds in the present study were submitted to CHS, and feed intake may be limited under hot conditions, according previous reports (Teeter et al., 1985;Dahlke et al., 2005;Ribeiro et al., 2005).In that sense, the increase in FI is a positive response, although weight gain and feed conversion were not enhanced.
No effect of the combined use of OSe and ISe (0.3 mg/kg ISe + 0.2 mg/kg OSe treatment) on performance was observed, which supports previous studies (Edens, 2001).Moreover, the level of 0.5 mg/kg Se (ISe) did not cause positive effects either, and this may be due to the fact that the birds do not require more than 0.3 mg/kg Se and any excess intake is promptly methylated and eliminated via urine and feces (Edens, 2001) and lungs (Rutz et al., 2005).
There were no significant effects of Se levels or sources on absolute or relative spleen and bursa weights (Table 3), which is consistent with the findings of Laganá et al. (2005) and Ribeiro et al. (2008), in which the supplementation of ISe or OSe, as well as of Zn, with and without vitamins C and E, had no impact on these parameters.According to Guimarães et al. (2003), broilers exposed to heat showed, at three weeks of age,  In the present study, bursa weights were much lower than those reported before by Laganá et al. (2005) and Ribeiro et al. (2008), despite the fact that bursas were weighed in the present experiment one week later than the mentioned studies.This effect may be partially explained by the use of female birds, and was certainly caused by vaccination against IBD.A similar finding was observed by Rubin et al. (2007).who vaccinated birds against IBD on day 14.
Bursal diameter was not affected by treatments (Table 3).According to FORT DODGE's Technical Manual (2008), the lesion score of the birds assessed at 35-40 days of age, if vaccinated on d 21, varies according to the invasion power of the viral strain.Highly pathogenic viruses cause massive destruction of the bursal corticalmedullary layer, which is followed by its replacement by fibrous tissue and atrophy, leading to a considerable decrease in diameter.Under these conditions, bursal diameters should range from 9.5 to 15.9 mm.The average diameter of the bursas found in this experiment was 12 mm, therefore showing efficient vaccination.
Table 4 presents bursal lymphocyte depletion results, which were significantly affected by the treatments (p<0.06).The contrast of mean values (not shown) reveals the lowest depletion score (p<0.01) in the bursas of birds fed with 0.3 mg/kg of OSe as compared to birds fed ISe.The results of Moraes et al. (2004) showed an association between "strong" vaccine strains and higher lymphocyte depletion: the more pathogenic the vaccine strain, the smaller the bursa.Our study suggests that the use of OSe in the diet may be a good alternative to attenuate this condition, possibly through a protective effect on that organ.Leng et al. (2003) reported that the use of a GPx enzyme complex improves the antioxidant and protective action of adequate Se levels on cells.Other researchers (Schrauzer, 2000;Edens, 2001;Yoon et al., 2007) found the same effect when OSe was used, therefore explaining why the cell damage caused by the vaccine in the bursal tissue was minimized in this experiment.Finally, reported findings  of studies in nutrigenomics (Dawson, 2006) showed the positive effect of OSe and ISe supplementation on gene transcription in intestinal and reproductive cells of rats, probably due to its capacity to enhance the activity of antioxidant mechanisms in these tissues.p<0.06 1 -Scores: 2, 31 to 50% depletion; 3, 51 to 69%; 4, 70 to 80% and 5, > 90% (Muskett, 1979).
No significant differences were found in inoculated and non-inoculated wattles weights among treatments (data not shown).The use of female birds, a single AT inoculation, and the wide variability in the results (CV= 72%), contrarily to Rubin et al. (2007), may explain the lack of response.
Table 5 shows the results of the hematological profile analysis.Although the ANOVA showed no difference among Se levels or sources, in the contrast analysis, 0.3 mg/kg of ISe produced higher hematocrit levels (p<0.07) as compared to 0.3 mg/of de OSe.However, these values are not outside the acceptable ranges for such parameters (Feldmann et al., 2000).According to Yahav et al. (1997), high hematocrit values indicate dehydration.A higher H:L ratio (p<0.10) was also observed in birds fed ISe.This parameter is mentioned in literature as an important sign of stress, particularly heat stress.Under these conditions, the corticoids released in the blood decrease the number of lymphocytes (Gross & Siegel, 1983).On the other hand, immune challenges increase the number of heterophils in the first 6 to 12 hours of the immune response, and this  (Harmon, 1998).Values of 0.91 and 0.60 in broilers submitted to CHS and thermoneutral environments, respectively were previously reported by Laganá et al. (2005), and the supplementation of organic Zn and Se was not sufficient to reverse this condition.In our experiment, the use of OSe seemed to partially attenuate the effects of CHS, lowering the H:L ratio to 0.73.No data were found in the literature challenging these findings.However, the explanation may lie in the influence of Se in maintaining the functionality of neutrophils, which are the heterophil counterparts in broilers (Harmon, 1998), as postulated by Arthur et al. (2003).
An important finding was that total leukocytes and lymphocytes were well below the normal standards, according Feldmann (2000), characterizing leukopenia and lymphopenia.Both were reported as partial responses to corticosteroids in some poultry species (Davison & Flack, 1981) and in birds under HS (Borges et al., 2003;Aengwanich et al., 2003).Lymphopenia was related to some viral diseases (Campbell, 2007).Oladele et al. (2005) reported lymphopenia in 4-week-old broilers and turkeys, 24h after intra-ocular inoculation with IBD virus.However, 72h after inoculation, the population of lymphocytes was already within normal ranges.In the present experiment, blood was collected 23 days after vaccination, allowing values to go back to normal prior to sampling.In addition, there are no reports associating vaccine strains, or even field strains, to leukopenia or lymphopenia in broilers.
Table 6 shows the results of the assessment of Ab titers against IBD and SRBC.There were no treatment effects on Ab response to IBD.Statistical differences were found for collection dates, and the highest titers    were obtained in the third collection, that is, 14d after vaccination.However, the contrast analysis revealed that ISe resulted higher titers (p<0.04) as compared to OSe.The level of 0.3 mg/kg ISe promoted the highest production of Ab against SRBC (p<0.05), with significant differences as compared to the other treatments.
The use of OSe decreased Ab production against SRBC and IBD.The action of this source of Se may be more related to tissue and organ integrity, and to the enhancement of metabolic systems that are key to the organic balance.Opposite results in terms of immunity are commonly reported in the literature (Lessard et al., 1997).Due to the wide range and specificity of actions of cells and molecules that regulate the immune system and their interactions, the decision for a given nutritional level or source will not always and equally affect all types of immune response.

CONCLUSIONS
Organic selenium improved the feed intake of broilers submitted to cyclic heat stress, an effect that can be useful during high temperatures commonly experienced in Brazilian practical conditions.Neither cellular immunity, nor spleen and bursa weight or bursal diameter were affected by Se levels or sources.However, broilers fed OSe presented lower scores of bursal lymphocyte depletion after vaccination against IBD, suggesting a protective effect on this tissue.Broilers receiving OSe also had the best H/L ratio.On the other hand, broilers fed diets with ISe were more efficient in the production of Ab against SRBC and IBD.
Organic and Inorganic Selenium on the Immune System of Growing Broilers Submitted to Immune Stimulation and Heat Stress higher cell death rates and hypotrophy in the bursa.

Table 1 -
Composition of feedstuffs and nutritional levels of the experimental diets.

Table 3 -
Absolute and relative weights of spleens and bursas, and bursal diameter of 42-d-old female broilers fed different levels and sources of Se in the diet and vaccinated against Infectious Bursal Disease.

Table 4 -
Frequency of bursal lymphocyte depletion of 42-d-old broilers vaccinated against Infectious Bursal Disease at 19 days of age and fed different selenium levels and sources in the diet.

Table 5 -
Hematological profile of 42-d-old broilers fed different Se levels and sources in the diet.

Table 6 -
Antibody titers against Infectious Bursal Disease1 (at 19, 29, 35and 42 days of age) and SRBC 2 (at 42 days of age) in broilers fed different selenium levels and sources in the diet. 1

Treatments 2 Antibody Titers Against Infectious Bursal Disease 3 Antibody Titers Against SRBC
Means followed by the same letter in the same column are not different (p>0.05) by LSmeans.1 -0.3 mg/kg ISe, 0.3 mg/kg ISe +0.2 mg/kg OSe, 0.5 mg/ kg ISe and 0.3 mg/kg OSe. 2 -Results obtained by ELISA (Flock Check IBD Kit, IDEXX Inc.).Results are considered negative when values are lower than 396.