Effects of probiotics and selenium combination on the immune and blood cholesterol concentration of pigs *

Ninety-six weanling piglets (four weeks old) were randomly assigned into four treatments as follows: 1. control group, 2. probiotics group, 3. probiotics combined with 0.3 ppm organic selenium and 4. carbadox group. Collected data was analysed by using the GLM program of SAS (1990), and each of the two groups was compared using Duncan's test, while the GLM analysis was significant. The experimental results revealed no influence on pig growth performance by the addition of probiotics alone or with the selenium. The immunoglobulin-G concentration of the probiotics with the 0.3 ppm selenium group was the highest among the four groups, and significantly (PO.05) higher than in the control group. The mortality of the piglets (all survived) and the very-low-density lipoprotein cholesterol (VLDL) concentration of the probiotics group was the lowest (PO.05) among the four groups for the probiotic and with selenium groups when compared to the control and carbadox groups.


INTRODUCTION
The widespread use of antibiotics in animals to control disease and promote growth has long caused consumer concern due to bacterial resistance and allergenic effects in humans (Williams and Heymann, 1998).Although the use of antimicrobials for growth promotion in livestock diets is still permitted in the United States, the European Union (EU) has decided to ban all growth-promoting antibiotics used in 626 PROBIOTICS AND Se -IMMUNE AND BLOOD CHOLESTEROL IN PIG animal feed, driving animal nutritionists and producers to devise natural alternatives for commercial pig farms to reduce the risk of higher mortality and poor growth performances following the ban on the use of antibiotics in feed.
Probiotics generally refers to a live culture of microorganisms, such as lactic acid bacteria (LAB), that exerts a beneficial effect on the host by improving the indigenous microbial balance.The most widely used probiotic LAB are lactobacilli and bifidobacteria that can survive in the intestine.Extensive studies on the beneficial effects on human health of these species have been reported (Perdigon et al., 1990;Benno et al., 1996).It has been confirmed that a culture of Lactobacillus acidophilus actively taking up cholesterol from laboratory media and it has beneficially influenced serum cholesterol levels (De Rodas et al., 1996).Besides, the use of Lactobacillus and Bifidobacterium sp. have been found functions include inhibiting pathogenic bacteria, antitumour and anticholesterolaemic activity, improving digestion, and stimulating the immune system (Piard and Desmazeaud, 1991;Adachi, 1992 ;Wu et al., 2001;Lin et al., 2002).Supplementing LAB to piglets also found promotes body weight gain, increases feed conversion, enhances the colonization of beneficial bacteria, and reduces levels of harmful intestinal bacteria.Our previous experiment found that Lactobacillus acidophilus, L. pentose and Bacillus subtilis mixture could modulate intestinal microbes, stimulate immune response, and reduce serum cholesterol (Lin et al., 2002).However, some other studies have failed to find any improvement in growth performance (Harper et al., 1983).
Additionally, selenium is recognized as an essential mineral for animals.Selenium has been added to farm animal diet in inorganic form as sodium selenite.However, because of its poorer bioavailability and the fact that it is an oxidant of inorganic selenium, sodium selenium could be replaced by organic selenium as a mineral supplement.Selenium deficiency is well known to reduce both cellular and humoral immune function in humans and laboratory animals (Combs and Combs, 1986), in animal reproduction performance (Keating and Caflfrey, 1989) as well as in failure of antioxidant membrane protection (Combs and Combs, 1986).The use of selenium in domestic animal diet has been confirmed to improve immune response in pigs (Lessard et al, 1991;Wuryastuti et al., 1993), decrease total cholesterol and triglyceride level (Kang et al., 2000), and decrease drip loss in broilers (Naylor et al., 2000).Due to both probiotics and selenium being immune stimulators, probiotics (such as L. acidophilus) can also concentrate selenium in cells, which is assumed to help the combination of probiotics and selenium to enhance animal immune responses.Additionally, researchers recently discovered an unexpected result, namely that selenium may act on lower low-density cholesterol (LDL) and raise high density cholesterol (HDL).This study therefore examines the combination of probiotics and selenium on pig immune responses, growth performances and blood cholesterol concentration.YU I.-T.ETAL.

Animals and diet treatments
Ninety-six four-week old weaned pigs were randomly assigned to four treatments, with four pigs per pen and six pens per treatment.The four treatments (TRT) were as follows: 1. TRT1 (control feed, maize-soyabean based diet, without additives), 2. TRT2: TRT1 added with 0.3% probiotics, a mixture consisting Lactobacillus acidophilus, Lactobacillus pentose and Bacillus subtilis, with the final concentration of each bacterial count being 10 9 CFU/g,  1 lists the ingredients and nutrient composition of the experimental diets.The probiotics mixture (spray-dried powder) of 0.3% was mixed with other ingredients (Table 1) in dry-mesh form before feeding.Feed was admimistered in dry-mesh form, and was completely consumed within five days after prepared.Before completely consumed, feeds were stored in room temperature in a cool, shady and dry place, and the survival rate of bacterial in feed after five days of storage in room temperature is still higher than 90% (unpublished data).Feeds and water were administered ad libitum, and pig body weight and feed intake were measured every two weeks to assess growth performance, including average daily gain (ADG), feed intake (FI) and feed conversion ratio (FCR).This trial ended when pig body weight reached 35 kg.

Blood withdrawal and immune response assay
Piglets were intramuscularly injected with 20 |ng/kg liveweight of lipopolysaccharides (LPS, serotype 055:B5, Sigma Chemical, St Louis, MO) one week before the end of the trial.LPS was injected intramuscular (im) in the neck behind the ears of pig to simulate the situation of piglets suffering E. coli attack.A blood sample (8 ml) was obtained from the superior vena cava of all piglets before LPS injection, and at 1 h, 2 h and 6 days following injection.After coagulation for 1 h at room temperature, blood clots were centrifuged (2500 g for 20 min at 4°C) and serum was removed and preserved at -20 °C for subsequent measurement of serum immunoglobulin-G (IgG) and tumor necrosis factor-a (TNF-a) concentrations.Total serum IgG concentrations were detected using an ELISA kit of porcine IgG (ICN Pharmaceuticals Inc., Ohio, USA).An ELISA porcine TNF-a kit (Endogen, Cambridge, MA) was used to assess total serum TNF-a.Serum samples were assayed in duplicate at either 1:1 or 1:10 dilution.The assay was sensitive to 10 pg/ml of TNF-a and had an intra-assay CV of 10.8%.

Blood cholesterol concentration analysis
Serum cholesterol content, including total cholesterol (TC), high-density lipoprotein cholesterol (HDL) and triglycerides (TRIG), was determined using enzymatic methods from commercially available kits (VITROS Cholesterol Kit, Ortho-Clinical Diagnostics, Johnson and Johnson Company, USA) that are suited to routine analysis, and the very-low-density lipoprotein cholesterol (VLDL) content, as obtained by TRIG divided by 5, and low-density lipoprotein cholesterol (LDL), were obtained by TC subtracts HDL and VLDL concentrations.

Statistical analysis
The least square means and their corresponding standard errors are obtained using the LSMEANS method (SAS, 1996).Moreover, the effects of dietary treatments on ADG, FI, FCR and on TNF-a, IgG concentrations, as well as on TC, HDL, LDL, VLDL and TRIC concentrations, are analysed using the General Linear Models and compared using the Duncan's new multiple range test procedure.

RESULTS AND DISCUSSION
Table 2 lists the growth performances and immune responses of piglets given probiotics alone, or given probiotics combined with 0.3 ppm selenium as well as carbadox alone.Different probiotics, including Lactobacillus spp., Bacillus spp., Streptococcus spp., the yeast Saccharomyces cerevisiae, and combinations of these organisms have been widely used in commercial farm animals.However, probiotics supplementation to piglets to improve growth performance has shown variable results, and the precise mode of action through which probiotics exert their positive influence remains uncertain (Turner et al., 2001).No difference in growth performance was observed for pigs fed diets with added Lactobacilli (Harper et al., 1983) or Bacilli (Kornegay and Risley, 1996).However, improved growth performance and reduced incidence of E. coli in faeces was noted when Lactobacilli or Bacillus spp.( Succi et al., 1995) was added to the diet.In this study, growth performance is not improved when the diet is supplemented with probiotics and 0.3 ppm selenium.Meanwhile, the FI of pigs fed diet supplemented with carbadox (TRT4) is significantly (P<0.05)lower than for those fed diet supplemented with TRT1 and TRT2, leading to a trend of improving the FCR for carbadox treatment.Carbadox is an antimicrobial and has been used to prevent dysentery and decrease disease as well as to improve growth performances of pigs for many years.The failure of carbadox to stimulate growth performance in this study was unexpected.However, in some situation, such as in clean or hygienic place, the beneficial effect of antimicrobials may be minor (Cromwell, 2001).The nursery building were cleaned with high pressure water and disinfected, so the pens were clean enough at the beginning of the study, which may have been a contributing factor.The similar result also appeared in the study of White et al. (2002).
Immunoglobulin -G (IgG) concentration is higher for TRT2, TRT3 and TRT4 than for TRT1 on the sixth day following LPS injection, and is significantly higher for TRT3 than for TRT1.As mentioned above, both probiotics and selenium stimulate immune responses.This work also found this result.Moreover, when combine probiotics with 0.3 ppm Se, the stimulation strength on immune tended to be enhanced (TRT3 compared with TRT2).TRT3 also tended to be higher than TRT4 (carbadox treatment), this observation result revealed the additional benefit of increasing serum IgG concentration when 0.3 ppm Se combined with probiotics was used in piglet diet.
TNF-a, a potent cytokine, was first identified as a serum protein that caused tumor necrosis in mice.The response of TNF-a concentration appears not always to accord with IgG concentration.When bacterial endotoxin prevails, macrophages excrete TNF-a, which controls different leukocyte activity, and regulates and optimizes the immune and inflammatory responses (Webel et al., 1997).TNF-a also regulates the acute phase response, induces the prosecution of other immunoregulatory cytokines, and activates T cells, B cells, NK cells, and neutrophils (Wells et al., 1999).In the current study, LPS injection sharply raised (P<0.05)TNF-a concentrations of all treatment groups to a level similar at lh post injection.No significant difference in TNF-a concentration was noted among the four treatments, although the concentration of TRT 2 (probiotics group) tended to be higher than that of TRT 1 (control group).The survival rate of the piglets in the whole study period is higher for TRT 2 (100%) than for the other three groups (83, 88 and 92% for TRT 1, 3 and 4, respectively), which seems to agree with the TNF-a and IgG concentration for TRT 2, but survival rate of piglets does not respond to TRT 3, which TNF-a concentration is tended but slightly lower than TRT 1.
Excessive cholesterol levels are associated with cardiovascular problems leading to coronary failure.High cholesterol content in pork disadvantaging consumers.Probiotics supplementation influences the concentration of some blood metabolites and the reduction of serum cholesterol and triglyceride (Noh et al., 1997).The proven in vitro cholesterol assimilation of Lacbacilli strains probably causes this in vivo observed lowering effect (Gilliland et al., 1985), and this assimilation of cholesterol and deconjugation of bile salt reduced serum cholesterol by affecting the enterohepatic cycle (De Rodas et al., 1996).Result of serum analysis in this study found that the addition of probiotics tended to reduce TC, LDL, TRIG and increase HDL, but only VLDL was significantly (PO.05) reduced by using probiotics.This result agreed with the findings of some in vitro (Gilliland et al., 1985;Brashears et al., 1998;Usman, 1999), or in vivo studies (De Rodas et al., 1996;Noh et al., 1997).
The beneficial influence of probiotics on serum cholesterol levels considered cholesterol incorporated into or attached to bacteria in the intestine is likely to be unavailable for absorption into the blood, and the ability to incorporate cholesterol into or attach it to bacteria has been equated with the ability to remove cholesterol from media (Kimoto et al., 2002).
In this study, selenium in combination with probiotics did not display any additive effect on lowering serum cholesterol concentration, although this tended (P>0.05) to reduce TC, VLDL, TRIG and increase HDL concentrations.Moreover, an in vivo study showed that selenium supplementation decreases total cholesterol and triglyceride levels in humans (Yilmaz et al., 1997); however, no similar results have been observed in pigs in vivo.This result seems to need further study to confirm.The function of RBC is known to be to carry oxygen from the lungs to the body tissue and organs, and to carry carbon dioxide back to the lungs where it is excreted.Excessively low density of RBC will cause anaemia and threaten health.Meanwhile, higher density of RBC in a normal range should benefit health.Blood cell characteristic analysis reveals that probiotics supplementation did not increase erythrocyte (RBC) concentration, but if combined with selenium, erythrocyte concentration tended to be enhanced.Notably, the RBC concentration is markedly higher (P<0.05) for carbadox treatment (TRT4) than for TRT 1.Although many data have demonstrated that the addition of carbadox can improve pig growth performance and reduce diarrhoea, no data has yet emerged demonstrating the increase in RBC concentration following carbadox addition.Whether this finding can explain any beneficial effect on pigs following the dietary addition of carbadox needs further investigation.

CONCLUSIONS
Probiotics inclusion in piglet diet demonstrated decreased (P<0.05)bad serum cholesterol (VLDL) concentration and tended (P>0.05) to reduce other cholesterols (LDL and TC) and increase HDL and serum IgG concentration.Moreover, the survival rate (100%) for the probiotics group is the highest among the four groups, but no effect was observed on growth performance and blood characteristics.The addition of 0.3 ppm selenium with probiotics in piglet diet exhibited an additive effect on increasing serum IgG concentration (P<0.05) six days following LPS injection, and tended (P>0.05) to decrease serum TC and VLDL concentration and increase HDL concentration.