Studies with Sanguinarine Like Alkaloids as Feed Additive in Broiler Diets

This research included two studies evaluating the live performance of broilers fed Sangrovit (minimum of 1.5% sanguinarine, a quaternary benzo[c]phenanthridine alkaloid extracted from Macleaya cordata). Both studies were conducted using Ross 308 female broiler chicks. Birds were fed corn-soybean meal all-vegetable diets without growth promoters with 5 treatments and 8 replications in each study. In the first study, treatments were composed of a Negative Control without feed additive and four diets with graded increases of Sangrovit of 12.5, 25, 37.5, and 50 ppm. In the second study, chicks received a similar diet from placement to 21 days of age and subsequently were given feeds with graded reductions in crude protein (CP) as follow: a Negative Control with 19.7% CP without sanguinarine, and then 19.7, 19.2, 18.8 and 18.3% CP supplemented with Sangrovit at 20 ppm. It was demonstrated that body weight was increased when birds were fed 50 ppm of Sangrovit at 21 d when compared to the Negative Control. Also comparatively to the Negative Control, cumulative feed conversion was improved for birds fed with Sangrovit at 37.5 ppm as well as feed intake from placement to 7 days at 12.5 ppm. No differences were observed in feed intake. Birds supplemented with Sangrovit and 18.8% CP had similar body weight gain and feed intake as the Negative Control with 19.7% CP. Mortality in both studies was not correlated with the treatments. Results from both studies indicate benefits of the supplementation of Sangrovit in diets for broilers.


INTRODUCTION
A great deal of controversy remains on the use of antimicrobials added to broiler feeds.Epidemiological events that result in antimicrobial resistance by bacteria are difficult to study, and several factors, including the overuse or misuse of antimicrobials in human and veterinary medicine, may intensify the severity of the problem (Shea, 2004).Growth promoters have been banned in European countries and voluntarily withdrawn by some producers in order to supply specific market demands.There has been an increase in the research on alternatives to growth promoters, including plant extracts.The use of natural antimicrobials produced from herbs and spices lends itself to more favorable acceptance by the general public as well as by countries that restrict the imports of products derived from animals fed antibiotics (Dickens & Ingram, 2001).Active compounds obtained from plants have been used for a variety of human needs for centuries; however, many commercially available additives based on natural extracts lack a definite mode of action in animal feeds.
Our experiments were aimed at evaluating the live performance of broilers fed corn-soybean meal allvegetable diets supplemented with sanguinarine like alkaloids produced from the extraction of Macleaya cordata.

MATERIAL AND METHODS
Two studies were conducted using steel-cage batteries (0.95 X 0.80 m).In Study 1, a titration with graded increases of Sangrovit ® was used, whereas in the Study 2, Sangrovit was added at a fixed dosis of 20 ppm in feeds given from 21 to 35 days of age.

Study 1
Four hundred one-day-old Ross X Ross 308 female broiler chicks (average individual weight = 43.9g), vaccinated for Marek's disease and infectious bronchitis were obtained from a commercial hatchery.Birds were randomly placed in 40 steel-cage batteries, with 10 birds per cage, in a continuous lighting program with feed and water provided ad libitum.
Corn-soybean meal all-vegetable mash diets without antibiotic growth promoters or anticoccidials were provided throughout the study.Feeding program consisted of starter feed from placement to 21 d, and grower feed from 21 to 35 d.Feeds had similar energy and nutrients levels after formulation according to Rostagno et al. (2005) as shown in Table 1.Treatments consisted of a Negative Control without feed additive and four graded levels of Sangrovit (12.5,25,37.5,and 50 ppm).Each treatment had 8 replications.
Birds were weekly weighed, and feed conversion ratio corrected for the weight of dead birds was calculated.Mortality was recorded daily.Data were analyzed using the Anova procedure of SAS (SAS, 2001).Tukey's test was used to assess differences between means, and values of P ≤ 0.05 were considered statistically significant.Mortality data was analyzed after arcsin transformation.

Study 2
Four hundred one-d-old Ross X Ross 308 female

RESULTS
The effects of treatments on performance parameters of studies 1 and 2 are presented in Tables 2 and 3.In both studies, mortality rate was considered normal for that time of the year and growth rate of the birds, and it was not affected by the treatments (grand means = 4.69 % in Study 1 and 1.50% in Study 2).
In study 1, body weight linearly increased at 21 d in birds fed Sangrovit (p ≤ 0.05).Effects were not observed in the other periods.Feed conversion ratio corrected for the weight of dead birds improved during the total experimental period when sanguinarine were added at 37.5 ppm to the feed in study 1.This response was quadratically adjusted (p ≤ 0.001) with the maximum response calculated as (-b/2a) at 32.84 ppm.Feed intake of birds fed Sangrovit improved during the first week,, whereas differences were not observed (p ≤ 0.05) during the other periods.
In Study 2, body weight and feed intake were not influenced by dietary protein reductions; however, feed conversion ration was negatively affected from 21 to 28 and from 21 to 35 d (Table 3).Mean differences were observed only between treatments with extremes in dietary protein, but the effects of the graded reductions in protein were linear and indicate that loss in feed conversion ratio caused by these reduction were not be recovered with the addition of Sangrovit at 20 ppm.

DISCUSSION
The results from Study 1 indicate a feed conversion ratio benefit with the use of sangrovit in broilers as previously shown in swine (Tschirner et al., 2003).It has been largely accepted that sub-therapeutic doses of antimicrobials are beneficial in intensive animal production systems.However, Niewold (2007) recently suggested that expected growth promoter effects of antimicrobials on animals are in fact mediated by antiinflammatory mechanisms.Previous research studies on QBAs suggested a combination of antimicrobial and anti-inflammatory effects as an explanation for their benefits on animal performance.QBAs have antimicrobial activity (Lenfeld et al., 1981), and a,b -Mean values without a common superscript are different using Tukey' test (P ≤ 0.05). 1 -At least 1.5% Sanguinarine.2 -Y = 0.00074 X + 0.81784, p = 0.0213, R 2 = 0.13. 3 -Y = -0.00003386X 2 + 0.00222 X + 0.10876, p = 0.0010, R 2 = 0.42.minimum inhibitory concentrations of sanguinarine against several bacteria were found in the oral cavity of humans (Dzink & Socransky, 1985).Many of these bacteria belong to species commonly found in the gastrointestinal tract of chickens.Therefore, sanguinarine effects against them are expected by analogy.In parallel, it is possible to speculate that sanguinarine activity as an anti-inflammatory substance may play a role in the partial improvements obtained in the broilers of Study 1.For instance, antiinflammatory activity has been largely demonstrated with various QBA´s, including sanguinarine (Lenfeld et al., 1981;Agarwal et al., 1991).In pig intestines, this activity is expected to be caused mostly by contact, since the bulk of these alkaloids are excreted in the feces, and only a very small proportion is absorbed (Kosina et al., 2004).
Sanguinarine has been shown to reduce intestinal decarboxylation of aromatic amino acids through the inhibition of L-amino acid decarboxylase in swine (Drsata et al., 1996).The rationale for reducing dietary protein in Study 2 was to investigate a possible similar effect in broilers.The performance obtained with the graded CP reductions appeared to be sustained to the level of 18.78% CP for feed conversion rate and of 18.32% for body weight and feed intake.However, protein retention measurements should be conducted to conclude that the impact of the reduction in protein was counteracted by the supplementation of the additive.
If Niewold's (2007) theory is correct, the search for alternatives to traditional antibiotic growth promoters does not necessarily mean a search for alternative antimicrobials.Sanguinarine, as some of other QBAs, has several interesting characteristics as a feed additive for broilers.Most of these characteristics support the benefits in body weight and feed conversion ratio observed in this study.

CONCLUSIONS
1. Dietary supplementation with sangrovit significantly improved body weight at 21 days, feed conversion ratio at 35 days, and feed intake at 7 days of age.While body weight was linearly affected by 50 ppm of Sangrovit at 21 days, feed conversion ratio at 35 days was maximized at the supplementation level of 32.8 ppm of Sangrovit.2. Body weight and feed intake were not affected when broilers were fed diets with graded reductions of crude protein; however, feed conversion ratio was negatively affected when with protein levels of 18.78% and lower.

Table 1 -
Composition of experimental diets, %. the study.Diets are shown in Table 1 and had similar energy and nutrient levels after formulation according to Rostagno et al. (2005).Treatments included a Negative Control with 19.7% crude protein (CP) without the feed additive, and diets with graded reductions of CP and supplemented with 20 ppm of Sangrovit as follows: 19.7, 19.2, 18.8, and 18.3%.Each treatment had 8 replications.Measurements and statistical analyses were performed as in Study 1.

Table 2 -
Growth performance of broilers fed diets supplemented with graded increases of Sangrovit 1 from 1 to 35 d (Study 1).