Reassessing flavophospholipol effects on broiler performance

The objective of this study was to evaluate bird responses to flavophospholipol at higher doses than those recommended by the Brazilian legislation. A trial was conducted with 900 male broilers divided into five doses of flavophospholipol: 0, 2, 4, 8 and 16 mg/kg. The performance evaluation was conducted weekly until 42 days of age. The evaluated parameters were: weight gain, feed conversion corrected for mortality, feed intake and mortality. At the end of experimental period, birds supplemented with growth promoter differed from the negative control for weight gain and feed conversion in the period from 1 to 42 days. Body weight gain and feed conversion were significantly higher for birds fed 16 mg/kg of flavophospholipol, compared with treatment without the growth promoter in the period from 1 to 21 days. In the accumulated period from 22 to 42 days, all doses were different for the negative control for feed conversion and body weight gain. The performance improvement was maximized at the dosage of 10.1 mg/kg for feed conversion ratio and 10.9 mg/kg for body weight gain by regression analysis. Flavomycin can be used as a growth promoter to improve feed conversion ratio and body weight gain in broilers from 1 to 42 days of age.


Introduction
Antimicrobials have been added to the diets of animals raised for the production of meat at sub therapeutic levels for many years.Consistent improvements in feed efficiency have justified this practice, which is supposedly mediated through modulation of the gut microbiota.Niewold (2007) suggested that the antibiotic growth promoters (AGP) actually reduce inflammatory responses by sparing the immune system, which leads to reduced morbidity and mortality as well as subclinical and clinical diseases.
Despite the well-recognized benefits, a trend to ban AGP from animal diets is now stronger than ever in many countries.Risk of resistance to antibiotics by pathogenic microorganisms has been a main driver to influence government authorities as they change the laws on the use of this class of product.Nevertheless, the association of bacterial resistance with AGP may be linked to questionable research (Cox et al., 2003;Dibner & Richards, 2005;Cervantes, 2011).It seems that, more than any long-term research, the precautionary principle has been the main reason for AGP banning in the European Union (World Health Association, 2003, 2004).
Flavomicyn, a glycophospholipid which has a known inhibiting capacity for Gram positive bacteria (Butaye et al., 2003a), can still be legally added at subtherapeutic doses in poultry diets in many countries.This molecule has never been used as a therapeutic drug for poultry and it lacks pharmacokinetic and pharmacodynamic activities (Pfaller et al., 2006).Inhibition of the enzyme transglycosylase, necessary for the formation of the bacterial cell wall and its consequent intestinal microbiota modulation promotes the development of Lactobacilli and Bifidobacteria in the intestinal tract, which are generally considered beneficial bacteria (Bolder et al., 1999).The establishment of a population of microorganisms that supports animal growth by flavophospholipol is based on the competitive exclusion effect, which has been observed to be accompanied by lower incidence of resistant genes and lower excretion of pathogenic bacteria, such as Salmonella, Clostridium and Campylobacter (Kissel, 1998;Butaye et al., 2003a;Pfaller et al., 2006).Worldwide, the inclusion of flavophospholipol varies from 1 to 20 mg/kg of feed.In Brazil, the use of growth promoters is regulated by MAPA (2008), which sets the use of flavophospholipol in birds at 1 to 2 mg/kg, which is similar to the regulation in the United States by the FDA (1985).
The purpose of this study was to reevaluate the effects of flavophospholipol in broiler diets since it has not been used in practice for many years, which may become a cheap alternative for rotating AGP programs.

Material and Methods
The procedures adopted throughout this study avoided unnecessary animal discomfort and followed the directives of the Committee of Ethics and Use of Animals of Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
A total of 900 one-day-old Cobb × Cobb 500 male broiler chicks originated from the University hatchery were placed in 60 floor pens, 1.70 × 1.65 m each, 15 birds per pen.Each pen had rice hulls as litter and was equipped with 3 nipple drinkers and a tube feeder.Individual one-day-old chick weight averaged 48±1 g and the birds had been vaccinated for Marek's and Gumboro disease at the hatchery.Average house temperature was 32 °C at placement, being reduced as animals aged to provide comfort throughout the study using heaters, fans and foggers whenever appropriate.Lighting was continuous until 7 d of age; 18 hours light:6 dark cycles were used afterwards.Feed and water were provided ad libitum.
Mash diets were provided for the birds in a 4-phase feeding program as follows: 1 to 7 d (pre-starter), 8 to 21 d (starter), 22 to 35 d (grower), and 36 to 42 d of age (finisher).Diets were formulated with corn, soybean meal and meat and bone meal as major ingredients.Treatments consisted of five graded inclusions of flavophospholipol (Flavomycin ® 80, Huvepharma, Sofia, Bulgaria): 0, 2, 4, 8 and 16 mg/kg (Table 1).A pooled sample of each treatment per phase feeding was analyzed for the contents of flavophospholipol as suggested by Gallo et al. (2010).
Live performance was evaluated weekly on a pen basis through the following measurements until 42 days of age: body weight gain (BWG), feed intake (FI), feed conversion 1 Each treatment was supplied to 9 replicate pens of 15 broilers at the beginning of the study.
The experimental design was completely randomized.Mortality data was analyzed after arcsine transformation (√x/100).Linear regressions were run using formulated concentrations of flavophospholipol with the PROC REG procedure of SAS (2001).Significance was accepted as α = 0.05, and mean differences were separated using Tukey's test.

Results and Discussion
Analyzed crude protein, calcium and total phosphorus for all diets and feed phases were as expected from feed formulation (Table 1).Analyzed flavophospholipol concentrations in all feeds were in accordance with expected levels from feed formulation; therefore, regression analysis was performed with formulated flavophospholipol in feeds.
Significant differences (P<0.05)occurred for BWG and FCR throughout the study and for FI from 1 to 21 days of age (Table 2).Adjustments obtained by linear regression analysis demonstrated improvements in BWG and FCR as flavophospholipol increased in the diet.Optimal responses obtained with flavophospholipol supplementation were calculated whenever a significant quadratic adjustment was possible.For BWG and FCR optimal responses were of 14.68 and 12.17 mg/kg of feed from 1 to 21 days (Figure 1), 9.83 and 10.31 mg/kg of feed from 22 to 42 days (Figure 2), and 10.10 and 10.90 mg/kg of feed from 1 to 42 days (Figure 3).Feed intake was adjusted by a quadratic model only from 1 to 21 days when the optimal response was achieved at 16 mg/kg (Figure 4).NS -not significant; SEM -standard error of the mean.
3 Individual feed conversion ratio corrected for mortality. 4 Individual body weight gain. 5 Optimal response based on calculated values of flavophospholipol in the predicted equation.
Figure 1 -Feed conversion ratio and body weight gain from 1 to 21 days of age.Mortality was not affected by the treatments and it was considered in an acceptable range for male broilers grown to the age used in this study (0.56±0.08).
Data from the present study show that birds experienced improvements in live performance whenever flavophospholipol was added to the diets.It was also demonstrated that benefits of adding flavophospholipol in the feeds could be obtained using levels much higher than those presently allowed to be used in commercial broiler production (maximum of 2 mg/kg of feed in Brazil (MAPA, 2008) and the USA (FDA, 1985).
Benefits of the inclusion of AGP in broiler diets on live performance have long been demonstrated (Miles et al., 2006;Cervantes et al., 2008;Hossain et al., 2008;Demir et al., 2008;Vieira et al., 2010).The mechanism of action of these compounds, however, is not easily explained.Flavophospholipol is a phosphoglycolipid antimicrobial produced by various strains of Streptomyces, active against Gram positive bacteria, such as Staphylococcus spp and Enterococcus spp (Aarestrup et al., 1998;Riedl et al., 2000;Butaye et al., 2003b).Therefore, it is expected that as harmful microorganisms are suppressed, the gut microbiota will come into balance towards a more benefic population.A well-balanced intestinal microbiota is considered to be an effective barrier against pathogenic bacteria, which besides positively affecting animal performance, may also result in positive effects for public health (Pfaller, 2006).For example, flavophospholipol was able to reduce the colonization and shedding of Salmonella spp and Clostridium perfringens (Cox et al., 2003;Bolder et al., 1999).Despite antimicrobial effects, live performance improvements with the use of AGP such as flavophospholipol, inhibition of inflammation effects have also been proposed as a mean of action for this type of compound (Niewold, In the present study, including flavophospholipol in the diets led to improvements in FCR and BW gain.These are benefits that can be translated into important amounts of feed saved in the process of animal production for human consumption.Therefore, the use of flavophospholipol as an additive in broiler nutrition is still an important tool in maintaining competitive production of broiler meat.

Conclusions
The use of flavophospholipol improves broiler live performance from 1 to 42 days of age.Optimal benefits vary with flavophospholipol concentrations from 9.83 to 16.00 mg/kg of feed.
followed by different letters differ by Tukey's Test (P>0.05).

Figure 2 -Figure 3 -Figure 4 -
Figure 2 -Feed conversion ratio and body weight gain from 22 to 42 days of age.

Table 1 -
Composition of experimental diets 1

Table 2 -
Live performance of male broilers fed diets with graded increases in flavophospholipol