Bone quality in layers fed aflatoxin, fumonisin and adsorbent

Introduction

Mycotoxins are a major factor reducing poultry productivity and the quality of poultry products. Nearly 25% of the grains produced worldwide is contaminated with mycotoxins (Mannon and Jonhson, 1985), so that the controlling of their impact is fundamental for the prevention economic losses.

The group of mycotoxins most broadly distributed throughout the world is that of aflatoxins, which are produced by the molds Aspergillus flavus, A. parasiticus and A. nominus (Kurtzman et al., 1987), that prosper in hot, humid weathers. Aflatoxin toxicity in broilers is characterized by decreased total blood protein, albumin, cholesterol, glucose, uric acid, inorganic phosphorus and calcium concentrations, as well as by an increase in the liver function indicator enzymes (Amer et al., 1998). Aflatoxins are also known to interfere with vitamin D metabolism, thus contributing to reduce bone strength, resulting in weakened legs (Hamilton, 1984).

Studies have shown that aflatoxins affect directly or indirectly the metabolism of calcium (Ca) and phosphorus (P) in birds (Glahn et al., 1991), two important inorganic elements representing 95 % of the mineral bone matrix. Ca, P and vid D are closely related with animal's metabolism and many times they interact in combination, mainly in bone formation (Vargas et al., 2004).

Glahn et al. (1991) explained that the effects of aflatoxins on Ca and P can be related with alterations in vit D and paratohormone (PTH) metabolism, since this particular mycotoxin can reduced the endogenous synthesis of this hormone and modify the sensitivity of the kidneys PTH, and even the synthesis of 1,25-dihydroxy vit D can be compromised, resulting in decreased renal excretion of P and increased Ca excretion. Aflatoxin reduces the intestinal absorption of vit D and liber damage prevent its conversion into the active form 25-hydroxycholecalciferol. Nevertheless, it is not clear if whether mycotoxins cause a specific metabolic deficiency of vit D3 or other nutrients, or if they simply affect the bird by reducing feed intake. In mycotoxin-contaminated feeds, particularly in the presence of Fusarium, increasing the levels of vit D3 is recommended.

Fumonisins are cytotoxic and carcinogenic, and their main effect is inhibiting the synthesis of sphingolipids, which are crucial for cell membrane integrity as well as for cell ion transport (Santurio, 2000). Layers can tolerate relatively high concentrations of fumonisin B1 (FB1) during long periods, without affecting health or performance (Kubena et al., 1999).

The objective of this study was to evaluate the rious effects of aflatoxin, fumonisin and their combination on the bone quality of commercial layers, and to analyze the efficacy of the glucan adsorbent in promoting the reduction or total prevention of these effects.

Materials and Methods

The experiment was performed in the Bio-Climate Chamber, FMVZ, UNESP, Botucatu Campus, Brazil. One hundred and sixty eight (168) Hisex Brown hens were used. Hens were 37 weeks old at experiment start. A completely-at-random design was used, with a 3 x 2 + 1 factorial arrangement (3 treatments con the addition of mycotoxins: aflatoxin (AF), fumonisin (FU) or both (AF+FU); treatments with or without the adsorbent (ADS) and a control feed with no mycotoxins or adsorbent, for a total of 7 treatments with 6 repetitions each. The experimental unit was one 4-bird cage, and the experiment lasted for 56 days, divided in two 28-day cycles. The toxic doses used were 1 ppm aflatoxin and 25 ppm fumonisin, while the adsorbent was a yeast cell wall-derived glucan, at the dose rate of 2 Kg/ton of feed.

Eggs were collected twice per day in order to record % lay. For the bone-quality study, 6 birds per treatment were euthanized, one per repetition, and the left tibiae were removed for later analysis. Bones were kept in their natural state (only the muscles were manually removed) for bone resistance analysis, performed using a specific cell coupled to a Texture Analyzer TA. XT Plus, to measure the force needed to brake all bones.

After the bone resistance analysis, bones were burned at 800°C in a muffle furnace for 3 hours in order to obtain bone ash. Ca and P quantification in the bone samples was performed by atomic absorption spectrometry, and visible ultra-violet spectrophotometry after sample de-mineralization (Neves et al., 2009; Moraes et al., 2009).

Results were statistically analyzed using General Linear Model (GLM), with the Statistical Analysis System (SAS, 2002) package. When significant differences were obtained (P<0.05), the means were compared among treatments using both Tukey´s test (means among treatments) and Dunnett´s test (treatment means compared with the controls).

Results and Discussion

Mean % lay, % bone ash, DM, Ca/P concentrations, and bone resistance results are shown in Table 1. No effects were seen (P>0.05) of mycotoxins or the absorbent on % bone ash or resistance to fracture. Birds in AF treatments showed lower (P = 0.0594) % lay, and these results match those reported by Oliveira et al. (2002), for concentrations of 100, 300 and 500 μg/kg AF and by Iqbal et al. (1983) for the concentrations from 100 to 500 μg/kg AFB1.

Table 1. Percent lay and bone quality traits of commercial layers in the different treatments

			MYCOTOXIN		P VALUES
TRAT¹	CONT	ADS	AF	FU	AF+FU	Mean	ADS	MYCO	INT	CONT	CV (%)
Lay (%)	94.79	No	76.72	86.76	77.38	80.29	0.5828	0.0594	0.5793	0.0430	14.77
Yes	83.93	90.77	73.51*	82.74
Mean	80.33 ab	88.77 a	75.45 b
Ash (%)	41.75	No	43.82	42.21	45.43	43.82	0.2805	0.1223	0.9415	0.3054	8.12
Yes	43.07	40.78	43.70	42.44
Mean	43.44	41.50	44.64
DM (%)	60.91	No	64.93	64.03	61.88 B	63.61	0.3067	0.9830	0.0465	0.0583	4.65
Yes	63.38	64.00	66.61 A	64.55
Mean	64.16	64.02	64.03
P (mg/l)	0.159	No	0.177	0.210 B	0.166	0.184	0.6549	0.7713	0.0283	0.0708	16.94
Yes	0.184	0.164 A	0.190	0.179
Mean	0.181	0.187	0.177
Ca (mg/l)	3.37	No	3.44	3.90	3.88	3.74	0.9845	0.0351	0.1355	0.0844	11.72
Yes	3.64	4.13	3.43	3.76
Mean	3.54 b	4.02 a	3.67 ab
Resistance to fracture (Kgf/mm)	9.12	No	11.22	10.16	10.12	10.50	0.2678	0.2362	0.0630	0.0837	20.79
Yes	10.16	10.35	13.59	11.24
Mean	10.69	10.26	11.70

^{¹ Treatments: CONT: control; AF: aflatoxin; FU: fumonisin; AF+FU: aflatoxin + fumonisin; ADS: adsorbent; MYCO: mycotoxin; INT: interaction.
* Differs from controls as per Dunnett´s test (P<0.05);
a, b Means in a line followed by different lower case letters are different as per Tukey's test (P<0.05);
A, B Means in a column followed by different capitol letters differ, as per Tukey's test (P<0.05).}

Interaction (P<0.05) was seen between the inclusion of mycotoxins and adsorbent for %(MS, since the AF+FU with ADS treatment had a higher (66.61%) MS content than the same treatment with no ADS (61.88%). The reduced % lay seen in with this last treatment (73.51%) mobilized less bone tissue, which can explain the increased %MS in the tibia. In fact, studies have shown that bone mobilization is related with egg production (Whitehead, 2004). Interaction was observed (P<0.05) also in P concentration with the FU treatment, with a higher value (0.210 mg/l) in the absence of ADS. Tibial Ca concentration differed between the mycotoxins. Birds in the FU treatment had higher concentrations (4.016 mg/L) than those in the AF treatment (3.54 mg/L).

AF is known to affect the liver and the kidneys, organs involved in the biosynthesis of active vit D (Hamilton, 1984), which is responsible for transferring dietary Ca and P to the bloodstream. Given the toxic effect of AF and therefore, the reduction in active vit D, decreased Ca absorption/bone deposition occurs. Being this the case, increased bone Ca was mobilized for eggshell formation, which can explain the lower content of Ca in the bones of the birds in the AF group.

Fumonisin does not seem to affect the synthesis of active vit D and, in this study, the intestinal absorption of dietary Ca and P would assure the fulfillment of requirements for eggshell formation, thus allowing for a higher concentration of these two minerals in the bones of the FU with no ADS treatment. Using the ADS resulted in a trend to increase % lay, which can be associated with the increased Ca/P demand for the egg. Nevertheless, perhaps the low FU concentration used herein was not enough to show more evident effects of this mycotoxin.

Conclusion

The changes observed in % lay are indicative of aflatoxin toxicity at the concentration of 1 ppm. Mycotoxins, either alone or in combination, had no effect on bone quality. Using the glucan adsorbent in the feed at the concentration of 2 kg/ton was not sufficient to prevent the effects of mycotoxins on egg production, and it did not affect bone quality.

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EV Siloto¹*, DRS Sartori², EFA Oliveira³, JR Sartori⁴, VB Fascina^5 -¹Alumna de doctorado del Programa de Posgrado de la Facultad de Medicina Veterinaria and Zootecnia, UNESP, Botucatu, SP, Brazil; ²Profra. del Departamento de Fisiología del Instituto de Biociencias, UNESP, Botucatu, SP, Brazil; ³Zootecnista, Facultad de Medicina Veterinaria and Zootecnia, UNESP, Botucatu, SP, Brazil; ⁴Profr. del Departamento de Mejoramiento and Nutrición Animal, Facultad de Medicina Veterinaria and Zootecnia, UNESP, Botucatu, SP, Brazil;

⁵Alumno de doctorado del Programa de Posgrado de la Facultad de Medicina Veterinaria and Zootecnia, UNESP, Botucatu, SP, Brazil.