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Determination of the effects of aflatoxin B1 givenin ovo on the proximal tibial growth plate ofbroiler chickens: histological, histometric andimmunohistochemical findingsY. Oznurlu a , I. Celik a , E. Sur a , T. Ozaydın a , H. Oğuz b & K. Altunbaş ca Department of Histology and Embryology , Faculty of Veterinary Medicine, University ofSelçuk , Konya , Turkeyb Department of Pharmacology and Toxicology , Faculty of Veterinary Medicine,University of Selçuk , Konya , Turkeyc Department of Histology and Embryology , Faculty of Veterinary Medicine, University ofAfyonkocatepe , Afyonkarahisar , TurkeyAccepted author version posted online: 02 Aug 2012.Published online: 14 Sep 2012.

To cite this article: Y. Oznurlu , I. Celik , E. Sur , T. Ozaydın , H. Oğuz & K. Altunbaş (2012) Determination of theeffects of aflatoxin B1 given in ovo on the proximal tibial growth plate of broiler chickens: histological, histometric andimmunohistochemical findings, Avian Pathology, 41:5, 469-477, DOI: 10.1080/03079457.2012.712673

To link to this article: http://dx.doi.org/10.1080/03079457.2012.712673

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Determination of the effects of aflatoxin B1 given in ovo onthe proximal tibial growth plate of broiler chickens:histological, histometric and immunohistochemical findings

Y. Oznurlu1*, I. Celik1, E. Sur1, T. Ozaydın1, H. Oguz2 and K. Altunbas3

1Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Selcuk, Konya, Turkey,2Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Selcuk, Konya, Turkey, and3Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Afyonkocatepe, Afyonkarahisar,Turkey

Detrimental effects of aflatoxin B1 (AFB1) on the embryonic development of broiler tibia and its proximalgrowth plate were determined by means of histological, histometric and immunohistochemical methods.For this, 420 fertile eggs from parent stocks of Ross 308 broiler chickens were divided into five groupsaccording to the proposed treatment: a control untreated group, a group injected with 30% ethanol and threefurther groups to be injected with 5, 15 or 40 ng AFB1. The eggs were injected into the air space prior toincubation. Five eggs from each group were opened at 9, 11, 13, 17, 19 and 21 days of incubation and tibialtissue samples were removed, dissected of muscle and connective tissues, and processed by means of routinehistological techniques. The cell proliferation rate of the epiphyseal growth plate cells was determined byimmunohistochemical assay of proliferating cell nuclear antigen (PCNA) expression. The results showedthat both proliferative and hypertrophic zones narrowed significantly (PB0.05), when compared with thecontrols, in all of the AFB1-treated groups whereas the transitional zone thickened, especially in the groupgiven 40 ng AFB1 group. The PCNA positivity indices of 15 and 40 ng AFB1-treated groups weresignificantly higher (PB0.05) on days 11, 13, 17, 19 and 21 of incubation. It was concluded that in ovo-administered AFB1 adversely affected embryonic development of the tibial growth plate, and that affectedhatched broilers might also be more susceptible to skeletal disorders during growth.

Introduction

Aflatoxins (AFs) are a group of closely related andextremely toxic metabolites of Aspergillus flavus andAspergillus parasiticus and can occur as natural con-taminants of poultry food (Oguz et al., 2000; Sur &Celik, 2003). Although 18 different AFs have beenidentified, only AFB1, AFB2, AFG1 and AFG2 havebeen detected in food and foodstuffs (Leeson et al.,1995). Among them, AFB1 is the most toxic for mostanimal species (Leeson et al., 1995; Celik et al., 2000).The toxic effects of AFs in poultry have been widelyinvestigated (Giambrone et al., 1978; Celik et al., 1996;Sur & Celik, 2003) and are a major concern in poultryproduction because of serious economic losses resultingfrom anorexia, poor feed utilization, decreased bodyweight gain, decreased egg production, increased sus-ceptibility to microbial and unspecified diseases andincreased mortality (Qureshi et al., 1998; Tessari et al.,2006; Oguz, 2011).

The dietary AFs and their metabolites have carcino-genic, teratogenic, mutagenic and growth inhibitoryeffects, and they also infiltrate and accumulate in mostof the soft tissues and fat depots of the chicken (Leesonet al., 1995; Bintvihok et al., 2002). AF residues are alsofound in the egg (Jacobson & Wiseman, 1974; Sudhakar,

1992; Qureshi et al., 1998). Jacobson & Wiseman (1974)found 9 ng AFB1/egg in chickens receiving diets contain-ing 100 mg/kg dietary AFB1 on day 10 of feeding.Trucksess et al. (1983) fed laying hens with a dietcontaining 8000 mg/kg AFB1 for 7 days and found thatlivers and ova contained the highest levels of AFB1 andits metabolite AF Ro. In previous reports (Trucksesset al., 1983; Qureshi et al., 1998), the carry-over ratioof AFB1 from layer hen’s food into eggs was between1/2000 and 1/2500. Although in Turkey the legal upperlimits in food for laying hens are 10 mg/kg for AFB1 and20 mg/kg for AF (Ministry of Agriculture of Turkey,1997), surveys have shown that these limits werefrequently exceeded (Nizamlioglu, 1996; Oguz et al.,2011). In poultry food and foodstuffs, AF contamina-tion and residues may be a serious problem in thepoultry industry because of the residues in fertilized eggs(Dietert et al., 1985). Moreover, the carry-over of AFfrom the feed to the fertilized egg might result ineconomic losses by decreasing embryo viability andhatchability (Qureshi et al., 1998), and by causing severalorgan malformations (Cilievici et al., 1980).

Bone growth occurs at regions of specializedcartilage*the growth plates*situated at both ends of

*To whom correspondence should be addressed. Tel: �90 332 2233571. Fax: �90 3322410063. E-mail: yoznurlu@selcuk.edu.tr

Avian Pathology (October 2012) 41(5), 469�477

Received 9 May 2012

ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/12/050469-09 # 2012 Houghton Trust Ltdhttp://dx.doi.org/10.1080/03079457.2012.712673

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all long bones. The growth plate comprises chondrocytesand their extracellular matrix where proteoglycans andcollagen type II predominate (Farquharson et al., 1992;Farquharson & Jefferies, 2000). During the longitudinalbone growth, progressive changes occur in the prolifera-tion rate, shape and size of the chondrocytes, and in thesynthesis and deposition of extracellular matrix compo-nents of the growth plate cartilage (Farquharson et al.,1995; Farquharson & Jefferies, 2000). These gradualchanges result in formation of the resting, proliferating,maturing/transitional, hypertrophic and degeneratingchondrocyte zones in the epiphyseal growth plate(Loveridge et al., 1993; Germiller & Goldstein, 1997).In the embryo, a classical ‘‘reserve zone’’ does not exist(Germiller & Goldstein, 1997). Many studies investigat-ing the rate of longitudinal bone growth have focused onproliferative and volumetric changes in the hypertrophicchondrocytes. In mammals, a linear relationship existsbetween the vertical height of the hypertrophic chon-drocyte in the direction of growth and the bone growthrate. This variation in cell height accounts for thedifferent growth rates between the animal species andthe different growth rates that occur at the proximal anddistal ends within the same long bone. This relationship,however, is not the case in birds, and it is the thickness ofthe avian growth plate that is related to the rate oflongitudinal bone growth. Further studies have indicatedthat variation in the size of the hypertrophic chondro-cytes in different avian species is small, and the largestfactor in determining the rate of growth is the size of theproliferative cell population within the growth plate(Leach & Gay, 1987; Kember et al., 1990; Germiller &Goldstein, 1997; Farquharson & Jefferies, 2000).

The proliferating cell nuclear antigen (PCNA), alsoknown as cyclin or DNA-polymerase delta auxiliaryprotein, is a 36-kb non-histone nuclear protein thatfunctions as the co-factor for DNA polymerase delta(Linden et al., 1992). PCNA levels increase rapidly in themid-G1 phase of the cell cycle, remain elevated through-out the S-phase and begin to decrease from the G2/M toG1 phase (Goldsworthy et al., 1993).

In this study, detrimental effects of AFB1 onthe embryonic development of tibia and its proximalgrowth plate were determined by means of histological,histometrical and immunohistochemical methods.

Materials and Methods

Preparation of AFB1 solutions. Pure AFB1 was obtained from Makor

Chemical Co. (Jerusalem, Israel). It was diluted in benzene to prepare a

stock solution containing 20 mg/ml. The solution was transferred into

vials to contain the desired concentrations of AFB1 for each dose group,

and left overnight for the benzene to evaporate. The AFB1 residue was

dissolved in absolute ethanol (99.9%), which was then reduced to 30%

with sterile double-distilled water (ETOH). The AFB1 concentration of

these solutions was measured in duplicate by a thin layer chromato-

graphy (TLC) densitometer equipped with a fluorescence detector

(MPF 43A; Perkin Elmer, Santa Clara, California, USA) at 365 nm

excitation and 425 nm emission wavelengths, and by an ultraviolet�visible recording spectrophotometer (UV 2100; Shimadzu, Nakagyo/

KU, Kyoto, Japan) using standards.

Treatment groups and injections. For the experiments, 420 fertile eggs of

Ross parent stock were used. The eggs were weighed and then divided

into five groups, as follows: Group 1, control group (45 eggs); Group 2,

ETOH-injected group (50 eggs); Group 3, 5 ng AFB1/egg group

(74 eggs); Group 4, 15 ng AFB1/egg group (99 eggs); and Group 5,

40 ng AFB1/egg group (152 eggs). Eggs were sanitized by fumigation

with 80 g potassium permanganate in 130 ml of 40% formaldehyde

solution/m3 for 20 min.

Treatments were performed just prior to placing the eggs in the

incubator. After drilling the egg shell at the blunt end, 20 ml test solution

was injected into the air space (Celik et al., 2000) using micropipettes

(Sealpette, Jencons, Finland) with sterile tips. After injection, the hole

was immediately sealed with melted paraffin. The eggs were placed in an

incubator (VGS 108; Kap, Istanbul, Turkey) maintained at 37.88C with

65% relative humidity and turned through 2708C every 2 h.

Determining the stage of embryonic development. From each group and

on each of days 9, 11, 13, 17, 19 and 21 of incubation, five randomly

selected eggs containing a developing embryo were weighed with a

digital balance (sensitivity: g90.01, PT 100; Sartorius Goettingen,

Germany) and opened. Their yolk sacs were also weighed. The

developmental stage of each embryo was determined according to the

Hamburger�Hamilton scale (Hamburger & Hamilton, 1951).

The mean relative embryo weight [(embryo weight/egg weight)�100]

and relative yolk sac weight [(yolk sac weight/egg weight)�100] of each

group was calculated.

Histological investigations. Tibias were removed whole, cleared of

muscle and connective tissues and weighed. Tibial length was measured

with a digital caliper (sensitivity: mm90.01).

For histology, the tissue samples were fixed in 10% buffered-formal

saline and decalcified through successive changes of ethylenediamine

tetraacetic acid solution, dehydrated, cleared and embedded in paraffin

blocks (Culling et al., 1985). For routine histological examination, tissue

sections were stained with Crossman’s trichrome (Culling et al., 1985)

and Alcian blue (Bancroft et al., 1994).

Immunohistochemical procedure. For immunohistochemical PCNA

staining of the samples, paraffin sections on glass slides coated with

poly-L-lysine were deparaffinized in xylene, hydrated and then placed in

phosphate-buffered saline (pH 7.6). Antigen retrieval was performed by

boiling for 10 min in citrate buffer (0.01 M). Sections were treated with

3% hydrogen peroxide for 20 min to quench endogenous peroxidase

activity, rinsed in deionized water and washed with phosphate-buffered

saline. Sections were incubated first with blocking serum (UHP 125;

ScyTek, Logan, Utah, USA) to reduce non-specific staining, and then

with a monoclonal antibody against PCNA (PC10, catalogue number

GTX71945; Genetex, Irvine, California, USA) at 378C in a moist

chamber for 60 min. The antibody was diluted 1:100 (v/v) with antibody

diluent solution (ABB125; ScyTek, Logan, Utah, USA). Antibody

detection was performed using a sensitive biotin�streptavidin detection

system (UHP 125; ScyTek) with 3,3?-diaminobenzidine tetrahydrochlor-

ide solution as chromogen (ACK125; ScyTek). Sections were counter-

stained with Mayer’s haematoxylin, dehydrated and then mounted. In

the negative control slides, tissue sections were incubated with

phosphate-buffered saline without the primary antibody. In PCNA

immunostaining, the positive chondrocytes were clearly distinguished

by their diffusely stained brownish-black nuclei.

Histomorphometry. The specimens were examined under a Nikon

Eclipse E-400 light microscope (Nikon Corporation, Chiyoda-ku,

Japan) equipped with a DS-5M digital camera and DS-L1 camera

control unit (Nikon Corporation). The percentage of PCNA-positive

chondrocytes was determined by counting the PCNA-expressing

chondrocytes in randomly selected triplicate fields of 10,000 mm2 areas.

Statistical analyses. The embryo weight, tibia weight, tibia length and

results of the histomorphometric analysis of the growth plate were

analysed by one-way analysis of variance and followed by post hoc

Duncan multiple comparisons tests using the Statistical Package for

Social Sciences (SPSS version 10.0; SPSS Inc., 145 Savery Hall,

University of Washington, USA. Differences were regarded as signifi-

cant at PB0.05.

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Results

Distribution of embryonic mortalities. The mean inferti-lity of all eggs used in the study was 7.85%. Mortality inthe control group was 6.97% (Table 1) and no develop-mental abnormalities were observed. In the 30% ETOH-injected group, mortality was 10.86%. Mortality rates inthe AFB1-injected groups increased in a dose-dependentmanner. Thus the mortality of 5, 15 and 40 ng AFB1/egggroups was 19.40%, 51.08% and 84.67%, respectively(Table 1).

Embryonic deaths occurred at earlier stages withincreasing doses of AFB1. They occurred mainlyduring the first 70 to 72 h (Hamburger�Hamilton scale19) of incubation and none of the embryos in the 40 ngAFB1/egg group survived to day 21 of incubation.

Tibia length and relative weights of embryo, yolk sac andtibia. The relative embryo weights, yolk sac weights, tibiaweights and tibia length on days 9, 11, 13, 17, 19 and 21of incubation are given in Figure 1. The mean relativeembryo weights of the control and solvent groups weresignificantly (PB0.05) higher than those of the AFB1

groups (Figures 1 and 4a). Relative yolk sac weights ofthe group given 40 ng AFB1/egg were found to besignificantly higher (PB0.05) on days 13, 17, 19 and 21

of incubation when compared with those of the control

group. The mean relative tibia weight and tibia length

decreased in a dose-dependent manner. The relative tibia

weights of the group treated with 40 ng AFB1/egg were

significantly (PB0.05) lower than those of the other

groups (Figures 1 and 4b).

Histological findings. Growth plates in the control groupdisplayed normal histology. The proliferative zone was

the most densely cellular zone of the growth plate. The

chondrocytes were typically aligned and densely pack-

aged in vertical columns. They were elliptical in shape

and had oval nuclei. The transitional zone, a narrow

zone adjacent to the distal edge of proliferative zone,

contained chondrocytes that were round to polygonal in

shape and not well organized into vertical columns.

Transitional chondrocytes typically had central vesicular

nuclei. The chondrocytes within the hypertrophic zone

appeared similar to those in the transitional zone except

that they were somewhat more organized into vertical

columns. They had more cytoplasmic vacuoles and

ragged cell boundaries.The most obvious changes in the AFB1-treated groups,

especially in the groups given 15 and 40 ng, involved the

proliferative and transitional zones. The chondrocytes of

these zones appeared necrotic (Figure 4c to f). The results

of the measurements of the growth plate showed that

both proliferative and hypertrophic zones significantly

(PB0.05) narrowed in the AFB1-treated groups whereas

the transitional zone thickened, especially in the group

given 40 ng AFB1 (Figure 2).PCNA-positive chondrocytes frequently located in

the proliferative, transitional and hypertrophic zones

(Figure 5a to f). PCNA positivity percentages of the 15

and 40 ng AFB1-treated groups were significantly higher

(PB0.05) on incubation days 11, 13, 17, 19 and 21

(Figure 3).

Table 1. Mean mortality values of the control and

experimental groups.

Group Number of eggs Mortality (%)

Control 45 6.97D

30% ETOH 50 10.86CD

5 ng AFB1/egg 74 19.40C

15 ng AFB1/egg 99 51.08B

40 ng AFB1/egg 152 84.67A

Values within a column with no common uppercase superscript

letter are significantly (PB0.05) different.

Figure 1. Relative embryo weights, relative yolk sac weights, relative tibia weights and tibia lengths of the groups. Results are means and

bars are standard deviations. No embryos survived on day 21 in the group given 40 ng AFB1.

The effects of AFB1 on the growth plate 471

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Discussion

AFs are important to the poultry industry because of

their frequent occurrence in feedstuffs, which produces

severe economic losses, and health problems in poultry

(Leeson et al., 1995; Santurio et al., 1999). Although the

maximum tolerable levels in Turkey in terms of total

AFs are 20 mg/kg for layer feed and 50 mg/kg for

feedstuffs (Ministry of Agriculture of Turkey, 1997),

some investigators have detected higher levels ranging

from 5 to 100 parts/109 in poultry food and foodstuffs

(Nizamlioglu, 1996; Oguz et al., 2011). Regarding the

carry-over ratio of 1/2000 and an AFB1 level of 10 mg/kg,an egg would contain 1.3 ng AFB1 if the hen consumed130 g food daily and laid every other day. Although theestimated level of AFB1 might not cause importantproblems in human health, it may cause serious pro-blems in the poultry.

Using the chick embryotoxicity screening test-I(CHEST-I), Jelinek et al. (1985) determined the embry-otoxicity limits for AFB1 as 0.3 to 30 ng/egg, and theteratogenicity limits as 3 to 30 ng/egg. In the presentstudy, relatively higher doses (5, 15 and 40 ng AFB1/egg)were used since the limits are frequently exceeded.

Figure 2. Mean widths (mm) of the growth plate zones of the groups. Results are means and bars are standard deviations. No embryos

survived on day 21 in the group given 40 ng AFB1. No measurements were made on day 11 for the group given 40 ng AFB1 because the

chondrocyte zones were not distinguished.

Figure 3. PCNA immune-positive cell percentages in growth plate chondrocytes of the groups. Results are means and bars are standard

deviations. There were no measurements on day 11 in the group given 40 ng AFB1 because the chondrocyte zones were not distinguished.

No embryos survived on day 21 in this group.

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In poultry food and foodstuffs, contamination and

residues may cause a serious poultry production problem

in fertilized eggs (Dietert et al., 1985). The carry-over of

AF from the feed to the fertilized egg causes serious

economic loss because the residual AF can affect embryo

viability and hatchability (Qureshi et al., 1998; Sur et al.,

2011) and causes organ malformations (Cilievici et al.,

1980).The mechanism of the effect of AFB1 in adults is well

established but there is less information about the toxicity

and detoxification mechanisms of embryonic cells. The

effect of AFB1 on DNA is the result of interaction of the

toxin with reactive sites of the macromolecule, and two

types of interaction are known to occur. One of them

results from a weak, reversible, non-covalent binding,

while the other is an irreversible covalent binding that

leads to the formation of AF�DNA adducts. Biotrans-

formation of AFB1 leads to the formation of a number of

metabolic products, particularly hydroxylated deriva-

tives. The metabolite AFB1-8,9-oxide is considered to

be responsible for the carcinogenic effect due to its high

ability to react with nucleophilic sites in macromolecular

components (Leeson et al., 1995). The compound is

extremely unstable and reacts with nuclear DNA and the

major adduct is formed at the N(7)-position of guanine.

This adduct could give rise to mutations inhibiting the

synthesis of RNA and hence enzymes and other proteins

(Jeffery et al., 1984; Hatch, 1988). The formation of

DNA�AF adducts results in malignant transformations

and deletions (Lafarge & Frayssinet, 1970) and sister

chromatid exchanges (Potchinsky & Bloom, 1993).Although there is less information about the toxicity

and detoxification mechanisms of the early embryonic

cells, the chick embryo gains detoxifying ability by day 5

to day 6 of incubation, shortly after which liver and

kidneys are functionally developed (Hamilton & Bloom,

1986). Further, in vitro studies using chick embryos

(Joshi & Joshi, 1981), rat embryos (Geissler & Faustman,

1988) and pre-implantation mouse embryo cell cultures

(Irvin et al., 1991) showed that the toxin arrested mitosis

Figure 4. 4a: Embryo from control group (left) and one from the 40 ng AFB1-treated group (right) at day 13 of incubation. 4b: Tibia of

a control embryo (left) and of an embryo from the 40 ng AFB1-treated group (right) at day 13 of incubation. 4c, 4d: Sections of tibial

growth plate of an embryo from the 40 ng AFB1-treated group at day 13 of incubation. Bar�1000 mm. 4e: Section of tibial growth plate

of an embryo from the 40 ng AFB1-treated group at day 17 of incubation. Bar�1000 mm. 4f: Section of tibial growth plate of an embryo

from the 40 ng AFB1-treated group at day 19 of incubation. Arrows indicate necrotic chondrocytes. Bar�100 mm: Alcian blue.

The effects of AFB1 on the growth plate 473

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and induced teratogenesis due to reduction in cell

proliferation during early morphogenesis. In this study,embryonic deaths occurred at the earlier stages of

incubation in AFB1-treated eggs. The higher mortality

of fertilized eggs occurred with increasing toxin doses ina positive dose�response manner (Table 1). These results

are similar to the results of previous studies (Dietertet al., 1985; Prelusky et al., 1987; Celik et al., 2000; Sur &

Celik, 2003).Aflatoxicosis in poultry is characterized by listlessness,

anorexia with lowered growth rate, poor food utiliza-tion, decreased weight gain, increased susceptibility to

environmental and microbial stresses and increased

mortality (Bailey et al., 1998; Parlat et al., 1999). Themost important effect of aflatoxicosis is poor body

weight gain, which directly affects profit. Aflatoxicosissignificantly depresses body weight gain (Huff et al.,

1986, 1988; Kubena et al., 1993, 1998; Scheideler,

1993; Oguz & Kurtoglu, 2000), and the mechanism for

this effect includes inhibition of RNA, DNA synthesis

as well as RNA polymerase activity (Hatch, 1988).A consequence of inhibition of RNA and DNA synth-

esis involves reduced protein synthesis, which ultimately

would reduce growth. In this study, mean relativeembryo weights of the AFB1-given groups were signifi-

cantly (PB0.05) reduced by AFB1 treatment (Figure 1).AF has also been reported to inhibit the development

and growth of bone tissue in broiler chickens, resulting inretardation of the skeletal system development, and the

effects are more pronounced in the tibia (Huff et al.,1980). These researchers also determined that breaking

strength and tibial diameters significantly (PB0.05)

decreased in the birds exposed to high levels of AF(5.0 and 10 mg/g) and ochratoxin (4.0 and 8.0 mg/g).

Chaudhry (1996) reported that the length of the femurand tibia, and the weight of the femur, tibia, radius and

ulna were significantly lower in birds fed continuously

with 5 mg/g AF in the feed for 6 weeks than birds that did

Figure 5. Chondrocytes expressing PCNA in sections of tibial growth plate demonstrated by immunostaining (arrows). 5a: Control

embryo at day 13 of incubation. 5b: Embryo from the 40 ng AFB1-treated group at day 13 of incubation showing increased numbers of

immunoreactive cells. 5c: Control embryo at day 17 of incubation. 5d: Embryo from the 40 ng AFB1-treated group at day 17 of incubation

showing increased numbers of immunoreactive cells. 5e: Control embryo at day 19 of incubation. 5f: Embryo from the 40 ng AFB1-treated

group at day 19 of incubation showing increased numbers of immunoreactive cells and also necrotic chondrocytes (arrowheads).

Bar�100 mm.

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not receive AF. In this study, retardation of skeletaldevelopment was evident especially at high levels ofAFB1. The results of tibia measurements have revealedthat AFB1-treated groups displayed lower mean tibialengths and weights when compared with the controlgroup (PB0.05; Figure 1).

Yolk, the main energy source for the developingembryo, supplies more than 90% of the total energyrequirements of the embryo by oxidation of yolk lipids(Speake et al., 1998). The weight and percentage of dry-matter accumulation of embryos are closely related tothe fatty acid composition of the yolk (Peebles et al.,1999). Thus, the yolk and its fatty acid content areessential for meeting the nutritional requirements ofdeveloping embryos (Yalcin et al., 2008). In this study,the yolk sac weight was significantly higher in AFB1-treated groups than those of the control birds. Our dataalso demonstrated that the embryo weights significantlydeclined in the AFB1-treated groups (Figure 1).

Bone abnormalities are yet another problem ofeconomic importance to the animal industry wheremycotoxin involvement is implicated. The epiphysealgrowth plate plays a key role in the skeletal development,and factors influencing the metabolism of bone tissuecan lead to skeletal abnormalities (Leach & Gay, 1987;Olkowski et al., 2011). In developing and fast-growingbones, such as the tibia, growth plate cartilage deter-mines the rate of longitudinal growth as well as theultimate length of bones (Orth, 1999). Previously,Fusarium roseum-contaminated corn was found toinduce a high Fusarium-induced osteochondrosis inci-dence of leg lesions in broiler chicks (Walser et al., 1982;Haynes et al., 1985; Farquharson, 1999). Chicks fed 2%F. roseum culture had a 90% incidence of tibialdyschondroplasia, which was well advanced by 4 to 6weeks of age (Walser et al., 1982; Haynes et al., 1985).Haynes et al. (1985) reported that the most consistentchanges in the proximal tibial growth plates from birdswith Fusarium spp.-induced tibial dyschondroplasiawere thickening of the transitional zone and increaseddistance between the tips of the metaphyseal vascularsprouts and proliferative/transitional junction. Similarly,Chaudhry (1996) reported that growth plate thicknesswas significantly reduced in 6-week-old birds given a dietcontaining 5 mg/g AFB1 when compared with birds givena control diet.

The results of the growth plate measurementsshowed that both proliferative and hypertrophic zonessignificantly narrowed (P B0.05; Figure 2) in the AFB1-treated groups whereas the transitional zone thickened,specifically in the group given 40 ng AFB1, whencompared with the controls. These results show thatsome skeletal deformities might be expected at furtherstages of the post-hatch period in the affected birds.

PCNA is a processing factor for DNA polymerasedelta auxiliary protein and has essential roles in thereplication and repair of damaged DNA (Shivji et al.,1992, 1998). PCNA immunohistochemistry has beenused as a potential tool for the study of proliferativeactivity of the tissues because a PCNA-positive cellindicates the replication and repair of damaged DNA.Banlunara et al. (2005) have reported that the percentageof PCNA-positive hepatocytes was significantly higherin ducklings given a diet containing AFB1 than in othergroups. In our study, PCNA positivity indices of the 15and 40 ng AFB1-treated groups were significantly

(PB0.05) higher on days 11, 13, 17, 19 and 21 of

incubation (Figure 3).In conclusion, this study has shown that in ovo

administrated AFB1 adversely affected embryonic devel-

opment of the tibial growth plate, and as a consequence

affected birds might be more susceptible to skeletal

disorders later in life. It was thought that the reason for

the increase of PCNA-positive cell indices in AFB1-

treated groups depended on the increase in DNA

polymerase activity. Because the increase in DNA

polymerase activity results from essential changes in

DNA behaviour, high PCNA indices might have arisen

from AFB1�DNA adduct formation and also DNA

damage.

Acknowledgements

This study was financed by SUBAP ‘‘Scientific Research

Projects Coordinating Office of Selcuk University’’

(Project No. 08401069).

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