Research in Veterinary Science 97 (2014) 297303

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Research in Veterinary Science

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Effects of dietary Crotalaria pallida seeds on the health and

performance of laying hens and evaluation of residues in eggs
Gonzalo J. Diaz a,*, Leidy X. Almeida a, Dale R. Gardner b
a
b

Laboratorio de Toxicologa, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Bogot, Colombia
ARS, Poisonous Plant Research Laboratory, USDA, 1150 E. 1400 N., Logan, UT 84341, USA

A R T I C L E

I N F O

Article history:
Received 18 February 2014
Accepted 28 June 2014
Keywords:
Crotalaria pallida
Pyrrolizidine alkaloids
Usaramine
Eggs
Laying hens

A B S T R A C T

The effect of three dietary concentrations of Crotalaria pallida (C. pallida) seeds (0, 1, 2, and 3% w/w) of
their normal diet were investigated in commercial laying hens during a 35 day feeding trial. All concentrations of C. pallida decreased body weight and feed intake (P < 0.05). Egg mass production and average
egg weight were decreased by feeding of 2% C. pallida seeds (P < 0.05). All concentrations of C. pallida
increased relative lung weight and serum activity of ALT, AST and LDH (P < 0.05); 3% C. pallida seeds decreased liver weight (P < 0.05). Analysis of the C. pallida seeds for dehydropyrrolizidine alkaloid content
detected usaramine and its N-oxide at a total alkaloid concentration of 0.18% (dry weight). Usaramine
was also detected in the eggs of all hens fed C. pallida seeds.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Pyrrolizidine alkaloids (PAs) are a large group of compounds
found in more than 6000 plant species (Chen et al., 2010). The 1,2dehydropyrrolizidine alkaloids (DHPAs) are considered most toxic
and are known to cause intoxication in animals and humans (Chen
et al., 2010; Mattocks, 1986). Most of the plants capable of causing
toxicosis in animals and humans belong to the genera Senecio,
Heliotropium, Symphytum and Crotalaria (Edgar et al., 2011). Crotalaria
spp. are commonly known as rattlebox or crotalaria, and are grown
as cover crops on sandy soils. Crotalaria spp. have been shown to
contain monocrotaline-type DHPAs, including the highly toxic
monocrotaline (Pilbeam et al., 1983) as well as fulvine, retusamine,
and trichodesmine (European Food Safety Authority, 2011). Crotalaria
spp. may also contain senecionine-type DHPAs (e.g. integerrimine,
jacobine, retrorsine, senecionine, seneciphylline) which are particularly common in Senecio spp. (European Food Safety Authority,
2011). Poisoning by crotalaria has been reported in horses (Rose et al.,
1957), rats (Copple et al., 2006), donkeys (Pessoa et al., 2013), sheep
(Nobre et al., 2005), chickens and pigs (Hooper and Scanlan, 1977).
DHPAs may also be transferred to animal products (meat, milk, eggs
and honey), causing an additional exposure risk for humans (Edgar
and Smith, 2000; European Food Safety Authority, 2011).

* Corresponding author. Tel.: +57 (1) 316-5000 Ext. 19450; fax: +57 (1) 316-5000
Ext. 19469.
E-mail address: gjdiazg@unal.edu.co (G.J. Diaz).
http://dx.doi.org/10.1016/j.rvsc.2014.06.011
0034-5288/ 2014 Elsevier Ltd. All rights reserved.

Crotalaria pallida Aiton (C. pallida, smooth crotalaria, striped

crotalaria, smooth rattlebox) is a less known species that has been
reported to be toxic for chickens (Diaz et al., 2003) and goats
(Hernndez and Canchila, 2001). C. pallida is a species native to Africa
and usually grows in warm, open areas and in arid and semiarid
regions (Fonseca et al., 2006). C. pallida is also used as green manure
in rotation with animal feed or human food crops (Uratani et al.,
2004). Consequently, there is potential for weedy re-growth that
could lead to contamination of the food crop with seeds and other
plant-parts of C. pallida. The present study investigated the effect
of contamination of chicken feed by seeds from C. pallida on the
health and productivity of the hens, and on the potential for transfer of toxic DHPAs into the human food chain via the eggs.

2. Materials and methods

Forty 32-week-old commercial laying hens (ISA Babcock Brown)
were placed in individual wire cages in an environmentally controlled room at 20 C with articial light provided 14 h per day. The
hens received feed and water ad libitum. The experiment was conducted following the welfare guidelines of the Poultry Research
Facility, College of Veterinary Medicine, National University of Colombia and was approved by the local ethics committee. A completely randomized experimental design was employed consisting
of four experimental groups with 10 hens per group. The four treatment diets were a commercial layer diet mixed with dried and
ground (>86% of particles <20 mesh) C. pallida seeds. In addition to
a negative control Group 1 receiving commercial layer diet, Groups

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G.J. Diaz et al./Research in Veterinary Science 97 (2014) 297303

2, 3 and 4 received the same diet but contaminated with 1%, 2%

or 3% (w/w) C. pallida seeds respectively. Seeds were collected
from specimens in a single eld, positively identied as C. pallida
by botanists of the Colombian National Herbarium of the National
University of Colombia.
The diets were fed for 5 weeks. Feed intake, egg mass production (g/hen), egg weight and number of eggs laid were monitored
at weekly intervals. Hens were individually weighed at the beginning of the experiment and at days 7, 14, 21, 28, and 35. Samples
of approximately 3 mL whole blood were collected from each hen
by venepuncture of the main brachial vein (vena brachialis) on days
0, 14 and 28 of the experiment. After centrifugation of the blood,
serum activities of aspartate aminotransferase (AST, P/N 41293),
alanine aminotransferase (ALT, P/N 41282), lactate dehydrogenase
(LDH, P/N 41222), gamma-glutamyltransferase (GGT, P/N 41293),
and creatine kinase (CK, P/N 41250) were determined by means of
a kinetic ultraviolet spectrophotometric method, using commercially available kits (Spinreact, Girona, Spain).
Eggs were collected the day before the start of the experiment
(day 0) and afterwards at weekly intervals (days 7, 14, 21, 28, and
35) for the analysis of DHPA content. Four or ve eggs per dietary
treatment were cracked open, their contents combined and homogenized and then freeze dried. In addition to these sampling
times, eggs were also collected from the remaining hens 1 week after
the end of the experiment and were analyzed for DHPAs.
At the end of the experiment, six hens from each dietary treatment were humanely euthanized in a CO2 chamber and examined
for gross lesions. The liver, lungs, heart, spleen, kidneys, proventriculus, gizzard and pancreas from each bird were removed and
weighed. Samples of liver and lung were xed in neutral buffered
10% formalin for 24 h, embedded in paran, sectioned at 5 m and
stained with hematoxylin and eosin for histological examination.

2.2. HPLC-MS analysis of DHPAs

The HPLC-(esi+)MS system included an Agilent 1260 binary pump,
an Agilent 1260 autosampler, a Synergi Hydro-RP column
(Phenomenex, 150 2.0 mm, with guard column) coupled to a Velos
Pro linear ion trap mass spectrometer (ThermoScientic) and a
heated electrospray ion source (HESI). A gradient mixture of acetonitrile (A) and 0.1% formic acid (B) was used for the mobile phase
at a ow rate of 0.4 mL/min. The timed gradient elution conditions were: 3% A (02 min); 370% A (linear gradient 215 min);
703% A (1518 min), and back to 3% A (1825 min). For the seed
analyses, the mass spectrometer used single stage MS detection with
a scan range of m/z 200800. As the C. pallida seed material was
found to contain usaramine as the major alkaloid, only usaramine
was targeted in the dried egg sample analyses using selected MS/
MS analysis. For the analysis of dried egg samples the mass spectrometer was set to monitor daughter ions (m/z 100400) from the
parent ion (m/z 352.2) in MS/MS mode using a relative collision value
of 35. Sample injection size was 5 L for all analyses. Quantitative
analyses were made by area under the curve of the selected ion trace
versus a calibration curve prepared from a standard solution of
usaramine (>95%, Poisonous Plant Research Laboratory collection). For plant analyses the calibration standards were prepared
by serial dilution to give standard concentrations of 6.5, 3.25, 1.63,
0.81 and 0.40 g/mL and using selected ion trace (m/z 325) for the
protonated usaramine (R2 = 0.9993 for linear regression over complete range of standards). For the analyses of dried egg samples the
calibration standards were prepared by serial dilution to give standard concentrations of 125, 62.5, 31.2, 15.6, 7.8, 2.0, and 0.5 ng/mL
and using combined selected ion trace (m/z 120, 138, 276, 324) for
usaramine (R2 = 0.9998 for linear regression over complete range
of standards). Limit of quantitation (LOQ) was estimated to be 0.5 ng/
mL by a S/N ~ 10 in sample solutions with a consequent calculated LOQ of 2 ng/g in dried egg samples.

2.1. Sample extractions for analysis of DHPAs

2.3. Statistical analysis
In duplicate, C. pallida ground and homogenized seed material
(0.100 g) was extracted with 4.0 mL of 0.05M H2SO4 in a 15 mL screw
cap glass tube by mechanical rotation for 1 h. The samples were centrifuged for 10 min (2500 g). A 0.2 mL aliquot of the acid extract
was applied to solid phase extraction columns (Phenomenex, Strata
SCX, 200 mg, pre-rinsed with 2 mL methanol, 2 mL water and 1 mL
0.05 M H2SO4). The columns were rinsed with 4 mL water and then
4 mL methanol. The alkaloid fraction was then eluted with 3 mL ammoniated methanol (10% saturated ammoniated methanol in methanol). The samples were evaporated to dryness under a ow of
nitrogen at 60 C and 2.0 mL of 50% methanol (0.1% formic acid) was
added to reconstitute the samples. Samples were then analyzed for
DHPAs by HPLC-(esi+)MS and MS/MS. Alkaloids were identied by
comparison of retention time and MS/MS spectra with those from
standard samples obtained from the Poisonous Plant Research
Laboratory private collection of DHPAs.
In a similar manner, the dried egg samples (0.250 g) were extracted with 5.0 mL of 0.05M H2SO4 in a 20 mL screw cap plastic
tube by mechanical rotation for 1 h. Acetonitrile (5.0 mL) was added
to aid in protein precipitation and the samples were then mixed for
an additional 15 min. The samples were centrifuged for 10 min
(2500 g). A 4.0 mL aliquot of the acid/acetonitrile extract was applied
to solid phase extraction columns (Phenomenex, Strata SCX, 200 mg,
pre-rinsed with 2 mL methanol, 2 mL water and 1 mL 0.05 M H2SO4).
The columns were rinsed with 4 mL water and then 6 mL methanol. The alkaloid fraction was then eluted with 5 mL ammoniated
methanol. The samples were evaporated to dryness under a ow
of nitrogen at 60 C and 0.50 mL of 5% methanol was added to reconstitute the samples. Samples were ltered through a 0.25 m
syringe lter prior to analysis of DHPAs by HPLC-MS.

Except for descriptions of gross and histological lesions, data for

response variables serum enzymes and relative organ weights were
subjected to analysis of variance (ANOVA) for a complete randomized design, using Statistix for Windows version 9 (2008). Variable means from treatments showing signicant differences in the
ANOVA were compared using Tukeys test. Statements of signicance are based on P < 0.05. The effect of the C. pallida treatment
on performance parameters measured at weekly intervals was estimated by analysis of covariance for repeated measurements (SAS
Institute, 2008; GLM procedure) using a mixed linear model with
class variables C. pallida level and time and their interaction.
3. Results
Analysis of the C. pallida seeds by HPLC-MS for PAs resulted in
the detection of usaramine and its corresponding N-oxide (Fig. 1).
The concentration of usaramine was found to be 0.16% as the free
base, and an estimated 0.02% as the N-oxide, for a total DHPA content
of about 0.18% (dry weight basis). The estimated daily usaramine
dose received by the hens fed 1, 2 and 3% C. pallida seeds was 0.54,
0.99, and 1.41 mg/kg, respectively.
No differences in the body weight recorded at weekly intervals
were seen at the beginning of the experiment or at day 7 (Fig. 2).
However, at days 14 and 21 of the experiment the hens receiving
2 and 3% C. pallida seeds had lower body weights than the controls (P < 0.05) and at days 28 and 35 all hens receiving C. pallida
seeds had lower body weights (P < 0.05). At the end of the experiment, differences in body weight were found among all experimental groups (P < 0.05). At this stage, the body weights of the hens

G.J. Diaz et al./Research in Veterinary Science 97 (2014) 297303

299

OH
OH

OH

OH
O
O
O

N
N

Usaramine N-oxide

Usaramine

Fig. 1. Chemical structure of usaramine, the dehydropyrrolizidine alkaloid identied in the Crotolaria pallida seeds, and its N-oxide.

2000

1900

Body weight (g)

1800

1700

1600
Control
1% CP seeds
2% CP seeds
3% CP seeds

1500

1400
0

14

21

28

35

Experimental days
Fig. 2. Effect of dietary Crotalaria pallida seeds on the body weight of laying hens.
Each point is the mean S.E.M. of 10 birds per treatment.

receiving 1, 2 and 3% C. pallida seeds were 8.7, 14.8, and 21.2% lower
than that of the control group, respectively. There was a signicant (P < 0.0019) time C. pallida level interaction in body weight
during the course of the experiment.
The performance parameters for the whole experiment (35 days)
are shown in Table 1. Data analyzed at weekly intervals are not
shown. Feed intake was lower in all groups fed C. pallida seeds
(P < 0.05) both when measured at weekly intervals and for the
5-week experimental period. There was a highly signicant
(P < 0.0001) time C. pallida level interaction for this variable. Egg
mass production was lower (P < 0.05) in the hens fed 2% C. pallida
seeds at weeks 4 and 5 (data not shown) and for the 5-week experimental period (P < 0.05; Table 1). Hens fed 3% C. pallida seeds
produced a lower egg mass during weeks 2 through 5 (P < 0.05, data
not shown) and for the 5-week experimental period (P < 0.05;
Table 1). Egg mass production was not affected by feeding 1%
C. pallida seeds. Hens fed 2 and 3% C. pallida seeds produced 13.0
and 25.0% less egg mass, respectively, during the 5 weeks of the experiment compared with controls. Further, there was a highly
signicant (P < 0.0001) time C. pallida level interaction for egg mass
production. Average egg weight did not differ among the four groups
either when analyzed at weekly intervals or for the 5-week experimental period. The average number of eggs laid per hen was lower
(P < 0.05) only in the group fed 3% CP. Feed conversion (kg of feed/g
of egg mass) was lower (P < 0.05) than the control group at week
1 for the groups fed 1 and 3% C. pallida seeds. At weeks 2 and 4, hens
fed 2 and 3% C. pallida seed had a lower feed conversion (P < 0.05)

Table 1
Effect of graded levels of dietary Crotalaria pallida (CP) seeds supplemented during 35 days (weeks 3236 of age) on laying hen performance.*
Treatment

1
2
3
4
P

Dietary CP
seeds (%)

Performance parameter
Feed intake
(g/hen/day)

Egg mass
production
(g/hen)

Average egg
weight (g)

Feed conversion
(kg of feed/kg
of egg mass)

Number of
eggs laid/hen

0
1
2
3

121.0 3.4a
96.4 4.0b
82.3 5.7c
72.5 3.2c
0.000

2054 67a
1969 46ab
1786 103b
1541 47c
0.000

61.6 1.5a
61.4 0.7a
58.8 1.1a
59.1 1.0a
0.175

2.08 0.05a
1.72 0.05b
1.61 0.05b
1.66 0.06b
0.000

33.3 0.7a
32.0 0.5a
29.7 2.0ab
26.2 1.0b
0.001

Means within a column lacking a common superscript letter differ (P < 0.05).
* Values are mean S.E.M. of 10 birds per treatment for the 35 experimental days.

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G.J. Diaz et al./Research in Veterinary Science 97 (2014) 297303

Table 2
Effect of graded levels of dietary Crotalaria pallida (CP) seeds on the serum enzyme activity (U/l) of selected enzymes of laying hens.*
Treatment

Dietary CP
seeds (%)

Days of exposure to the experimental diets

0

14

28

Alanine amino transferase

1
2
3
4
P

9.5 0.8a
9.0 0.9a
8.1 0.8a
8.8 0.9a
0.680

0
1
2
3

5.9 0.7a
9.7 1.5b
11.4 1.3b
9.5 1.1b
0.049

14

28

Aspartate amino transferase

15.6 1.5a
13.2 1.6a
13.5 1.5a
16.2 2.0a
0.489

82.3 3.7a
86.5 3.9a
81.2 3.9a
73.0 4.2a
0.144

73.6 2.4a
75.6 4.4a
82.7 4.3a
90.7 8.7a
0.157

14

28

Lactate dehydrogenase
74.6 7.5a
106.3 8.9b
109.5 15.8b
101.0 6.4b
0.047

2069 275a
2194 275a
2107 275a
1582 292a
0.442

1252 117a
1617 229a
1607 189a
1134 88a
0.103

1604 205a
2195 214b
2377 195b
2108 213b
0.040

Means within a column lacking a common superscript letter differ (P < 0.05).
* Values are mean S.E.M. of 10 birds per treatment.

compared with controls, whereas at week 3 all groups fed C. pallida

seeds had a lower (P < 0.05) feed conversion ratio and at week 5 no
differences were observed. Overall, there was a lower (P < 0.05) feed
conversion rate for the 5-week experimental period for all hens fed
C. pallida seeds (Table 1). Further, there was a signicant (P < 0.007)
time C. pallida level interaction for feed conversion.
No differences were observed in the ve serum enzymes evaluated at day 0 (Table 2). Further, no differences in CK or GGT activity were found at any of the three sampling points (days 0, 14 and
28 of the experiment, data not shown). However, compared with
the control group, ALT serum activity was higher (twofold) in all
groups fed C. pallida seeds at day 14 (P < 0.05), while AST and LDH
activities were higher (P < 0.05) in the same groups at day 28 (1.31and 1.48-fold, respectively).
Organ weights taken at the end of the experiment showed differences only in the relative weights of liver and lungs (Table 3).
Hens receiving 3% C. pallida seeds had a lower (P < 0.05) liver weight
compared with controls (22.4% less), while all groups receiving
C. pallida seeds had greater (P < 0.05) lung weights compared with
control hens. Hens receiving 1, 2 and 3% C. pallida seeds had a 33.3,
25.0, and 30.6% increase in lung weight compared with controls.
Usaramine was not detected in the lyophilized egg samples taken
at weekly intervals in the control group at any of the sampling times
tested or in the eggs from day 0 in any treatment (Fig. 3). Usaramine,
however, was detected in the eggs of all hens receiving C. pallida
seeds in their diet at all sampling times. The maximum detected
concentration was 885 ng/g for the eggs collected at day 14 from
the hens receiving 3% C. pallida. The lowest concentration was
290 ng/g and was found at day 7 in the eggs from the hens receiving 1% C. pallida. Eggs collected 1 week after the end of the experiment (day 42) still contained detectable concentrations of usaramine
(11.5, 9.5 and 43.5 ng/g for the hens receiving 1, 2 and 3% C. pallida,
respectively). HPLC-(esi+)MS/MS chromatograms of control eggs (day
0 of the experiment) and from egg samples taken at day 28 from
the hens fed 2% C. pallida are shown in Fig. 4. No usaramine was
detected in the control sample, whereas a peak identied as
usaramine is shown in the egg sample from day 28.
The post-mortem examination of the six hens per treatment
selected at the end of the experiment showed no abnormalities in

control hens or those receiving 1% C. pallida. However, hens fed 2

and 3% C. pallida showed smaller livers compared with controls. The
livers of these hens also showed an abnormal discoloration (a
mottled brown-yellow color) and were rmer than normal. In addition, the hens fed 2 and 3% C. pallida had distended gallbladders.
Right ventricular dilation was observed in hens fed 2 and 3% C. pallida.
Some of the hens fed 2 and 3% C. pallida also had complete ovarian
follicular regression indicating that they had ceased laying (2/6 and
4/6 for the hens fed 2 and 3% C. pallida, respectively). Histological
examination of the lung samples revealed no abnormalities in any
treatment. However, liver samples from hens fed C. pallida showed
mild to moderate hepatocyte necrosis, megalocytosis and bile duct
proliferation. Liver samples from hens receiving 3% C. pallida also
showed marked hepatocyte anisokaryosis.
No mortality was recorded in any of the treatments during the
experiment.
4. Discussion
The importance of crotalaria seed contamination in animal feed
is now fully recognized and the European Communities through
Directive 2202/32/EC (2002) have regulated the maximum content
of Crotalaria spp. in all feedingstuffs. According to this directive, the
maximum content of Crotalaria spp. shall be 100 mg/kg (ppm), relative to a feedingstuff with a moisture content of 12%. This directive, however, does not specify whether this gure refers to 100 ppm
of seeds, seed material or Crotalaria spp. alkaloids.
The adverse effects of DHPAs from several plant species have been
well characterized in poultry (Hooper and Scanlan, 1977; Pass, 1982;
Pass et al., 1979; Pereira et al., 2011; Ross and Tucker, 1977). In the
case of Crotalaria spp., Crotalaria retusa and Crotalaria spectabilis
appear to be more toxic than C. pallida for chickens. In a study conducted by Hooper and Scanlan (1977), chickens fed 0.5% C. retusa
seeds containing 4.04.4% monocrotaline showed mortality 12 days
after the start of the experiment and by day 45 all chickens had died.
In another study, supplementation of 0.5% C. retusa seeds containing 4.4% monocrotaline for 8 weeks caused high mortality and
reduced feed intake, weight gain and feed conversion eciency (Ross
and Tucker, 1977). Laying hens fed 0.02, 0.04 and 0.06% C. spectabilis

Table 3
Effect of graded levels of dietary Crotalaria pallida (CP) seeds on selected relative organ weights of laying hens.*
Treatment

Dietary CP
seeds (%)

Relative organ weight (% body weight)

Liver

Heart

Lungs

Spleen

Kidney

Pancreas

1
2
3
4
P

0
1
2
3

2.01 0.09a
1.85 0.09ab
1.77 0.15ab
1.56 0.12b
0.039

0.41 0.02a
0.40 0.01a
0.43 0.02a
0.46 0.01a
0.112

0.36 0.02a
0.48 0.07b
0.45 0.05b
0.47 0.03b
0.001

0.09 0.02a
0.08 0.01a
0.11 0.03a
0.11 0.03a
0.123

0.70 0.08a
0.65 0.10a
0.64 0.07a
0.62 0.07a
0.401

0.18 0.01a
0.20 0.03a
0.20 0.05a
0.21 0.04a
0.584

Means within a column lacking a common superscript letter differ (P < 0.05).
* Values are mean S.E.M. of six birds per treatment.

G.J. Diaz et al./Research in Veterinary Science 97 (2014) 297303

1000
900

Usaramine concentration (ng/g)

800
700
600
500
400
300
Control
1% CP seeds
2% CP seeds
3% CP seeds

200
100
0
0

14

21

28

35

42

Experimental days
Fig. 3. Usaramine concentration (ng/g) in pooled lyophilized egg samples taken at
weekly intervals and determined by HPLC-MS/MS.

seeds in their diets for 28 days showed 5, 25 and 40% mortality

(Pereira et al., 2011), while no mortality was recorded in the present
experiment. The apparent difference in toxicity among these three
Crotalaria spp. might be related not only to the specic DHPAs present
in each species (monocrotaline in C. retusa and C. spectabilis vs
usaramine in C. pallida) but also to the concentration of DHPAs in
their seeds (3.55.0% in C. spectabilis (Hooper and Scanlan, 1977)
4.04.4% in C. retusa, and 0.18% in C. pallida).
Adult birds also appear to be more resistant to C. pallida seeds
since 1-day-old broiler chickens fed the same dietary levels used
in the present study for 21 days showed 2.1, 6.2 and 16.7% mortality for the 1, 2 and 3% C. pallida seed supplementation, respectively (Diaz et al., 2003).
The inclusion of 2 or 3% C. pallida in the diets of laying hens
signicantly affected egg production, an effect that has been reported previously. For example, hens fed ground aerial parts of
Senecio vernalis containing 0.14% total DHPA had a signicant decrease in feed intake and egg production when levels were 2%
(Erksz et al., 2003). Interestingly, dietary supplementation of
C. pallida seeds signicantly improved (lowered) feed conversion rate
in all treatments. This effect was probably caused by the decrease
in voluntary feed intake caused by the presence of C. pallida seeds
that was not accompanied by a proportional decrease in egg mass
production. It is well known that Crotalaria spp. are generally unpalatable (Sippel, 1964), which might explain the decrease in voluntary feed intake observed in the present experiment. The decrease
in feed intake could also be associated with the progression of the
DHPA toxicosis. The signicant (P < 0.05) time vs. body weight, feed
intake, egg mass production and feed conversion interactions indicate that the effects caused by C. pallida exposure are highly dependent on time of exposure. This nding corroborates previous
studies showing that the time of exposure determines the severity of the effect (Erksz et al., 2003).
Serum enzymatic activities are normally measured to evaluate
toxic-related effects on different organs. As expected, CK serum levels
were not signicantly different at any sampling time since CK is
mostly used to detect muscle damage. This nding is consistent with

301

previous studies showing no increase in CK activity associated with

liver damage (Diaz et al., 1999). On the other hand, three of the four
enzymes associated with liver damage (ALT, AST and LDH) were signicantly high in the hens receiving C. pallida seeds, although only
at one of the two sampling times tested after the start of the experiment. The increase in ALT activity found at day 14 but not at
day 28 might be due to a subacute liver response associated with
hepatocyte necrosis at day 14 vs. a more chronic response with less
enzyme leakage at day 28. Signicant increases in serum enzymes
associated with liver damage have been previously reported in chickens fed C. pallida seeds (Diaz et al., 2003). Also, signicantly higher
levels of ALT, AST and LDH have been reported in laying hens prone
to suffer from liver damage (Diaz et al., 1999). GGT was not increased in the present experiment, although feeding C. pallida seeds
caused a signicant increase in serum GGT in chickens (Diaz et al.,
2003). The greatest amount of cellular GGT is in the brush borders
of renal and bile duct epithelium (Kramer and Hoffmann, 1997). It
might be possible that mature birds (laying hens) are more resistant than young chickens (21 days of age) to the adverse effects of
DHPAs on bile duct epithelium. These ndings suggest that the measurement of serum enzyme activities needs to be done repeatedly
in order to increase the possibility to detect changes and that other
liver function tests should also be included.
The gross pathological ndings observed at the end of the experiment are in agreement with those reported in similar experiments where laying hens were exposed to DHPAs in their diets
(Erksz et al., 2003; Hooper and Scanlan, 1977; Pereira et al., 2011).
The change in the heart (right ventricular dilation) results from increased venous pressure associated with the liver damage. Pulmonary hypertension can result from right-ventricular failure which
could explain the increased relative weight of the lungs. The damage
to the liver is caused by the direct toxic action of the DHPA on the
hepatic cell (Edgar et al., 2011), while the distended gallbladders
are usually the result of cholestasis from decreased feed intake. Cessation of laying can be explained by the decrease in triglycerides
intake or a consequence of the liver damage, since the liver is responsible for the synthesis of the egg yolk components (Johnson,
1986). The histological changes observed in the liver samples analyzed are also in agreement with previous studies (Pereira et al.,
2011) and reect the direct damage caused by the DHPA to the hepatocyte. These changes also agree with the increase (P < 0.05)
in the serum activities of ALT, AST, and LDH found in at least one
sampling point.
Usaramine, the only DHPA detected in the C. pallida seeds used
in the present study, had been previously reported as the main DHPA
of C. pallida (Fletcher et al., 2009; Silva Flores et al., 2009). Usaramine
was rst isolated from seeds of Crotalaria usaramoensis (Culvenor
and Smith, 1966) and later reported in Senecio vulgaris plant material (Pieters and Vlietinck, 1988), other Senecio spp. (Gardner et al.,
2006; Krebs et al., 1996) and Pericallis spp. (Domnguez et al., 2008).
Usaramine is a geometrical isomer of retrorsine and belongs to the
cyclic diester group of DHPAs. Toxic DHPAs may exist as monoesters or diesters and the diester linkages may be cyclic or acyclic. Cyclic
diesters, such as usaramine, typically exhibit the highest toxicity
of the three forms; acyclic diesters are less toxic than cyclic diesters but more toxic than monoesters (Chen et al., 2010). The toxicity of usaramine estimated from the present study corresponds
to a highly toxic compound (150 mg/kg) according to the relative toxicities classication of Osweiler et al. (1985).
The transfer of usaramine from the seeds to the eggs is a very
signicant nding due to the hepatotoxic effects of DHPAs in humans.
Further, this study conrms the transfer of a DHPA from the diet
of hens to the eggs: hens receiving up to 3% dietary C. pallida containing 0.18% usaramine for 5 weeks showed egg concentrations of
290885 ng usaramine/g dried sample. In a previous eld case report,
laying hens fed a ration contaminated with Heliotropium europaeum

302

G.J. Diaz et al./Research in Veterinary Science 97 (2014) 297303

Fig. 4. HPLC-(esi+)MS/MS analysis of dried egg samples and detection of usaramine. (A) Total ion chromatogram (TIC) for egg sample from group 3 day 0 of treatment (control).
(B) TIC from the analysis of the eggs taken from group 3 (2% CP seed supplement) at day 28 with possible detection of usaramine at 6.81 min. (C,D) Reconstructed ion chromatogram for m/z (120, 138, 276, 324) selected for usaramine and the corresponding MS/MS spectrum.

seeds at an estimated concentration of 0.6% (w/w) laid eggs containing 49l68 ng/g total DHPAs (Edgar and Smith, 2000). Assuming an average water content of 73.5% in a whole egg (Sugino et al.,
1996), the usaramine content in the fresh eggs from the present experiment would range from 77 to 235 ng/g, values close to those
found by Edgar and Smith (2000). Since the average weight of the
egg contents is 51.7 g (Sugino et al., 1996), the total DHPA content
per egg would range from 4.0 to 12.1 g. The maximum allowed concentration of DHPAs in herbal medicines on sale in Germany is 0.1 g
per daily dose and this regulation provides a basis for assessing food
safety in relation to DHPA contamination (Edgar and Smith, 2000).
The estimated DHPA content of each egg from the present study

corresponds to 40- to 121-fold the maximum recommended 0.1 g

daily dose. Usaramine was still present in trace concentrations in
the eggs 7 days after cessation of exposure to the contaminated feed.
This may have some implications for determining a suitable withdrawal period before eggs from exposed hens can be accepted as
free of DHPAs.
In summary, the present study demonstrates that feeding laying
hens 2% C. pallida seeds causes signicant adverse effects on both
health and performance. Dietary levels of 1% C. pallida cause mild
adverse effects that do not affect performance; however, all C. pallida
levels tested lead to signicant accumulation of usaramine in the
eggs.

G.J. Diaz et al./Research in Veterinary Science 97 (2014) 297303

Acknowledgments
Thanks are due to Amparo Corts from the Toxicology Laboratory of the College of Veterinary Medicine, National University of
Colombia for her assistance and help with the in vivo trial. Thanks
are also due to Dr. Hermann Boermans and Dr. Bryan Stegelmeyer
for their constructive comments on the manuscript and to Prof. Nhora
Martnez-Rueda for her help with the statistical analysis.
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