Content uploaded by Christi A Yoder
Author content
All content in this area was uploaded by Christi A Yoder
Content may be subject to copyright.
Available via license: CC BY-NC-ND 4.0
Content may be subject to copyright.
Molecular Effects of Nicarbazin on Avian Reproduction
C. A. Yoder,*
1
J. K. Graham,† and L. A. Miller*
*National Wildlife Research Center, 4101 LaPorte Avenue, Fort Collins, Colorado 80521-2154;
and †Department of Biomedical Sciences/Physiology, Colorado State University, Fort Collins 80523
ABSTRACT Nicarbazin (NCZ) is an anticoccidial drug
routinely used in the poultry industry that can negatively
affect reproduction by reducing egg production, egg
weight, and egg hatchability. The molecular mechanisms
by which NCZ affects reproduction are unknown. Lipo-
protein lipase, vitellogenin, transglutaminase, and cal-
cium are all involved in egg formation and embryogene-
sis. Therefore, in vitro assays were used to evaluate 4
potential mechanisms of action of NCZ on egg formation
and embryogenesis. First, a lipoprotein lipase assay was
conducted to determine if NCZ increases lipoprotein li-
pase activity. Second, vitellogenin phosphorylation was
evaluated to determine if NCZ acts as a vitellogenin phos-
phatase. Third, transglutaminase activity was measured
to determine if NCZ inhibits transglutaminase activity.
Finally, bull sperm was used as a model to determine if
specific channel-mediated calcium uptake can be blocked
by NCZ. Nicarbazin increased the activity of lipoprotein
Key words: calcium ionophore, lipoprotein lipase, nicarbazin, transglutaminase, vitellogenin
2006 Poultry Science 85:1285–1293
INTRODUCTION
Nicarbazin (NCZ) is an anticoccidial drug routinely
used in the poultry industry since the 1950s to control
protozoan cecal and intestinal infections by Eimeria spe-
cies in broiler chickens. It is an equimolar complex con-
sisting of 4,4′-dinitrocarbanilide (DNC) and 2-hydroxy-
4,6-dimethylpyrimidine (HDP). The function of HDP is
to increase absorption of the material in the gut, whereas
DNC is the active anticoccidial drug (Cuckler et al., 1955;
Rogers et al., 1983). When fed to laying hens, NCZ affects
reproduction by reducing egg production, egg weight,
and egg hatchability (Jones et al., 1990b; Hughes et al.,
1991; Chapman, 1994).
Although the mechanisms by which NCZ reduces egg
production and egg weight are unknown, NCZ may be
preventing ova from maturing (Baker et al., 1957). Nec-
ropsy of hens fed a ration containing 90 ppm NCZ re-
2006 Poultry Science Association Inc.
Received January 27, 2006.
Accepted March 9, 2006.
1
Corresponding author: christi.yoder@aphis.usda.gov
1285
lipase in vitro at 3.9 and 7.8 g of NCZ/mL. Nicarbazin
increased intracellular calcium levels in bull sperm, sug-
gesting it also acts as a calcium ionophore. The portion
of the NCZ molecule responsible for the increase in intra-
cellular calcium is 2-hydroxy-4,6-dimethylpyrimidine.
Nicarbazin affected vitellogenin phosphorylation but
only at a concentration many times higher than expected
plasma values. Nicarbazin also inhibited transglutami-
nase activity in vitro. Whereas the 4,4′-dinitrocarbanilide
portion of the NCZ molecule inhibited transglutaminase
activity, the 2-hydroxy-4,6-dimethylpyrimidine portion
increased transglutaminase activity. All of these assays
were conducted in vitro; therefore these results should
be viewed as preliminary findings to aid in directing
further research on the effect of NCZ on reproduction in
vivo. Because NCZ increases lipoprotein lipase activity
and acts as a calcium ionophore, future experiments
should investigate these effects in particular.
vealed that the largest follicle was absent from the ovary
with no signs of recent ovulation or atresia (Baker et al.,
1957). Luck (1979) also found ovaries without a follicular
hierarchy and less well-developed oviducts in laying hens
treated with 375 ppm NCZ in their feed. Although NCZ
did not affect luteinizing hormone levels or pituitary re-
sponsiveness to luteinizing hormone releasing hormone,
it decreased the sensitivity of the chicken hypothalamus
to exogenous progesterone (Luck, 1979). Luck (1979) sug-
gested that egg production is decreased because yolk
deposition in the follicles is prevented. White Leghorn
hens fed 400 to 700 ppm NCZ in feed exhibited reduced
egg production concomitant with a 2-fold rise in plasma
cholesterol concentrations, supporting Luck’s hypothesis
(Weiss, 1979).
Egg yolk is comprised of very low density lipoprotein
(VLDL) and vitellogenin (VTG), both of which are pro-
duced in the liver in response to estrogen stimulation
(Hillyard et al., 1956; Kudzma et al., 1979; Green, 1980;
Shapiro, 1982; Wallace, 1985). The main constituent of
egg yolk is VLDL. Although nonlaying hens and roosters
have small amounts of serum VLDL, it is a major serum
component in laying hens (Burley et al., 1984). One com-
ponent of VLDL, apoVLDL-II, provides the VLDL parti-
YODER ET AL.1286
cles some resistance to degradation by lipoprotein lipase
(LL; Griffin et al., 1982; Schneider et al., 1990). In addition,
laying hen VLDL particles contain less apoC-II, a LL acti-
vator (Griffin et al., 1982; Griffin and Perry, 1985).
Vitellogenin is comprised of 1 lipovitellin and 2 phos-
vitin polypeptides (Deely et al., 1975; Chistmann et al.,
1977). One of the posttranslational modifications VTG
undergoes is the phosphorylation of serine residues on
phosvitin (Wang and Williams, 1982). The phosphates
confer a negative charge that allows phosvitin to bind
calcium and iron (Allerton and Perlmann, 1965; Clark,
1970; Taborsky, 1980). Because of this, VTG is the main
carrier of calcium and iron to the egg yolk (Morgan, 1975;
Grunder et al., 1980; Lopez-Berjes et al., 1981). Dephos-
phorylation of these serine residues on phosvitin prevents
the uptake of VTG into the follicle (Miller et al., 1982).
Hens treated with 400 ppm NCZ in their feed exhibit
reduced calcium binding by calcium binding protein and
blood hypercalcemia (Bar and Hurwitz, 1971). This indi-
cates that although VTG is produced, it is altered by NCZ
somehow, preventing it from binding calcium.
Upon release into the blood stream, VLDL and VTG
pass out of the capillaries surrounding the oocyte (Perry
et al., 1978a,b) and through the granulosa cell layer of
the oocyte. Once they reach the oolemma, they bind to
the same 95 kDa receptor (George et al., 1987; Stifani et al.,
1990; Barber et al., 1991). Clusters of occupied receptors
induce the formation of clathin coated pits that are en-
gulfed by the oolemma to become clathin coated vesicles
(Wyburn et al., 1965; Schjeide et al., 1969). Transglutami-
nase assists in the formation of clathin coated pits, and
inhibition of the enzyme prevents uptake of VTG (Tucci-
arone and Lanclos, 1981).
The molecular mechanism by which NCZ reduces egg
hatchability is also unknown. However, NCZ may change
the permeability of the vitelline membrane, creating an
unfavorable environment for embryonic development
(Polin, 1957; van Tienhoven et al., 1958; Cunningham,
1977). Laying hens fed NCZ produce eggs with mottled
yolks (Baker et al., 1957: Polin et al., 1957, Jones et al.,
1990a). Mottled yolks show a decrease in yolk solids (Cun-
ningham, 1976), and exhibit an increase in albumen (Cun-
ningham, 1977). In addition, yolk components such as
fat, protein, calcium, phosphorus, and iron decrease in
mottled yolks but increase in the albumen of eggs with
mottled yolks (Cunningham, 1976: Cunningham, 1977).
We hypothesized that if NCZ increases LL activity, it
could cause the degradation of VLDL in the blood prior
to reaching the egg. Because VLDL is the major compo-
nent of egg yolk, egg weight and egg production would
decrease as a result. Phosphorylation of serine residues
on VTG is necessary for calcium and iron binding and
binding to the 95 kDa receptor. Therefore, we hypothe-
sized that if NCZ acts as a phosphatase, it would prevent
VTG from binding to the receptor, thus reducing egg
weight and production. Additionally, there would be less
calcium and iron available to the embryo, which could
affect egg hatchability. We hypothesized that if NCZ in-
hibited transglutaminase (TG) activity, clathin coated pits
could not form and uptake of yolk components would
not occur, resulting in reduced egg weight and produc-
tion. We also hypothesized that if NCZ acts as a calcium
channel blocker, it could disrupt crucial ion gradients
needed for proper egg formation and embryogenesis.
We chose to focus on the mechanisms described in
the previous paragraph as potential targets of NCZ. The
objective of this study was to determine the molecular
mechanisms by which NCZ affects egg hatchability and
egg production. We accomplished this by testing 4
hypotheses as follows: 1) NCZ increases LL activity; 2)
NCZ acts as a VTG phosphatase; 3) NCZ inhibits TG
activity; and 4) NCZ acts as a calcium channel blocker.
METHODS AND MATERIALS
Lipoprotein Lipase Assay
The LL assay was based on the principle that LL will
cleave dibutyrlfluorescein (DBF), releasing fluorescein
that can then be measured in a spectrofluorometer (Del
Prado et al., 1994). Dibutyrlfluorescein was prepared as
described previously by Del Prado et al. (1994). Briefly,
10 mL of pyridine (P4036, Sigma Chemical Co., St. Louis,
MO), 30 mL of butyric anhydride (150540, Sigma Chemi-
cal Co.), and 10 mg of fluorescein (F6377, Sigma Chemical
Co.) were mixed at 23°C for 8 min and then incubated
in the dark for 24 h at room temperature. To this mixture
was added 30 mL of 100% ethanol (111000200, Pharmco
Products Inc., Brookfield, CT), and the mixture was incu-
bated at −20°C for 23 h. The mixture was thawed at 23°C
for 15 min, then mixed on a vortex mixer for 15 min to
break up the crystals.
Solvent was removed using a vacuum flask and 5.5 cm,
grade 362 filter paper (F2215-55, Baxter). The filtrate was
washed with 95% ethanol until the solvent ran clear, and
the DBF was stored in the dark at 4°C. A DBF stock
solution was made by dissolving 10 mg of DBF in 50
mL of ethylene glycol monomethyl ether (EGME; E2632,
Sigma Chemical Co.). A DBF working solution was made
by mixing 5 mL of DBF stock solution with 100 mL of
low potassium phosphate buffer (291 mOsm, pH = 7.09).
Disposable 12 × 75 mm borosilicate glass tubes (60825-
913, VWR International, Aurora, CO) were used for the
assay. To each test tube was added 1 mL of DBF working
solution and 15 gofLL(1g LL/L of Dulbecco’s PBS).
In 2 separate tubes, 10 L of LL inhibitors, AA861 (0.03
M; A3711, Sigma Chemical Co.), or nordihydroguaiaretic
acid (0.1 M; N5023, Sigma Chemical Co.) in EGME were
added as negative controls. To 1 of 4 other tubes was
added 10 Lof1,2,4,or8g of NCZ (Phibro Animal
Health Inc., Fairfield, NJ)/10 L of dimethyl sulfoxide
(DMSO; D5879, Sigma Chemical Co.). Two tubes con-
taining 10 L of DMSO or EGME served as controls for
the NCZ or inhibitor tubes, respectively. A test tube with
only DBF and LL served as a positive control. A test
tube with DBF and no LL served as a blank to monitor
background fluorescence. The solution in each test tube
MOLECULAR MECHANISMS OF NICARBAZIN 1287
was mixed briefly on a vortex mixer, then incubated in
a water bath at 37°C.
Tubes were removed after 1 min of incubation, and
the amount of fluorescein released was determined by
measuring fluorescence with a Turner model 450 spec-
trofluorometer. The spectrofluorometer was zeroed first
using a blank tube consisting of DBF working solution
only, and the gain was set to 1. The excitation wavelength
was set at 490 nm, and the emission wavelength was set
at 535 nm. After obtaining readings, the test tubes were
returned to the water bath. Tubes were removed for sub-
sequent readings at 2 min intervals until 11 min of incuba-
tion time had passed. The experiment was replicated 5
times.
Vitellogenin Phosphorylation Assay
Phosphorylation of VTG was assessed using a pur-
chased phosphoprotein stain (Molecular Probes Inc., Eu-
gene, OR). Plasma samples were obtained by drawing 3
mL of blood from the brachial vein of laying and nonlay-
ing chickens. Plasma samples were pooled to standardize
the amount of VTG in each sample. The plasma of nonlay-
ing chickens was used as a negative control. Positive
controls consisted of laying hen plasma only or laying
hen plasma plus 10 L of DMSO. Just prior to starting
the assay, fresh NCZ, DNC (390151, Aldrich Chemical
Co., Milwaukee, WI), and HDP (22588-6, Aldrich Chemi-
cal Co.) solutions were made. To 100 L of laying hen
plasma was added 10 Lof1,2,4,or8g of NCZ/10
L of DMSO; 1, 2, 4, or 8 g of DNC/10 L of DMSO;
or 1, 2, 4, or 8 g of HDP/10 L of water. Samples were
mixed and incubated at 4°C for 30 min. After incubation,
all plasma samples were diluted 1:100 in PBS (P4417,
Sigma Chemical Co.). A standards solution was prepared
by mixing 2 L of PeppermintStick standard (P33350,
Molecular Probes Inc.) with 38 L of ultra pure water.
A1× SDS-Tris-glycine running buffer was made by
adding 70 mL of 10× SDS-glycine (161-0732, BioRad Labo-
ratories, Hercules, CA) to 630 mL of ultra pure water.
Fixing solution consisted of 100 mL of methanol (A433P-
4, Fisher Scientific, Fair Lawn, NJ), 20 mL of acetic acid
(45726, Sigma-Aldrich, St. Louis, MO), and 80 mL of de-
ionized water. A ProQ Diamond destaining solution was
made by mixing 187.5 mL of deionized water, 50 mL of
acetonitrile (494445, Sigma-Aldrich), and 12.5 mL of 1 M
sodium acetate (110191, Aldrich Chemical Co.).
Sample buffer (3×,20L; 87703S, New England BioLabs
Inc., Ipswich, MA) was added to 40 L of plasma dilutions
and to the standards solution. Samples were mixed briefly
and centrifuged for 5 s at 8 to 10 × G. Samples were
heated for 5 min at 95°C, then centrifuged again for 5 s
at8to10× G.A4to20%Tris-glycine-SDS minigel (81002-
006, VWR International, Aurora, CO) was covered with
1× SDS-Tris-glycine running buffer. Each well was loaded
with 50 L of sample, and the plasma proteins were
separated by gel electrophoresis at 150 V for 90 min.
The gel was removed from the electrophoresis appara-
tus, covered with 100 mL of fixing solution, and incubated
by gently agitating at 23°C for 30 min. The gel was washed
twice by covering it with 100 mL of ultra pure water and
gently agitating at 23°C for 10 min. The gel was then
covered with 50 mL of ProQ Diamond phosphoprotein
stain (P33300, Molecular Probes Inc.) and incubated in
the dark with gentle agitation at 23°C for 2 h. The phos-
phoprotein stain was removed, and 80 mL of ProQ Dia-
mond destaining solution was added to the gel. The gel
was incubated in the dark with gentle agitation at 23°C
for 1 h. The destaining step was repeated once.
Images of the gel were produced on an Epichemi3
Darkroom 2UV benchtop transilluminator (UVP Bio-
imaging Systems, Ultraviolet Products Ltd., Cambridge,
UK) using an ethidium bromide filter (excitation = 365
nm, emission = 570 to 640 nm). Digital images were ana-
lyzed by densitometry using Scion Image for Windows
(Scion Corporation, Frederick, MD). The experiment was
replicated 5 times.
A Western blot was used to confirm the presence and
position of VTG on the gel. Briefly, plasma samples from
a laying hen and from a male were diluted 1:100 in PBS
and applied to a 4 to 20% Tris-glycine-SDS minigel with
sample buffer. Proteins were separated for 90 min at 150
V using SDS-PAGE. Proteins were transferred to a nitro-
cellulose membrane in transfer buffer for 60 min at 100V.
The membrane was blocked for 30 min at 23°C with gentle
agitation using blocking buffer consisting of Tris buffered
saline (TBS) and 5% milk powder. The membrane was
then incubated with 1:1000 rabbit anti-VTG antibody (D.
Williams, Pharmacological Sciences, SUNY, Stony Brook,
NY) in blocking solution for 2 h at 23°C with gentle agita-
tion. The membrane was washed once with TBS con-
taining 0.05% Tween 20 (vol/vol; P1379, Sigma Chemical
Co.), and twice with TBS.
The membrane was then incubated with alkaline phos-
phatase labeled goat anti-rabbit IgG antibody (1:1,000;
A7778, Sigma Chemical Co.) in blocking buffer for 60 min
at 23°C with gentle agitation. The membrane was washed
once with TBS-Tween 20 and twice with TBS. Color was
developed by incubating the membrane in alkaline phos-
phatase substrate (pH 9.5; B5655, Sigma Chemical Co.)
containing 0.15 mg of 5-bromo-4-chloro-3-indolyl phos-
phate/mL, 0.3 mg of Nitro blue tetrazolium/mL, 100 mM
Tris buffer, and 5 mM magnesium chloride. The reaction
was stopped after 10 min by washing the membrane in
deionized water.
TG Assay
The TG activity was assessed using an assay previously
described by Lilley et al. (1997). The assay measures the
protein crosslinking activity of TG based upon incorpora-
tion of biotin-labeled casein into unlabeled casein that is
bound to microtiter plates.
Casein was biotinylated using a procedure previously
described for labeling antibodies with biotin (Harlow and
Lane, 1988). A 0.1 M sodium borate buffer was prepared
by dissolving 7 g of boric acid (B6768, Sigma Chemical
Co.) and 10 g of sodium tetraborate (B0127, Sigma Chemi-
YODER ET AL.1288
cal Co.) in 1 L of deionized water and titrating the solution
to pH 8.8. A solution of 3 mg of N′,N′-dimethylcasein/
mL (C9801, Sigma Chemical Co.) was prepared in 0.1 M
sodium borate buffer. A solution of 3 mg of N-hydroxy-
succinimide biotin/mL (H1759, Sigma Chemical Co.) was
prepared in DMSO.
The casein and biotin solutions were combined in a 9:1
casein:biotin ratio and incubated at 23°C for 4 h. Ammo-
nium chloride (1 M; A4515, Sigma Chemical Co.) was
added to the biotin ester solution at a rate of 20 L per
250 g of biotin ester. The solution was incubated for 10
min at 23°C, then dialyzed against PBS overnight in #3
Spectra/Por dialysis tubing (132724, Spectrum Medical
Industries, Los Angeles, CA). The biotinylated casein was
stored at −70°C until use.
Flat bottom 96 well microtiter plates (3455, Thermo
LabSystems, Franklin, MA) were coated with 50 ng/well
N,N′dimethylcasein in 50 mM sodium carbonate buffer
(C3041, Sigma Chemical Co.) at pH 9.8 (100 L/well) and
incubated at 37°C for 1 h. Plates were washed twice with
PBS containing 0.05% Tween 80 (vol/vol; P8074, Sigma
Chemical Co.), and twice with deionized water.
Plates were blocked with 300 L/well BSA (1 mg/mL)
in 50 mM sodium carbonate buffer for 30 min at 23°C.
Plates were washed twice with PBS-Tween 80, twice with
deionized water, and once with 100 mM Tris-HCl (pH
8.5; T3253, Sigma Chemical Co.). Each plate was then
incubated overnight at 37°C with 100 L/well 100 mM
Tris-HCl containing 5 mM calcium chloride (C4901,
Sigma Chemical Co.), 10 mM dithiotheitol (D0632, Sigma-
Aldrich), 37.5 mM putrescine (D13208, Aldrich Chemical
Co.), and 0.25% TG (wt/vol; T5398, Sigma Chemical Co.).
Plates were removed from the incubator and washed
twice with PBS-Tween 80, twice with deionized water,
and once with 100 mM Tris-HCl.
To each plate was added 100 L/well 100 mM Tris-
HCl containing 5 mM calcium chloride, 10 mM dithiothei-
tol, 0.75 g/mL biotinylated casein, and 0.5% TG (wt/
vol). In addition, 10 L/well of Tris-HCl, DMSO, 1:200
goat anti-TG antibody (T7066, Sigma Chemical Co.), NCZ,
DNC, or HDP were added to the appropriate wells. The
NCZ and DNC solutions consisted of 1, 2, 4, or 8 gof
NCZ or DNC/10 L of DMSO, and the HDP solutions
consisted of 1, 2, 4, or 8 g of HDP/10 L of water. All
NCZ, DNC, and HDP solutions were made just prior to
starting the assay. Each plate had 6 wells per treatment
group. Wells containing only Tris-HCl were used as nega-
tive controls. Plates were incubated for 1 h at 37°C. Plates
were washed twice with PBS-Tween 80, twice with deion-
ized water, and once with 100 mM Tris-HCl.
A 1:625 dilution of extravidin peroxidase (E2886, Sigma
Chemical Co.) was added to each well (100 L/well), and
plates were incubated at 37°C for 45 min. Plates were
washed twice with PBS-Tween 80, twice with deionized
water, and once with 0.05 M phosphate-citrate buffer (pH
5.0; P9305, Sigma Chemical Co.) containing 0.014% hydro-
gen peroxide.
A 3,3′,5,5′-tetramethylbenzidine (T3405, Sigma Chemi-
cal Co.) solution was made by dissolving 1 3,3′,5,5′-tetra-
methylbenzidine tablet per 10 mL of 0.05 M phosphate-
citrate buffer. The 3,3′,5,5′-tetramethylbenzidine solution
was added to each well (100 L/well), and color was
allowed to develop. The reaction was terminated after 2
to 3 min using 100 L/well 2 M sulfuric acid. Plates
were read at 450 nm on an Ultramark Microplate Imaging
System (170-9500, BioRad Laboratories). The experiment
was replicated 5 times, with 1 plate per replication.
Calcium Channel Assay
Bull sperm were used as a model for this experiment
because sperm contain L-type calcium channels similar
to those found in avian follicular cells (Schwartz et al.,
1989; Goodwin et al., 2000). In addition, millions of sperm
can be obtained without extensive purification, which
can alter a cell’s membrane function. A large influx of
intracellular calcium though calcium channels occurs in
sperm during capacitation, and this influx can be induced
in vitro by incubating sperm with progesterone (Kobiri
et al., 2000). The influx of intracellular calcium can be
monitored using flow cytometry.
Bull tyrodes solution was made by dissolving 5.69 g of
sodium chloride (S7653, Sigma Chemical Co.), 0.23 g of
potassium chloride (P3911, Sigma Chemical Co.), 0.04 g
of sodium phosphate (S0876, Sigma Chemical Co.), 2.09
g of sodium bicarbonate (S5761, Sigma Chemical Co.),
0.29 g of calcium chloride dihydrate (C5080, Sigma Chem-
ical Co.), and 0.08 g of magnesium chloride hexahydrate
(M2670, Sigma Chemical Co.) in nanopure water. Bull
Tyrode’s albumin-lactate-pyruvate diluent was made by
dissolving 0.0022 g of sodium pyruvate (P2256, Sigma
Chemical Co.), 0.368 mL of sodium lactate (L1375, Sigma
Chemical Co.), 0.09 g of glucose (G7528, Sigma Chemical
Co.), 0.238 g of N-(2-hydroxyethyl)piperazine-N′2-eth-
anesulfonic acid (H3375, Sigma Chemical Co.), and 0.3 g
of BSA (A2153, Sigma Chemical Co.) in 100 mL of bull
tyrodes solution.
Bull sperm were diluted to 50 × 10
6
cells/mL in bull
Tyrode’s albumin-lactate-pyruvate diluent, and 2 mL of
the diluted sperm was added to each sample tube. Sperm
in all sample tubes except the control tubes were stained
with 10 M Fluo-3 AM (an intracellular calcium indicator;
F1241, Invitrogen, Carlsbad, CA) and 5 M propidium
iodide (a stain to detect dead cells; P1304MP, Invitrogen).
There were 3 control tubes consisting of Fluo-3 AM stain
only, propidium iodide stain only, and both stains.
To each sample tube, 20 L of DMSO, 80 M nifedipine
(calcium channel inhibitor; N7634, Sigma Chemical Co.),
4.75 M A23187 (calcium ionophore; C5149, Sigma-Ald-
rich), NCZ, DNC, or HDP solutions were added. The
NCZ solutions consisted of 1, 2, 4, or 8 g of NCZ/20
L of DMSO. The DNC solution consisted of 8 gof
DNC/20 L of DMSO. The HDP solution consisted of 8
g of HDP/20 L of water.
Tubes were mixed briefly using a vortex mixer, then
incubated for 20 min at 23°C in the dark. A 0.5-mL sub-
sample was analyzed on an Epics V flow cytometer
(Coulter Electronics, Miami, FL) with the argon laser
MOLECULAR MECHANISMS OF NICARBAZIN 1289
tuned to 488 nm to excite Fluo-3 AM and propidium
iodide. The filter setup included a 457 to 505-nm laser
blocker, a 550-nm dichoic beam splitter, a 525 to 560-nm
band-pass filter to detect Fluo-3 AM, and a 610-nm long-
pass filter to detect propidium iodide. To the remainder
of the samples was added 800 Lof40M progesterone
in DMSO. The samples were mixed briefly on a vortex
mixer and then were incubated at 37°C for 1 h. Subsam-
ples (0.5 mL) were taken every 15 min during the hour
of incubation for analysis on the flow cytometer. This
experiment was replicated 5 times.
Statistical Analysis
Lipoprotein Lipase Assay. The absorbance value for
the blank test tube was subtracted from the absorbance
for all other tubes in the same time period. The difference
between the absorbance of the LL positive control and
the DMSO control was subtracted from all test tubes con-
taining DMSO in the same time period. The difference
between the absorbance of the LL positive control and
the EGME control was subtracted from all test tubes con-
taining EGME in the same time period. The adjusted
absorbances were used to standardize the data by calcu-
lating a percent of the LL positive control. Absorbances
for each tube were divided by the absorbance for the LL
positive control in the same time period, and the result
was multiplied by 100 to obtain a percentage of the posi-
tive control. The standardized percentages were analyzed
as a mixed effects model (PROC MIXED, SAS Institute
Inc., Cary, NC), and significance was defined as P ≤ 0.05.
Means separations were carried out using PDMIX800
(Saxton, 1998).
Vitellogenin Phosphorylation Assay. To obtain a
mean background value for each gel, the mean optical
density of the area of the gel corresponding to the VTG
band was averaged for the nonlaying chicken plasma and
PeppermintStick standard lanes. The mean background
value was subtracted from the mean optical density of
the VTG bands for each gel to create an adjusted density.
The adjusted density for each VTG band was compared
with the adjusted density of the VTG band for laying
chicken plasma on the same gel to obtain a percentage
of the control. The percentages of the control were ana-
lyzed by ANOVA (PROC GLM, SAS Institute), and sig-
nificance was defined as P ≤ 0.05. Means were separated
using the least significant difference.
Transglutaminase Assay. The absorbances of the
blank wells were averaged, and the mean absorbance was
subtracted from the absorbance of each well to eliminate
background fluorescence. For each plate, all 6 wells in
each treatment group were averaged. The average ab-
sorbance for each treatment group was divided by the
average absorbance for the DMSO control group for that
plate. The result was multiplied by 100 to obtain a percent-
age of the DMSO control. The percentages of the DMSO
control were used for analysis by ANOVA (PROC GLM,
SAS Institute), and significance was defined as P ≤ 0.05.
Table 1. Effect across time of addition of 10 Lof1,2,4,or8g/10
L of nicarbazin (NCZ) in dimethyl sulfoxide (DMSO), lipoprotein
lipase (LL) inhibitors AA861 (0.03 M), and nordihydroguaiaretic acid
(NDGA; 0.1 M) in ethylene glycol monomethyl ether (EGME), or DMSO
to test tubes containing 1 mL of dibutyrlfluorescein (DBF) and 15 g
of LL on LL activity in vitro after 1, 3, 5, 7, 9, and 11 min of incubation
at 37°C
Mean percentage
Treatment n of DMSO control
DMSO control 30 100.0
d
0.03 M AA861 30 −17.7
e
0.1 M NDGA 30 10.9
d
1 g of NCZ 30 107.3
c
2 g of NCZ 30 108.6
c
4 g of NCZ 30 169.4
b
8 g of NCZ 30 233.4
a
SEM 8.4
a–e
Means within the column with different subscripts are significantly
different (P ≤ 0.05).
Means were separated using the least significant dif-
ference.
Calcium Channel Assay. Data were standardized by
calculating a percentage of the DMSO control for the
percentage of cells with low intracellular calcium, the
percentage of cells with high intracellular calcium, and
the percentage of dead cells. The percentage of cells with
low intracellular calcium in each group was divided by
the percentage of cells with low intracellular calcium in
the DMSO control group for the same time period. The
result was multiplied by 100 to obtain a percentage of
the DMSO control. The same procedure was used to calcu-
late a percentage of the DMSO control for the percentage
of cells with high intracellular calcium and the percentage
of dead cells. The standardized percentages were ana-
lyzed as a mixed effects model (PROC MIXED, SAS Insti-
tute), and significance was defined as P ≤ 0.05. Means
separations were carried out using PDMIX800 (Saxton,
1998).
RESULTS
Nicarbazin significantly increased LL activity (Table 1).
However, LL activity decreased over time (P ≤ 0.05); most
of the change occurred during the first 3 min of incuba-
tion. A significant treatment × period interaction also ex-
isted (P ≤ 0.05). Changes in LL activity during the remain-
der of the incubation period were slight. Both AA861
and NDGA inhibited LL activity, giving 100 and 89%
inhibition, respectively.
Vitellogenin phosphorylation differed among treat-
ments (Table 2). Whereas DMSO decreased the amount
of VTG phosphorylation by 19.5% compared with the
control, NCZ and DNC were not significantly different
from the control or from DMSO. Although treatment with
1, 2, and 4 g of HDP decreased the amount of VTG
phosphorylation compared with the control, they were
not different from DMSO.
Transglutaminase activity also differed among treat-
ments (Table 3). Whereas HDP tended to increase the
activity of TG by 30 to 40%, NCZ and DNC tended to
YODER ET AL.1290
Table 2. Effect of addition of 10 Lof1,2,4,or8g/10 L of nicarbazin
(NCZ) in dimethyl sulfoxide (DMSO), 1, 2, 4, or 8 g/10 Lof4,4′-
dinitrocarbanilide (DNC) in DMSO, 1, 2, 4, or 8 g/10 L of 4,6-dimeth-
ylpyrimidine (HDP) in water, or DMSO to 100 L of chicken plasma
on phosphorylation of vitellogenin
1
Mean percentage
Treatment n of laying hen control
Laying control 12 100.0
a
DMSO 5 80.5
bcd
1 g of NCZ 5 93.6
ab
2 g of NCZ 5 86.3
abcd
4 g of NCZ 5 92.6
abc
8 g of NCZ 5 89.3
abc
1 g of DNC 5 83.3
bcd
2 g of DNC 5 88.0
abcd
4 g of DNC 5 90.6
abc
8 g of DNC 5 92.9
abc
1 g of HDP 5 79.9
bcd
2 g of HDP 5 79.2
cd
4 g of HDP 5 75.3
d
8 g of HDP 5 93.1
abc
SEM 4.9
a–d
Means within the column with different subscripts are significantly
different (P ≤ 0.05).
1
Plasma was diluted 1:100 in PBS, and proteins were separated on a
4 to 20% Tris-glycine minigel for 90 min at 150 V. Phosphoproteins were
stained with ProQ Diamond phosphoprotein stain (33300, Molecular
Probes, Eugene. OR).
decrease the activity of TG by 30 to 61.5%. The anti-TG
antibody inhibited TG activity by 66%.
As shown in Table 4, there was a significant treatment
effect on the percentages of cells having high intracellular
calcium but not on the percentages of cells having low
intracellular calcium (P = 0.4737). The percentage of cells
having low intracellular calcium tended to increase over
time (P ≤ 0.05), whereas the percentage of cells having
high intracellular calcium tended to decrease over time
(P ≤ 0.05). There was a significant treatment × time interac-
Table 3. Effect of addition of 10 Lof1,2,4,or8g/10 L of nicarbazin
(NCZ) in dimethyl sulfoxide (DMSO), 4,4′-dinitrocarbanilide (DNC) in
DMSO, 4,6-dimethylpyrimidine ′HDP) in water, 1:200 goat antitransglu-
taminase antibody in PBS, or DMSO to microtiter plates containing 100
L/well Tris-HCl solution consisting of 5 mM calcium chloride, 10 mM
dithiotheitol, 0.75 g/mL of biotinylated casein, and 0.5% transglutami-
nase (w:v) on transglutaminase (TG) activity in vitro
Mean percent
Treatment n of DMSO control
DMSO control 5 100.0
b
Anti-TG antibody 5 33.6
e
1 g of NCZ 5 70.2
c
2 g of NCZ 5 55.6
cd
4 g of NCZ 5 38.5
de
8 g of NCZ 5 43.2
de
1 g of DNC 5 65.0
d
2 g of DNC 5 55.1
cd
4 g of DNC 5 51.0
cde
8 g of DNC 5 51.9
cde
1 g of HDP 5 141.1
a
2 g of HDP 5 131.2
a
4 g of HDP 5 132.4
a
8 g of HDP 5 135.2
a
SEM 7.2
a–e
Means within the column with different subscripts are significantly
different (P ≤ 0.05).
Table 4. Effect of addition of 20 Lof1,2,4,or8g/20 L of nicarbazin
(NCZ) in dimethyl sulfoxide (DMSO), 8 g/20 L of 4,4′-dinitrocarbani-
lide (DNC) in DMSO, 8 g/20 L of 4,6-dimethylpyrimidine (HDP) in
water, 80 M nifedipine (calcium channel inhibitor), 4.75 M A23187
(calcium ionophore), or DMSO to test tubes containing 2 mL of bull
sperm in Tyrode’s albumin-lactate-pyruvate diluent (50 × 106 cells/mL)
stained with 10 M Fluo-3 AM and 5 M propidium iodide on the
percentage of sperm cells having high intracellular calcium 15 min after
the addition of 800 Lof40M progesterone and incubation at 37°C
0 min 15 min
Treatment n Mean
1
n Mean
DMSO control 5 100.0
defghijk
5 100.0
defghijk
Nifedipine 5 97.8
defghijk
5 60.0
jk
A23187 5 237.5
b
5 297.5
a
1 g of NCZ 5 143.3
cdf
5 113.1
cdefghij
2 g of NCZ 5 142.7
cdef
5 127.2
cdefghi
4 g of NCZ 5 134.9
cde
5 84.3
fghijkl
8 g of NCZ 5 131.9
cdefg
5 112.8
cdefghij
8 g of DNC 4 114.0
cdefghij
4 86.1
defghijk
8 g of HDP 4 167.5
c
4 114.1
cdefghij
SEM 22.5
a–j
Means within columns with the different subscripts are significantly
different (P ≤ 0.05).
1
Means are percentage of the DMSO control.
tion effect for the percentage of cells having high intracel-
lular calcium (P ≤ 0.05; Table 4). The effects of NCZ on
intracellular calcium levels occurred within the first 15
min of incubation. Treatment significantly affected the
percentage of dead cells (P ≤ 0.05), with A23187 and nifed-
ipine inducing the highest percentages of dead cells. The
percentages of dead cells in the NCZ, DNC, and HDP
groups were not different from the controls with and
without DMSO. As expected, the percentage of dead cells
increased over time (P ≤ 0.05)
DISCUSSION
Nicarbazin increased the activity of LL in vitro in the
4 and 8 g treatment groups. The total assay volume in
each test tube was 1.025 mL, giving a concentration of
3.9 g/mL and 7.8 g/mL in the 4 and 8 g of NCZ
treatment groups, respectively. The entire NCZ molecule
has a molecular weight of 426.38, whereas the DNC por-
tion has a molecular weight of 292.25 (Wells, 1999), which
is 68.5% of the NCZ molecule. Therefore, 4 gofNCZ
contains 2.74 g of DNC and 8 g of NCZ contains 5.48
g of DNC. The concentration of DNC in the assay was
therefore 2.67 and 5.35 g/mL in the 4 and 8 g of NCZ
treatment groups, respectively. These values are within
the range expected in the plasma of waterfowl fed NCZ-
treated bait at 31 to 49 mg of NCZ/kg of BW. A study
of mallards fed at these dose levels showed peak plasma
DNC levels were 2.7 to 5.4 g/mL (Yoder et al., 2006a).
Chickens fed 400 mg of NCZ/kg of feed had peak
plasma DNC levels of approximately 3 g/mL and a 71%
reduction in egg production (Ott et al., 1956). Several
studies found feeding 125 mg of NCZ/kg of feed reduced
egg production significantly (Baker et al., 1957; McLough-
lin et al., 1957; Jones et al., 1990c). A comparative gavage
study showed treatment of chickens with NCZ at 125
MOLECULAR MECHANISMS OF NICARBAZIN 1291
ppm produced a peak plasma DNC level of 2.9 g/mL
(Yoder et al., 2005). These plasma DNC levels are compa-
rable to the concentrations used in the in vitro assay.
The increased activity of LL due to NCZ could cause
premature degradation of VLDL while in the blood, re-
sulting in a decrease of lipid being deposited into the yolk,
thereby decreasing overall egg weight and production.
Baker et al. (1957) suggested NCZ might prevent ova
from maturing. Necropsy of hens fed a ration containing
90 ppm NCZ revealed that the largest follicle was absent
with no signs of recent ovulation or atresia (Baker et al.,
1957). Luck (1979) also found ovaries without a follicular
hierarchy and less well-developed oviducts in laying hens
treated with 375 ppm NCZ in feed.
Nicarbazin did not affect luteinizing hormone levels or
pituitary responsiveness to luteinizing hormone releasing
hormone but decreased the sensitivity of the chicken hy-
pothalamus to exogenous progesterone (Luck, 1979).
Luck (1979) suggested that egg production is decreased
because yolk deposition in the follicles is prevented.
White Leghorns fed 400 to 700 ppm NCZ in feed exhibited
reduced egg production concomitant with a 2-fold rise
in plasma cholesterol concentrations, supporting Luck’s
hypothesis (Weiss, 1979). These studies support our hy-
pothesis that the increased LL activity due to NCZ causes
premature degradation of VLDL. Future studies should
investigate the effect of NCZ treatment on the activity of
LL in vivo.
Although a statistically significant effect of NCZ on
phosphorylation of VTG was found, this effect is probably
not biologically significant. The total assay volume of
plasma plus treatment was 0.11 mL. If DNC comprises
68.5% of the NCZ molecule, then HDP must comprise
31.5% of the NCZ molecule. Using these figures, the con-
centrations of DNC and HDP in this assay ranged from
9.1 to 72.7 g/mL in the DNC and HDP groups. The
concentration of DNC in the NCZ groups ranged from
6.2 to 49.8 g/mL, and the concentration of HDP in the
NCZ groups ranged from 2.8 to 22.9 g/mL. This is many
times higher than what would be expected in plasma. A
decrease in VTG phosphorylation was caused by DMSO
by itself. Although the NCZ and DNC groups had VTG
with a greater degree of phosphorylation than the DMSO
group, they were not significantly different from the
DMSO group. The HDP group appeared to have no effect
except at the 8-g level.
There was a very large amount of VTG on the gels,
which might make it difficult to detect small changes in
phosphorylation. The assay could be rerun with plasma
diluted at least 1:1,000 in PBS. However, such small
changes would not likely be biologically significant. A
more appropriate experiment would be to treat laying
hens with NCZ and compare the phosphorylation of VTG
from the plasma of treated and control hens.
Nicarbazin did have an inhibitory effect on TG in vitro.
The portion of the NCZ molecule that appears to be re-
sponsible for this effect is DNC. Both DNC and NCZ
decreased TG activity compared with the DMSO control,
whereas HDP increased TG activity.
The total assay volume used in each well was 0.11 mL.
Again, the concentrations of DNC and HDP ranged from
9.1 to 72.7 g/mL in the DNC and HDP groups, much
higher than what would be expected in plasma. The con-
centration of DNC in the NCZ groups ranged from 6.2
to 49.8 g/mL, and the concentration of HDP in the NCZ
groups ranged from 2.8 to 22.9 g/mL. The same inhibi-
tory effect might not occur at lower levels. Only the 4
and 8 g of NCZ and DNC groups produced a decrease
in TG activity similar to the anti-TG antibody. We chose
to use the higher concentrations of NCZ, DNC, and HDP
for this experiment because of the difficulty of accurately
measuring such small quantities of NCZ, DNC, and HDP.
The total assay volume used in the calcium assays was
2.82 mL. The concentrations of DNC used ranged from
0.2 to 1.9 g/mL in the NCZ groups and was 2.8 g/
mL in the DNC group. The concentrations of DNC in the
DNC and 8 g of NCZ groups are comparable to what
is expected in plasma, whereas the concentrations in the
remaining NCZ groups are lower than expected plasma
values. The concentrations of HDP ranged from 0.1 to 0.9
g/mL in the NCZ groups and 2.8 g/mL in the HDP
group. The concentration of HDP in the 8 g of NCZ
group is close to expected plasma values, whereas the
concentrations in the remaining NCZ groups are lower
than expected. The concentration in the HDP group is
higher than expected. Wells (1999) reported a range of
HDP concentrations from 1.07 to 2.07 g/mL in chickens
fed 125 ppm NCZ for 7 d.
The effects of NCZ on intracellular calcium levels oc-
curred within the first 15 min of incubation. No significant
effects on intracellular calcium levels were observed after
30 min of incubation. The HDP group consistently had a
greater percentage of sperm cells with high intracellular
calcium than the DMSO control, indicating it is acting as
an ionophore. However, as has already been pointed out,
the concentration of HDP used in that group is slightly
higher than expected plasma values. Fifteen minutes after
the addition of progesterone, the NCZ groups had a
higher percentage of cells with high intracellular calcium
than the DMSO control. The percentage of cells with high
intracellular calcium was comparable in the NCZ and
HDP groups. Because the concentrations of HDP in the
NCZ groups were lower than expected plasma values, it
seems reasonable to conclude that NCZ acts as an iono-
phore. The portion of the NCZ molecule responsible for
ionophore activity is HDP. As compared with the DMSO
control, the DNC group had comparatively fewer sperm
cells with high intracellular calcium, indicating it may act
as a weak calcium channel blocker.
The apparent activity of NCZ as an ionophore may
help explain damage to the vitelline membrane in NCZ-
treated hens that leads to egg yolk mottling and a reduc-
tion in egg hatchability. As an ionophore, NCZ could
insert itself into the vitelline membrane, making the mem-
brane more permeable. Evidence that vitelline mem-
branes from NCZ-treated hens are more permeable was
shown by Cunningham (1976, 1977). Cunningham found
that mottled yolks from NCZ-treated hens exhibited a
YODER ET AL.1292
decrease in yolk solids (1976) and an increase in egg
albumen (1977). The percentages of fat, protein, ash, cal-
cium, phosphorus, and iron are also reduced in mottled
yolks (Cunningham, 1976), but these components in-
creased in the egg albumen (Cunningham, 1977). Mottled
yolks also contain the egg white proteins ovalbumin and
conalbumin (Cunningham, 1976). In addition, vitelline
membranes from NCZ-treated hens show degeneration
at the microscopic level (Yoder et al., 2006b).
Although these assays examined the effects of NCZ in
vitro, they provide some clues as to the mechanism by
which NCZ affects reproduction. One of the main effects
of NCZ on reproduction is to increase the activity of LL,
thereby decreasing the amount of VLDL deposited into
the follicle. The other main effect is the activity of NCZ as
an ionophore to increase the permeability of the vitelline
membrane. These assays should be viewed as preliminary
studies to aid in directing further research on the effect
of NCZ on reproduction in vivo.
ACKNOWLEDGMENTS
P. Nash and J. Pilon provided technical assistance. K.
Bynum and K. Fagerstone provided comments on earlier
drafts of the manuscript.
REFERENCES
Allerton, S. E., and G. E. Perlmann. 1965. Chemical characteriza-
tion of the phosphoprotein phosvitin. J. Biol. Chem.
240:3892–3898.
Baker, R. C., F. W. Hill, A. van Tienhoven, and J. H. Bruckner.
1957. The effect of nicarbazin on egg production and egg
quality. Poult. Sci. 36:718–726.
Bar, A., and S. Hurwitz. 1971. Relationship of calcium-binding
protein to calcium absorption in the fowl. Isr. J. Med. Sci.
7:382–383.
Barber, D. L., E. J. Sanders, R. Aebersold, and W. J. Schneider.
1991. The receptor for yolk lipoprotein deposition in the
chicken oocyte. J. Biol. Chem. 266:18761–18770.
Burley, R. W., R. W. Sleigh, and F. S. Shenstone. 1984. Lipopro-
teins from the blood and egg yolk of the hen. The transfer
of very-low-density lipoprotein to egg yolk and possible
changes to apoprotein B. Eur. J. Biochem. 142:171–176.
Chapman, H. D. 1994. A review of the biological activity of
the anticoccidial drug nicarbazin and its application for the
control of coccidiosis in poultry. Poult. Sci. Rev. 5:231–243.
Chistmann, J. L., M. J. Grayson, and R. C. C. Huang. 1977.
Comparative study of hen yolk phosvitin and plasma vitello-
genin. Biochemistry 16:3250–3256.
Clark, R. C. 1970. The isolation and composition of two phospho-
proteins from hen’s egg. Biochem. J. 118:537–542.
Cuckler, A. C., C. M. Malanga, A. J. Basso, and R. C. O’Neill.
1955. Antiparasitic activity of substituted carbanilide com-
plexes. Science 122:244–245.
Cunningham, F. E. 1976. Composition and functional properties
of mottled yolks. Poult. Sci. 55:994–998.
Cunningham, F. E. 1977. Properties of albumen from eggs hav-
ing mottled yolks. Poult. Sci. 56:1819–1821.
Deely, R. G., K. P. Mullnix, W. Wetekam, H. Kronenberg, M.
Meyers, J. D. Eldridge, and R. F. Goldberger. 1975. Vitello-
genin synthesis in the avian liver: Vitellogenin is the precur-
sor of the egg yolk phosphoproteins. J. Biol. Chem.
250:9060–9066.
Del Prado, M., H. Hernandez-Montes, and S. Villalpando. 1994.
Characterization of a fluorometric method for lipoprotein
lipase. Arch. Med. Res. 25:331–335.
George, R., D. L. Barber, and W. J. Schneider. 1987. Characteriza-
tion of the chicken oocyte receptor for low and very low
density lipoproteins. J. Biol. Chem. 262:16838–16847.
Goodwin, L. O., D. S. Karabinus, R. G. Pergolzzi, and S. Benoff.
2000. L-type voltage-dependent calcium channel alpha-1C
subunit mRNA is present in ejaculated human spermatozoa.
Mol. Hum. Reprod. 6:127–136.
Green, C. D. 1980. The formation of yolk proteins and their
endocrine control. Oxf. Rev. Reprod. Biol. 2:97–122.
Griffin, H. D., G. Grant, and M. M. Perry. 1982. Hydrolysis of
plasma triacylglycerol-rich lipoproteins from immature and
laying hens (Gallus domesticus) by lipoprotein lipase in vitro.
Biochem. J. 206:647–654.
Griffin, H. D., and M. M. Perry. 1985. Exclusion of plasma lipo-
proteins of intestinal origin from avian egg yolk because of
their size. Comp. Biochem. Physiol. 82B:321–325.
Grunder, A. A., R. B. Guyer, E. G. Buss, and C. O. Clagett. 1980.
Calcium-binding proteins in serum: Quantitative differences
between thick and thin shell lines of chickens. Poult. Sci.
59:828–884.
Harlow, E., and D. Lane. 1988. Labeling antibodies with biotin.
Pages 340–341 in Antibodies: A Laboratory Manual. Cold
Spring Harbor Laboratory, New York, NY.
Hillyard, L. A., C. Entenman, and I. L. Chaikoff. 1956. Concentra-
tion and composition of serum lipoproteins of cholesterol-
fed and stilbestrol-injected birds. J. Biol. Chem. 223:359–368.
Hughes, B. L., J. E. Jones, J. E. Toler, J. Solis, and D. J. Castaldo.
1991. Effects of exposing broiler breeders to nicarbazin con-
taminated feed. Poult. Sci. 70:476–482.
Jones, J. E., B. L. Hughes, J. Solis, D. J. Castaldo, and J. E. Toler.
1990a. Effect of nicarbazin on brown-egg layer-breeders.
Appl. Agric. Res. 5:149–152.
Jones, J. E., J. Solis, B. L. Hughes, D. J. Castaldo, and J. E.
Toler. 1990b. Production and egg-quality responses of White
Leghorn layers to anticoccidial agents. Poult. Sci. 69:378–387.
Jones, J. E., J. Solis, B. L. Hughes, D. J. Castaldo, and J. E.
Toler. 1990c. Reproduction responses of broiler-breeders to
anticoccidial agents. Poult. Sci. 69:27–36.
Kobiri, H., S. Miyazaki, and Y. Kuwabara. 2000. Characterization
of intracellular Ca++ increase in response to progesterone
and cyclic nucleotides in mouse spermatozoa. Biol. Reprod.
63:113–120.
Kudzma, D. J., J. B. Swaney, and E. N. Ellis. 1979. Effects of
estrogen administration on the lipoproteins and apoproteins
of the chicken. Biochim. Biophys. Acta 572:257–268.
Lilley, G. R., M. Griffin, and P. L. R. Bonner. 1997. Assays for the
measurement of tissue transglutaminase (type II) mediated
protein crosslinking via e-(
G
-glutamyl) lysine and N′,N′-
bis(
G
-glutamyl) polyamine linkages using biotin labeled ca-
sein. J. Biochem. Biophys. Methods 34:31–43.
Lopez-Berjes, M. A., M. Recio, and J. Planas. 1981. Plasma varia-
tion of transferrin-iron and phosvitin-iron during the laying
period in chicken hens. Poult. Sci. 60:1951–1956.
Luck, M. R. 1979. The adverse effects of nicarbazin on reproduc-
tive activity in the hen. Br. Poult. Sci. 20:605–607.
McLoughlin, D. K., E. E. Weh, and R. Rubin. 1957. Egg shell
color and egg production in New Hampshire laying hens as
affected by nicarbazin medication. Poult. Sci. 36:880–884.
Miller, S. M., A. Kato, and S. Nakai. 1982. Sedimentation equilib-
rium study of the interaction between egg white lysozyme
and ovomucin. J. Agric. Food Chem. 30:1127–1132.
Morgan, E. H. 1975. Plasma iron transport during egg laying
and after oestrogen administration in the domestic fowl. Q.
J. Exp. Physiol. 60:233–247.
Ott, W. H., S. Kuna, C. C. Porter, and A. C. Cuckler. 1956.
Biological studies on nicarbazin, a new anticoccidial agent.
Poult. Sci. 35:1355–1367.
MOLECULAR MECHANISMS OF NICARBAZIN 1293
Perry, M. M., A. B. Gilbert, and A. J. Evans. 1978a. Electron
microscope observations on the ovarian follicle of the domes-
tic fowl during the rapid growth phase. J. Anat. 127:481–497.
Perry, M. M., A. B. Gilbert, and A. J. Evans. 1978b. The structure
of the germinal disc region of the hen’s ovarian follicle during
the rapid growth phase. J. Anat. 127:379–392.
Polin, D. 1957. Biochemical and weight changes of mottled yolks
in eggs from hens fed nicarbazin. Poult. Sci. 36:831–835.
Polin, D., W. H. Ott, and O. H. Siegmund. 1957. The incidence
and degree of yolk mottling in eggs from hens fed diets with
and without nicarbazin. Poult. Sci. 36:524–528.
Rogers, E. F., R. D. Brown, J. E. Brow, D. M. Kazazis, W. J.
Leanza, J. R. Nichols, D. A. Ostlind, and T. M. Rodino. 1983.
Nicarbazin complex yields dinitrocarbanilide as ultrafine
crystals with improved anticoccidial activity. Science
222:630–632.
Saxton, A. M. 1998. A macro for converting mean separation
output to letter groupings in Proc Mixed. Pages 1243–1246
in Proc. 23rd SAS Users Group Int., SAS Institute, Cary, NC.
Schjeide, O. A., R. I.-S. Lin, E. A. Grellert, F. R. Galey, and J. F.
Mead. 1969. Isolation and preliminary chemical analysis of
coated vesicles from chicken oocytes. Physiol. Chem. Phys.
1:141–163.
Schneider, W. J., R. Carroll, D. L. Severson, and J. Nimpf. 1990.
Apolipoprotein VLDL-II inhibits lipolysis of triglyceride-rich
lipoproteins in the laying hen. J. Lipid Res. 31:507–513.
Schwartz, J. L., E. K. Asm, G. A. Mealing, B. K. Tsang, E. C.
Rousseau, J. F. Whitfield, and M. D. Payet. 1989. T- and
L-calcium channels in steroid-producing chicken granulosa
cells in primary culture. Endocrinology 125:1973–1982.
Shapiro, D. J. B. 1982. Steroid hormone regulation of vitellogenin
gene expression. CRC Crit. Rev. Biochem. 12:187–203.
Stifani, S., D. L. Barber, J. Nimpf, and W. J. Schneider. 1990. A
single chicken oocyte plasma membrane protein mediates
uptake of very low density lipoprotein and vitellogenin. Proc.
Natl. Acad. Sci. USA 87:1955–1959.
Taborsky, G. 1980. Iron binding by phosvitin and its conforma-
tional consequences. J. Biol. Chem. 255:2976–2985.
Tucciarone, L. M., and K. D. Lanclos. 1981. Evidence for the
involvement of transglutaminase in uptake of vitellogenin
by Xenopus laevis oocytes. Biochem. Biophys. Res. Commun.
99:221–227.
van Tienhoven, A., F. W. Hill, A. Prock, and R. C. Baker. 1958.
The effect of nicarbazin on yolk quality. Poult. Sci. 37:129–132.
Wallace, R. A. 1985. Vitellogenesis and oocyte growth in non-
mammalian vertebrates. In Developmental Biology. Vol. 1.
L. W. Browder, ed. Plenum, New York, NY.
Wang, S., and D. L. Williams. 1982. Biosynthesis of the vitello-
genins. Identification and characterization of nonphosphory-
lated precursors to avian vitellogenin I and vitellogenin II.
J. Biol. Chem. 257:3837–3846.
Weiss, H. S. 1979. Nicarbazin induced hypercholesterolemia in
the hen. Proc. Soc. Exp. Biol. Med. 103:49–53.
Wells, R. J. 1999. Residues of some veterinary drugs in animals
and foods: Nicarbazin. FAO Food and Nutrition Paper 41/11.
Wyburn, G. M., H. S. Johnston, and R. N. C. Aitken. 1965.
Specialized plasma membranes in the preovulatory follicle
of the fowl. Z. Zellforsch. 68:70–79.
Yoder, C. A., L. A. Miller, and K. S. Bynum. 2005. Comparison
of nicarbazin absorption in chickens, mallards, and Canada
geese. Poult. Sci. 84:1491–1494.
Yoder, C. A., J. K. Graham, L. A. Miller, K. S. Bynum, J. J.
Johnston, and M. J. Goodall. 2006a. Effect of method of deliv-
ering nicarbazin to mallards on plasma 4,4′-dinitrocarbani-
lide levels and reproduction. Poult. Sci. 85:(in press).
Yoder, C. A., J. K. Graham, L. A. Miller, K. S. Bynum, J. J.
Johnston, and M. J. Goodall. 2006b. Evaluation of nicarbazin
as a potential waterfowl contraceptive using mallards as a
model. Poult. Sci. 85:
