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Molecular Effects of Nicarbazin on Avian Reproduction

Authors:
  • Center for Genocide Research and Education

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. Lipoprotein lipase, vitellogenin, transglutaminase, and calcium are all involved in egg formation and embryogenesis. 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 lipase activity. Second, vitellogenin phosphorylation was evaluated to determine if NCZ acts as a vitellogenin phosphatase. 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 lipase in vitro at 3.9 and 7.8 microg of NCZ/mL. Nicarbazin increased intracellular calcium levels in bull sperm, suggesting it also acts as a calcium ionophore. The portion of the NCZ molecule responsible for the increase in intracellular 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 transglutaminase 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.
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,Ndimethylcasein 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-N2-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.
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... Ott et al. [1] reported that feeding laying hens with 100 ppm or more of NCZ caused depigmentation of the brown shell and reduced hatchability in approximately 60%. Although the mode of action of NCZ for contraceptive activity is unknown, micrographs of vitelline membranes in mallards showed severe degenerative changes and preliminary in vitro studies indicated that it may increase intracellular calcium levels and early degradation of very low-density lipoproteins that comprise egg yolk Yoder et al. [3]. However, the effect of NCZ has not been studied in Creole hens of Mexico. ...
... Even though there are previous reports of the negative effect of nicarbazin on incubation parameters of birds Yoder et al. [3] and Avery et al. [10], there is not information with respect to birds that are not raised under industrial condition environments, as the Creole chickens of Mexico. Results of the current investigation agree with those of Hughes et al. [11] and Jones et al. [12] who found reduced hatchability in eggs from birds exposed to nicarbazin. ...
... As stated by Chapman [13], affection of the integrity of the vitelline membrane is the mechanism by which the coccidiostat affects the correct development of the chick embryo, so that early mortality is expected. On the other hand, given what is known about its mechanism of action Yoder et al. [3] the first stages of development are the most negatively affected by nicarbazin. In line with this fact, our results agree with Ott et al. [1] who found a 3.8-fold increase in early embryo mortality when White Rock breeder hens where fed nicarbazin. ...
... The adjuvant is added to increase DNC absorption at the intestinal level (Cuckler et al., 1955). Nicarbazin acts inside the eggs and increases the permeability of the yolk membrane causing the yolk and the albumin to mix, interrupting embryo development (Yoder et al., 2006a). When nicarbazin was provided to rock pigeons, the number of chicks was reduced by 59% and in the case of Canada geese eggs hatching decreased by 56% (Bynum et al., 2007;Avery et al., 2008). ...
... The magnitude of the reduction on successful nestlings in our study was similar to that described by Bynum et al. (2007) and Avery et al. (2008), who reported reductions in nestling numbers of 59% for rock pigeons and up to 56% in eggs hatching in Canada geese, respectively. Nicarbazin, used as contraceptive, has an effect on egg development at the time of onset of follicle development (Avery et al., 2008;Yoder et al., 2006a). Yoder et al. (2006a) found that nicarbazin increases the activity of lipoprotein lipase, reducing the amount of very low-density lipoprotein deposited in the follicle and consequently decreases the production of eggs and their weight. ...
... Nicarbazin, used as contraceptive, has an effect on egg development at the time of onset of follicle development (Avery et al., 2008;Yoder et al., 2006a). Yoder et al. (2006a) found that nicarbazin increases the activity of lipoprotein lipase, reducing the amount of very low-density lipoprotein deposited in the follicle and consequently decreases the production of eggs and their weight. ...
Article
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Eared doves (Zenaida auriculata) are responsible for substantial losses in cereal and oil crops as well as in dairy and feedlot production in the southern cone of South America. Various strategies have been shown to be effective in reducing damage at the farm scale, but in some scenarios, it is necessary to also incorporate population control methods due to excessive bird population size. An alternative approach to reduce pest bird populations is the use of contraceptive methods, minimizing the impact on the environment and non-target populations. Nicarbazin is registered in the United States as a contraceptive for Branta canadensis and Columba livia. The aim of this study was to measure the effect of nicarbazin on the reproductive performance of eared doves in captivity. This study included eleven caged pairs of nesting eared doves in three experimental phases (pre-treatment, treatment, recovery). Each pair was exposed to nicarbazin bait for four hours per day. The contraceptive used was OvoControlP® (0.5% nicarbazin) ground with a millstone into particles of 0.5 to 3.0 mm. Daily bait consumption and reproductive variables per pair (egg laying and 14-day-old fledgling) were recorded, and levels of 4,4'dinitrocarbanilide were measured in feces and unhatched eggs. Median consumption was 4.2 g of bait/pair/day. We observed a 62% reduction in the number of viable eggs and successful nestlings in the treatment phasein contrast to pre-treatment (V=36; p= 0.006). There were no significant differences (V= 0; p= 1) in the number of viable eggs between the pretreatment and recovery phases. Median daily bait consumption by pairs producing zero or one nestling (4.4 and 5.0 g/pair/day respectively) was significantly higher than that of pairs that had two nestlings (3.4 g/pair) during the treatment phase (t= 2.0; p= 0.002). Nicarbazin was effective in reducing reproductive performance of eared doves, and its effect was reversible when the treatment finished.
... A nivel molecular hay indicios de que el nicarbazin actúa aumentando la actividad de las enzimas lipasas de lipoproteínas y funciona como un ionóforo de calcio, aumentando los niveles de calcio intracelular en el esperma. Además produce inhibición de la actividad de la enzima transglutaminasa (Yoder et al., 2006a). La actividad ionófora del nicarbazin permitiría explicar el cambio de permeabilidad de la membrana del saco vitelino. ...
... El mismo conduce a que la yema y el albumen se mezclen (Figura 6) generando un ambiente extremo para el embrión y reduciendo el éxito de la eclosión de los huevos. Por otro lado el aumento de la actividad lipasa podría llevar a una reducción en sangre de lipoproteínas de baja densidad disminuyendo la cantidad de esta proteína que puede depositarse en la yema (Yoder et al., 2006a). ...
Research
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Actividades humanas como la agricultura introducen cambios en los ambientes naturales que en ocasiones provocan un incremento de las poblaciones de aves y mamíferos. Estas poblaciones superabundantes pueden generar conflictos entre los seres humanos y los animales silvestres. Problemas como el daño a cultivos, transmisión de enfermedades, riesgo de colisiones con vehículos, accidentes aéreos, degradación ambiental, etc. Hasta mediados de los 50 el control letal mediante el envenenamiento con cebos tóxicos, caza o trampas era la técnica utilizada como medio de regular las poblaciones abundantes. Sin embargo estos métodos son deficientes y presentan limitaciones legales, sanitarias, ambientales y concernientes a la opinión pública, por lo que se hace necesario el desarrollo de técnicas alternativas. Entre estas técnicas se desarrolla a principios de los 60 y 70 el control poblacional mediante el uso de quimioesterilizantes. Desarrollados principalmente para mamíferos, los contraceptivos han presentado un éxito relativo. Con un enfoque de la investigación cada vez mayor en el desarrollo de contraceptivos, y un mejor conocimiento de los sistemas y los comportamientos reproductivos de los animales, el control de la fertilidad como tecnología ha avanzado rápidamente. Actualmente la investigación en mamíferos está dirigida al desarrollo de vacunas contraceptivas. En aves el campo está orientado al desarrollo de contraceptivos orales como el nicarbazin, ácido linoleico conjugado y el diazacolesterol. El presente trabajo realiza una revisión de los trabajos publicados concernientes a los contraceptivos en aves. Esta publicación es fruto de un proyecto desarrollado en el marco de un acuerdo de vinculación tecnológica entre INIA-DGSA-MTO-ALUR, con financiación PACC-ALUR.
... Unspecified mode of action: for some compounds, it was not possible to identify a mode of action. Nicarbazin is one of the first synthetic compounds with a broad spectrum of activity; however, it is not indicated in summer, because it can increase the risk of heat stress, and in laying hens it negatively affects egg production [5,73]. The mode of action seems to occur by inhibition of succinate and ATP transhydrogenases and accumulation of intracellular calcium in second generation schizonts [74]. ...
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Avian coccidiosis is a disease causing considerable economic losses in the poultry industry. It is caused by Eimeria spp., protozoan parasites characterized by an exogenous–endogenous lifecycle. In vitro research on these pathogens is very complicated and lacks standardization. This review provides a description of the main in vitro protocols so far assessed focusing on the exogenous phase, with oocyst viability and sporulation assays, and on the endogenous phase, with invasion and developmental assays in cell cultures and in ovo. An overview of these in vitro applications to screen both old and new remedies and to understand the relative mode of action is also discussed.
... Nicarbazine wordt sinds 1950 gebruikt als coccidiostaticum bij kippen (Jones et al.,1990). De bijwerking van deze stof bij vogels is dat de ontwikkeling van eieren verstoord wordt, afhankelijk van de toegediende dosis (Sherwood et al.,1956;Yoder et al., 2006). Aanvankelijk werd van deze eigenschap gebruik gemaakt om vrijlevende, residentiële populaties canadaganzen (Branta canadensis) in te tomen (Avery et al., 2008;MacDonald en Wolf, 2013). ...
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Gebruik van ypozane bij de reu Anticonceptie voor duiven
... 8,9 Nicarbazin has the ability to reduce egg production because it interferes in cholesterol metabolism, which affects formation of the vitreous membrane, destroying the separation between egg yolk and egg white. 10,11 We refer to the product based on nicarbazin, used in Europe to control pigeon populations, as NP1 (see Material and Methods). NP1 consists of corn seeds covered with nicarbazin (800 ppm) and a water-repellent film. ...
Article
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BACKGROUND Nicarbazin is an anti‐coccidial product sometimes used as a contraceptive to reduce the size of feral pigeon populations. However, its effectiveness in reducing pigeon population size in cities has generated some controversy. Here, we evaluate its effectiveness in Barcelona city. RESULTS We set 23 feeding stations in which we provided nicarbazin and 10 feeding stations with a placebo (untreated corn). Censuses were carried out before and after one year of treatment, in radii of 200 m around each feeder. We additionally censused 28 circles of 200 m of radius distributed randomly 200 m away from the feeders and 28 circles distributed >500 m away from the feeders, which acted as controls. Population size across the whole city was also evaluated pre‐ and post‐treatment. We found that feral pigeon density did not change after one year of treatment, either in the circles around feeding stations with nicarbazin or in the areas around control stations distributed at 200 and > 500 m from the feeders. Population size in placebo circles rose after a year by 10%. The pigeon census for the whole city of Barcelona showed a 10% increase. CONCLUSION Overall, our results indicate that the nicarbazin treatment had no effect on the feral pigeon population size, and we advise against its use as a pigeon control method, at least in large cities. This article is protected by copyright. All rights reserved.
... The 4,4′-dinitrocarbanilide component of nicarbazin inhibits transglutaminase activity, whereas the 2-hydroxy-4,6-dimethylpyrimidine portion increases transglutaminase activity. In addition, nicarbazin increases lipoprotein lipase activity and acts as a calcium ionophore (Yoder et al. 2006). Nicarbazin and narasin show synergistic activity (Challey and Jeffers 1973) and a combination product of these drugs was developed. ...
Article
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Coccidiosis is a parasitic disease of a wide variety of animals caused by coccidian protozoa. The coccidia are responsible for major economic losses of the livestock industry. For example, the annual cost due to coccidiosis to the global poultry industry has been estimated to exceed US$ 3 billion annually. Currently available drugs for the control of this disease are either polyether ionophorous antibiotics that are derived from fermentation products, or synthetic compounds, produced by chemical synthesis. Unfortunately, no new drugs in either category have been approved for use for decades. Resistance has been documented for all those of the drugs currently employed and therefore the discovery of novel drugs with unique modes of action is imperative if chemotherapy is to remain the principal means to control this disease. This chapter aims to give an overview of the efficacy and mode of action of the current compounds used to control coccidiosis in livestock and provides a brief outlook of research needs for the future.
Chapter
Coccidiosis is a parasitic disease of the intestinal tract of poultry that is caused by protozoan parasites of the genus Eimeria. This disease is of worldwide occurrence and every year costs the poultry industry many millions of dollars to control. The parasites have an oral-fecal life cycle with no intermediate host and, depending upon the extent of exposure and environmental factors, can cause extensive morbidity and mortality. Coccidiosis is primarily a disease of young birds but can occur in older stock if they have not acquired immunity to infection. Unfortunately, there is little research on the effects of coccidiosis in birds reared for consumer egg production or broiler breeders. The emphasis of control strategies, especially in birds reared to produce eggs, has been to provide protection to young chicks while allowing them to acquire immunity that will protect them in later life. These strategies can be achieved by chemotherapy or vaccination.
Chapter
The popularity of birds as pets has increased substantially over the past several years. The fact that most pet birds are confined to the home environment exposes them to toxicants, such as the pyrolysis products, that poultry and wild birds are unlikely to come into contact with. Alternatively, pet birds can be exposed to toxicants to which poultry and wild birds are also exposed, but which are in different forms or from different sources. A diagnosis is generally based upon a history of exposure and characteristic postmortem lesions. Presently, there is no analytical test available to confirm exposure to the pyrolysis products. The rapidity of onset of severe signs and subsequent death most often precludes treatment. Awareness of the hazard and avoiding housing birds near polytetrafluoroethylene coatings is the best prevention. Other gases such as hydrogen sulfide and carbon dioxide generally do not present significant intoxication risks for birds. Interestingly, chickens are less sensitive to hydrogen sulfide than humans or dogs.
Nicarbazin is an effective and wide spread recognized substance for controlling the protozoal diseases with low number of short term adverse reactions. There are some molecular mechanisms proposed for its adverse effect but no one has conducted a research in vivo. Also there is no evidence found which can illustrate the long term adverse effects of the drug. It may hypothesized that Nicarbazin can affect the oxidative situation of cells by lipoprotein lipase activation. This study investigated the relationship between using Nicarbazin or its ingredients and oxidative stress situation by monitoring changes in the oxidative enzymes activity:Catalase, Glutathione peroxidase, Superoxide dismutase and specific biomarkers of oxidation: Dityrosine and Malondialdehyde. The study also investigates Nicarbazin's effects on colony stimulating factors in lung cells. In conclusion it found that Nicarbazin can worsen oxidative stress and increase colony stimulating factors which may have affect on long term adverse reactions of drug.
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NICARBAZIN effectively controls experimentally induced coccidiosis in chickens. Barber (1955) reported that levels of 0.02, 0.0125, and 0.0075 percent in mash gave protection against both Eimeria tenetta and E. acervulina. The regimen was not accompanied by toxicity or interference with development of immunity. Ott et al. (1955) reported that this drug, when incorporated in the feed of growing chicks at levels of 0.01 and 0.02 percent, did not interfere with normal growth or feed utilization. Likewise, these authors stated that it did not interfere with sexual maturity, egg production, or egg fertility. However, the feeding of the drug to New Hampshire laying hens was reported by these authors to be associated with a change in the color of the egg shells from brown to white and with reduced hatchability. McClary (1955) observed that nicarbazin at 0.0125 percent in the diet of White Rock hens was responsible for changing the color . . .
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Albumen from normal eggs and from eggs having mottled yolks was examined for composition and for functional capacity. Yolk mottling was induced by including 0.02% Nicarbazin in the hen’s ration. Albumen from eggs having mottled yolks was quite different in composition from albumen collected from normal eggs. Moisture was lower, but percentage of protein, fat, ash, and mineral content was higher in albumen from mottled eggs than from normal albumen. Fat content was significantly greater in albumen from eggs having mottled yolks, indicating that lipid material had passed from mottled yolks, through the vitelline membrane, into the albumen. Electrophoretic analysis indicated very little difference in protein composition between the two albumen samples; however, there were indications of lipid contamination in the albumen sample from eggs with motded yolks. There was considerable difference in the functional performance of albumen from eggs with mottled yolks and albumen from eggs having normal yolks. Albumen from eggs with motded yolks had impaired foaming capacity and produced poor angel cakes.
Article
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INTRODUCTION YOLK mottling has been observed for many years but the physical and chemical alterations of the yolk associated with this phenomenon have not received particular attention. The only data were those which characterized a sample of mottled areas, removed from boiled yolks, as having a higher protein-fat ratio than a sample of non-mottled yolks (Almquist, 1933). When nicarbazin, a coccidiostat, was fed at levels above 0.005 percent to laying hens, there was an increase in the incidence and degree of yolk mottling above that observed in eggs from non-medicated hens (Polin, Ott and Siegmund, 1957). Some of the yolks were analyzed to determine the changes associated with mottling, and the results are reported herein. EXPERIMENTAL The eggs used in this experiment were obtained from White Plymouth Rock and White Leghorn hens. Some of these birds were kept in single cage batteries at the laboratory while others were at a . . .
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NICARBAZIN, an equimolecular complex of 4,4′-dinitrocarbanilide and 2-hydroxy-4,6-dimethylpyrimidine, has been studied extensively and found to be an effective anticoccidial agent for chickens. This paper summarizes the presently available information on this anticoccidial agent. CHEMICAL NATURE OF NICARBAZIN During the investigation of the therapeutic potentialities of arylureas, Cuckler, Malanga, Basso and O’Neill (1955) observed that molecular complexes of certain substituted carbanilides possessed antiparasitic activity. The complex formed with 4,4′-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP) was the most effective anticoccidial compound (Figure 1). In addition to HDP, several other compounds form equimolecular complexes with DNC. These include 2-hydroxy pyrimidine, 3-amino-as-triazine, 2-hydroxypyridine, 2-mercapto-4,6-dimethyl pyrimidine, formamide, dimethylacetamide, dimethylformamide, tetramethylurea, acetylpiperidine, and the hydrochlorides of pyridine and trimethylamine. Although the complexes formed with DNC and these various compounds had demonstrable anticoccidial activity, none was more potent than nicarbazin. Furthermore, several structural analogues of DNC were either ineffective as anticoccidial agents, or failed to form a complex . . .
Article
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DURING the winter of 1956 several field cases were reported to the Experiment Station of flocks of hens in which a large percentage of the freshly laid eggs showed abnormalities of the yolk. The yolks seen in these field cases and subsequently produced experimentally were blemished by a combination of translucent and opaque areas with the result that the color of the yolk surface was not uniform. In many eggs, whitish-yellow “curdled” areas were evident, and in severe cases the yolk had a brownish-orange color and was streaked. These yolks were apparently similar to those described by Jeffery (1945). The studies reported here were undertaken to determine the possible cause of the yolk damage observed in the field cases. MATERIALS AND METHODS During the experiments described below, eggs laid the previous day were broken out on a glass plate supported between mirrors so that all the surfaces of the yolk . . .
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IN A previous paper by Baker et al. (1957) the effects of feeding Nicarbazin to laying hens on the yolk quality of their eggs was described. Neither the nature of the damage to the yolk nor the mechanisms involved in causing the damage were known. Experiments were designed to study the possible mechanisms involved. MATERIALS AND METHODS Eggs were obtained from hens fed Nicarbazin. The scoring system used to estimate yolk damage was the same as described previously (Baker et al., 1957). Water uptake of the yolk was determined according to the method of Orru (1939). The intact yolk was dried on filter paper, placed in a tared beaker and covered with the experimental solution. The system was incubated at 37°C. for 3 hours, the fluid drained off and the last traces of solution removed with filter paper by rolling the yolk over the paper. It proved to be practically . . .
Article
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INTRODUCTION ALTHOUGH yolk mottling in fresh and stored eggs was reported almost 25 years ago (Almquist, 1933), and for years has been a generally recognized problem, little is known of its incidence in farm flocks and the nature of its occurrence. It is seen in held eggs more frequently than in fresh eggs (Romanoff and Romanoff, 1949). Preliminary studies conducted at the Merck Institute showed that the drug nicarbazin recommended as a coccidiostat for young chickens could cause an increase in the incidence and degree of yolk mottling if fed to laying hens. Subsequently, field and laboratory studies were initiated to determine the minimum level of nicarbazin in rations fed to hens that would cause the mottling effect. The data of these studies are reported herein. A preliminary report (Polin et al., 1956a) has already been published. EXPERIMENTAL White Plymouth Rock (W.P.R.) and New Hampshire (N.H.) hens were used in . . .
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Yolk mottling was induced by feeding 0.02% Nicarbazin to 6-month old White Leghorn pullets. A second group of pullets, fed a standard layer ration, served as the control. Eggs were collected for three months on a daily basis, but the yolks were pooled on a weekly basis for analysis. Composition and functional properties of both mottled and normal yolks were determined. The percent solids of normal yolks was significantly higher than mottled yolks. The pH of normal yolks was lower than mottled yolks but not significantly. Normal yolks contained higher percentage fat, protein, and ash than mottled yolks; however, the percent carbohydrate was lower in normal yolks. There was no difference in the cholesterol content of mottled and normal yolks. Normal yolks contained significantly higher percent calcium, phosphorus, and iron than mottled yolks. Emulsifying capacity and viscosity of normal yolks were greater than for mottled yolks. Foam volume and sponge cake volume were always larger for mottled yolks than for normal yolks, but normal yolks produced more stable foam and better cake texture. Electrophoretic separation of the components of mottled and normal yolks indicated that two egg white proteins were present in mottled yolks but not apparent in normal yolks. The two proteins are believed to be ovalbumin and conalbumin.
Article
1. Phosvitin extracted from domestic hen's-egg yolk was resolved on Sephadex G-100 into two phosphoprotein components. 2. The major component has a molecular weight of about 3.4×104 and alanine as an N -terminal residue. Glucosamine is present, but tyrosine is virtually absent. 3. The minor component has a molecular weight of about 2.8×104 and lysine as an N -terminal residue. Missing residues are glucosamine, methionine and leucine. Lysine, histidine, threonine, glycine, phenylalanine and tyrosine contents differ significantly from those of the major component. 4. Sephadex G-100 also removes small amounts of an impurity with a much higher molecular weight.