Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
83
THE DETECTIONOF CALCIFICATION IN KIDNEYS OF THE
LAYING HENS(GALLUS GALLUSDOMESTICUS) BY USING THE
HISTOLOGICAL AND IMMUNOHISTOCHEMICAL
TECHNIQUES
Ahmed Mahdi Al- badri *,Ali Fayadh Bargooth**,Esraa Abdul khaliq
Zegyer*
*Department of Biology, College of Science, University of Wasit, Wasit,Iraq.
**Department of Animal Resources,College of Agriculture,University of
Wasit,Wasit, Iraq.
Keywords: Calcification, Kidneys, Von Kossa, Immunohistochemistry, laying hens
ABSTACT
The present research was planned to realize and determine the calcification in
the kidneys of two ages (18 months and 24 months) of laying hens. There are no clear
differences in histological appearance by using (H&E) stain between to ages, but
there are higher significant differences in the height of epithelium cells and lower
significant differences in the lumen of renal tubules in the kidneys of laying hens for
(24 months) than in the (18 months). By using special dyes (Van Kossa)
revelateddeposits of calcium salts and using immunohistochemistry method, the
results showed presence of calcification in form of small foci in varying sizes bearing
within them brown to dark color calcium deposits in the glomerulus, the epithelium
layer and basement membrane of the renal tubules and between them. In general the
calcified foci in different areas of kidneys in the 24 month age increase than in the 18
months age.
INTRODUCTION
In mammals and birds, the kidneys are main organ participate with keeping
the stable nature of the internal environment by saving balancebetween glomerular
filtration andvolume of renal tubular secretion in addition to the osmolality,ionic
content and pH of the body fluids, there are high important role of the kidney in
maintaining homeostasis [1].
In birds, the urinarysystem have large paired kidneys, located symmetrically
at the sides of the vertebral column, the kidneys drained the secretion by ureters.The
uerters open into the urodeum of the cloacae, urinary bladder is absent in birds [2].
There are tiny toughen structures with shoaly drooping between them covering
the kidney. Each one of these structure is composed unite of cortical tissue of kidney
[3]. About8 or 10 of the cortical unites consistence collection urine into one medullary
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
84
cone. These areas of kidney tissue are recognize as lobules composed from little
elongated collectives of cortical tissue conjointly the one medullary cone to the urine
stream. Medullary lobule is necessary etcher encompassed with connective tissue
covering join to cortex at broad ending and related to the main urethral department at
the other [4].
Avian kidney lobe is expressing cutting into lobules, the lobules had cortex
and medulla [5,6,7]. The nephron is the active unite of the kidney and its structure has
considerable varies amongst variant vertebrates; also its different between species.
There are two types of nephron in birds (reptilian type and mammalian type). The
reptilian type is smaller than the mammalian and have no loop of henle[8,2].
There are no differences in glomerulus between the avian and mammalian
except in size, the mammalian glomerulus is larger and has complex system of
capillaries loops. The proximal convoluted tubules(P.C.T) are variant from distal
convoluted tubules(D.C.T) in height of epithelium cells which is approximately
higher cuboidal than the P.C.T. and there are no brush border in DCT.[3,4].
Calcium is the metal which is consider highest concentration in laying hens,
it's about 1.5% of the body weight. In the adult bird, the calcium is diversify higher
than one third of all mineral [9]. The higher concentration of calcium ions is taking
through the lifetime until reach to the egg production to begin decrease in
concentration [10].The calcium salts are reabsorption through the proximal and distal
tubules of nephrons in kidneys. There are participate in some parts of these structure
as calcium phosphate in the epithelium and there are calcium carbonate in the renal
interstitium[11].Egg laying and shell calcification need high quality ion
calcium(metastasis). The calcium carbonate is compose 95% from the egg shell [12].
The incidence of calcification is occur by series of complex and regulation
developments in parts of organs [13]. The principle of the causality of calcification
include "age, hormone, nutrition calcium, vitamin D and the parathyroid hormone
plays chief part in calcification [14].The broiler chicks which feeding 3.27% calcium in
diet for 15 weeks starting with calcification in kidneys [15]
MATERIALS AND METHODS
Animals and tissue preparation:
Twelve birds of laying hens selected from poultry fields of the kut town.
According to the age, the bird were classified into two groups (18 months and 24
months), six laying hens chicken in each class. These birds were well health and it's
were not undergo from any disease. The birds were anesthized by using the
chloroform, then the kidneys were rise and taken samples from different regions of it.
The samples were fixated with 10% of neutral buffered formalin for (72 hours), after
that the specimens washed by tap water 4-6 hours. The processes of the technical
histology were utilized which include "dehydration, clearing, infiltration, embedding,
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
85
cutting, and staining". There were two dyes using hematoxylin and eosin (H&E) for
showing the general structures, and Van Kossa stain torevelate the deposit of calcium
salts in tissues of the kidney of laying hens[16].The morphometric measurements
were done includes: diameters of PCT and DCT[17]."Statistical analysis was
obtained as standard deviation and standard error for many parameters in kidneys.
Analysis of T-test forstatistical differences of variables of two sets was done [18]".
Immunohistochemistry technique:
The standard biotin free one- step HRP polymer anti-mouse, rat and rabbitIgG
(H+L) with DAB immunostaining procedure were used to detect calcium deposits
inthe kidney. The dewaxed, hydration of the series of alcohol solution and washed up
time of distilled waterwere evented for sections of embedded in laying hens. Antigen
retrieval by submerge slides in jar having "citrate buffer solution". Slides were
washed with "phosphate buffer saline with pH 7.2 for 5 minute". "The sections were
incubated with peroxidase block for 5-10 minutes at room temperature RT, and then
were washed with distilled water 3times, also slides were washed with PBS.
Subsequently, sections were incubated with protein blocking solution 5-10 minutes at
RT, and then incubated with primary antibody (diluted of 1:500) for 30 minutes at
RT. Slides were washed with PBS 5-7 times, and then incubated with one -step HRP
polymer for 30 minutes at RT, then slides were washed with PBS, also slides were
washed with distilled water 2-3 times afterward, add few drops of ready to use DAB
reagent on tissue slides (was used by mixed well 1 ml of reagent buffer & substrate
(BS) and 50 microliter of reagent C chromogen) for 6-10 minutes at RT, then washed
with PBS and were washed in distilled water. Later, section was incubated with
hematoxylin stain 30- 60 seconds subsequently; slides were washed with distilled
water and mounted with D. P. X. mounting medium"[19]and the photographs were
takenbya canon digital camera with 18.0 mega pixel
RESULTS AND DISCUSSION
Histological study:
In the cross section of kidneys in the two groupsof the laying hens, the
microscopic examination were showed that the kidneys covered by capsule composed
of smooth muscle with some of collagen fibers. The kidneys had many lobules, these
lobules had two layers (medullary tissue and cortical tissue) the delineation between
cortical and medullary tissue are no cleared as in mammalian. There are found the
cortical type and medullary type of nephrons (Fig.1, 2).The same finding was showed
by [20].
The current result revealed that the nephrons had renal corpuscles composed
of the glomerulus surrounding with Bowmans capsule which is separated from it by
Bowman space. The distal convoluted tubules(DCT) were showed different from
proximal convoluted tubules(PCT) which the epithelium lining without brush border
and the cells was lighter staining, the shape of the DCT was approximately cuboidal.
The limbs of henle lining with simple cuboidal epithelium cells, the thick and thin
limbs of henle passed between the collecting ducts(Fig.1, 2).This finding was in
agreement with that of [21].
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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In both ages, our results showed with Van kossa stain, there were presence of
calcification as few small blot deposited in epithelium, basement membranes of renal
tubules and between them, also can be observed in the glomerulus. This calcification
represented the calcium salts which has brown to dark in color. The calcification in
the 18 age relatively decrease than in the 24 age(Fig.3, 4, 5, 6).This is may be because
increase intake of calcium in fed of laying hens for long period lead to calcification in
kidney, this opinion was concordant to what has been mentioned by many authors as
[22] that the broiler chicks which feeding 3.27% calcium in diet for 15 weeks starting with
calcification in kidneys,[23] remained that the nephrocalcinosis in young ostriches occur
when intake (13.48% calcium & 0.82% phosphorous) this is diagnosis through radio
graphically and appeared as multiple radio opacities throughout the renal parenchyma. For
verification, [24] observed that the high dietary calcium and low dietary phosphorous diet of
pullet and laying hens cause calcification &urolithiasis.
The diameters of tubules and height of epithelial cells of proximal convoluted
tubules and distal convoluted tubules in 18 months age of laying hens were higher
significant differences than in 24 months age(Table 1). We suggest that the presence
of calcification in the epithelium and basement membrane of the tubules which more
abundant in the 24 month age than the 18 month age lead to thickening in these parts,
this cause decrease of the lumens of tubules. This suggestion agree with [25] who
mention that the occurrence of calcification in kidney of mice after giving the calcium
gluconate caused thickening of epithelium and basement membrane of renal tubules
and there are no presence of calcification in the nucleus of cells.
This occurrence of increasing of calcification in the kidneys of laying hens
through forward of aging is agree with [15]who show the calcification is increase with
the progression of aging in many organs(heart, trachea and kidneys) of chicken and
ostrich, and there are many authors refers for that as [26] whom appeared that the
calcification in the trachea increasing with the progression of aging in laying hens by
using the immunohistochemical methods.
Table(1):Show diameter of PCT and DCT with the height of epithelial lining inthe
two ages of laying hens:
Aging 18 month 24 month
Diameter
of tubule
PCT 45.15 ± 1.132
A
42.12 ± 1.211
B
DCT 29.23 ± 1.211
A
25.34 ± 1.113
B
Height of
epithelial
PCT 11.32 ± 0.145
A
14.16 ± 0.121
B
DCT 7.422 ± 0.312
A
9.532 ± 0.226
B
"The values represent the Mean ± Standard Error S.E.) The different capital letters mean significant differences at level of
p<0.05"
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Fig(1):Histological section of kidney in laying hens (at 18 months age) showno cleared
delineation of cortical and medullary layers. capsule (Ca) Cortex (C), medulla (M) cortical
nephrons (Cn) medullary nephrons (Mn), proximal convoluted tubules (PCT),distal convoluted
tubules (DCT), collecting duct(CD) and loop of henle (LH). (H & E stain 20 X ).
Fig(2):Histological section of kidney in laying hens (at 18 months age) showno cleared delineation
of cortical and medullary layers. capsule (Ca), Cortex (C), medulla (M) cortical nephrons (Cn),
medullary nephrons (Mn) proximal convoluted tubules (PCT) ,distal convoluted tubules (DCT),
collecting duct(CD) and loop of henle (LH). (H & E stain 20 X ).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Fig(3):Histological section of kidney in laying hens (at 18 months age) showcalcification in form
of group's of small foci (F): in glomerulus (1),epithelium and basement membrane of proximal
convoluted tubules (2), epithelium and basement membrane of distal convoluted tubules (3) and
between renal tubules (4) (Von Kossa 20 X ).
Fig(4):Histological section of kidney in laying hens (at 24 months age) showcalcification in form
of group's of small foci (F): in glomerulus (1),epithelium and basement membrane of proximal
convoluted tubules (2), epithelium and basement membrane of distal convoluted tubules (3) and
between renal tubules (4) (Von Kossa 20 X ).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Fig(5):Histological section of kidney in laying hens (at 18 months age) showcalcification in form
of group's of small foci (F) in different areas of kidney (Von Kossa 20 X ).
Fig(6):Histological section of kidney in laying hens (at 24 months age) show increase of
calcification than in (18 months age). The calcification was in form of group's of small foci (F) in
different areas of kidney. (Von Kossa 20 X ).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Immunohistochemistry study:-
A- At 18 months age:
At this stage, the current study revealed that the calcification was detected in
the glomerulus of Bowmans corpuscles in form of foci has brown to dark deposits
calcium salts inside it(Fig.7, 8). These resultant of calcification in the glomerulus of
Bowman capsules relatively was appreciated [22]. On the other hand, these foci of
calcium salts was scattered in the epithelium, basement membrane of proximal
convoluted tubules and distal convoluted tubules and between these tubules. The
amount of these areas relatively valued (++)
B- At 24 months age:-
In this precede age, the present study found that the calcification was demonsted
more proportion from previous age in all regions of kidneys which occurrence the
calcification(Fig.9, 10)..Comparatively with the previous age can appreciated the
calcification in this age at ( +++)in glomerulus, (+++)in proximal convoluted tubules
and distalconvoluted tubules
This occurrence of increasing of calcification in the kidneys of laying hens
through forward of aging is agree with [15]who show the calcification is increase with
the progression of aging in many organs(heart, trachea and kidneys) of chicken and
ostrich, and there are many authors refers for that as [24] whom appeared that the
calcification in the trachea increasing with the progression of aging in laying hens by
using the immunohistochemical methods
Fig(7):immunohistochemicalsection of kidney in laying hens (at 18 months age) appeared
calcification in form of group's of small foci (F) in different areas of kidney.Proximal
convoluted tubules(PCT) and dital convoluted tubules(DCT).(20 X ).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Fig(8):Immunohistochemicalsection of kidney in laying hens (at 18 months age) appeared
calcification in form of group's of small foci (F)in different areas of kidney.Glomerulus (G).40 X
Fig(9):Immunohistochemicalsection of kidney in laying hens (at 24months age) appeared
relatively valued calcification more than in the previous age. The calcification in form of group's
of small foci (F) in different areas of kidney.Proximal convoluted tubules(PCT) and dital
convoluted tubules(DCT). 20 X
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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بواسطة (Gallus gallusdomesticus) الکشف عن التکلس فی کلیة الدجاج البیاض
استخدام التقنیات النسیجیة والکیمیاء النسیجیة المناعیة
احمد مھدی صالح*، علی فیاض برغوث**، أسراء عبدالخالق زغیر*
*قسم علوم الحیاة، کلیة العلوم، جامعة واسط، واسط، العراق.
**قسم الثروة الحیوانیة، کلیة الزراعة، جامعة واسط، واسط، العراق.
الخلاصة
ھر و 24 رین( 18 ش ده للعم اض وتحدی دجاج البی ة ال ی کلی تکلس ف ن ال ق م ث للتحق ذا البح مم ھ ص
بغة تخدام ص د اس رین عن لا العم ی ک یجی ف المظھر النس ات ب ود اختلاف دم وج ائج ع رت النت ھر). اَظھ ش
دجاج ة ال ی کلی ة ف ات البولی ة للنبیب ا الظھاری اع الخلای الھیموتوکسلینوالایوسینفی حین وجد فرق معنوی فی ارتف
ار ی اقط وی ف رق معن ائج ف جلت النت ا س البیاض عند عمر 24 شھر اعلى مما ھی علیھ فی عمر 18 شھر بینم
بغة ة( ص بغات خاص تخدام ص م اس ھر. ت ر 24 ش النبیبات البولیة فی عمر 18 شھر اعلى مما ھی علیھ فی عم
ائج رت النت یوم، اظھ لاح الکالس بات ام ن ترس الفان کوسا) وطریقة الفحص بالکیمیاء النسجیة المناعیة للکشف ع
ة ی الطبق ة وف ی الکبیب وجود تکلسات على شکل بؤر صغیرة مختلفة الاحجام وذات لون بنی الى اسود مترسبة ف
اطق ی المن ة ف البؤر المتکلس ة کانت ورة عام ات. بص ک النبیب ین تل ة وب ات الکلوی دی للنبیب اء القاع ة والغش الطلائی
المختلفة من الکلیة فی عمر 24 شھر اکثر مما ھی علیھ بعمر 18 شھر.
REFERENCES
Fig(10):Immunohistochemicalsection of kidney in laying hens (at 18 months age) appeared
calcification in form of group's of small foci (F) in different areas of kidney.Glomerulus (G)40X
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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[1]-Hall, L. W. (1983). Kidney function. In: Veterinary Nephrology 1st ed., BAS
printers limited. Girton College, University of Cambridge. pp:27 – 56.
[2]-Hodges, R. D. (1974).The renal system. In the histology of the fowl.2nd ed.
Academic press. London and New York. pp:489 -524.
[3]-Siller, W. G. (1981).The kidney. In: Renal pathology of the fowl. 2nd ed. academic
press. London. pp:193 – 220.
[4]-Farner, D. S. and King, J. R. (1972). The urinary system of the birds. In: Avian
biology . Academic press. New York and London . vol. ІІІ. pp:527-572.
[5]-Bohle, M.A. and Christensen, J.A. (1985). Structure of the avian kidney. Anat.
Record., 212: 33-40.
[6]-Casotti, G.; Lindberg, K. K. and Braun, E. J. (2000). Functional morphology of
the avian medullar cone. Am. J. Physiol. Regul. Integr. Comp. Physiol., 279: 1722-
1730.
[7]-Braun, E. J. (1999). Integration of organ systems in avian osmoregulation. J. Exp.
Zoolo, 283:702-707.
[8]-Reece, W.O. (2004). Dukes’ Physiology of Domestic Animals. 12thed. Cornell
University Press, Ithaca, PP: 107-113.
[9]-Klasing, K.C. (1998). Amino acid. In: Comparative avian nutrition. Wallingford.
CAB International,PP: 133-170
[10]- Luck, M. R. and Scanes, C. G. (1979): Plasma levels of ionized calcium in the
laying hen (Gallus domesticus). Comp. Biochemical. Physiol. 63: 177-181.
[11]-. Gottschalk, C. W.; Lassiter, W. E., and Mlle, M. (I960).Localization of urine
acidification in the mammalian kidney. Am. J. Physiol., , 98:580-585.
[12]-Arie, B. R. (2008). Review: Calcium homeostasis and vitamin D metabolism and
expression in strongly calcifying laying birds. Part:Molecular & Integrative
Physiology.In:Comparative Biochemistry and Physiology.Institute of Animal Science,
ARO, The Volcani Ctr., Bet Dagan.P.p: 477-490.
[13]-Gilmcher, M.J. (1984). Recent studies of mineral phase in bone and its possible
linkage to the organic matrix by protein-bound phosphate bonds. Philosophical
Transactions of the Royal Society of London, Series Biological Sciences. 304, 479-
508
[14] Perry, T. W. (1984): Animal Life-Cycle Feeding and Nutrition. Academic Press,
INC. New York, USA. Pp: 22-27.
[15]- Morrow, C. J. (1997). Hypophosphatemic rickets and nephrocalcinosis in ostrich
chicks brooded and reared on limestone sand. Vet. Rec. 140:531-532.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
94
[16]—Bancroft, J. D. and Stevens, A. (2013). Theory and Practice of Histology
Techniques. 8th ed. Churchil Livingstone. pp:127-129.
[17]-kayer, S. R. and Banchero, N. (1985). Myocardial capillary in acclimation to
hypoxia. P. flugars, arch. 44:391-325.
[18]-Joda, M. (2008). The progressive statistical analysis by using spss. 1st edit. Wales
house edition, Amman. Jordon. pp: 100-103.
[19]-Robertson,G. J. and Wilhelm, S. I. (2008). Syringeal anatomy and allometry in
murres (Alcidae:Uria). Ornithol. J. 149: 545-554.
[20]-Morild, I.; Bohle, A. and Christensen, J. A. (1985). Structure of the avian
kidney . J. of anatomy Research. 212(1): 33–40.
[21]-Riddell, C. (1999). Histology and physiology of the chicken. In: Comparative
anatomy. 3ed Ed. University of Saskatchewan. University press, Canada. pp:102-110
[22]- Chandra, M. (1984). Hematologic changes in nephritis in poultry induced
bydiets high in protein, high in calcium, containing urea or deficient in vitamin A.
Poultry Sci. 63:710-716.
[23]-Wideman, R.F.;Mallinson, E.T. and Rothenbacher, H. (1983): Kidney function
of pullets and laying hens during outbreaks of urolithiasis. Poult Sci. 62:1954-1970.
[24]-Cualfield, J. B.and Schrag, P. E. (2000). Electron Microscope study of Renal
Calcification. VoI.44,NO. 3
[25]-TRAvis, D. (1990). Structural features of mineralization from tissue to
macromolecular levels of organization in the decapod crustacea. In: Comparative biology
of calcified tissues. Ann. New York Acad. Sc. (In press).
[26]-Al- badri, A.M. andAl-Salman, A. A. (2016).Histological and
Immunohistochemistry Studies of Trachea Calcification in the Laying Hens (Gallus
gallusdomesticus). International Journal of Scientific Engineering and Research
(IJSER).4(2) : 11-16.
