This document summarizes a study on the scanning electron microscopic structure and chemical composition of urinary calculi (stones) found in geriatric dogs. Microscopic examination of urine samples revealed increased numbers of blood cells, epithelial cells, pus cells, casts, bacteria and crystals of various shapes, predominantly struvite, calcium oxalate dihydrate and monohydrate, and ammonium urate. Scanning electron microscopy showed perpendicular columnar strata of struvite crystals and wavy phases of uric acid. Chemical analysis identified calcium phosphate, calcium oxalate and urea stones. The study characterized the microscopic and electron microscopic appearance of crystals and chemical composition of urinary calculi in geriatric dogs.

1. Scanning Electron Microscopic Structure and Composition of Urinary Calculi of Geriatric Dogs
IJVSAH
Scanning Electron Microscopic Structure and Composition
of Urinary Calculi of Geriatric Dogs
*1Karlapudi Satish Kumar* and 2Kandula Srikanth
1Department of Veterinary Medicine, College of Veterinary Science, Rajendranagar, PV Narsimha Rao Telangana
Veterinary University, Hyderabad, Telangana State, India
2Department of Veterinary Medicine, College of Veterinary Science, Korutla, Jagityal dist, Telangana State, India
Microscopic examination of urine samples collected from geriatric dogs revealed increased
numbers of erythrocytes, leucocytes, epithelial cells and pus cells along with casts, bacteria,
spermatozoa and crystals of various shapes. Among the different crystals, triple phosphate or
struvite were predominant, followed by calcium oxalate dihydrate, calcium oxalate monohydrate
and ammonium urate or biurate. The struvite crystals were, coffin-lid shape and while calcium
oxalate dihydrate were octahedron or envelope and monohydrate crystals demonstrated “picket
fence” and “dumbbell” and “hemp seed” appearance. Brown or yellow-brown spherical bodies
with irregular borders with thorn-apple appearance were shown by ammonium urate or biurate
crystals. SEM aspects of magnesium ammonium phosphate crystals revealed perpendicular
columnar strata, few with scattered hexa or octa-hedral coffin-lid shaped crystals and calcium
phosphate crystals were like cracked eggshells. Presence of wavy phases with sundry areas (uric
acid), picket fence (calcium oxalate monohydrate) and typical envelope (calcium oxalate
dehydrate) were electron microscopic appearance of various crystals.
Key words: crystalluria, urinary calculi, scanning electron microscope, composition, geriatric dog
INTRODUCTION
Urinary incontinence and/or inappropriate urination
caused by cystitis or cystic calculi is commonly reported in
geriatric dogs and is a major reason for ascending infection
that may progress to renal insufficiency (Davies, 1996)
Urolithiasis, particularly struvite uroliths have been
reported as the most common and recurrent clinical
problem in dogs (Houston et al., 2004 and Sosnar et al.
,2002). Literature also revealed an equalization of calcium
oxalate and struvite-containing calculi, and subsequent
increase in calcium oxalate-containing uroliths in the
present scenario (Picavet et al., 2007 and Ling et al.,
2003). Though urolithiasis is predominantly noticed
among all the breeds, calcium oxalate crystals are
common in small breeds and whereas, struvite formation
is predominant among large breeds. Struvite and calcium
oxalate uroliths were recorded to be most common with a
significant increase in calcium oxalate crystals over a
period of time; most commonly noticed among male while
struvite in female (Doreen et al., 2017). Though the
reasons are still obscure, certain predisposing factors, viz.,
geographical preference to few breed, demographic and
nutritional changes might be the causes for difference in
mineral composition in stone formation (Dijcker et al.,
2012). Microscopic evaluation of urine, particularly crystals
might help in decision making for dietary adjustments.
Present paper puts on record about scanning electron
microscopic aspects and chemical composition of crystals
from urine samples of geriatric dogs.
MATERIALS AND METHODS
Urine sample collection: Urine samples were collected
from 50 geriatric renal insufficiency dogs that were
presented to Teaching Veterinary Clinical Complex of
College of Veterinary Science, Hyderabad, by bladder
expression or ultrasound guided cystocentesis (Kurien et
al., 2004) or by catheterisation, for routine urinalysis.
*Corresponding Author: Karlapudi Satish Kumar,
Ph.D., Professor and University Head, Department of
Veterinary Medicine, College of Veterinary Science,
Rajendranagar, PV Narsimha Rao Telangana Veterinary
University, Hyderabad – 500030, Telangana State, India.
Email: drsatish.ksk@gmail.com
Co-Author Email: srikanth0649@gmail.com
Research Article
Vol. 7(1), pp. 081-087, March, 2021. © www.premierpublishers.org ISSN: 8991-0338
International Journal of Veterinary Science and Animal Husbandry

2. Scanning Electron Microscopic Structure and Composition of Urinary Calculi of Geriatric Dogs
Kumar and Srikanth. 082
Microscopic examination: Collected urine sample was
processed to obtain urine sediment and microscopic
examination was carried out by the procedure mentioned
by Carolyn and Weinstein (2012). The sample was
centrifuged in a test tube at 2,000 rpm for 5 minutes until a
moderately cohesive button is produced at the bottom of
the tube. The supernatant is decanted and a volume of 0.2
to 0.5 ml is left inside the tube. The sediment is re-
suspended in the remaining supernatant by flicking the
bottom of the tube several times. A drop of re-suspended
sediment is poured onto a glass slide and cover slip was
placed. It was then examined by light microscope under
both low-power and high-power field (Kesson et al., 1978).
Electron microscopy: Sediment suspensions were dried
overnight at 370C, isolated under stereozome, mounted on
double sided sticky carbon tape, and exposed / fixed with
1% osmium tetroxide as a fume fixation. The processed
samples were mounted over the stubs with double-sided
carbon conductivity tape, and a thin layer of gold coat over
the samples were done by using an automated sputter
coater using JEOL JFC – JSM 5600 electron microscope
unit, at required magnification as per the standard
procedures given by John . (1998), at RUSKA laboratory,
College of Veterinary Science, Rajendranagar,
Hyderabad.
Chemical analysis of uroliths: Various uroliths that were
collected from urinary bladder and urethra during the study
were subjected for qualitative chemical analysis as per
procedure given by Hodgkinson (1971).
RESULTS
Examination of cellular components and casts: Urine
sample from renal insufficiency dogs was centrifuged at
2000rpm/5min and the sediment was examined under
microscope at low power field then changed to high power
field for quantitative microscopic examination. Complete
examination of urinary sediment requires proper
identification of cells (RBCs, WBCs and epithelial cells),
casts, organisms (bacterial, fungal), crystals, mucus and
artifacts or contaminants. Numbers of casts are recorded
per lpf and numbers of RBCs, WBCs and epithelial cells
are recorded per hpf. The sample revealed erythrocytes,
leucocytes, epithelial cells and pus cells whose mean
values were 18, 5, 8 & 6 per high power field among renal
insufficiency dogs against 1-2, 0, 0-2 & 0 among
apparently healthy adult dogs. Microscopic examination of
urine also revealed moderate (15 per hpf) to many (22 per
hpf) crystals of various types, few coccobacilli, casts and
debris along with occasional spermatozoa.
Examination of urinary crystals: Various types of
crystals identified during microscopic examination of urine
were formed by precipitation of solutes, specifically
inorganic salts, organic compounds or iatrogenic
compounds. Crystals are more likely to form in
concentrated urine samples. In the present study crystals
of various types, shape and colour were identified.
Predominantly these crystals were of triple phosphate or
struvite (41.68%), followed by calcium oxalate dihydrate
(23.24%), calcium oxalate monohydrate (18.80%) and
ammonium urate or biurate (16.28%). Triple phosphate
(magnesium ammonium phosphate) or struvite crystals
were colourless, three to six sided prisms with oblique
ends “coffin-lid” (fig. 1). Calcium oxalate dihydrate and
monohydrate crystals appeared in different sizes in solitary
or in aggregation. Calcium oxalate dihydrate crystals
appeared as colourless squares whose corners were
connected by intersecting lines with characteristic shape
of octahedron or envelope (fig. 2) and whereas, calcium
oxalate monohydrate crystal varied in size with two
dimentional spindle, oval and various forms like “picket
fence” and “dumbbell” and “hemp seed” shape (fig. 3).
Ammonium urate or biurate crystals appeared as brown or
yellow-brown spherical bodies with irregular borders
"thorn-apples" (fig. 4). The most common site of
predilection of calculi were urinary bladder/urethra in
males and only urinary bladder in females. The calculi of
various colour, texture, shape and size recovered (fig. 5)
through cystotomy or urethrotomy were subjected for
chemical analysis and scanning electron microscopy.
Chemical analysis: In the present study chemical
analysis of the different stones (uroliths) retrieved from
urinary bladder and or urethra of the renal insufficiency
geriatric dogs revealed calcium phosphate (struvite),
calcium oxalate and ureate (urea) stones (fig. 4 & 5). The
major mineral component of calculi in urinary bladder was
struvite and that in urethra and multiple locations was
calcium oxalate.
Scanning Electron Microscopy (SEM): When
topographic features of various calculi were investigated
with scanning electron microscopy (SEM), perpendicularly
cracked fragments showed concentric laminations
composed of alternatively packed compact and loose
strata (fig. 6 and 7) suggesting struvite which was
confirmed on chemical analysis as consisted to be
magnesium ammonium phosphate (MAP). Few crystals
with smooth or porous surfaces and scattered hexa or
octa-hedral coffin-lid shaped crystals in the loosely packed
strata (fig. 8 and 9) was also seen. The surfaces of few
stones were like cracked eggshells (fig. 10) and revealed
calcium phosphate on chemical analysis. Further, the
surface of few calculi demonstrated wavy phases with
sundry areas (fig. 11) that were chemically identified as
uric acid. Whereas, SEM aspects of few crystals that
revealed calcium oxalate monohydrate on chemical
analysis appeared as ‘picket fence appearance’ and
‘dumbbell’ and ‘hemp seed’ shape (fig. 12) and calcium
oxalate dihydrate that were retractile, 3dimensional
octahedron, mail type envelope (fig. 13).

3. Scanning Electron Microscopic Structure and Composition of Urinary Calculi of Geriatric Dogs
Int. J. Vet. Sci. Anim. Husb. 083
Discussion
Uroliths are solid structures which arise from disturbances
of the physico-chemical balance and/or of the
hydrodynamic system of the urine and the urinary tract.
These calculi or crystals and microscopic minimally and is
composed of crystalline and amorphous organic and/or
inorganic. components, which may be mixed with a non-
crystalline high molecular substance (Schneider, 1982)
Presence of increased epithelial cell counts in the urine of
dogs is suggestive of renal impairment (Osborne et al.,
1972). Squamous epithelial cells observed in healthy as
well as renal patient have no diagnostic value as they can
be normally in less numbers, but also in large numbers
among females during estrus. Whereas, increased
number of transitional epithelial cells may present in the
urine sediment with infection, mechanical trauma
(urolithiasis), or neoplasia of the urinary tract (Chew et al.,
2011). Various cellular components observed in the
present study viz., red blood cells, white blood cells,
epithelial cells, crystals, casts, bacteria/yeasts,
spermatozoa and amorphous debris are in accordance
with (Carolyn and Nicole, 2012).
Casts are cylindrical molds of the renal tubules composed
of aggregated protein matrix with or without embedded
cells. Casts form by precipitation of protein and any intact
cells, intracellular organelles, brush border, or cellular
debris that present in the tubular lumen. In the present
study several types of casts were identified in the urine
sediment of most dogs affected with renal failure.
Significantly higher count of urinary casts in renal affected
dogs could be due to active pathologic process at loop of
henle, distal tubule and collecting tubule. The absence of
cast does not rule out the disease and number is not a
reliable index of severity, duration and reversibility or
irreversibility of disease (Osborne and Finco, 1995). RBC
casts are indicative of glomerular disease and WBC casts
were indicative of tubular disease, especially
pyelonephritis. Epithelial casts are associated with acute
tubular necrosis or pyelonephritis, granular casts are
suggestive of tubule-interstitial disease and waxy casts in
many cases suggested renal stasis or nephron obstruction
and represented serious disease and referred to as renal
failure casts (Chew et al., 2011; Ringsrud, 2001 and Patel,
2006).
Urinary calculi are a result of the crystalisation from
inorganic and organic components of urine. In spite of the
fact that they are normally present, the process of their
diagnostics is still not fully established (Ryall, 2011).
Urolithiasis in the present study was evaluated in dogs
beyond 8 years and was more common in lower urinary
tract (Gleaton et al., 2001) of males. Over saturation of
urine with calcium oxalate leads to precipitation of crystals
and formation of urinary calculi, inspite of certain inhibitors
like magnesium that can withhold the process of super
saturation. However, this process of initiation of calculi
formation can be ascribed to the presence of citric acid and
some ions (Ryall, 2011). Magnesium chelates with
oxalates to form magnesium oxalate, more soluble than
calcium oxalate and inhibits the crystallisation of Calcium
oxalate (LI et al., 1985; Doremus et al., 1978 and Ryall et
al., 1981). Oxalates are highly toxic agents may cause
renal insufficiency by inducing severe tubular injury and
renal cellular sloughing (Bibilash et al., 2010 and Marengo
et al., 2004). Super saturation is followed by formation of a
solid crystal phase (nucleation) in the renal tissue that
permit further deposition and aggregation of crystals
(Finlayson and Reid, 1978). Urinary macromolecules also
prevent this step in the process of calculi formation by
binding or adsorption of crystal surface to induce
degradation of crystal faces and edges (Addadi et al.,
2001).
Proteins, lytic enzymes and other organic matrix present
in normal urine also cause dissolution and degradation of
the urinary crystals, which gives irregular shapes of
calcium oxide monohydrate and dehydrate crystals under
SEM and decrease crystals chance for deposition on the
renal tissue; for instance osteopontin is incriminated in
crystals erosion and the appearance of cracked surfaces
(Thurgood et al., 2010). Further, other substances
secreted by the kidneys like undifferentiated proteins, and
glycosaminoglycans inhibit retention of crystals and
nucleation by reducing the ability of attachment of Calcium
oxide monohydrate to renal epithelial cell membranes
(Grover et al., 2010). Other crystals like, calcium oxalate
cause discontinuities within the mineral phase, increase
the non uniform crystals and reduce the average size of
their crystallites component due to presence of
intracrystalline proteins within them (Fleming et al., 2003).
Isolated clusters of calcium oxalate crystals, isolated or
clumps of crystals appearing tetrahedral shape of calcium
oxide dihydrate were observed under light microscope and
SEM. While, appearance of atypical shapes of calcium
oxide monohydrate crystals with rounded edges and bow–
tie appearance were demonstrated by SEM (Walaa et al.,
2014) and columnar shaped aggregation of crystallites,
suggesting calcium oxalates (Tufani et al., 2017).
CONCLUSION
Microscopic and electron microscopic examination of urine
samples from renal insufficiency geriatric dogs revealed
increased number of blood cells, epithelial cells and pus
cells along with casts and calculi predominantly, triple
phosphate or struvite, followed by calcium oxalate
dihydrate, calcium oxalate monohydrate and ammonium
urate or biurate. Coffin-lid and picket fence were various
shapes, while perpendicular columnar strata, wavy phases
with sundry areas and cracked eggshell appearance were
SEM characters of different crystals.

4. Scanning Electron Microscopic Structure and Composition of Urinary Calculi of Geriatric Dogs
Kumar and Srikanth. 084
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5. Scanning Electron Microscopic Structure and Composition of Urinary Calculi of Geriatric Dogs
Int. J. Vet. Sci. Anim. Husb. 085
Fig. 1 Coffin-lid shaped Struvite crystals
(40X)
Fig. 2 Envelop or octahedron shaped
colourless Calcium oxalate dihydrate crystals
(40X)
Fig. 3 Picket fence shape calcium oxalate
monohydrate crystals (40X)
Fig. 4 Ammonium urate stones with
Thorn-apple appearance
Fig. 5 Struvite and other types of calculi
retrieved from bladder and urethra
Fig. 6 Closely packed strata with
perpendicular laminar columns – Struvite
(1400X)

6. Scanning Electron Microscopic Structure and Composition of Urinary Calculi of Geriatric Dogs
Kumar and Srikanth. 086
Fig. 7 Closely and loosely packed strata of
struvite - magnesium ammonium
phosphate (2000X)
Fig. 8 Hexa - hedral coffin-lid shaped
crystals in the loosely packed strata
(900X)
Fig. 9 Octahedral shaped crystals (1000X) Fig. 10 Cracked eggshell appearance of
calcium phosphate crystal (70X)
Fig. 11 Uric acid calculi demonstrating
wavy phases with sundry areas (70X)
Fig. 12 Typical ‘picket fence appearance’
of calcium oxalate monohydrate
crystals (150X)

7. Scanning Electron Microscopic Structure and Composition of Urinary Calculi of Geriatric Dogs
Int. J. Vet. Sci. Anim. Husb. 087
Accepted 13 March 2021
Citation: Kumar KS, Srikanth K (2021). Scanning Electron Microscopic Structure and Composition of Urinary Calculi of
Geriatric Dogs. International Journal of Veterinary Science and Animal Husbandry 7(1): 081-087.
Copyright: © 2021 Kumar and Srikanth. This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are cited.
Fig. 13 Calcium oxalate dihydrate
crystals appearing as 3d octahedron,
mail type envelope (2000X)