Diagnosis of sexual cycle by means

of vaginal smear method in the chinchilla

( Chinchilla lanigera )
Tayfur Bekyürek1, Narin Liman2 & Güner Bayram3
1 2
Department of Obstetrics and Gynecology, Department of Histology and Embryology, Faculty of
Veterinary Medicine, University of Erciyes, Kayseri, Turkey and 3 University of Erciyes, Training College
. . .
of SaŽ ye ÇikrikçiogÏ lu, Kayseri, Turkey

Summary
An investigation was made as to whether the sexual cycle and pregnancy can be determined
by means of vaginal smear in chinchillas. T his study represents the ®rst attem pt to record
changes which occur in the pattern of exfoliated cells in chinchilla’s vaginal smear during
anoestrus, proestrus, oestrus, metoestrus and pregnancy. Fifteen female chinchillas aged from
8 months to 3 years and bred through harem breeding method were used. T he major change
during proestrus was an increase in the proportion of super®cial cells, with a corresponding
decrease in other cells. Goblet cells were observed in the smears prepared by strong aspiration
during this cycle. Neutrophils, small and large intermediates and parabasal cells were not
found in the smear during oestrus and the smear consisted of super®cial cells only. In the
proportion of neutrophils, small and large intermediates and parabasal cells increased during
metoestrus. In addition, metoestrum and foam cells were found in this cycle. In anoestrus;
super®cial and parabasal cells were present in small numbers. Also small and large
intermediate cells as well as neutrophils were present. Traces of foam and metoestrum cells
were found. During pregnancy, neutrophils generally of medium density were present,
parabasal; small and large intermediate cells were present at low or medium density, and
super®cial cells were only present in trace amounts.

Keywords Vaginal smears; chinchilla; sexual cycles

T he chinchilla, which sleeps during the day
and is active at night, is a herbivorous South
American rodent of the suborder Hystrico-
morpha and family Chinchillidae (Bingam i &
Beach 1968 ). T he species is best known as
fur-beari ng animals valued throughout the
world for their remarkabl y soft fur. Chin-
chillas are also kept at homes as pets and
used as experimental anim als at laborat ories.
T here are three different species: C h inch illa
la ni ge ra , C . c o sti na and C . b re vic a ud a ta .
C . la nige ra is the species commercially bred
C o rre spond e nc e to : T. Be k yuÈ re k
Accepted 22 May 2001
as a fur animal (Weir 1970, 1976, CË alõs;kaner
1993 ). In spite of having a lifespan of 20 years,
their reproductive performance continues for
9 to 10 years. Chinchillas are bred through
the harem breeding method, with a male
being matc hed with four females in an ideal
breeding system. Matin g lasts only a short
tim e and usually occurs at night. T he
presence of a copulation plug and fallen
batch es of hair on the straw are indicative of
successful mating.
T he copulation plug is initially white and
soft but later becomes harder and transparent
yellow in colour (Weir 1970, 1976, Puzder &
Novikmec 1992, T hiede 1994 ). T he Chin-
# Laboratory Animals Ltd. Laboratory Animals (2002) 36, 51–60
52
chilla’s gestation period is usually between
105±115 days and the period is 111 days in
C . la nige ra (Weir 1966, 1970, 1976, Kuroiwa
& Imamichi 1977, Neira e t a l. 1989, Puzder
& Novikmec 1992, CË alõs;kaner 1993, T hiede
1994) and 128 days in C . b re vic a ud a ta
(CË alõs;kaner 1993).
Female chinchillas reach puberty at 4 to 5
months of age but they reach mating matur-
ity at 8 to 9 months of age. Chinchillas are
seasonally polyoestrus and their sexual
activities increase at the beginning of
November until the end of May. During
summer and early autumn anoestrus occurs
(Weir 1970, Kuroiwa & Imamichi 1977,
Jakubow e t a l. 1984, Puzder & Novikm ec
1992, T hiede 1994 ). T he durati on of the oes-
trus cycle is usually 28 to 35 days (Kuroiwa &
Imamichi 1977, CË alõs;kaner 1993, T hiede
1994). T he period of the oestrus lasts for 3 to
4 days. T he vulva is closed by a hym en (Weir
1966, 1969, 1970, 1973, Kuroiwa & Imamichi
1977, Puzder & Novikm ec 1992, T hiede
1994).
Little is known about the diagnosis of the
sexual cycle and pregnancy in chinchilla.
T he criterion of vaginal perforat ion is used
generally as a primary indicati on of oestrus.
Ultrasonography and radiography, and obser-
vations of abdom inal palpation or increases
in live weight, have reported been used to
diagnose pregnancy in the female chinchilla.
Diagnosis by ultrasonography involves shav-
ing the hair, reducing fur qualit y. Moreover,
some such tests do not always yield accurate
results, since the increase in live weight is
distinctive only after 50 to 60 days of preg-
nancy (Weir 1970, Puzder & Novikmec 1992 ).
In addition, the radiography method is
expensive and can cause early embryonic
deaths or foetal malformations during
development.
Female chinchilla will accept a male in the
oestrus cycle only if she likes him. Females
may becom e aggressive and attac k males if
the fem ales refuse to mate and the male is
insistent. Fighting between males and
fem ales may result in the death of the male
(Bingam i & Beach 1968, CË alõs;kaner 1993 ). In
order to prevent this, arti®cial fertilization
procedures are performed. T herefore, the
determination of sexual cycle and, particu-
Laboratory Animals (2002) 36
Bekyürek, Liman & Bayram
larly, measurements of oestrus are increas-
ingly im portant. T his study was designed
because vaginal cytology is an effective
method for determining sexual cycle and
pregnancy (without affect ing fur qualit y).
Chinchillas are economically im portant and
their breeding is progressively increasing in
Turkey.
Material and methods
Anim a ls
Fifteen female C . La nige ra aged between
8 months to 3 years (300±500 g) were used
in this study. Males were placed in harem
breeding units with four fem ales. T his
arrangement allows the male to travel at will
between the individual ly-housed females.
Females are collared to prevent their entry
into the common male runway, thus avoid-
ing their access to other female cages.
T he females were housed individually in
stainless-steel cages. T he cages measured
51651630 cm. T he chinchillas were fed
with pellet chinchilla grain and alfal fa
a d lib itum . Clean water was given dail y in
glass bottles. Vitamin complex was added
twice a week to the water. Chinchillas were
kept in a well-aired environment with the
temperature between 10±18 C. T hey were
hot exposed to direct sunlight, but kept in a
room, which was illuminated for 12 h and
dark for 12 h a day.
Ha nd ling
Chinchillas were taken from their cages by
their tail. For vaginal exam inations, the
animals were held by the tai l and their
forepaws rested on the observer’s chest.
Th e c o lle c tio n o f va gina l sm e a r sa m ple s
Vaginal smear samples were collected by the
aspiration technique for 2 years at 15-day
intervals over the period of sexual activity
(between November and May) and during
summer (anoestrus). A small column of
sterile saline (approximately 1 ml) was drawn
up into a Pasteur pipette and its full length
was slipped into the caudal vagina, takin g
care not to squeeze the bulb. T he bulb was
Sexual cycle diagnosis by vaginal smear in the chinchilla 53

then squeezed quickly and gently several (4 ) 0.5% aqueous eosin

times, to make the ¯uid column wash Eosin (aqueous) 0.5 g
rapidly bac k and forth and in doing so, pick Distilled water 100 ml
up cells. A small drop of ¯uid was placed on a
microscopic slide near the labelled end. T he Te ch niq ue
slide was then vertically tipped to allow the Alcohol-®xed smears are washed well in
drop to run down the length of the slide. T he water.
excess ¯uid was blott ed from the end of the Stain with Harris’ haematoxyl in for
slide before it was stood upright to air-dry. one minute.
Wash in water and differentiat e in acid±
Sta ining te c h niq ue alcohol until the nuclei are sharply
stained blue and the bac kground is
T hese sam ples were stained by haemat oxylin relatively unstained. An average
& eosin (Bourne 1990) and by modi®ed differentiat ion time is 2±6 s.
Ayoub-Shklar’s for prekeratin and keratin Wash in water and blue in the bicarbonate
staining techniques (Luna 1968 ), and were solution for 10±20 s.
examined under light microscopy. T he Wash well in water.
Ayoub-Shklar’s staining technique was Stain with eosin for 10±20 s.
combined with Harris’s and Weigert’s Wash in water, dehydrate with 96%
haematoxylin stains. alcohol and absolute alcohol and clear in
xylene, two changes each.
Ha e m a to xylin & e o sin te c h niq ue Mount with Entellan.
So luti o ns:
Ayo ub -Sh k la r’s fo r pre k e ra tin a nd k e ra tin
(1 ) Harris’ haemat oxylin
staining te c h niq ue
Haematoxylin 5g
Ethyl alcohol 50 ml Solutions for Harris’s haematoxylin (1), (2 ), &
Potassium or ammonium 100 g (3 ) were prepared as above.
alum So lutio ns fo r We ige rt’s iro n h a e m a to xylin
Distilled water 950 ml (1 ) Weigert’s iron haematoxylin: T his is
Mercuric oxide 2.5 g normally stored as two separate solutions
Glacial acetic acid 40 ml
So lutio n A
Dissolve the haematoxyl in in the alcohol Haem at oxylin 1g
using gentle heat (using a 56 C oven or water Ethyl alcohol 100 ml
bath ) and dissolve the alum in the distilled Dissolve with gentle heat
water using a Bunsen with frequent stirring.
So lutio n B
Whilst the aqueous alum solution is still hot,
30% aqueous ferric chloride 4 ml
add the alcoholic haemat oxylin solution and
Conc. hydrochloric acid 1 ml
bring to the boil, stirring frequently. Turn off
Distilled water
the Bunsen just before adding the mercuric
oxide, as the resultant effervescence may Add together equal volumes of A and B prior
cause spillage. Cool quickly by plunging the to use, and ®lter before use
container into cold water, add the acetic acid (2 ) Methyl carbonate
and ®lter. T he solution is ready for immedi- Distilled water 125 ml
ate use but will need re®ltering. Methanol 125 ml
(2 ) 1% hydrochloric acid in 70% alcohol Sodium carbonate 0.5 g
70% alcohol 100 ml 5% Acid fuchsin solution*
Conc. hydrochloric acid 1 ml Acid fuchsin 5.0 g
(3 ) 3.2% aqueous sodium bicarbonate Distilled water 100.0 ml
Sodium bicarbonat e (aqueous) 2 g Aniline blue-Orange G solution*
Distilled water 100 ml Aniline blue, water soluble 0.5 g
Laboratory Animals (2002) 36
54
Orange G 2.0 g
Phosphotungstic acid 1.0 g
Distilled water 100 ml
* For consistent staining results use fresh
solutions.
Sta ini ng pro c e d ure
T he smears can be held aft er air drying
and without ®xation for an inde®nite
time. T hey can be ®xed by placing in 96%
ethyl alcohol for 3 min.
Alcohol-®xed smears wash well in water.
Stain with Weigert’s iron haematoxylin
for 5 min or Harris’ haematoxyl in for
one minute.
After staining with Weigert’s iron
haematoxylin:
Wash in water for 5 min and differentiat e in
methyl carbonate for one minute and then
wash in water for 5 min.
After staining with Harris’ haematoxylin :
Wash in water and differenti ate in acid alco-
hol until the nuclei are sharply stained blue
and the bac kground is relatively unstained.
Wash in water and blue in the bicarbonate
solution 10±20 s.
Wash in distilled water.
Stain with acid fuchsin solution for 3 min.
Transfer slides directly to aniline blue-
orange G solution for 45 min.
Transfer slides directly to 96% alcohol for
three changes.
Dehydrate with absolute alcohol and clear
in xylene, two changes each.
Mount with Entellan.
Th e c la ssi®c a tio n o f c e lls
Cell types found in vaginal smears were
classi®ed as parabasal, small and large inter-
mediate, super®cial (partl y and completely
corni®ed cells), foam cells, metoestrum cells
and neutrophils, according to their morpho-
logical charac teristics.
Pa ra b a sa l ce lls : T hese are the smallest
vaginal epithelial cells. T hey are small ovoid
cells with a large nuclear-to-c ytoplasmic
rati o.
Laboratory Animals (2002) 36
Bekyürek, Liman & Bayram
Sm a ll in te rm e d ia te c e lls: T hese cells vary
considerably in size. T hey are the growing,
transitional cells between the spherical
parabasal cells and the larger, more mature,
¯attened cells into which they develop as
they move further away from the basal
layers. T hey vary in shape from nearly
round to oval; and most are ellipsoid.
T he cell outline is very regular. As with
parabasal cells, the nucleus is large and
vesicular, but it is less basophilic.
La rge inte rm e d ia te c e lls : T hese cells are
characterized by their shape and nuclear
appearance rather than their size. T hey
represent a transitional stage between the
larger of the regular-shaped, variabl e-sized,
small intermediate cells and the irregular-
shaped, squamous, super®cial cells. T hey are
squam ous in shape with an active, round,
normal-sized nucleus.
Supe r®c ia l ce lls : T hese are large and ¯at.
T heir nucleus is very fai nt or pyknotic and
dense; it may be absent. T he intensity of
cytoplasmic stain can also vary, depending on
the degree of corni®cation and=or degenera-
tion. T hese cells have keratin incorporated
into the cytoplasm . In the category of par-
tially corni®ed super®cial cells we include
cells with distinct, dark pyknotic nuclei or
with fai nt staining nuclei. In the category of
fully corni®ed super®cial cells we include
cells with no nuclei or with an indistinct and
condensed nucleus. Super®cial cells are
extremely angulated, folded, and irregular
in shape.
Me to e strum c e lls : Small intermediate cells
or parabasal cells with a neutrophil in the
cytoplasm have been termed `m etoestrum
cells’.
Fo a m ce lls : Small intermediate cells or
parabasal cells with multiple, clear
cytoplasmic vacuoles have been termed
`foam cells’.
T he densities of these cells in the smear
sam ples were tak en into consideration in the
determ ination of sexual cycles. T he density
of cell types found in vaginal smears were
evaluated as traces (‡ =¡ ), little (‡ ), medium
Sexual cycle diagnosis by vaginal smear in the chinchilla 55

(‡ ‡ ) or dense (‡ ‡ ‡ ). In the study, cell
counting was not performed for the diagnosis
of sexual cycles in chinchilla. However 1±5
cells were determined as trace, 10±15 as lit-
tle, about 40±50 cells as medium , and when
dominant in smear, as `dense’. T he densities
of neutrophils were dealt with separately.
Th e c linic a l o b se rva tio ns
Weir (1973 ) stat ed that vaginal perforati on is
a reliable indicat ion of oestrus in hystrico-
morph rodents. In this study, the criterion of
vaginal perforation was used as a primary
indication of oestrus. Clinically, the mating
behaviours in all of the animals, the abdom -
inal palpation ®ndings, and the dates when
birt hs took place were also recorded. T he
appearance in the cage of a copulation plug
and fallen batc hes of hair were used as
indications of successful mating.
super®cial and large intermediat e cells were
found in trace am ounts. At this stage of the
cycle, goblet cells were observed in the
smears prepared by strong aspiration (Fig 1).
During mid-proestrus, a decline in the num-
ber of parabasal and small intermediat e cells,
and a less rapid increase in large intermediate
cells and at higher rate in super®cial cells,
were observed (Fig 2).
In late proestrus, the small and large
intermediates disappeared (large inter-
mediate cells were occasionally present in
trace am ounts), and parabasal cells declined
to trace am ounts. T here were few
neutrophils. Partly or completely corni®ed
super®cial cells were present at a moderate
density (Fig 3).

Results
All cell cytoplasms were stained pink, the
nuclei purple, and the super®cial cell types in
the same colour in all the smears stained by
haematoxylin & eosin. However, with the
modi®ed Ayoub-Shklar’s staining method,
the intensity of the cytoplasmic stain of
super®cial cells varied depending on the
degree of corni®cation and=or degeneration,
in colours ranging from blue and dark red
to orange, and this was particularly more
distinctive during oestrus. When the Ayoub-
Shklar’s staining method was combined with Fig 1 Goblet cells (g) at the onset proestrus, 640
Harris haematoxylin, it was found that the
nuclei of neutrophils were stained more pale,
but when combined with Weigert’s haema-
toxylin, both neutrophils and metoestrum
cells were distinctively stained. In both
haematoxylin combinations, cytoplasms of
small and large intermediate cells were
stained pale blue and their nuclei orange;
cytoplasms of parabasal cells were stained
blue and their nuclei purple, being darker
with Weigert’s haematoxylin.

Pro e strus
At the onset of proestrus, densities of neu- Fig 2 During the mid proestrus, partly (S) and
trophils, parabasal and small intermediate completely (Sc) corniŽ ed superŽ cial, large
cells increased from little to medium , and intermediate (L) cells and neutrophils (n), 620

Laboratory Animals (2002) 36

56 Bekyürek, Liman & Bayram

O e strus
In the smear samples obtained during this
period, super®cial cell types were dominant,
but all other cell types appeared (Fig 4). At the
onset of metoestrus, parabasal and inter-
mediate cells and neutrophils began to
reappear and they existed among super®cial
cells in only a few cases.

Meto e strus
During mid-m etoestrus, starting with the
reappearance of neutrophils, parabasal and Fig 5 At the metoestrus, the parabasal (p), small (i)
intermediate cells, it was observed that large and large (L) intermediate, completely corniŽ ed
superŽ cial (Sc), metoestrum (m) cells and neutrophils
intermediate cells and neutrophils were
(n), 640
dense, however parabasal and small inter-
mediate cells were at medium density and
there were only a few super®cial cells (Fig 5).
In addition, at this stage, metoestrum and
foam cells were found (Fi g 6).

Fig 6 At the metoestrus, the metoestrum (m), foam

(f) cells and intracytoplasmic granules (arrows), 6100

Fig 3 During the late proestrus, completely corniŽ ed

superŽ cial (Sc), large intermediate (L) cells and
neutrophils (n), 620

Fig 7 Anoestrus, the parabasal (p), small inter-

Fig 4 At the oestrus, the partly (S) and completely mediate (i), partly (S) and completely (Sc) corniŽ ed
(Sc) corniŽ ed superŽ cial cells, 640 superŽ cial cells and neutrophils (n), 620

Laboratory Animals (2002) 36

Sexual cycle diagnosis by vaginal smear in the chinchilla 57

Ano e strus Discussion

During this phase of the cycle, super®cial
and parabasal cells occurred and small and
large intermediate cells and neutrophils were
present in low densities (Fig 7). Foam and
metoestrum cells were rare.
Pre gna nc y
During pregnancy neutrophils were generally
of medium density but occasionally this rate
varied from little to dense. Parabasal and
small and large intermediate cells were
present at low or medium densities, and
super®cial cells were present in trace or little
amounts (Fi g 8).
T he changes in the vaginal smear of
chinchillas related to the phases of sexual
cycle and pregnancy are shown in Tabl e 1.
Fig 8 At the pregnancy, the parabasal (p), small (i)
and large (L) intermediate (i), partly corniŽ ed
superŽ cial (S) cells and neutrophils (n), 620
T he gonadal hormones (progesterone and
particularly, oestrogen) play important roles
in the regulation of the sexual cycle and
maintenance of gestation. Oestrogen causes
the histological changes in vaginal epithe-
lium. Prior to puberty and during anoestrus
in adult s, the vaginal epithelium is only a
few cell layers thick. With oestrogen
stimulation, the vaginal mucosa becom es
a strati®ed squamous epithelium comprised
of many layers of cells (Bell e t a l. 1973,
Concannon & Digregorio 1986, Vrcic e t a l.
1991 ). Oestrogen affec ts the vaginal
epithelium in three ways, by proliferation,
maturat ion and exfoliation (Montes & Lugue
1988 ). Vaginal corni®cation has been used
as an indicator of biological activity in many
animal species (Fowler e t a l. 1971 ). Vaginal
smears re¯ect the histological features of
the vaginal epithelium. Vaginal cytology is
an important aid for the diagnosis of sexual
cycle and pregnancy and reproductive dis-
orders (Wright 1990 ).
No research on the classi®cation of cycle
in chinchillas has been reported in the
literature. In the present study, the sexual
cycle of the chinchilla was divided into four
periods named as proestrus, oestrus,
metoestrus, and anoestrus, and vaginal smear
®ndings were evaluated accordingly.
In our study, vaginal smear ®ndings
obtai ned during the proestrus cycle in
chinchillas resembled those in dogs (Fowler

Table 1 The changes for all phases of the sexual cycle and pregnancy in vaginal smear of Chinchilla laniger

SuperŽ cial cell

Small Large
Parabasal intermediate intermediate Partly Completely Foam Metoestrum Goblet
Neutrophils cell cell cell corniŽ ed corniŽ ed cell cell cell

Early ‡‡‡ ‡‡ ‡‡ ‡ =¡ ‡ =¡ ‡ =¡ ¡ ¡ ‡
proestrus
Proestrus ‡‡ ‡ ‡ ‡ ‡ ‡ ¡ ¡ ¡
Late ‡ ‡ =¡ ¡ ¡ , ‡ =¡ ‡‡ ‡‡ ¡ ¡ ¡
proestrus
Oestrus ¡ ¡ ¡ ¡ ‡‡‡ ‡‡‡ ¡ ¡ ¡
Metoestrus ‡‡‡ ‡‡ ‡‡ ‡‡‡ ‡ ‡ ‡ =¡ ‡ =¡ ¡
Anoestrus ‡ ‡ ‡ ‡ ‡ =¡ ‡ =¡ ‡ =¡ ‡ =¡ ¡
Pregnancy ‡‡ ‡‡ ‡‡ ‡ ‡ ‡ ¡ ¡ ¡
(‡ =¡ ): trace, (‡ ): little, (‡ ‡ ): medium, (‡ ‡ ‡ ): dense

Laboratory Animals (2002) 36

58
e t a l. 1971, Bell e t a l. 1973, Roszel 1977,
Concannon & Digregorio 1986, Wright 1990 ),
cats (UÈ nal e t a l. 1997 ), rats (Montes & Lugue
1988, Oba 1988 ) and mice (Vrcic e t a l. 1991 ),
in terms of general characteristics. T hey dif-
fered from dogs in the absence of erythrocytes,
as is the case in other rodents. T he presence
of goblet cells observed in the smears of
chinchillas at the onset of proestrus indicates
that there is mucinous transformation of the
super®cial two or three layers, which is a
unique feature of the rodent oestrus cycle
(Vrcic e t a l. 1991 ).
T he oestrus phase of the sexual cycle is
marked by super®cial corni®cat ion of the
squamous epithelium. T he neutrophils are
absent and super®cial cells are dominant in
smears obtained during oestrus in dogs
(Fowler e t a l. 1971, Bell e t a l. 1973, Roszel
1977, Concannon & Digregorio 1986, Wright
1990), rats (Montes & Lugue 1988, Oba 1988 )
and mice (Vrcic e t a l. 1991 ). T he incidence of
intermediate cells in the vaginal smear of cat s
during oestrus is reported as 17% (UÈ nal e t a l.
1997). In the smear samples collected during
oestrus in chinchillas, ®ndings comparable to
those of dogs (Fowler e t a l. 1971, Bell e t a l.
1973, Roszel 1977, Concannon & Digregorio
1986, Wright 1990 ), rats (Mont es & Lugue
1988, Oba 1988 ) and mice (Vrcic e t a l. 1991 )
were observed. In addit ion, the oestrus cycle
was evident in most of the smear samples
obtained from chinchillas if they have a
vaginal aperture. T herefore, the presence of a
vaginal aperture is a determinant of oestrus
in chinchillas (as reported by Weir 1970,
Kuroiwa & Imamichi 1977, Puzder &
Novikmec 1992 ).
In the present study, metoestrus changes
observed were comparable to those in other
anim al species (Fowler e t a l. 1971, Bell e t a l.
1973, Concannon & Digregorio 1986, Montes
& Lugue 1988, Wright 1990). However,
metoestrum and foam cells, which were
reported to be present in dogs (Christie e t a l.
1972, Concannon & Digregorio 1986 ) were
observed during metoestrus in chinchillas.
To our knowledge, no inform ati on on their
presence in other anim al species has been
given in the literature. It has also been stated
that some intermediate cells in dogs contain
cytoplasmic granules (Holst 1986). In this
Laboratory Animals (2002) 36
Bekyürek, Liman & Bayram
study, purple stained round granules of
different sizes in some intermediat e cells
were found.
Although smear ®ndings obtained from
chinchillas during the anoestrus period were
similar to those reported in dogs (Fowler e t a l.
1971, Bell e t a l. 1973, Roszel 1977,
Concannon & Digregorio 1986, Wright 1990 )
and cats (UÈ nal e t a l. 1997 ). Generally cell
count was reduced, as compared to other
cycle periods. Also, during this period
metoestrum and foam cells, reported to be
present in dogs, were observed in trace
amounts (Christie e t a l. 1972, Concannon &
Digregorio 1986 ).
T he study revealed that smear samples
from pregnant chinchillas have character-
istics similar to those observed in other
animal species (Fowler e t a l. 1971, Bell e t a l.
1973, Roszel 1977, DogÆaneli e t a l. 1979,
Concannon & Digregorio 1986, Montes &
Lugue 1988, Oba 1998, Wright 1990, UÈ nal
e t a l. 1997). In dogs, pregnancy has the same
characteristics reported in metoestrus and
therefore may be confused with this stage
(Roszel 1997 ). Also, it is suggested that the
changes observed in smears obtained during
the ®rst month of pregnancy in sheep are
clear but anoestrus levels do not differ during
the subsequent months (DogÆaneli e t a l.
1979 ). It is possible to distinguish between
pregnancy and metoestrus by the appearance
of metoestrum and foam cells.
T he trichrome staining methods have an
im portant place in endocrine cytology. As the
vaginal epithelial cells respond to oestrogen,
in the sexual cycle the cytoplasm changes
from blue to orange because of the develop-
ment of precursors of keratin. T his tinctorial
property emphasizes the period of maximum
oestrogenic effect. As a result, oestrus is
easily recognized in the smear which con-
tains alm ost exclusively large epithelial cells
with orange cytoplasm s (Roszel 1977, Bourne
1990 ). T he applicability of another trichrome
staining method in vaginal cytology was also
researched. T he staining method used by
Ayoub-Shkl ar’s method for prekeratin and
keratin demonstration (Luna 1968) was
applied in the smears in three different ways.
In the ®rst method, when applied without
changing the procedure, it was seen that the
Sexual cycle diagnosis by vaginal smear in the chinchilla
nuclei of neutrophils and parabasal cells were
not stained and it was dif® cult to distinguish
one from the other; Harris and Weigert’s
haematoxylin were used as nuclei stain in
the second and third methods, respectively.
Successful results were achieved in both
stainings, but it was determined that the
nuclei of the cells were darker in smears with
Weigert’s haematoxylin stain. It was
observed that distribution of prekeratin and
keratin in super®cial cells could be demon-
strated distinctively with Ayoub-Shk lar’s
staining method, and greater success was
achieved in distinguishing between partly
and completely corni®ed epithelial cells,
which are the subtypes of super®cial cells. It
was observed that this method has many
advan tages: it is economical and easily
applicable in every laboratory and it does not
require the use of a special ®xative. If the
smear is allowed to dry, various art efacts do
not occur. It yields very good results even
with smears which have been dried and kept
for a long while. It has a short staining period
and few steps; the preparation of the stain is
easy and fast and there is limited use of stain
types.
In this present research, oestrus was con-
®rmed as a result of a co-evaluation of clin-
ical ®ndings and vaginal smear, and it was
concluded that the vaginal smear method is a
reliable and practical method of diagnosi ng
the sexual cycle in chinchillas. In addition,
the Ayoub-Shklar’s staining technique (a tri-
chrome staining method) was used for the
®rst time in this study and it was demon-
strated that this technique is feasible in the
evaluation of vaginal cytology and that this
method can be used as an alternative staining
method in vaginal cytology.
Ac k no w le d gm e nts T his study is supported by the
Research Institute of Erciyes University and the State
of Planning Organization (DPT ).
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