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Diagnosis of sexual cycle by means of vaginal smear method in the chinchilla ( Chinchilla lanigera )

Tayfur Bekyu rek1, Narin Liman2 & Gu ner Bayram3


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 . . . lu, Kayseri, Turkey of Sa ye C ikrikc iog

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 chinchillas 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 supercial 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 supercial 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; supercial 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 supercial 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 Hystricomorpha 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. Chinchillas 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 yu re k Accepted 22 May 2001

as a fur animal (Weir 1970, 1976, C als ;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, 5160


Bekyu rek, Liman & Bayram

chillas gestation period is usually between 105115 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, C als ;kaner 1993, T hiede 1994) and 128 days in C . b re vic a ud a ta (C als ;kaner 1993). Female chinchillas reach puberty at 4 to 5 months of age but they reach mating maturity 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 oestrus cycle is usually 28 to 35 days (Kuroiwa & Imamichi 1977, C als ;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 observations of abdom inal palpation or increases in live weight, have reported been used to diagnose pregnancy in the female chinchilla. Diagnosis by ultrasonography involves shaving 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 pregnancy (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, C als ;kaner 1993 ). In order to prevent this, articial fertilization procedures are performed. T herefore, the determination of sexual cycle and, particuLaboratory Animals (2002) 36

larly, measurements of oestrus are increasingly 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 (300500 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 avoiding their access to other female cages. T he females were housed individually in stainless-steel cages. T he cages measured 51 651 630 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 1018 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 observers 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


then squeezed quickly and gently several times, to make the uid column wash rapidly bac k and forth and in doing so, pick up cells. A small drop of uid was placed on a microscopic slide near the labelled end. T he slide was then vertically tipped to allow the drop to run down the length of the slide. T he excess uid was blott ed from the end of the slide before it was stood upright to air-dry.

(4 ) 0.5% aqueous eosin Eosin (aqueous) Distilled water

0.5 g 100 ml

Sta ining te c h niq ue T hese sam ples were stained by haemat oxylin & eosin (Bourne 1990) and by modied Ayoub-Shklars for prekeratin and keratin staining techniques (Luna 1968 ), and were examined under light microscopy. T he Ayoub-Shklars staining technique was combined with Harriss and Weigerts haematoxylin stains. Ha e m a to xylin & e o sin te c h niq ue
So luti o ns: (1 ) Harris haemat oxylin Haematoxylin Ethyl alcohol Potassium or ammonium alum Distilled water Mercuric oxide Glacial acetic acid

Te ch niq ue Alcohol-xed smears are washed well in water. Stain with Harris haematoxyl in for one minute. Wash in water and differentiat e in acid alcohol until the nuclei are sharply stained blue and the bac kground is relatively unstained. An average differentiat ion time is 26 s. Wash in water and blue in the bicarbonate solution for 1020 s. Wash well in water. Stain with eosin for 1020 s. Wash in water, dehydrate with 96% alcohol and absolute alcohol and clear in xylene, two changes each. Mount with Entellan.

5g 50 ml 100 g 950 ml 2.5 g 40 ml

Ayo ub -Sh k la rs fo r pre k e ra tin a nd k e ra tin staining te c h niq ue

Solutions for Harriss haematoxylin (1), (2 ), & (3 ) were prepared as above. So lutio ns fo r We ige rts iro n h a e m a to xylin (1 ) Weigerts iron haematoxylin: T his is normally stored as two separate solutions So lutio n A Haem at oxylin Ethyl alcohol Dissolve with gentle heat So lutio n B 30% aqueous ferric chloride Conc. hydrochloric acid Distilled water 1g 100 ml

Dissolve the haematoxyl in in the alcohol using gentle heat (using a 56 C oven or water bath ) and dissolve the alum in the distilled water using a Bunsen with frequent stirring. Whilst the aqueous alum solution is still hot, add the alcoholic haemat oxylin solution and bring to the boil, stirring frequently. Turn off the Bunsen just before adding the mercuric oxide, as the resultant effervescence may cause spillage. Cool quickly by plunging the container into cold water, add the acetic acid and lter. T he solution is ready for immediate use but will need reltering. (2 ) 1% hydrochloric acid in 70% alcohol 70% alcohol 100 ml Conc. hydrochloric acid 1 ml (3 ) 3.2% aqueous sodium bicarbonate Sodium bicarbonat e (aqueous) 2 g Distilled water 100 ml

4 ml 1 ml

Add together equal volumes of A and B prior to use, and lter before use (2 ) Methyl carbonate Distilled water Methanol Sodium carbonate 5% Acid fuchsin solution* Acid fuchsin Distilled water 125 ml 125 ml 0.5 g 5.0 g 100.0 ml

Aniline blue-Orange G solution* Aniline blue, water soluble 0.5 g

Laboratory Animals (2002) 36


Bekyu rek, Liman & Bayram

Orange G Phosphotungstic acid Distilled water

2.0 g 1.0 g 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 indenite time. T hey can be xed by placing in 96% ethyl alcohol for 3 min. Alcohol-xed smears wash well in water. Stain with Weigerts iron haematoxylin for 5 min or Harris haematoxyl in for one minute. After staining with Weigerts 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 alcohol until the nuclei are sharply stained blue and the bac kground is relatively unstained. Wash in water and blue in the bicarbonate solution 1020 s. Wash in distilled water. Stain with acid fuchsin solution for 3 min. Transfer slides directly to aniline blueorange 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.

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 irregularshaped, squamous, supercial cells. T hey are squam ous in shape with an active, round, normal-sized nucleus. Supe rc 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 cornication and=or degeneration. T hese cells have keratin incorporated into the cytoplasm . In the category of partially cornied supercial cells we include cells with distinct, dark pyknotic nuclei or with fai nt staining nuclei. In the category of fully cornied supercial cells we include cells with no nuclei or with an indistinct and condensed nucleus. Supercial 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

Th e c la ssic a tio n o f c e lls Cell types found in vaginal smears were classied as parabasal, small and large intermediate, supercial (partl y and completely cornied cells), foam cells, metoestrum cells and neutrophils, according to their morphological 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

Sexual cycle diagnosis by vaginal smear in the chinchilla


( ) or dense ( ). In the study, cell counting was not performed for the diagnosis of sexual cycles in chinchilla. However 15 cells were determined as trace, 1015 as little, about 4050 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 hystricomorph 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.

supercial 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 number of parabasal and small intermediat e cells, and a less rapid increase in large intermediate cells and at higher rate in supercial cells, were observed (Fig 2). In late proestrus, the small and large intermediates disappeared (large intermediate cells were occasionally present in trace am ounts), and parabasal cells declined to trace am ounts. T here were few neutrophils. Partly or completely cornied supercial cells were present at a moderate density (Fig 3).

All cell cytoplasms were stained pink, the nuclei purple, and the supercial cell types in the same colour in all the smears stained by haematoxylin & eosin. However, with the modied Ayoub-Shklars staining method, the intensity of the cytoplasmic stain of supercial cells varied depending on the degree of cornication and=or degeneration, in colours ranging from blue and dark red to orange, and this was particularly more distinctive during oestrus. When the AyoubShklars staining method was combined with Harris haematoxylin, it was found that the nuclei of neutrophils were stained more pale, but when combined with Weigerts haematoxylin, 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 Weigerts haematoxylin.

Fig 1

Goblet cells (g) at the onset proestrus, 640

Pro e strus At the onset of proestrus, densities of neutrophils, parabasal and small intermediate cells increased from little to medium , and

Fig 2 During the mid proestrus, partly (S) and completely (Sc) corni ed super cial, large intermediate (L) cells and neutrophils (n), 620
Laboratory Animals (2002) 36


Bekyu rek, Liman & Bayram

O e strus In the smear samples obtained during this period, supercial cell types were dominant, but all other cell types appeared (Fig 4). At the onset of metoestrus, parabasal and intermediate cells and neutrophils began to reappear and they existed among supercial cells in only a few cases. Meto e strus During mid-m etoestrus, starting with the reappearance of neutrophils, parabasal and intermediate cells, it was observed that large intermediate cells and neutrophils were dense, however parabasal and small intermediate cells were at medium density and there were only a few supercial cells (Fig 5). In addition, at this stage, metoestrum and foam cells were found (Fi g 6).

Fig 5 At the metoestrus, the parabasal (p), small (i) and large (L) intermediate, completely corni ed super cial (Sc), metoestrum (m) cells and neutrophils (n), 640

Fig 6 At the metoestrus, the metoestrum (m), foam (f) cells and intracytoplasmic granules (arrows), 6100 Fig 3 During the late proestrus, completely corni ed super cial (Sc), large intermediate (L) cells and neutrophils (n), 620

Fig 4 At the oestrus, the partly (S) and completely (Sc) corni ed super cial cells, 640
Laboratory Animals (2002) 36

Fig 7 Anoestrus, the parabasal (p), small intermediate (i), partly (S) and completely (Sc) corni ed super cial cells and neutrophils (n), 620

Sexual cycle diagnosis by vaginal smear in the chinchilla


Ano e strus During this phase of the cycle, supercial 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 supercial 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.

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 epithelium. 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 stratied 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 cornication has been used as an indicator of biological activity in many animal species (Fowler e t a l . 1971 ). Vaginal smears reect the histological features of the vaginal epithelium. Vaginal cytology is an important aid for the diagnosis of sexual cycle and pregnancy and reproductive disorders (Wright 1990 ). No research on the classication 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

Fig 8 At the pregnancy, the parabasal (p), small (i) and large (L) intermediate (i), partly corni ed super cial (S) cells and neutrophils (n), 620

Table 1

The changes for all phases of the sexual cycle and pregnancy in vaginal smear of Chinchilla laniger
Super cial cell Small Large Parabasal intermediate intermediate Partly Completely Foam Metoestrum Goblet Neutrophils cell cell cell cell cell cell corni ed corni ed

Early proestrus Proestrus Late proestrus Oestrus Metoestrus Anoestrus Pregnancy

= , =

= =

= =

= =

= =

( = ): trace, ( ): little, ( ): medium, ( ): dense Laboratory Animals (2002) 36


Bekyu rek, Liman & Bayram

e t a l. 1971, Bell e t a l . 1973, Roszel 1977, Concannon & Digregorio 1986, Wright 1990 ), nal e t a l . 1997 ), rats (Montes & Lugue cats (U 1988, Oba 1988 ) and mice (Vrcic e t a l . 1991 ), in terms of general characteristics. T hey differed 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 supercial 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 supercial cornicat ion of the squamous epithelium. T he neutrophils are absent and supercial 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 nal e t a l . during oestrus is reported as 17% (U 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

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 ) nal e t a l . 1997 ). Generally cell and cats (U 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 characteristics similar to those observed in other animal species (Fowler e t a l . 1971, Bell e t a l . aneli e t a l . 1979, 1973, Roszel 1977, Dog Concannon & Digregorio 1986, Montes & nal Lugue 1988, Oba 1998, Wright 1990, U 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 aneli e t a l . the subsequent months (Dog 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 development 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 contains 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 ars 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 Weigerts 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 Weigerts haematoxylin stain. It was observed that distribution of prekeratin and keratin in supercial cells could be demonstrated distinctively with Ayoub-Shk lars staining method, and greater success was achieved in distinguishing between partly and completely cornied epithelial cells, which are the subtypes of supercial 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 conrmed as a result of a co-evaluation of clinical 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-Shklars staining technique (a trichrome staining method) was used for the rst time in this study and it was demonstrated 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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