Bulgarian Journal of Veterinary Medicine, 2016 ONLINE FIRST ISSN 1311-1477; DOI: 10.15547/bjvm.997 Review APPLICATION OF EXFOLIATIVE VAGINAL CYTOLOGY IN CLINICAL CANINE REPRODUCTION – A REVIEW A. L. ANTONOV Department of Obstetrics, Reproduction and Reproductive Disorders, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, 6000, Bulgaria Summary Antonov, A. L., 2016. Application of exfoliative vaginal cytology in clinical canine reproduction – a review. Bulg. J. Vet. Med. (online first). Vaginal cytology has many practical applications in the evaluation of both the normal and abnormal bitch. The objective of this review is to describe the use of exfoliative vaginal cytology as a diagnostic tool in clinical canine reproduction. Key words: bitch, exfoliative vaginal cytology INTRODUCTION respect to the optimal breeding time (Tur- malaj et al., 2011). Exfoliative vaginal cytology is the most popular diagnostic method as a part of the gynaecological examination in the bitch PROCEDURES FOR VAGINAL CELL (Dreier, 1975; Gunzel-Apel & Koivisto, COLLECTION AND OBTAINING 1984; Linde & Karlson, 1984; Soderberg, SMEARS 1986a,b; Tammer et al., 1994; Johnson, Vaginal cells could be collected using a 2006; Wehrend, 2007; Trasch, 2008; sterile speculum and a saline-moistened Turmalaj et al., 2011; Groppetti et al., cotton swab (Wrobel et al., 1975; Wright 2012; Leigh et al., 2013; Wehrend et al., & Parry, 1989; Gunzel-Apel, 1993). It is 2013). It is based on determination of important to prevent contact of the swab cyclic cellular changes occurring in the with the vestibule, because its cells do not vaginal epithelium as a result of reproduc- react as quickly to an increase in the blood tive hormone levels, especially estrogens estrogen concentration as the vaginal (Wright & Parry, 1989). The method is mucous membrane (Röttger, 2010). simple and useful even in physiological or Collection of vaginal cells may be pathological conditions of the bitch repro- done using only a cotton swab, without a ductive system, most frequently the deter- speculum (Feldman & Nelson, 1996; Bo- mination of the estrous cycle stages with wen, 2000; Johnston et al., 2001; Kustritz, Application of exfoliative vaginal cytology in clinical canine reproduction – a review 2006; Aydin et al., 2011). The cotton- two solutions of Diff-Quick® stain. These tipped end of the swab is passed into the smears could be stored for years and dorsal commissure of the vulva, pressed examined later (Johnston et al., 2001; gently in the caudodorsal direction until it Chatdarong et al., 2002). passes over the ischial arch and then the Aydin et al. (2011) have examined swab is rotated through a complete revo- vaginal smears by a direct technique lution in each direction and withdrawn comparing it with classical staining to (Aydin et al., 2011). The cotton tip is determine the stages of the sexual cycle of lightly rolled from one end of a glass mic- the bitch and found it reliable only in roscope slide to the other (Feldman & detecting the estrus stage. Nelson, 1996; Johnston et al., 2001; Kus- tritz, 2006). EVALUATION OF VAGINAL SMEARS Vaginal cells may be collected by inoculation in the vaginal cavity and after- The evaluation of vaginal smears is wards aspiration of sterile saline using a performed with a light microscope at mag- plastic catheter (Olson et al., 1988). The nifications of 100 to 400×. A minimum of liquid is applicated on a glass microscope 10 observation fields should be examined slide, spread into a thin film and air-dried. (Theise, 2002). The method is minimally invasive, but so- There are different vaginal cell types metimes a change in cell morphology and (Ehlers, 2000). According to most authors, lower absolute cell count are observed the cells from the vaginal wall are diffe- (Olson et al., 1984a,b; Guyant, 1988). rentiated into basal, parabasal, intermedia- The smears could be stained using a te, superficial and squamous cells (Rieck trichrome or Papanicolaou stain (Papani- & Kratzheller, 1955; Schutte, 1967; Drei- colaou, 1942), but the technique is labou- er, 1975; Guyant, 1988; Maneke, 2002; rious (England & Concannon, 2002). Theise, 2002; Johnson, 2006). Many authors reported its use after redu- It is generally accepted that vaginal cing some steps of the staining (Barret, cell types in the smear are related to the 1976; Kubicek, 1978; Dumon & Morel, stage of the estrous cycle (Bell et al., 1989). 1973), which makes the vaginal exfolia- New methylene blue stained smears tive cytology a valuable add-on test in could be viewed immediately after dye ap- reproductive clinical diagnostics (Christie plication, but red blood cells are not stai- & Bell, 1973; Ehlers, 2000). ned and the smears can not be stored for Basal cells are the smallest cells (10– examination at a later time (Johnston et 20 μm) in the vaginal wall. They are al., 2001). Other classical staining techni- occupied almost entirely by the nucleus ques use stains such as May-Grunwald, and are rarely identified in the smears Boehringer Mannheim, Pappenheim and because they are on the basement memb- Testsimplets® (Gunzel-Apel & Koivisto, rane (Olson et al., 1984a; Wright & Parry, 1984). 1989). Diff-Quick® or Haemacolor® (Merck Parabasal cells are small (15–25 μm), KGaA) is a rapid modified Wright-Giem- round or ovoid with marginal big nucleus. sa stain easily applicable in the clinical Occasionally, they contain cytoplasmic practice. The smears are fixed with vacuoles. Parabasal cells may also contain methanol and then are stained with the neutrophil granulocytes in the cytoplasm 2 BJVM, ××, No × A. L. Antonov and then are called metestrus cells, al- (Rieck & Kratzheller, 1955; Christie et though they can occur in other cycle al., 1972; Dreier, 1975; Concannon & Di- stages (Wright & Parry, 1989) or vaginitis gregorio, 1986; Johnston et al., 2001; (Olson et al., 1984b). Maneke, 2002; Johnson, 2006). Intermediate cells exhibit great varia- Squamous cells are large cornified tions in diameter, so they are differentia- superficial cells which underwent degene- ted as small (20 μm) and large type (30 ration to become dead anucleated cells μm) (Rieck & Kratzheller, 1955; Dreier, (Simmons & Olson, 1989). They usually 1975; Olson, 1989; Maneke, 2002). Both stain dark blue-purple during the estrus types have a well shaped nucleus. Small (Johnston et al., 2001). intermediate cells are round to elliptical, Besides the vaginal cells, other types but may also have polygonal outline whe- of cells observed in vaginal smears are red reas the large type has an irregular and blood cells; neutrophil granulocytes (leu- angulated cytoplasmic border (Christie et kocytes); bacteria; tumour cells; clitoral al., 1972). Intermediate cells have a pro- fossa epithelial cells; spermatozoa; giant minent nucleus. The large type is someti- trophoblastic cells and debris (Maneke, mes confused with superficial cells becau- 2002). se they are of similar size (Johnston et al., 2001). CLINICAL APPLICATION OF VAGI- Superficial cells are large cells with a NAL EXFOLIATIVE CYTOLOGY diameter ranging between 30 μm (Christie et al., 1972) and 75 μm (Concannon & Determination of optimum time for Digregorio, 1986). They have irregular or breeding or artificial insemination angulated borders and dark, pyknotic or faint nucleus. Superficial cells attain their Fertility in the bitch is considered to be of maximum at the time of estrogen peak great socio-economic importance (Leigh Fig. 1. Vaginal smear of a bitch in proestrus: 1 – red blood cell; 2 – small intermediate cell; 3 – large intermediate cell; 4 – superficial cell; 5 – squamous cell (original, Haemacolor®, ×100). BJVM, ××, No × 3 Application of exfoliative vaginal cytology in clinical canine reproduction – a review ←2 ←1 Fig. 2. Vaginal smear of a bitch in estrus: 1 – superficial cell; 2 – squamous cell (original, Haemacolor®, ×100). et al., 2013). The majority of the bitches breeding occurs 3 to 10 days before the presented with history of infertity are in onset of cytologic diestrus. The detection fact fertile (Concannon et al., 1989). The of the first day of cytologic diestrus most common cause of conception failure allows determining whether the breeding with reported incidence between 40 and was done at the appropriate time 50% is mistimed breeding time (Zoldag et (Johnston et al., 2001). al., 1993; Johnston et al., 1994). Exfoliative vaginal cytology is additio- A gradual shift from parabasal and nally applied in cases of silent heat, i. e. intermediate to superficial cells occurs “white heat”, without a vulvar haemorrha- during the proestrus. Also, large numbers gic discharge in the presence of superfici- of red blood cells are present (Fig. 1). The al and squamous cells in vaginal smears optimum time for natural breeding or (Wright & Parry, 1989; Gunzel-Apel, artificial insemination is the estrus stage, 1993). when the percentage of superficial and Predicting the whelping date squamous cells (Fig. 2) in vaginal smears is above 80% (Simmons, 1970; Johnston If breeding or insemination are done at the et al., 2001; Srinivas et al., 2004). Bree- optimum time for ultimate conception ding or insemination should be done in a rates, it is possible to predict with a great two or three days interval until diestrus accuracy the time of whelping if a series occurs, as recognised by the appearance of vaginal smears are taken and the first of parabasal cells, neutrophils (Simmons, day of cytologic diestrus is determined. 1970; Srinivas et al., 2004) and the num- The anticipated time of whelping is 56–58 ber of superficial and squamous cells days, most commonly (93% of bitches) 57 decreases by at least 20% (Holst & Phe- days after the onset of cytologic diestrus mister, 1974) (Fig. 3). Conception rates (Holst & Phemister, 1974; Johnston et al., after a single mating are above 95% if 2001). 4 BJVM, ××, No × A. L. Antonov Fig. 3. Vaginal smear of a bitch on the first day of diestrus: 1 – red blood cell; 2 – small intermediate cell; 3 – large intermediate cell; 4 – squamous cells; 5 – neutrophil granulocyte (original, Haemacolor®, ×100).