RPCV (2003) 98 (547) 135-138 REVISTA PORTUGUESA DE CIÊNCIAS VETERINÁRIAS Vaginal cytology in queens with estrus induced with equine chorionic gonadotrophin Citologia vaginal em gatas com estro induzido por gonadotrofina coriônica eqüina Marcos Renato Franzosi Mattos*a, Lucilene Simões-Mattosb, Lúcia Daniel Machado da Silvab Universidade Estadual do Ceará (UECE), Faculdade de Veterinária (FAVET), Programa de Pós-Graduação em Ciências Veterinárias (PPGCV) Av. Paranjana, 1700, Campus do Itaperi, Fortaleza, Ceará, Brazil. CEP 60.715-100 Summary: In order to determine the indirect influence of seve- Introduction ral doses of equine chorionic gonadotrophin (eCG) on vaginal In feline and mainly canine species, vaginal cyto- cytology, 42 queens were randomly allotted to four groups for treatment with 0.9% saline (control) or with 25, 40 or 60 IU/Kg logy examination is a current method for the assess- of eCG injected IM. Epithelial and blood cells examined in 135 ment of the stage of estrous cycle and of endocrine or vaginal smears showed the normal cell pattern of feline estrus. reproductive pathologies (Herron, 1977; Roszel, 1977; Mucus was seen in all smears. However, small intermediate cells Toniollo et al., 1995; Vannucchi et al., 1997; Nasci- were rarely found and the presence of erythrocytes and neutro- mento e Lopez, 1999; Erunal-Maral et al., 2000). This phils was considered negligible. In conclusion, the cell pattern of the vaginal epithelium of queens with estrus induced with is due to the high responsiveness of the vaginal epi- different doses of eCG was similar to that of queens with natu- thelium to hormonal fluctuations, especially estrogens, ral estrus. Thus, we may speculate that the hormonal stimulus causing changes in the cellular profile. Increasing occurring in the vaginal epithelium of eCG-induced queens is estrogen concentrations determine cell proliferation, the same as that occurring in natural estrus, or that this epithe- with thickening of the vaginal epithelium layers and lium responds similarly to different dose of eCG. On this basis, vaginal cytology may be not an efficient tool for the assessment consequent cell differentiation (Papanicolaou, 1942; of the hormonal environment of queens with induced estrus. Roszel, 1977; Concannon e Digregorio, 1986; Johns- ton et al., 2001). Resumo: Para determinar a influência indirecta de várias doses Currently, assisted reproductive technologies (ART) de gonadotrofina coriônica eqüina (eCG) na citologia vaginal, such as estrus synchronization, ovulation induction 42 gatas foram distribuídas aleatoriamente em quatro grupos tratados com solução salina 0,9% (controle) ou com 25, 40 ou and/or superovulation have been developed using the 60 UI/Kg de eCG via IM. De 135 esfregaços vaginais, foram domestic cat as model for wild felids (Dresser et al., examinadas as células epiteliais e sangüíneas, que apresenta- 1987; Wildt et al., 1992; Swanson et al., 1995. 1998; ram padrão celular normal para o estro felino. Houve presença Mattos et al., 2001a; Aguiar et al., 2002). However, de muco em todos os esfregaços. Por outro lado, células do low reproductive success is obtained in domestic cats tipo pequena intermediária foram raramente encontradas, e a presença de eritrócitos e neutrófilos foi considerada irrelevante. submitted to ART (Pope, 2000) mainly with gona- Podemos concluir que o padrão celular do epitélio vaginal de dotrophin therapy (Verstegen et al., 1993; Graham gatas com o estro induzido por diferentes doses de eCG foi et al., 2000; Mattos et al., 2001a). It is known that semelhante ao de gatas em estro natural. Assim, podemos espe- plasma estradiol-17β concentrations are enhanced cular que o estímulo hormonal sobre o epitélio vaginal em gatas in queens with gonadotrophin-induced estrus, toge- que receberam eCG é o mesmo do das gatas em estro natural, ou que este epitélio responde da mesma maneira frente a diferentes ther with an early increase in plasma progesterone doses de eCG. Neste sentido, a citologia vaginal pode não ser concentrations causing hormonal disruption. These uma ferramenta eficiente para a avaliação do ambiente hormonal hormonal alterations are considered to be responsible de gatas com estro induzido. for the low reproductive success by interfering with physiological status (Goodrowe et al., 1986; Roth et al., 1997; Barnes, 2000; Graham et al., 2000). Several studies using different evaluation methods to deter- mine how and where this hormonal alteration causes a low reproductive success have obtained different and inconclusive results (Herron and Sis, 1974; Goodrowe * Corresponding author: e-mail [email protected], et al., 1986; Orosz et al., 1992; Verstegen et al., 1993; telephone. +55-85-299-2760, fax +55-85-299-2740. Schramm and Bavister, 1995; Roth et al., 1995,1997; aFUNCAP fellowship bCNPq fellowship Barnes, 2000; Graham et al., 2000; Silva et al., 2001). 135 Mattos, M.R.F. et al. RPCV (2003) 98 (547) 135-138 In rats, the vaginal epithelium is highly sensitive to maintained in natural light (approximately 12 h light / hormone administration, permitting a reliable endpoint 12 h dark). The queens were randomly allotted to four assessment of hormonal imbalance (Banik and Herr, groups receiving different doses of eCG (IU/kg/IM) as 1969; Nagaoka et al., 2002). In contrast, the indirect follows: 0 (control group), 25, 40 and 60. The estrous influence of exogenous gonadotrophins causing the cycles were daily monitored, with evaluation of the steroidal hormones imbalance after ART procedures peculiar signs of each phase according to previous on the cellular pattern of the vaginal epithelium of studies (Banks, 1986; Johnston et al., 2001; Silva et cats is poorly known. Indeed, due to the sensitivity of al., 2001). On the fifth day of interestrus, the queens the vaginal epithelium to steroid hormones, vaginal received a single injection of eCG. The control group cytology may be an indirect parameter for the deter- was considered as natural estrus and received only mination of the hormonal environment of gonadotro- 1 mL of saline solution (0.9%). After hormonal or phin-induced estrus in cats. Thus, it is very important placebo injection, the queens were exposed daily to to establish reliable endpoints for the assessment of the toms until male acceptance, although at that time the individual hormonal milieu of queens with gona- mating was prevented by us. Twenty-four and 48 h dotrophin-induced estrus queens in order to assess the after the first day of male acceptance by queens, four consequences of ART procedures. mating episodes were allowed to occur over a period Thus, as the action of equine chorionic gonadotro- of two hours. It is important to point out that this study phin (eCG) and FSH are similar concerning the relea- is part of a wide trial which evaluates the effects of sing of estrogens, the purpose of the present study was gonadotrophins on different reproductive parameters to access the possibility of the indirect influence on the (data not shown). This explains the mating procedures vaginal cellular profile of queens by ovarian stimula- cited above. Thus, only vaginal cytology findings will tion with progressive increasing doses of eCG. be mentioned. Materials and Methods Vaginal cytology Animals and treatments Ten to 15 minutes after the last mating, vaginal cells were obtained by gently passing a sterile cotton-tipped Forty-seven adult intact undefined breed cats Felis( swab into the vaginal canal followed by a quick 180º catus) were used in this trial, 42 queens (body weight rotation. The cells were transferred to one or two 2.5 to 4.8 kg), and five toms (3.5 to 5.8 kg). The ani- slides, air dried, and immediately fixed with 100% mals were housed in individual cages (80 x 60 x 60 ethanol. The smears were stained with hematoxylin cm) and fed a dry maintenance cat food (Eukanuba and eosin, modified (Mattos et al., 2001b). A total Chiken and Rice Formula, Kitekat and Gatsy) and of 135 vaginal smears were obtained from the four water ad libitum throughout the study. In addition, they different treatments on the second and third days of were kept free in a commun solarium with sufficient permitted mating. Twenty-six slides were prepared for material for distraction for at least four hours/day. The the control group, 36 for the 25 IU group, 34 for the 40 experiment was accomplished from February to June IU group, and 39 for the 60 IU group. of 2002, in the Fortaleza city, Northeastern Brazil, situated at 3º 43’ 47” South and 38º 30’ 37” West. In Evaluation of vaginal cells this region there are few differences in number of hour per day along the year and the queens show estrous Smears were examined under a light microscope at cycle during all year. In this work, the queens were magnifications of X32, X100, X400 and X1000. Two- Figure 1 - Percentage of epithelial vaginal cells (mean ± SEM) of the Figure 2 - Percentage of epithelial vaginal cells (mean ± SEM) of exfo- vaginal smears from 42 domestic queen (Felis catus) in natural (control) liates vaginal smears of domestic queen (Felis catus) at first and second or induced estrus at different doses of eCG. PB - Parabasal; SI - Small days of mating. PB - Parabasal; SI - Small intermediate; LI - Large intermediate; LI - Large intermediate; NS - Nucleated superficial; AS - intermediate; NS - Nucleated superficial; AS - Anucleated superficial. p Anucleated superficial. p ≤ 0.01. ≤ 0.01. 136 Mattos, M.R.F. et al. RPCV (2003) 98 (547) 135-138 hundred epithelial cells from each slide were evaluated this respect, examination of the cell profile by vaginal and classified according to previous studies (Concan- cytology during the estrous cycle may be an inefficient non e Digregorio, 1986; Shille et al., 1979; Mattos et tool for the evaluation of the hormonal environment in al., 2001b) as: parabasal (PB), small intermediate (SI), cats with induced estrus. large intermediate (LI), nucleated superficial (NS) In this trial, the profile of the epithelial cells detected and anucleated superficial (AS).