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Cervical patency during non-ovulatory and ovulatory estrus cycles in domestic cats
Theriogenology 66 (2006) 804–810 www.journals.elsevierhealth.com/periodicals/the
Cervical patency during non-ovulatory and ovulatory estrus cycles in domestic cats Kaywalee Chatdarong a,*, Chainarong Lohachit a, Soonthorn Kiartmanakul b, Eva Axne´r c, Catharina Linde Forsberg c a
Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand b Department of Surgery, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand c Department of Clinical Sciences, P.O. Box 7054, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden Received 16 August 2005; received in revised form 16 January 2006; accepted 20 January 2006
Abstract The cervix functions as a barrier to spermatozoa. Vaginal artificial insemination in cats is, therefore, likely to be successful only at the period of estrus when the cervix is open. This study aimed to define the period of cervical patency in cats in both non-ovulatory and ovulatory estrus cycles. A total of 15 reproductive cycles were studied in six cats during the estrous stage. Cervical patency was monitored with the cats under sedation, by infusing 2 mL of Iohexol contrast medium via a 3.5 French tomcat catheter into the cranial vagina during estrus. Day one of estrus was defined as the first day the cats showed estrous behavior. Non-ovulatory cycles were characterized by a serum progesterone concentration on days 11–15 that was below 5 nmol/L and a normal interestrus interval of 7–14 days. Ovulatory cycles were characterized by a serum progesterone concentration on days 11–15 that was above 5 nmol/L and an interestrus interval that exceeded 30 days. The cervix was considered to be open when the contrast medium was seen to enter the uterus, and to be closed when the contrast medium remained in the vagina. Blood samples were collected at each examination and were assayed for estradiol-17b and progesterone concentrations. The cervix was open on the first day of standing estrus at a mean estradiol-17b serum concentration of 87.4 21.8 pmol/L (range 14 to 180 pmol/L) and closed at an estradiol concentration of 47.1 12.4 pmol/L (range 4 to 180 pmol/L). In the ovulatory cycles the cervix was closed at a progesterone concentration of 9.8 4.4 nmol/L (range 0.6–28.4 nmol/L). There was no difference in the duration of cervical patency in non-ovulatory and ovulatory cycles (5.5 1.2 days and 5.2 0.5 days, respectively) ( p > 0.05). The higher overall mean concentrations of estradiol17b seen in the ovulatory cycles than in the non-ovulatory cycles, indicate that a high level of estradiol is necessary for induction of ovulation. Ovulation in 60% of unmated females in this study indicates that the techniques used for evaluation of cyclus stage and cervical opening have the potential to induce ovulation in the cat. This study demonstrates that cervical patency is not influenced by the occurrence of ovulation, but is due to individual variations between cats. # 2006 Elsevier Inc. All rights reserved. Keywords: Feline; Radiography; Artificial insemination
1. Introduction * Corresponding author at: Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand. Tel.: +66 2 218 9644; fax: +66 2 255 3910. E-mail address: [email protected] (K. Chatdarong). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.01.053
The cervix functions as a primary barrier of the reproductive tract to the external environment as well as to spermatozoa in animals depositing the semen in the vagina during mating. Changes in cervical patency
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occur during the various stages of the estrus cycle. The dog, unlike the cat, is a spontaneous ovulator. In the bitch the cervix has been observed to open on the day of the maximal value of the estradiol:progesterone ratio, whereas the closure of the cervix occurred 2.6 1 days prior to the first sign of cytological metestrus [1]. Using positive contrast vagino-uterography, the cervix of the bitch was found to close 6.7 1.4 days [1] and 6.9 1.1 days [2] after the estimated time of the LH peak, when most bitches were still in estrus. Thus, cervical closure was suggested to be a limiting factor for reproductive success both by natural mating and intravaginal insemination in the dog [2]. This is supported by the significantly better results obtained by intrauterine insemination compared to vaginal insemination in the dog [3]. In cats, trans-cervical catheterization is difficult to perform due to the anatomy of the cervix, which is narrow, and nondistensible [4]. Although trans-cervical catheterization has been reported to be successful in every stages of estrus cycle [5], modifications of the vaginal and cervical anatomy found during estrus have been shown to contribute to a more difficulty in trans-cervical catheterization in the cats [6]. However, trans-cervical insemination using fresh and frozen semen has been successful in the cat [7]. Intravaginal insemination is easy to perform but the results are guarded. In the cat, the conception rate by vaginal insemination has been reported to be only 6.6% using 20 106 spermatozoa, 33.3% using 40 106 spermatozoa and was 77.8% when 80 106 spermatozoa were used [8], whereas by surgical intrauterine insemination a conception rate of 80% was obtained using only 8 106 fresh spermatozoa [9]. This shows that in the cat, like in the dog, the cervix is a crucial barrier for spermatozoa to enter the uterus. This is further supported by the similar findings in a previous study of sperm distribution at different times after natural mating in cats [10]. For vaginal insemination to be successful it should, thus, be performed at a time during the estrous period when the cervix is open. During the non-ovulatory cycle the cervix of the cat has been shown to be functionally open throughout the time of maximum cornification of the vaginal mucosa [11]. The effect of ovulation on the cervical opening has, however, not been assessed. In cats, conflicting evidence exist as to whether mating and ovulation influence estrus length or not. Paape et al. [12] reported that the duration of estrus was shortened by the occurrence of ovulation while, in contrast, Shille et al. [13] found that coitus increased the duration of estrus. Others found no difference in estrus length between ovulating and non-
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ovulating cats, and no effect of coitus on the length of estrus [14,15]. Thus, information about the occurrence and duration of cervical patency in both non-ovulatory and ovulatory cycles would improve our knowledge of feline reproductive physiology and be of importance for the determination of the optimal period for semen deposition both by natural mating and by vaginal artificial insemination in the cat. This study aimed to define the periods of cervical opening during estrus in non-ovulatory and ovulatory cycles in the domestic cat. 2. Materials and methods 2.1. Animals Six nulliparous female domestic cats of mixed breed (cats no. 1–6) aged 2–3 years and weighing 2.5– 3.0 kg were housed together in a 7 m 5 m 3 m (W L H) wire enclosure at Chulalongkorn University, Bangkok, Thailand. The colony was maintained under an artificial light schedule (14 h light: 10 h dark) supplemented with ambient light from the windows. A separate wire enclosure allowed visualization and olfaction of an adult male cat adjacent to the females. The animals were provided with a commercial cat food (Scient Diet, Hill’s Pet Nutrition, Topeka, KS, USA) and had free access to water. 2.2. Estrus detection Fifteen reproductive cycles in the six cats (1–3 each) were examined from June 2003 to March 2004. The onset of estrus was detected by daily behavioral observations and confirmed by vaginal cytology at each examination during estrus. Vaginal cytology was performed using a 2 mm-diameter cotton swab (Fo¨rbandsmaterial AB, Partille, Sweden) moistened with isotonic saline. The vaginal cells were smeared onto a glass slide and stained with modified Wright’s Giemsa (Dip Quik, Clinag, Bangkok, Thailand). A vaginal smear with a clearing of the background, a reduction of cellular debris and a proportion of superficial cells of 80% or more were considered as an estrous smear. The cats were considered to have ovulatory cycles when serum progesterone concentrations were 5 nmol/L on days 11–15 of estrus and the interestrus interval was more than 30 days. Cycles with progesterone concentrations remaining at <5 nmol/L and with an interestrus interval of 7–14 days were considered as non-ovulatory. Of the fifteen examined estrus cycles, six were considered as non-ovulatory and nine as ovulatory cycles.
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Table 1 Cervical patency and serum concentration of estradiol-17b and progesterone of the individual non-ovulatory estrous cycles from the first day the cats showed estrous behavior Cat Day of estrus (estradiol-17b (pmol/L), progesterone (nmol/L)) number 1 2 3 4 5 1 2 3 4 5 6
O O O O O O
Length of cervical opening (days) (80, 0.6) O (41, 0.7) O (26, 0.7) O (40, 0.7) C (10, 0.7) 4 (71, 0.6) O (21, 0.6) O (16, 0.6) C (24, 0.6) 3 (>180, 0.8) – – – – – O (32, 0.6) O (18, 0.6) C (9, 0.6) 8 (43, 0.6) – O (69, 0.6) – – O (87, 0.6) O (13, 0.6) C (21, 0.6) 7 (134, 0.6) – O (37, 0.6) – C (15, 0.6) – (64, 0.6) – – O (23, 0.8) – C (7, 0.6) – 6
7
8
9
O = open cervix, C = closed cervix, – = examination was not performed.
open, the shape of the uterine horns and the endometrial lining was examined [16]. Examinations were pursued until there was no evidence that the contrast medium was no longer seen to enter the uterus. In one cat (omitted from the study), examination was discontinued when an interestrous vaginal smear had been observed for 5 days while the cervix was still open. It was attempted to conduct the examinations daily. However, there was a limitation of the availability to utilize the radiography equipment which was set for the client animals as a priority. The frequency of examinations performed during estrus in individual cats is illustrated in Tables 1 and 2.
2.3. Assessment of the cervical patency, and hysterography Cervical patency was assessed using radiography with the cats sedated with acepromazine maleate (Sedastress, Farvet, Bladel, The Netherlands) 0.1 mg/ kg intramuscularly. A volume of 2 mL Iohexol 300 mg iodine/mL (Omnipaque, Nycomed AB, Roskilde, Denmark) was used as contrast medium and infused slowly through an open-end 3.5 mm French tomcat catheter (Sherwood, St. Louis) inserted into the cranial vagina until no further cranial movement could be achieved. The cats were positioned in dorsal recumbency and with the hindquarters elevated at an angle of approximately 158. The infusion was stopped when efflux of the contrast was observed in the vagina. Radiographs (Picker, Picker International Inc., Cleveland, USA) were taken immediately thereafter, in lateral and ventrodorsal projections. The cervix was defined as open when the contrast was seen to enter the uterus, or as closed when the contrast remained in the cranial vagina. When the cervix was shown to be functionally
2.4. Hormonal analyzes Blood was collected via cephalic venipuncture at the time of each examination during estrus and once between days 11–15 after the onset of estrus. The serum was separated by centrifugation and stored at 20 8C until assayed. The hormonal analysis was performed at the Department of Biomedical Sciences and Veterinary
Table 2 Cervical patency and serum concentration of estradiol-17b and progesterone of the individual ovulatory estrous cycles from the first day the cats showed estrous behavior Cat Day of estrus (estradiol-17b (pmol/L), progesterone (nmol/L)) number 1 2 3 4 5
6
1 2 3a 3b 4 5a 5b 5c 6
4 6 4 4 O (114, 0.8) O (39, 12.1) C (134, 32.7) 7 C (9, 50.9) – C (63, 4.6) 5 C (65, 6.9) – C (9, 33.4) 5
O O O O O O O O O
(117, 0.6) (81, 1.1) (86, 0.6) (139, 0.6) (70, 0.6) (139, 0.6) (87, 0.6) (44, 0.6) (96, 0.6)
– O (35, 0.6) – O (177, 2.0) O (73, 0.6) O (80, 0.6) – O (>180, 1.1) O (109, 1.0) O (103, 0.6) O (42, 0.6) O (41, 0.9) – O (116, 0.6) – – – O (43, 0.9)
O O O O O O O O O
(147, 0.6) (>180, 1.1) (84, 4.7) (84, 4.7) (135, 1.0) (34, 7.7) (61, 0.6) (131, 0.6) (20, 9.2)
C (92, 0.6) O (80, 1.7) C (88, 16.3) C (88, 16.3) O (47, 0.6) – O (135, 0.9) – O (11, 20.3)
O (57, 8.8)
7
8
Length of cervical opening (days)
C (16, 28.2)
a, b, c = repeating examination performed in the same cat, O = open cervix, C = closed cervix, – = examination was not performed.
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Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden. The serum was analyzed for estradiol-17b by radioimmunoassay using enhanced luminescence (Amerlite Estradiol 60-assay, OrthoClinical Diagnostics, Amersham, UK). The inter- and intra-assay coefficients of variation for estradiol-17b were: 33.5% and 18.1% at 6.5 pmol/L; 1.0% and 1.0% at 49.0 pmol/L; and 20.3% and 10.1% at 133.4 pmol/L. The maximum level of estradiol-17b was 180 pmol/L. The minimal assay sensitivity of estradiol-17b was 2.4 pmol/L. Progesterone was determined by a luminescence immunoassay (Immulite Progesterone, Diagnostic Products Corporation, LA, USA). The inter-and intra-assay coefficients of variation for progesterone were: 13.2% and 2.5% at 2.3 nmol/L; 6.7% and 1.2% at 24.6 nmol/; and 3.6% and 1.7% at 54.2 nmol/L. The minimal assay sensitivity of progesterone was 0.6 nmol/L. 2.5. Statistical analysis Periods of cervical opening and serum concentrations of estradiol-17b and progesterone are shown as mean S.E. Comparisons of the duration of cervical opening and serum concentrations of estradiol and progesterone between non-ovulatory and ovulatory cycles and between days of estrus were analyzed using paired t-test. One set of data from each female and type of estrus cycle was used for the statistical calculations. The non-ovulatory cycle of all cats and the ovulatory cycles of cats 1, 2, 3a, 4, 5b, and 6 were selected for the analysis of comparison of the duration of cervical patency. The level of significance was set at p 0.05. 3. Results The cervix of one cat permitted entry of the contrast medium throughout both estrous and interestrous periods. It was, therefore, excluded from the study. The patency of the cervix to the contrast medium and the respective hormonal concentrations in non-ovulatory and ovulatory estrous cycles in the individual cats (n = 6) are shown in Tables 1 and 2. In two of the cats (nos. 3 and 5) more than one ovulatory cycle was examined (Table 2). On the first day the cervix was open, maximal cornification of the vaginal cells was found and on the day the cervix was closed, the cornification had begun to decline in all of the seven cats. When only the cats with complete data were evaluated in a non-ovulatory cycle (cats no. 1–4) and in an ovulatory cycle (cats no. 1, 2, 3a, 4, 5b and 6), the cervix was found to be open for 3–8 days (5.5 1.2
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days) in the non-ovulatory cycle and 4–7 days (5.2 0.5 days) in the ovulatory cycle. Also when including the incomplete data the duration of cervical patency in the non-ovulatory cycle was found not to differ significantly from that in the ovulatory cycle irrespective of whether the cervix would have been closed ( p = 0.47) or open ( p = 0.64) the day before actually observed closure. Closing of the cervix was seen earliest on day 4 (n = 1) and day 5 (n = 3) of the non-ovulatory and ovulatory cycle, respectively, when the serum concentration of estradiol began to decline. However, variations between individual cats were observed. The cervix was seen to be open on the first day of standing estrus, at serum concentrations of estradiol17b of between 14 and 180 pmol/L (average 87.4 21.8 pmol/L) and closed at serum concentrations of estradiol-17b of between 4 and 180 pmol/L (average 47.1 12.4 pmol/L). The overall mean serum estradiol concentrations during estrus were higher in the ovulatory cycles than in the non-ovulatory cycles ( p < 0.05) (Fig. 1). The concentration of estradiol in the non-ovulatory cycles (cats no. 1–4) was significantly lower on the first day the cervix was found to be closed compared to the preceding day when the cervix was still open, whereas no such difference was observed in the ovulatory cycles (cats no. 1, 2, 3a, 3b, 4, 5b and 6) ( p > 0.05). In the ovulatory cycles, the initial rise in serum progesterone was observed on day 4 of estrus (Fig. 2) and reached 71.0 8.9 nmol/L during days 11– 15 of estrus. Data from the individual cats demonstrated the rise in serum progesterone was on day 4 (cats no. 3a, 3b, 5a and 6), day 6 (cats no. 2, 5b and 5c), and day 7 (cat no. 4) of estrus (Table 2). The hysterographic appearance in all of the six cats examined during estrus demonstrated a normal slight
Fig. 1. Serum concentrations of estradiol17-b in non-ovulatory (- -*- -) (n = 6) (cats no. 1, 2, 3, 4, 5 and 6) and ovulatory (—&—) (n = 9) (cats no. 1, 2, 3a, 3b, 4, 5a, 5b, 5c and 6) estrus cycles (mean S.E.). Values with different letters differ significantly ( p < 0.05).
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inner contour during interestrus (Fig. 4) and a wavy uterine lumen similar to in the other cats during estrus, indicating normal uterine horns for the stage. 4. Discussion
Fig. 2. Serum concentration of progesterone in six cats (cats no. 1, 2, 3a, 3b, 4, 5a, 5b, 5c and 6) during ovulatory estrus cycles (mean S.E.).
curved uterine shape with a wavy uterine lumen and a smooth inner contour typical for the stage (Fig. 3). The hysterography of the cat excluded from the study demonstrated a straight uterine lumen with a smooth
Fig. 3. Hysterographic appearance in the cats during estrus demonstrated a slight curved uterine shape with a wavy uterine lumen and a smooth inner contour.
Fig. 4. Hysterographic appearance during interestrus, in the cat excluded from the study demonstrates a straight uterine lumen with a smooth inner contour.
In addition to the correlations between expression of estrus behavior, sexual receptivity, cornified vaginal cytology and elevated serum estradiol concentrations described previously [12], the present study demonstrated that the cervix is open on the first day of estrus behavior in the cat. Controversial data have been published on whether the estrous period of the cat is shortened [12] or prolonged [13] by the occurrence of ovulation. However, in a recent study in 14 female cats, estrus was found to last for 7.4 4.4 days in non-bred (n = 15) and 4.4 1.6 days in bred cycles (n = 23), which was not significantly different [15]. The nonsignificant difference in the duration of cervical opening between the non-ovulatory and ovulatory cycles in the present study indicates that the duration of cervical opening is not affected by ovulation, but is due to individual variations between cats. In the cats, the corpora lutea secrete increasing amount of progesterone, starting 1–2 days after ovulation [12] and ovulation occurred 48–52 h after the LH peak [14]. The rise in progesterone concentration on days 4, 6 and 7 in the cats in this study, indicating that ovulation seemed to occur on day 2 (cats no. 3a, 3b, 5a and 6), day 4 (cats no. 2, 5b and 5c) and day 5 (cats no. 4), respectively. With the limited numbers of cats in this study, it could not be concluded that period of ovulation at early-, mid- and late-estrus, affected the duration of cervical patency. However, there was a trend that the later the ovulation, the longer the duration of cervical opening, demonstrated by that the cervix of cats no. 2 and 4 (ovulated at mid-estrus) opened longer than that of cat no. 3 (ovulated at early-estrus (6 and 7 versus 4)). Although the number of cats was limited, strength of the present study is that the same cats were compared in both a non-ovulatory and an ovulatory cycle as there were considerable variations between cats. When the individual cats that had complete sets of data from both types of cycles were evaluated (n = 4), it was found that the period of cervical opening was the same in both types of cycles in two cats (cats no. 1 and 4), whereas the period of cervical opening was shorter in the non-ovulatory cycle than in the ovulatory cycle in one cat (cat no. 2) while the opposite was found to be the case in cat no. 3. The duration of cervical patency in two ovulatory cycles observed in cat no. 3 were equal, likely demonstrating a consistency of the cervical dynamic in the same cat.
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The concentrations of estradiol-17b in the individual cats in this study varied greatly on the same days of the estrous cycle. On the first day of standing estrus the cervix was observed to be open and the estradiol concentration ranged from basal level to exceeding the maximal concentration limit of the assay (180 pmol/L). In the nonovulatory cycle, the closure of the cervix seems to be an effect of the decline in estradiol, as the estradiol concentration on the first day the cervix was closed was significantly lower than on the preceding day when the cervix was still open. In contrast, the closure of the cervix in the ovulatory cycle is likely influenced by the increase in progesterone concentration, as there were no differences in estradiol concentrations on the first day the cervix was closed and the last day it was open. The mechanism of the progesterone regulated closure of the cervix in the ovulatory cycle in the cat in this study, thus, appears to be similar to that observed in the dog [1]. The procedures used in this study appeared to have the potential to stimulate ovulation in some estrus cycles, perhaps related to high concentrations of estradiol. Generally, vaginal stimulation by mating or manipulation is transmitted via a spinal afferent nervous pathway to the hypothalamus. There it is converted to a hormonal signal of GnRH, which in turn induces release of FSH and LH, and thereby ovulation [17]. The cat requires several days of priming by increasing concentrations of estrogen over a period of time after coitus to initiate a surge of LH sufficient to induce ovulation [18]. Although some cats require multiple coital or mechanical vaginal stimulations for successful ovulation [19], a single copulation on the third day of estrus, when the estradiol concentration is markedly high has been reported to be adequate for a sufficient LH release to induce ovulation [20,21]. As significantly higher estradiol-17b concentrations were observed in the ovulatory cycles than in the non-ovulatory cycles, it is possible that a high concentration of estradiol-17b was a prerequisite for the ovulation to occur. Another possibility is that vaginal stimulation during sampling caused a release of GnRH and in turn a release of FSH and LH, which induced final maturation of follicles and ovulation in these cycles. Granulosa cells of follicles stimulated by the release of FSH and LH might have produced more estradiol. Ovulation occurring in 60% (9/15) of the estrus cycles in this study was most likely induced by the vaginal swabbing and/or the procedures used for infusion of the contrast medium. Either mechanical vaginal stimulation using a smooth rod or a cotton swab, or stroking on the back of the queen may suffice to induce ovulation [22,23]. Moreover, ovulation might
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have occurred spontaneously due to the presence of the male cat in the adjacent cage. The visualization of a male has been reported to increase the incidence of spontaneous ovulation in young group-housed cats in the range of from 0–22% to 33–57% [24]. However, ovulation was not observed in cats undergoing a similar examination for cervical patency in a previous study [11], the only difference being that those cats were sedated using 100–120 mg/kg b.w. medetomidine. Medetomidine offers advantages over acepromazine with respect to the degree of sedation, analgesic effect, muscle relaxation and arousal reactions to external stimuli [25,26]. The cats sedated with acepromazine in this study were found to be more struggling during the insertion of the tomcat catheter into the cranial vagina and toward the cervix, compared to the cats in the previous study. Therefore, the movements by the cats as a reaction to the insertion of the catheter seemed to induce a strong neural firing in the vagina sufficient to stimulate ovulation in some estrus cycles. The slightly curved uterine shape with a wavy uterine lumen and a smooth inner contour observed in the hysterographies in the females in estrus in this study demonstrated that they had normal uterine horns for the follicular stage of the reproductive cycle [16]. In cat omitted from the study, passage of the contrast medium to the uterus was observed in all stages of the cycle, which must be considered cervical functional abnormal based on the results of this study and a previous one [11]. The hysterographic image of this cat during interestrus was, however, normal with straight uterine horns with a smooth inner contour [16]. 5. Conclusions The duration of cervical patency in the cat is not affected by ovulation, but is likely due to the period of ovulation at early-, mid- or late-estrus. Considerable individual variations in the opening and closing of the cervix were observed. The cervix of the cat was found to be open at the onset of estrous behavior. The techniques used in this study to evaluate the cervical opening have the potential of inducing ovulation in the cat when a light degree of sedation is used. Especially when the female has an estrus cycle with a high estradiol concentration, a single vaginal stimulation seems to be adequate for inducing ovulation. Acknowledgements The authors are grateful for the financial support by Rachadapiseksompotch fund at Chulalongkorn Uni-
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versity and the Department of Biomedical Science and Veterinary Public Health, Swedish University of Agricultural Science. The cat food was provided by Hill’s Pet Nutrition Inc., USA. The authors thank Assistance Professor Padet Tummaruk for the assistance of statistical analyzes. References [1] Silva LDM, Onclin K, Verstegen JP. Cervical opening in relation to progesterone and oestradiol during heat in beagle bitches. J Reprod Fertil 1995;104:85–90. [2] Verstegen JP, Silva LDM, Onclin K. Determination of the role of cervical closure in fertility regulation after mating or artificial inseminatin in Beagle bitches. J Reprod Fertil Suppl 2001; 57:31–4. [3] Linde-Forsberg C, Stro¨m Holst B, Govette G. Comparison of fertility data from vaginal vs. intrauterine insemination of frozen-thawed dog semen: a retrospective study. Theriogenology 1999;52:11–23. [4] Swanson WF, Godke RA. Transcervical embryo transfer in the domestic cat. Lab Anim Sci 1994;44:228–91. [5] Chatdarong K, Lohachit C, Ponglowhapan S, Linde-Forsberg C. Transcervical catheterization and cervical patency during the estrus cycle in domestic cat. J Reprod Fertil 2001;57(Suppl): 353–6. [6] Zambelli D, Buccioli M, Castagnetti C, Belluzzi S. Vaginal cervical anatomic modification during the oestrus cycle in relation to transcervical catheterization in the domestic cat. Reprod Domest Anim 2004;39:76–80. [7] Zambelli D, Castagnetti C. Transcervical insemination with fresh or frozen semen in the domestic cat: new technique and preliminary results. In: Fifth annual conference of the European society for domestic animal reproduction (ESDAR); 2001. p. 34. [8] Tanaka A, Takagi Y, Nakagawa K, Fujimoto Y, Hori T, Tsutsui T. Artificial intravaginal insemination using fresh semen in cats. J Vet Med Sci 2000;62:1163–7. [9] Tsutsui T, Tanaka A, Takagi Y, Nakagawa K, Fujimoto Y, Murai M, et al. Unilateral intrauterine horn insemination of fresh semen in cats. J Vet Med Sci 2000;62:1241–5. [10] Chatdarong K, Lohachit C, Linde Forsberg C. Distribution of spermatozoa in the female reproductive tract of the domestic cat in relation to ovulation induced by natural mating. Theriogenology 2004;62:1027–241. [11] Chatdarong K, Kampa N, Axne´r E, Linde Forsberg C. Investigation of cervical patency and uterine appearance in domestic cats by fluoroscopy and scintigraphy. Reprod Domest Anim 2002; 37:275–81.
[12] Paape SR, Shille VM, Seto H, Stabenfeldt GH. Luteal activity in the pseudopregnant cat. Biol Reprod 1975;13:470–4. [13] Shille VM, Lundstro¨m KE, Stabenfeldt GH. Follicular function in the domestic cat as determined by estradiol-17b concentrations in plasma: relation to estrous behaviour and cornification of exfoliated vaginal epithelium. Biol Reprod 1979;21:953–63. [14] Wildt DE, Chan S, Seager SWJ, Chakraborty PK. Ovarian activity, circulating hormones and sexual behavior in the cat. I. Relationships during the coitus-induced luteal phase and the oestrous period without mating. Biol Reprod 1981;25: 15–28. [15] Root MV, Johnston SD, Olson PN. Estrous length, pregnancy rate, gestation and parturition lengths, litter size, and juvenile mortality in the domestic cat. J Am Anim Hosp Assoc 1995; 31:429–33. [16] Chatdarong K, Rungsipipat A, Axne´r E, Linde Forsberg C. Hysterographic appearance and uterine histology at different stages of the reproductive cycle and after progestagen treatment in the domestic cat. Theriogenology 2005;64:12–29. [17] Shille VM, Sojka NJ. Feline reproduction. In: Ettinger SJ, Feldman EC, editors. Textbook of veteirnary internal medicine. Philadelphia, WB: Saunders; 1995. p. 1690–8. [18] Banks DR, Stabenfeldt GH. Luteinizing hormone release in the cat in response to coitus on consecutive days of estrus. Biol Reprod 1982;26:603–11. [19] Concannon PW, Hodgson B, Lein D. Reflex LH release in estrous cats following single and multiple copulations. Biol Reprod 1980;23:111–7. [20] Shille VM, Munro C, Farmer SW, Papkoff H. Ovarian and endocrine responses in the cat after coitus. J Reprod Fertil 1983;69:29–39. [21] Tsutsui T, Stabenfeldt GH. Biology of ovarian cycles, pregnancy and pseudopregnancy in the domestic cat. J Reprod Fertil Suppl 1993;47:29–35. [22] Greulich WW. Artificially induced ovulation in the cat (Felis domestica). Anat Rec 1934;58:217–24. [23] Feldman E, Nelson R. Feline reproduction. In: Feldman E, Nelson R, editors. Canine and feline endocrinology and reproduction. 2nd ed., Philadelphia, WB: Saunders; 1996. p. 741–68. [24] Gudermuth DF, Newton L, Daels P, Concannon PW. Incidence of spontaneous ovulation in young, group housed cats based on serum and fecal concentrations of progesterone. J Reprod Fertil Suppl 1997;51:177–84. [25] Verstegen JP, Fargetton X, Donnay I, Ectors F. An evaluation of medetomidine/ketamine and other drug combinations for anaesthesia in cats. Vet Rec 1991;128:32–5. [26] Kojima K, Nishimura R, Mutoh T, Takao K, Matsunaga S, Mochizuki M, et al. Comparison of sedative effects of medetomidine-midazolam, acepromazine-butorphanol and midazolambutorphanol in dogs. Zentralbl Veterinarmed A 1999;46:141–8.
Cervical patency during non-ovulatory and ovulatory estrus cycles in domestic cats Kaywalee Chatdarong a,*, Chainarong Lohachit a, Soonthorn Kiartmanakul b, Eva Axne´r c, Catharina Linde Forsberg c a
Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand b Department of Surgery, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand c Department of Clinical Sciences, P.O. Box 7054, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden Received 16 August 2005; received in revised form 16 January 2006; accepted 20 January 2006
Abstract The cervix functions as a barrier to spermatozoa. Vaginal artificial insemination in cats is, therefore, likely to be successful only at the period of estrus when the cervix is open. This study aimed to define the period of cervical patency in cats in both non-ovulatory and ovulatory estrus cycles. A total of 15 reproductive cycles were studied in six cats during the estrous stage. Cervical patency was monitored with the cats under sedation, by infusing 2 mL of Iohexol contrast medium via a 3.5 French tomcat catheter into the cranial vagina during estrus. Day one of estrus was defined as the first day the cats showed estrous behavior. Non-ovulatory cycles were characterized by a serum progesterone concentration on days 11–15 that was below 5 nmol/L and a normal interestrus interval of 7–14 days. Ovulatory cycles were characterized by a serum progesterone concentration on days 11–15 that was above 5 nmol/L and an interestrus interval that exceeded 30 days. The cervix was considered to be open when the contrast medium was seen to enter the uterus, and to be closed when the contrast medium remained in the vagina. Blood samples were collected at each examination and were assayed for estradiol-17b and progesterone concentrations. The cervix was open on the first day of standing estrus at a mean estradiol-17b serum concentration of 87.4 21.8 pmol/L (range 14 to 180 pmol/L) and closed at an estradiol concentration of 47.1 12.4 pmol/L (range 4 to 180 pmol/L). In the ovulatory cycles the cervix was closed at a progesterone concentration of 9.8 4.4 nmol/L (range 0.6–28.4 nmol/L). There was no difference in the duration of cervical patency in non-ovulatory and ovulatory cycles (5.5 1.2 days and 5.2 0.5 days, respectively) ( p > 0.05). The higher overall mean concentrations of estradiol17b seen in the ovulatory cycles than in the non-ovulatory cycles, indicate that a high level of estradiol is necessary for induction of ovulation. Ovulation in 60% of unmated females in this study indicates that the techniques used for evaluation of cyclus stage and cervical opening have the potential to induce ovulation in the cat. This study demonstrates that cervical patency is not influenced by the occurrence of ovulation, but is due to individual variations between cats. # 2006 Elsevier Inc. All rights reserved. Keywords: Feline; Radiography; Artificial insemination
1. Introduction * Corresponding author at: Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand. Tel.: +66 2 218 9644; fax: +66 2 255 3910. E-mail address: [email protected] (K. Chatdarong). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.01.053
The cervix functions as a primary barrier of the reproductive tract to the external environment as well as to spermatozoa in animals depositing the semen in the vagina during mating. Changes in cervical patency
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occur during the various stages of the estrus cycle. The dog, unlike the cat, is a spontaneous ovulator. In the bitch the cervix has been observed to open on the day of the maximal value of the estradiol:progesterone ratio, whereas the closure of the cervix occurred 2.6 1 days prior to the first sign of cytological metestrus [1]. Using positive contrast vagino-uterography, the cervix of the bitch was found to close 6.7 1.4 days [1] and 6.9 1.1 days [2] after the estimated time of the LH peak, when most bitches were still in estrus. Thus, cervical closure was suggested to be a limiting factor for reproductive success both by natural mating and intravaginal insemination in the dog [2]. This is supported by the significantly better results obtained by intrauterine insemination compared to vaginal insemination in the dog [3]. In cats, trans-cervical catheterization is difficult to perform due to the anatomy of the cervix, which is narrow, and nondistensible [4]. Although trans-cervical catheterization has been reported to be successful in every stages of estrus cycle [5], modifications of the vaginal and cervical anatomy found during estrus have been shown to contribute to a more difficulty in trans-cervical catheterization in the cats [6]. However, trans-cervical insemination using fresh and frozen semen has been successful in the cat [7]. Intravaginal insemination is easy to perform but the results are guarded. In the cat, the conception rate by vaginal insemination has been reported to be only 6.6% using 20 106 spermatozoa, 33.3% using 40 106 spermatozoa and was 77.8% when 80 106 spermatozoa were used [8], whereas by surgical intrauterine insemination a conception rate of 80% was obtained using only 8 106 fresh spermatozoa [9]. This shows that in the cat, like in the dog, the cervix is a crucial barrier for spermatozoa to enter the uterus. This is further supported by the similar findings in a previous study of sperm distribution at different times after natural mating in cats [10]. For vaginal insemination to be successful it should, thus, be performed at a time during the estrous period when the cervix is open. During the non-ovulatory cycle the cervix of the cat has been shown to be functionally open throughout the time of maximum cornification of the vaginal mucosa [11]. The effect of ovulation on the cervical opening has, however, not been assessed. In cats, conflicting evidence exist as to whether mating and ovulation influence estrus length or not. Paape et al. [12] reported that the duration of estrus was shortened by the occurrence of ovulation while, in contrast, Shille et al. [13] found that coitus increased the duration of estrus. Others found no difference in estrus length between ovulating and non-
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ovulating cats, and no effect of coitus on the length of estrus [14,15]. Thus, information about the occurrence and duration of cervical patency in both non-ovulatory and ovulatory cycles would improve our knowledge of feline reproductive physiology and be of importance for the determination of the optimal period for semen deposition both by natural mating and by vaginal artificial insemination in the cat. This study aimed to define the periods of cervical opening during estrus in non-ovulatory and ovulatory cycles in the domestic cat. 2. Materials and methods 2.1. Animals Six nulliparous female domestic cats of mixed breed (cats no. 1–6) aged 2–3 years and weighing 2.5– 3.0 kg were housed together in a 7 m 5 m 3 m (W L H) wire enclosure at Chulalongkorn University, Bangkok, Thailand. The colony was maintained under an artificial light schedule (14 h light: 10 h dark) supplemented with ambient light from the windows. A separate wire enclosure allowed visualization and olfaction of an adult male cat adjacent to the females. The animals were provided with a commercial cat food (Scient Diet, Hill’s Pet Nutrition, Topeka, KS, USA) and had free access to water. 2.2. Estrus detection Fifteen reproductive cycles in the six cats (1–3 each) were examined from June 2003 to March 2004. The onset of estrus was detected by daily behavioral observations and confirmed by vaginal cytology at each examination during estrus. Vaginal cytology was performed using a 2 mm-diameter cotton swab (Fo¨rbandsmaterial AB, Partille, Sweden) moistened with isotonic saline. The vaginal cells were smeared onto a glass slide and stained with modified Wright’s Giemsa (Dip Quik, Clinag, Bangkok, Thailand). A vaginal smear with a clearing of the background, a reduction of cellular debris and a proportion of superficial cells of 80% or more were considered as an estrous smear. The cats were considered to have ovulatory cycles when serum progesterone concentrations were 5 nmol/L on days 11–15 of estrus and the interestrus interval was more than 30 days. Cycles with progesterone concentrations remaining at <5 nmol/L and with an interestrus interval of 7–14 days were considered as non-ovulatory. Of the fifteen examined estrus cycles, six were considered as non-ovulatory and nine as ovulatory cycles.
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Table 1 Cervical patency and serum concentration of estradiol-17b and progesterone of the individual non-ovulatory estrous cycles from the first day the cats showed estrous behavior Cat Day of estrus (estradiol-17b (pmol/L), progesterone (nmol/L)) number 1 2 3 4 5 1 2 3 4 5 6
O O O O O O
Length of cervical opening (days) (80, 0.6) O (41, 0.7) O (26, 0.7) O (40, 0.7) C (10, 0.7) 4 (71, 0.6) O (21, 0.6) O (16, 0.6) C (24, 0.6) 3 (>180, 0.8) – – – – – O (32, 0.6) O (18, 0.6) C (9, 0.6) 8 (43, 0.6) – O (69, 0.6) – – O (87, 0.6) O (13, 0.6) C (21, 0.6) 7 (134, 0.6) – O (37, 0.6) – C (15, 0.6) – (64, 0.6) – – O (23, 0.8) – C (7, 0.6) – 6
7
8
9
O = open cervix, C = closed cervix, – = examination was not performed.
open, the shape of the uterine horns and the endometrial lining was examined [16]. Examinations were pursued until there was no evidence that the contrast medium was no longer seen to enter the uterus. In one cat (omitted from the study), examination was discontinued when an interestrous vaginal smear had been observed for 5 days while the cervix was still open. It was attempted to conduct the examinations daily. However, there was a limitation of the availability to utilize the radiography equipment which was set for the client animals as a priority. The frequency of examinations performed during estrus in individual cats is illustrated in Tables 1 and 2.
2.3. Assessment of the cervical patency, and hysterography Cervical patency was assessed using radiography with the cats sedated with acepromazine maleate (Sedastress, Farvet, Bladel, The Netherlands) 0.1 mg/ kg intramuscularly. A volume of 2 mL Iohexol 300 mg iodine/mL (Omnipaque, Nycomed AB, Roskilde, Denmark) was used as contrast medium and infused slowly through an open-end 3.5 mm French tomcat catheter (Sherwood, St. Louis) inserted into the cranial vagina until no further cranial movement could be achieved. The cats were positioned in dorsal recumbency and with the hindquarters elevated at an angle of approximately 158. The infusion was stopped when efflux of the contrast was observed in the vagina. Radiographs (Picker, Picker International Inc., Cleveland, USA) were taken immediately thereafter, in lateral and ventrodorsal projections. The cervix was defined as open when the contrast was seen to enter the uterus, or as closed when the contrast remained in the cranial vagina. When the cervix was shown to be functionally
2.4. Hormonal analyzes Blood was collected via cephalic venipuncture at the time of each examination during estrus and once between days 11–15 after the onset of estrus. The serum was separated by centrifugation and stored at 20 8C until assayed. The hormonal analysis was performed at the Department of Biomedical Sciences and Veterinary
Table 2 Cervical patency and serum concentration of estradiol-17b and progesterone of the individual ovulatory estrous cycles from the first day the cats showed estrous behavior Cat Day of estrus (estradiol-17b (pmol/L), progesterone (nmol/L)) number 1 2 3 4 5
6
1 2 3a 3b 4 5a 5b 5c 6
4 6 4 4 O (114, 0.8) O (39, 12.1) C (134, 32.7) 7 C (9, 50.9) – C (63, 4.6) 5 C (65, 6.9) – C (9, 33.4) 5
O O O O O O O O O
(117, 0.6) (81, 1.1) (86, 0.6) (139, 0.6) (70, 0.6) (139, 0.6) (87, 0.6) (44, 0.6) (96, 0.6)
– O (35, 0.6) – O (177, 2.0) O (73, 0.6) O (80, 0.6) – O (>180, 1.1) O (109, 1.0) O (103, 0.6) O (42, 0.6) O (41, 0.9) – O (116, 0.6) – – – O (43, 0.9)
O O O O O O O O O
(147, 0.6) (>180, 1.1) (84, 4.7) (84, 4.7) (135, 1.0) (34, 7.7) (61, 0.6) (131, 0.6) (20, 9.2)
C (92, 0.6) O (80, 1.7) C (88, 16.3) C (88, 16.3) O (47, 0.6) – O (135, 0.9) – O (11, 20.3)
O (57, 8.8)
7
8
Length of cervical opening (days)
C (16, 28.2)
a, b, c = repeating examination performed in the same cat, O = open cervix, C = closed cervix, – = examination was not performed.
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Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden. The serum was analyzed for estradiol-17b by radioimmunoassay using enhanced luminescence (Amerlite Estradiol 60-assay, OrthoClinical Diagnostics, Amersham, UK). The inter- and intra-assay coefficients of variation for estradiol-17b were: 33.5% and 18.1% at 6.5 pmol/L; 1.0% and 1.0% at 49.0 pmol/L; and 20.3% and 10.1% at 133.4 pmol/L. The maximum level of estradiol-17b was 180 pmol/L. The minimal assay sensitivity of estradiol-17b was 2.4 pmol/L. Progesterone was determined by a luminescence immunoassay (Immulite Progesterone, Diagnostic Products Corporation, LA, USA). The inter-and intra-assay coefficients of variation for progesterone were: 13.2% and 2.5% at 2.3 nmol/L; 6.7% and 1.2% at 24.6 nmol/; and 3.6% and 1.7% at 54.2 nmol/L. The minimal assay sensitivity of progesterone was 0.6 nmol/L. 2.5. Statistical analysis Periods of cervical opening and serum concentrations of estradiol-17b and progesterone are shown as mean S.E. Comparisons of the duration of cervical opening and serum concentrations of estradiol and progesterone between non-ovulatory and ovulatory cycles and between days of estrus were analyzed using paired t-test. One set of data from each female and type of estrus cycle was used for the statistical calculations. The non-ovulatory cycle of all cats and the ovulatory cycles of cats 1, 2, 3a, 4, 5b, and 6 were selected for the analysis of comparison of the duration of cervical patency. The level of significance was set at p 0.05. 3. Results The cervix of one cat permitted entry of the contrast medium throughout both estrous and interestrous periods. It was, therefore, excluded from the study. The patency of the cervix to the contrast medium and the respective hormonal concentrations in non-ovulatory and ovulatory estrous cycles in the individual cats (n = 6) are shown in Tables 1 and 2. In two of the cats (nos. 3 and 5) more than one ovulatory cycle was examined (Table 2). On the first day the cervix was open, maximal cornification of the vaginal cells was found and on the day the cervix was closed, the cornification had begun to decline in all of the seven cats. When only the cats with complete data were evaluated in a non-ovulatory cycle (cats no. 1–4) and in an ovulatory cycle (cats no. 1, 2, 3a, 4, 5b and 6), the cervix was found to be open for 3–8 days (5.5 1.2
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days) in the non-ovulatory cycle and 4–7 days (5.2 0.5 days) in the ovulatory cycle. Also when including the incomplete data the duration of cervical patency in the non-ovulatory cycle was found not to differ significantly from that in the ovulatory cycle irrespective of whether the cervix would have been closed ( p = 0.47) or open ( p = 0.64) the day before actually observed closure. Closing of the cervix was seen earliest on day 4 (n = 1) and day 5 (n = 3) of the non-ovulatory and ovulatory cycle, respectively, when the serum concentration of estradiol began to decline. However, variations between individual cats were observed. The cervix was seen to be open on the first day of standing estrus, at serum concentrations of estradiol17b of between 14 and 180 pmol/L (average 87.4 21.8 pmol/L) and closed at serum concentrations of estradiol-17b of between 4 and 180 pmol/L (average 47.1 12.4 pmol/L). The overall mean serum estradiol concentrations during estrus were higher in the ovulatory cycles than in the non-ovulatory cycles ( p < 0.05) (Fig. 1). The concentration of estradiol in the non-ovulatory cycles (cats no. 1–4) was significantly lower on the first day the cervix was found to be closed compared to the preceding day when the cervix was still open, whereas no such difference was observed in the ovulatory cycles (cats no. 1, 2, 3a, 3b, 4, 5b and 6) ( p > 0.05). In the ovulatory cycles, the initial rise in serum progesterone was observed on day 4 of estrus (Fig. 2) and reached 71.0 8.9 nmol/L during days 11– 15 of estrus. Data from the individual cats demonstrated the rise in serum progesterone was on day 4 (cats no. 3a, 3b, 5a and 6), day 6 (cats no. 2, 5b and 5c), and day 7 (cat no. 4) of estrus (Table 2). The hysterographic appearance in all of the six cats examined during estrus demonstrated a normal slight
Fig. 1. Serum concentrations of estradiol17-b in non-ovulatory (- -*- -) (n = 6) (cats no. 1, 2, 3, 4, 5 and 6) and ovulatory (—&—) (n = 9) (cats no. 1, 2, 3a, 3b, 4, 5a, 5b, 5c and 6) estrus cycles (mean S.E.). Values with different letters differ significantly ( p < 0.05).
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inner contour during interestrus (Fig. 4) and a wavy uterine lumen similar to in the other cats during estrus, indicating normal uterine horns for the stage. 4. Discussion
Fig. 2. Serum concentration of progesterone in six cats (cats no. 1, 2, 3a, 3b, 4, 5a, 5b, 5c and 6) during ovulatory estrus cycles (mean S.E.).
curved uterine shape with a wavy uterine lumen and a smooth inner contour typical for the stage (Fig. 3). The hysterography of the cat excluded from the study demonstrated a straight uterine lumen with a smooth
Fig. 3. Hysterographic appearance in the cats during estrus demonstrated a slight curved uterine shape with a wavy uterine lumen and a smooth inner contour.
Fig. 4. Hysterographic appearance during interestrus, in the cat excluded from the study demonstrates a straight uterine lumen with a smooth inner contour.
In addition to the correlations between expression of estrus behavior, sexual receptivity, cornified vaginal cytology and elevated serum estradiol concentrations described previously [12], the present study demonstrated that the cervix is open on the first day of estrus behavior in the cat. Controversial data have been published on whether the estrous period of the cat is shortened [12] or prolonged [13] by the occurrence of ovulation. However, in a recent study in 14 female cats, estrus was found to last for 7.4 4.4 days in non-bred (n = 15) and 4.4 1.6 days in bred cycles (n = 23), which was not significantly different [15]. The nonsignificant difference in the duration of cervical opening between the non-ovulatory and ovulatory cycles in the present study indicates that the duration of cervical opening is not affected by ovulation, but is due to individual variations between cats. In the cats, the corpora lutea secrete increasing amount of progesterone, starting 1–2 days after ovulation [12] and ovulation occurred 48–52 h after the LH peak [14]. The rise in progesterone concentration on days 4, 6 and 7 in the cats in this study, indicating that ovulation seemed to occur on day 2 (cats no. 3a, 3b, 5a and 6), day 4 (cats no. 2, 5b and 5c) and day 5 (cats no. 4), respectively. With the limited numbers of cats in this study, it could not be concluded that period of ovulation at early-, mid- and late-estrus, affected the duration of cervical patency. However, there was a trend that the later the ovulation, the longer the duration of cervical opening, demonstrated by that the cervix of cats no. 2 and 4 (ovulated at mid-estrus) opened longer than that of cat no. 3 (ovulated at early-estrus (6 and 7 versus 4)). Although the number of cats was limited, strength of the present study is that the same cats were compared in both a non-ovulatory and an ovulatory cycle as there were considerable variations between cats. When the individual cats that had complete sets of data from both types of cycles were evaluated (n = 4), it was found that the period of cervical opening was the same in both types of cycles in two cats (cats no. 1 and 4), whereas the period of cervical opening was shorter in the non-ovulatory cycle than in the ovulatory cycle in one cat (cat no. 2) while the opposite was found to be the case in cat no. 3. The duration of cervical patency in two ovulatory cycles observed in cat no. 3 were equal, likely demonstrating a consistency of the cervical dynamic in the same cat.
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The concentrations of estradiol-17b in the individual cats in this study varied greatly on the same days of the estrous cycle. On the first day of standing estrus the cervix was observed to be open and the estradiol concentration ranged from basal level to exceeding the maximal concentration limit of the assay (180 pmol/L). In the nonovulatory cycle, the closure of the cervix seems to be an effect of the decline in estradiol, as the estradiol concentration on the first day the cervix was closed was significantly lower than on the preceding day when the cervix was still open. In contrast, the closure of the cervix in the ovulatory cycle is likely influenced by the increase in progesterone concentration, as there were no differences in estradiol concentrations on the first day the cervix was closed and the last day it was open. The mechanism of the progesterone regulated closure of the cervix in the ovulatory cycle in the cat in this study, thus, appears to be similar to that observed in the dog [1]. The procedures used in this study appeared to have the potential to stimulate ovulation in some estrus cycles, perhaps related to high concentrations of estradiol. Generally, vaginal stimulation by mating or manipulation is transmitted via a spinal afferent nervous pathway to the hypothalamus. There it is converted to a hormonal signal of GnRH, which in turn induces release of FSH and LH, and thereby ovulation [17]. The cat requires several days of priming by increasing concentrations of estrogen over a period of time after coitus to initiate a surge of LH sufficient to induce ovulation [18]. Although some cats require multiple coital or mechanical vaginal stimulations for successful ovulation [19], a single copulation on the third day of estrus, when the estradiol concentration is markedly high has been reported to be adequate for a sufficient LH release to induce ovulation [20,21]. As significantly higher estradiol-17b concentrations were observed in the ovulatory cycles than in the non-ovulatory cycles, it is possible that a high concentration of estradiol-17b was a prerequisite for the ovulation to occur. Another possibility is that vaginal stimulation during sampling caused a release of GnRH and in turn a release of FSH and LH, which induced final maturation of follicles and ovulation in these cycles. Granulosa cells of follicles stimulated by the release of FSH and LH might have produced more estradiol. Ovulation occurring in 60% (9/15) of the estrus cycles in this study was most likely induced by the vaginal swabbing and/or the procedures used for infusion of the contrast medium. Either mechanical vaginal stimulation using a smooth rod or a cotton swab, or stroking on the back of the queen may suffice to induce ovulation [22,23]. Moreover, ovulation might
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have occurred spontaneously due to the presence of the male cat in the adjacent cage. The visualization of a male has been reported to increase the incidence of spontaneous ovulation in young group-housed cats in the range of from 0–22% to 33–57% [24]. However, ovulation was not observed in cats undergoing a similar examination for cervical patency in a previous study [11], the only difference being that those cats were sedated using 100–120 mg/kg b.w. medetomidine. Medetomidine offers advantages over acepromazine with respect to the degree of sedation, analgesic effect, muscle relaxation and arousal reactions to external stimuli [25,26]. The cats sedated with acepromazine in this study were found to be more struggling during the insertion of the tomcat catheter into the cranial vagina and toward the cervix, compared to the cats in the previous study. Therefore, the movements by the cats as a reaction to the insertion of the catheter seemed to induce a strong neural firing in the vagina sufficient to stimulate ovulation in some estrus cycles. The slightly curved uterine shape with a wavy uterine lumen and a smooth inner contour observed in the hysterographies in the females in estrus in this study demonstrated that they had normal uterine horns for the follicular stage of the reproductive cycle [16]. In cat omitted from the study, passage of the contrast medium to the uterus was observed in all stages of the cycle, which must be considered cervical functional abnormal based on the results of this study and a previous one [11]. The hysterographic image of this cat during interestrus was, however, normal with straight uterine horns with a smooth inner contour [16]. 5. Conclusions The duration of cervical patency in the cat is not affected by ovulation, but is likely due to the period of ovulation at early-, mid- or late-estrus. Considerable individual variations in the opening and closing of the cervix were observed. The cervix of the cat was found to be open at the onset of estrous behavior. The techniques used in this study to evaluate the cervical opening have the potential of inducing ovulation in the cat when a light degree of sedation is used. Especially when the female has an estrus cycle with a high estradiol concentration, a single vaginal stimulation seems to be adequate for inducing ovulation. Acknowledgements The authors are grateful for the financial support by Rachadapiseksompotch fund at Chulalongkorn Uni-
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versity and the Department of Biomedical Science and Veterinary Public Health, Swedish University of Agricultural Science. The cat food was provided by Hill’s Pet Nutrition Inc., USA. The authors thank Assistance Professor Padet Tummaruk for the assistance of statistical analyzes. References [1] Silva LDM, Onclin K, Verstegen JP. Cervical opening in relation to progesterone and oestradiol during heat in beagle bitches. J Reprod Fertil 1995;104:85–90. [2] Verstegen JP, Silva LDM, Onclin K. Determination of the role of cervical closure in fertility regulation after mating or artificial inseminatin in Beagle bitches. J Reprod Fertil Suppl 2001; 57:31–4. [3] Linde-Forsberg C, Stro¨m Holst B, Govette G. Comparison of fertility data from vaginal vs. intrauterine insemination of frozen-thawed dog semen: a retrospective study. Theriogenology 1999;52:11–23. [4] Swanson WF, Godke RA. Transcervical embryo transfer in the domestic cat. Lab Anim Sci 1994;44:228–91. [5] Chatdarong K, Lohachit C, Ponglowhapan S, Linde-Forsberg C. Transcervical catheterization and cervical patency during the estrus cycle in domestic cat. J Reprod Fertil 2001;57(Suppl): 353–6. [6] Zambelli D, Buccioli M, Castagnetti C, Belluzzi S. Vaginal cervical anatomic modification during the oestrus cycle in relation to transcervical catheterization in the domestic cat. Reprod Domest Anim 2004;39:76–80. [7] Zambelli D, Castagnetti C. Transcervical insemination with fresh or frozen semen in the domestic cat: new technique and preliminary results. In: Fifth annual conference of the European society for domestic animal reproduction (ESDAR); 2001. p. 34. [8] Tanaka A, Takagi Y, Nakagawa K, Fujimoto Y, Hori T, Tsutsui T. Artificial intravaginal insemination using fresh semen in cats. J Vet Med Sci 2000;62:1163–7. [9] Tsutsui T, Tanaka A, Takagi Y, Nakagawa K, Fujimoto Y, Murai M, et al. Unilateral intrauterine horn insemination of fresh semen in cats. J Vet Med Sci 2000;62:1241–5. [10] Chatdarong K, Lohachit C, Linde Forsberg C. Distribution of spermatozoa in the female reproductive tract of the domestic cat in relation to ovulation induced by natural mating. Theriogenology 2004;62:1027–241. [11] Chatdarong K, Kampa N, Axne´r E, Linde Forsberg C. Investigation of cervical patency and uterine appearance in domestic cats by fluoroscopy and scintigraphy. Reprod Domest Anim 2002; 37:275–81.
[12] Paape SR, Shille VM, Seto H, Stabenfeldt GH. Luteal activity in the pseudopregnant cat. Biol Reprod 1975;13:470–4. [13] Shille VM, Lundstro¨m KE, Stabenfeldt GH. Follicular function in the domestic cat as determined by estradiol-17b concentrations in plasma: relation to estrous behaviour and cornification of exfoliated vaginal epithelium. Biol Reprod 1979;21:953–63. [14] Wildt DE, Chan S, Seager SWJ, Chakraborty PK. Ovarian activity, circulating hormones and sexual behavior in the cat. I. Relationships during the coitus-induced luteal phase and the oestrous period without mating. Biol Reprod 1981;25: 15–28. [15] Root MV, Johnston SD, Olson PN. Estrous length, pregnancy rate, gestation and parturition lengths, litter size, and juvenile mortality in the domestic cat. J Am Anim Hosp Assoc 1995; 31:429–33. [16] Chatdarong K, Rungsipipat A, Axne´r E, Linde Forsberg C. Hysterographic appearance and uterine histology at different stages of the reproductive cycle and after progestagen treatment in the domestic cat. Theriogenology 2005;64:12–29. [17] Shille VM, Sojka NJ. Feline reproduction. In: Ettinger SJ, Feldman EC, editors. Textbook of veteirnary internal medicine. Philadelphia, WB: Saunders; 1995. p. 1690–8. [18] Banks DR, Stabenfeldt GH. Luteinizing hormone release in the cat in response to coitus on consecutive days of estrus. Biol Reprod 1982;26:603–11. [19] Concannon PW, Hodgson B, Lein D. Reflex LH release in estrous cats following single and multiple copulations. Biol Reprod 1980;23:111–7. [20] Shille VM, Munro C, Farmer SW, Papkoff H. Ovarian and endocrine responses in the cat after coitus. J Reprod Fertil 1983;69:29–39. [21] Tsutsui T, Stabenfeldt GH. Biology of ovarian cycles, pregnancy and pseudopregnancy in the domestic cat. J Reprod Fertil Suppl 1993;47:29–35. [22] Greulich WW. Artificially induced ovulation in the cat (Felis domestica). Anat Rec 1934;58:217–24. [23] Feldman E, Nelson R. Feline reproduction. In: Feldman E, Nelson R, editors. Canine and feline endocrinology and reproduction. 2nd ed., Philadelphia, WB: Saunders; 1996. p. 741–68. [24] Gudermuth DF, Newton L, Daels P, Concannon PW. Incidence of spontaneous ovulation in young, group housed cats based on serum and fecal concentrations of progesterone. J Reprod Fertil Suppl 1997;51:177–84. [25] Verstegen JP, Fargetton X, Donnay I, Ectors F. An evaluation of medetomidine/ketamine and other drug combinations for anaesthesia in cats. Vet Rec 1991;128:32–5. [26] Kojima K, Nishimura R, Mutoh T, Takao K, Matsunaga S, Mochizuki M, et al. Comparison of sedative effects of medetomidine-midazolam, acepromazine-butorphanol and midazolambutorphanol in dogs. Zentralbl Veterinarmed A 1999;46:141–8.