THERIOGENOLOGY

DETERMINATION OF OVULATION TIME IN BITCHES BASED ON TEASING, VAGINAL CYTOLOGY, AND ELISA FOR PROGESTERONE G.F. Bouchard, N. Solorzan~,~ P.W. Concannon,* R.S. Youngquist and C.J. Bierschwal Department of Medicine and Surgery University of Missouri, Columbia, MO 65211 Qepartrnent of Physiology, NYCVM Cornell University, Ithaca, NY 14853 Received for publication: September 18, 1990
1990

Accept& Nc?vember 27,

ABSTRACT

The estrous cycle of 16 mature mongrel female dogs was monitored to evaluate the accuracy of teasing, vaginal cytology and quantitative ELISA progesterone assay to determine ovulation. The dogs were presented to male, and blood samples and vaginal swabs were taken daily during proestrus and es&us. Selected serum samples collected during estrus were assayed for endogenous LH by radioimmunoassay @IA). Plasma samples collected during proestrus and estrus were assayed for progesterone with a commercially available ELISA kit. Ovulation was considered to take place 48 h after the preovulatory LH peak. Vaginal cytology smears were stained with Wrights stain and evaluated for the percentage of superficial squamous cells. Day 1 of die&us (Day 1) was defined as a drop of 20% or more in the total number of superficial cells. Two standard curves (linear and best fitted curves) commonly used with ELISA were compared together and with the RIA progesterone assay. Ovulation was estimated to occur when progesterone concentration was 4.9 + 1.O rig/ml (mean + SD, n = 1.5), with a range of 3.4 to 6.6 nglml. Based on vaginal cytology, ovulation took place 6.9 & 1.6 d (n = 15) after 80% of the squamous cells were superficial and 6.8 f 1.4 d (n = 16) before Day 1. Ovulation took place 2.1 + 3.9 d (n = 11) after the first day of standing estrus and 8.8 &- 1.5 d (n = 10) before the last day of receptivity. The two standard curves were found parallel to each other and to the RIA progesterone assay. Based on the results of the present study, ELISA progesterone assay and determination of the first day of estrus by vaginal cytology are reliable methods for predicting ovulation, whereas the last day of receptivity as determined by teasing and Day 1 as determined by vaginal cytology are reliable methods to retrospectively estimate ovulation time. Key words: bitch, ovulation, teasing, cytology, progesterone

Acknowledgements This investigation was supported by the Veterinary Medicine Committee on Research Grant # C2-003.51 and partly by NIH Grant # RR03622. The authors wish to thank Mr. C. Trenton Boyd for library assistance and Dr. Gary F. Krause for his guidance with the statistics.

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INTRODUCTION The development of a rapid and reliable method for predicting ovulation in dogs would have a large impact on canine reproduction. Such a method would improve the fertility and the cost-efticiency of semen preservation techniques (freezing and cooling). It would also find application in breeding and infertility management and diagnostic, research and embryo transfer. Various methods are commonly used to monitor the bitchs estrous cycle. Teasing and secondary signs of estrus are widely used by breeders to determine mating time (1). The credibility of this technique to determine ovulation is jeopardized by subjectivity of interpretation and variability among dogs (2). Vaginal cytology is commonly used clinically to monitor canine estrous cycles. This technique defines adequately the most fertile period, but it estimates ovulation time only retrospectively (3). Hormonal assays have been found to accurately determine the time of ovulation. Elevation of progesterone concentrations prior to ovulation is peculiar to bitches. A serum progesterone concentration of 5.44 + 0.93 nglml (mean + SEM) as measured by RIA was found to correspond to ovulation (4). In this study, ovulation was determined by microscopic examination of sections of the ovary at specific times after the luteinizing hormone (LH) surge. The LH surge occurs about 48 h before ovulation (2). Determination of the LH surge by RIA is the preferred method for estimating ovulation because it is reliable and not invasive. Since the assay can only be performed by specialized laboratories, this technique is limited to research purposes only. Laparoscopy has also been used to determine ovulation. However, the need to open the ovarian bursa to examine the ovary and the limited number of examinations possible during estrus limit the use of this technique (5). Vaginoscopy can readily determine the enlargement of the vaginal mucosal folds occurring during proestrus and estrus (6). The fertile period can be estimated by this technique, but no fertility trial has been conducted as yet. More recently, ultrasonography has been proposed to detect ovulation (7,8), but more research is needed before this technique can be advocated. Recently, a quantitative enzymoimmunologic technique (ELISA) has been developed to determine progesterone concentration in bovine serum, plasma, or milk. This technique, unlike radioimmunoassay, is rapid and can be performed by an unspecialized laboratory. An ELISA kit (Ovucheck TM , Cambridge Veterinary Science, Cambridge, England) has been validated for measuring progesterone concentrations in dog plasma (9). In the past, two different standard curves (linear and beat fitted curves) have been used to determine the progesterone concentration from the spectrophotometric absorbance value. The relative effectiveness of the two curves has never been compared. Qualitative ELISA for progesterone kits have also been introduced to the dog market and are of great value to the practitioner. However, there is no data available in the literature regarding the efficiency of these products to predict ovulation. The objectives of this project were 1) to determine the best standard curve to evaluate progesterone concentration from spectrophotometric absorption values and 2) to evaluate the adequacy of teasing, vaginal cytology, and quantitative ELISA progesterone assays for the determination of the time of ovulation in dogs.

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MATERIALS AND METHODS Sixteen mature, mixed breed female dogs were used for this experiment. Vaccination and deworming were current. The dogs were housed in a controlled environment with 12 h of light daily and 22C ambient temperature the year around. The experiment was conducted from September 1988 to July 1989. The reproductive cycle of each bitch was followed twice weekly by vaginal cytology and teasing by a male with good libido to determine the onset of proestrus. Bitches in proestrus and estrus were teased daily. Daily serum, plasma and vaginal cytology samples were obtained until the end of estrus. Standing for the male, displaying the vulva, and deviating the tail to the side were regarded as physical signs of estrus. A total of 15 ml of blood was withdrawn daily between 1400 and 1600 h by jugular venipuncture into evacuated tubes containing either no anti-coagulant (serum) or EDTA (plasma). Serum and plasma samples were obtained by centrifugation and stored frozen until assayed. A glass speculum was introduced into the anterior vagina and cotton tipped swabs (six inches long) were used to collect vaginal cytology samples. Swabs were gently rolled onto precleaned glass slides to obtain smears that were stained with Wrights stain. The bitch was considered in estrus when more than 90% of the cells were superficial keratinized epithelial cells. We also measured the interval from the first day when more than 80% of the epithelial cells were superficial keratinized and ovulation. Day one of diestrus (Day 1) was defined as a drop of 20% or more of the total number of superficial cells (10). Serum samples collected daily between Days -6 and -11 from Day 1 (based on vaginal cytology) were assayed by RIA for endogenous LH. Luteinizing hormone in serum samples was measured in triplicate using a heterologous double-antibody RIA previously described (11). The within-assay and between-assay coefficients of variation ranged from 8 to 14% and 12 to lS%, respectively. Plasma samples collected daily during proestrus and estrus were assayed for progesterone in duplicate with a commercial ELISA kit. Progesterone standards used were 0.5, 1, 5 and 10 ng/ml. Spectrophotometric absorbance readings were transformed into progesterone concentrations by the use of the best fitted curve and the linear curve. The best fitted curve is constructed of different third-degree polynomials arranged together to form a single curve. A third-degree polynomial is calculated between two adjacent points, A total of N-l (N = number of standard points) polynomials are computed and assembled together to form a smooth single curve. This curve fitting technique is known as spline interpolation (12). The first degree equation is of the type y = a+bx, where y = absorbance, a = Y axis intercept, b = slope and x = ng/ml of progesterone. Progesterone concentrations below 0.5 ng/ml were rounded to 0.5 nglml with both standard curves. Samples with progesterone concentration over 10 nglml were either expressed as greater than 10 nglml (best-fitted curve) or estimated by extrapolation (linear curve). The day when ovulation took place was estimated to be 48 h after the preovulatory LH peak (13). None of the samples were below 0.5 nglml or above 10 nglml at the time of ovulation. Mean progesterone concentrations and standard deviation was computed at the day ovulation took place. The onset and the last day of es&-us as determined by vaginal cytology or teasing were also calculated relative to the day ovulation took place. Progesterone concentrations measured by the two different ELISA standard curves or with RIA were compared. Randomly selected serum samples (n = 64) with ELISA-

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measured progesterone concentrations between 0.5 and 10 nglml were assayed for progesterone by RIA. Serum samples were assayed for progesterone using a commercial solid-phase progesterone RIA (Coat-A-Count ProgesteroneTM,Diagnostic Products Corp., Los Angeles, CA, USA). All the samples were assayed in duplicate. The progesterone standards used were 0. 0.1, 0.5, 2, 10, 20 and 40 nglml. The within-assay and between-assay coefticients of variation ranged from 3 to 8% and 5 to 696, respectively. The two standard curves were compared to each other and to the RIA. Only samples with ELISA-measured progesterone concentrations between 0.5 and 10 nglml were used for comparison. The Student-t test and regression analysis were applied to determine the best standard curve. RESULTS The ELISA iinear standard curve, the ELLSA best fitted standard curve, and the IUA assay were different (P < 0.05, n = 111) when the Student-t test was applied. However, the linear correlation coefficient computed by regression analysis between the linear and best fitted standard curves (rz = 0.998, n = 64) was highly significant (P < 0.00001). The linear correlation coefficient was also highly significant (P < 0.0001) between the ELISA linear standard curve and RIA assay (9 = 0.896, n = 64) and between the best fitted curve standard curve and RIA assay (r = 0.893, n = 64). Ovulation was estimated to occur when progesterone concentration was 4.9 f 1.O nglml (mean f SD, n = 15) with a range of 3.4 to 6.6 nglml. The sample from Bitch 149 at the time of ovulation was misplaced. The concentration of progesterone at the time of the LH surge was 2.1 f 0.7 ng/ml (n = 16). These progesterone concentrations were computed with the linear ELISA standard curve. Progesterone concentrations obtained by the three methods of measurement (RIA assay, linear and best fitted ELISA standard curves) are presented in Table 1. Figure I illustrates serial means for progesterone and LH concentrations and the estimated time of ovulation. Based on vaginal cytology, ovulation took place 6.9 + 1.6 d (n = 15) after 80% of the vaginal epithelial cells were superficial keratinized and 6.8 f 1.4 d (n = 16) before Day 1. The LH surge occurred 8.8 f 1.4 d (n = 16) before Day 1. Figure 2 represents serial means for superficial keratinized cell percentages and LH concentrations and the estimated time of ovulation. Estrus was estimated to last 13.8 + 1.6 d (n=15) from the first day 80% of the vaginal epithelial cells were superficial keratinized or 13.2 f 1.7 d (n= 15) when 90% or more of the vaginal epithelial cells were superficial keratinized. Based on receptivity, ovulation took place 2.1 + 3.9 d (n = 11) after the first day of standing estrus and 8.8 f. 1.5 d (n = 13) before the last day of receptivity. One dog did not stand for the male but vaginal cytology indicated estrus. Therefore, data collected from this dog were not used. Also, data regarding the first and last day of receptivity were not available for all dogs.

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15

ct
3
0

-7-6-5-4-3-Z-l Figure 1

1
Time

(days)

Relatioship between LH surge and progesterone concentrations as measured by ELISA.

12 10 _ x > \5 I J 0 6460 40 B100 .-

80

E g d x ci

ET > 20
0 ; -10
I
l

1 8

I 10

-8

-6

-4

-2

2
Time

4
(days)

I 12

Figure

2.

Relationship superficial

between keratinized

LH surge and percentage of cells on vaginal cytology.

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Table 1.

Progesterone concentrations at ovulation and the luteinizing hormone peak

Linear Standard Curve

Best fitted Standard Curve

RIA assay

Ovulation LH peak

4.9*1.0 2.1 f0.7

4.7*1.1 2.1kO.7

3.3f0.8 1.7kO.5

Progesterone concentrations are expressed in ng/ml with their standard deviations. DISCUSSION The two ELISA standard curves were found different with the Student-t test, but the correlation coefftcient was excellent between the two curves. Therefore, a close relationship exists between the two standard curves, but one should indicate which curve was used to compute the progesterone concentration. The two ELISA standard curves were also different from RIA, but the correlation coefficient was also very good (P < 0.0001). Independently of the ELISA standard curve, there was still a close relationship between ELISA and RIA assays. Other authors have reported a high correlation between ELISA and RIA progesterone assays (9,14) In the present study, the time of ovulation was estimated by an indirect method. Previous reports refer to the LH peak as an accurate method for estimating the time of ovulation in bitches. Determination of the LH peak to predict ovulation also has the advantage of being noninvasive and therefore should not disturb the process of ovulation. Our observations indicate that the LH peak occurred 8.8 days (SD = 1.4) before Day 1, which is similar to that reported previously (2,3). Increased progesterone concentrations were measured by the time of the LH peak. Progesterone concentrations were also consistent around the time of ovulation (4.9 nglml, SD = 1.0). As shown in Figure 1, the increase of progesterone concentrations is rapid after the LH peak (slope = 2.1 nglmllday). This rapid elevation of the progesterone concentration narrows the margin of error in determining the ovulation time. Similar results were reported by others using RIA (4,13). However, the increase of the progesterone concentration after the LH peak was more pronounced when measured by ELISA than when measured by RIA progesterone assay, although the standard deviations were similar (Table 1). This indicates that the ELISA progesterone assay is more likely to differentiate the LH peak from ovulation time. Recently, the use of RIA for progesterone to improve fertility in bitches has been investigated (15-17). Two investigations evaluated the conception rate of large number of bitches when insemination was timed based on a rapid RIA progesterone assay. Okkens et al. (1985) reported that 38 of 41 (93%) bitches became pregnant following breeding (single or multiple) after progesterone concentration increased above 5 nglml (16). Van Haaften et al. (1989) reported that 81 of 104 (78%) subfertile bitches and 105 of 112 (94%) fertile bitches became pregnant following breeding (single or multiple) timed with the aid of RIA

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progesterone assay. In their study, breeding was recommended within 9 h when the progesterone concentration exceeded 12 ng/ml, within 9 to 33 h, when it was between 6 and 12 nglml, and within 33 to 57 h when the progesterone level was between 5 and 6 ng/ml (15). Subfertile bitches were selected based on their history. Bitches with conception failure following more than half of the breeding attempts or following a single breeding attempt were considered subfertile. The results of these two studies emphasize the usefulness of progesterone assays to enhance conception rate in bitches. As we found in our investigation, there is a good correlation between the results of RIA and ELISA progesterone assay (9,14). For this reason, we feel confident in recommending ELISA progesterone assay to monitor the estrous cycle and the breeding management of bitches. However, fertility trials are indicated to confirm this conclusion. As reported previously, time of ovulation can be estimated retrospectively with vaginal cytology (2). Our observations indicate that the time of ovulation can also be determined from the first day of cytologic estrus with similar accuracy. Ovulation occurs 6.8 d (SD = 1.4) before Day 1 and 6.9 d (SD = 1.6) after the first day of e&us. We defined the first day of estrus as the first day on which 80% or more of the vaginal epithelial cells are keratinized Therefore, we suggest that vaginal cytology is a good alternative to progesterone assay. This technique is rapid and easy to perform and provides a fairly accurate estimation of the time of ovulation both prospectively and retrospectively. Okkens et al. (1985) reported that only 30% of 20 bitches became pregnant when vaginal cytology was used as the sole method for detecting ovulation. Details regarding the breeding schedule were omitted in that study. Another study did not support the use of vaginal cytology based on the variability of the estrous cycle of two bitches (17). Our results do not agree with these previous investigators, probably because we have a different perspective on the use of vaginal cytology. Fertility needs to be evaluated when the first days of estrus correspond to 80% or more of superficial cells on vaginal cytology. We found that teasing is not an appropriate method for determining the fertile period, regardless of the approach used. This agrees with previous reports (2,513). In many clinical situations, receptivity is a satisfactory method for scheduling breeding because of the long life of sperm (4 to 6 d; 18,19), the spread of ovulation (between 6 and 48 h; 4,13), post-ovulatory maturation of oocytes (approximately 2 to 3 d; 20), and viability of mature oocytes (1 to 2 d; 17,20). However, more critical situations such as insemination with frozen or chilled semen or management of infertility require a more precise determination of the fertile period than teasing can offer. We therefore do not advise using teasing as the sole method for determining the time of ovulation. REFERENCES 1. Evans, J.M. and White, K. Breeding from your bitch. Henston Ltd., Guildford, England, 1988, pp 59-124. In: The Book of the Bitch.

2.

Holst, P.A. and Phemister, R.D. Onset of diestrus in the Beagle bitch: significance. Am. J. Vet. Res. &iO1406 (1974).

Definition and

3.

Holst, P.A. and Phemister, R.D. Temporal sequence of events in the estrous cycle of the bitch. Am. J. Vet. Res. j&705-706 (1975).

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Concarmon, P., Hansel, W. and McEntee, K. Change in LH, progesterone and sexual behavior and associated with preovulatory luteinization in the bitch Biol. Reprod. ~604-613 (1977). Wildt, D.E., Chakraborty, P.K., Panko, W.B. and Seager, S.W.J. Relationship of reproductive behavior, serum luteinixing hormone, and time of ovulation in the bitch. Biol. Reprod. &561-570 (1978). Concannon, P. and Lein, D.H. Hormonal and clinical correlates of ovarian cycles, ovulation, pseudopregnancy, and pregnancy in dogs. Ig: Kirk, R.W. (cd.), Current Veterinary Therapy. W. B. Saunders Company, Philadelphia, PA, 1989, pp.l2691282. Inaba, T., Matsui, N., Shimizu, R. and Imori, T. Use of echography in bitches for detection of ovulation and pregnancy. Vet. Rec. m:276-277 (1984). England, G.C.W. and Allen, W.E. Real-time ultrasonic imaging of the ovary and uterus of the dog. J. Reprod. Fertil. z(Suppl.):91-100 (1989). Eckersall, P.D. and Harvey, M.J.A. The use of a bovine plasma progesterone ELISA kit to measure progesterone in equine, ovine, and canine plasmas. Vet. Rec. 1205-8 (1987). Olson, P.N., Thrall, M.A., Wykes, P.M., Nett, T.M. and Sawyer, H.R. Vaginal cytology. Part I. A useful tool for staging the canine estrous cycle. Compend. Cont. Ed. 6:288-298 (1984). Concannon, P. W. Induction of fertile oestrus in anoestrous dogs by constant infusion of GnRH agonist. J. Reprod. Fertil.~(Suppl.):149-160 (1989). Sedgewick, R. Curve fitting. In: Algorithms. Inc., New York, NY, 1983, pp. 67-77. Addison-Wesley Publishing Company,

5.

6.

7.

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9.

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11.

12.

13.

Phemister, R.D., Holst, J.S., Spano J.S. and Hopwood, M.L. the Beagle bitch. Biol. Reprod. &74-82 (1973).

Time of ovulation in

14.

England, G.C.W., Allen, W.E. and Porter, D. J. A comparison of radioimmunoassay with quantitative and qualitative enzyme-linked immunoassay for plasma progestogen detection in bitches. Vet. Rec. 125: 107-108 (1989). van Haaften, B., Dieleman, S.J., Okkens, A. C. and Willemse, A. H. Timing the mating of dogs on the basis of blood progesterone concentration. Vet. Rec. m:524526 (1989). Okkens, A. S., Dieleman, S.J. in the dog a comparison of the cytology. In: Voorjaarsdagen Ass. (Amsterdam, Netherlands) and Vogel, F.. Determination of the ovulation period rapid progesterone assay, vaginoscopy and vaginal 1985 Proceedings of the Netherlands Sm. Anim. Vet. pp. 26-27 (1985).

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Jeffcoate, LA. and Lindsay, F.E.F. Ovulation detection and timing of insemination based on hormone concentrations, vaginal cytology and the endoscopic appearance of the vagina in domestic bitches. J. Reprod. Fertil. s(Suppl.):277-287 (1989). Concannon, P., Whaley, S., Lein, D. and Wissler, R. Canine gestation length: variation related to time of mating and fertile life of sperm. Am. J. Vet. Res. 44:18191821 (1983). Doak, R.L., Hall, A. and Dale, H.E. Longetivity of spermatozoa in the reproductive tract of the bitch. J. Reprod. Fertil. 1351-58 (1967). Olson, P.N. and Husted, P. W. Breeding management for optimal reproductive effkiency in the bitch and the stud dog. &: Morrow, D.A. (ed.), Current Therapy in Theriogenology. W. B. Saunders Company, Philadelphia, PA, 1986, ~~463-468.

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