determination of ovulation time in bitches based on teasing, vaginal cytology, and elisa for...

8/13/2019 Determination of Ovulation Time in Bitches Based on Teasing, Vaginal Cytology, And Elisa for Progesterone

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THERIOGENOLOGY

DETERMINATION OF OVULATION TIME IN BITCHES BASED ONTEASING, VAGINAL CYTOLOGY, AND ELISA FOR PROGESTERONE

G.F. Bouchard, N. Solorzan~,~ P.W. Concannon,* R.S. Youngquistand C.J. Bierschwal

Department of Medicine and SurgeryUniversity of Missouri, Columbia, MO 65211

Qepartrnent of Physiology, NYCVMCornell University, Ithaca, NY 14853

Received for publication: September 18, 1990Accept Nc?vember 27, 1990ABSTRACT

The estrous cycle of 16 mature mongrel female dogs was monitored to evaluate theaccuracy of teasing, vaginal cytology and quantitative ELISA progesterone assay to determineovulation. The dogs were presented to male, and blood samples and vaginal swabs weretaken daily during proestrus and es&us. Selected serum samples collected during estrus wereassayed for endogenous LH by radioimmunoassay @IA). Plasma samples collected duringproestrus 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. Vaginalcytology smears were stained with Wrights stain and evaluated for the percentage ofsuperficial squamous cells. Day 1 of die&us (Day 1) was defined as a drop of 20% or morein 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 Orig/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 superficialand 6.8 f 1.4 d (n = 16) before Day 1. Ovulation took place 2.1 + 3.9 d (n = 11) afterthe 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 progesteroneassay.

Based on the results of the present study, ELISA progesterone assay and determinationof 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 byvaginal cytology are reliable methods to retrospectively estimate ovulation time.Key words: bitch, ovulation, teasing, cytology, progesterone

AcknowledgementsThis investigation was supported by the Veterinary Medicine Committee on ResearchGrant 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 thestatistics.

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INTRODUCTIONThe 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 andthe cost-efticiency of semen preservation techniques (freezing and cooling). It would alsofind application in breeding and infertility management and diagnostic, research and embryotransfer.

Various methods are commonly used to monitor the bitchs estrous cycle. Teasing andsecondary signs of estrus are widely used by breeders to determine mating time (1). Thecredibility of this technique to determine ovulation is jeopardized by subjectivity ofinterpretation and variability among dogs (2). Vaginal cytology is commonly used clinicallyto 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 serumprogesterone concentration of 5.44 + 0.93 nglml (mean + SEM) as measured by RIA wasfound to correspond to ovulation (4). In this study, ovulation was determined by microscopicexamination 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 LHsurge by RIA is the preferred method for estimating ovulation because it is reliable and notinvasive. Since the assay can only be performed by specialized laboratories, this technique islimited 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 ofexaminations possible during estrus limit the use of this technique (5). Vaginoscopy canreadily determine the enlargement of the vaginal mucosal folds occurring during proestrusand estrus (6). The fertile period can be estimated by this technique, but no fertility trial hasbeen 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 todetermine progesterone concentration in bovine serum, plasma, or milk. This technique,unlike radioimmunoassay, is rapid and can be performed by an unspecialized laboratory. AnELISA kit (Ovucheck TM Cambridge Veterinary Science, Cambridge, England) has beenvalidated for measuring progesterone concentrations in dog plasma (9). In the past, twodifferent standard curves (linear and beat fitted curves) have been used to determine theprogesterone concentration from the spectrophotometric absorbance value. The relativeeffectiveness of the two curves has never been compared. Qualitative ELISA forprogesterone kits have also been introduced to the dog market and are of great value to thepractitioner. However, there is no data available in the literature regarding the efficiency ofthese products to predict ovulation.

The objectives of this project were 1) to determine the best standard curve to evaluateprogesterone concentration from spectrophotometric absorption values and 2) to evaluate theadequacy of teasing, vaginal cytology, and quantitative ELISA progesterone assays for thedetermination of the time of ovulation in dogs.

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MATERIALS AND METHODSSixteen 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 hof light daily and 22C ambient temperature the year around. The experiment was conductedfrom September 1988 to July 1989.

The reproductive cycle of each bitch was followed twice weekly by vaginal cytologyand teasing by a male with good libido to determine the onset of proestrus. Bitches inproestrus and estrus were teased daily. Daily serum, plasma and vaginal cytology sampleswere obtained until the end of estrus. Standing for the male, displaying the vulva, anddeviating the tail to the side were regarded as physical signs of estrus. A total of 15 ml ofblood was withdrawn daily between 1400 and 1600 h by jugular venipuncture into evacuatedtubes containing either no anti-coagulant (serum) or EDTA (plasma). Serum and plasmasamples were obtained by centrifugation and stored frozen until assayed. A glass speculumwas introduced into the anterior vagina and cotton tipped swabs (six inches long) were usedto collect vaginal cytology samples. Swabs were gently rolled onto precleaned glass slides toobtain smears that were stained with Wrights stain. The bitch was considered in estrus whenmore than 90% of the cells were superficial keratinized epithelial cells. We also measuredthe interval from the first day when more than 80% of the epithelial cells were superficialkeratinized and ovulation. Day one of diestrus (Day 1) was defined as a drop of 20% ormore of the total number of superficial cells (10).

Serum samples collected daily between Days -6 and -11 from Day 1 (based on vaginalcytology) were assayed by RIA for endogenous LH. Luteinizing hormone in serum sampleswas 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% and12 to lS%, respectively.

Plasma samples collected daily during proestrus and estrus were assayed forprogesterone in duplicate with a commercial ELISA kit. Progesterone standards used were0.5, 1, 5 and 10 ng/ml. Spectrophotometric absorbance readings were transformed intoprogesterone concentrations by the use of the best fitted curve and the linear curve. The bestfitted curve is constructed of different third-degree polynomials arranged together to form asingle curve. A third-degree polynomial is calculated between two adjacent points, A totalof N-l (N = number of standard points) polynomials are computed and assembled togetherto form a smooth single curve. This curve fitting technique is known as splineinterpolation (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. Progesteroneconcentrations 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 than10 nglml (best-fitted curve) or estimated by extrapolation (linear curve). The day whenovulation took place was estimated to be 48 h after the preovulatory LH peak (13). None ofthe samples were below 0.5 nglml or above 10 nglml at the time of ovulation. Meanprogesterone concentrations and standard deviation was computed at the day ovulation tookplace. The onset and the last day of es&-us as determined by vaginal cytology or teasing werealso calculated relative to the day ovulation took place.

Progesterone concentrations measured by the two different ELISA standard curves orwith 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 forprogesterone by RIA. Serum samples were assayed for progesterone using a commercialsolid-phase progesterone RIA (Coat-A-Count ProgesteroneTM,Diagnostic Products Corp., LosAngeles, CA, USA). All the samples were assayed in duplicate. The progesterone standardsused were 0. 0.1, 0.5, 2, 10, 20 and 40 nglml. The within-assay and between-assaycoefticients 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 sampleswith ELISA-measured progesterone concentrations between 0.5 and 10 nglml were used forcomparison. The Student-t test and regression analysis were applied to determine the beststandard curve.RESULTS

The ELISA iinear standard curve, the ELLSA best fitted standard curve, and the IUAassay 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 bestfitted standard curves (rz = 0.998, n = 64) was highly significant (P < 0.00001). Thelinear correlation coefficient was also highly significant (P < 0.0001) between the ELISAlinear standard curve and RIA assay (9 = 0.896, n = 64) and between the best fitted curvestandard curve and RIA assay (r = 0.893, n = 64).Ovulation was estimated to occur when progesterone concentration was 4.9 f 1 Onglml (mean f SD, n = 15) with a range of 3.4 to 6.6 nglml. The sample from Bitch 149at 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 computedwith the linear ELISA standard curve. Progesterone concentrations obtained by the threemethods of measurement (RIA assay, linear and best fitted ELISA standard curves) arepresented in Table 1. Figure I illustrates serial means for progesterone and LHconcentrations and the estimated time of ovulation.Based on vaginal cytology, ovulation took place 6.9 + 1.6 d (n = 15) after 80% of thevaginal 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 serialmeans for superficial keratinized cell percentages and LH concentrations and the estimatedtime 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 ofstanding estrus and 8.8 f. 1.5 d (n = 13) before the last day of receptivity. One dog did notstand for the male but vaginal cytology indicated estrus. Therefore data collected from thisdog were not used. Also, data regarding the first and last day of receptivity were notavailable for all dogs.

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15

7 6 5 4 3 Z l 0 1 2 3 4 5 6 7 8

Figure 1

12 -

10 -

_ B-x>\5

6-IJ 4-0

ct3 .

Time days)Relatioship between LH surge and progesteroneconcentrations as measured by ELISA.

100.

80 Eg

60 -dxci40 ET>

20

0 ; I I I I I I I I 1 I I10 8 -6 -4 -2 0 2 4 6 8 10 12

Time days)Figure 2. Relationship between LH surge and percentage of

superficial keratinized cells on vaginal cytology.

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Table 1. Progesterone concentrations at ovulation and the luteinizing hormone peak

Ovulation

Linear Best fittedStandard Curve Standard Curve

4.9*1.0 4.7*1.1

RIAassay

3.3f0.8LH peak 2.1 f0.7 2.1kO.7 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 thecorrelation coefftcient was excellent between the two curves. Therefore, a close relationshipexists between the two standard curves, but one should indicate which curve was used tocompute the progesterone concentration. The two ELISA standard curves were also differentfrom RIA, but the correlation coefficient was also very good (P < 0.0001). Independentlyof the ELISA standard curve, there was still a close relationship between ELISA and RIAassays. Other authors have reported a high correlation between ELISA and RIA progesteroneassays (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 ofovulation in bitches. Determination of the LH peak to predict ovulation also has theadvantage 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 theLH 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 reportedby others using RIA (4,13). However, the increase of the progesterone concentration afterthe LH peak was more pronounced when measured by ELISA than when measured by RIAprogesterone assay, although the standard deviations were similar (Table 1). This indicatesthat the ELISA progesterone assay is more likely to differentiate the LH peak from ovulationtime.Recently, the use of RIA for progesterone to improve fertility in bitches has beeninvestigated (15-17). Two investigations evaluated the conception rate of large number ofbitches when insemination was timed based on a rapid RIA progesterone assay. Okkens etal. (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 etal. (1989) reported that 81 of 104 (78%) subfertile bitches and 105 of 112 (94%) fertilebitches 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 theprogesterone concentration exceeded 12 ng/ml, within 9 to 33 h, when it was between 6 and12 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 failurefollowing more than half of the breeding attempts or following a single breeding attemptwere considered subfertile. The results of these two studies emphasize the usefulness ofprogesterone 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 theestrous cycle and the breeding management of bitches. However, fertility trials are indicatedto confirm this conclusion.

As reported previously, time of ovulation can be estimated retrospectively with vaginalcytology (2). Our observations indicate that the time of ovulation can also be determinedfrom 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 firstday of estrus as the first day on which 80% or more of the vaginal epithelial cells arekeratinized Therefore, we suggest that vaginal cytology is a good alternative to progesteroneassay. This technique is rapid and easy to perform and provides a fairly accurate estimationof the time of ovulation both prospectively and retrospectively.

Okkens et al. (1985) reported that only 30% of 20 bitches became pregnant whenvaginal cytology was used as the sole method for detecting ovulation. Details regarding thebreeding schedule were omitted in that study. Another study did not support the use ofvaginal cytology based on the variability of the estrous cycle of two bitches (17). Our resultsdo not agree with these previous investigators, probably because we have a differentperspective on the use of vaginal cytology. Fertility needs to be evaluated when the firstdays 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 manyclinical situations, receptivity is a satisfactory method for scheduling breeding because of thelong 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 matureoocytes (1 to 2 d; 17,20). However, more critical situations such as insemination with frozenor chilled semen or management of infertility require a more precise determination of thefertile period than teasing can offer. We therefore do not advise using teasing as the solemethod for determining the time of ovulation.

REFERENCES1. Evans, J.M. and White, K. Breeding from your bitch. In: The Book of the Bitch.

Henston Ltd., Guildford, England, 1988, pp 59-124.2. Holst, P.A. and Phemister, R.D. Onset of diestrus in the Beagle bitch: Definition and

significance. Am. J. Vet. Res. &iO1406 (1974).3. Holst, P.A. and Phemister, R.D. Temporal sequence of events in the estrous cycle ofthe 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 sexualbehavior 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 ofreproductive 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.), CurrentVeterinary Therapy. W. B. Saunders Company, Philadelphia, PA, 1989, pp.l269-1282.Inaba, T., Matsui, N., Shimizu, R. and Imori, T. Use of echography in bitches fordetection 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 anduterus 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 ELISAkit 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. Vaginalcytology. 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 infusionof GnRH agonist. J. Reprod. Fertil.~(Suppl.):149-160 (1989).Sedgewick, R. Curve fitting. In: Algorithms. Addison-Wesley Publishing Company,Inc., New York, NY, 1983, pp. 67-77.Phemister, R.D., Holst, J.S., Spano J.S. and Hopwood, M.L. Time of ovulation inthe Beagle bitch. Biol. Reprod. &74-82 (1973).

England, G.C.W., Allen, W.E. and Porter, D. J. A comparison of radioimmunoassaywith quantitative and qualitative enzyme-linked immunoassay for plasma progestogendetection in bitches. Vet. Rec. 125: 107-108 (1989).van Haaften, B., Dieleman, S.J., Okkens, A. C. and Willemse, A. H. Timing themating of dogs on the basis of blood progesterone concentration. Vet. Rec. m:524-526 (1989).Okkens, A. S., Dieleman, S.J. and Vogel, F.. Determination of the ovulation periodin the dog a comparison of the rapid progesterone assay, vaginoscopy and vaginalcytology. In: Voorjaarsdagen 1985 Proceedings of the Netherlands Sm. Anim. Vet.Ass. (Amsterdam, Netherlands) pp. 26-27 (1985).

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17. Jeffcoate, LA. and Lindsay, F.E.F. Ovulation detection and timing of inseminationbased on hormone concentrations, vaginal cytology and the endoscopic appearance ofthe vagina in domestic bitches. J. Reprod. Fertil. s(Suppl.):277-287 (1989).

18. 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. 44:1819-1821 (1983).

19. Doak, R.L., Hall, A. and Dale, H.E. Longetivity of spermatozoa in the reproductivetract of the bitch. J. Reprod. Fertil. 1351-58 (1967).

20. Olson, P.N. and Husted, P. W. Breeding management for optimal reproductiveeffkiency in the bitch and the stud dog. &: Morrow, D.A. (ed.), Current Therapy inTheriogenology. W. B. Saunders Company, Philadelphia, PA, 1986, ~~463-468.

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determination of ovulation time in bitches based on teasing, vaginal cytology, and elisa for...

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