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Accuracy of canine parturition date prediction from the initial rise in preovulatory progesterone concentration
Theriogenology 60 (2003) 1187–1196
Accuracy of canine parturition date prediction from the initial rise in preovulatory progesterone concentration Michelle A. Kutzlera,*, Hussni O. Mohammedb, Stephen V. Lambb, Vicki N. Meyers-Wallena,c a
Department of Biomedical Sciences, Cornell University, Ithaca, NY, 14853 USA Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY, 14853 USA c J.A. Baker Institute for Animal Health, Cornell University, Ithaca, NY, 14853 USA
b
Received 6 May 2002; accepted 3 February 2003
Abstract Accurate prediction of parturition date is useful for clinical management of canine parturition. For nearly all normal canine pregnancies, parturition occurs 64–66 days from the LH peak, the timing of which cannot be differentiated from the initial sharp rise in serum progesterone (P4) concentrations. We sought to determine by retrospective analysis if prebreeding serum progesterone concentrations could accurately predict parturition date. Serum progesterone concentrations recorded as serial samples from 63 bitches (19 breeds) were analyzed. Progesterone concentrations were measured by radioimmunoassay (RIA) or chemiluminescent immunoassay (CLIA). The CLIA method was validated for use in determining P4 concentrations in canine serum and results were comparable to those obtained with RIA. Bitches were grouped by nonpregnant body weight (BW) and litter size (LS). Day 0 (D0), the day of preovulatory rise in serum P4, was defined as the day that P4 concentration rose to l.5 ng/ml and was at least twice the baseline concentration. The predicted parturition date, 65 days following the day of preovulatory rise in serum P4 (D65), was compared to actual parturition date, the day the first pup was delivered. We determined that mean P4 concentration at D0 for all BW groups was 2:02 0:18 ng/ml and there was significant variation in P4 concentrations between BW groups after D1. In addition, we determined that the accuracy of parturition date prediction within a 1, 2, and 3 day interval using prebreeding serum progesterone concentrations was 67, 90, and 100%, respectively, and that the accuracy was not affected by body weight or litter size. # 2003 Elsevier Science Inc. All rights reserved. Keywords: Dog; LH surge; Pregnancy; Progesterone
* Corresponding author. Present address: Department of Clinical Sciences, College of Veterinary MedicineOregon State University, Corvallis, OR 97331, USA. Tel.: þ1-541-737-6952; fax: þ1-541-737-8651. E-mail address: [email protected] (M.A. Kutzler).
0093-691X/$ – see front matter # 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0093-691X(03)00109-2
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1. Introduction Accurate prediction of canine parturition date is useful for managing parturition and planning cesarean sections. Prediction of parturition date from breeding dates is not accurate. Actual gestation length in the bitch is 65 1 days when timed from the preovulatory LH surge in peripheral blood [1] However, apparent gestation length can vary by as much as 14 days (57–72 days) when timed from the first of multiple matings [1] or by as much as 11 days (58–69 days) when timed from a single mating [2]. The disparity between apparent and actual gestation length in the dog is due to the longevity of behavioral estrus (3–21 days) [3] and of sperm viability in the female reproductive tract (as much as 6 days) [1]. The day of the LH surge is a reliable physiologic event in canine reproduction, from which ovulation, oocyte maturation, implantation, fetal development, and parturition date can be determined. As stated earlier, gestation length determined from the day of the LH surge shows very little variation [4]. In the same study, Concannon et al. also illustrated that the initial rises in LH and progesterone could not be dissociated [4], and in a later study this finding was supported [5]. Thus, the preovulatory rise in progesterone levels as well as LH have been shown to be reliable physiologic events in normal dogs. In clinical practice, various methods have been used to monitor serum progesterone (P4) concentrations to determine the optimal time for insemination, since this is more convenient and economical than monitoring serum LH. Previous studies of canine reproductive physiology indicate that P4 concentrations can vary widely after the LH surge. Reports using quantitative results from P4 radioimmunoassay (RIA) to estimate ovulation (2 days after LH surge; P4 ¼ 5–6 ng/ml) or the time of insemination (4–7 days after LH surge; P4 ¼ 6–12 ng/ml), have predicted gestation lengths ranging from 58 to 70 days [6–11]. However, little variation was reported in peripheral P4 concentrations (0:8 0:1, 1:6 0:2, and 2:6 0:2) near the time of the preovulatory LH surge (day before, day of, and day following the LH surge, respectively) [4]. Thus, we hypothesized that parturition date could be accurately predicted from the initial rise in progesterone concentration that occurs at the time of the preovulatory LH surge [4]. We also hypothesized that chemiluminescent immunoassay (CLIA), a nonisotopic quantitative method for determining hormone concentrations that is less labor intensive, technically difficult, and dangerous for personnel compared to RIA, would accurately measure progesterone levels. The objectives of this study were to determine: (a) the accuracy and precision of CLIA compared to RIA for progesterone quantification, (b) the accuracy of parturition date prediction when timed from the initial rise in peripheral P4 concentrations indicative of Day 0 (D0), and (c) whether maternal body weight or litter size had an effect on the prediction accuracy.
2. Materials and methods From 1992 to 2001, serial blood samples were collected from clinic patients (Small Animal Fertility and Infertility Service, Cornell University Hospital for Animals, College of Veterinary Medicine, Ithaca, NY) or research bitches (Department of Clinical Sciences,
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College of Veterinary Medicine and J.A. Baker Institute for Animal Health, Cornell University, Ithaca, NY) for breeding management, with the first sample collected within 1–2 days of the onset of proestrus clinical signs (i.e. vulvar swelling, serosanguineous discharge). Blood samples were collected daily or every other day during proestrus and estrus as defined by vaginal cytology (proestrus: 20%, but <90% cornified epithelial cells, estrus: 90% cornified epithelial cells). Serum was separated from blood collected in glass tubes without separating (SST) gel and P4 concentrations were measured by RIA (Coat-A-Count1, Diagnostic Product Corporation, Los Angeles, CA) or CLIA (Immulite1, Diagnostic Product Corporation, Los Angeles, CA) using commercially available reagents. The detection limit for the RIA and CLIA, defined as the concentration that is 2 S.D. below the response at zero dose, was 0.02 and 0.2 ng/ml, respectively. The specificity, accuracy, and precision of the CLIA were prospectively evaluated. Immunologic specificity was verified by measuring serum P4 in samples serially diluted in 7% bovine serum albumin/phosphate-buffered saline (Sigma-Aldrich, St Louis, MO). Biological specificity was verified by measuring P4 in daily serum samples taken from dogs at the onset of proestrus until the first mating. The accuracy of the CLIA was evaluated using two methods. First, P4 results obtained with CLIA were compared with results from the previously validated RIA used in this study [12]. Second, known amounts of progesterone (Sigma-Aldrich, St Louis, MO), determined gravimetrically and by assay, were added to two canine serum samples and one combined serum and plasma pool with low endogenous P4 concentrations. Interassay precision was evaluated by determining the percent coefficient of variation (%CV) for pooled canine serum and plasma samples tested in separate assays over a 30-day period. Intra-assay precision was evaluated by testing eight canine samples 10 times consecutively. Dogs selected to participate in the retrospective study were those for which (1) a minimum of three serum samples were assayed per estrous cycle, with at least one sample containing a P4 concentration of <1.0 ng/ml and one subsequent sample of >2.5 ng/ml, and (2) delivery date of the first pup and litter size at birth were known from the medical record or direct communication with the owner. Pregnancies in which medical treatment was given prior to delivery of the first pup or an elective cesarean section was performed were excluded from consideration for this study. In addition, bitches that had preovulatory P4 concentrations between 1 and 2 ng/ml for 3 days were excluded. Bitches were divided into four body weight (BW) groups based on nonpregnant body weight: small (9 kg), medium (>9–20 kg), large (>20–40 kg), and giant (>40 kg). Mean litter size and S.D. were calculated for each group. A normal litter size for each BW group was defined as being within 1 S.D. of the mean. Small and large litter sizes were defined as those that were less or greater than 1 S.D. from the mean of their BW group, respectively. The day of the initial rise in P4, designated as D0, was defined as the first day that serum P4 concentration was 1.5 ng/ml, which has previously been determined as the day coincident with the LH surge [10]. For bitches sampled every other day, D0 was defined as the first day that serum P4 concentration was 1.5 ng/ml and was followed by a P4 concentration >3.0 ng/ml on the next day sampled. Gestation length was calculated as days following D0 and parturition was predicted to occur on D65. Actual parturition date was recorded as the day that the first pup was delivered. The differences between the predicted and actual parturition dates 1 and 2 days were evaluated using Chi-square
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tests. Gestation lengths between BW groups and between LS groups were compared by one-way analysis of variance (ANOVA). The difference in the rate of change of the serum P4 rise between BW groups was compared using linear regression. All analyses were made using statistical software (Statistix1, Analytical Software, St. Paul, MN). The level of significance for all statistical tests was considered at P < 0:05.
3. Results 3.1. CLIA validation Serial dilutions of canine serum samples inhibited binding of the enzyme-linked P4 in a manner that was parallel with the standard curve, demonstrating immunologic sensitivity of the assay (Table 1). Biologic specificity was verified by results reflecting normal established progesterone concentrations for dogs. Accuracy was verified by comparing measurements of samples using both procedures. The CLIA results were highly correlated (r 2 ¼ 0:975) with the previously validated RIA. The regression equation for canine Table 1 Concentrations of progesterone (P4; ng/ml) in serial dilutions of canine serum samples determined by the chemiluminescent immunoassay Sample ID
Dilution
Actual P4
Corrected P4
488-01
1:1 1:2 1:4 1:8 1:16
17.00 17.00 9.84 19.68 5.00 20.00 2.36 18.88 0.94 15.10 Mean actual/expected ¼ 108%
179-00
1:1 1:2 1:4 1:8 1:16
17.40 17.40 10.30 20.60 5.52 22.08 2.69 21.52 1.44 23.04 Mean actual/expected ¼ 125%
134-00
1:1 1:2 1:4 1:8 1:16
17.80 17.80 9.22 18.44 4.52 18.08 2.53 20.24 1.13 18.08 Mean actual/expected ¼ 105%
059-00
1:1 1:2 1:4 1:8 1:16
20.20 20.20 10.80 21.60 4.79 19.16 2.94 23.52 1.27 20.32 Mean actual/expected ¼ 105%
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Table 2 Concentrations of progesterone (P4; ng/ml) recovered from three canine samples to which four quantities of P4 were added, as measured by chemiluminescent immunoassay Sample ID
[E]
Concentration of P4 added
Mean %R
þ0.79 (ng/ml)
þ2.28 (ng/ml)
þ3.78 (ng/ml)
þ8.00 (ng/ml)
605-01 490-01 Canine pool
0.00 0.39 1.14
1.15 1.64 1.70
1.92 2.55 2.83
3.08 4.92 3.94
8.59 8.15 8.60
105 113 86
Mean %R
0.51
1.50 115
2.43 87
3.98 93
8.45 99
101 99
Percent of P4 recovered (%R) was determined by dividing the sample with the added P4 of a known quantity and the endogenous concentration ([E]).
samples (n ¼ 72) ranging from 0.05 to 22.30 ng/ml was: y ¼ 0:951x 0:16 [where y ¼ CLIA (ng/ml) and x ¼ RIA (ng/ml)]. Recovered percentages of known amounts of P4 added to canine samples (Table 2) also indicate the CLIA is sufficiently accurate. The interassay %CV of the two dog pools having mean P4 concentrations of 0.96 and 9.52 ng/ ml was 11.9 and 6.7%, respectively, which was comparable to the manufacturer’s human matrix controls that were assayed in parallel (Table 3). The intra-assay precision in the range of interest (1.0–2.0 ng/ml) was 11% (Table 4). Table 3 Mean concentrations of progesterone (P4; ng/ml) and interassay percent coefficient of variation (%CV) for dog and human samples (n) each tested separately over a 30-day period using chemiluminescent immunoassay Sample ID
Species
P4
%CV
n
IMM1-2 DPC-CON418 DPC-CON518 IMM1-3
Canine Human Human Canine
0.96 1.60 2.95 9.52
11.9 12.8 9.3 6.7
22 22 22 22
Table 4 Concentrations of progesterone (P4; ng/ml) and intra-assay percent coefficient of variation (%CV) for canine samples (n) measured by chemiluminescent immunoassay Sample ID
P4
%CV
n
065-00.2 736-01.4.5 699-01.2 761-01.4.3 345.5-761.7-01 820-01.2 699-01.3 134-00.2
0.48 1.55 1.56 1.96 2.99 5.09 10.81 15.97
20.6 8.9 16.8 7.2 9.9 3.9 5.8 5.1
10 10 10 10 10 10 10 10
Mean
9.8
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Table 5 Bitches studied to predict parturition using the initial rise in preovulatory progesterone (P4) concentrations BW group
Breed
NC
NB
Small
Jack Russell Terrier Scottish Terrier Tibetan Spaniel Tibetan Terrier Wirehaired Fox Terrier
2 1 4 2 1
Medium
Beagle Boxer Brittany Spaniel Shetland Sheepdog Standard Poodle
Large
LS range
Mean LS (S.D.)
2 1 3 1 1
1–7
4 (2)
1 1 1 1 3
1 1 1 1 2
1–12
6 (3)
Bulldog Doberman German Shepherd Dogs German Shorthaired Pointer Golden Retriever Greyhound Labrador Retriever Labrador Retriever/Greyhound cross
1 1 23 2 2 6 12 4
1 1 17 1 1 4 10 4
1–13
7 (3)
Giant
Great Dane Newfoundland
2 12
2 8
2–14
8 (4)
Total
19 breeds þ 1 cross breed
82
63
1–14
NC: number of estrous cycles, NB: number of bitches, LS: litter size. Bitches were divided into four groups based on nonpregnant body weight (BW): small (9 kg), medium (>9–20 kg), large (>20–40 kg), and giant (>40 kg). Litter size was recorded at birth.
Table 6 Parturition date was predicted to occur 65 days after the initial rise in preovulatory progesterone (P4) concentrations All bitches
Parturition date prediction interval 65 1 days 65 2 days 65 3 days
% Delivering in that interval
67
90
100
BW groups
Small
Medium
Large
Actual gestation length, mean (S.D.) in days LS groups Actual gestation length, mean (S.D.) in days
Giant
65.8 (1.23) 65.7 (1.67) 64.0 (1.02) 64.9 (1.79) Small
Average
Large
65.1 (1.76) 65.1 (1.64) 64.4 (1.5)
Actual gestation length was defined as the interval from the initial rise in P4 to birth of the first pup. There was no significant difference between the mean actual gestation length for bitches grouped by nonpregnant body weight (BW) or litter size (LS).
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Fig. 1. The initial rise in preovulatory progesterone (P4; D0) was defined as the first day that P4 1:5 ng/ml and at least twice the baseline concentration. The slope of the rise in mean P4 after D0 was significantly different between small and large BW groups when compared to medium and giant BW groups (P < 0:05), being 2.62, 1.07, 2.30, and 1.13 for small to giant BW groups, respectively.
3.2. Parturition date prediction Sixty-three bitches from 19 breeds and one group of cross bred bitches differing in body weight and litter size were evaluated (Table 5). Mean P4 concentrations for small to giant BW groups is plotted in Fig. 1. Equal variance was found in the slopes of the P4 curves for BW groups, allowing statistical comparison. The slopes differed significantly between small and large BW groups when compared to medium and giant BW groups. The means of actual gestation length and the accuracy of parturition date prediction were not significantly different within BW or LS groups (Table 6). From the initial rise in preovulatory P4 concentrations, the overall accuracy for predicting parturition within 3 days (62–68 days) was 100% for all bitches studied.
4. Discussion Progesterone CLIA is available through human clinical and veterinary diagnostic laboratories and is comparable in cost to the RIA. In addition, CLIA has the advantage of continuous processing allowing for rapid turnaround time without the disadvantages associated with isotopic assays (e.g. radioactivity, sample batching). Compared to RIA, the intra-assay precision for CLIA in the range of interest for determining the initial rise in P4 (1–2 ng/ml) is lower and the detection limit is higher. However,
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we did not find these differences to be clinically relevant to applications in canine reproduction. Estimation of gestational age in bitches is feasible because the rate of canine embryonic development is synchronous and closely correlated to the time of the preovulatory LH surge [13]. Therefore, gestational age, and hence parturition date, can be determined accurately when timed from the preovulatory LH surge. A previous study of Beagles reported a mean gestation length of 65.1 (0.1) days when defined as the interval from the preovulatory LH surge to parturition [4]. For all bitches in the present study, using preovulatory serum progesterone concentrations to define D0, the accuracy of parturition date prediction within the interval of 65 1, 2, and 3 days was 67, 90, and 100%, respectively. Furthermore, accurate predictions were obtained independent of maternal body weight or litter size. These results are supported by Linde-Forsberg and Forsberg [8], who reported that canine gestation length is not influenced by maternal body weight or litter size. It is likely that results of the present study differ from some earlier reports [10,11] because gestation in the present study was timed from a reliable physiologic event (i.e. the initial rise in progesterone), which occurs simultaneous with the LH surge [4,5]. The differences between studies in methods of timing gestation have led to different conclusions regarding variation in gestation length and the ability to accurately predict parturition date. Previous reports suggested that maternal body weight and litter size had an effect on gestation length when (1) gestation length was defined as the interval from mating to parturition, and (2) the day of mating was determined by plasma P4 concentrations between 6 and 12 ng/ml on the day before mating and >12 ng/ml on the day of mating [10]. We estimate that the day of mating in those studies was likely to be D2 from the initial preovulatory rise in P4 (Fig. 1, Table 7). Interbreed variation in gestation length was reported to be 3–6 days, with Alsatians having a shorter gestation length compared to other breeds studied [8]. However, estimation of gestation length in that manner is likely to be inaccurate due to the inherent variability in P4 concentrations after D1 (Fig. 1). Table 7 Comparison of mean (S.D.) progesterone (P4) concentrations (ng/ml) from previous studies (reported as prior to the LH surge, on the day of LH surge, and on the day of ovulation) and from the present study (where Day 0 is defined by the initial rise in P4) Prior to LH surge
LH surge
Wright [14] <1.0 Badinand et al. [6] Present study
3 days
Small Medium Large Giant Mean (S.D.), all groups
Ovulation
2–4 (range) 5.5 >1.0 >5 2 days
1 day
0.92 0.85 0.53 0.57
0.78 0.84 0.68 0.61
1.46 1.26 1.01 0.96
2.14 1.79 2.18 1.97
0.72 (0.2)
0.73 (0.1)
1.17 (0.23)
2.02 (0.18)
Day 0 (0.05) (0.20) (0.09) (0.08)
1 day
2 days
3 days
3.46 3.10 3.38 3.99
11.86 4.10 5.18 4.62
15.13 5.00 8.27 6.98
3.48 (0.37) 6.44 (3.64) 8.84 (4.4)
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There have been many reports describing P4 concentrations at various times near the onset of the LH surge (Table 7). Wright reported P4 concentrations during anestrus and proestrus, at the time of the LH surge, and at the time of ovulation [14]. Badinand et al. [6] reported P4 concentrations at the time of the LH surge, 0–24 h after the LH peak, and on the first day of diestrus. Best agreement between P4 values in these reports and the present study is in the values prior to the LH surge. Mean (S.D.) P4 concentration at day of the initial rise in preovulatory P4 concentrations (D0) for all BW groups in the present study was 2.02 (0.18) ng/ml. The rate of the rise in P4 concentration between D4 and D0 did not vary significantly, but did differ significantly between bitches of varying body weights following D1 (Fig. 1, Table 7). Previously reported P4 concentrations on the day of ovulation (2 days after LH surge) do not reflect the significant variation documented in the present study, and will lead to inaccurate predictions based on such estimation. Prediction of parturition date within the interval of 65 3 days, based on the initial rise in P4, is more accurate than any previous report, having a predictive accuracy of 100% irrespective of body weight or litter size. To make an accurate prediction using the P4 method, it is important to: (1) obtain serum samples at least every other day, beginning in proestrus, until the initial rise in P4 (D0) is identified, (2) use a quantitative P4 assay that is validated for dogs, (3) if using CLIA, avoid using collection tubes with serum separating gel, and (4) time gestation from the initial rise in P4, rather than from P4 concentrations occurring after the LH surge. The rationale for sampling at the first signs of proestrus is based upon previous studies indicating that there is little correlation between the onset of estrus and LH surge [2,5,14–16]. For example, Concannon et al. [5] reported that the LH surge occurred between the fifth day of proestrus and the second day of estrus. Our clinical experiences support those observations as we could not predict P4 concentration based upon vaginal cytology. Using the initial rise in preovulatory P4 concentrations to predict parturition date is particularly suitable for clinical practice because it can be applied to all pregnancies, regardless of maternal body weight or litter size, and requires no correction factors or additional technical skill. A method of comparable accuracy, transabdominal ultrasonography to estimate gestational age from fetal measurements [17], requires investment in ultrasound equipment, some technical skill in ultrasonography, and use of correction factors for small and giant breeds. However, there is a strong correlation between these two methods (McNemar w2 ¼ 1:00) and both are more accurate in predicting parturition date than previously reported methods. Using this method to time gestation accurately, further studies can be performed to determine the earliest gestational age at which a fetus can be delivered and remain viable without medical intervention.
Acknowledgements We thank S. Long, A.J. Williams, A. Lanfair, and T. Powell for their technical support and assistance; C. Wilker, J. Ellington, C. Schweizer, C. Gradil, and R. Wheeler for professional services rendered in association with clinical patients reported herein; and T. Reimers for technical expertise in assay validation.
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References [1] Concannon PW, Whaley S, Lein D, et al. Canine gestation length: variation related to time of mating and fertile life of sperm. Am J Vet Res 1983;44(10):1819–21. [2] Holst PA, Phemister RD. Onset of diestrus in the Beagle bitch: definition and significance. Am J Vet Res 1974;35:401–6. [3] Concannon PW, McCann JP, Temple M. Biology and endocrinology of ovulation, pregnancy and parturition in the dog. J Reprod Fertil Suppl 1989;39:3–25. [4] Concannon PW, Hansel W, Visek WJ. The ovarian cycle of the bitch: plasma estrogen, LH and progesterone. Biol Reprod 1975;13(1):112–21. [5] Concannon P, Hansel W, McEntee K. Changes in LH, progesterone and sexual behavior associated with preovulatory luteinization in the bitch. Biol Reprod 1977;17:604–13. [6] Badinand F, Fontbonne A, Maurel MC, et al. Fertilization time in the bitch in relation to plasma concentration of estradiol, progesterone and luteinizing hormone and vaginal smears. J Reprod Fertil Suppl 1993;47:63–7. [7] Jeffcoate IA, Lindsay FEF. 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 Suppl 1989;39:277–87. [8] Linde-Forsberg C, Forsberg M. Results of 527 controlled artificial inseminations in dogs. J Reprod Fertil Suppl 1993;47:313–23. [9] Linde-Forsberg C, Stro¨ m Holst B, Govette G. Comparison of fertile data from vaginal vs. intrauterine insemination of frozen–thawed dog semen: a retrospective study. Theriogenology 1999;52(1):11–23. [10] Okkens AC, Hekerman TW, de Vogel JW, et al. Influence of litter size and breed on variation in length of gestation in the dog. Vet Q 1993;15(4):160–1. [11] Okkens AC, van Osch W, Teunissen JM, et al. The influence of breed on gestation. In: Proceedings from the Advances in Dog, Cat and Exotic Carnivore Reproduction, Oslo, Norway. 2000. p. 85. [12] Reimers TJ, Lamb SV, Bartlett SA, et al. Effects of hemolysis and storage on quantification of hormones in blood samples from dogs, cattle, and horses. Am J Vet Res 1991;52:1075–80. [13] Bysted BV, Dieleman SJ, Hyttel P, et al. Embryonic developmental stages in relation to the LH-peak in the bitch. In: Proceedings from the Advances in Dog, Cat and Exotic Carnivore Reproduction, Oslo, Norway. 2000. p. 75. [14] Wright PJ. Application of vaginal cytology and plasma progesterone determinations to the management of reproduction in the bitch. J Small Anim Pract 1990;31:335–40. [15] Phemister RD, Holst PA, Spano JS, et al. Time of ovulation in the Beagle bitch. Biol Reprod 1973;8: 74–82. [16] Tsutsui T. Studies on the physiology of reproduction in the dog. II. Observation on the time of ovulation. Jpn J Anim Reprod 1973;18:137–42. [17] Kutzler MA, Yeager AE, Mohammed HO, et al. Accuracy of canine parturition date using fetal measurements obtained by ultrasonography to estimate gestational age. Theriogenology [resubmitted].
Accuracy of canine parturition date prediction from the initial rise in preovulatory progesterone concentration Michelle A. Kutzlera,*, Hussni O. Mohammedb, Stephen V. Lambb, Vicki N. Meyers-Wallena,c a
Department of Biomedical Sciences, Cornell University, Ithaca, NY, 14853 USA Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY, 14853 USA c J.A. Baker Institute for Animal Health, Cornell University, Ithaca, NY, 14853 USA
b
Received 6 May 2002; accepted 3 February 2003
Abstract Accurate prediction of parturition date is useful for clinical management of canine parturition. For nearly all normal canine pregnancies, parturition occurs 64–66 days from the LH peak, the timing of which cannot be differentiated from the initial sharp rise in serum progesterone (P4) concentrations. We sought to determine by retrospective analysis if prebreeding serum progesterone concentrations could accurately predict parturition date. Serum progesterone concentrations recorded as serial samples from 63 bitches (19 breeds) were analyzed. Progesterone concentrations were measured by radioimmunoassay (RIA) or chemiluminescent immunoassay (CLIA). The CLIA method was validated for use in determining P4 concentrations in canine serum and results were comparable to those obtained with RIA. Bitches were grouped by nonpregnant body weight (BW) and litter size (LS). Day 0 (D0), the day of preovulatory rise in serum P4, was defined as the day that P4 concentration rose to l.5 ng/ml and was at least twice the baseline concentration. The predicted parturition date, 65 days following the day of preovulatory rise in serum P4 (D65), was compared to actual parturition date, the day the first pup was delivered. We determined that mean P4 concentration at D0 for all BW groups was 2:02 0:18 ng/ml and there was significant variation in P4 concentrations between BW groups after D1. In addition, we determined that the accuracy of parturition date prediction within a 1, 2, and 3 day interval using prebreeding serum progesterone concentrations was 67, 90, and 100%, respectively, and that the accuracy was not affected by body weight or litter size. # 2003 Elsevier Science Inc. All rights reserved. Keywords: Dog; LH surge; Pregnancy; Progesterone
* Corresponding author. Present address: Department of Clinical Sciences, College of Veterinary MedicineOregon State University, Corvallis, OR 97331, USA. Tel.: þ1-541-737-6952; fax: þ1-541-737-8651. E-mail address: [email protected] (M.A. Kutzler).
0093-691X/$ – see front matter # 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0093-691X(03)00109-2
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1. Introduction Accurate prediction of canine parturition date is useful for managing parturition and planning cesarean sections. Prediction of parturition date from breeding dates is not accurate. Actual gestation length in the bitch is 65 1 days when timed from the preovulatory LH surge in peripheral blood [1] However, apparent gestation length can vary by as much as 14 days (57–72 days) when timed from the first of multiple matings [1] or by as much as 11 days (58–69 days) when timed from a single mating [2]. The disparity between apparent and actual gestation length in the dog is due to the longevity of behavioral estrus (3–21 days) [3] and of sperm viability in the female reproductive tract (as much as 6 days) [1]. The day of the LH surge is a reliable physiologic event in canine reproduction, from which ovulation, oocyte maturation, implantation, fetal development, and parturition date can be determined. As stated earlier, gestation length determined from the day of the LH surge shows very little variation [4]. In the same study, Concannon et al. also illustrated that the initial rises in LH and progesterone could not be dissociated [4], and in a later study this finding was supported [5]. Thus, the preovulatory rise in progesterone levels as well as LH have been shown to be reliable physiologic events in normal dogs. In clinical practice, various methods have been used to monitor serum progesterone (P4) concentrations to determine the optimal time for insemination, since this is more convenient and economical than monitoring serum LH. Previous studies of canine reproductive physiology indicate that P4 concentrations can vary widely after the LH surge. Reports using quantitative results from P4 radioimmunoassay (RIA) to estimate ovulation (2 days after LH surge; P4 ¼ 5–6 ng/ml) or the time of insemination (4–7 days after LH surge; P4 ¼ 6–12 ng/ml), have predicted gestation lengths ranging from 58 to 70 days [6–11]. However, little variation was reported in peripheral P4 concentrations (0:8 0:1, 1:6 0:2, and 2:6 0:2) near the time of the preovulatory LH surge (day before, day of, and day following the LH surge, respectively) [4]. Thus, we hypothesized that parturition date could be accurately predicted from the initial rise in progesterone concentration that occurs at the time of the preovulatory LH surge [4]. We also hypothesized that chemiluminescent immunoassay (CLIA), a nonisotopic quantitative method for determining hormone concentrations that is less labor intensive, technically difficult, and dangerous for personnel compared to RIA, would accurately measure progesterone levels. The objectives of this study were to determine: (a) the accuracy and precision of CLIA compared to RIA for progesterone quantification, (b) the accuracy of parturition date prediction when timed from the initial rise in peripheral P4 concentrations indicative of Day 0 (D0), and (c) whether maternal body weight or litter size had an effect on the prediction accuracy.
2. Materials and methods From 1992 to 2001, serial blood samples were collected from clinic patients (Small Animal Fertility and Infertility Service, Cornell University Hospital for Animals, College of Veterinary Medicine, Ithaca, NY) or research bitches (Department of Clinical Sciences,
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College of Veterinary Medicine and J.A. Baker Institute for Animal Health, Cornell University, Ithaca, NY) for breeding management, with the first sample collected within 1–2 days of the onset of proestrus clinical signs (i.e. vulvar swelling, serosanguineous discharge). Blood samples were collected daily or every other day during proestrus and estrus as defined by vaginal cytology (proestrus: 20%, but <90% cornified epithelial cells, estrus: 90% cornified epithelial cells). Serum was separated from blood collected in glass tubes without separating (SST) gel and P4 concentrations were measured by RIA (Coat-A-Count1, Diagnostic Product Corporation, Los Angeles, CA) or CLIA (Immulite1, Diagnostic Product Corporation, Los Angeles, CA) using commercially available reagents. The detection limit for the RIA and CLIA, defined as the concentration that is 2 S.D. below the response at zero dose, was 0.02 and 0.2 ng/ml, respectively. The specificity, accuracy, and precision of the CLIA were prospectively evaluated. Immunologic specificity was verified by measuring serum P4 in samples serially diluted in 7% bovine serum albumin/phosphate-buffered saline (Sigma-Aldrich, St Louis, MO). Biological specificity was verified by measuring P4 in daily serum samples taken from dogs at the onset of proestrus until the first mating. The accuracy of the CLIA was evaluated using two methods. First, P4 results obtained with CLIA were compared with results from the previously validated RIA used in this study [12]. Second, known amounts of progesterone (Sigma-Aldrich, St Louis, MO), determined gravimetrically and by assay, were added to two canine serum samples and one combined serum and plasma pool with low endogenous P4 concentrations. Interassay precision was evaluated by determining the percent coefficient of variation (%CV) for pooled canine serum and plasma samples tested in separate assays over a 30-day period. Intra-assay precision was evaluated by testing eight canine samples 10 times consecutively. Dogs selected to participate in the retrospective study were those for which (1) a minimum of three serum samples were assayed per estrous cycle, with at least one sample containing a P4 concentration of <1.0 ng/ml and one subsequent sample of >2.5 ng/ml, and (2) delivery date of the first pup and litter size at birth were known from the medical record or direct communication with the owner. Pregnancies in which medical treatment was given prior to delivery of the first pup or an elective cesarean section was performed were excluded from consideration for this study. In addition, bitches that had preovulatory P4 concentrations between 1 and 2 ng/ml for 3 days were excluded. Bitches were divided into four body weight (BW) groups based on nonpregnant body weight: small (9 kg), medium (>9–20 kg), large (>20–40 kg), and giant (>40 kg). Mean litter size and S.D. were calculated for each group. A normal litter size for each BW group was defined as being within 1 S.D. of the mean. Small and large litter sizes were defined as those that were less or greater than 1 S.D. from the mean of their BW group, respectively. The day of the initial rise in P4, designated as D0, was defined as the first day that serum P4 concentration was 1.5 ng/ml, which has previously been determined as the day coincident with the LH surge [10]. For bitches sampled every other day, D0 was defined as the first day that serum P4 concentration was 1.5 ng/ml and was followed by a P4 concentration >3.0 ng/ml on the next day sampled. Gestation length was calculated as days following D0 and parturition was predicted to occur on D65. Actual parturition date was recorded as the day that the first pup was delivered. The differences between the predicted and actual parturition dates 1 and 2 days were evaluated using Chi-square
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tests. Gestation lengths between BW groups and between LS groups were compared by one-way analysis of variance (ANOVA). The difference in the rate of change of the serum P4 rise between BW groups was compared using linear regression. All analyses were made using statistical software (Statistix1, Analytical Software, St. Paul, MN). The level of significance for all statistical tests was considered at P < 0:05.
3. Results 3.1. CLIA validation Serial dilutions of canine serum samples inhibited binding of the enzyme-linked P4 in a manner that was parallel with the standard curve, demonstrating immunologic sensitivity of the assay (Table 1). Biologic specificity was verified by results reflecting normal established progesterone concentrations for dogs. Accuracy was verified by comparing measurements of samples using both procedures. The CLIA results were highly correlated (r 2 ¼ 0:975) with the previously validated RIA. The regression equation for canine Table 1 Concentrations of progesterone (P4; ng/ml) in serial dilutions of canine serum samples determined by the chemiluminescent immunoassay Sample ID
Dilution
Actual P4
Corrected P4
488-01
1:1 1:2 1:4 1:8 1:16
17.00 17.00 9.84 19.68 5.00 20.00 2.36 18.88 0.94 15.10 Mean actual/expected ¼ 108%
179-00
1:1 1:2 1:4 1:8 1:16
17.40 17.40 10.30 20.60 5.52 22.08 2.69 21.52 1.44 23.04 Mean actual/expected ¼ 125%
134-00
1:1 1:2 1:4 1:8 1:16
17.80 17.80 9.22 18.44 4.52 18.08 2.53 20.24 1.13 18.08 Mean actual/expected ¼ 105%
059-00
1:1 1:2 1:4 1:8 1:16
20.20 20.20 10.80 21.60 4.79 19.16 2.94 23.52 1.27 20.32 Mean actual/expected ¼ 105%
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Table 2 Concentrations of progesterone (P4; ng/ml) recovered from three canine samples to which four quantities of P4 were added, as measured by chemiluminescent immunoassay Sample ID
[E]
Concentration of P4 added
Mean %R
þ0.79 (ng/ml)
þ2.28 (ng/ml)
þ3.78 (ng/ml)
þ8.00 (ng/ml)
605-01 490-01 Canine pool
0.00 0.39 1.14
1.15 1.64 1.70
1.92 2.55 2.83
3.08 4.92 3.94
8.59 8.15 8.60
105 113 86
Mean %R
0.51
1.50 115
2.43 87
3.98 93
8.45 99
101 99
Percent of P4 recovered (%R) was determined by dividing the sample with the added P4 of a known quantity and the endogenous concentration ([E]).
samples (n ¼ 72) ranging from 0.05 to 22.30 ng/ml was: y ¼ 0:951x 0:16 [where y ¼ CLIA (ng/ml) and x ¼ RIA (ng/ml)]. Recovered percentages of known amounts of P4 added to canine samples (Table 2) also indicate the CLIA is sufficiently accurate. The interassay %CV of the two dog pools having mean P4 concentrations of 0.96 and 9.52 ng/ ml was 11.9 and 6.7%, respectively, which was comparable to the manufacturer’s human matrix controls that were assayed in parallel (Table 3). The intra-assay precision in the range of interest (1.0–2.0 ng/ml) was 11% (Table 4). Table 3 Mean concentrations of progesterone (P4; ng/ml) and interassay percent coefficient of variation (%CV) for dog and human samples (n) each tested separately over a 30-day period using chemiluminescent immunoassay Sample ID
Species
P4
%CV
n
IMM1-2 DPC-CON418 DPC-CON518 IMM1-3
Canine Human Human Canine
0.96 1.60 2.95 9.52
11.9 12.8 9.3 6.7
22 22 22 22
Table 4 Concentrations of progesterone (P4; ng/ml) and intra-assay percent coefficient of variation (%CV) for canine samples (n) measured by chemiluminescent immunoassay Sample ID
P4
%CV
n
065-00.2 736-01.4.5 699-01.2 761-01.4.3 345.5-761.7-01 820-01.2 699-01.3 134-00.2
0.48 1.55 1.56 1.96 2.99 5.09 10.81 15.97
20.6 8.9 16.8 7.2 9.9 3.9 5.8 5.1
10 10 10 10 10 10 10 10
Mean
9.8
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Table 5 Bitches studied to predict parturition using the initial rise in preovulatory progesterone (P4) concentrations BW group
Breed
NC
NB
Small
Jack Russell Terrier Scottish Terrier Tibetan Spaniel Tibetan Terrier Wirehaired Fox Terrier
2 1 4 2 1
Medium
Beagle Boxer Brittany Spaniel Shetland Sheepdog Standard Poodle
Large
LS range
Mean LS (S.D.)
2 1 3 1 1
1–7
4 (2)
1 1 1 1 3
1 1 1 1 2
1–12
6 (3)
Bulldog Doberman German Shepherd Dogs German Shorthaired Pointer Golden Retriever Greyhound Labrador Retriever Labrador Retriever/Greyhound cross
1 1 23 2 2 6 12 4
1 1 17 1 1 4 10 4
1–13
7 (3)
Giant
Great Dane Newfoundland
2 12
2 8
2–14
8 (4)
Total
19 breeds þ 1 cross breed
82
63
1–14
NC: number of estrous cycles, NB: number of bitches, LS: litter size. Bitches were divided into four groups based on nonpregnant body weight (BW): small (9 kg), medium (>9–20 kg), large (>20–40 kg), and giant (>40 kg). Litter size was recorded at birth.
Table 6 Parturition date was predicted to occur 65 days after the initial rise in preovulatory progesterone (P4) concentrations All bitches
Parturition date prediction interval 65 1 days 65 2 days 65 3 days
% Delivering in that interval
67
90
100
BW groups
Small
Medium
Large
Actual gestation length, mean (S.D.) in days LS groups Actual gestation length, mean (S.D.) in days
Giant
65.8 (1.23) 65.7 (1.67) 64.0 (1.02) 64.9 (1.79) Small
Average
Large
65.1 (1.76) 65.1 (1.64) 64.4 (1.5)
Actual gestation length was defined as the interval from the initial rise in P4 to birth of the first pup. There was no significant difference between the mean actual gestation length for bitches grouped by nonpregnant body weight (BW) or litter size (LS).
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Fig. 1. The initial rise in preovulatory progesterone (P4; D0) was defined as the first day that P4 1:5 ng/ml and at least twice the baseline concentration. The slope of the rise in mean P4 after D0 was significantly different between small and large BW groups when compared to medium and giant BW groups (P < 0:05), being 2.62, 1.07, 2.30, and 1.13 for small to giant BW groups, respectively.
3.2. Parturition date prediction Sixty-three bitches from 19 breeds and one group of cross bred bitches differing in body weight and litter size were evaluated (Table 5). Mean P4 concentrations for small to giant BW groups is plotted in Fig. 1. Equal variance was found in the slopes of the P4 curves for BW groups, allowing statistical comparison. The slopes differed significantly between small and large BW groups when compared to medium and giant BW groups. The means of actual gestation length and the accuracy of parturition date prediction were not significantly different within BW or LS groups (Table 6). From the initial rise in preovulatory P4 concentrations, the overall accuracy for predicting parturition within 3 days (62–68 days) was 100% for all bitches studied.
4. Discussion Progesterone CLIA is available through human clinical and veterinary diagnostic laboratories and is comparable in cost to the RIA. In addition, CLIA has the advantage of continuous processing allowing for rapid turnaround time without the disadvantages associated with isotopic assays (e.g. radioactivity, sample batching). Compared to RIA, the intra-assay precision for CLIA in the range of interest for determining the initial rise in P4 (1–2 ng/ml) is lower and the detection limit is higher. However,
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we did not find these differences to be clinically relevant to applications in canine reproduction. Estimation of gestational age in bitches is feasible because the rate of canine embryonic development is synchronous and closely correlated to the time of the preovulatory LH surge [13]. Therefore, gestational age, and hence parturition date, can be determined accurately when timed from the preovulatory LH surge. A previous study of Beagles reported a mean gestation length of 65.1 (0.1) days when defined as the interval from the preovulatory LH surge to parturition [4]. For all bitches in the present study, using preovulatory serum progesterone concentrations to define D0, the accuracy of parturition date prediction within the interval of 65 1, 2, and 3 days was 67, 90, and 100%, respectively. Furthermore, accurate predictions were obtained independent of maternal body weight or litter size. These results are supported by Linde-Forsberg and Forsberg [8], who reported that canine gestation length is not influenced by maternal body weight or litter size. It is likely that results of the present study differ from some earlier reports [10,11] because gestation in the present study was timed from a reliable physiologic event (i.e. the initial rise in progesterone), which occurs simultaneous with the LH surge [4,5]. The differences between studies in methods of timing gestation have led to different conclusions regarding variation in gestation length and the ability to accurately predict parturition date. Previous reports suggested that maternal body weight and litter size had an effect on gestation length when (1) gestation length was defined as the interval from mating to parturition, and (2) the day of mating was determined by plasma P4 concentrations between 6 and 12 ng/ml on the day before mating and >12 ng/ml on the day of mating [10]. We estimate that the day of mating in those studies was likely to be D2 from the initial preovulatory rise in P4 (Fig. 1, Table 7). Interbreed variation in gestation length was reported to be 3–6 days, with Alsatians having a shorter gestation length compared to other breeds studied [8]. However, estimation of gestation length in that manner is likely to be inaccurate due to the inherent variability in P4 concentrations after D1 (Fig. 1). Table 7 Comparison of mean (S.D.) progesterone (P4) concentrations (ng/ml) from previous studies (reported as prior to the LH surge, on the day of LH surge, and on the day of ovulation) and from the present study (where Day 0 is defined by the initial rise in P4) Prior to LH surge
LH surge
Wright [14] <1.0 Badinand et al. [6] Present study
3 days
Small Medium Large Giant Mean (S.D.), all groups
Ovulation
2–4 (range) 5.5 >1.0 >5 2 days
1 day
0.92 0.85 0.53 0.57
0.78 0.84 0.68 0.61
1.46 1.26 1.01 0.96
2.14 1.79 2.18 1.97
0.72 (0.2)
0.73 (0.1)
1.17 (0.23)
2.02 (0.18)
Day 0 (0.05) (0.20) (0.09) (0.08)
1 day
2 days
3 days
3.46 3.10 3.38 3.99
11.86 4.10 5.18 4.62
15.13 5.00 8.27 6.98
3.48 (0.37) 6.44 (3.64) 8.84 (4.4)
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There have been many reports describing P4 concentrations at various times near the onset of the LH surge (Table 7). Wright reported P4 concentrations during anestrus and proestrus, at the time of the LH surge, and at the time of ovulation [14]. Badinand et al. [6] reported P4 concentrations at the time of the LH surge, 0–24 h after the LH peak, and on the first day of diestrus. Best agreement between P4 values in these reports and the present study is in the values prior to the LH surge. Mean (S.D.) P4 concentration at day of the initial rise in preovulatory P4 concentrations (D0) for all BW groups in the present study was 2.02 (0.18) ng/ml. The rate of the rise in P4 concentration between D4 and D0 did not vary significantly, but did differ significantly between bitches of varying body weights following D1 (Fig. 1, Table 7). Previously reported P4 concentrations on the day of ovulation (2 days after LH surge) do not reflect the significant variation documented in the present study, and will lead to inaccurate predictions based on such estimation. Prediction of parturition date within the interval of 65 3 days, based on the initial rise in P4, is more accurate than any previous report, having a predictive accuracy of 100% irrespective of body weight or litter size. To make an accurate prediction using the P4 method, it is important to: (1) obtain serum samples at least every other day, beginning in proestrus, until the initial rise in P4 (D0) is identified, (2) use a quantitative P4 assay that is validated for dogs, (3) if using CLIA, avoid using collection tubes with serum separating gel, and (4) time gestation from the initial rise in P4, rather than from P4 concentrations occurring after the LH surge. The rationale for sampling at the first signs of proestrus is based upon previous studies indicating that there is little correlation between the onset of estrus and LH surge [2,5,14–16]. For example, Concannon et al. [5] reported that the LH surge occurred between the fifth day of proestrus and the second day of estrus. Our clinical experiences support those observations as we could not predict P4 concentration based upon vaginal cytology. Using the initial rise in preovulatory P4 concentrations to predict parturition date is particularly suitable for clinical practice because it can be applied to all pregnancies, regardless of maternal body weight or litter size, and requires no correction factors or additional technical skill. A method of comparable accuracy, transabdominal ultrasonography to estimate gestational age from fetal measurements [17], requires investment in ultrasound equipment, some technical skill in ultrasonography, and use of correction factors for small and giant breeds. However, there is a strong correlation between these two methods (McNemar w2 ¼ 1:00) and both are more accurate in predicting parturition date than previously reported methods. Using this method to time gestation accurately, further studies can be performed to determine the earliest gestational age at which a fetus can be delivered and remain viable without medical intervention.
Acknowledgements We thank S. Long, A.J. Williams, A. Lanfair, and T. Powell for their technical support and assistance; C. Wilker, J. Ellington, C. Schweizer, C. Gradil, and R. Wheeler for professional services rendered in association with clinical patients reported herein; and T. Reimers for technical expertise in assay validation.
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