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Department of Dairy Science, University of Wisconsin-Madison, Madison 53706
1 Corresponding author: pmfricke{at}wisc.edu
| ABSTRACT |
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injection of Presynch and the first GnRH injection of Ovsynch, and cows with serum progesterone
1.0 ng/mL in one or both samples were classified as cycling, whereas cows with serum progesterone <1.0 ng/mL in both samples were classified as anovular. For the practical method, transrectal ultrasonography (U/S) was used to determine the presence or absence of a corpus luteum (CL) at the first GnRH injection of Ovsynch, and cows without CL were classified as anovular, whereas cows with CL were classified as cycling. Statistical agreement (kappa) between the RIA and U/S methods to identify cycling cows was 0.66. Sensitivity, specificity, positive predictive value, and negative predictive value of U/S to identify anovular status were 85.7, 87.7, 64.7, and 95.9%, respectively. We conclude that assessing the presence or absence of CL at the first GnRH injection of a Presynch + Ovsynch protocol using U/S is a reliable and practical method for identifying the cyclicity status of cows before first TAI, but may slightly overestimate the proportion of anovular cows compared with the RIA method.
Key Words: dairy cow anovular ultrasound
| INTRODUCTION |
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Identification of anovular cows using a variety of methods has been reported in the scientific literature. Cartmill et al. (2001) reported that 16.2% of cows were anovular using 3 blood samples collected 7 and 5 d apart before initiation of Ovsynch (synchronization regimen using sequential injections of GnRH and PGF2
to precisely time ovulation for TAI), whereas Gümen et al. (2003) used 2 consecutive weekly ultrasound (U/S) examinations and blood samples to detect the presence of luteal tissue and serum progesterone (P4) and reported that 20.2% of cows were anovular. Another frequently reported method is to assess serum P4 from blood samples collected 10 to 14 d apart before or during a hormonal protocol for synchronization of ovulation and TAI (Cordoba and Fricke, 2002; Cerri et al., 2004; Stevenson et al., 2006) with the rationale that cycling cows should have serum P4
1 ng/mL at one or both of these serum samples, whereas anovular cows would have serum P4 < 1 ng/mL in both serum samples.
Presynchronization protocols (Presynch; postpartum regimen using 2 injections of PGF2
administered 14 d apart to synchronize estrous cycles before applying Ovsynch) have been used to manipulate the reproductive cycle so that cows are in the mid-luteal phase of the estrous cycle at initiation of Ovsynch, thereby increasing fertility to TAI (Moreira et al., 2001; Navanukraw et al., 2004). Cycling cows receiving a Presynch + Ovsynch protocol for first postpartum TAI should have an ultrasonographically detectable CL at the first GnRH injection of Ovsynch, whereas anovular cows would not. Presynchronization increased the proportion of cows with greater P4 (
1.0 ng/mL) at the second PGF2
injection of Presynch and the first GnRH injection of Ovsynch (60.0 vs. 52.0% and 72.0 vs. 59.0%, respectively; El-Zarkouny et al., 2004). Indeed, 26% of cows submitted for first postpartum TAI using Presynch + Ovsynch were classified as anovular based on the absence of a detectable CL by U/S at the first GnRH injection of Presynch + Ovsynch (Sterry et al., 2006); however, this method has not been compared with other published methods for identifying anovular cows.
Our objective was to determine the accuracy of using the presence or absence of a CL based on a single U/S examination of ovaries at the first GnRH injection of a Presynch + Ovsynch protocol compared with a published method using serum P4 profiles from 2 blood samples collected 12 d apart. Our hypothesis was that the U/S method would be a practical and accurate method for identifying anovular cows under field conditions compared with more intensive blood sampling and RIA technique.
| MATERIALS AND METHODS |
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(Lutalyse, 25 mg i.m., Pfizer Animal Health, New York, NY) administered 14 d apart (39 ± 3 and 53 ± 3 d postpartum). Ovsynch was initiated 12 d after the second PGF2
injection of Presynch as follows: GnRH (Cystorelin, 100 µg i.m., Merial, Duluth, GA) 65 ± 3 d postpartum; PGF2
, 72 ± 3 d postpartum; GnRH 54 h after PGF2
. All cows received TAI approximately 16 h after the second GnRH injection of Ovsynch.
Cows were enrolled into the experiment weekly beginning 53 ± 3 d postpartum on the day of the second PGF2
injection of the Presynch + Ovsynch protocol. A total of 891 lactating Holstein cows were initially enrolled into the experiment, with 842 of these cows included in the final analysis (28 cows did not receive the correct hormone injections during Presynch + Ovsynch or were sold or died, and 21 cows had one or more missing blood samples). Body condition score (1 = emaciated and 5 = obese; Wildman et al., 1982) was evaluated by the same individual throughout the study at the time of enrollment, and cows with a BCS
2.0 (n = 13 cows) were not enrolled into the study based on breeding criteria set by the farm manager.
Collection and Processing of Serum Samples
Blood samples were collected via venipuncture of the median caudal vein or artery into evacuated tubes (Vacutainer; BD, Franklin Lakes, NJ). Blood samples were allowed to clot for 12 h at 4°C, centrifuged (1,935 x g for 15 min), and serum was harvested and stored at 20°C until assayed for P4 concentration using a solid-phase, no-extraction RIA (Coat-a-Count, Diagnostic Products Cooperation, Los Angeles, CA). Interassay and intraassay coefficients of variation were 10.8 and 8.9% and 2.0 and 6.2% for low (2.0 ng/mL) and high (10.0 ng/mL) quality control samples, respectively.
Determination of Cyclicity Status
Two methods were compared to assess cyclicity status before first postpartum TAI. For the standard method (RIA), blood samples were collected at the second PGF2
injection of Presynch and at the first GnRH injection of Ovsynch (Figure 1
). Cows with serum P4
1.0 ng/mL in one or both serum samples were classified as cycling, whereas cows with serum P4 <1.0 ng/mL in both samples were classified as anovular as described previously (Moreira et al., 2001; Stevenson et al., 2006).
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Cycling cows subjected to a Presynch + Ovsynch protocol for first postpartum TAI should have an ultrasonically detectable CL at the first GnRH injection of Ovsynch 12 d after the second PGF2
injection of Presynch, whereas anovular cows should not. Accordingly, for the U/S method, cows without a detectable CL at the first GnRH injection of Presynch + Ovsynch were classified as anovular, whereas cows in which a CL was detected were classified as cycling.
To further assess the accuracy of using U/S to determine functional luteal status, the presence of a CL based on U/S was compared with the presence of a functional CL based on serum P4 in a blood sample collected at the U/S examination. Cows with serum P4
1.0 ng/mL were classified as having a functional CL present, whereas cows with serum P4 <1.0 ng/mL were classified as lacking a functional CL as described previously (Rivera et al., 2005).
Statistical Analyses
The kappa statistic of PROC FREQ (SAS Institute, 1999) was used to determine agreement between U/S and RIA to determine cyclicity status (Martin et al., 1987; Noordhuizen et al., 2001). Kappa is a measure of repeatability in which a value of 1 indicates perfect agreement, and 0 indicates the level of agreement expected to occur by chance alone. In the comparison of tests, a kappa value of 0.4 to 0.5 indicates a moderate level of agreement, 0.5 to 0.6 indicates good agreement, and >0.6 indicates a high level of agreement (Martin et al., 1987).
A 2 x 2 contingency table was constructed to determine the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the U/S method to detect anovular cows (Martin et al., 1987; Table 1
). Although the "true" cyclicity status of cows in the study was not established (i.e., the RIA method may result in errors), the P4 concentration determined by RIA was used as the gold standard to determine cyclicity status of cows for assessing the U/S method. Sensitivity was calculated as the proportion of anovular cows that were correctly classified by U/S (number of true anovular cows/number of true anovular cows + number of false cycling cows), and the PPV was the proportion of all cows classified anovular by U/S that truly were anovular (number of true anovular cows/number of true anovular cows + number of false anovular cows). Specificity was the proportion of cycling cows correctly classified by U/S (number of true cycling cows/number of true cycling cows + number of false anovular cows), and NPV was the proportion of all cows classified cycling that truly were cycling (number of true cycling cows/number of true cycling cows + number of false cycling cows). A similar 2 x 2 contingency table was constructed to determine sensitivity, specificity, PPV, and NPV to estimate the presence of a functional CL. Sensitivity, specificity, PPV, and NPV calculations were performed using Win Episcope 2.0 (Thrusfield et al., 2001).
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2 analysis using the PROC FREQ statistic of SAS (SAS Institute, 2003). Regression analyses were performed to determine the best fitted line between percentage of anovular cows and BCS using PROC REG in SAS (SAS Institute, 2003). Treatment differences with P < 0.05 were considered significant, and differences between P > 0.05 and P < 0.15 were considered statistical tendencies. | RESULTS AND DISCUSSION |
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10 mm, and the average P4 concentration for these cows was 0.30 ± 0.05 ng/mL. It is possible that these cows were undergoing luteal regression at the time of assessment using ultrasound. Although ultrasonographic assessment of CL is a viable alternative to determine circulating P4 concentrations, luteal tissue regresses at a slower rate than the decrease in plasma P4 concentration during luteal regression (Kastelic et al., 1990). Furthermore, the RIA method used to determine cyclicity status in this and other studies may not be as accurate as more intensive methods that incorporate a combination of more frequent and sequential U/S and RIA for P4 (Wiltbank et al., 2002).
To assess the use of a 1.0-ng/mL cutoff point for serum P4 using the RIA method, sensitivity, specificity, PPV, NPV, prevalence, and accuracy of U/S compared with RIA outcomes were calculated using various cutoff points. Varying the P4 cutoff point used to assess anovular cows for the RIA method from 0.5 to 1.0 to 1.2 ng/mL resulted in similar values for sensitivity, specificity, PPV, NPV, true prevalence, and accuracy of the U/S method, whereas increasing the cutoff point to
1.5 ng/mL resulted in a gradual decrease in sensitivity, NPV, and accuracy (Table 4
).
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1.0 ng/mL, respectively, at the U/S examination. The 35 cows with P4
1.0 ng/mL at the first GnRH injection of Ovsynch (i.e., cycling based on RIA) and lacking a CL at this time based on U/S were classified as anovular using U/S; the mean P4 concentration for these cows was 3.3 ± 0.3 ng/mL. In addition, 75 (9.0% of all cows) cows had serum P4
1.0 ng/mL at the second PGF2
injection of Presynch and <1.0 ng/mL at the first GnRH injection of Ovsynch (i.e., cycling based on RIA), and 47 (5.6% of all cows) of these cows had no CL at the first GnRH injection of Ovsynch (i.e., anovular based on U/S). Although a variety of physiologic scenarios could explain these discrepancies, we cannot determine the exact source of disagreement in each case, and at least some of this disagreement is likely due to technical error in interpreting the U/S examinations. Most important, although the U/S method used in the present study to determine cyclicity status slightly overestimated the proportion of anovular cows compared with the RIA method, all but 25 anovular cows based on the RIA method were identified using the U/S method (Table 1
Another consideration for the disagreement between U/S and RIA was that cows with ovarian cysts were considered anovular using U/S in the present study. Fluid-filled cystic structures
25 mm in diameter identified at the scanning session conducted at the time of the second GnRH injection were classified as ovarian cysts based on ultrasonographic morphology (Fricke and Wiltbank, 1999). No attempt was made to further classify cystic structures as follicular, luteal, or benign based on ultrasonographic morphology. When luteal cysts were differentiated from follicular cysts based on P4 concentrations >0.5 ng/mL, mean P4 concentration was 1.6 ± 0.1 ng/mL (Farin et al., 1990), whereas when the cutoff point to determine luteal cysts was 0.9 ng/mL, mean P4 concentrations were 3.6 ± 0.5 to 3.9 ± 0.6 ng/mL (Ribadu et al., 1994; Douthwaite and Dobson, 2000). Thus, cystic cows lacking a CL and having high P4 may have reduced the sensitivity of the U/S method to identify anovular cows in the present study.
Based on the P4 concentrations at the second PGF2
injection of Presynch and the first GnRH injection of Ovsynch (high,
1.0 ng/mL; low, <1.0 ng/mL), cows were classified into high-high, high-low, low-high, and low-low groups resulting in a distribution of 55.3 (466/842), 8.9 (75/842), 15.0 (126/842), and 20.8% (175/842), respectively. The mean P4 concentration for anovular cows identified using the RIA method was 0.08 ± 0.01 ng/mL at the second PGF2
injection of Presynch and 0.10 ± 0.01 ng/mL at the first GnRH injection of Ovsynch compared with a mean P4 concentration of 1.20 ± 0.12 ng/mL at the second PGF2
injection and 0.60 ± 0.08 ng/mL at the first GnRH injection of Ovsynch for anovular cows identified using the U/S method.
Assessment of Luteal Status
In the present study, statistical agreement (kappa) between the presence or absence of a CL using U/S and serum P4 concentration at the U/S examination was 0.74 (95% confidence interval = 0.69 to 0.79) indicating a high level of agreement between the methods (Martin et al., 1987). Sensitivity, specificity, PPV, and NPV of using U/S to assess the presence of a CL were 94.1, 78.8, 91.3, 84.9%, respectively (Table 3
). Our results agree with previous studies reporting a kappa agreement between U/S and plasma P4 concentration to detect CL of 0.71 and 0.74 (Battocchio et al., 1999; McDougall and Rhodes, 1999). Another study reported a kappa value >0.75 among skilled U/S operators, and a kappa value of >0.9 between an experienced operator and ovarian dissection to determine the presence of CL (Lean et al., 1992). Pierson and Ginther (1987) reported 100% agreement between ultrasound and ovarian dissection for identification of the presence of a CL in Holstein heifers.
Anovular Cows by BCS
The proportion of anovular cows submitted for TAI is of concern because fertility is reduced and pregnancy loss is greater for anovular cows submitted for TAI compared with cycling herdmates (Gümen et al., 2003; Santos et al., 2004b; Stevenson et al., 2006). An inverse relationship between BCS and cyclicity status has been reported in which cows with the lowest BCS have the greatest proportion of anovular cows (Moreira et al., 2001; Gümen et al., 2003; Santos et al., 2004a). Moreover, studies reported that cows with lower BCS (
2.5) have reduced fertility after Ovsynch and TAI (Moreira et al., 2000; Souza, 2005). Based on logistical regression analysis of BCS categories, both methods yielded a similar proportion of anovular cows except for cows with a BCS of 3.00 in which the proportion of anovular cows was greater (P < 0.01) for the U/S method compared with the RIA method (Figure 2
). Based on a linear regression analysis of these data, a quadratic effect was fitted in which the proportion of anovular cows decreased as BCS increased up to 3.25 and then increased. This increase after a BCS of 3.25 was based on the derivatives of the quadratic equations. For the U/S method, the R2 = 0.95 and R2adj= 0.92 (y = 301.5 168.1x +24.9 x2; P < 0.01), whereas for the RIA method, R2 = 0.97 and R2adj = 0.95 (y = 381.8 224.5x + 33.8 x2; P < 0.01), where y = percentage of cows not cycling and x = BCS. Despite this relationship, only 38.4% of all cows classified as anovular using the U/S method and 48.6% of all cows classified as anovular using the RIA method had a BCS
2.5 at the second PGF2
injection of Presynch. Indeed, the majority of cows classified anovular (61.6% using the U/S method and 51.4% using the RIA method) in the present study had a BCS >2.5 at the second PGF2
injection of Presynch. Thus, physiologic conditions other than BCS must play a role in determining cyclicity status of lactating dairy cows (Wiltbank et al., 2002). From a practical perspective, identification of anovular cows based on the criterion of low BCS alone would be an ineffective strategy for identifying and treating anovular cows before first postpartum TAI.
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| CONCLUSIONS |
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| ACKNOWLEDGEMENTS |
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Received for publication November 21, 2006. Accepted for publication March 13, 2007.
| REFERENCES |
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