- Email: [email protected]
Effectiveness of a Commercial Early Conception Factor Test for Use in Cattle
The Professional ScientistTest 17:51–53 Case Study: Evaluation Animal of a Pregnancy Kit in Cows
51
CCommercial S : Effectiveness of a Early Conception ASE TUDY
Factor Test for Use in Cattle C. S. WHISNANT1, L. A. PAGELS, and M. G. DAVES Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621
Abstract The purpose of this study was to test the effectiveness of a commercially available early pregnancy test in cows. The test early conception factor (ECF™) was designed to distinguish between open and pregnant cows by detection of a protein present in blood serum or milk of pregnant cows. All tests were conducted on blood serum according to instructions of the manufacturer. Blood samples were taken via tail vein puncture between 6 and 15 d post-insemination from 67 Holstein or Jersey cows. Serum was placed in a test cassette and the results recorded after 2 h by three independent observers. Ultrasonography was performed on d 25 to 30 post-insemination, or rectal palpation was performed 35 to 42 d post-insemination by experienced personnel. These results were compared with the test results. The test results corresponded with the results of the ultrasound or rectal palpation 51% of the time. Of the 49% that disagreed, 25% were diagnosed pregnant by the ECF test but were found to be open by ultrasound or palpation, and 24% were diagnosed open by the test and found to be pregnant by ultrasound or palpation. These results
1To
whom correspondence should be addressed: [email protected]
able, early conception factor (ECFTM, Concepto-Diagnostics, Knoxville, TN), that is reported to be able to (Key Words: Early Conception Factor, identify a protein found in serum or milk of pregnant cows. The literature Pregnancy Test, Cows.) accompanying the kit states that the factor can be detected in blood or milk of pregnant cows from 48 h to Pregnancy diagnosis in cattle has 20 d after breeding. A small amount depended upon rectal palpation for of serum or milk is placed on the test many years. The earliest time that cassette followed by a wash solution. pregnancy can be detected by an The presence of two lines on the test experienced palpator is ca. 30 to 35 d indicates pregnancy while one line with accuracy increasing with time indicates an open cow. It is reported post-insemination (2). In the past 10 to have an accuracy rate of 93% in to 15 yr, ultrasonography has been identifying open cows (data taken used to detect pregnancy. With this from Web site, www.conceptotechnology it is possible to consisdiagnostics.com). No independent tently detect pregnancy in cows by verification of this accuracy has been about 25 d after breeding and slightly reported. The purpose of this experiearlier in heifers (personal experience ment was to test the accuracy of the of authors). However, the technology kit using ultrasonography between d is expensive, and skill and experience 25 to 30 post-insemination or palpaare required for early pregnancy tion on d 35 to 42 post-insemination detection as with palpation. for comparison. Early pregnancy tests are available for some species, including humans, which are easy to use and relatively Blood samples were taken by tail inexpensive. These depend upon the vein puncture from 67 heifers or cows presence of a factor in pregnant at either the North Carolina State animals that is not found in nonUniversity Dairy Educational Unit in pregnant females. Some such factors have been identified in cows, includ- Raleigh or the North Carolina Department of Agriculture Umstead ing interferon tau and pregnancyDairy Unit in Butner. Test animals specific protein B (1, 5). Recently, a included both Jersey and Holstein kit has become commercially availsuggest that the test does not perform at the necessary level of accuracy.
Introduction
Materials and Methods
52
cows and heifers. All animals were between 6 and 15 d post-insemination. This research was approved by the North Carolina State University Animal Care and Use Committee. The test used was the ECF™ test. All tests were conducted according to the instructions provided by the manufacturer. All test reagents were stored, refrigerated, and allowed to come to room temperature before testing. Blood samples were allowed to clot, and a single drop of serum was placed into the receptacle of the test cassette using the disposable pipettes provided. According to the manufacturer’s instructions, the cassette contains a membrane to which is attached antibodies to a protein referred to as ECF. After application of the sample, the wash solution was placed on the receptacle, and the tests were allowed to sit for 2 h at room temperature before being examined for the results. With this test, two lines on the cassette indicated a positive test for the presence of the ECF, and a single line indicated a negative test. The single upper line on the cassette indicated that the test membrane had not been damaged in shipping or storage. All cassettes were examined by three individuals who independently recorded their results. Transrectal ultrasonography was performed on animals from the Dairy Educational Unit on d 25 to 30 postinsemination using an Aloka 500V (Aloka, Wallingford, CT) equipped with a 7.5-MHz rectal probe to detect the presence or absence of embryonic vesicles. At the Umstead unit, rectal palpation for pregnancy detection was performed between d 35 and 42 by experienced personnel from the North Carolina State University College of Veterinary Medicine. If any ultrasound or palpation exam was questionable, the cow was examined again 1 wk later. Statistical analysis was performed with the Chi square test using SAS (4). For the test, sensitivity was defined as the ability of the kit to detect pregnant cows, that is, the number of pregnant diagnoses that
Whisnant et al.
TABLE 1. Comparison of results from ECFTM test (Concepto-Diagnosticsa) and ultrasonographyb or palpation. Number examined ECFTM result, no. Ultrasound/palpation result, no. ECFTM false diagnoses, no. (%) ECFTM correct diagnoses, no. (%)
67 67
Pregnant
Open
39 38 17 (43%) 22 (58%)
28 29 16 (57%) 12 (41%)
aConcepto-Diagnostics
(Knoxville, TN). was performed using an Aloka 500V (Aloka, Wallingford, CT) scanner with a 7.5-MHz rectal probe and performed on d 25 to 30 post-insemination. bUltrasonography
were confirmed as pregnant by ultrasonography or palpation. Specificity was defined as the number of cows diagnosed as open by the kit that were open when examined by ultrasonography or palpation. Accuracy was defined as the number of correct diagnoses (either pregnant or nonpregnant) divided by the total number of cows diagnosed. A false positive was defined as an animal that was diagnosed pregnant by the kit but was found to be open by ultrasonography or palpation. A false negative was defined as an animal that was diagnosed open by the kit but found to be pregnant by ultrasonography or palpation. Additionally cows were divided into those tested on d 6 to 10 after insemination and those tested on d 11 to 15, in order to determine if accuracy of the test differed with time post-insemination.
Results and Discussion According to the instructions, the presence of a second line, however faint, was to be regarded as a sign of conception. Some of the cassettes were quite faint, but there was agreement between all three observers in all cases as to whether or not a second line was present. The test results corresponded with the results of the ultrasound examination or palpation (accuracy) 51% of the time (Table 1). There was a significant difference (P<0.01) between the results of the ECF™ and palpation or ultrasonography. Of the 49% that
disagreed, 25% were diagnosed pregnant by the test and were found to be open by palpation or ultrasonography, and 24% were diagnosed as open by the test and were later found to be pregnant. Sensitivity of the test kit (correct pregnant diagnoses divided by total pregnant) was 57.9%. Specificity of the test kit (correct nonpregnant diagnoses divided by total nonpregnant) was 41.4% (Table 1). These results were different (P<0.001) than expected if the ECF™ kit had an accuracy of 93% as reported on the Web site. Few details of that test were given, but cows on a commercial dairy were checked using the kit between d 1 and 15 after insemination and then palpated 31 d after insemination. There was no difference in the current study (P>0.10) between cows tested on d 6 to 10 and those tested on d 11 to 15 post-insemination. Sensitivity and specificity for cows in the d 6 to 10 group were 44.4 and 38.5%, respectively. For cows in the d 11 to 15 group, sensitivity and specificity were 45 and 43.8%, respectively. A significant level of early embryonic death occurs in cattle (6). As reviewed by Zavy, the precise timing of this embryo mortality differs between studies, but all place the majority of it occurring by d 18 of pregnancy. This would be between the time the ECF™ test was conducted and when the ultrasonography or palpation was performed. This could account for the cows with a positive ECF™ test result (25%) that
53
Case Study: Evaluation of a Pregnancy Test Kit in Cows
were found to be open when examined by palpation or ultrasonography. Unfortunately, no other method exists for determining pregnancy in cows at this early stage of gestation (6 to15 d). However, embryonic death cannot explain the cows (24%) that had a negative ECF™ test and were found to be pregnant at the later ultrasonography or palpation. No other inseminations were performed on these cows prior to examination for pregnancy, and no bulls were present on either farm. This falsenegative result presents a more serious problem with the test. Under many management systems, such cows would have been injected with prostaglandin F2a to allow for rebreeding, which would have aborted the pregnancy. Development of a test for identifying open cows early after insemination would be a benefit to cattle producers. Factors secreted by the early bovine conceptus include interferon tau, pregnancy-specific protein B, and pregnancy-associated glycoprotein (bPAG). Interferon tau is secreted by the bovine conceptus during d 14 to 17 of pregnancy (1). Pregnancy- specific protein B has been reported to be detectable in serum as early as d 15 of pregnancy (3) but is more reliable at d 30 (5). Pregnancy-associated glycoprotein was detected as early as d 22 in serum of some cows and d 30 of pregnancy in all cows (7). None of these factors could be the protein detected by the ECF™ test based on its described molecular weight of 200,000 daltons as reported on the company Web site. Furthermore, none of these molecules appears to be produced or detectable as early as the ECF.
Implications The accuracy of the ECF™ test reported in this study is not sufficient to recommend the use of this product. It was expected that some cows diagnosed pregnant by the test would be found open later as embryonic death is known to occur, especially during this stage of gestation. However, the fact that 24% were diagnosed as open and found to be pregnant indicates a need for improved accuracy. The open cows under many management systems would have been injected with prostaglandin F2a to allow for rebreeding and the pregnancy would have been aborted. Early diagnosis of open cows would be a benefit to producers, but the ECF™ test examined in this experiment does not possess the level of accuracy needed to recommend use of the product.
Acknowledgments The authors thank Kevin Walden and Johnny Brooks and other personnel at the North Carolina State University Dairy Educational Unit and the North Carolina Department of Agriculture Umstead Dairy for their assistance in this project and Steve Washburn and Billy Flowers for suggestions to the manuscript.
Literature Cited 1. Bartol, F. F., R. M. Roberts, F. W. Bazer, G. S. Lewis, J. D. Godkin, and W. W. Thatcher. 1985. Characterization of proteins produced in vitro by periattachment bovine conceptuses. Biol. Reprod. 32:681. 2. Bearden, H. J., and J. W. Fuquay. 2000. Applied Animal Reproduction. 5th edition. Prentice Hall, Upper Saddle River, NJ, p. 278. 3. Butler, J. E., W. C. Hamilton, R. G. Sasser, C. A. Ruder, G. M. Hass, and R. J. Williams. 1981. Detection and partial characterization of two bovine pregnancy-specific proteins. Biol. Reprod. 26:925. 4. SAS. 1997. SAS 6.12 User’s Guide. SAS Institute, Inc., Cary, NC. 5. Sasser, R. G., C. A. Ruder, K. A. Ivani, J. E. Butler, and W. C. Hamilton. 1986. Detection of pregnancy by radioimmunoassay of a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation. Biol. Reprod. 35:936. 6. Zavy, M. T. 1994. Embryonic Mortality in Cattle. In Embryonic Mortality in Domestic Species. M. T. Zavy and R. D. Geisert (Eds.). p. 99. CRC Press, Boca Raton, FL. 7. Zoli, A. P., L. A. Guilbault, P. Delahaut, W. B. Ortiz, and J. F. Beckers. 1992. Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy diagnosis. Biol. Reprod. 46:83.
51
CCommercial S : Effectiveness of a Early Conception ASE TUDY
Factor Test for Use in Cattle C. S. WHISNANT1, L. A. PAGELS, and M. G. DAVES Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621
Abstract The purpose of this study was to test the effectiveness of a commercially available early pregnancy test in cows. The test early conception factor (ECF™) was designed to distinguish between open and pregnant cows by detection of a protein present in blood serum or milk of pregnant cows. All tests were conducted on blood serum according to instructions of the manufacturer. Blood samples were taken via tail vein puncture between 6 and 15 d post-insemination from 67 Holstein or Jersey cows. Serum was placed in a test cassette and the results recorded after 2 h by three independent observers. Ultrasonography was performed on d 25 to 30 post-insemination, or rectal palpation was performed 35 to 42 d post-insemination by experienced personnel. These results were compared with the test results. The test results corresponded with the results of the ultrasound or rectal palpation 51% of the time. Of the 49% that disagreed, 25% were diagnosed pregnant by the ECF test but were found to be open by ultrasound or palpation, and 24% were diagnosed open by the test and found to be pregnant by ultrasound or palpation. These results
1To
whom correspondence should be addressed: [email protected]
able, early conception factor (ECFTM, Concepto-Diagnostics, Knoxville, TN), that is reported to be able to (Key Words: Early Conception Factor, identify a protein found in serum or milk of pregnant cows. The literature Pregnancy Test, Cows.) accompanying the kit states that the factor can be detected in blood or milk of pregnant cows from 48 h to Pregnancy diagnosis in cattle has 20 d after breeding. A small amount depended upon rectal palpation for of serum or milk is placed on the test many years. The earliest time that cassette followed by a wash solution. pregnancy can be detected by an The presence of two lines on the test experienced palpator is ca. 30 to 35 d indicates pregnancy while one line with accuracy increasing with time indicates an open cow. It is reported post-insemination (2). In the past 10 to have an accuracy rate of 93% in to 15 yr, ultrasonography has been identifying open cows (data taken used to detect pregnancy. With this from Web site, www.conceptotechnology it is possible to consisdiagnostics.com). No independent tently detect pregnancy in cows by verification of this accuracy has been about 25 d after breeding and slightly reported. The purpose of this experiearlier in heifers (personal experience ment was to test the accuracy of the of authors). However, the technology kit using ultrasonography between d is expensive, and skill and experience 25 to 30 post-insemination or palpaare required for early pregnancy tion on d 35 to 42 post-insemination detection as with palpation. for comparison. Early pregnancy tests are available for some species, including humans, which are easy to use and relatively Blood samples were taken by tail inexpensive. These depend upon the vein puncture from 67 heifers or cows presence of a factor in pregnant at either the North Carolina State animals that is not found in nonUniversity Dairy Educational Unit in pregnant females. Some such factors have been identified in cows, includ- Raleigh or the North Carolina Department of Agriculture Umstead ing interferon tau and pregnancyDairy Unit in Butner. Test animals specific protein B (1, 5). Recently, a included both Jersey and Holstein kit has become commercially availsuggest that the test does not perform at the necessary level of accuracy.
Introduction
Materials and Methods
52
cows and heifers. All animals were between 6 and 15 d post-insemination. This research was approved by the North Carolina State University Animal Care and Use Committee. The test used was the ECF™ test. All tests were conducted according to the instructions provided by the manufacturer. All test reagents were stored, refrigerated, and allowed to come to room temperature before testing. Blood samples were allowed to clot, and a single drop of serum was placed into the receptacle of the test cassette using the disposable pipettes provided. According to the manufacturer’s instructions, the cassette contains a membrane to which is attached antibodies to a protein referred to as ECF. After application of the sample, the wash solution was placed on the receptacle, and the tests were allowed to sit for 2 h at room temperature before being examined for the results. With this test, two lines on the cassette indicated a positive test for the presence of the ECF, and a single line indicated a negative test. The single upper line on the cassette indicated that the test membrane had not been damaged in shipping or storage. All cassettes were examined by three individuals who independently recorded their results. Transrectal ultrasonography was performed on animals from the Dairy Educational Unit on d 25 to 30 postinsemination using an Aloka 500V (Aloka, Wallingford, CT) equipped with a 7.5-MHz rectal probe to detect the presence or absence of embryonic vesicles. At the Umstead unit, rectal palpation for pregnancy detection was performed between d 35 and 42 by experienced personnel from the North Carolina State University College of Veterinary Medicine. If any ultrasound or palpation exam was questionable, the cow was examined again 1 wk later. Statistical analysis was performed with the Chi square test using SAS (4). For the test, sensitivity was defined as the ability of the kit to detect pregnant cows, that is, the number of pregnant diagnoses that
Whisnant et al.
TABLE 1. Comparison of results from ECFTM test (Concepto-Diagnosticsa) and ultrasonographyb or palpation. Number examined ECFTM result, no. Ultrasound/palpation result, no. ECFTM false diagnoses, no. (%) ECFTM correct diagnoses, no. (%)
67 67
Pregnant
Open
39 38 17 (43%) 22 (58%)
28 29 16 (57%) 12 (41%)
aConcepto-Diagnostics
(Knoxville, TN). was performed using an Aloka 500V (Aloka, Wallingford, CT) scanner with a 7.5-MHz rectal probe and performed on d 25 to 30 post-insemination. bUltrasonography
were confirmed as pregnant by ultrasonography or palpation. Specificity was defined as the number of cows diagnosed as open by the kit that were open when examined by ultrasonography or palpation. Accuracy was defined as the number of correct diagnoses (either pregnant or nonpregnant) divided by the total number of cows diagnosed. A false positive was defined as an animal that was diagnosed pregnant by the kit but was found to be open by ultrasonography or palpation. A false negative was defined as an animal that was diagnosed open by the kit but found to be pregnant by ultrasonography or palpation. Additionally cows were divided into those tested on d 6 to 10 after insemination and those tested on d 11 to 15, in order to determine if accuracy of the test differed with time post-insemination.
Results and Discussion According to the instructions, the presence of a second line, however faint, was to be regarded as a sign of conception. Some of the cassettes were quite faint, but there was agreement between all three observers in all cases as to whether or not a second line was present. The test results corresponded with the results of the ultrasound examination or palpation (accuracy) 51% of the time (Table 1). There was a significant difference (P<0.01) between the results of the ECF™ and palpation or ultrasonography. Of the 49% that
disagreed, 25% were diagnosed pregnant by the test and were found to be open by palpation or ultrasonography, and 24% were diagnosed as open by the test and were later found to be pregnant. Sensitivity of the test kit (correct pregnant diagnoses divided by total pregnant) was 57.9%. Specificity of the test kit (correct nonpregnant diagnoses divided by total nonpregnant) was 41.4% (Table 1). These results were different (P<0.001) than expected if the ECF™ kit had an accuracy of 93% as reported on the Web site. Few details of that test were given, but cows on a commercial dairy were checked using the kit between d 1 and 15 after insemination and then palpated 31 d after insemination. There was no difference in the current study (P>0.10) between cows tested on d 6 to 10 and those tested on d 11 to 15 post-insemination. Sensitivity and specificity for cows in the d 6 to 10 group were 44.4 and 38.5%, respectively. For cows in the d 11 to 15 group, sensitivity and specificity were 45 and 43.8%, respectively. A significant level of early embryonic death occurs in cattle (6). As reviewed by Zavy, the precise timing of this embryo mortality differs between studies, but all place the majority of it occurring by d 18 of pregnancy. This would be between the time the ECF™ test was conducted and when the ultrasonography or palpation was performed. This could account for the cows with a positive ECF™ test result (25%) that
53
Case Study: Evaluation of a Pregnancy Test Kit in Cows
were found to be open when examined by palpation or ultrasonography. Unfortunately, no other method exists for determining pregnancy in cows at this early stage of gestation (6 to15 d). However, embryonic death cannot explain the cows (24%) that had a negative ECF™ test and were found to be pregnant at the later ultrasonography or palpation. No other inseminations were performed on these cows prior to examination for pregnancy, and no bulls were present on either farm. This falsenegative result presents a more serious problem with the test. Under many management systems, such cows would have been injected with prostaglandin F2a to allow for rebreeding, which would have aborted the pregnancy. Development of a test for identifying open cows early after insemination would be a benefit to cattle producers. Factors secreted by the early bovine conceptus include interferon tau, pregnancy-specific protein B, and pregnancy-associated glycoprotein (bPAG). Interferon tau is secreted by the bovine conceptus during d 14 to 17 of pregnancy (1). Pregnancy- specific protein B has been reported to be detectable in serum as early as d 15 of pregnancy (3) but is more reliable at d 30 (5). Pregnancy-associated glycoprotein was detected as early as d 22 in serum of some cows and d 30 of pregnancy in all cows (7). None of these factors could be the protein detected by the ECF™ test based on its described molecular weight of 200,000 daltons as reported on the company Web site. Furthermore, none of these molecules appears to be produced or detectable as early as the ECF.
Implications The accuracy of the ECF™ test reported in this study is not sufficient to recommend the use of this product. It was expected that some cows diagnosed pregnant by the test would be found open later as embryonic death is known to occur, especially during this stage of gestation. However, the fact that 24% were diagnosed as open and found to be pregnant indicates a need for improved accuracy. The open cows under many management systems would have been injected with prostaglandin F2a to allow for rebreeding and the pregnancy would have been aborted. Early diagnosis of open cows would be a benefit to producers, but the ECF™ test examined in this experiment does not possess the level of accuracy needed to recommend use of the product.
Acknowledgments The authors thank Kevin Walden and Johnny Brooks and other personnel at the North Carolina State University Dairy Educational Unit and the North Carolina Department of Agriculture Umstead Dairy for their assistance in this project and Steve Washburn and Billy Flowers for suggestions to the manuscript.
Literature Cited 1. Bartol, F. F., R. M. Roberts, F. W. Bazer, G. S. Lewis, J. D. Godkin, and W. W. Thatcher. 1985. Characterization of proteins produced in vitro by periattachment bovine conceptuses. Biol. Reprod. 32:681. 2. Bearden, H. J., and J. W. Fuquay. 2000. Applied Animal Reproduction. 5th edition. Prentice Hall, Upper Saddle River, NJ, p. 278. 3. Butler, J. E., W. C. Hamilton, R. G. Sasser, C. A. Ruder, G. M. Hass, and R. J. Williams. 1981. Detection and partial characterization of two bovine pregnancy-specific proteins. Biol. Reprod. 26:925. 4. SAS. 1997. SAS 6.12 User’s Guide. SAS Institute, Inc., Cary, NC. 5. Sasser, R. G., C. A. Ruder, K. A. Ivani, J. E. Butler, and W. C. Hamilton. 1986. Detection of pregnancy by radioimmunoassay of a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation. Biol. Reprod. 35:936. 6. Zavy, M. T. 1994. Embryonic Mortality in Cattle. In Embryonic Mortality in Domestic Species. M. T. Zavy and R. D. Geisert (Eds.). p. 99. CRC Press, Boca Raton, FL. 7. Zoli, A. P., L. A. Guilbault, P. Delahaut, W. B. Ortiz, and J. F. Beckers. 1992. Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy diagnosis. Biol. Reprod. 46:83.