- Email: [email protected]
Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle
G Model
ARTICLE IN PRESS
ANIREP 5197 1–7
Animal Reproduction Science xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle
2
3
4
5
6 7 8 9 10 11 12 13
Q1
Aly Karen a,b,∗ , Noelita Melo De Sousa c , Jean-Franc¸ois Beckers c , Árpád Csaba Bajcsy a,d , János Tibold e , István Mádl e , Ottó Szenci a,d a
Clinic for Large Animals, Faculty of Veterinary Science, Szent István University, Üllo-Dóra major, 2225, Hungary ˝ Department of Theriogenology, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516 Kafrelsheikh, Egypt Laboratory of Animal Endocrinology and Reproduction, Laboratory of Animal Endocrinology and Reproduction, Faculty of Veterinary Medicine, University of Liege, Liege, 4000 Belgium d MTA-SZIE Large Animal Clinical Research Group, Üllo-Dóra major, 2225, Hungary ˝ e Agroproduct Zrt, Pápa, 8500, Hungary b c
14
15
a r t i c l e
i n f o
a b s t r a c t
16 17 18 19 20 21
Article history: Received 28 November 2014 Received in revised form 13 April 2015 Accepted 7 May 2015 Available online xxx
22 23 24 25 26
Keywords: Transrectal ultrasonography Bovine pregnancy proteins Pregnancy diagnosis
The present study aimed to compare the accuracy of a commercial PAG-ELISA test (Bovine Preg Test 29) and bovine pregnancy-associated glycoprotein radioimmunoassay (PAG-RIA) for diagnosing pregnancy at Day 28 after insemination in dairy cows. Transrectal ultrasonography (TRUS) was performed in 100 Holstein-Friesian cows at Day 28 after artificial insemination (AI; Day 0) to diagnose pregnancy. After TRUS examination, blood sample was collected from the coccygeal vessels of each cow to measure the concentrations of bPAGs by PAG-RIA test and Bovine Preg Test 29. Milk samples were collected at Days 0, 21 and 28 for measurement of progesterone (P4) by ELISA test. The cows were reexamined by TRUS at Day 42 to confirm the pregnancy diagnoses. The actual gold standard was based on TRUS outcomes at Day 28 that agreed with the outcomes of PAG-RIA test or PAG-ELISA test. If the outcomes of TRUS at Day 28 and PAG-RIA test and PAG-ELISA test did not agree, the gold standard was based on the outcome of TRUS at Day 42. Out of 100 inseminated cows, 41 were confirmed pregnant at Day 28 after AI. Based on the actual gold standard, the sensitivity of TRUS, PAG-ELISA and PAG-RIA tests for diagnosing pregnant cows at Day 28 were 92.7%, 90.2% and 100%, while the specificity of the three tests for diagnosing non-pregnant cows were 91.5%, 98.3% and 94.4%, respectively. The overall accuracy of the three tests were 92%, 95% and 97%, respectively. The degree of agreement (Kappa ± S.E.) between PAG-RIA and PAG-ELISA test was 0.90 ±0.04. The degrees of agreement between PAG-RIA and PAG-ELISA and TRUS at Day 28 were 0.80 ±0.5 and 0.76 ± 0.06, respectively. In conclusion, the commercial PAG-ELISA test is a highly accurate method for diagnosing early pregnancy in dairy cows on Day 28 after AI and may be used as an alternative method to the TRUS and the PAG-RIA test. © 2015 Elsevier B.V. All rights reserved.
∗ Corresponding author at: Department of Theriogenology, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516 Kafrelsheikh, Egypt. Tel.: +20 1002773137; fax: +20 473231311. E-mail address: [email protected] (A. Karen). http://dx.doi.org/10.1016/j.anireprosci.2015.05.005 0378-4320/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
G Model ANIREP 5197 1–7
A. Karen et al. / Animal Reproduction Science xxx (2015) xxx–xxx
2 27
ARTICLE IN PRESS
1. Introduction
Q2
Early pregnancy diagnosis is a management tool 29 required for shortening the calving interval in dairy cows. 30 In addition identification of non-pregnant cows as early 31 as possible after insemination reduces inter-insemination 32 interval for herds performing synchronization and resyn33 chronization without estrus detection (Lucy et al., 2011). 34 The diagnostic method used for early pregnancy diag35 nosis should be accurate, practical, fast, and inexpensive. 36 Transrectal palpation (TRP) is widely used for pregnancy 37 diagnosis in cows; however; with a moderate amount of 38 experience, a reliable diagnosis of pregnancy is possible 39 after Days 35–39 (Youngquist, 2007). Transrectal ultraso40 nography (TRUS) is an accurate method for early pregnancy 41 diagnosis from Day 29 after insemination of dairy cows 42 (Romano et al., 2006), however, it requires a specialized 43 equipment and a trained person to perform the scanning 44 (Lucy et al., 2011). Alternatively, blood or milk chemi45 cal tests can be used before Day 29 after insemination. 46 These tests are: progesterone (P4) and bovine pregnancy47 associated glycoproteins (bPAGs). Progesterone-ELISA test 48 (cow-side test) can be applied in the milk at Days 21–24 49 after insemination with a 100% negative predictive value; 50 however the test has relatively low (60–94%) positive pre51 dictive value (Nakao et al., 1982; Sauer et al., 1986). 52 Bovine pregnancy-associated glycoproteins (bPAGs) or 53 pregnancy specific protein B (bPSPB) are mainly secreted by 54 trophoblastic binucleate cells (Wooding et al., 2005) and 55 they have been detected in the blood of some pregnant 56 cows as early as Days 15–22 after AI (Sasser et al., 1986; 57 Zoli et al., 1992; Green et al., 2005). Therefore, these 58 glycoproteins have been used as a marker of pregnancy 59 and feto-placental well-being in cattle (Breukelman et al., 60 2012). Radioimmunoassays (RIA) for bPSPB or bPAG-1 have 61 a high sensitivity (95–100%) for identifying pregnant cattle 62 from Day 30 after breeding onwards. However, the speci63 ficity of these tests for identifying non-pregnant cows in 64 most studies is greatly variable ranging from 69.3% to 93% 65 (Sasser et al., 1986; Humblot et al., 1988; Zoli et al., 1992; 66 Skinner et al., 1996; Szenci et al., 1998) due to the differ67 ence in the incidence of embryonic mortalities which might 68 have occurred between days of blood test and day of con69 firmation of pregnancy by TRUS or transrectal palpation 70 (gold standard) among studies. More recently, ELISA assays 71 for bPSPB and bPAG have been developed and became 72 commercially available. These commercial ELISA assays 73 eliminate the hazard of radiation and enable the producer 74 to apply the test on the farm. The sensitivity of these assays 75 ranged from 93.9% to 100% and their specificity ranged from 76Q3 66.7% to 95.5% at Days 27–38 after breeding (Silva et al., 77 2007; Pare et al., 2008; Howard et al., 2007; Romano and 78 Larson, 2010). 79 To evaluate a new method of pregnancy diagnosis, a 80 contemporary accurate method should be used as a gold 81 standard since a substantial percentage of dairy cows expe82 rienced late embryonic mortalities. TRUS has been used as 83 a gold standard in previous studies since it is safe, non84 invasive and give immediate results. However, its accuracy 85 for pregnancy diagnosis has not reached 100% before Day 86 30 in most field studies (Fricke, 2002). The accuracy of the 28
RIA and ELISA tests for bPSPB or bPAG for early pregnancy diagnosis have not been compared in a same study. Therefore, the present study aimed to compare the accuracy of a commercial PAG-ELISA test (Bovine Preg Test 29) with the PAG-RIA test for diagnosing pregnancy at Day 28 after insemination in dairy cows.
87 88 89 90 91 92
2. Materials and methods
93
2.1. Animals and management
94
One-hundred Holstein Friesian cows belonging to a dairy farm in Hungary were used in the present study from March to June 2010. The parity of the cows ranged between 1 and 8 with an average of 2.4 ± 1.4. The cows were between 55 and 475 days in milk with an average of 163 ± 92.7 days. The animal protocols were approved by the local committee (Faculty Committee for Animal Experiments, Faculty of Veterinary Science, Szent Istvan University, Hungary). According to the routine reproductive management, each non-pregnant cow with a mature corpus luteum (diameter ≥17 mm, Repasi et al., 2003) determined by TRUS, received 500 g of cloprostenol sodium (2 mL of PGF Veyx® Forte A.U.V. inj, Veyx-Pharma GmbH, Schwarzenborn, Germany) intramuscularly. The cows were free from mastitis, laminitis and clinical endometritis. The cow was considered as free from clinical endometritis by absence of purulent or mucopurulent vaginal discharge and absence of uterine fluid by TRUS (Sheldon and Lewis, 2006). Three days later, all of these cows, not inseminated during the first and second day after PGF2␣ treatment, were examined by TRP by one of the inseminators and if the cow showed clinical signs (erected uterus, estrus discharge, and having a palpable follicle) of estrus was inseminated and then 100 g of gonadorelin [6D-Phe] acetate (2 mL of GonavetVeyx® , Veyx-Pharma GmbH) was administered intramuscularly. The semen used for artificial insemination (AI) was chosen by the herd managers as part of the routine management of the herd. The day of AI was considered as Day 0 for calculating the gestation period. 2.2. Ultrasonographic examinations Transrectal ultrasonography was carried out by the same operator using a real time B-mode ultrasound scanner (Falco Vet, Esaote/Pie Medical, Maastricht, The Netherlands) equipped with a 6–8 MHz linear endo-rectal transducer. Transrectal ultrasonography was performed for each cow at Day 28 after AI. Positive diagnoses of pregnancy by transrectal ultrasonography depended on the detection of anechoic allantoic fluid and/or the embryo proper (Szenci et al., 1998). The ovaries were scanned for presence of the corpus luteum. The cows were re-examined by TRUS at Day 42 to confirm the pregnancy diagnoses on the basis of detection of an embryo with a beating heart. 2.3. Milk and blood sampling Milk samples were collected from each cow at Days 0, 21 and 28 after morning milking. After discarding the first 5 squirts of milk from each quarter, an equal amount
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123
124
125 126 127 128 129 130 131 132 133 134 135 136
137
138 139 140
G Model ANIREP 5197 1–7
ARTICLE IN PRESS A. Karen et al. / Animal Reproduction Science xxx (2015) xxx–xxx
152
of milk was collected from each quarter in a clean plastic tube (10 mL). The collected milk was frozen at −20 ◦ C until assayed for progesterone (P4). Immediately after ultrasound scanning at Day 28, blood and milk samples were collected from each cow. The blood sample was withdrawn from the coccygeal blood vessels of each cow using vacutainers (S-Monovette® 9 mL Z, Sarstedt, Nümbrecht, Germany). Blood samples were kept in an icebox for maximum 2 h and then were centrifuged at 2000 × g for 10 min. The collected serum was stored at −20 ◦ C until the assessment of bPAG-1 and bPAG by radioimmunoassay and ELISA, respectively.
153
2.4. Progesterone assay
141 142 143 144 145 146 147 148 149 150 151
163
Progesterone concentrations in the whole milk were determined by using an ELISA test (Ovucheck® milk, Product # C006, Biovet Inc., Saint-Hyacinthe, Canada). Progesterone concentrations were obtained from absorbance micro-plate reader and washer (ELX800 and ELX50, BioTek U.S., Winooski, VT, USA). Intra- and inter-assay coefficients of variation were 5.10% and 8.15%, respectively and the minimum detection limit of the assay was 1 ng/mL. Milk P4 at Day 0 < 3 ng/mL and >7 ng/mL at Days 21 and 28 were threshold for indicating pregnancy (Garmo et al., 2008).
164
2.5. Bovine PAG-1 radioimmunoassy assay
154 155 156 157 158 159 160 161 162
178
Serum concentrations of bPAG-1 were estimated by using a double-antibody radioimmunoassay (RIA-706) as previously described by Perényi et al. (2002a). Rabbit polyclonal antiserum AS#706 was raised against co-purified caprine PAG 55 kDa and PAG 62 kDa (accession numbers P80935 and P80933). The minimum detection limit of the RIA-706 was 200 pg/mL. The intra-assay and inter-assay coefficients of variation were 3.5% and 7.6%, respectively. More details concerning RIA-706 validation (specificity, parallelism, accuracy) were previously reported by different authors (Perényi et al., 2002a,b). Bovine PAG-1 concentration of 800 pg/mL in the plasma (Szenci et al., 1998) was taken as the cut-off point for diagnosing pregnancy.
179
2.6. Bovine PAG-ELISA test
165 166 167 168 169 170 171 172 173 174 175 176 177
180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195
Serum PAG concentrations were determined with a competitive two-step immune-enzymatic assay (Bovine Preg Test 29® , product catalogue number TRM-505, Biovet Inc., Saint-Hyacinthe, Canada). The standard curve ranges from 0 to 3500 pg/mL. The micro-wells were pre-coated with specific antibodies providing a solid phase for capture of PAG in the serum or the standards. After incubation of standards and samples for 1 h at 37 ◦ C in the dark, the wells were thoroughly washed to remove all components other than those bound to antibodies. Pregnancy protein antibodies coupled to horseradish peroxydase (HRPRO) were then added to the wells and allowed to bind the immobilized pregnancy protein during a second incubation (for 1 h in dark place at 37 ◦ C). After washing the wells to remove unbound conjugate, HRPRO substrate was added to the wells and allowed to react during final incubation step for
3
30 min. Thereafter the stop solution was added. The optical density of the well was measured at 450 nm 15 min after the addition of the stop solution using a spectrophotometer (ELX800 BioTek, U.S., Winooski, VT, USA). The cut-off value for diagnosing pregnancy was 500 pg/mL (Paré et al., 2008). At this cut-off value, there was the least difference between the sensitivity and specificity of the test for pregnancy diagnosis in the present study. The inter-assay coefficients of variation of low (15.93 pg/mL) and high (1701 pg/mL) quality control concentrations were 9.1% and 4.2%, respectively. The intra-assay coefficients of variation for both quality control concentrations were less than 5%. The minimum detection limit of the assay was 1.46 pg/mL. 2.7. Gold standard for pregnancy The actual gold standard was based on TRUS outcomes at Day 28 that agreed with the outcomes of PAG-RIA test or PAG-ELISA test. If the outcomes of TRUS at Day 28 and PAGRIA test and PAG-ELISA test did not agree, the gold standard was based on the outcome of TRUS at Day 42 (Silva et al., 2007) and in certain conditions milk P4 profile at Days 0, 21 and 28 after AI, was also considered i.e.: (a) If a cow was found positive by TRUS on Day 28 and the P4 concentrations were <0.3 ng/mL at Day 0 and >7 ng/mL at Days 21 and 28 and she was non-pregnant on PAG ELISA and PAG-RIA tests at Day 28 and found open by TRUS at Day 42, this cow was considered pregnant. (b) If a cow was found open by TRUS on Day 28, but she was positive on PAG ELISA and PAG-RIA test and the P4 concentrations were <0.3 ng/mL at Day 0 and >7 ng/mL at Days 21 and 28 and found open by TRUS at Day 42, this cow was considered pregnant. (c) If a cow was positive by TRUS on the basis of detection of intrauterine fluid, but was negative by PAG test and P4 profile and thereafter was found open by TRUS at Day 42, this cow was considered open at Day 28 after AI. (d) If a cow was found positive by one or both PAG tests but was found negative by TRUS at Day 28 and P4 profile and thereafter was found open by TRUS at Day 42, this cow was considered open at Day 28 after AI. Thus the incorrect TRUS outcomes at Day 28 after AI were adjusted by the pregnancy outcomes based on TRUS at Day 42 and P4 profile at Days 0, 21 and 28 after AI, respectively. 2.8. Statistical analyses Based on the adjusted pregnancy outcomes at Day 28, the results of PAG-RIA and PAG-ELISA tests were classified as: correct positive diagnoses (a), incorrect positive diagnoses (b), correct negative diagnoses (c), and incorrect negative diagnoses (d). From these values, the following diagnostic criteria of PAG-RIA and PAG-ELISA tests and TRUS were calculated (Martin et al., 1987):
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
196 197 198 199 200 201 202 203 204 205 206 207 208
209
210 211 212 213 214 215 216
217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236
237 238 239 240
241
242 243 244 245 246 247 248
G Model
ARTICLE IN PRESS
ANIREP 5197 1–7
A. Karen et al. / Animal Reproduction Science xxx (2015) xxx–xxx
4
Table 1 Comparison of transrectal ultrasonography (TRUS), commercial pregnancy-associated glycoprotein ELISA test and pregnancy-associated glycoprotein-1 radioimmunoassay (PAG-RIA) test for early pregnancy diagnosis in dairy cows at Day 28 after AI based on the actual gold standard. Evaluated criteria
TRUS
PAG-ELISA
PAG-RIA
Correct positive diagnoses (a) Incorrect positive diagnoses (b) Correct negative diagnoses (c) Incorrect negative diagnoses (d) Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Overall accuracy (%)
38 5 54 3 92.7 (84.7–100%) 91.5 (84.4–98.6) 88.4 (78.4 –98.0%) 94.7 (88.9–100%) 92 (86.7–97.3%)
37 1 58 4 90.2 (81.2–99.3%) 98.3 (95.0–100%) 97.4 (92.3–100%) 93.7 (87.4–99.7%) 95 (90.7–99.3)
41 3 56 0 100 (100–100%) 94.9 (89.3–100%) 93.2 (85.7–100%) 100 (100–100%) 97 (93.7–100%)
Percentages within parenthesis indicate 95% confidence interval.
249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266
(i) The sensitivity (Se) — — number of correct positive pregnancy diagnoses diagnoses/all pregnant cows based on the actual gold standard × 100 (a/a + d × 100); (ii) The specificity (Sp) = number of correct negative pregnancy diagnoses by the test/all nonpregnant cows based on the actual gold standard × 100 (c/c + b × 100); (iii) the positive predictive (PPV) = number of correct positive pregnancy diagnoses/number of diagnosed pregnant by blood test or TRUS × 100 (a/a + b × 100); (iv) the negative predictive value (NPV) = number of correct negative pregnancy diagnoses/number of diagnosed non pregnant by blood test or TRUS × 100 (c/c + d × 100) and (v) the overall accuracy (Ac) = number of correct positive pregnancy diagnoses + number of correct negative pregnancy diagnoses/total number of cows tested by diagnostic tests × 100 (a + c/a + b + c + d × 100). Also, 95%-confidence interval of each accuracy parameter of the diagnostic tests was determined.
272
Estimates of the agreement between TRUS, PAG-RIA and PAG-ELISA were determined by using Kappa statistic in GraphPad Software. A Kappa value of 0.4–0.5 indicates a moderate degree of agreement, 0.5–0.6 indicates good agreement and >0.6 indicates a high level of agreement (Martin et al., 1987).
273
3. Results
267 268 269 270 271
274 275 276 277 278 279 280
Based on the actual gold standard, 41 of 100 inseminated cows were pregnant at Day 28 after AI. Nine of the 41 pregnant cows were considered as experienced late embryonic mortalities after Day 28. These 9 cows were positive at Days 28 by the TRUS, the PAG-RIA and the P4 tests, respectively. By the PAG-ELISA test, 7 of these 9 cows were positive at Day 28. Thereafter, these 9 cows were
open by TRUS at Day 42 after AI. Therefore these 9 cows included in the analyses as pregnant cows at Day 28 after AI. The embryo proper with a beating heart was observed by TRUS only in 41.5% (17/41) pregnant cows at Day 28 after AI. The accuracy values of the TRUS, the PAG-RIA and the PAG-ELISA tests are presented in Table 1. By TRUS, three pregnant cows were diagnosed negative at Day 28 after AI; neither the allantoic fluid nor the embryo was detected. In these cows, the bPAG concentrations measured by the PAGRIA and the PAG-ELISA tests and milk P4 concentrations were higher than the thresholds for diagnosing pregnancy and they were pregnant by TRUS at Day 42. In addition 5 non-pregnant cows, based on the actual gold standard, were diagnosed positive by ultrasonography. In these 5 cows, only non-echogenic fluid was observed in the uterus by TRUS. In these false positive ultrasonographic diagnoses, the bPAG concentrations measured by the RIA and the ELISA tests at Day 28 after AI were less than the threshold for diagnosing pregnancy. Furthermore, the P4 concentrations were lower than 3 ng/mL at Day 0 and <7 ng/mL at Day 21 in 3 cows, while they were >3 ng/mL at Day 0 and >7 ng/mL at Day 21 in 2 cows (Table 2). In addition, these five cows were open by TRUS at Day 42 after AI. Regarding the PAG-ELISA test, four pregnant cows (false negative diagnosis) had PAG concentrations lower than the threshold for diagnosing pregnancy. These cows were positive by the TRUS, the PAG-RIA and the P4 tests, two of them were diagnosed pregnant at Day 42 and the other 2 cows were non-pregnant by Day 42. Regarding the PAG-RIA test, all pregnant cows had bPAG-1 concentrations at Day 28 after AI higher than the threshold for diagnosing pregnancy, however, 3 nonpregnant cows had bPAG-1 concentrations higher than the threshold for diagnosing pregnancy. These cows were diagnosed non-pregnant by the TRUS and the P4 test.
Table 2 Pregnancy-associated glycoprotein (PAG) concentrations measured by radioimmunoassay (RIA) and ELISA tests at Day 28 and milk progesterone (P4) concentrations at Days 0, 21 and 28 after AI in cows with false positive ultrasonographic diagnoses at Day 28 after AI. Cow ID
A B C D E
PAG tests at Day 28
P4 test (ng/mL)
RIA (pg/mL)
ELISA (pg/mL)
Day 0
Day 21
Day 28
200 200 200 300 200
25 2.0 79 61 68
2.5 1.8 1.3 3.2 5.7
1.0 2.3 3.0 24.9 21.2
17.8 9.3 17.9 30.6 3.1
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315
G Model ANIREP 5197 1–7
ARTICLE IN PRESS A. Karen et al. / Animal Reproduction Science xxx (2015) xxx–xxx
323
Also, two of these cows were negative by the PAG-ELISA test while, the third one was positive (false positive diagnosis). These cows were in milk for 83, 87 and 98 days after calving, respectively. The degree of agreement (Kappa ± S.E.) between the PAG-RIA and the PAG-ELISA tests was 0.90 ± 0.04. The degrees of agreement of TRUS at Day 28 with the PAG-RIA and the PAG-ELISA tests were 0.80 ± 0.5 and 0.76 ± 0.06, respectively.
324
4. Discussion
316 317 318 319 320 321 322
325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374
In the present study, the sensitivity (92.7%) and the specificity (91.5%) of TRUS at Day 28 after AI were slightly less than those (96.5% and 93.4%, respectively) reported for TRUS at Day 27 when the outcomes of TRUS was adjusted to the outcome of the PAG-ELISA test at Days 27 and TRUS at Day 32 (Silva et al., 2007). The specificity of TRUS at Day 28 obtained in the present study is greater than those (54–87.4%) reported in other studies in which the TRUS outcomes were confirmed by either TRUS at Days 32–35 or rectal palpation between Days 50 and 90 (Pieterse et al., 1990; Badtram et al., 1991; Hanzen and Laurent, 1991; Nation et al., 2003; Romano et al., 2006). Unlike the abovementioned studies, TRUS was carried out concurrent with PAG-ELISA and PAG-RIA tests at Day 28 after AI in the present study which enabled us to diagnose late embryonic mortalities occurred after Day 28 till Day 42. Since cows suffered from late embryonic mortalities (n = 9) were confirmed pregnant by the PAG-ELISA and the TRUS tests at Day 28, they were included in the analyses as pregnant cows and consequently decreased the false positive diagnoses and a higher specificity of TRUS obtained in the present study. Five cows which were incorrectly diagnosed pregnant by TRUS at Day 28 were non-pregnant at the time of the examination, whereas, the PAG values were less than the threshold for diagnosing pregnancy and P4 values were either less that than the threshold for diagnosing pregnancy at Days 21 and 28 (n = 3) or it was higher than 3 ng/mL at Day 0 (n = 2) indicating that the cows were incorrectly inseminated at Day 0. Some of these cows (n = 3) might have been in estrus at the ultrasonographic examination and the detection of a mature ovarian follicle was overlooked due to the limitation of time imposed by the cow flow in this large dairy farm (1000 cows milked daily). Similarly, Nation et al. (2003) observed intrauterine fluid by TRU in 4 cows at Days 28–35 and diagnosed open when re-examined 7 days later. The embryo proper with a beating heart was observed by TRUS only in 41.5% of pregnant cows in the present study. Similar percentage (43.1%) was reported by Silva et al. (2007) at Day 27 after AI. The reason of this low percentage of detection of embryo by TRUS at Day 28 might be the small mass embryo and the time constraints for individual cow diagnoses imposed by the cow flow on the commercial dairy (Silva et al., 2007). The sensitivity of the PAG-ELISA test (Bovine Preg Test 29) for diagnosing pregnant cows in the present study (90.2%) was lower than that (99.4%) reported in a study using the same test but samples were withdrawn somewhat later between Days 29 and 36 after AI (Pare et al., 2008). The sensitivity of the PAG-ELISA test increased with increasing the time interval after AI (Green et al., 2009;
5
Friedrich and Holtz, 2010; Sinedino et al., 2014). Also it was lower than those (93.9% to 98.8%) reported in other studies using different commercial PAG-ELISA tests between Days 25 and 29 after AI (Silva et al., 2007; Green et al., 2009; Romano and Larson, 2010; Sinedino et al., 2014). This difference could be attributed to the difference in the PAG subsets used for the development of antisera in the different commercial tests whereas it is important to mention that there are more than 22 bovine transcribed genes encoding PAG expression that are expressed temporally at varying levels throughout gestation (Green et al., 2000). The specificity of the PAG-ELISA test for diagnosing nonpregnant cows obtained in the present study (98.3%) was much higher than those (77–90.7%) reported in the literature evaluating the accuracy of the PAG-ELISA test at Days 26–30 on the basis of TRUS or palpation per rectum performed at different time intervals from PAG-ELISA tests (Friedrich and Holtz, 2010; Fernandez et al., 2013; Sinedino et al., 2014). On the other hand, the specificity of the test in the present study was similar or slightly higher than those (91.1–97.2%) reported in those studies in which the TRUS was carried out concurrent with the PAG-ELISA test (Silva et al., 2007; Green et al., 2009; Romano and Larson, 2010; Piechotta et al., 2011). Performing the PAG-ELISA test concurrent with the TRUS enabled us to diagnose late embryonic mortalities which occurred after Day 28 till Day 42. Since cows suffered from late embryonic mortalities were confirmed pregnant by the PAG-ELISA and the TRUS tests at Day 28, they were included as pregnant cows and consequently decreased the false positive diagnoses. On the other hand, performing the PAG-ELISA test without contemporary TRUS could not differentiate between pregnant cows at Day 28 and those suffered from embryonic mortalities before testing (false positive diagnoses). Bovine PAG has about a 3-day half-life after experimentally induced embryonic mortality in heifers (Szenci et al., 2003) therefore residual quantities of the proteins may persist in the maternal circulation till the time of sampling. This explanation is supported by the finding of a study using the same commercial kit used in the present study in which the specificity of the test was 100% in non-inseminated cows, while it was 66.7% in the inseminated cows (Pare et al., 2008). The sensitivity (100%) and the specificity (94.4%) of the PAG-RIA test were higher than those (95% and 69.3%, respectively) reported in a study performed at Days 29–30 after AI (Szenci et al., 1998). The higher sensitivity in the present study is attributed to the use of heterologous RIA system (RS#706; a combination of caprine PAG 55 kDa and 62 kDa antisera) which is better than the homologous RIA system (RS #497 bPAG-1 67 kDa antibody) used in the study of Szenci et al. (1998) for recognizing PAG secreted by the bovine placenta between Days 21 and 51 of gestation (Perényi et al., 2002b). The higher specificity of PAG-RIA test in this study is attributed to the use of adjusted TRUS outcome at Day 28 as a basis for calculating the accuracy of PAG-RIA test, while in the other study (Szenci et al., 1998), the outcomes of TRUS at Day 58 was used as a gold standard for calculating the accuracy of the PAG-RIA test. The three false positive diagnoses made by the PAG-RIA test were negative by TRUS and by the P4 test at Days 21 and 28, and
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435
G Model ANIREP 5197 1–7
A. Karen et al. / Animal Reproduction Science xxx (2015) xxx–xxx
6
436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473
474
475
476
477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497
ARTICLE IN PRESS
two of them were negative by the PAG-ELISA test, while one was positive (Day in milk was 83) confirming that these cows were non-pregnant at Day 28 after AI. The residual amount of PAG from previous calving (Days 83 and 98 days in milk, respectively) might be the reason of the higher PAG values measured by the RIA test (Zoli et al., 1992). In addition, the bovine placental PAG measured by bovine Preg 29 test might have disappeared earlier after calving than that measured by the RIA test. The high degrees of agreement among the TRUS, the PAG-RIA and the PAG-ELISA tests confirm that the three tests had similar accuracies for diagnosing early pregnancy in dairy cows. There are advantages of TRU when compared with the PAG blood tests. Transrectal ultrasonography provides an instantaneous diagnosis of pregnancy. It also has the ability to evaluate the uterine and ovarian morphology of non-pregnant cows and identify the dead embryo and nonviable pregnancy (Lucy et al., 2011). These advantages must be weighed against the cost of ultrasound equipment, the technical skill required when performing the ultrasound procedure and whether or not the farm has access to trained person who can do early pregnancy diagnosis with ultrasound (Fricke, 2002). The PAG-ELISA test does not need a specialized lab for performing the RIA test and skilled practitioner to carry out transrectal ultrasonography. Pregnancy diagnosis by PAG-ELISA test at Day 28 would enable implementation of earlier re-synchrony of the open cows. However, about 6.3% (false negative diagnoses) of diagnosed non pregnant cows by PAG-ELISA test in the present study would suffer from iaterogenic abortion if they would have been involved in resynchronization program which might offset the advantage of early resynchrony. In conclusion, the commercial PAG-ELISA test (Bovine Preg Test 29® ) is a highly accurate method for diagnosing early pregnancy in dairy cows on Day 28 after AI and may be used as an alternative method to the TRUS and the PAG-RIA test. Conflict of interest The authors declare no conflict of interest. References Badtram, G.A., Gains, J.D., ThomasCB, Bosu, W.T.K., 1991. Factors influencing the accuracy of early pregnancy detection in cattle by real-time ultrasound scanning of the uterus. Theriogenology 35, 1153–1167. Breukelman, S.P., Perényi, Z., Taverne, M.A.M., Jonker, H., van der Weijden, G.C., Vos, P.L.A.M., de Ruigh, L., Dieleman, S.J., Beckers, J.F., Szenci, O., 2012. Characterization of pregnancy losses after embryo transfer by measuring plasma progesterone and bovine pregnancy-associated glycoprotein-1. Vet. J. 194, 71–76. Fernandez, G.I., Moncebaez, J., Elinzondo, C., Hernandez, H., Ulloa-Arvizu, R., Rojas, S., 2013. Negative prediction value of pregnancy-associated glycoprotein contributes to reduce the days during which nonpregnant Holstein cows are subjected to diverse strategies of hormonal synchronization. Int. J. Anim. Vet. Adv. 5, 160–164. Fricke, P.M., 2002. Scanning the future: ultrasonography as a reproductive management tool for dairy cow. J. Dairy Sci. 85, 1918–1926. Friedrich, M., Holtz, W., 2010. Establishment of an ELISA for measuring bovine pregnancy associated glycoprotein in serum or milk and its application for early pregnancy detection. Reprod. Dom. Anim. 45, 142–146. Garmo, R.T., Refsdal, A.O., Karlberg, K., Ropstad, E., Waldmann, A., Beckers, J.F., Reksen, O., 2008. Pregnancy incidence in Norwegian red cows
using nonreturn to estrus, rectal palpation, pregnancy associated glycoproteins and progesterone. J. Dairy Sci. 91, 3025–3033. Green, J.A., Xie, S., Quan, X., Bao, B., Gan, X., Mathialagan, N., Beckers, J.F., Roberts, M.R., 2000. Pregnancy-associated bovine and ovine glycoproteins exhibit spatially and temporally distinct expression patterns during pregnancy. Biol. Reprod. 62, 1624–1631. Green, J.A., Parks, E.T., Avalle, P.M., Telugu, P.B., McLain, L.A., Peterson, J., Mc Millan, W., Mathialagan, N., Hook, P.R., Xie, S., Roberts, M., 2005. The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers. Theriogenology 63, 1481–1503. Green, J.C., Volkmann, D.H., Poock, S.E., McGarth, M.F., Ehrhadt, M., Moseley, A.E., Lucy, M.C., 2009. Technical note: a rapid enzyme linked immunosorbent assay blood test for pregnancy in dairy and beef cattle. J. Dairy Sci. 92, 3819–3824. Hanzen, C., Laurent, Y., 1991. Application de l‘échographie bidimensionnelle au diagnostic de gestation et l‘évaluation de l‘incidence de la mortalité, embryonnaire dans l’espèce bovine. Ann. Med. Vet. 134, 481–487. Howard, J., Gabor, G., Gray, T., Passavant, C., Ahmadzadeh, A., Sasser, N., Pals, D., Sasser, G., 2007. BioPRYN, a blood-based pregnancy test for managing breeding and pregnancy in cattle. In: Proceeding, Western Section, American Society of Animal Science, vol. 58, pp. 295–298. Humblot, P., Camous, S., Martal, J., Charlery, J., Jeanguyot, N., Thibier, M., Sasser, R.G., 1988. Diagnosis of pregnancy by radioimmunoassay of a pregnancy specific protein in the plasma of dairy cows. Theriogenology 30, 257–267. Lucy, M., Green, J., Poock, S., 2011. Pregnancy determination in cattle: a review of available alternatives. In: Proceedings, Applied Reproductive Strategies in Beef Cattle, August 31–September 1, 2011, Joplin, MO, pp. 367–375. Martin, S.W., Meek, A.H., Willeberg, P., 1987. Veterinary Epidemiology, Principles and Methods. Iowa State University Press, Ames, IA, pp. 63–71. Nakao, T., Sugihashi, A., Ishibashi, Y., Tosa, E., Nakagawa, Y., Yuto, H., Nomura, T., Ohe, T., Ishimi, S., Takahashi, H., Kowia, M., Tusnoda, N., Kawata, K., 1982. Use of milk progesterone enzymeimmunoassay for early pregnancy diagnosis in cows. Theriogenology 18, 267–274. Nation, D.P., Malmo, J., Davis, G.M., Macmillan, K.L., 2003. Accuracy of bovine pregnancy detection using transrectal ultrasonography at 28 to 35 days after insemination. Aust. Vet. J. 81, 63–65. Paré, J., Audet-Grenier, M.-H., Rouillier, Sirard, M.-A., 2008. Evaluation of the DG29 test for early detection of pregnancy in cattle. Can. Vet. J. 49, 1119–1121. Perényi, Z., Szenci, O., Sulon, J., Drion, P.V., Beckers, J.F., 2002a. Comparison of the ability of three radioimmunoassays to detect pregnancyassociated glycoproteins in bovine plasma. Reprod. Domest. Anim. 37, 100–104. Perényi, Z., Szenci, O., Drion, P.V., Banga-Mboko, H., Sousa, N.M., El Amiri, B., Beckers, J.F., 2002b. Aspartic proteinase members secreted by the ruminant placenta: specificity of three radioimmunoassay systems for the measurement of pregnancy-associated glycoproteins. Reprod. Domest. Anim. 37, 324–329. Piechotta, M., Bollwien, J., Friedrich, M., Heilkenbrinker, T., Passavant, C., Branen, J., Sasser, G., Hoedemaker, M., Bollwein, H., 2011. Comparison of commercial ELISA blood tests for early pregnancy detection. J. Reprod. Dev. 57, 72–75. Pieterse, M.C., Szenci, O., Wiilemse, A.H., Bajcsy, A.C., Dieleman, S.J., Taverne, M.A.M., 1990. Early pregnancy diagnosis in cattle by means of linear-array real-time ultrasound scanning of the uterus and a quantitative and qualitative milk progesterone test. Theriogenology 33, 697–707. Repasi, A., Beckers, J.F., Sulon, J., Perenyi Zs, Reiczigel, J., Szenci, O., 2003. Effect of different doses of prostaglandin on the area of corpus luteum, the largest follicle and progesterone concentration in the dairy cow. Reprod. Dom. Anim. 38, 1–6. Romano, J.E., Larson, J.E., 2010. Accuracy of pregnancy specific protein-B test for early pregnancy diagnosis in dairy cattle. Theriogenology 74, 932–939. Romano, J.E., Thompson, J.A., Forrest, D.W., Westhusin, M.E., Tomaszweski, M.A., Kraemer, D.C., 2006. Early pregnancy diagnosis by transrectal ultrasonography in dairy cattle. Theriogenology 66, 1034–1041. Sasser, R.G., Ruder, C.A., Ivani, K.A., Butler, J.E., Hamilton, W.C., 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, 936–942. Sauer, M.J., Foulkes, J.A., Worsfold, A., Morris, B.A., 1986. Use of progesterone II-glucuronide-alkaline phosphatase conjugate in a sensitive microtitre-plate enzyme immunoassay of progesterone in milk and
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576
G Model ANIREP 5197 1–7
ARTICLE IN PRESS A. Karen et al. / Animal Reproduction Science xxx (2015) xxx–xxx
577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593
its application to pregnancy testing in dairy cattle. J. Reprod. Fertil. 76, 375–391. Sheldon, I.M., Lewis, G., LeBlanc, S., Gilbert, R., 2006. Defining postpartum uterine disease in dairy cattle. Theriogenology 65, 1516–1530. Silva, E., Sterry, R.A., Kolb, D., Mathialagan McGarth, M.F., Ballam, J.M., Fricke, P.M., 2007. Accuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy status of lactating dairy cows twenty seven days after timed artificial insemination. J. Dairy Sci. 90, 4612–4622. Sinedino, L.D.P., Lima, F.S., Bisinotto, R.S., Cerri, R.L.A., Santos, J.E.P., 2014. Effect of early or late resynchronization based on different methods of pregnancy diagnosis on reproductive performance of dairy cows. J. Dairy Sci. 97, 1–10. Skinner, J.G., Gray, D., Gebbie, F.E., Beckers, J.F., Sulon, J., 1996. Field evaluation of pregnancy diagnosis using bovine pregnancy-associated glycoproetein (bPAG). Cattle Pract. 4, 281–284. Szenci, O., Beckers, J.F., Humblot, P., Sulon, J., Sasser, G., Taverne, M.A.M., Varga, J., Baltusen, R., Schekk, Gy., 1998. Comparison of
7
ultrasonography, bovine pregnancy-specific protein B, and bovine pregnancy-associated glycoprotein 1 tests for pregnancy detection in dairy cows. Theriogenology 50, 77–88. Szenci, O., Beckers, J.F., Sulon, J., Bevers, M.M., Börzsönyi, L., Fodor, L., Kovács, F., Taverne, M.A.M., 2003. Effect of induction of late embryonic mortality on plasma profiles of pregnancy associated glycoproteins in heifers. Vet. J. 165, 307–313. Wooding, F.B., Roberts, R.M., Green, J.A., 2005. Light and electron microscope immunocytochemical studies of the distribution of pregnancy-associated glycoproteins (PAGs) throughout pregnancy in the cow: possible functional implications. Placenta 26, 807–827. Youngquist, R.S., 2007. Pregnany diagnosis. In: Youngquist, R.S., Threlfall, W.R. (Eds.), Current Therapy in Large Animal Theriogenology. , 2nd edition. Saunder, Missouri, USA, pp. 294–303. Zoli, A.P., Guilbault, L.A., Delahaut, P., Ortiz, W.B., Beckers, J.F., 1992. Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy diagnosis. Biol. Reprod. 46, 83–92.
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611
ARTICLE IN PRESS
ANIREP 5197 1–7
Animal Reproduction Science xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle
2
3
4
5
6 7 8 9 10 11 12 13
Q1
Aly Karen a,b,∗ , Noelita Melo De Sousa c , Jean-Franc¸ois Beckers c , Árpád Csaba Bajcsy a,d , János Tibold e , István Mádl e , Ottó Szenci a,d a
Clinic for Large Animals, Faculty of Veterinary Science, Szent István University, Üllo-Dóra major, 2225, Hungary ˝ Department of Theriogenology, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516 Kafrelsheikh, Egypt Laboratory of Animal Endocrinology and Reproduction, Laboratory of Animal Endocrinology and Reproduction, Faculty of Veterinary Medicine, University of Liege, Liege, 4000 Belgium d MTA-SZIE Large Animal Clinical Research Group, Üllo-Dóra major, 2225, Hungary ˝ e Agroproduct Zrt, Pápa, 8500, Hungary b c
14
15
a r t i c l e
i n f o
a b s t r a c t
16 17 18 19 20 21
Article history: Received 28 November 2014 Received in revised form 13 April 2015 Accepted 7 May 2015 Available online xxx
22 23 24 25 26
Keywords: Transrectal ultrasonography Bovine pregnancy proteins Pregnancy diagnosis
The present study aimed to compare the accuracy of a commercial PAG-ELISA test (Bovine Preg Test 29) and bovine pregnancy-associated glycoprotein radioimmunoassay (PAG-RIA) for diagnosing pregnancy at Day 28 after insemination in dairy cows. Transrectal ultrasonography (TRUS) was performed in 100 Holstein-Friesian cows at Day 28 after artificial insemination (AI; Day 0) to diagnose pregnancy. After TRUS examination, blood sample was collected from the coccygeal vessels of each cow to measure the concentrations of bPAGs by PAG-RIA test and Bovine Preg Test 29. Milk samples were collected at Days 0, 21 and 28 for measurement of progesterone (P4) by ELISA test. The cows were reexamined by TRUS at Day 42 to confirm the pregnancy diagnoses. The actual gold standard was based on TRUS outcomes at Day 28 that agreed with the outcomes of PAG-RIA test or PAG-ELISA test. If the outcomes of TRUS at Day 28 and PAG-RIA test and PAG-ELISA test did not agree, the gold standard was based on the outcome of TRUS at Day 42. Out of 100 inseminated cows, 41 were confirmed pregnant at Day 28 after AI. Based on the actual gold standard, the sensitivity of TRUS, PAG-ELISA and PAG-RIA tests for diagnosing pregnant cows at Day 28 were 92.7%, 90.2% and 100%, while the specificity of the three tests for diagnosing non-pregnant cows were 91.5%, 98.3% and 94.4%, respectively. The overall accuracy of the three tests were 92%, 95% and 97%, respectively. The degree of agreement (Kappa ± S.E.) between PAG-RIA and PAG-ELISA test was 0.90 ±0.04. The degrees of agreement between PAG-RIA and PAG-ELISA and TRUS at Day 28 were 0.80 ±0.5 and 0.76 ± 0.06, respectively. In conclusion, the commercial PAG-ELISA test is a highly accurate method for diagnosing early pregnancy in dairy cows on Day 28 after AI and may be used as an alternative method to the TRUS and the PAG-RIA test. © 2015 Elsevier B.V. All rights reserved.
∗ Corresponding author at: Department of Theriogenology, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516 Kafrelsheikh, Egypt. Tel.: +20 1002773137; fax: +20 473231311. E-mail address: [email protected] (A. Karen). http://dx.doi.org/10.1016/j.anireprosci.2015.05.005 0378-4320/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
G Model ANIREP 5197 1–7
A. Karen et al. / Animal Reproduction Science xxx (2015) xxx–xxx
2 27
ARTICLE IN PRESS
1. Introduction
Q2
Early pregnancy diagnosis is a management tool 29 required for shortening the calving interval in dairy cows. 30 In addition identification of non-pregnant cows as early 31 as possible after insemination reduces inter-insemination 32 interval for herds performing synchronization and resyn33 chronization without estrus detection (Lucy et al., 2011). 34 The diagnostic method used for early pregnancy diag35 nosis should be accurate, practical, fast, and inexpensive. 36 Transrectal palpation (TRP) is widely used for pregnancy 37 diagnosis in cows; however; with a moderate amount of 38 experience, a reliable diagnosis of pregnancy is possible 39 after Days 35–39 (Youngquist, 2007). Transrectal ultraso40 nography (TRUS) is an accurate method for early pregnancy 41 diagnosis from Day 29 after insemination of dairy cows 42 (Romano et al., 2006), however, it requires a specialized 43 equipment and a trained person to perform the scanning 44 (Lucy et al., 2011). Alternatively, blood or milk chemi45 cal tests can be used before Day 29 after insemination. 46 These tests are: progesterone (P4) and bovine pregnancy47 associated glycoproteins (bPAGs). Progesterone-ELISA test 48 (cow-side test) can be applied in the milk at Days 21–24 49 after insemination with a 100% negative predictive value; 50 however the test has relatively low (60–94%) positive pre51 dictive value (Nakao et al., 1982; Sauer et al., 1986). 52 Bovine pregnancy-associated glycoproteins (bPAGs) or 53 pregnancy specific protein B (bPSPB) are mainly secreted by 54 trophoblastic binucleate cells (Wooding et al., 2005) and 55 they have been detected in the blood of some pregnant 56 cows as early as Days 15–22 after AI (Sasser et al., 1986; 57 Zoli et al., 1992; Green et al., 2005). Therefore, these 58 glycoproteins have been used as a marker of pregnancy 59 and feto-placental well-being in cattle (Breukelman et al., 60 2012). Radioimmunoassays (RIA) for bPSPB or bPAG-1 have 61 a high sensitivity (95–100%) for identifying pregnant cattle 62 from Day 30 after breeding onwards. However, the speci63 ficity of these tests for identifying non-pregnant cows in 64 most studies is greatly variable ranging from 69.3% to 93% 65 (Sasser et al., 1986; Humblot et al., 1988; Zoli et al., 1992; 66 Skinner et al., 1996; Szenci et al., 1998) due to the differ67 ence in the incidence of embryonic mortalities which might 68 have occurred between days of blood test and day of con69 firmation of pregnancy by TRUS or transrectal palpation 70 (gold standard) among studies. More recently, ELISA assays 71 for bPSPB and bPAG have been developed and became 72 commercially available. These commercial ELISA assays 73 eliminate the hazard of radiation and enable the producer 74 to apply the test on the farm. The sensitivity of these assays 75 ranged from 93.9% to 100% and their specificity ranged from 76Q3 66.7% to 95.5% at Days 27–38 after breeding (Silva et al., 77 2007; Pare et al., 2008; Howard et al., 2007; Romano and 78 Larson, 2010). 79 To evaluate a new method of pregnancy diagnosis, a 80 contemporary accurate method should be used as a gold 81 standard since a substantial percentage of dairy cows expe82 rienced late embryonic mortalities. TRUS has been used as 83 a gold standard in previous studies since it is safe, non84 invasive and give immediate results. However, its accuracy 85 for pregnancy diagnosis has not reached 100% before Day 86 30 in most field studies (Fricke, 2002). The accuracy of the 28
RIA and ELISA tests for bPSPB or bPAG for early pregnancy diagnosis have not been compared in a same study. Therefore, the present study aimed to compare the accuracy of a commercial PAG-ELISA test (Bovine Preg Test 29) with the PAG-RIA test for diagnosing pregnancy at Day 28 after insemination in dairy cows.
87 88 89 90 91 92
2. Materials and methods
93
2.1. Animals and management
94
One-hundred Holstein Friesian cows belonging to a dairy farm in Hungary were used in the present study from March to June 2010. The parity of the cows ranged between 1 and 8 with an average of 2.4 ± 1.4. The cows were between 55 and 475 days in milk with an average of 163 ± 92.7 days. The animal protocols were approved by the local committee (Faculty Committee for Animal Experiments, Faculty of Veterinary Science, Szent Istvan University, Hungary). According to the routine reproductive management, each non-pregnant cow with a mature corpus luteum (diameter ≥17 mm, Repasi et al., 2003) determined by TRUS, received 500 g of cloprostenol sodium (2 mL of PGF Veyx® Forte A.U.V. inj, Veyx-Pharma GmbH, Schwarzenborn, Germany) intramuscularly. The cows were free from mastitis, laminitis and clinical endometritis. The cow was considered as free from clinical endometritis by absence of purulent or mucopurulent vaginal discharge and absence of uterine fluid by TRUS (Sheldon and Lewis, 2006). Three days later, all of these cows, not inseminated during the first and second day after PGF2␣ treatment, were examined by TRP by one of the inseminators and if the cow showed clinical signs (erected uterus, estrus discharge, and having a palpable follicle) of estrus was inseminated and then 100 g of gonadorelin [6D-Phe] acetate (2 mL of GonavetVeyx® , Veyx-Pharma GmbH) was administered intramuscularly. The semen used for artificial insemination (AI) was chosen by the herd managers as part of the routine management of the herd. The day of AI was considered as Day 0 for calculating the gestation period. 2.2. Ultrasonographic examinations Transrectal ultrasonography was carried out by the same operator using a real time B-mode ultrasound scanner (Falco Vet, Esaote/Pie Medical, Maastricht, The Netherlands) equipped with a 6–8 MHz linear endo-rectal transducer. Transrectal ultrasonography was performed for each cow at Day 28 after AI. Positive diagnoses of pregnancy by transrectal ultrasonography depended on the detection of anechoic allantoic fluid and/or the embryo proper (Szenci et al., 1998). The ovaries were scanned for presence of the corpus luteum. The cows were re-examined by TRUS at Day 42 to confirm the pregnancy diagnoses on the basis of detection of an embryo with a beating heart. 2.3. Milk and blood sampling Milk samples were collected from each cow at Days 0, 21 and 28 after morning milking. After discarding the first 5 squirts of milk from each quarter, an equal amount
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123
124
125 126 127 128 129 130 131 132 133 134 135 136
137
138 139 140
G Model ANIREP 5197 1–7
ARTICLE IN PRESS A. Karen et al. / Animal Reproduction Science xxx (2015) xxx–xxx
152
of milk was collected from each quarter in a clean plastic tube (10 mL). The collected milk was frozen at −20 ◦ C until assayed for progesterone (P4). Immediately after ultrasound scanning at Day 28, blood and milk samples were collected from each cow. The blood sample was withdrawn from the coccygeal blood vessels of each cow using vacutainers (S-Monovette® 9 mL Z, Sarstedt, Nümbrecht, Germany). Blood samples were kept in an icebox for maximum 2 h and then were centrifuged at 2000 × g for 10 min. The collected serum was stored at −20 ◦ C until the assessment of bPAG-1 and bPAG by radioimmunoassay and ELISA, respectively.
153
2.4. Progesterone assay
141 142 143 144 145 146 147 148 149 150 151
163
Progesterone concentrations in the whole milk were determined by using an ELISA test (Ovucheck® milk, Product # C006, Biovet Inc., Saint-Hyacinthe, Canada). Progesterone concentrations were obtained from absorbance micro-plate reader and washer (ELX800 and ELX50, BioTek U.S., Winooski, VT, USA). Intra- and inter-assay coefficients of variation were 5.10% and 8.15%, respectively and the minimum detection limit of the assay was 1 ng/mL. Milk P4 at Day 0 < 3 ng/mL and >7 ng/mL at Days 21 and 28 were threshold for indicating pregnancy (Garmo et al., 2008).
164
2.5. Bovine PAG-1 radioimmunoassy assay
154 155 156 157 158 159 160 161 162
178
Serum concentrations of bPAG-1 were estimated by using a double-antibody radioimmunoassay (RIA-706) as previously described by Perényi et al. (2002a). Rabbit polyclonal antiserum AS#706 was raised against co-purified caprine PAG 55 kDa and PAG 62 kDa (accession numbers P80935 and P80933). The minimum detection limit of the RIA-706 was 200 pg/mL. The intra-assay and inter-assay coefficients of variation were 3.5% and 7.6%, respectively. More details concerning RIA-706 validation (specificity, parallelism, accuracy) were previously reported by different authors (Perényi et al., 2002a,b). Bovine PAG-1 concentration of 800 pg/mL in the plasma (Szenci et al., 1998) was taken as the cut-off point for diagnosing pregnancy.
179
2.6. Bovine PAG-ELISA test
165 166 167 168 169 170 171 172 173 174 175 176 177
180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195
Serum PAG concentrations were determined with a competitive two-step immune-enzymatic assay (Bovine Preg Test 29® , product catalogue number TRM-505, Biovet Inc., Saint-Hyacinthe, Canada). The standard curve ranges from 0 to 3500 pg/mL. The micro-wells were pre-coated with specific antibodies providing a solid phase for capture of PAG in the serum or the standards. After incubation of standards and samples for 1 h at 37 ◦ C in the dark, the wells were thoroughly washed to remove all components other than those bound to antibodies. Pregnancy protein antibodies coupled to horseradish peroxydase (HRPRO) were then added to the wells and allowed to bind the immobilized pregnancy protein during a second incubation (for 1 h in dark place at 37 ◦ C). After washing the wells to remove unbound conjugate, HRPRO substrate was added to the wells and allowed to react during final incubation step for
3
30 min. Thereafter the stop solution was added. The optical density of the well was measured at 450 nm 15 min after the addition of the stop solution using a spectrophotometer (ELX800 BioTek, U.S., Winooski, VT, USA). The cut-off value for diagnosing pregnancy was 500 pg/mL (Paré et al., 2008). At this cut-off value, there was the least difference between the sensitivity and specificity of the test for pregnancy diagnosis in the present study. The inter-assay coefficients of variation of low (15.93 pg/mL) and high (1701 pg/mL) quality control concentrations were 9.1% and 4.2%, respectively. The intra-assay coefficients of variation for both quality control concentrations were less than 5%. The minimum detection limit of the assay was 1.46 pg/mL. 2.7. Gold standard for pregnancy The actual gold standard was based on TRUS outcomes at Day 28 that agreed with the outcomes of PAG-RIA test or PAG-ELISA test. If the outcomes of TRUS at Day 28 and PAGRIA test and PAG-ELISA test did not agree, the gold standard was based on the outcome of TRUS at Day 42 (Silva et al., 2007) and in certain conditions milk P4 profile at Days 0, 21 and 28 after AI, was also considered i.e.: (a) If a cow was found positive by TRUS on Day 28 and the P4 concentrations were <0.3 ng/mL at Day 0 and >7 ng/mL at Days 21 and 28 and she was non-pregnant on PAG ELISA and PAG-RIA tests at Day 28 and found open by TRUS at Day 42, this cow was considered pregnant. (b) If a cow was found open by TRUS on Day 28, but she was positive on PAG ELISA and PAG-RIA test and the P4 concentrations were <0.3 ng/mL at Day 0 and >7 ng/mL at Days 21 and 28 and found open by TRUS at Day 42, this cow was considered pregnant. (c) If a cow was positive by TRUS on the basis of detection of intrauterine fluid, but was negative by PAG test and P4 profile and thereafter was found open by TRUS at Day 42, this cow was considered open at Day 28 after AI. (d) If a cow was found positive by one or both PAG tests but was found negative by TRUS at Day 28 and P4 profile and thereafter was found open by TRUS at Day 42, this cow was considered open at Day 28 after AI. Thus the incorrect TRUS outcomes at Day 28 after AI were adjusted by the pregnancy outcomes based on TRUS at Day 42 and P4 profile at Days 0, 21 and 28 after AI, respectively. 2.8. Statistical analyses Based on the adjusted pregnancy outcomes at Day 28, the results of PAG-RIA and PAG-ELISA tests were classified as: correct positive diagnoses (a), incorrect positive diagnoses (b), correct negative diagnoses (c), and incorrect negative diagnoses (d). From these values, the following diagnostic criteria of PAG-RIA and PAG-ELISA tests and TRUS were calculated (Martin et al., 1987):
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
196 197 198 199 200 201 202 203 204 205 206 207 208
209
210 211 212 213 214 215 216
217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236
237 238 239 240
241
242 243 244 245 246 247 248
G Model
ARTICLE IN PRESS
ANIREP 5197 1–7
A. Karen et al. / Animal Reproduction Science xxx (2015) xxx–xxx
4
Table 1 Comparison of transrectal ultrasonography (TRUS), commercial pregnancy-associated glycoprotein ELISA test and pregnancy-associated glycoprotein-1 radioimmunoassay (PAG-RIA) test for early pregnancy diagnosis in dairy cows at Day 28 after AI based on the actual gold standard. Evaluated criteria
TRUS
PAG-ELISA
PAG-RIA
Correct positive diagnoses (a) Incorrect positive diagnoses (b) Correct negative diagnoses (c) Incorrect negative diagnoses (d) Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Overall accuracy (%)
38 5 54 3 92.7 (84.7–100%) 91.5 (84.4–98.6) 88.4 (78.4 –98.0%) 94.7 (88.9–100%) 92 (86.7–97.3%)
37 1 58 4 90.2 (81.2–99.3%) 98.3 (95.0–100%) 97.4 (92.3–100%) 93.7 (87.4–99.7%) 95 (90.7–99.3)
41 3 56 0 100 (100–100%) 94.9 (89.3–100%) 93.2 (85.7–100%) 100 (100–100%) 97 (93.7–100%)
Percentages within parenthesis indicate 95% confidence interval.
249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266
(i) The sensitivity (Se) — — number of correct positive pregnancy diagnoses diagnoses/all pregnant cows based on the actual gold standard × 100 (a/a + d × 100); (ii) The specificity (Sp) = number of correct negative pregnancy diagnoses by the test/all nonpregnant cows based on the actual gold standard × 100 (c/c + b × 100); (iii) the positive predictive (PPV) = number of correct positive pregnancy diagnoses/number of diagnosed pregnant by blood test or TRUS × 100 (a/a + b × 100); (iv) the negative predictive value (NPV) = number of correct negative pregnancy diagnoses/number of diagnosed non pregnant by blood test or TRUS × 100 (c/c + d × 100) and (v) the overall accuracy (Ac) = number of correct positive pregnancy diagnoses + number of correct negative pregnancy diagnoses/total number of cows tested by diagnostic tests × 100 (a + c/a + b + c + d × 100). Also, 95%-confidence interval of each accuracy parameter of the diagnostic tests was determined.
272
Estimates of the agreement between TRUS, PAG-RIA and PAG-ELISA were determined by using Kappa statistic in GraphPad Software. A Kappa value of 0.4–0.5 indicates a moderate degree of agreement, 0.5–0.6 indicates good agreement and >0.6 indicates a high level of agreement (Martin et al., 1987).
273
3. Results
267 268 269 270 271
274 275 276 277 278 279 280
Based on the actual gold standard, 41 of 100 inseminated cows were pregnant at Day 28 after AI. Nine of the 41 pregnant cows were considered as experienced late embryonic mortalities after Day 28. These 9 cows were positive at Days 28 by the TRUS, the PAG-RIA and the P4 tests, respectively. By the PAG-ELISA test, 7 of these 9 cows were positive at Day 28. Thereafter, these 9 cows were
open by TRUS at Day 42 after AI. Therefore these 9 cows included in the analyses as pregnant cows at Day 28 after AI. The embryo proper with a beating heart was observed by TRUS only in 41.5% (17/41) pregnant cows at Day 28 after AI. The accuracy values of the TRUS, the PAG-RIA and the PAG-ELISA tests are presented in Table 1. By TRUS, three pregnant cows were diagnosed negative at Day 28 after AI; neither the allantoic fluid nor the embryo was detected. In these cows, the bPAG concentrations measured by the PAGRIA and the PAG-ELISA tests and milk P4 concentrations were higher than the thresholds for diagnosing pregnancy and they were pregnant by TRUS at Day 42. In addition 5 non-pregnant cows, based on the actual gold standard, were diagnosed positive by ultrasonography. In these 5 cows, only non-echogenic fluid was observed in the uterus by TRUS. In these false positive ultrasonographic diagnoses, the bPAG concentrations measured by the RIA and the ELISA tests at Day 28 after AI were less than the threshold for diagnosing pregnancy. Furthermore, the P4 concentrations were lower than 3 ng/mL at Day 0 and <7 ng/mL at Day 21 in 3 cows, while they were >3 ng/mL at Day 0 and >7 ng/mL at Day 21 in 2 cows (Table 2). In addition, these five cows were open by TRUS at Day 42 after AI. Regarding the PAG-ELISA test, four pregnant cows (false negative diagnosis) had PAG concentrations lower than the threshold for diagnosing pregnancy. These cows were positive by the TRUS, the PAG-RIA and the P4 tests, two of them were diagnosed pregnant at Day 42 and the other 2 cows were non-pregnant by Day 42. Regarding the PAG-RIA test, all pregnant cows had bPAG-1 concentrations at Day 28 after AI higher than the threshold for diagnosing pregnancy, however, 3 nonpregnant cows had bPAG-1 concentrations higher than the threshold for diagnosing pregnancy. These cows were diagnosed non-pregnant by the TRUS and the P4 test.
Table 2 Pregnancy-associated glycoprotein (PAG) concentrations measured by radioimmunoassay (RIA) and ELISA tests at Day 28 and milk progesterone (P4) concentrations at Days 0, 21 and 28 after AI in cows with false positive ultrasonographic diagnoses at Day 28 after AI. Cow ID
A B C D E
PAG tests at Day 28
P4 test (ng/mL)
RIA (pg/mL)
ELISA (pg/mL)
Day 0
Day 21
Day 28
200 200 200 300 200
25 2.0 79 61 68
2.5 1.8 1.3 3.2 5.7
1.0 2.3 3.0 24.9 21.2
17.8 9.3 17.9 30.6 3.1
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315
G Model ANIREP 5197 1–7
ARTICLE IN PRESS A. Karen et al. / Animal Reproduction Science xxx (2015) xxx–xxx
323
Also, two of these cows were negative by the PAG-ELISA test while, the third one was positive (false positive diagnosis). These cows were in milk for 83, 87 and 98 days after calving, respectively. The degree of agreement (Kappa ± S.E.) between the PAG-RIA and the PAG-ELISA tests was 0.90 ± 0.04. The degrees of agreement of TRUS at Day 28 with the PAG-RIA and the PAG-ELISA tests were 0.80 ± 0.5 and 0.76 ± 0.06, respectively.
324
4. Discussion
316 317 318 319 320 321 322
325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374
In the present study, the sensitivity (92.7%) and the specificity (91.5%) of TRUS at Day 28 after AI were slightly less than those (96.5% and 93.4%, respectively) reported for TRUS at Day 27 when the outcomes of TRUS was adjusted to the outcome of the PAG-ELISA test at Days 27 and TRUS at Day 32 (Silva et al., 2007). The specificity of TRUS at Day 28 obtained in the present study is greater than those (54–87.4%) reported in other studies in which the TRUS outcomes were confirmed by either TRUS at Days 32–35 or rectal palpation between Days 50 and 90 (Pieterse et al., 1990; Badtram et al., 1991; Hanzen and Laurent, 1991; Nation et al., 2003; Romano et al., 2006). Unlike the abovementioned studies, TRUS was carried out concurrent with PAG-ELISA and PAG-RIA tests at Day 28 after AI in the present study which enabled us to diagnose late embryonic mortalities occurred after Day 28 till Day 42. Since cows suffered from late embryonic mortalities (n = 9) were confirmed pregnant by the PAG-ELISA and the TRUS tests at Day 28, they were included in the analyses as pregnant cows and consequently decreased the false positive diagnoses and a higher specificity of TRUS obtained in the present study. Five cows which were incorrectly diagnosed pregnant by TRUS at Day 28 were non-pregnant at the time of the examination, whereas, the PAG values were less than the threshold for diagnosing pregnancy and P4 values were either less that than the threshold for diagnosing pregnancy at Days 21 and 28 (n = 3) or it was higher than 3 ng/mL at Day 0 (n = 2) indicating that the cows were incorrectly inseminated at Day 0. Some of these cows (n = 3) might have been in estrus at the ultrasonographic examination and the detection of a mature ovarian follicle was overlooked due to the limitation of time imposed by the cow flow in this large dairy farm (1000 cows milked daily). Similarly, Nation et al. (2003) observed intrauterine fluid by TRU in 4 cows at Days 28–35 and diagnosed open when re-examined 7 days later. The embryo proper with a beating heart was observed by TRUS only in 41.5% of pregnant cows in the present study. Similar percentage (43.1%) was reported by Silva et al. (2007) at Day 27 after AI. The reason of this low percentage of detection of embryo by TRUS at Day 28 might be the small mass embryo and the time constraints for individual cow diagnoses imposed by the cow flow on the commercial dairy (Silva et al., 2007). The sensitivity of the PAG-ELISA test (Bovine Preg Test 29) for diagnosing pregnant cows in the present study (90.2%) was lower than that (99.4%) reported in a study using the same test but samples were withdrawn somewhat later between Days 29 and 36 after AI (Pare et al., 2008). The sensitivity of the PAG-ELISA test increased with increasing the time interval after AI (Green et al., 2009;
5
Friedrich and Holtz, 2010; Sinedino et al., 2014). Also it was lower than those (93.9% to 98.8%) reported in other studies using different commercial PAG-ELISA tests between Days 25 and 29 after AI (Silva et al., 2007; Green et al., 2009; Romano and Larson, 2010; Sinedino et al., 2014). This difference could be attributed to the difference in the PAG subsets used for the development of antisera in the different commercial tests whereas it is important to mention that there are more than 22 bovine transcribed genes encoding PAG expression that are expressed temporally at varying levels throughout gestation (Green et al., 2000). The specificity of the PAG-ELISA test for diagnosing nonpregnant cows obtained in the present study (98.3%) was much higher than those (77–90.7%) reported in the literature evaluating the accuracy of the PAG-ELISA test at Days 26–30 on the basis of TRUS or palpation per rectum performed at different time intervals from PAG-ELISA tests (Friedrich and Holtz, 2010; Fernandez et al., 2013; Sinedino et al., 2014). On the other hand, the specificity of the test in the present study was similar or slightly higher than those (91.1–97.2%) reported in those studies in which the TRUS was carried out concurrent with the PAG-ELISA test (Silva et al., 2007; Green et al., 2009; Romano and Larson, 2010; Piechotta et al., 2011). Performing the PAG-ELISA test concurrent with the TRUS enabled us to diagnose late embryonic mortalities which occurred after Day 28 till Day 42. Since cows suffered from late embryonic mortalities were confirmed pregnant by the PAG-ELISA and the TRUS tests at Day 28, they were included as pregnant cows and consequently decreased the false positive diagnoses. On the other hand, performing the PAG-ELISA test without contemporary TRUS could not differentiate between pregnant cows at Day 28 and those suffered from embryonic mortalities before testing (false positive diagnoses). Bovine PAG has about a 3-day half-life after experimentally induced embryonic mortality in heifers (Szenci et al., 2003) therefore residual quantities of the proteins may persist in the maternal circulation till the time of sampling. This explanation is supported by the finding of a study using the same commercial kit used in the present study in which the specificity of the test was 100% in non-inseminated cows, while it was 66.7% in the inseminated cows (Pare et al., 2008). The sensitivity (100%) and the specificity (94.4%) of the PAG-RIA test were higher than those (95% and 69.3%, respectively) reported in a study performed at Days 29–30 after AI (Szenci et al., 1998). The higher sensitivity in the present study is attributed to the use of heterologous RIA system (RS#706; a combination of caprine PAG 55 kDa and 62 kDa antisera) which is better than the homologous RIA system (RS #497 bPAG-1 67 kDa antibody) used in the study of Szenci et al. (1998) for recognizing PAG secreted by the bovine placenta between Days 21 and 51 of gestation (Perényi et al., 2002b). The higher specificity of PAG-RIA test in this study is attributed to the use of adjusted TRUS outcome at Day 28 as a basis for calculating the accuracy of PAG-RIA test, while in the other study (Szenci et al., 1998), the outcomes of TRUS at Day 58 was used as a gold standard for calculating the accuracy of the PAG-RIA test. The three false positive diagnoses made by the PAG-RIA test were negative by TRUS and by the P4 test at Days 21 and 28, and
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435
G Model ANIREP 5197 1–7
A. Karen et al. / Animal Reproduction Science xxx (2015) xxx–xxx
6
436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473
474
475
476
477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497
ARTICLE IN PRESS
two of them were negative by the PAG-ELISA test, while one was positive (Day in milk was 83) confirming that these cows were non-pregnant at Day 28 after AI. The residual amount of PAG from previous calving (Days 83 and 98 days in milk, respectively) might be the reason of the higher PAG values measured by the RIA test (Zoli et al., 1992). In addition, the bovine placental PAG measured by bovine Preg 29 test might have disappeared earlier after calving than that measured by the RIA test. The high degrees of agreement among the TRUS, the PAG-RIA and the PAG-ELISA tests confirm that the three tests had similar accuracies for diagnosing early pregnancy in dairy cows. There are advantages of TRU when compared with the PAG blood tests. Transrectal ultrasonography provides an instantaneous diagnosis of pregnancy. It also has the ability to evaluate the uterine and ovarian morphology of non-pregnant cows and identify the dead embryo and nonviable pregnancy (Lucy et al., 2011). These advantages must be weighed against the cost of ultrasound equipment, the technical skill required when performing the ultrasound procedure and whether or not the farm has access to trained person who can do early pregnancy diagnosis with ultrasound (Fricke, 2002). The PAG-ELISA test does not need a specialized lab for performing the RIA test and skilled practitioner to carry out transrectal ultrasonography. Pregnancy diagnosis by PAG-ELISA test at Day 28 would enable implementation of earlier re-synchrony of the open cows. However, about 6.3% (false negative diagnoses) of diagnosed non pregnant cows by PAG-ELISA test in the present study would suffer from iaterogenic abortion if they would have been involved in resynchronization program which might offset the advantage of early resynchrony. In conclusion, the commercial PAG-ELISA test (Bovine Preg Test 29® ) is a highly accurate method for diagnosing early pregnancy in dairy cows on Day 28 after AI and may be used as an alternative method to the TRUS and the PAG-RIA test. Conflict of interest The authors declare no conflict of interest. References Badtram, G.A., Gains, J.D., ThomasCB, Bosu, W.T.K., 1991. Factors influencing the accuracy of early pregnancy detection in cattle by real-time ultrasound scanning of the uterus. Theriogenology 35, 1153–1167. Breukelman, S.P., Perényi, Z., Taverne, M.A.M., Jonker, H., van der Weijden, G.C., Vos, P.L.A.M., de Ruigh, L., Dieleman, S.J., Beckers, J.F., Szenci, O., 2012. Characterization of pregnancy losses after embryo transfer by measuring plasma progesterone and bovine pregnancy-associated glycoprotein-1. Vet. J. 194, 71–76. Fernandez, G.I., Moncebaez, J., Elinzondo, C., Hernandez, H., Ulloa-Arvizu, R., Rojas, S., 2013. Negative prediction value of pregnancy-associated glycoprotein contributes to reduce the days during which nonpregnant Holstein cows are subjected to diverse strategies of hormonal synchronization. Int. J. Anim. Vet. Adv. 5, 160–164. Fricke, P.M., 2002. Scanning the future: ultrasonography as a reproductive management tool for dairy cow. J. Dairy Sci. 85, 1918–1926. Friedrich, M., Holtz, W., 2010. Establishment of an ELISA for measuring bovine pregnancy associated glycoprotein in serum or milk and its application for early pregnancy detection. Reprod. Dom. Anim. 45, 142–146. Garmo, R.T., Refsdal, A.O., Karlberg, K., Ropstad, E., Waldmann, A., Beckers, J.F., Reksen, O., 2008. Pregnancy incidence in Norwegian red cows
using nonreturn to estrus, rectal palpation, pregnancy associated glycoproteins and progesterone. J. Dairy Sci. 91, 3025–3033. Green, J.A., Xie, S., Quan, X., Bao, B., Gan, X., Mathialagan, N., Beckers, J.F., Roberts, M.R., 2000. Pregnancy-associated bovine and ovine glycoproteins exhibit spatially and temporally distinct expression patterns during pregnancy. Biol. Reprod. 62, 1624–1631. Green, J.A., Parks, E.T., Avalle, P.M., Telugu, P.B., McLain, L.A., Peterson, J., Mc Millan, W., Mathialagan, N., Hook, P.R., Xie, S., Roberts, M., 2005. The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers. Theriogenology 63, 1481–1503. Green, J.C., Volkmann, D.H., Poock, S.E., McGarth, M.F., Ehrhadt, M., Moseley, A.E., Lucy, M.C., 2009. Technical note: a rapid enzyme linked immunosorbent assay blood test for pregnancy in dairy and beef cattle. J. Dairy Sci. 92, 3819–3824. Hanzen, C., Laurent, Y., 1991. Application de l‘échographie bidimensionnelle au diagnostic de gestation et l‘évaluation de l‘incidence de la mortalité, embryonnaire dans l’espèce bovine. Ann. Med. Vet. 134, 481–487. Howard, J., Gabor, G., Gray, T., Passavant, C., Ahmadzadeh, A., Sasser, N., Pals, D., Sasser, G., 2007. BioPRYN, a blood-based pregnancy test for managing breeding and pregnancy in cattle. In: Proceeding, Western Section, American Society of Animal Science, vol. 58, pp. 295–298. Humblot, P., Camous, S., Martal, J., Charlery, J., Jeanguyot, N., Thibier, M., Sasser, R.G., 1988. Diagnosis of pregnancy by radioimmunoassay of a pregnancy specific protein in the plasma of dairy cows. Theriogenology 30, 257–267. Lucy, M., Green, J., Poock, S., 2011. Pregnancy determination in cattle: a review of available alternatives. In: Proceedings, Applied Reproductive Strategies in Beef Cattle, August 31–September 1, 2011, Joplin, MO, pp. 367–375. Martin, S.W., Meek, A.H., Willeberg, P., 1987. Veterinary Epidemiology, Principles and Methods. Iowa State University Press, Ames, IA, pp. 63–71. Nakao, T., Sugihashi, A., Ishibashi, Y., Tosa, E., Nakagawa, Y., Yuto, H., Nomura, T., Ohe, T., Ishimi, S., Takahashi, H., Kowia, M., Tusnoda, N., Kawata, K., 1982. Use of milk progesterone enzymeimmunoassay for early pregnancy diagnosis in cows. Theriogenology 18, 267–274. Nation, D.P., Malmo, J., Davis, G.M., Macmillan, K.L., 2003. Accuracy of bovine pregnancy detection using transrectal ultrasonography at 28 to 35 days after insemination. Aust. Vet. J. 81, 63–65. Paré, J., Audet-Grenier, M.-H., Rouillier, Sirard, M.-A., 2008. Evaluation of the DG29 test for early detection of pregnancy in cattle. Can. Vet. J. 49, 1119–1121. Perényi, Z., Szenci, O., Sulon, J., Drion, P.V., Beckers, J.F., 2002a. Comparison of the ability of three radioimmunoassays to detect pregnancyassociated glycoproteins in bovine plasma. Reprod. Domest. Anim. 37, 100–104. Perényi, Z., Szenci, O., Drion, P.V., Banga-Mboko, H., Sousa, N.M., El Amiri, B., Beckers, J.F., 2002b. Aspartic proteinase members secreted by the ruminant placenta: specificity of three radioimmunoassay systems for the measurement of pregnancy-associated glycoproteins. Reprod. Domest. Anim. 37, 324–329. Piechotta, M., Bollwien, J., Friedrich, M., Heilkenbrinker, T., Passavant, C., Branen, J., Sasser, G., Hoedemaker, M., Bollwein, H., 2011. Comparison of commercial ELISA blood tests for early pregnancy detection. J. Reprod. Dev. 57, 72–75. Pieterse, M.C., Szenci, O., Wiilemse, A.H., Bajcsy, A.C., Dieleman, S.J., Taverne, M.A.M., 1990. Early pregnancy diagnosis in cattle by means of linear-array real-time ultrasound scanning of the uterus and a quantitative and qualitative milk progesterone test. Theriogenology 33, 697–707. Repasi, A., Beckers, J.F., Sulon, J., Perenyi Zs, Reiczigel, J., Szenci, O., 2003. Effect of different doses of prostaglandin on the area of corpus luteum, the largest follicle and progesterone concentration in the dairy cow. Reprod. Dom. Anim. 38, 1–6. Romano, J.E., Larson, J.E., 2010. Accuracy of pregnancy specific protein-B test for early pregnancy diagnosis in dairy cattle. Theriogenology 74, 932–939. Romano, J.E., Thompson, J.A., Forrest, D.W., Westhusin, M.E., Tomaszweski, M.A., Kraemer, D.C., 2006. Early pregnancy diagnosis by transrectal ultrasonography in dairy cattle. Theriogenology 66, 1034–1041. Sasser, R.G., Ruder, C.A., Ivani, K.A., Butler, J.E., Hamilton, W.C., 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, 936–942. Sauer, M.J., Foulkes, J.A., Worsfold, A., Morris, B.A., 1986. Use of progesterone II-glucuronide-alkaline phosphatase conjugate in a sensitive microtitre-plate enzyme immunoassay of progesterone in milk and
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576
G Model ANIREP 5197 1–7
ARTICLE IN PRESS A. Karen et al. / Animal Reproduction Science xxx (2015) xxx–xxx
577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593
its application to pregnancy testing in dairy cattle. J. Reprod. Fertil. 76, 375–391. Sheldon, I.M., Lewis, G., LeBlanc, S., Gilbert, R., 2006. Defining postpartum uterine disease in dairy cattle. Theriogenology 65, 1516–1530. Silva, E., Sterry, R.A., Kolb, D., Mathialagan McGarth, M.F., Ballam, J.M., Fricke, P.M., 2007. Accuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy status of lactating dairy cows twenty seven days after timed artificial insemination. J. Dairy Sci. 90, 4612–4622. Sinedino, L.D.P., Lima, F.S., Bisinotto, R.S., Cerri, R.L.A., Santos, J.E.P., 2014. Effect of early or late resynchronization based on different methods of pregnancy diagnosis on reproductive performance of dairy cows. J. Dairy Sci. 97, 1–10. Skinner, J.G., Gray, D., Gebbie, F.E., Beckers, J.F., Sulon, J., 1996. Field evaluation of pregnancy diagnosis using bovine pregnancy-associated glycoproetein (bPAG). Cattle Pract. 4, 281–284. Szenci, O., Beckers, J.F., Humblot, P., Sulon, J., Sasser, G., Taverne, M.A.M., Varga, J., Baltusen, R., Schekk, Gy., 1998. Comparison of
7
ultrasonography, bovine pregnancy-specific protein B, and bovine pregnancy-associated glycoprotein 1 tests for pregnancy detection in dairy cows. Theriogenology 50, 77–88. Szenci, O., Beckers, J.F., Sulon, J., Bevers, M.M., Börzsönyi, L., Fodor, L., Kovács, F., Taverne, M.A.M., 2003. Effect of induction of late embryonic mortality on plasma profiles of pregnancy associated glycoproteins in heifers. Vet. J. 165, 307–313. Wooding, F.B., Roberts, R.M., Green, J.A., 2005. Light and electron microscope immunocytochemical studies of the distribution of pregnancy-associated glycoproteins (PAGs) throughout pregnancy in the cow: possible functional implications. Placenta 26, 807–827. Youngquist, R.S., 2007. Pregnany diagnosis. In: Youngquist, R.S., Threlfall, W.R. (Eds.), Current Therapy in Large Animal Theriogenology. , 2nd edition. Saunder, Missouri, USA, pp. 294–303. Zoli, A.P., Guilbault, L.A., Delahaut, P., Ortiz, W.B., Beckers, J.F., 1992. Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy diagnosis. Biol. Reprod. 46, 83–92.
Please cite this article in press as: Karen, A., et al., Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.005
594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611