Effect of early or late resynchronization based on ...

J. Dairy Sci. 97:1–10 http://dx.doi.org/10.3168/jds.2013-7887 © American Dairy Science Association®, 2014.

Effect of early or late resynchronization based on different methods of pregnancy diagnosis on reproductive performance of dairy cows L. D. P. Sinedino,* F. S. Lima,* R. S. Bisinotto,* R. L. A. Cerri,† and J. E. P. Santos*1 *Department of Animal Sciences, University of Florida, Gainesville 32611 †Faculty of Land and Food Systems, University of British Columbia, Vancouver, BC VT6 1Z4, Canada

that were resynchronized and received timed AI was greater for ER than for LR (30.0 vs. 7.6%). Cows in ER had a shorter interval between inseminations when inseminated following spontaneous estrus (21.7 ± 1.1 vs. 27.8 ± 0.8 d) or after timed AI (35.3 ± 1.2 vs. 55.2 ± 1.4 d). Nevertheless, the ER did not affect the rate of pregnancy (adjusted hazard ratio = 1.23; 95% confidence interval = 0.94 to 1.61) or the median days postpartum to pregnancy (ER = 132 vs. LR = 140). A total of 2,129 PAG ELISA were evaluated. Overall, sensitivity, specificity, and positive and negative predictive values averaged 95.1, 89.0, 90.1, and 94.5%, respectively, and the accuracy was 92.1%. In conclusion, PAG ELISA for early diagnosis of pregnancy had acceptable accuracy, but early resynchronization after nonpregnancy diagnosis with PAG ELISA did not improve the rate of pregnancy or reduce days open in dairy cows continuously observed for estrus. Key words: dairy cow, pregnancy-associated glycoprotein, reproduction, resynchronization

ABSTRACT

The aim of this study was to compare the reproductive performance of dairy cows subjected to early (ER) or late (LR) resynchronization programs after nonpregnancy diagnoses based on either pregnancy-associated glycoproteins (PAG) ELISA or transrectal palpation, respectively. In addition, the accuracy of the PAG ELISA for early pregnancy diagnosis was assessed. Lactating Holstein cows were subjected to a PresynchOvsynch protocol with timed artificial insemination (AI) performed between 61 and 74 DIM. On the day of the first postpartum AI, 1,093 cows were blocked by parity and assigned randomly to treatments; however, because of attrition, 452 ER and 520 LR cows were considered for the statistical analyses. After the first postpartum AI, cows were observed daily for signs of estrus and inseminated on the same day of detected estrus. Cows from ER that were not reinseminated in estrus received the first GnRH injection of the Ovsynch protocol for resynchronization 2 d before pregnancy diagnosis. On d 28 after the previous AI (d 27 to 34), pregnancy status was determined by PAG ELISA, and nonpregnant cows continued on the Ovsynch protocol for reinsemination. Pregnant cows had pregnancy status reconfirmed on d 46 after AI (d 35 to 52) by transrectal palpation, and those that lost the pregnancies were resynchronized. Cows assigned to LR had pregnancy diagnosed by transrectal palpation on d 46 after AI (d 35 to 52) and nonpregnant cows were resynchronized with the Ovsynch protocol. Blood was sampled on d 28 after AI (d 27 to 34) from cows in both treatments that had not been reinseminated on estrus and again on d 46 after AI (d 35 to 52) for assessment of PAG ELISA to determine the accuracy of the test. Cows were subjected to treatments for 72 d after the first insemination. Pregnancy per AI (P/AI) at first postpartum timed AI did not differ between treatments and averaged 28.9%. The proportion of nonpregnant cows

INTRODUCTION

Reproductive performance is a major component of the economic viability in dairy herds and shortening the reinsemination interval for nonpregnant cows is expected to reduce the time to pregnancy and improve the pregnancy rate (Meadows et al., 2005; De Vries, 2006; Ribeiro et al., 2012). From 55 to 70% of lactating dairy cows fail to become pregnant in response to AI and need to be reinseminated (Santos et al., 2004). Programs for synchronization of the estrous cycle and timed AI have been widely used to increase submission to AI and to reduce the interval between inseminations (Fricke et al., 2003). However, nonpregnant cows need to be identified before being subjected to resynchronization protocols and the development of methods for early pregnancy diagnosis is critical to further reduce the interbreeding interval. The diagnosis of pregnancy in cattle is traditionally performed starting around d 35 to 40 after insemination by transrectal palpation of the reproductive tract for identification of the amniotic vesicle (Warnick et

Received December 30, 2013. Accepted May 5, 2014. 1 Corresponding author: [email protected]

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al., 1995), or at approximately 27 d after insemination with the aid of ultrasonography (Silva et al., 2007). Alternatively, chemical methods that detect the presence of conceptus-derived antigens can be used to identify pregnant cows early after AI (Green et al., 2009, 2011). Modern pregnancy-associated glycoproteins (PAG) are members of a large family of aspartic proteinases expressed by binucleate trophoblast cells in even-toed ungulates (Xie et al., 1991). These proteins are released into the uterine stroma after the fusion between binucleate and endometrial cells (Xie et al., 1991) and can be observed in the maternal circulation throughout gestation (Sasser et al., 1986; Green et al., 2005). The development of monoclonal antibodies specific for PAG produced during initial stages of gestation have allowed for the use of immunoassays such as ELISA for early pregnancy detection that can be performed in cows after 60 d postpartum (Silva et al., 2007; Green et al., 2009). Nevertheless, the concentrations of PAG in the blood of pregnant cows increase from d 24 to 30 after AI and then decrease toward d 60 of gestation (Thompson et al., 2010), suggesting that the accuracy of determining pregnancy status varies depending on the day after AI in which the blood is collected (Szenci et al., 1998; Green et al., 2009). Previous studies have shown that diagnosing pregnancy starting on d 25 after AI based on PAG concentrations resulted in acceptable accuracy (Silva et al., 2007; Green et al., 2009) and reduced interbreeding interval and time to pregnancy in lactating cows resynchronized exclusively using timed AI (Silva et al., 2009). Nonetheless, the combination of estrous detection and timed AI for reinsemination of nonpregnant cows is used in the majority of dairy herds, as it often results in greater submission to AI and reduced time to pregnancy (Ribeiro et al., 2012; Giordano et al., 2013). A large portion of cows are expected to be observed in estrus and reinseminated before d 32 after insemination (Chebel et al., 2003; Bartolome et al., 2005; Galvão et al., 2007); therefore, the need for early pregnancy diagnosis will vary according to the ability to identify nonpregnant cows that return to estrus before any pregnancy diagnosis testing. The main hypothesis of the present study was that an early resynchronization (ER) program starting at 28 d after AI using the PAG ELISA for pregnancy diagnosis and decision on resynchronization would reduce the interval between inseminations and, therefore, the time to pregnancy compared with the use of transrectal palpation in a late resynchronization protocol starting 46 d after AI. A second hypothesis was that the PAG ELISA would result in acceptable accuracy to determine pregnancy status in lactating dairy cows. Therefore, the objectives were to compare the time to Journal of Dairy Science Vol. 97 No. 8, 2014

pregnancy in cows subjected to ER versus late resynchronization (LR) programs based on nonpregnancy diagnosis using PAG ELISA or transrectal palpation, respectively, and to evaluate the accuracy of the PAG ELISA for pregnancy diagnosis. MATERIALS AND METHODS Animals, Housing, and Feeding

A total of 1,093 lactating Holstein cows from a commercial dairy herd located in central California were enrolled in this experiment from December 2004 to April 2005. Primiparous and multiparous cows were housed separately in freestall barns equipped with sprinklers and fans. Cows had ad libitum access to water and were fed a TMR to meet or exceed the requirements of lactating Holstein cows weighing 650 kg and producing 45 kg of milk per day with 3.5% fat and 3.2% true protein (NRC, 2001). Cows were fed multiple times daily and were milked 3 times daily. Reproductive Management and Treatments

Weekly cohorts of cows were subjected to the Presynch-Ovsynch protocol (Moreira et al., 2001) for the first postpartum timed AI (Figure 1). Briefly, cows received an i.m. injection of PGF2α (Lutalyse sterile solution, 25 mg of dinoprost as tromethamine salt, Zoetis, Madison, NJ) given on d 32 (25 to 38) and again on d 46 (39 to 52) postpartum. The Ovsynch protocol was initiated at 58 (51 to 64) DIM with an i.m. injection of GnRH (Cystorelin, 100 μg of gonadorelin diacetate tetrahydrate; Merial Ltd., Duluth, GA), which was followed 7 d later by an injection of PGF2α and a final GnRH injection of PGF2α 48 h later. Cows were inseminated 24 h after the last GnRH injection (61 to 74 DIM). The body condition of cows was scored using a 1 to 5 scale (0 = emaciated and 5 = obese; Ferguson et al., 1994) at 54 ± 3 DIM and those with BCS >2.00 were considered eligible for enrollment in the study. For purposes of statistical analyses, cows were categorized as having low (≤2.75) or moderate (≥3.00) BCS. The study followed a randomized complete block design. On the day of the first AI postpartum, cows were blocked by parity and, within each block, assigned randomly to either an ER (n = 548) or LR (n = 545) program (Figure 1). After the first AI postpartum, cows from both programs were observed for signs of estrus based on removal of tail chalk and those in estrus were inseminated on the same day. Cows assigned to ER had pregnancy diagnosed weekly and they received the first GnRH injection of the Ovsynch protocol for resynchronization, on average, on

RESYNCHRONIZATION PROGRAMS FOR DAIRY COWS

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Figure 1. Diagram of the study. All cows underwent a Presynch-Ovsynch protocol and received the first AI at 68 DIM (range: 61–74 DIM; study d 0). Early Resynch: cows were subjected to early pregnancy diagnosis by pregnancy-associated glycoprotein (PAG) ELISA on d 28 after AI (27 to 34 d), and nonpregnant cows continued on the resynchronization protocol. Late Resynch: cows were subjected to late pregnancy diagnosis by transrectal palpation on d 46 after AI (35 to 52 d) and nonpregnant cows received the resynchronization protocol starting on the day of pregnancy diagnosis. Cows observed in estrus were inseminated on the same day. The study lasted 72 d from the first insemination. Cows were considered pregnant at the end of the study based on palpation performed on d 46 after AI. BS = blood sample collected for analysis of concentrations of progesterone; G = injection of gonadotropin-releasing hormone; PAG = PAG ELISA pregnancy diagnosis; PG = injection of PGF2α; TAI = timed AI;  = blood sample collected for measurements of progesterone concentrations in plasma;  = blood sample collected for analysis of PAG by ELISA.

d 26 after the previous AI. Two days later, on d 28 after AI (d 27 to 34), pregnancy status was determined by PAG ELISA. Ultrasonography was performed on the same day of blood collection to serve as the gold standard for PAG accuracy determination. Because cows in ER were evaluated weekly, it was expected that all would have the blood test performed between 27 and 32 d after AI. However, a small number of cows had to have a second blood sample collected 2 d later because of disagreement between the PAG ELISA and the ultrasonography. Blood was collected from the coccygeal vessels into 3 mL evacuated tubes containing K2 EDTA (Vacutainer; Becton Dickinson, Franklin Lakes, NJ). Tubes were placed on ice immediately after collection and shipped to Monsanto Co. (St. Louis, MO) using an overnight shipping courier (FedEx Corp., Memphis, TN). Cows diagnosed as nonpregnant based on the PAG assay received the remaining injections of the Ovsynch protocol and were reinseminated 8 d after the blood testing. Conversely, cows diagnosed as pregnant according to the PAG assay did not receive any further treatment and had pregnancy status reconfirmed on d 46 after AI (d 35 to 52) by transrectal palpation of the reproductive tract and its contents. Throughout the

manuscript, the first pregnancy diagnosis will be defined as on d 28, which refers to d 27 to 34 after AI, and the final pregnancy diagnosis will be defined as on d 46, which refers to d 35 to 52 after insemination. Cows with pregnancies reconfirmed completed the study, whereas cows that lost the pregnancy were resynchronized with the Ovsynch protocol. Pregnancy diagnosis in LR cows was performed every 14 d by transrectal palpation of the uterus and contents. Because a few cows did not have a definitive diagnosis, some were reevaluated a few days later, which resulted in an interval that was longer than 14 d (d 35 to 52 after the previous AI). The 14-d interval reflected the management already implemented on the farm in which half of the herd was evaluated for pregnancy in one week, and the other half in the subsequent week. Throughout the manuscript, the day of pregnancy diagnosis for LR cows is defined as d 46, which refers to the average day for this treatment. Cows diagnosed as pregnant completed the study, whereas nonpregnant cows were resynchronized with the Ovsynch protocol starting on the day of nonpregnancy diagnosis. The study lasted 72 d from the first AI. A cow was considered pregnant for the study in both treatments based Journal of Dairy Science Vol. 97 No. 8, 2014

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on the results of transrectal palpation performed on d 46 after AI. Progesterone Concentrations and Definition of Estrous Cyclic Status

Estrous cyclic status was defined based on the concentration of progesterone in plasma collected on the day of the second PGF2α injection of the presynchronization (46 DIM) and the day of the first GnRH injection of the Ovsynch protocol (58 DIM). Blood was sampled by puncture of the coccygeal vessels into 10-mL evacuated tubes containing K2 EDTA (Vacutainer; Becton Dickinson). Samples were immediately placed on ice and arrived at the laboratory within 6 h of collection. Blood tubes were centrifuged at 3,000 × g for 15 min at 4°C for plasma separation. Plasma samples were frozen at −25°C until assayed. The concentration of progesterone was determined by an ELISA validated by Cerri et al. (2004); samples were analyzed in duplicate, with every microplate containing the standards 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 ng/mL. The coefficient of variation was assessed with 2 known samples containing 1.5 and 3.0 ng/mL. Intraassay and interassay coefficients of variation for all microplates were 5.7 and 9.1%, respectively. Individual samples with a coefficient of variation greater than 15% were reanalyzed. Cows were classified as estrous cyclic when the concentration of progesterone was ≥1 ng/mL in at least 1 of the samples. Conversely, cows with progesterone concentration 0.10. Treatment was forced in all final models. Time to pregnancy was analyzed by the the Cox proportional hazard model with PROC PHREG of SAS. The time variable considered was the number of days between the first timed AI and the day of AI that resulted in pregnancy or the day in which cows were censored from the analyses (i.e., cows that were sold, died, or remained nonpregnant until reaching the end of study). Models included the fixed effects of treatment, parity, BCS, estrous cyclic status, and the interactions between treatment and covariates. A backward stepwise elimination method was used to continuously

remove independent variables with P > 0.10 and treatment was forced in all final models. The median and mean days to pregnancy were obtained from PROC LIFETEST of SAS. A survival plot was generated with MedCalc software (version 12.7.7.0; MedCalc Software bvba, Mariakerke, Belgium). Estimates between the agreement of pregnancy outcomes of transrectal ultrasonography or palpation and PAG ELISA were calculated by kappa statistic in PROC FREQ of SAS and 95% confidence intervals were determined. Treatment differences with P ≤ 0.05 were considered significant and those with 0.05 < P ≤ 0.10 were considered as tendencies. RESULTS

One-hundred twenty-one cows were removed from the data analyses. Ninety-one cows from the ER treatment and respective block mates were excluded from the study because of issues with the PAG ELISA results originated from a malfunction of the 96-well plate washer or an inconclusive PAG result by the laboratory, which prevented them from being resynchronized or completing the study. Additional cows were removed from the study because of death (n = 6), culling from the herd based on farm personnel decision (n = 18), adherence of the reproductive tract (n = 3), and failure to follow the experimental protocol (n = 3). Therefore, from the 1,093 cows initially enrolled in the study, 972 (n = 452 of ER and n = 520 of LR) were considered for the statistical analyses. The proportion of primiparous cows and the average lactation number did not differ (P > 0.30) between ER (35.5% and 2.23 ± 0.06) and LR groups (38.2% and 2.16 ± 0.06). Similarly, the proportion of cows classified as having low BCS and the average BCS did not differ (P > 0.60) between ER (61.2% and 2.84 ± 0.01) and LR groups (60.5% and 2.84 ± 0.01). The proportion of cows classified as estrous cyclic at the initiation of the Ovsynch protocol for the first postpartum AI did not differ (P = 0.13) between treatments (ER = 64.7 vs. LR = 69.3%). Accuracy of PAG ELISA for Early Pregnancy Diagnosis

A total of 2,129 test diagnostics were performed at different intervals after AI. The overall accuracy of the PAG ELISA for early diagnosis of pregnancy was high and showed little variation with the day after insemination when the blood was sampled (Table 1). The overall kappa value was 0.82 (95% CI = 0.79–0.84). The Se averaged 95.1% and the Sp averaged 89.0%. Journal of Dairy Science Vol. 97 No. 8, 2014

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Table 1. Measures of reliability of using pregnancy-associated glycoprotein (PAG) ELISA for early pregnancy diagnosis at different intervals after AI Measures of reliability1 Days after AI, % (no./total no.) 27 28 to 30 31 to 35 >35 Overall

Sensitivity 94.6 96.1 98.7 94.4 95.1

(318/336) (173/180) (77/78) (474/502) (1,042/1,096)

Specificity 89.9 90.7 88.1 85.2 89.0

(437/486) (204/225) (111/126) (167/196) (919/1,033)

Positive predictive value 86.6 89.2 83.7 94.2 90.1

(318/367) (173/194) (77/92) (474/503) (1,042/1,156)

Negative predictive value 96.0 96.7 99.1 85.6 94.5

(437/455) (204/211) (111/112) (167/195) (919/973)

Kappa (95% CI)

Accuracy 91.8 93.1 92.2 91.8 92.1

(755/822) (377/405) (188/204) (641/698) (1,961/2,129)

0.79 0.83 0.82 0.79 0.82

(0.75–0.84) (0.78–0.89) (0.74–0.90) (0.74–0.84) (0.79–0.84)

1 Sensitivity = (number of pregnant cows correctly diagnosed by PAG ELISA/number of pregnant cows diagnosed by ultrasonography or palpation) × 100; specificity = (number of nonpregnant cows correctly diagnosed by PAG ELISA/number of nonpregnant cows diagnosed by ultrasonography or palpation) × 100; positive predictive value = (number of cows correctly testing pregnant/number of total pregnant cows by PAG ELISA) × 100; negative predictive value = (number of cows correctly testing nonpregnant/number of total nonpregnant cows by PAG ELISA) × 100; accuracy = (number of correct PAG ELISA/number of total tests performed) × 100.

Pregnancy per AI, Pregnancy Loss, and Time to Pregnancy

Pregnancy per AI (P/AI) at the first AI diagnosed on d 46 after insemination did not differ between treatments and averaged 28.9% (Table 2). Cows with moderate BCS had greater (P = 0.003) P/AI than those with low BCS (34.6 vs. 25.3%). Estrous cyclic cows tended to have (P = 0.06) greater P/AI compared with those that were anovular at the initiation of the Ovsynch protocol (31.0 vs. 24.5%). Pregnancy to the first AI postpartum was not affected by parity or by the interactions between treatment and parity, BCS, and estrous cyclic status. The risk of pregnancy loss at first postpartum insemination between the initial pregnancy diagnosis using transrectal ultrasound on 28 and the transrectal palpation on d 46 of gestation did not differ between treatments (Table 2). Multiparous cows had greater (P = 0.02) pregnancy loss than primiparous cows (19.7 vs. 12.5%). Estrous cyclic status and BCS did not influence (P > 0.40) the risk of pregnancy loss at first AI.

The proportion of nonpregnant cows that were resynchronized and received timed AI was greater (P < 0.001) for ER than for LR (Table 2). Parity, estrous cyclic status, and BCS did not affect the proportion of cows that were not observed in estrus and received timed AI. The day when pregnancy diagnosis was performed in cows not observed in estrus in ER and LR is depicted in Figure 2. By design, the day of pregnancy diagnosis for cows not detected in estrus was 14 d earlier (P < 0.001) in those enrolled in the ER than LR (28.2 ± 0.1 vs. 45.6 ± 0.1 d). The earlier pregnancy diagnosis resulted in a shorter interval between inseminations (P < 0.001) for cows in the ER than LR (28.5 ± 1.0 vs. 41.5 ± 0.9 d). This shorter interval occurred in cows inseminated on estrus (ER = 21.7 ± 1.1 vs. LR = 27.8 ± 0.8 d) or following timed AI (ER = 35.3 ± 1.2 vs. LR = 55.2 ± 1.3 d). Pregnancy per AI after the first insemination was not affected by treatment (P = 0.32) and averaged 18.4 and 17.6% for ER and LR, respectively; however, P/AI was greater (P = 0.02) for cows reinseminated on estrus than for those receiving

Table 2. Pregnancy per AI and pregnancy loss at the first timed AI, submission to timed AI after the first insemination, and interval between AI in Holstein cows subjected to early or late resynchronization protocols Treatment1 Parameter Cows Pregnant at first postpartum timed AI,2 % d 28 d 46 Pregnancy loss, 28 to 46 d,3 % Submission to resynchronized timed AI,4 % Interval between AI, d (LSM ± SEM) 1

Early Resynch 452 35.4 28.3 20.0 30.0 28.5 ± 0.9

Late Resynch 520 34.6 29.4 15.3 7.6 41.5 ± 1.0

P-value — 0.80 0.78 0.31 0.60 indicates a high level of agreement (Martin et al., 1987). The measures of reliability of the PAG ELISA varied slightly according to the day after insemination when blood was sampled, and was highest between 31 to 35 d after AI. Szenci et al. (1998) and Green et al. (2009) also demonstrated that measures of reliability of chemical tests varied slightly with day after AI. The differences with day after insemination probably occurred because of the changes in PAG concentration throughout gestation (Green et al., 2005; Thompson et al., 2010; Giordano et al., 2012), and that very early pregnancy diagnosis typically results in inflated values for pregnancy loss (Santos et al., 2004). Collectively, these factors would likely influence the accuracy of test diagnostics. Green et al. (2005) reported that PAG became detectable by ELISA as early as d 22 of gestation in a small percentage of the pregnant cows. Green et al. (2005) also showed that, from d 24 to 28, PAG concentrations increased rapidly, reaching an average of 8.75 ± 3.04 ng/mL by d 28 of gestation. In the same study, the average concentration of PAG increased to 12.3 ± 4.08 ng/mL in wk 5 and then declined to 6.8 ± 3.8 ng/mL in wk 8 of gestation. A similar pattern was observed by Thompson et al. (2010), who followed the plasma profile of pregnant dairy cows and observed that PAG concentrations increased from d 24 to 30, reaching approximately 5.0 ng/mL, and then decreased to approximately 2.0 ng/mL by d 60 after AI. Giordano et al. (2012) also followed the dynamics of PAG concentration from 1 to 49 d of gestation and observed a rapid increase from 22 to 32 d, reaching a peak of 4.4 ± 1.8 ng/mL at approximately d 42, followed by a steadily decline to reach the lowest value of 2.5 ± 0.9 ng/mL on d 49 after timed AI. From these studies, it seems plausible that the accuracy of PAG as a method of pregnancy diagnosis will suffer from small variations in the timing of blood sampling and testing. Sensitivity, which is the ability of the test to identify a pregnant cow, was highest (98.7%) when blood was collected from 31 to 35 d after AI. In the same period, the NPV was 99.1%, which represented the proportion of nonpregnant outcomes provided by PAG ELISA that were from truly nonpregnant cows. A possible reason for the high NPV is that temporal PAG profile that peaks around the fifth week of gestation (Green et al.,

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2005; Thompson et al., 2010), possibly allowing for a more accurate diagnosis around this time. These results indicate that a PAG ELISA would likely identify almost all pregnant cows from 31 to 35 d after AI. They also indicate that, from every 100 nonpregnancy diagnosis, only 0.9% would be represented by a pregnant cow. The PPV averaged 90.1%, meaning that 9.9% of the pregnant outcomes by the PAG ELISA were actually nonpregnant cows that would require a later reconfirmation of pregnancy to identify these misdiagnosed animals. It is likely that a portion of these misdiagnoses were the result of pregnancy loss. In fact, not every cow diagnosed as pregnant by ultrasonography is truly pregnant (Silva et al., 2007), and some cows that have signs of a viable pregnancy will fail to maintain gestation in the following days (Santos et al., 2004; Giordano et al., 2012). Santos et al. (2004) summarized numerous studies and found 12.8% late-embryonic mortality in lactating dairy cows in the first 15 d after the initial pregnancy diagnosis starting on d 27 after AI. Giordano et al. (2012) reported that although concentrations of PAG declined within 1 to 2.5 d after induction of pregnancy loss with PGF2α or intrauterine infusion of hypertonic saline solution, it took an average of 9.5 d for concentrations to become equal to those of nonpregnant cows. Therefore, is it expected that during the process of pregnancy loss, some cows might be falsely diagnosed as pregnant because the amount of PAG in plasma is still elevated and above the threshold value for detection of pregnancy. In fact, only 5 d after induction of embryonic mortality, the concentration of PAG was below the cut-off value for pregnancy (Giordano et al., 2012). This delay is in part because the half-life for some PAG in the maternal circulation is 2.7 to 3.9 d, and induction of pregnancy loss caused the PAG concentration to decrease below the cut-off value for pregnancy only after 4 to 8 d (Szenci et al., 2003). On the other hand, the false-negative results are caused by some pregnant cows with PAG concentrations below the threshold for pregnancy detection. Indeed, some cows exhibit individual variations with low PAG concentration (Szenci et al., 1998; Green et al., 2005; Giordano et al., 2012). Nevertheless, these cases are exceptions. The majority of the cows exhibit a temporal pattern of PAG expression during gestation (Green et al., 2005; Giordano et al., 2012). CONCLUSIONS

Implementing an early diagnosis of pregnancy followed by resynchronization in cows observed for estrus resulted in a small proportion of resynchronized inseminations performed by timed AI, which reduced the interval between breedings, but was unable to reduce Journal of Dairy Science Vol. 97 No. 8, 2014

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days open or the interval from first insemination to pregnancy. The lack of benefit from early resynchronization was likely caused by the high detection of estrus before the day of pregnancy diagnosis. The PAG ELISA for early pregnancy diagnosis had high measures of reliability at different intervals after AI, with the highest Se and NPV observed between 30 and 35 d after AI, indicating that this interval resulted in the lowest proportion of missed pregnancies and the lowest risk for inducing an iatrogenic abortion with the use of prostaglandins. When herds have high detection of estrus, the interval after AI when pregnancy is diagnosed seems to have little effect on time to pregnancy. ACKNOWLEDGMENTS

The authors thank the owner and staff of Rancho Teresita Dairy (Tulare, CA) for the use of their cows and facilities. Our appreciation is extended to Monsanto Co. (St. Louis, MO) for performing the chemical tests. Financial support for this study was provided by a grant from Monsanto Co. REFERENCES Bartolome, J. A., A. Sozzi, J. McHale, P. Melendez, A. C. M. Arteche, F. T. Silvestre, D. Kelbert, K. Swift, L. F. Archbald, and W. W. Thatcher. 2005. Resynchronization of ovulation and timed insemination in lactating dairy cows. II: Assigning protocols according to stages of the estrous cycle, or presence of ovarian cysts or anestrus. Theriogenology 63:1628–1642. Cerri, R. L. A., J. E. P. Santos, S. O. Juchem, K. N. Galvão, and R. C. Chebel. 2004. Timed artificial insemination with estradiol cypionate or insemination at estrus in high-producing dairy cows. J. Dairy Sci. 87:3704–3715. Chebel, R. C., J. E. P. Santos, R. L. A. Cerri, K. N. Galvão, S. O. Juchem, and W. W. Thatcher. 2003. Effect of resynchronization with GnRH on day 21 after artificial insemination on pregnancy rate and pregnancy loss in lactating dairy cows. Theriogenology 60:1389–1399. De Vries, A. 2006. Economic value of pregnancy in dairy cattle. J. Dairy Sci. 89:3876–3885. Ferguson, J. D., D. T. Galligan, and N. Thomsen. 1994. Principal descriptors of body condition score in Holstein cows. J. Dairy Sci. 77:2695–2703. Fricke, P. M., D. Z. Caraviello, K. A. Weigel, and M. L. Welle. 2003. Fertility of dairy cows after resynchronization of ovulation at three intervals following first timed insemination. J. Dairy Sci. 86:3941–3950. Galvão, K. N., J. E. P. Santos, R. L. A. Cerri, R. C. Chebel, H. M. Rutigliano, R. G. Bruno, and R. C. Bicalho. 2007. Evaluation of methods of resynchronization for insemination in cows of unknown pregnancy status. J. Dairy Sci. 90:4240–4252. Giordano, J. O., P. M. Fricke, and V. E. Cabrera. 2013. Economics of resynchronization strategies including chemical tests to identify nonpregnant cows. J. Dairy Sci. 96:949–961. Giordano, J. O., J. N. Guenther, G. Lopes Jr., and P. M. Fricke. 2012. Changes in serum pregnancy-associated glycoprotein, pregnancyspecific protein B, and progesterone concentrations before and after induction of pregnancy loss in lactating dairy cows. J. Dairy Sci. 95:683–697. Green, J. A., T. E. Parks, M. P. Avalle, B. P. Telugu, A. L. McLain, A. J. Peterson, W. McMillan, N. Mathialagan, R. R. Hook, S. Xie, Journal of Dairy Science Vol. 97 No. 8, 2014

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