Introduction

Early identification of nonpregnant dairy cows and heifers post breeding can improve reproductive efficiency and pregnancy rate by decreasing the interval between AI services and increasing AI service rate. Thus, new technologies to identify nonpregnant dairy cows and heifers early after artificial insemination (AI) may play a key role in management strategies to improve reproductive efficiency and profitability on commercial dairy farms. Transrectal palpation is the oldest and most widely used method for early pregnancy diagnosis in dairy cattle (Cowie, 1948). However, a newer technology may someday replace transrectal palpation as the method of choice for pregnancy diagnosis in the dairy industry. Before this transition can occur, two events must transpire. First, a technology must be developed that exceeds transrectal palpation in one or more of the characteristics of the ideal early pregnancy test. Second and no less important, this new technology must be practically integrated into a systematic on-farm reproductive management strategy and empirically demonstrated to exceed the status quo of the industry (i.e., transrectal palpation) in reproductive performance. This overview will focus on transrectal ultrasonography due to the recent increase in adoption of this technology by bovine practitioners.

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Return to Estrus as a Diagnostic Indicator of Pregnancy Status

Return to estrus from 18 to 24 days after AI is often considered by dairy farmers the easiest and least costly method for determining nonpregnancy in dairy cattle early post breeding. This assumption, however, is being challenged by new research and long-recognized reproductive problems. First, estrous detection efficiency is estimated to be less than 50% on most dairy farms in the United States (Senger, 1994). This is likely a result of the short duration of estrus behavior reported for lactating cows (Dransfield et al., 1998) and because cows display estrus behavior poorly when housed on concrete flooring (Vailes and Britt, 1990), a common housing situation for dairy cattle in many regions of the US and other countries. Second, estrous cycle duration varies widely among lactating dairy cows from the standard 21-day interval and averaged around 24 days with a high degree of variability among animals lactating dairy cows (Sartori et al., 2004). This variability makes it difficult to target detection of return to estrus for groups of animals receiving AI on the same day. Finally, the high rate of pregnancy loss in dairy cows can increase the interval from insemination to return to estrus for cows that maintain a pregnancy then loose that pregnancy later during gestation (Fricke et al., 2003). The rate of pregnancy loss occurring during the period of gestation when dairy cattle are submitted for pregnancy examinations using ultrasonography or rectal palpation is high and, therefore, is a key factor for understanding the implementation and implications of methods for early pregnancy diagnosis.

Pregnancy Loss in Dairy Cattle

Pregnancy loss contributes to reproductive inefficiency because fertility assessed at any point during pregnancy is a function of both conception rate and pregnancy loss (Fricke, 2002). Since the widespread implementation of transrectal ultrasonography for reproductive research in cattle (Griffin and Ginther, 1992), several studies have reported rates of pregnancy loss during early gestation under field conditions. Table 1 summarizes reported rates of pregnancy loss in lactating dairy cows from an initial pregnancy diagnosis conducted 27 to 30 days post breeding to a subsequent pregnancy reassessment 14 to 42 days later. Taken together, average pregnancy loss reported in these studies exceeded 15%. Vasconcelos et al. (1997) characterized pregnancy loss at various stages of gestation using transrectal ultrasonography and reported pregnancy losses of 11% from 28 to 42 d, 6% from 42 to 56 d, and 2% from 56 to 98 d post AI, suggesting that the rate of loss is higher early during gestation, then decreases as gestation proceeds.

Early pregnancy diagnosis can improve reproductive performance by decreasing the interval between successive AI services and coupling a nonpregnancy diagnosis with an aggressive strategy to rapidly rebreed these animals (Fricke, 2002). Conversely, it has long been accepted that pregnancy status should be determined in dairy cattle as soon as possible after insemination but without having the diagnosis confounded by subsequent pregnancy loss (Studer, 1969; Melrose, 1979). Pregnancy loss diminishes the benefit of early pregnancy diagnosis in two ways. First, because of the high rate of pregnancy loss that occurs around the time during gestation that most direct and indirect pregnancy tests are performed (Table 1); the magnitude of pregnancy loss detected is greater the earlier post breeding that a positive diagnosis is made. Thus, the earlier that pregnancy is diagnosed post breeding, the fewer nonpregnant cows are identified to which a management strategy can be implemented to rebreed them. Second and more important, cows diagnosed pregnant earlier post breeding have a greater risk for pregnancy loss compared to cows diagnosed later post breeding. If left unidentified, cows diagnosed pregnant early post breeding that subsequently loose that pregnancy reduce reproductive efficiency by extending the interval from calving to the conception that results in a full-term pregnancy.

To compensate for pregnancy loss, cows diagnosed pregnant early post breeding must undergo one or more subsequent pregnancy reconfirmations to identify and rebreed cows that experience pregnancy loss. Thus, dairy mangers who have implemented early pregnancy diagnoses must consider the timing and frequency of subsequent pregnancy examinations to maintain the reproductive performance of the herd. Problems caused by pregnancy loss apply to all currently available methods for assessing pregnancy status early post breeding, and may relegate pregnancy testing before 30 to 40 days post breeding an untenable management strategy unless pregnancy diagnoses can be made continually on a daily basis or at each milking until the rate of pregnancy loss decreases, or until the underlying causes of pregnancy loss are understood and mitigated.

Attributes of the Ideal Pregnancy Test

For successful integration into a reproductive management system, an ideal early pregnancy test for dairy cattle would be 1) sensitive (i.e., correctly identify pregnant animals) 2) specific (i.e., correctly identify nonpregnant animals), 3) inexpensive, 4) simple to conduct under field conditions, and 5) able to determine pregnancy status at the time the test is performed. Most currently available methods for pregnancy diagnosis exhibit one or more of these attributes, but none currently available or under development exhibit all of them. A final attribute of an ideal test would be the ability to determine pregnancy status without the need to physically handle the animal to administer the test. Such a test may overcome the inherent limitations of current tests caused by pregnancy loss and may make pregnancy diagnosis before 30 to 40 days postpartum in dairy cattle an economically viable reproductive management strategy. Although rectal palpation and transrectal ultrasonography both require animal handling to administer the test, future strategies and technologies for early pregnancy diagnosis may someday realize this goal.

Transrectal Palpation

Transrectal palpation of the uterus for pregnancy diagnosis in cattle was first described in the 1800’s (Cowie, 1948) and is the oldest and most widely used method for early pregnancy diagnosis in dairy cattle today. Palpation technique can vary among practitioners. Transrectal palpation of the amniotic vesicle as an aid in determining pregnancy status in dairy cattle was described by Wisnicky and Cassida (1948), whereas slipping of the chorioallantoic membranes between the palpator’s thumb and forefinger beginning on about day 30 of gestation was described by Zemjanis (1970). Veterinary schools across the US and in other countries continue to train their students in the art of transrectal palpation for diagnosis of pregnancy in dairy cattle.

Because pregnancy in cattle can be terminated by manual rupture of the amnionic vesicle (Ball and Carroll, 1963), many studies have investigated the extent of iatrogenic pregnancy loss induced by transrectal palpation. Several studies have suggested that examining pregnant cows early in gestation by transrectal palpation increases the risk of iatrogenic pregnancy loss (Abbitt et al., 1978; Franco et al., 1987; Paisley et al., 1978; Valliancourt et al., 1979; White et al., 1989), whereas other studies have suggested that cows submitted for transrectal palpation earlier during gestation had a decreased risk for abortion or that palpation had no effect on subsequent embryonic losses (Studer, 1969; Thurmond and Picanso, 1993). Although controversy still exists regarding the extent of iatrogenic pregnancy loss induced by transrectal palpation, other factors have a greater influence on calving rates than pregnancy examination by transrectal palpation (Thompson et al., 1994). Furthermore, because the risk of pregnancy loss is high during the period of gestation when cows are diagnosed pregnant by transrectal palpation (Table 1), and because most cows within a herd are submitted for pregnancy examination, it is impossible for dairy producers and veterinarians to distinguish between iatrogenic losses occurring due to transrectal palpation and spontaneous losses that would normally have occurred in these cows.

Because of its widespread use and the number of bovine practitioners trained to perform the procedure, transrectal palpation will likely remain a mainstay for pregnancy diagnosis in dairy cattle until a newer method for pregnancy diagnosis is developed that exceeds the technique in one or more of the attributes of the ideal pregnancy test. Furthermore, because of its widespread use, high accuracy, and relatively low cost per animal, transrectal palpation is the industry standard that newer methods for pregnancy diagnosis in dairy cattle must displace as the method of choice for pregnancy diagnosis.

B-Mode Ultrasonography

Applications of and detailed methods for performing transrectal ultrasonography for reproductive research have been reviewed and described in detail (Ginther, 1998; Griffin and Ginther, 1992). Most veterinary students continue to be taught that ultrasound is a secondary technology for bovine reproductive work; however, the information-gathering capabilities of ultrasonic imaging far exceed those of transrectal palpation (Ginther, 1995). Although early pregnancy diagnosis is among the most practical application for reproductive management using transrectal ultrasonography, additional information gathered using the technology that may be useful for reproductive management include evaluation of ovarian structures, identification of cows carrying twin fetuses, and determination of fetal sex (Fricke, 2002). A fetal heartbeat can be visualized at around 21 d of gestation under controlled experimental conditions and using a high-quality scanner and transducer (Curran et al., 1986), and represents the definitive characteristic for positive confirmation of a viable pregnancy using transrectal ultrasonography. Although the rate of pregnancy loss is significant in studies using ultrasound to assess the rate of loss (Table 1), the technique itself has not been implicated as a direct cause of pregnancy loss in cattle (Ball and Logue, 1994; Baxter and Ward, 1997). Ultrasound is a less invasive technique for early pregnancy diagnosis than is transrectal palpation (Paisley et al., 1978; Vaillancourt et al., 1979) and may minimize the rare incidence of palpation-induced abortions.

Under most on-farm conditions, pregnancy diagnosis can be rapidly and accurately diagnosed using ultrasound as early as 26 d post AI (Filteau and DesCôteaux, 1998; Kastelic et al., 1991). When conducted between 21 and 25 d post breeding, sensitivity and specificity of pregnancy diagnosis using ultrasound was 44.8% and 82.3%, respectively, but increased to 97.7% and 87.7%, respectively, when conducted between 26 and 33 d post AI (Pieterse et al., 1990). Sensitivity and specificity of pregnancy diagnosis in lactating dairy cows based on ultrasonographic detection of uterine fluid as well as embryonic membranes from 28 to 35 days after AI was 96% and 97%, respectively (Nation et al., 2003). Pregnancy diagnosis in dairy heifers based on the presence of intraluminal uterine fluid before Day 16, however, is unreliable because small amounts of fluid are present in non-inseminated heifers as early as 10 days after estrus (Kastelic et al., 1991). For lactating dairy cows, ultrasonographic detection of uterine fluid as well as embryonic membranes from 28 to 35 days after AI was an accurate estimation of the presence of an embryo at the time of observation (Nation et al., 2003). Although ultrasound conducted at ≥ 45 days post breeding did not increase accuracy of pregnancy diagnosis for an experienced palpator, it may improve diagnostic accuracy of a less experienced one (Galland et al., 1994).

As a pregnancy diagnosis method, transrectal ultrasonography is accurate and rapid, and the outcome of the test is known immediately at the time the test is conducted. Veterinary-grade ultrasound machines equipped with one rectal transducer are expensive and cost $8,000 to $16,000, and the cost of this technology may limit its practical implementation (Fricke, 2002). Although dairy producers can purchase an ultrasound scanner and conduct pregnancy examinations on their own cows, they generally lack the knowledge, training, and experience required to accurately perform pregnancy examinations (Fricke, 2002). Transrectal ultrasonography is slowly being incorporated into reproductive management schemes in dairies primarily by bovine practitioners who have adopted this technology. The extent to which transrectal ultrasonography will displace transrectal palpation as the primary direct method for pregnancy diagnosis in dairy cattle remains to be seen. Because many experienced bovine practitioners can accurately diagnose pregnancy as early as 35 days post breeding using transrectal palpation, pregnancy examination using transrectal ultrasonography at 26 to 28 days post breeding only reduces the interval from insemination to pregnancy diagnosis by 7 to 9 days. The rate of pregnancy loss and the efficacy of strategies to rebreed cows at various stages post breeding also play a role in determining the advantages and disadvantages on the timing of pregnancy diagnosis and resynchronization (Fricke et al., 2003).

On Farm Implementation of Early Nonpregnancy Diagnosis

Synergies between new reproductive management technologies hold the key to maximizing reproductive efficiency on dairy farms. However, reproductive management protocols that allow for synchronization of ovulation and subsequent identification and resynchronization of nonpregnant cows must be practical to implement within the day to day operation of a dairy farm or the protocol will fail due to lack of compliance (Fricke et al., 2003). This is especially true for larger farms that must schedule and administer artificial inseminations, hormone injections, and pregnancy tests for a large number of animals on a daily or weekly basis. Identification of nonpregnant cows early post breeding can only improve reproductive efficiency when coupled with a management strategy to rapidly submit nonpregnant cows for a subsequent AI service. Thus, any method for early pregnancy diagnosis must be integrated as a component of the overall reproductive management strategy in place on the farm. The various component technologies of the reproductive management system will in turn determine the timing of the events as they occur on a daily or weekly basis. As stated previously, it has long been accepted that pregnancy status should be determined in dairy cattle as soon as possible after insemination but without having the diagnosis confounded by subsequent pregnancy loss (Studer, 1969; Melrose, 1979). New research on the practical implementation of early pregnancy diagnosis using transrectal ultrasonography into a systematic synchronization and resynchronization system has confirmed this notion and illustrated the pitfalls and limitations of early pregnancy diagnosis (Fricke et al., 2003).

Field Trial: Integrating Systematic Synchronization with Transrectal Ultrasonography

Two recently adopted technologies for reproductive management of dairy cattle include hormonal protocols such as Ovsynch (Pursley et al., 1995, 1997) and Presynch/Ovsynch (Moreira et al., 2001; Navanukraw et al., 2004), and use of transrectal ultrasonography for early identification of nonpregnant cows (Fricke, 2002). We conducted a field trial to compare three intervals from first TAI to resynchronization of ovulation on a dairy incorporating transrectal ultrasonography as a method for early pregnancy diagnosis (Fricke et al., 2003). The objective was to compare conception rate to first TAI service after a modified Presynch protocol with conception rates after resynchronization of ovulation using Ovsynch at three intervals post TAI (Resynch) coupled with pregnancy diagnosis using transrectal ultrasonography. Lactating dairy cows on a commercial dairy farm were enrolled into this study on a weekly basis.

All cows received a modified Presynch protocol to receive first postpartum TAI as follows: 25 mg PGF2α (d 32 ± 3; d 46 ± 3); 50 μg GnRH (d 60 ± 3); 25 mg PGF2α (d 67 ± 3) and 50 μg GnRH (d 69 ± 3) postpartum (Navanukraw et al., 2004). All cows received TAI immediately after the second GnRH injection of the Presynch protocol (d 0) as per a Cosynch TAI schedule. At first TAI, cows were randomly assigned to each of three treatment groups for resynchronization of ovulation (Resynch) using Ovsynch [50 μg GnRH (d -9); 25 mg PGF2α (d -2) and 50 μg GnRH + TAI (d -0)] to induce a second TAI for cows failing to conceive to first TAI service. All cows (n=235) in the first group (D19) received a GnRH injection on d 19 post TAI and continued the Ovsynch protocol if diagnosed nonpregnant using transrectal ultrasound on d 26 post TAI. Cows (n=240) in the second (D26) and cows (n=236) in the third (D33) groups initiated the Ovsynch protocol if diagnosed nonpregnant using transrectal ultrasound on d 26 post-TAI or d 33 post-TAI, respectively. Submission of cows for first postpartum TAI service was scheduled so that the first four injections of the Presynch plus Ovsynch protocol occurred on Tuesdays followed by the second GnRH injection and TAI occurring on Thursdays (Table 2). Initiation times for Resynch for each of the three treatment groups in this study were chosen to occur on Tuesdays so that injection schedules would remain consistent for all cows assigned to weekly breeding groups at any given time. To adhere to the Tuesday/Thursday schedule, all pregnancy examinations were conducted on Tuesdays. To fit the reproductive management system, the first pregnancy examination using transrectal ultrasound was conducted 26 d after TAI for the D19 and D26 cows and 33 d after TAI for the D33 cows (Figure 1).

Figure 1. Diagram of resynchronization treatment groups from Fricke et al. (2003). Pregnancy rate per artificial insemination (PR/AI) and pregnancy loss from first pregnancy evaluation to a second reconfirmation were evaluated to determine the best method for integration of early pregnancy diagnosis using transrectal ultrasonography.

Implicit to the experimental design, first assessment of pregnancy status was not conducted at the same interval after the Ovsynch TAI among the three treatment groups. Pregnancy status after the Ovsynch TAI was first assessed 26 d after TAI for cows in the D19 and D26 groups, whereas pregnancy status was assessed 33 d post Ovsynch TAI for cows in the D33 group. Overall fertility to Ovsynch was 40% and was greater for D19 and D26 cows than for D33 cows (Table 3). This difference is likely due to a greater period in which pregnancy loss can occur in the D33 cows due to the increased interval from TAI to pregnancy diagnosis (26 vs. 33 d). When pregnancy status was reassessed for all treatment groups at 68 d after Ovsynch TAI, overall PR/AI to Ovsynch was 31% and did not differ among treatments (Table 3). Thus, differences in PR/AI at the first pregnancy exam and pregnancy losses between the first and second pregnancy exams among treatment groups likely represent an artifact of time of assessment of pregnancy GnRH + TAI status after TAI inherent to the experimental design rather than to treatment differences. Overall PR/AI to Resynch was 32% and was greater for D26 and D33 cows than for D19 cows (Table 4).

The Challenges for Early Pregnancy Diagnosis

Data from Tables 3 and 4 illustrate the limitations of integrating early pregnancy diagnosis into a reproductive management program. First, the system with the most aggressive early nonpregnancy diagnosis and resynchronzation schedule (i.e., the D19 treatment) was not a viable management strategy based on the poor fertility after the Resynch TAI (Table 4) probably due to follicular and luteal dynamics at the stage post breeding that the synchronization protocol was initiated. Furthermore, these results suggest the counterintuitive notion that delaying pregnancy diagnosis from 26 to 33 days post TAI may improve reproductive efficiency when using a hormonal protocol for timed AI to program nonpregnant cows for rebreeding due to the high rate of pregnancy loss occurring in cows diagnosed pregnant at 26 vs. 33 days post TAI (Table 3).

Conclusion

Although coupling a nonpregnancy diagnosis with a management decision to quickly reinitiate AI service may improve reproductive efficiency by decreasing the interval between AI services, early pregnancy loss and the effectiveness of hormonal ovulation and estrus control protocols initiated at certain physiologic stages post breeding may limit the effectiveness of many methods for early pregnancy diagnosis currently under development, especially when compared to
transrectal palpation. These limitations make the benefits of many currently available methods for early pregnancy diagnosis questionable and require that all animals diagnosed pregnant early after insemination be scheduled for rechecks at later times during gestation to identify animals experiencing pregnancy loss. It remains to be seen whether a new test will replace transrectal palpation as the primary method used for pregnancy diagnosis in dairy cattle.

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Author

Paul M. Fricke
Department of Dairy Science
University of Wisconsin-Madison
Madison, WI 53706
pmfricke@wisc.edu