Maintaining a successful reproductive program on today’s dairies is an arduous task. While it is rewarding to hear a pronouncement of pregnancy, there is not much the manager can do with that diagnosis. Except wait. Wait to recheck later to make sure the calf is growing. Wait for a calf to be born. So while it is tempting to focus on a positive pregnancy diagnosis, it is important to remember that an early non – pregnancy diagnosis is valuable information to act upon.
The sooner non-pregnant cows can be identified, the sooner they can be re – inseminated. Coupling a non-pregnancy diagnosis with a management decision to quickly reinitiate AI service improves reproductive efficiency and pregnancy rate. Repeat breeders who fail to become or stay pregnant also offer management the option to make a culling decision.
There are direct and indirect methods used to determine pregnancy status. Direct methods involve the direct detection of the tissues and/or associated fluids of the conceptus either manually by transrectal palpation or visualized by transrectal ultrasound. Indirect methods measure reproductive hormones at specific stages after AI or the detection of conceptus specific substances in maternal body fluids.
Direct Determinations
The watch and wait method is the gold standard of measuring pregnancy status. This direct method involves the direct visualization of the calf being born. This method is both sensitive and specific. She either was pregnant and delivered a calf, or she was not; and a calf is not born.
Bumping the calf through the cow’s abdominal wall is another direct method of determining pregnancy status. Depending on the size and stature of the cow and the experience of the clinician, the calf may be felt during the last trimester of pregnancy. This is accomplished by vigorously bumping against the cow’s right flank with a closed fist, to feel the skeletal mass of the calf. Calf bumping is not the best method of non – pregnancy diagnosis; the calf may be small, or positioned dorsally in the caudal abdomen. Repeated calf bumping on different days at different times may help to improve accuracy of the diagnosis using this method.
Perhaps the oldest form of directly determining pregnancy status involves watching for return to estrus. The bovine estrous cycle is approximately 21 days long, with a range of 17 – 24 days. Absence of estrous activity every three weeks until parturition provides strong evidence that conception occurred and the cow did not suffer pregnancy loss. Gestation length in Holsteins is approximately 282 days (9 months).
The need to do better
Successful reproductive managers quickly realize the need for an accurate early determination of pregnancy status so that action can be taken on non – pregnant cows. Managers also need to have confidence in the test result.
While the “watch and wait” method is highly accurate, it is the least time sensitive. Considering the average gestation is around 282 days, that’s potentially 9 months a cow could be not pregnant and management doesn’t know and can’t act upon it. Likewise, estrous detection is an important skill but can be fraught with inaccuracies. Failure alone to observe estrus is not enough to confirm pregnancy. There’s always the possibility a cow was in estrus, and management failed to observe it.
Dairy workers should know the signs of estrous behavior. These include: vaginal mucous discharge, increased restlessness and increased vocalization, smelling of other cows, following other cows and attempting to mount them, allowing themselves to be mounted, and a ruffled tail head or mud on their hind flanks from being mounted. Reproductive management plans must include what to do with cows exhibiting estrus.
Palpating or visualizing the developing fetus and placenta is a direct method of determining pregnancy status. Skilled veterinarians performing transrectal palpation are able to diagnose pregnancy status beginning approximately 35 days post breeding. Accuracy is achieved with experience. Physical attributes of the cow also have an effect. For example, cows with excessive pelvic fat are harder to palpate. During transrectal palpation, the veterinarian renders a positive diagnosis of pregnancy when he/she palpates a fluid filled uterine horn, the presence of a mature corpus luteum (CL) ipsilateral to the fluid filled horn, presence of amniotic vesicle (approximately 35 days post conception,) and/or placenta, (approximately 40 days post conception). Palpation also allows evaluation of uterine and ovarian pathology.
When using transrectal ultrasonography, visualization of a corpus luteum on the ovary ipsilateral to the fluid – filled uterine horn containing an embryo with a heartbeat is the basis for a positive pregnancy diagnosis. This can be confidently visualized 30 days post breeding. Ultrasound allows the visualization of dead embryos, ovarian pathology, uterine morphology and twins. Fetal sex can be accurately diagnosed at fetal age 55 to 60 days.
Researchers have concluded the overall accuracy of transrectal ultrasonography for determination of pregnancy status 27 days after timed AI to be 95% when the previously determined cows were rechecked at a later date. The positive predictive value was 90%. The negative predictive value was correct for 98% The positive predictive value is lower because of the normal early pregnancy loss; these cows may have been pregnant at the early ultrasound determination, only to be found open at re – check. Twins decrease the positive predictive value of the diagnosis. Pregnancy loss is three – fold higher for cows carrying twins versus singletons (Silva et al.).
How well does the test outcome predict true status of the pregnancy?
Positive predictive value: the test correctly identified those truly pregnant; determination of pregnancy was confirmed with recheck by another method and/or at a later date.
Negative predictive value: the test correctly identified those truly not pregnant; determination of non – pregnancy was confirmed with recheck by another method and/or at a later date.
Indirect Determinations
Measuring the reproductive hormone progesterone is an example of an indirect method. Progesterone (P4) monitoring is used to identify non – pregnant cows. Cow – side kits which measure P4 in milk are currently available. P4 monitoring has a 95% to 100% negative predictive value, low progesterone levels on day 21, or better yet on day 23 after last estrous (post – AI) indicate the cow is open. Mastitis can disguise progesterone; milk from mastitic quarters yields low P4. The positive predictive value of milk P4 is 80%. This low positive predictive value is due to embryonic mortality. P4 will remain high with pyometra. (Rhodes).
The hormone progesterone is secreted by the cor- pus luteum (CL) and is responsible for maintaining the pregnancy. If cow is pregnant, or a fluid filled uterine horn signals pregnancy, progesterone per- sists beyond the next heat cycle. At best, you can use milk progesterone tests to tell you if cow is open 21 – 24 days after last breeding. Monitoring milk progesterone may help you perform better heat detection. Serial monitoring would give you a good indication of her reproductive cycle.
Bovine pregnancy-associated glycoprotein (PAG) is secreted by bi – nucleate cells present in cotyledons. Over 20 different bovine PAGs have been identified. While researchers do not understand the function of these PAGs, better testing techniques now allow them to be used as a chemical marker for pregnancy diagnosis.
An ELISA blood test was developed to detect six PAGs as a method for early pregnancy diagnosis. Consistent measurable PAG levels in maternal blood plasma occurs 15 days after conception and peaks at day 32 post conception. PAG levels then sharply decline to a nadir from day 53 to 60 days post conception, gradually increasing to 74 to 102 days. Increasing levels are measured as the cotyledon mass grows while supporting the growing fetus, and remain high until 60 days after calving. (Ricci et al, 2015).
Primiparous cows have greater PAG levels than multiparous cows. Researchers do not know why the six PAGs measured by ELISA drop to a low level 53 days after conception and then why they rebound. It is unknown what is happening with the other PAGs not measured by the available test.
Commercial blood plasma ELISA PAG tests currently being offered by various private veterinary clinic and commercial animal health laboratories include BioPRYN, DG29 and the IDEXX Bovine Pregnancy Test. When mailed overnight, it takes approximately 36 hours from sample collection to receive outcomes from the lab. Scheduling with mail services must be taken into account when submitting samples.
Overall accuracy of determining pregnancy status when measuring plasma PAG at 32 days post AI was found to be 95%. The positive predictive value using plasma PAG was 91%, the negative predictive value was 97%. Prediction of non – pregnancy is highly accurate, just as it is for transrectal ultrasonography. Early pregnancy loss contributes to the lower accuracy of an early pregnancy diagnoses (Silva et al).
PAGs secreted in maternal blood are transferred to milk; however, milk PAG levels are approximately two – fold less than plasma PAG levels. IDEXX Laboratories has developed a commercial milk ELISA to measure PAG. Milk PAG levels peak 32 to 39 days post – conception. Milk PAG levels then slowly decline to a nadir from 46 to 67, rebounding to higher levels 74 to 102 days post conception. Milk PAG drops sooner and remains lower longer than does plasma PAG. PAGs are measurable in milk for 60 days after calving.
Plasma and milk PAG levels are negatively correlated with milk production for both primiparous and multiparous cows (Ricci et al, 2015). Dilution of milk PAG by increased milk production is not the reason for why milk PAG declines, because plasma PAG also declines as milk production increases. While scientists speculate the reasons for this negative correlation, those evaluating test outcomes must bear in mind the cows’ milk production may cause a false negative test result. Rechecks may be needed.
Accuracy of both plasma and milk PAG testing compared to transrectal ultrasonography was evaluated from 25 to 102 days after AI . Overall accuracy for correctly identifying 32 day pregnancy status to recheck status was 92% for plasma and 89% for milk PAG levels. The plasma PAG positive predictive value was 84% while its negative predictive value was 100%. The milk PAG positive predictive value was 78% and its negative predictive value was 99% (Ricci et al).
The importance of Re-checks
It has long been recommended that pregnancy status should be determined in dairy cows as soon as possible after AI, but without having the diagnosis confounded by subsequent pregnancy loss. Various research trials confirm a normal 13% pregnancy loss occurring from 27 to 31 days and 38 to 50 days in gestation (Ricci, et al, 2014). An early diagnosis of pregnancy (less than 50 days after AI) must be re – confirmed at a later date to identify cows that experience early embryonic death.
In Summary
Managers have several direct and indirect methods available for determining bovine pregnancy status. It is important for managers to have confidence in the determination of a non – pregnant outcome so they can act on that information. Accuracy is correlated to the days post service the determination is made. Coupling a non – pregnancy diagnosis with a management decision to quickly re – inseminate the cow improves reproductive efficiency and pregnancy rate.
| *Days post AI | Pregnant | Recheck | Non-pregnant |
| Visual return to estrus | Does not return to heat until after calves | Does not return to heat every 21 days | Returns to heat every 21 days |
| Progesterone assay | High P4 on day 23 | Low P4 on day 23 | |
| Transrectal palpation | Fetus day 50-60 | CL, membrane slip/amniotic vesicle day 35-40 | Non conceptus day 35-40, no CL, developing follicle |
| Transrectal ultrasound | Fetal sex day 58 | Amniotic vesicle/fetal heartbeat day 30 | No conceptus day 30, no CL, developing follicle |
| Blood PAG | Positive day 74 | Positive day 32
False negative with high milk production |
Negative day 32 |
| Milk PAG | Positive day 74 | Positive day 32-39
False negative with high milk production |
Negative day 32-39 |
| Blood or Milk PAG |
False positive: < 60 days since last pregnancy False negative: blood: 53-60 days milk: 46-67 days |
Source: UW Extension
