Pregnancy testing in cattle! Which options are available?

Pregnancy testing in cattle is crucial for effective herd management and economic efficiency. By identifying pregnant cows early, farmers can ensure that these animals receive the appropriate management for dry-off decision, the correct nutrition and care needed for a healthy pregnancy and successful calving. 

Early pregnancy diagnosis helps in detecting non-pregnant cows, allowing for prompt rebreeding decisions or eventually culling, which can significantly reduce the costs associated with maintaining non-productive animals. Additionally, early pregnancy detection can help in planning for labor and resources around calving, ultimately leading to improved herd health and productivity. Overall, regular pregnancy testing is a key practice for optimizing reproductive performance and maximizing the profitability of cattle operations.

Rectal palpation

One of the earliest methods applied in large scale in Bovine Veterinary medicine for the determination of pregnancy is surely the rectal palpation of the uterine content and of the ovaries. Mastering this skill has been a relevant milestone in the profession for most veterinary students and new graduates which have been approaching the practice of bovine medicine.

Traditionally, cows are appointed to be examined no earlier than 40 to 60 days of supposed gestation, depending upon the skill of the examiner and the age and size of the dam. This method of pregnancy diagnosis, upon the skills of the operator, can be performed as early as day 30 of pregnancy and can be used thereafter until term.

Due to several changes in the size, texture, location, and content of the uterus during pregnancy, the examiner must find at least one of these four univocal signs of pregnancy before making a definitive diagnosis, each of these can only be present in a pregnant animal at different stages of gestation (Zemjanis, 1970):

• Palpation of the amniotic vesicle,
• Sensing of the fetal membrane slip,
• Palpation of the placentomes (union of cotyledons and caruncles),
• Palpation of part of the fetus and its movements,

Rectal palpation also allows for estimates of the embryonic/fetal age and detection of ovarian structures such as a corpus luteum or follicles, when they are of relevant or marked shape end size. The procedure when performed by experienced operators does not bring potential risks of embryonic losses (Romano et al 2017).

Ultrasonography

The adoption of transrectal real time ultrasonography represents a significant revolution in the study of bovine reproduction. It is an extremely valuable tool for real time evaluations for practitioners and researchers that permits detection of pregnancy potentially as early as 25 days of gestation. Additionally, gives the best clues for the decision of application of the correct protocols for the re-synchronisation of open cows, according to the presence or absence of follicles and corpora lutea at the time of negative pregnancy diagnosis, in this the ultrasound brings the most accurate information in fact clinicians can visually detect structures that would be hard or even impossible to detect by rectal palpation (Fricke et al., 2005).

Pregnancy status (pregnancy stage, potential pathological conditions, the diagnosis of twinning), the fetal sex, the ovarian function and uterine morphology can all be accurately assessed with the use of ultrasound, making it a very valuable and flexible tool inside farms’ management.

In some of the preliminary work performed in Madison Wisconsin, at Dr. O.J Ginther laboratory, fetal heartbeat has been detected at approximately day 21 of gestation in cattle (Kastelic et al., 1988), but it is more easily and routinely detectable after day 25 of pregnancy and is the “gold standard” for proof of presence of a viable conceptus along with uterine morphological examinations and embryo measurements (crown rump length). 
Ultrasonography raises the level of accuracy of pregnancy detection as the death of the embryo can be clearly diagnosed by:

1. The absence of the embryo/fetus heartbeat,
2. Membranes detachment or disorganization,
3. Reduced amount of amniotic or allantoic fluid
4. Echoic floating structures, including remnants of the conceptus
   (Lopez-Gatius F.; Garcia-Ispierto, 2010).

The disadvantage of ultrasound is that elevated accuracy with good operative rhythms and high sensibility and specificity, is limited before 28-30 days of gestation and that the pregnancy status is only guaranteed upon the time of diagnosis.

In large scale dairy farms, where a reproductive routine is implemented, it is advisable to confirm the pregnancy status of cows after the first pregnancy-check, normally around day 60 of gestation. With this working routine, it is possible to accurately confirm the pregnancy of twins and the evaluation of the sex of the foetus/foetuses.

Sex is determined by evaluating the morphology and location of a fetal component called the genital tubercle using ultrasound. It is a reliable and accurate method for sex determination beginning on day 55 to 60 of gestation. Attempts to determine fetal sex earlier can be inaccurate due to incomplete migration of the genital tubercle. This bilobated hyperechoic structure which is initially developing medially on the abdominal wall, between the hombelicus and the tail of the embryo, migrates cranially towards the umbilicus in the male fetuses and towards the tail in the female fetuses around 2 months of gestation.

Pregnancy associated chemicals determination

Pregnancy associated glycoproteins (PAGs) were identified by researchers looking for a pregnancy specific marker that could be used for pregnancy diagnosis in cattle. While PAGs have proven to be a reliable marker for pregnancy, their physiological role is still unknown; although it has been hypothesized that PAGs may help assist with adhesion actions between the uterus and placenta (Wallace et al., 2015).

PAGs are synthesised by mononucleate and binucleate trophoblast cells of the placenta and secreted in the maternal bloodstream. PAGs enter the maternal circulation as early as day 22 to 24 and reach levels currently acceptable for accurate pregnancy diagnosis at day 28.

Pregnancy specific protein B (PSPB) or PAG-1 was the first identified member of the PAG family, which encompass more than 20 individual proteins and two dozen genes.

The ELISA was demonstrated to detect pregnant or non-pregnant cows via PAGs at day 28 post insemination. Studies comparing the efficacy of the PAG ELISA, PAG Radio immune assay and transrectal ultrasonography revealed comparable results for the diagnosis of pregnancy in cattle at day 28 of gestation although some differences were identified in the ability of certain assays to detect non-pregnant animals (Karen et al., 2015).

Today, commercial PAG testing is extremely accurate providing 98 % true positive (pregnant) reading and false positive (reported as pregnant but actual non-pregnant) rates from 1-5%; however, some false positives may be due to embryonic mortality.

Tests currently available include BioPRYN (BioTracking LLC. Moscow, ID USA), IDEXX Bovine pregnancy test (IDEXX Laboratories Inc. Westbrook, ME USA) and DG29 pregnancy test (Genex Cooperative Inc. Shawano, WI USA). BioPRYN accepts blood samples from heifers 25 days post breeding and cows 28 days post breeding, IDEXX recommends day 28 blood or milk samples and DG29 has been validated using day 29 blood samples.

The disadvantage of the PAGs is that they are secreted at a very high levels even after parturition and they are still circulating in the cow’s system, in high levels, until 60 days post partum;but this generally should not coincide with the timing of retesting for these cows.

Milk or blood PAGs are accurate to detect an early pregnancy between day 28 and 39 of gestation, then they physiologically decline and become detectable at accurate levels again from day 74 onwards. For this reason, in order to confirm a pregnancy and evaluate potential embryonic losses, cows should be re-tested not before day 74 (Ricci et al., 2015).

Conclusions

Pregnancy diagnosis is one of the key clinical procedures that veterinarians have provided to cattle farms for generations, this practice really supports the successful management of dairy and beef herds. Several options are available, the early detection of non-pregnant animals gives a unique opportunity to make the most sustainable decision for the farm.

More technological solutions such as ultrasonography bring some advantages to the farm such as the possibility of detecting embryonic mortality, twinning and eventually the gender of the fetus, allowing for potentially more management decisions to be made. Pregnancy associated glycoproteins in either blood or milk can help screening for pregnancy where the other methods might be complex to apply.

References

Zemjanis R. Pregnancy examination. In: Zemjanis R, ed. Diagnostic and therapeutic techniques in animal reproduction. 2nd ed. Baltimore: The Williams & Wilkins Co, 1970;29 46.

Romano JE, Fahning ML. Effects of early pregnancy diagnosis by per rectal palpation of the amniotic sac on pregnancy loss in dairy cattle. J Am Vet Med Assoc 2013;243:1462–7

Fricke, P. M., & Lamb, G. C. (2005). Potential applications and pitfalls of reproductive ultrasonography in bovine practice. The Veterinary clinics of North America. Food animal practice, 21(2), 419–436. https://doi.org/10.1016/j.cvfa.2005.02.005

Kastelic J, Curran S, Pierson R, Ginther O. Ultrasonic evaluation of the bovine conceptus. Theriogenology. 1988;29(1):39-54. http://dx.doi.org/10.1016/0093-691X(88)90030-1

Lopez-Gatius F.; Garcia-Ispierto, I. 2010. Ultrasound and endocrine findings that help to assess the risk of late embryo/early foetal loss by non-infectious cause in dairy cattle. Reprod Domest Anim 45: 9.

Wallace, R. M., K. G. Pohler, M. F. Smith, and J. A. Green. 2015. Placental PAGs: gene origins, expression patterns, and use as markers of pregnancy. Reproduction 149: R115-126.

Karen, A. et al. 2015. 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 159: 31-37.

Pohler, K.G. et al 2019., Pregnancy diagnosis in a beef herd, Proceedings, Applied Reproductive Strategies in Beef Cattle; August 20-21, 2019; Knoxville, TN., 158-167.

Ricci, A., Carvalho, P. D., Amundson, M. C., Fourdraine, R. H., Vincenti, L., & Fricke, P. M. (2015). Factors associated with pregnancy-associated glycoprotein (PAG) levels in plasma and milk of Holstein cows during early pregnancy and their effect on the accuracy of pregnancy diagnosis. Journal of dairy science, 98(4), 2502–2514. https://doi.org/10.3168/jds.2014-8974