**7. Accurate diagnosis of pregnancy and pregnancy loss in dairy cow**

Pregnancy diagnosis plays an essential role in decreasing days open on dairy farms. Therefore, it is essential to select an accurate method for diagnosing early pregnancy and pregnancy loss (late embryonic and early fetal mortality) because the cost of each day open 100 days after calving may reach \$4.00 [165] or €2.5–6.5 [166], respectively. Besides traditional pregnancy diagnosis (rectal palpation of the uterus or progesterone tests) [167–169], there are several new possibilities to diagnose early pregnancy on dairy farms. However, before introducing any new diagnostic test on our dairy farm, we must evaluate the accuracy of that particular test. Their results must be confirmed by the old diagnostic method to decrease the adverse effects of false-negative diagnoses. This can be caused by prostaglandin treatment to reduce the interval to the next AI service [167] or by using new resynchronization protocols in our dairy farm [170–172].

One of the most recent techniques for diagnosing early pregnancy on the dairy farm is B-mode ultrasonography. Under field conditions, ultrasonography may achieve accurate results from Days 25 to 30 after AI [173–175]. However, the accuracy of the transrectal ultrasonographic diagnoses greatly depends on the frequency of the transducer used, the surgeon's skill, the criterion used for a positive pregnancy diagnosis, and the position of the uterus in the pelvic inlet [176]. For example, if during ulrasonographic examinations performed between Days 24 and 38 after AI, we can find a uterus far cranial to the pelvic inlet compared with those cases when the uterus is located within or close to the pelvic inlet, we can make more incorrect nonpregnancy diagnoses [177].

Nonpregnant animals can be selected accurately by evaluating blood flow in the corpus luteum around Day 20 after AI, meaning we can substantially improve the reproductive efficiency of our herd [169].

Pregnancy protein RIA assays such as pregnancy-specific protein B/PSPB/, pregnancy-associated glycoprotein/PAG/, and PSP60, commercial ELISA, or rapid visual ELISA tests may provide an alternative method to ultrasonography for determining early pregnancy and pregnancy loss in dairy cows. However, the relatively long half-life after calving and pregnancy loss may limit the effectiveness of these laboratory methods for early pregnancy diagnosis in the field, especially when compared with a direct method such as transrectal ultrasonography [176]. Linear array/sector B-mode [178] and Doppler ultrasonography [169] may exceed the other diagnostic methods in the amount of information collected from each animal during scanning. However, their accuracies greatly depend on the operator's proficiency and availability [178].

A new technology (in-line milk analysis system) has already made the automatic collection of milk samples at milking robots or in the milking parlor to analyze progesterone to detect early pregnancy and pregnancy loss, respectively [113, 179, 180]. Bruinjé and Ambrose [113] reported high sensitivity (>95%) from Day 27 after AI, while the specificity was somewhat lower before Day 40 after AI. Any new biomarkers discovered for early pregnancy diagnosis may make it possible to diagnose

#### *Animal Husbandry*

pregnancy loss much earlier, which may significantly contribute to increasing reproductive efficiency in our dairy herds. The importance of this technology would also be emphasized by its ability to identify pregnant and nonpregnant animals on time with no animal handling because even a simple transrectal examination of dairy cows can lead to increased plasma and salivary cortisol concentrations and changes in heart rate, heart rate variability, and behavior that are indicative of pain [181].

Although fertilization in the cow can be detected by measuring the early pregnancy factor with the rosette inhibition test, it is not a practical method; therefore, it needs further development. Recently found biomarkers such as interferon-tau-stimulated genes or microRNAs may help us diagnose early pregnancy in dairy cows; however, these tests need further development before their general use in the dairy practice [176].
