These markers of PD are also secreted by the non-pregnant animal. Thus, the usefulness of these factors to detect pregnancy in animals is mostly dependent on their concentration in the biological fluid. Since these markers are also present in the non-pregnant animal at one or other stage, these markers usually give false positive results and thus their use is limited for PD. Nevertheless, these are still useful markers of PD if they are used in combination with some other method to confirm pregnancy.

Progesterone

Progesterone, a steroid hormone, is one of the most commonly used non-pregnancy specific markers used for the PD. It is used to detect PD on the basis of its concentration in the blood/plasma/serum/milk. Progesterone is secreted by corpus luteum (CL) to maintain the pregnancy in the animal. It is also secreted post ovulation and thus is also used for estrus detection. One of the main limitations with the use of progesterone is higher rate of false positive, where it identifies a non-pregnant animal as pregnant. This is the reason that progesterone has not been accepted widely for the PD in animals as the results are not consistent.

Miscellaneous Markers

Since the fetus is semi-allogeneic, the biological changes must occur in females to accept the incoming fetus and thus its implantation must be associated with the secretion of signals which may be used as markers for PD. One such important signal is interferon tau (IFNT) which is secreted as maternal recognition factor, which occurs day 15 to 17 of gestation, for carrying the fetus to full term. But due to the difficulty in assaying the quantity of IFNT in the blood of animals, a reliable test or kit was not developed. Therefore the focus was shifted towards the quantification of genes associated with the IFNT signaling in the peripheral blood mononuclear cells and were collectively termed as interferon stimulated genes (ISGs). The most commonly used ISGs for the differentiation of pregnant and cows are ISG15, Mx1 and Mx2. Since these genes are stimulated by IFNT, which is also an antiviral protein, virus infection in animals results in false positive signals.

In summary, non-specific tests are not reliable for PD in cattle and buffaloes, but their use may be recommended with some other tests. The commercial test or kits have also not been developed with these markers and their use is mainly limited to the laboratories. But still these may be helpful in future as tests are further refined and standardized.

These markers are expressed in the pregnant animals only and thus are more useful in developing kits for PD in cattle and buffaloes.

Early Pregnancy Factors (EPFs)

EPFs are supposed to suppress the immune system to make the uterus and immune system of animals ready to accept the semi-allogeneic fetus for implantation. EPF was used for PD tests due to its target sampling time from 2 to 7 days. But like progesterone the results are not specific and cannot identify the non-pregnant animals. Also, most of the pregnancy loss occurs during post day 2 of fertilization therefore the use of EPF for PD is limited for PD in cattle and buffaloes.

Estrone Sulphate

Estrone sulphate is present in bovine placentomes and is a conjugated steroid product of estrogen. It is mainly present in amniotic and allantoic fluids and maternal plasma. It is detected after 52 days of pregnancy. However, ES can be used reliably only after 100 days of pregnancy. This is the main reason that its use was avoided for the PD.

Pregnancy Associated Glycoproteins (PAGs)

PAGs are identified as markers for pregnancy in cattle and buffaloes. It is suggested that the PAGS may play a role in the adhesion of the uterus and placenta during implantation, though their role is largely unknown. PAGs are secreted by binucleate trophoblast cells of the placenta and may be detected in the blood as early as day 22 of gestation. Some of the most commonly used PAGs for the detection of pregnancy are PAG-1, PAG-4, PAG-6, PAG-7, and PAG-17. PAG-ELISA tests have also been developed for the PD. Some commercially available PD tests based on PAGs have accuracy level of 95-99% in detecting the pregnant animal positively and false positive rate is only 1-5% which may be due to the early embryonic mortality. Recently, few farmer friendly kits as strip based assays have been developed by using PAGs by NDRI, Karnal and are under trial for the PD in cattle and buffaloes. Some of the currently available PD tests are BioPRYN (BioTracking LLC. Moscow, ID USA), DG29 pregnancy test (Genex Co-operative Inc. Shawano, USA) and IDEXX Bovine pregnancy test (IDEXX Laboratories Inc. Westbrook, USA).

Circulating miRNAs

miRNAs are the non-coding RNAs of 18-22 nucleotides in length and are involved in regulating the function of genes. miRNAs may be detected in the blood, serum, urine and milk of animals. These are released into the biological fluids in the form of extracellular vesicles and thus may be detected for pregnancy diagnosis. Some of the miRNAs like bta-mir 140, miR-XX1 and miR-XX2, have been predicted as the early markers of PD.

In summary, the pregnancy specific markers of pregnancy have more promising future and some commercial kits have already been developed for PD on the basis of PAGs. In future, we may see some kits for the PD being developed on miRNAs.