Journal of Animal Sciences and Livestock Production Open Access

Keywords

BioPRYN Rapid visual Pregnancy Test (Bio-PRD); BioPRYN Flex ELISA; Optical Density (OD); Pregnancy Specific Protein B (PSPB); Pregnancy –Associated Glycoprotein (PAG); Prepared Enhancer solution; TMB substrate; Stop solution; Ultrasonography; Rectal palpation; Artificial Insemination (AI).

Introduction

Early pregnancy diagnosis is critical for maximizing herd productivity in the cattle industry. Current methods for pregnancy diagnosis include: observation for return to estrus, rectal palpation, trans-rectal ultrasonography, and blood tests for specific antigens.

These blood tests include detection of Pregnancy-Associated Glycoproteins (PAGs) which belongs to a large family of aspartic peptidase of which Pregnancy-Specific Protein B (PSPB) was the first member to be discovered [1]. These glycoproteins are produced exclusively by specialized trophoblastic giant cells in ruminant placenta [2] , which migrates from trophectoderm to fuse with maternal uterine epithelial cells, and release their granular content ( containing PSPB and PAG ) into the maternal circulation [3]. The PAGs consist of several isoelectric and molecular protein variants that can be partially purified from other placental proteins. Antibodies made against the purified proteins can be used to detect the presence of these proteins in the peripheral circulation of cattle. Because these proteins are specific to the placental tissue, it is possible to use detection of PAGs in the maternal circulation as indicator of pregnancy [4].

Trans-rectal palpation, B-mode ultrasonography and measurement of blood PSPB and PAG concentration are commonly used for early pregnancy diagnosis. Serum PSPB and PAG concentrations increase progressively from 30 days of pregnancy, peak 1-5 days before calving and decline there after [5,6]. The first blood –based pregnancy- specific assay for ruminant was described by Sasser et al. on the basis of radioimmunoassay for PSPB [5,7]. Pregnancy associated glycoproteins such as PBSP B [8, 9] and other PAGs [6,10,11] have been used for early detection of pregnancy in dairy cattle, starting as early as 21 days after AI.

More recently, ELISA assays for PBSB [4] and PAG [10] became commercially available. The PSPB ELISA is available under the trade name BioPRYN and provides a qualitative pregnancy classification based on measurement of PBSB in serum of pregnant ruminants and has been commercially available since 2003 in USA and applied 25 days post breeding in heifers and 28 days in adult cows while PAGs was developed by IDEXX, USA.

BioPRYN (Bio Tracking) has recently manufactured Bio-RPD kits to be inspected visually with shorter incubations and utilized on-farm or in areas without access to laboratory equipment. Cattle can be tested 28 days post breeding or greater. The manufacture claimed that the sensitivity is (99.9%) and matches with BioPRYN-flex ELISA assay, but with visual interpretation and human subjectivity there were a small percentage (1.5%) of none pregnant animals that were identified as pregnant.

Therefore the objective of the present study was to evaluate and compare the results of Bio-RPD with BioPRYN flex ELISA assay as early pregnancy determination in dairy cattle.

Materials and Methods

The study was performed at Al Safi dairy farm Al Kharj, Saudi Arabia. 92 cows sera were used for both Bio-RPD and BioPRYN flex ELISA and the results obtained by both assays were compared.

Bio-RPD Visual test:

The test was performed as per BioPRYN instructions. 100 microliters of cow’s sera were added to the well of the plate. The standards were added after all the samples have been loaded in duplicates (Hi and low standards). Gently swirl and allow the plate for at least 10 minutes after the last standard is added. Then add 50 microliters of detector Buffer (#1) in the well with the samples and standards and gently swirl and incubate for 10 minutes at room temperature. Wash 4 times with water and then add 100 microliters of prepared Enhancer solution (#2) to all wells and incubate for 10 minutes. Wash 4 times with water and add 100 microliters of TMB substrate (#3) to all wells and incubate for 10 minutes at room temperature. Add 50 microliters of stop buffer (#4) to all well without removing TMB solution. Then visually read the results of the plate. Any sample above the Hi standard is considered pregnant and any samples below the low standard is considered not pregnant. Any sample between the Hi and Low standard is considered Recheck. This means the animal has a lower than expected level protein and should be rechecked in a week to determine the actual status.

BioPRYN Flex ELISA Assay:

BioPRYN Flex ELISA (Bio Tracking LLC, Moscow, ID, USA) was also used in the study and it was performed according to the instruction of the manufacture. It is provide a qualitative classification based on comparison of a sample optical density with three threshold optical density values (high, low and cut-off) calculated for each plate. The threshold values were calculated from the Optical Density (OD, wave length 450nm) values of duplicates test well for two PSPB (high and low). If the OD exceeded the high threshold the animal was categorized as pregnant. An OD less than threshold, was designated as not pregnant.

Result

Test results of 92 tested sera by Bio-RPD and BioPRYN Flex were illustrated in (Table 1, Table 2, Figure 1, Figure 2).

Figure 1: BioPRYN visual results.

Figure 2: BioPRYN ELISA results.

Table 1: Bovine serum BioPRYN ELISA and BioPRYN visual pregnancy results.

Table 2: Pregnancy results.

The results showed animals defined as pregnant, recheck, not pregnant (open) and false positive. Out of 92 tested sera, 66 were categorized by BioPRYN Flex ELISA as pregnant (71.7%) and 63 were defined by Bio-RPD as pregnant (68, 5%).

Twenty six recorded as not pregnant (open) using BioPRYN Flex (28.3%) and Twenty five were categorized not pregnant using Bio- RPD (27.2%). Three samples were considered recheck by Bio-RPD as their color reading is between the Hi and Low standards.

With BioPRYN Flex none were defined false positive (0%) while with Bio-RPD 1 is diagnosed as false positive 1.1% as it is not pregnant (open) by BioPRYN Flex. The sensitivity of Bio-RPD is 99% when compared with BioPRYN Flex ELISA.

Discussion

Pregnancy-Specific Protein B (PSPB) was first detected in bovine placenta (Butler et al., 1982). Antibodies made against these purified proteins (PSPB) can be used to detect the presence of these proteins in the peripheral circulation of the cows. And because these proteins are specific to the placenta tissue, they become visible in the maternal blood and can be determined with high accurateness from 28 to 30 days post breeding as an indicator of pregnancy.

The rapid visual Bio-RPD and BioPRYN ELISA were used in this study to measure PBSB values in dairy cows under field condition and their results were compared together.

In our study, a higher accuracy and sensitivity of Bio-RPD were recorded (99%) when compared with BioPRYN ELISA. These results seemed consistent with those reported by the manufacture who claimed that the sensitivity is (99.9%) and matches with BioPRYNflex ELISA assay.

Any samples between the Hi and Low standards mean the animal has a lower than expected protein and should be rechecked in a week to determine the actual status. One limitation of the present study was the fact that 3 cow samples were recorded as recheck, and could not be used for determination of accuracy and sensitivity calculation because no chance for testing them again after one week to check the level of the protein.

In summary, the rapid Bio-RPD assay, based on visual inspection with shorter incubation for determine PSPB and utilized on farms without access to laboratory, had extremely high and similar sensitivity for early pregnancy diagnosis in dairy cows compared with PSPB ELISA.

Conclusion

Test results showed that the sensitivity of Bio-RPD visual test is 99% compared to BioPRYN Flex ELISA. Thus it is recommended to be utilized as rapid tool for pregnancy diagnosis on farms that have no laboratory equipment or transrectal ultrasonography.

References

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