Interferon

1. The Detection of Pregnancy Associated Glycoproteins (PAG) in Routine Milk Recording Samples as an Indicator of Pregnancy in Dairy Cattle
T.M.Byrem1, K. Velek2 & H.L. Pearse2 INTRODUCTION

Accurate and timely detection of pregnancy in dairy cows is an essential component of any bovine reproductive management programme. Good reproductive performance has multiple financial benefits. Traditionally, pregnancy is determined through rectal palpation of the cow or by transrectal ultrasound (TU).

An enzyme-linked Immunoassay (ELISA) for the detection of pregnancy-associated glycoproteins (PAGs) in bovine milk samples and has been developed to provide a laboratory-based method for the accurate detection of pregnancy which could offer veterinarians and dairy farmers an important tool for the identification of open cows in the dairy herd.

Reproductive performance and efficiency in dairy herds depends greatly on the accurate and timely diagnosis of pregnancy (Oltenacu et al., 1990; De Vries 2006).

Recently, several immunological detection assays for the quantification of pregnancy associated glycoproteins (PAG) in blood serum have been developed as accurate, cost-effective and convenient alternatives (Sasser et al., 1986; Sousa et al., 2006; Whitlock & Maxwell, 2008, Silva et al., 2009).

However, data from previous studies have indicated a significant reduction in performance when existing serum-based assays were applied to milk samples (Friedrich & Holtz, 2008; Gajewski et al., 2008).

The IDEXX Bovine Pregnancy test for use on serum or EDTA plasma is based on the target antigen, PAG. They are expressed specifically in the maternal and embryonic regions of the placenta and are a sub-group of the aspartic protease family. More than 22 bovine transcribed genes have been identified and these are expressed temporally at varying levels throughout gestation. IDEXX has developed a lab-based immunological detection assay (ELISA) which can reliably and accurately detect PAG in bovine milk samples in less than 4 hours.

MATERIALS AND METHODS

DIAGNOSTIC METHOD

Serum samples were run on the IDEXX Bovine Pregnancy Test and milk samples on the IDEXX Milk Pregnancy Test at IDEXX Inc., (One IDEXX Drive, Westbrook, Maine).
The IDEXX ELISA uses monoclonal antibodies directed against PAG, which are set on a microplate that will capture the PAG present in the sample. Secondary anti-PAG antibodies, coupled to a signal amplification system, are used as the detection reagent. Enzyme substrate (TMB) is used as a coloured indicator to reveal the PAG contained in the sample. After stopping the reaction the optical density of each well is read at a wavelength of 450 nm. Results are calculated and expressed as sample – negative (S-N).
For serum samples, if the result is ≥0.3 samples are classed as positive (pregnant), and below 0.30 classed as negative (not pregnant).
For milk samples, if the result is ≥0.15 the samples are classed as positive (pregnant), and below 0.10 classed as negative (not pregnant).
Results falling between 0.10 and 0.15 are classified as re-check in the milk assay.

TEMPORAL STUDY

Milk samples (n=291) were collected from thirteen cows at regular intervals during lactation to assess changes in milk PAG levels throughout gestation.

POST-CALVING DECLINE IN MILK PAG LEVELS

Milk samples were obtained from 134 cows at varying days post-calving, but prior to service to analyze speed of decline in milk PAG levels after parturition and to determine the day post-calving at which PAGs are no longer detected.

TEST PERFORMANCE EVALUATION

From Herd A, 192 paired milk and serum samples were collected from individual lactating Holstein cattle greater than 60 days post-partum. Samples were collected for cows throughout gestation from 40 days post breeding up to a maximum of 227 days post breeding.
From Herd B, 120 paired milk and serum samples were collected from lactating Holstein cows greater than 60 days post-partum. Samples were collected for cows throughout gestation from 40 days post breeding up to a maximum of 243 days post breeding.
In addition to milk and serum samples, all cows were tested by rectal palpation or ultrasound to confirm pregnancy status at the time of sample collection.
In order to determine diagnostic specificity of the test, 290 paired milk and serum samples were collected from five geographically diverse herds of lactating Holstein cattle. Cows greater than 60 days post-partum but not bred or not pregnant where included in the sample set. In addition to milk and serum samples, all cows were tested by rectal palpation or ultrasound to confirm pregnancy status at the time of sample collection.
RESULTS

TEMPORAL STUDY

Results from the temporal study show the presence of PAG in milk throughout gestation (Figure 1). Whilst a high degree of variability in PAG levels was observed for different cows, PAGs are detectable as early as 24 days of pregnancy and remain elevated throughout gestation. Assay response declines slightly from day 40 through day 90 of gestation, but PAG levels steadily increase during mid and late gestation.

POST-CALVING DECLINE IN MILK PAG LEVELS

For the 134 cows sampled post-calving, but prior to breeding, milk PAG levels were below the cut-off for all cows by 60 days post-calving (Figure 2). The results show a rapid decline in PAGs after parturition with an observed specificity of 100% at 60 days post-calving but prior to breeding.
TEST PERFORMANCE EVALUATION

Based on test results of the 192 cows from Herd A, test sensitivity for the milk ELISA was 99.4% (95% CI: 96.0-100%) and specificity was 100% (95% CI: 87.3-100%) for cows greater than forty days post-breeding (Table 1). Agreement between the blood and milk assays was 94.5%.

Figure 3 shows the results of the milk PAG ELISA on 290 open cows (not bred or confirmed open by ultrasound or palpation) from five geographically diverse herds. Overall specificity was 96.7% (95% CI: 94.9-98.9%. Fewer open samples are available in late gestation, but specificity after day 90 of gestation was 100%.

DISCUSSION

Data from the current study show that the IDEXX Milk Pregnancy Test is able to accurately detect pregnancy status using milk samples from dairy cows throughout gestation. Compared to tests used for milk PAG analysis in previous studies (Friedrich & Holtz, 2008; Gajewski et al., 2008), the current test was able to consistently detect the presence of PAG in milk from pregnant cows much earlier in gestation (24 days vs 150 days).
Although milk PAG levels varied markedly between individual animals throughout gestation, sensitivity and specificity of the current milk PAG ELISA was similar to that reported for commercially available serum PAG ELISA (Paré et al., 2008; Breed et al., 2009).
As with serum PAG levels, the current data show that milk PAG levels are highest near parturition and it is important to ensure that PAG from the previous pregnancy do not interfere with the current pregnancy diagnosis.

CONCLUSION
Using the IDEXX Milk Pregnancy Test, the presence of PAG in milk can be detected with suitable analytical accuracy for pregnancy detection in dairy herds. The detection of PAG in milk throughout gestation offers great scope for the application of the test through herd recording schemes.
2. Pregnancy-associated glycoproteins as a new diagnostic tool in cattle reproduction
Gajewski, Z; Pertajtis, M; Sousa, N M; Beckers, J F; Pawlinski, B; Janett, F
Introduction

The discovery of pregnancy-associated glycoproteins (PAG) brought a revolution in cattle’s reproductive techniques (Butler et al., 1982; Humblot et al., 1988; Zoli et al.,
1991; Mialon et al.,1993).
Structural and biochemical differentiation in a range of PAG group
In international literature PAG was described as: pregnancy-specific protein B (PSPB), pregnancy-specific protein 60 (PSP-60), or SBU-3 antigen (Humblot et al., 1988; Xie et al., 1991; Zoli et al., 1991; Zoli et al., 1992; Atkinson et al., 1993; Mialon et al., 1993; Xie et al., 1995).

Variability of amino acid sequences, the carbohydrate component and degree of glycosylation in several molecules result in great differences between various types of those glycoproteins (Zoli et al., 1991; Atkinson et al., 1993; Beckers et al., 1994; Klisch et al., 2006).

A similar situation was observed in primates and equids with the existence of the more or less glycosylated forms of the human chorionic gonadotrophin (hCG) and the equine chorionic gonadotrophin (eCG).
Another feature, which characterizes pregnancy-associated glycoproteins, is the molecular mass, which reaches the level of 70kDa, which is twice more than other plasma glycoproteins (Beckers et al., 1994; Patel et al., 2004; Klisch et al., 2006).
Successive investigations revealed correlations between the level of PAGs in maternal blood and in milk. Recent tests demonstrated that the level of milk production can modify the concentration of PAGs circulating in blood.
The more “liters of milk” is being produced, the less PAGs can be collected from maternal blood (Lopez-Gatius et al., 2007c). At the same time the level of PAGs in milk is growing (Tainturier et al., 1996; Gajewski et al., 2008a; Gajewski et al., 2008b).
Pregnancy-associated glycoproteins belong to a group of proteolytic enzymes, known as aspartic proteinases (AP). PAGs contain more than 50% of amino acid sequences identical to pepsin, cathepsin D and cathepsin E, but the vast majority of them are enzymatically inactive (Xie et al., 1991; Roberts et al., 1995; Szafranska et al., 1995; Xie et al., 1995; Perényi et al., 2002). Only some of them can bind themselves to pepstatin A, which is an inhibitor of AP (Green et al., 1998; Green et al., 2000).

The bovine PAG family

The first discovered bovine PAG was boPAG-1, also known as boPAG-67kDa (Green et al., 1998). The rest of the molecules belonging to this family got the following numbers as their names, which was accepted in international nomenclature. So far recent investigations have proved the existence and sequences of 22 bovine PAGs, named appropriately boPAG-1 – boPAG-22. On the basis of biochemical analysis and biological properties of individual glycoproteins PAGs werepartitioned into three groups:

1) The pepsinogen-like PAG (boPAG-8) group
2) The gonadotrophin-like PAG (boPAG-2) group
3) The major bovine PAG family (boPAG-1) group

1) The pepsinogen-like PAG (boPAG-8) group
The boPAG-8 group is the family generally detected among animals which belong to Placentalia. Those specific peptides were also discovered in horses, cats, zebras, mice and rats (Green et al., 1998). In theory these glycoproteins are enzymatically active, but so far this has not been scientifically confirmed (Szafranska et al., 1995; Beckers et al., 1999).

2) The gonadotrophin-like PAG (boPAG-2) group
Several studies raised suspicions about LH-like activity substances existing in trophoblastic cells (Beckers et al., 1988; Beckers et al., 1994; Beckers et al., 1998).
The following research on cows, in which the pituitary gonadotropic activity was reduced, demonstrated that the corpus luteum is still supported by additional luteotropic substances (Szafranska et al., 1995; Beckers et al., 1999).
It was a breakthrough discovery in cattle breeding.
The next experiments using radioreceptor assay method made by Ailenberg and Shemesh (1953) showed that these molecules can stimulate the bovine granulose cell layer to produce progesterone (Beckers et al., 1994; Beckers et al., 1998; Sousa et al., 2006).
The gonadotrophin-like PAGs are built from a polypeptide, which contains 372 amino acids (Beckers et al. 1998; Beckers et al. 1999).

3) The major bovine PAG family (boPAG-1) group
The first discovered bovine PAG isolated from cotyledons was boPAG-67kDa, also known as boPAG-1. This group also contains the proteins: boPAG-56kDa, boPAG- 67kDa, boPAG-75kDa (Klisch et al.,2006).
The major bovine PAG family’s molecules are expressed at the stage of early blastocyst to delivery and even a few days after (Green et al., 2000; Ushizawa et al., 2004). The synthesis in such early stage of pregnancy suggests that PAGs are needed in the implantation process and placentogenesis (Wooding et al., 2005).
An unexpected phenomenon occurred during research. Antigens, immunologically similar to boPAG-67kDa, were found in testicular tissue and in ovarian extracts.
There is no confirmation that this interesting discovery has anything to do with PAG level in blood, and there are no other studies about it. However, we may presume, that there are more places, apart from the trophoblast, where PAG can be synthesized.

Diagnostic methods

Research Article

3. Characterization and In Silico Analysis of Pregnancy-Associated Glycoprotein-1 Gene of Buffalo (Bubalus bubalis)
Jerome A.,1 S. K. Singh,1 S. K. Agarwal,1 Mohini Saini,2 and Ashwin Raut3
1Division of Animal Reproduction, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India
2Centre for Wildlife, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India
3Division of Animal Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India
Correspondence should be addressed to Jerome A., [e-mail]

INTRODUCTION

Pregnancy-Associated Glycoproteins (PAGs) are trophoblastic proteins belonging to the Aspartic proteinase family secreted by different placental cells of many mammalian species. They play a pivotal role in placentogenesis, foetomaternal unit remodeling, and implantation.
The identification of the genes encoding those proteins will be helpful to unravel the intricate embryogenomic functions during pregnancy establishment. Considering importance of these proteins, the present study was undertaken to characterize the pregnancy associated glycoprotein-1 gene of buffalo.

Pregnancy is established and maintained by the two-way communication between the conceptus and the mother. These intricate dialogues which are initiated after fertilization are crucial as these signals are considered potential markers for effective placental remodeling, pregnancy recognition, and successful implantation.

These interactions between the conceptus and maternal system emphasize the importance of both the components in maternal recognition of pregnancy and embryonic development [1].

These important signals to the maternal system to sustain pregnancy are mediated by numerous molecules which include steroid hormones, peptide hormones, cytokines, and growth factors [1, 2].

Conceptus-derived substances are considered to be precise and reliable markers of pregnancy and fetal well-being. Pregnancy-associated glycoproteins are one such large family of protein molecules produced by conceptus for the recognition by the mother. Pregnancy-associated glycoproteins (PAGs) are acidic glycoprotein belonging to the Aspartic Proteinase superfamily sharing more than 50% amino acid sequence identity with Pepsin, Cathepsin D, and E [3, 4].

Pregnancy-Associated Glycoprotein-1 (PAG-1) also known as Pregnancy Specific Protein B (PSPB), PSP-60, and SBU3, is secreted by the binucleate cells of the conceptus trophectoderm [6].

PAG-1 is detectable in maternal blood soon after implantation as binucleate cells migrate from the trophectoderm and fuse with uterine epithelial cells and hence it is considered as a potential signal from the conceptus [7].
The products of binucleate cells in maternal circulation have also been reported to be associated with placental mass, fetal number, twins, and neonatal birth weight in cattle [8, 9].

Based on the evolutionary study and phylogenetic linkage bovine pregnancy associated glycoproteins family has been segregated as ancient (bovine PAG-2, bovine PAG-8) and modern (bovine PAG-1) [13–15].

But there is no report on the characterization and phylogenetic analysis of pregnancy-associated glycoprotein-1 gene of buffalo. Moreover, identification of gene encoding buffalo Pregnancy-Associated Glycoprotein-1 may provide an avenue for producing recombinant protein which will be helpful to develop diagnostics for early pregnancy diagnosis and marker for embryonic development [16].

MATERIALS AND METHODS

2.1. Sample Collection and RNA Isolation.
2.2. cDNA Synthesis and Buffalo PAG-1 Gene Amplification.
2.3. Buffalo PAG-1 Gene Cloning.
2.4. Sequencing Buffalo PAG-1 Gene.

RESULTS AND DISCUSSION

An 1181 base pairs buffalo Pregnancy Associated Glycoprotein PAG-1 gene was PCR amplified from the RNA obtained from the fetal cotyledons. BLAST analysis of the buffalo PAG-1 sequence retrieved a total of 20 cattle, 5 goat, and 4 sheep PAG sequences, exhibiting more than 80% similarity. Buffalo PAG-1 gene contained an uninterrupted open reading frame of 1140 base pairs encoding 380 amino acids that possess a 15 amino acid signal peptide and mature peptide of 365 amino acids. The phylogenetic study of the buffalo PAG-1 gene revealed buffalo PAG-1 is more related to cattle, goat, and sheep PAG-1 sequences. By this study characterization of buffalo PAG-1 gene and its evolutionary relationship was deduced for the first time.

Phylogenetic Analysis of the buffalo PAG-1 sequences with other domestic species revealed buffalo PAG-1 is more related with bovine, caprine, and ovine species and less similar to equine, porcine, deer, and zebra due to various nonsynonymous substitutions in the entire sequence in the latter species.
Porcine PAG-1 forms an entire different clade as it consists of variant amino acids residues when compared with ruminant species.

Phylogram constructed between buffalo PAG-1 and different bovine PAGs sequences revealed buffalo PAG-1 was more related with bovine PAG-1 thereby belonging to the recently duplicated PAG gene group (PAG-1 group) and differing significantly from the ancient PAG group consisting of PAG-2 and 8 [13–15]. CONCLUSION

Buffalo Pregnancy-Associated Glycoprotein-1 cDNA encoding buffalo PAG-1 protein of 380 amino acids was characterized and found to be recently duplicated group members of the aspartic proteinase family being proteolytically inactive due to key mutations close to the active site.

J. Dairy Sci. 90:4612–4622 doi:10.3168/jds.2007-0276 American Dairy Science Association, 2007.

4.Accuracy of a Pregnancy-Associated Glycoprotein ELISA to Determine Pregnancy Status of Lactating Dairy Cows Twenty-Seven Days After Timed Artificial Insemination
E. Silva,* R. A. Sterry,* D. Kolb,† N. Mathialagan,‡ M. F. McGrath,‡ J. M. Ballam,‡ and P. M. Fricke*1
*Department of Dairy Science, University of Wisconsin, Madison 53706
†Lodi Veterinary Clinic, Lodi, WI 53555
‡Monsanto Agricultural Company, St. Louis, MO 63167

ABSTRACT

To determine the accuracy of a pregnancy-associated glycoprotein (PAG) ELISA in identifying pregnancy status 27 d after timed artificial insemination (TAI), blood samples were collected from lactating Holstein cows (n = 1,079) 27 d after their first, second, and third postpartum TAI services. Pregnancy diagnosis by transrectal ultrasonography (TU) was performed immediately after blood sample collection, and pregnancy outcomes by TU served as a standard to test the accuracy of the PAG ELISA.

Pregnancy outcomes based on the PAG ELISA and TU that agreed were considered correct, whereas the pregnancy status of cows in which pregnancy outcomes between PAG and TU disagreed were reassessed by TU 5 d later.

The accuracy of pregnancy diagnosis was less than expected when using TU 27 d after TAI (93.7 to 97.8%), especially when pregnancy outcomes were based on visualization of chorioallantoic fluid and a corpus luteum but when an embryo was not visualized.

The accuracy of PAG ELISA outcomes 27 d after TAI was 93.7, 95.4, and 96.2% for first, second, and third postpartum TAI services, respectively.

Statistical agreement (kappa) between TU and the PAG ELISA 27 d after TAI was 0.87 to 0.90.

Pregnancy outcomes based on the PAG ELISA had a high negative predictive value, indicating that the probability of incorrectly administering PGF2α to pregnant cows would be low if this test were implemented on a commercial dairy.

INTRODUCTION

Efficient and aggressive reproductive management of lactating dairy cows can be achieved if an accurate early nonpregnant diagnosis is combined with a resynchronization protocol, resulting in acceptable fertility.
Palpation per rectum is routinely used to determine pregnancy status in dairy cattle; however, with this method it can be difficult to diagnose pregnancy status accurately earlier than 30 to 35 d after AI (Momont, 1990; Youngquist, 2007).

Use of transrectal ultrasonography (TU) is gaining popularity among bovine practitioners (Fricke, 2002), but accuracy of pregnancy diagnosis by TU decreases at a gestational age of less than 33 d (Pieterse et al., 1990; Badtram et al., 1991; Romano et al., 2006).
Laboratory assays for detecting proteins originating from binucleate cells of the embryonic trophoblast have been developed to determine pregnancy status in cattle (Sasser et al., 1986; Zoli et al., 1992; Green et al., 2005).

Pregnancy-specific protein-B was the first pregnancy specific protein identified in cattle (Butler et al., 1982) and was later found to have the same N-terminal AA sequence as pregnancy-associated glycoprotein (PAG; Xie et al., 1991; Lynch et al., 1992).

Both pregnancy specific protein-B and PAG have subsequently been reclassified as boPAG-1, and an ELISA was developed to detect PAG as a method of early pregnancy diagnosis in cattle (Green et al., 2005).

Mean PAG concentrations in cattle increase from 15 to 35 d in gestation; however, variation in serum PAG levels among cows precludes PAG as a reliable indicator of pregnancy until about 26 to 30 d in gestation (Zoli et al., 1992; Humblot, 2001).

Coupling an early nonpregnant diagnosis with a management strategy to rapidly reinitiate AI can improve reproductive efficiency by decreasing the interval between AI services, thereby improving the AI service rate (Fricke, 2002).

The objective of this study was to compare the accuracy of plasma PAG ELISA with TU for determining the pregnancy status of lactating dairy cows 27 d after timed AI (TAI). When a new test is evaluated, it is necessary to analyze the accuracy and feasibility of the new test compared with existing methods (Bossuyt et al., 2006).

A “gold standard” is a quality control that provides the basis for determining the value of a diagnostic test.
The sensitivity of TU (the gold standard) to detect pregnant cows and heifers from 23 to 33 d after AI varies from 61.5 to 97.7%, whereas the specificity of TU to detect nonpregnant animals varies from 76.6 to 87.8% (Pieterse et al., 1990; Badtram et al., 1991).

Nonetheless, TU is the most reliable method of determining pregnancy status 27 d after TAI under farm conditions and can be performed concurrently with blood sample collection for the PAG ELISA 27 d after TAI.