Topics: Cattle, Milk, Human chorionic gonadotropin Pages: 13 (3638 words) Published: March 18, 2013
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


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.



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.


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


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.


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...
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