|dc.description.abstracteng||The aim of the PhD dissertation was to examine the reliability of a PAG polyclonal ELISA developed by Friedrich and Holtz (2010) in goats, different cattle breeds and bovine milk. The dissertation comprised three studies. In the first study, pregnancy-associated glycoprotein (PAG) pattern and pregnancy detection in Boer goats were determined using an ELISA with different antisera. The second study investigated PAG profiles in dairy, dual purpose and beef cows. The effects of milk fat content, milk treatments, milk type and milk preservatives on PAG ELISA in bovine milk was determined in the third study.
The first study addressed the question to what extent plasma PAG determination may serve as a means of early pregnancy detection in goats in a similar way as it is practiced in cows, and whether an ovine or bovine PAG-ELISA may be utilized to this end. Blood samples were collected from eight pregnant pluriparous Boer goat does twice weekly during the first seven weeks and the last four weeks of pregnancy and weekly in-between and during four weeks following parturition. Plasma PAG concentrations (mean ± SEM) were determined using a competitive enzyme-linked immunosorbent assay. Assays were conducted with polyclonal antisera raised in rabbits against purified preparations of caprine (AS#706), ovine (AS#780) and bovine PAG (AS#726). In the assay systems, purified bovine PAG served as standard and tracer and goat anti-rabbit IgG served as coating antibody. With the antibody raised against caprine PAG (AS#706) a steep increase to a climax of 69 ± 9 ng/ml on day 56 of pregnancy was followed by a gradual decline to 16 ± 3 ng/ml at parturition and 0.3 ± 0.07 ng/ml four weeks postpartum. The results achieved with the anti-ovine PAG (AS#780) showed close similarity, a maximum of 92 ± 14 ng/ml being reached at 56 days of pregnancy. With anti-bovine PAG (AS#726), the PAG level increased to a maximum of 3.1 ± 0.2 ng/ml on day 105 of pregnancy and fluctuated around 3 ng/ml until the end of pregnancy. The difference between pregnant and non-pregnant does reached a significant level 21 days after conception, one week earlier than with caprine and ovine antisera.
In the second study, the PAG pregnancy profiles of a dual-purpose (Simmental) and two beef breeds (Uckermark and Aubrac) were compared with the profile of a specialized dairy breed (Holstein-Friesian). The Holstein-Friesian cows were sampled weekly; the levels of the other breeds were presented at three-week intervals. The overall significant breed difference recorded (P=0.013) was obtained from deviations in PAG levels of Simmental and Uckermark cows from the Holstein-Friesian and Aubrac breed during the initial three weeks and from 23 weeks of pregnancy onward. During the time between 4 and 22 weeks, when the detection of pregnancy is an issue, PAG levels of the studied breeds were in close agreement (P>0.05). No significant effect of body mass of cow or calf (relative to mass of dam) was detected. These findings imply that the PAG pregnancy test may be executed irrespective of breed or type of cow, affirming the suitability of the test as a valuable asset in cattle husbandry.
The third study was conducted to investigate the effect of milk preservatives, milk fat and different milk treatments on PAG concentrations in milk measured by PAG ELISA. The effect of milk preservatives, milk type and storage duration was investigated using fresh milk (non-homogenized, non-pasteurized), organic milk (non-homogenized, pasteurized) and ultra-high temperature treated (UHT) milk (homogenized, sterilized) with 3.5 % fat. The milk was skimmed by centrifugation and treated either with bronopol, sodium azide, potassium dichromate or left without treatment as a control. Known PAG concentrations were added 2 days, 1 day and shortly before the assay. The effect of milk fat content was studied using UHT milk with two different fat contents (1.5 and 0.3 %) spiked with known concentrations of PAG. The effects of pasteurization and sonication were tested using pooled milk from dairy cows after about 2 weeks of calving. Following milk skimming, samples were pasteurized at 63°C for 30 minutes, sonicated or left without treatment as a control. Samples were then stored at three different temperatures (room temperature, 4°C and -20°C) for different durations (1, 3, 5 and 7 days). In the first experiment, fresh and UHT milk showed the best correlation coefficients between measured and expected PAG concentrations. All milk samples can be stored up to three days before the PAG analysis without drastic change in the measured PAG content. When milk preservatives were added, bronopol gave the best correlation between expected and measured PAG values. Differences in PAG recovery rates between different fat contents of UHT milk were not statistically significant (P>0.05). Recovery rates at lower PAG concentrations were significantly higher (P<0.05) than those with higher PAG content and freshly prepared samples were significantly higher (P<0.05) than those stored for 1 or 2 days. The effect of ultrasonic treatment was less destructive to milk PAG content compared to pasteurization, although the results in the pasteurization group were more uniform throughout storage time. The cooled milk samples did not show drastic change in the recovery rates in the first 3 days of storage. To determine PAG concentration in milk samples using the PAG ELISA, skimmed fresh or skimmed UHT milk was preferred for the preparation of assay standards and control. When considering milk preservatives, bronopol was found to be the most desirable. The milk samples could be pasteurized and/or kept cooled for 3 days without noticeable changes in PAG content.||de