Detection of Progesterone in Cow’s Milk for the Prediction of Reproductive Performance

Abstract

Reproductive management is important for the profitability of dairy farms. The correct detection of estrus plays an important role in the success of insemination, conception and further reproductive outcomes. Conventional methods to detect estrus are often laborious or erroneous. Progesterone (P4) is one of the hormones that regulates the estrous cycles of female animals and therefore, the determination of P4 levels is one of the most accurate means for the detection and verification of the onset of estrus. However, to measure progesterone in cow’s milk one must overcome the milk matrix effects, negating the interference of the various components in milk on the measurement. The aim of the thesis is to develop a sensitive and practical test to measure P4 in cow’s milk for potential application in estrus detection. To reduce the milk matrix effect, magnetic beads conjugated with anti-P4 monoclonal antibodies (mAb) were used to separate P4 from milk. Subsequently, P4 concentrations were determined by surface plasmon resonance (SPR) inhibition assays. Initially, low binding was observed, and thought to arise due to the conjugation of mAb to the relatively large magnetic beads causing steric hindrance of the binding between conjugated mAb and P4 immobilized on the SPR sensor surface. This can result in low detection sensitivity or even failure of tests. Therefore, the SPR sensor surfaces and the magnetic bead conjugates were optimized (described below) to reduce the steric hindrance of binding and improve the sensitivity of the test. P4 concentrations of cow’s milk collected on dairy farms were measured using the developed tests. Additionally, a newly selected P4 aptamer was explored for its potential to replace mAb in P4 tests, the aptamer possessing advantages of lower production costs and long-term stability. The P4 test that was developed using mAb and magnetic bead conjugates on an SPR platform achieved a limit of detection of 0.020 ng/ml P4 in the buffer solutions and 0.036 ng/ml P4 in diluted raw milk. The high sensitivity is likely to result from: (1) improved attachment chemistry by using a new, low molecular weight coating on an SPR gold chip formed by a self-assembled monolayer of a carboxylated terthiophene; (2) more accessible P4 on sensor surfaces created by in situ synthesis of P4-linker-ovalbumin conjugates on the SPR surfaces; and (3) better oriented mAbs on the magnetic bead surfaces via conjugation to sulfhydryl groups of mAb using bifunctional crosslinkers. The P4 concentrations of raw milk samples determined by the developed test showed good correlation (p<0.05) with the levels measured by a commercial milk P4 test kit based on enzyme linked immunoassays, and exhibited expected trends that followed cows’ estrous cycles, although the assay conditions and procedures of the developed test would require further optimization to further improve the accuracy and precision of concentration determination. The P4 aptamer tested showed much lower binding affinity to P4 than the mAb, which will most likely lead to low detection sensitivity of P4. Therefore, it was deemed not suitable for replacing mAb in the current assays. Further research is necessary to provide fundamental information such as the binding affinity and binding sequence of this aptamer, which may lead to the improvement of its binding affinity to P4, and design of assays, for better performance in P4 detection. It is demonstrated here that using mAb and magnetic bead conjugates to separate P4 before measurement can successfully reduce milk matrix effects and enable the detection of P4 directly in milk using an SPR sensor for estrus detection. To our knowledge, this P4 test was the first SPR P4 test using antibody and magnetic bead conjugates to reduce the matrix effect. This format can be adapted for other SPR tests which suffer from matrix effects, for example tests on food and environmental samples, and other biofluids for food safety control, environmental monitoring and disease diagnosis. Meanwhile, the novel gold coating material presented here can be used to functionalize gold surfaces other than SPR sensor surfaces, and the new approach of immobilizing small molecules on solid supports as well as the new method to conjugate antibody and magnetic beads are both easy facile and versatile in biosensor development and other possible applications.