novel approaches to expression and detection of oestrus in dairy cows

and time of mount, duration of mount, any other relevant information
relating to position. In POC 3 both CCTV (Smart Witness Wireless System
SWC101S; Maplin, Notts, UK) and visual observation of cow behaviour
were used to confirm oestrus by UWB recording.
5.2.6 Data Analysis
5.2.6.1 Assay for Milk Progesterone
Milk progesterone concentration was determined using a 96 well microtitre
plate-based enzyme-linked immunosorbent assay (ELISA) supplied as a
commercially available kit (Ridgeway Science Ltd., Alvingdon, UK).
Microtitre plates coated with antibody stored at 4°C were warmed to room
temperature (25°C) prior to use. The foil seal was removed, wells emptied
and plate blotted dry onto tissue paper. Prior to assay reagents, whole milk
standards; 0, 1, 2, 5, 10, 20 and 50ng/ml, and quality controls; 2ng/ml
and 5ng/ml, (progesterone in milk from an ovariectomized animal or cow in
oestrus) supplied with the kit, and samples were brought to room
temperature and thoroughly vortexed to ensure homogeneity of the
samples. A volume of 10μl of standards, quality controls and samples were
added to the wells in duplicate. 200μl of progesterone enzyme label
(supplied) was added to each well and the plate incubated at room
temperature for 1 hour 30 minutes. After incubation, wells were emptied
and washed 3 times with cold water, tap drying on paper between each
wash. The substrate solution supplied was then added to each well at a
volume of 200μl and incubated in the dark at 25°C for 20 minutes to allow
the colour to develop. Strong colour denoted low levels of progesterone
(heat or not pregnant) and weak colour, high levels of progesterone (mid
cycle or pregnant). The immunosorbance of each well was recorded at a
wavelength of 570nm using an automated plate reader (Labsystems
Multiskan Ascent 354) and processed using specific software to read the
absorbance and transform into progesterone concentration (Ascent
Software Version 2.6, Thermo Labsystems). The assay kit was capable of a
sensitivity of 5pg/ml although a sensitivity of this scale was not required
for this purpose and therefore the lowest standard was 1ng/ml. The interassay
coefficient of variation was 12.6%.
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