novel approaches to expression and detection of oestrus in dairy cows

was achieved. PCR products were removed from the gel using the QIAquick
Gel Extraction Kit (Qiagen, Sussex, UK). The DNA band was excised using a
scalpel under a UV light machine and dissolved at 50˚C on a heat block
(Techne DRI-BLOCK DB.3A) in 3 volumes of Buffer QG (supplied with the
kit) to bind the DNA. Samples in solution were added to a QIAquick spin
column in a 2ml collection tube (both supplied with the kit), and samples
centrifuged (MiniSpin, Eppendorf, Stevenage, UK) at 10000rpm for 1
minute. 700µl of Buffer PE (supplied with the kit) was then added to the
column, centrifuged at 10000rpm for 1 minute, and the process repeated
to wash the DNA. The empty column and collecting tube were then
centrifuged for 1 minute to remove the residual alcohol, before eluting the
DNA with 30µl of 2mM Tris solution, pH 7.0-8.5 and centrifuging for 1
minute to collect the purified PCR product.
3.2.4.4 DNA Sequencing
Samples were sent for sequencing (Beckman CEQ8000 Sequencer) with
the PCR primers detailed in section 3.2.4.2 for the promoter region of the
bovine GnRH receptor gene, which identified the SNPs at positions 966 and
1189.
3.2.5 Statistical Analyses
Statistical analysis was carried out using Genstat 14th edition (VSN
International Ltd, Hemel Hempstead, UK). Activity data were analysed as
generalized linear mixed models (GLMM) using the residual maximum
likelihood (REML) procedure, with Poisson distribution and logarithmic link
function. The model fitted fixed effects for SNPs (variates; wildtype
homozygote, 0; heterozygote, 1; mutant homozygote, 2). For the random
effects of the model, individual cows represented subjects to allow for
multiple oestruses per cow but only 1 SNP per cow. The significance of
fixed effects was assessed by Wald tests. The resulting model was:
Y il = µ + S i + C l + ε il
Other variables were then included into the statistical model fitted as fixed
effects for SNPs (variates; wildtype homozygote, 0; heterozygote, 1;
mutant homozygote, 2), parity (classified according to lactation number as
1, 2 and ≥3) and oestrus time period (classified as Jan-Mar, 1; Apr-Jun, 2;
Jul-Sept, 3; Oct-Dec, 4). This was to account for the combined effects on
activity, eliminating external cow factors and seasonal effects to
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