novel approaches to expression and detection of oestrus in dairy cows

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Table 3.1 Cont. Peroxysome proliferatoractivated PPARGC1A C1892t/c c.1892 + T>C in intron 9 Association with milk fat yield (Weikard et al., 2005) receptor-γ coactivator-1α intron9 Prolactin PRL 89398 g/a R G8398A Milk yield and fat percentage in 1 st (Brym et al., 2005) lactation Prolactin Receptor PRLR Ser18Asn Ser18Asn in signal peptide Associated with milk protein and (Viitala et al., 2006) fat yields Ribosomal Protein S6 rs29019569CT BTA2, C>T, Base pair position Effects on feed efficiency (Sherman et al., 2008b) Kinase 316 880 Signal Transducer and STAT1 c3141t C3141T in 3’ UTR Allele C associated with increases (Cobanoglu et al., 2006) Activator of Transcription 1 in milk fat and protein percentages Signal Transducer and Activator of Transcription 5A STAT5A g12195c G12195C in exon 8 Associated with decreases in milk protein and fat percentage Associated with embryonic survival rate (Khatib et al., 2008b;Khatib et al., 2009) 72
3.2.4 Sequencing of DNA in the Laboratory Four blood samples were sequenced in the laboratory undergoing DNA extraction, polymerase chain reaction (PCR) amplification, gel extraction for purification and then sequencing of the gondaotrophin releasing hormone receptor (GnRH-R) gene. The GnRH-R gene was chosen for sequencing in the laboratory for a learning exercise because primers for this gene had been previously optimised in the laboratory. 3.2.4.1 DNA Purification Frozen samples were incubated at 37˚C (Mini 18L CLAD Incubator) and defrosted rapidly to aid red blood cell lysis. 3ml of blood was then purified using the Gentra Puregene Blood Kit (Qiagen, Sussex, UK). The of blood was added to 50ml tubes containing 9ml of Red Blood Cell (RBC) Lysis Solution, supplied with the kit, vortexed and centrifuged (DuPont Sorvall RC5C) at 10000rpm for 15 minutes. The supernatant was removed, the pellet resuspended in 9ml RBC solution and the process repeated twice more, to ensure ample lysis of cells. After final centrifugation, the supernatant was discarded, the pellet resuspended in the remaining residual and 3ml of Cell Lysis Solution (supplied with the kit) added to dissolve all remaining structures into solution, aided by incubation at 37˚C (Mini 18L CLAD Incubator) for 1 hour. Post incubation samples were cooled on ice to aid precipitation of the proteins. 50ml chloroform and isoamylalcohol (both sourced from Sigma- Aldrich, Dorset, UK) was prepared (49:1) and 3ml added to 50ml phase lock gel tubes (Eppendorf, Stevenage, UK), along with 3ml phenol (10mM Tris HCl, pH 8.0, 1mM EDTA; sourced from Sigma-Aldrich, Dorset, UK). DNA in the Cell Lysis Solution (from previous step) was transferred to the phase lock gel tubes, vortexed and centrifuged (DuPont Sorvall RC5C) at 10000rpm for 15 minutes. Using the phase separation technique allowed differentiation between the DNA and the protein. DNA in the upper aqueous phase was separated from the protein fraction in the lower organic phase. The DNA sample was transferred to clean 50ml phase lock gel tubes and phase separation carried out again using 5ml chloroform isoamylalcohol. After the second centrifugation the DNA aqueous layer was transferred to a new 50ml tube containing 1ml of Protein Precipitation Solution (supplied with the kit) and centrifuged again for 15 minutes. 73
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NOVEL APPROACHES TO EXPRESSION AND
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TABLE OF CONTENTS TABLE OF CONTENTS
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2.2.3 Statistical Analyses ........
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5.3 RESULTS .......................
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Furthermore, automated software was
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LIST OF FIGURES Figure 1.1 Hormonal
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LIST OF TABLES Table 1.1 Trends in
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LIST OF ABBREVIATIONS ˚ ˚C μM 2D
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CHAPTER 1 - Introduction & Literatu
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the past 50 years and duration of o
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Oestrus growing follicle (Staigmill
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ecomes the main inhibitor of FSH an
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calf at 40-50 days post partum; inv
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al., 2006). However, aged sperm hav
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can occur within 2-3 days, but if t
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educes the incidence of problem cow
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oestradiol, the LH surge and ovulat
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The secondary signs of oestrus can
Page 37 and 38: There are also changes in normal be
Page 39 and 40: engage in more natural behaviours i
Page 41 and 42: 1983). Exact explanations and mecha
Page 43 and 44: 2006) and disruption of LH secretio
Page 45 and 46: 1.4.3.2 Milk Yield and Nutrition Di
Page 47 and 48: influence the ability of the ovary
Page 49 and 50: cyclicity can be delayed if dietary
Page 51 and 52: indication for the optimal time to
Page 53 and 54: calving), 3) pre-breeding heat date
Page 55 and 56: 1.5.2 Physiological Changes Physiol
Page 57 and 58: 1.5.2.3 Body and Milk Temperature T
Page 59 and 60: physical activity and stage of the
Page 61 and 62: caused by the general environment t
Page 63 and 64: may be gained. This is because data
Page 65 and 66: In summary the objective was to for
Page 67 and 68: diet, with concentrates at milking.
Page 69 and 70: oestrus was defined as 3 consecutiv
Page 71 and 72: The interaction between parity and
Page 73 and 74: individual oestrus was not signific
Page 75 and 76: Table 2.4 The effects of the intera
Page 77 and 78: (361 vs. 578 points, respectively,
Page 79 and 80: oestrous expression with increasing
Page 81 and 82: the blood (Sangsritavong et al., 20
Page 83 and 84: CHAPTER 3 - Single Nucleotide Polym
Page 85 and 86: population owing to previous select
Page 87: Table 3.1 Cont. Follicle Stimulatin
Page 91 and 92: was achieved. PCR products were rem
Page 93 and 94: 3.4 DISCUSSION The objectives of th
Page 95 and 96: fertility and oestrous expression.
Page 97 and 98: CHAPTER 4 - Development of a Novel
Page 99 and 100: In summary UWB seems a good option
Page 101 and 102: Initial tests were carried out to i
Page 103 and 104: Therefore this demonstrates that X
Page 105 and 106: which is most important for achievi
Page 107 and 108: Figure 4.9 Horizontal - Vertical Di
Page 109 and 110: that UWB is matching the ‘truth
Page 111 and 112: mounting cow. For example height ch
Page 113 and 114: Backpack 1 st put on in AI stalls E
Page 115 and 116: Cows’ behaviour was assessed at 5
Page 117 and 118: or that the cows were in an area of
Page 119 and 120: plane, but mostly in achieving high
Page 121 and 122: Develop techniques for analysis of
Page 123 and 124: in elastic silicone moulded over a
Page 125 and 126: and time of mount, duration of moun
Page 127 and 128: observed matched with increases in
Page 129 and 130: 5.3 RESULTS Results demonstrate pos
Page 131 and 132: Figure 5.2 Graph showing mounting b
Page 133 and 134: Table 5.1 Results from POC 2 showin
Page 135 and 136: Table 5.3 Efficiency and accuracy o
Page 137 and 138: oestrus and oestrus it is clear to
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P4 Concentration, ng/ml P4 Concentr
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Activity Activity Activity Activity
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5.4 DISCUSSION The aim of this work
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averaged 70% which was lower than t
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when the cows lie down, especially
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the level of oestrous activity was
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technology larger herds must be mon
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monitoring could also monitor non-l
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antennae which could affect accurac
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predispose cows to illness e.g. met
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CHAPTER 6 - Overall Discussion & Co
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oestrus, h 2 =0.27 (Lovendahl et al
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The final section of this thesis de
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ate. By using UWB in a commercial s
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provides an insight the potential o
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BLOWEY, R. (2005) Factors associate
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ovine gonadotrophin releasing hormo
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GINTHER, O. J., KOT, K., KULICK, L.
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associated with embryonic survival
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ody condition at parturition on end
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Signs in a 24-h Tie-Stalled Dairy H
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SHERMAN, E. L., NKRUMAH, J. D., MUR
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Influence of negative energy balanc
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novel approaches to expression and detection of oestrus in dairy cows

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