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研究生:李河維
研究生(外文):Aswah Ridhowi
論文名稱:乳牛白細胞介素-8基因多態性與產乳性能相關研究
論文名稱(外文):Study on Association Between Interleukin-8 Gene Polymorphism and Milk Production in Dairy Cows
指導教授:張秀鑾 博士蘇亞迪 博士
指導教授(外文):Hsiu-Luan Chang, Ph.DSuyadi, Dr. Agr. Sci.
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:動物科學與畜產系所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2012
畢業學年度:101
論文頁數:78
中文關鍵詞:荷蘭母牛白細胞介素-8基因產乳性狀多態性
外文關鍵詞:Holstein cowsInterleukin-8 geneMilk production traitsPolymorphism
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Genomic selection on individual genes is a promising method to genetically improve economically important traits in dairy cattle. The objectives of this study were to identify the polymorphism of Interleukin-8 (IL8) gene and its association with milk production traits, and to compare the variation of milk production traits during lactation period among various IL8 genotypes. Sixty-eight Holstein cows with 171 milking records were genotyped and were evaluated for the impact of polymorphism of IL8 gene on milk production traits. Fixed effects model was used for analysis. Traits evaluated were 305-2X-ME, daily milk yield (MY), fat%, protein%, lactose%, total solid%, somatic cell count (SCC), and somatic cell score (SCS). Genotypic frequencies of CC, CT and TT were 0.22, 0.47, and 0.31, respectively for single nucleotide polymorphism (SNP) of IL8-T2862C. No significant difference was found among genotypes for 305-2X-ME, fat%, protein%, total solid%, and SCS. However, significant effects did shown on MY and SCC (P &;lt; 0.05), and lactose% (P &;lt; 0.01). Cows with genotype CC had less in SCC compared with CT and TT genotype. This might imply that CT and TT genotype cows had low potency to activate neutrophil for phagocytosis when compared with those of CC ones. Although cows with CC genotype showed the lowest SCC, the MY in this genotype was still lower than those of other genotype ones. This seemed to strongly indicate that the number of cells secreting milk in cows with CC genotype was lower compared with CT and TT genotype. Estimates of additive effect showed IL8-T2862C was associated with 0.70 kg increase in MY and 3.24 (104 cells/mL) more in SCC. Additive effect also tended to be associated with 0.18 increases in SCS (P &;lt; 0.1). Results from dominant effect evaluation showed significant effects in MY (+1.22 kg, P &;lt; 0.05) and lactose% (+0.07%, P &;lt; 0.01). These results confirm that IL8 gene plays main role in phenotypic traits, and it might be used in marker assisted selection to improve milk production traits. Significant association between variation of MY and milk component during lactation period on IL8-T2862C genotype of cows was also observed. CT cows showed the most variation in MY and milk component, followed by TT and CC genotypes. Although the association of IL8-T2862C polymorphism with the activation of neutrophil for phagocytosis required further confirmed, the SNP studied did correlate with milk production traits in Holstein cows.
Genomic selection on individual genes is a promising method to genetically improve economically important traits in dairy cattle. The objectives of this study were to identify the polymorphism of Interleukin-8 (IL8) gene and its association with milk production traits, and to compare the variation of milk production traits during lactation period among various IL8 genotypes. Sixty-eight Holstein cows with 171 milking records were genotyped and were evaluated for the impact of polymorphism of IL8 gene on milk production traits. Fixed effects model was used for analysis. Traits evaluated were 305-2X-ME, daily milk yield (MY), fat%, protein%, lactose%, total solid%, somatic cell count (SCC), and somatic cell score (SCS). Genotypic frequencies of CC, CT and TT were 0.22, 0.47, and 0.31, respectively for single nucleotide polymorphism (SNP) of IL8-T2862C. No significant difference was found among genotypes for 305-2X-ME, fat%, protein%, total solid%, and SCS. However, significant effects did shown on MY and SCC (P &;lt; 0.05), and lactose% (P &;lt; 0.01). Cows with genotype CC had less in SCC compared with CT and TT genotype. This might imply that CT and TT genotype cows had low potency to activate neutrophil for phagocytosis when compared with those of CC ones. Although cows with CC genotype showed the lowest SCC, the MY in this genotype was still lower than those of other genotype ones. This seemed to strongly indicate that the number of cells secreting milk in cows with CC genotype was lower compared with CT and TT genotype. Estimates of additive effect showed IL8-T2862C was associated with 0.70 kg increase in MY and 3.24 (104 cells/mL) more in SCC. Additive effect also tended to be associated with 0.18 increases in SCS (P &;lt; 0.1). Results from dominant effect evaluation showed significant effects in MY (+1.22 kg, P &;lt; 0.05) and lactose% (+0.07%, P &;lt; 0.01). These results confirm that IL8 gene plays main role in phenotypic traits, and it might be used in marker assisted selection to improve milk production traits. Significant association between variation of MY and milk component during lactation period on IL8-T2862C genotype of cows was also observed. CT cows showed the most variation in MY and milk component, followed by TT and CC genotypes. Although the association of IL8-T2862C polymorphism with the activation of neutrophil for phagocytosis required further confirmed, the SNP studied did correlate with milk production traits in Holstein cows.
Abstract I
Acknowledgement III
Contents V
Figures and tables of contents VIII
Abbreviations XI
1. Introduction 1
1.1 Objectives 2
2. Literature review 3
2.1 Somatic cell count (SCC) 3
2.2 The effect of SCC on milk production and milk component 3
2.3 Bovine defense mechanisms 6
2.3.1 Anatomical defense 6
2.3.2 Cellular defense 7
2.3.3 Soluble defense 9
2.4 Interleukin-8 (IL8) receptor 10
2.5 Association between polymorphism of IL8 receptor with neutrophil 11
2.6 Interleukin-8 (IL8) gene 12
2.7 IL8 gene polymorphisms and its association with milk production traits 15
3 Materials and methods 24
3.1 Animals 24
3.2 Milk production traits evaluated 24
3.3 DNA extraction 25
3.4 DNA quantification and quality analysis 26
3.5 Genotyping of IL8 27
3.5.1 Primer design 27
3.5.2 PCR-RFLP 28
3.5.4 Loading sample in the gel 29
3.6 Cloning and sequencing of IL8 fragment 30
3.6.1 Purification (Gel extraction) 30
3.6.2 DNA ligation 31
3.6.3 Transformation exogenous DNA 31
3.6.4 Extraction plasmid DNA 32
3.6.5 EcoR I Restriction enzyme cut confirmation of exsogen recombinant DNA 32
3.6.6 Target fragment sequencing analysis and sequence alignment 33
3.7 Statistical analysis 34
4. Results and discussions 35
4.1 Genotypic and allelic information of IL8 gene in Holstein cows 35
4.2 Factor affecting milk production traits in Holstein cows 39
4.3 The effect of IL8 genotype on milk production in Holstein cows 42
4.4 The effect of IL8 genotype on udder health in Holstein cows 43
4.5 The effect of IL8 genotype on milk component in Holstein cows 45
4.6 Additive and dominance effect of IL8 genotype on milk production traits in Holstein cows 49
4.7 The effect of IL8 genotype on variation of milk production traits during lactation in Holstein cows 50
4.8 Additive and dominance effect of IL8 genotype on variation of milk production traits in Holstein cows 52
4.9 The effect of season and parity on milk production traits in Holstein cows 55
4.10 The effect of season on variation of milk production traits during lactation period in Holstein cows 60
58
4.11 The effect of parity on variation of milk production traits during lactation period in Holstein cows
60
4.12 Estimate of parity effect on variation of milk production traits in Holstein cows 62
5. Conclusions and recommendations 67
5.1 Conclusions 67
5.2 Recommendations 68
6. References 69
Author profile 78

Figure and Table of Contents

Figure 1 Output of IL8-T2862C genotyping via PCR-RFLP using HpyCH4V endonuclease 35
Figure 2 Family test of IL8-T2862C alleles by PCR-RFLP in Holstein cows 37
Figure 3 Sequence profile of IL8-T2862C 38
Figure 4 Phenotypic trend of 305-2X-ME (kg) in Taiwan Holstein cows born during 1999 and 2009 40
Figure 5 Phenotypic trend of daily milk yield (kg/d) in Taiwan Holstein cows born during 1999 and 2009 41
Figure 6 Phenotypic trend of SCC (104cells/mL) in Taiwan Holstein cows born during 1999 and 2009 41
Figure 7 Phenotypic trend of SCS in Taiwan Holstein cows born during 1999 and 2009 41

Table 1 Relationship between somatic cell score, somatic cell count and milk lost per lactation 4
Table 2 Milk yield and milk composition in cows with SCC ≤ 200,000 cells/mL and SCC ≥ 200,000 cell/mL 5
Table 3 Innate immunity of bovine mammry gland 6
Table 4 Immune response of bovine mammary gland to respond the various bacteria 10
Table 5 Comparison of concentrations of total lipids, free fatty acids, phospholipids, interleukin-8, somatic cell count, and sodium in milk produced by breasts with symptoms of mastitis versus milk produced by contralateral asymptomatic breasts 15
Table 6 The genetic information of IL8 gene in Chinese Holstein 16
Table 7 Association of polymorphism of IL8 gene with milk traits and SCS in Chinese Holstein (1/2) 17
Table 8 Association of polymorphism of IL8 gene with milk traits and SCS in Chinese Holstein (2/2)
19
Table 9 Association of the SNPs with estimated breeding values for milk yield, fat yield, protein yield, udder depth and somatic cell score (SCS) in Canadian Holstein 20
Table 10 Genotype frequencies, minor allele frequencies (MAF) and significance of deviation from Hardy-Weinberg equilibrium (HWE) for the SNPs investigated within the Holstein-Friesian bull cohort 21
Table 11 Allele substitution effect (standard error in parenthis) between SNPs and milk performance within Holstein Friesian sires 22
Table 12 Relative expression of the genes in crossbreed cows with mastitis in relation to the animals without mastitis
23
Table 13 The genotypic and allelic information of IL8 gene in Holstein cows 37
Table 14 ANOVA for effects of birth year, farm, season, parity and IL8 genotype on 305-2X-ME, daily milk yield, fat, protein, lactose, total solid, SCC and SCS in Holstein cows 39
Table 15 The effect of IL8 genotype on 305-2X-ME, daily milk yield, SCC and SCS and milk component (fat, protein, lactose, total solid) in Holstein cows 47
Table 16 The effect of IL8 genotype on variation of milk production traits during lactation in Holstein cows 48
Table 17 Additive and dominance effects of IL8 genotype on 305-2X-ME, daily milk yield, fat, protein, lactose, total solid, SCC and SCS in Holstein cows

49
Table 18 Additive and dominance effects of IL8 genotype on variation of 305-2X-ME, daily milk yield, fat, protein, lactose, total solid, SCC and SCS in Holstein cows 54
Table 19 Additive and dominance effects of IL8 genotype on 305-2X-ME, daily milk yield, fat, protein, lactose, total solid, SCC and SCS in Holstein cows

57
Table 20 Estimate of parity effect on 305-2X-ME, daily milk yield, fat, protein, lactose, total solid, SCC and SCS in Holstein cows
58
Table 21 The effect of season on variation of 305-2X-ME, daily milk yield, SCC, SCS and milk component (fat, protein, lactose, total solid) in Holstein cows 64
Table 22 The effect of parity on variation of 305-2X-ME, daily milk yield, SCC, SCS and milk component (fat, protein, lactose, total solid) in Holstein cows 65
Table 23 Estimate of parity effect on variation of 305-2X-ME, daily milk yield, fat, protein, lactose, total solid, SCC and SCS in Holstein cows 66


Abbreviations
bp – Base pair
BSA – Bovine serum albumin
BTA6 – Bos taurus autosome 6
CCL2 – Chemokine ligand-2
CCR2 – Chemokine receptor-2
CD14 – Cluster of differentiation-14
CSF – Colony stimulating factors
CXCR1 – Chemokine receptor-1
CXCR2 – Chemokine receptor-2
DNA – Dioxyribonucleic acid
DYD – Daughter yield deviations
FEZL – Forebrain embryonic zinc finger-like
FFA – Free fatty acid
GLM – General linear model
HWE – Hardy-weinberg equilibrium
IFN – Interferons
IFNγ - Interferon gamma
Ig – Immunoglobulins
IL1β – Interleukin-1 β
IL2 – Interleukin-2
IL6 – Interleukin-6
IL8 – Interleukin-8
IL8RA – Interleukin-8 receptor
IMI – Intra mammary infection
LS Means – Least square means
LBP – Lipopolisaccharide binding protein
MHC – Major histocompatibility complex
MAF – Minor allele frequencies
mRNA – Messenger ribonucleic acid
NK – Natural killer
PIC – Polymorphism information contents
PL – Phospholipid
pmCD14 – plasma membrane receptor
PMN – Polymorphonclear
QTL – Quantitative trait loci
SCS – Somatic cell score
SEMA5A – Semaphorin-5A
SSC – Somatic cell count
SSCP – Single-strand conformation polymorphism
Std Err – Standard error
TLR2 – Toll-like receptor-2
TLR4 – Toll-like receptor-4
TNF-α – Tumor necrosis factor-α

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