Genetic variation in genes associated with milk production traits and fatty acids composition in New Zealand dairy cattle : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Field: Agricultural and Biological Sciences (Life Sciences)

The myostatin gene (MSTN), alternatively known as the growth and differentiation factor 8 gene (GDF8), encodes the myostatin protein (MSTN). It has pleiotropic effects, and it is expressed in skeletal muscle as well as the mammary gland. Its expression has been associated with increased skeletal muscle mass, and decreased adipogenesis as a result of the reduced secretion of leptin. Leptin is a product of the obese (ob) gene. It is a 16-kDa protein hormone mainly secreted from white adipose tissue and it regulates feed intake, energy partitioning and metabolism, as well as lactogenesis. Using Polymerase Chain Reaction (PCR) amplification, coupled with Single Strand Conformation Polymorphism (SSCP) analyses and subsequent nucleotide sequencing, an extended region of the bovine MSTN and leptin (LEP) genes were investigated for genetic variations in a variety of cattle breeds from New Zealand and Nigeria. Five regions of MSTN were investigated. These included exons 1, 2 and 3, and parts of introns 1 and 2; and they were studied in 883 cattle of varied breeds from New Zealand (Hereford, Angus, Charolais, Simmental, Red Poll, South Devon, Shorthorn, Murray Grey, cross-bred Holstein-Friesian × Jersey cattle (NZ HF × J-cross, or Kiwicross™) and from Nigeria (Sokoto Gudali, Red Bororo, White Fulani and cross bred Holstein-Friesian × White Fulani). Eight PCR-SSCP banding patterns were observed in the intron 1 region amplified, four in the intron 2 region amplified and none in the exon regions. A total of seventeen single-nucleotide substitutions (nine in intron 1 and eight in a region spanning the intron 2 - exon 3 boundary) and one nucleotide deletion were detected. For the leptin gene (LEP), three regions (intron 1/exon 2, part of intron 2 and the entire exon 3) were examined in 657 cattle from a variety of breeds farmed in New Zealand and Nigeria. These included NZ Hereford, Angus, Shorthorn, and NZ HF × J-cross cows; and the Nigerian Sokoto Gudali, Red Bororo, White Fulani, and cross-bred Holstein-Friesian × White Fulani cattle. Four PCR-SSCP patterns were detected in the intron 1/exon 2 region with four nucleotide sequence variations, three patterns in intron 2 with three sequence variations, and three patterns in exon 3 with five nucleotide sequence variations. The effect of intron 1 MSTN variation on milk production traits and the composition of milk FA were investigated. The saturated fatty acids (SFAs) were grouped into three groups based on the length of their carbon chain; Short chain fatty acid (SCFA: C4:0 to C8:0), medium chain fatty acid (MCFA: C10:0 to C14:0) and long chain fatty acid (LCFA: C15:0 to C24:0). The unsaturated fatty acids (UFAs) with only one double bond (monounsaturated fatty acid: MUFA) ranged from C10:1 to C22:1 and were grouped as MUFA, whereas those with two or more double bonds (polyunsaturated fatty acid: PUFA) ranged from C18:2 to C22:5 and were grouped as PUFA. Using General Linear Mixed-effect Model (GLMM) analyses, single variant presence/absence models revealed the presence of variant D to be associated with reduced levels of C8:0, C10:0, C12:0, C12:1, and the grouped MCFA level; but an increased level of C16:1 cis-9. Variant B was associated with decreased C15:0 iso level, whereas the presence of C was associated with increased levels of C20:3 cis-8, 11, 14 and C22:1, trans-13. Only the NZ HF × J-cross cows with the following MSTN genotypes; AA (n = 151), AB (n = 92), AC (n = 53) and AD (n = 65) were studied in the milk FA genotype association analyses; with the remaining genotypes, AE (n = 15), BB (n = 7), BC (n = 7), BD (n = 17), CC (n = 6) CD (n = 10) and DD (n = 7) having frequencies less than 5% each, and not being analysed. These genotype model results were consistent with the single variant presence/absence models, with genotype AD being associated with reduced C10:0, C12:0, and C12:1 levels. For the leptin gene, the effect of exon 3 variation on milk production traits and FA composition in NZ HF × J-cross dairy cows were investigated. Association studies using GLMMs revealed the presence of variant A3 (the most common variant) decreased the levels of C15:1 C22:0, C24:0, C18:1 trans-11, C18:1 trans-9, cis-12 and C18:1 all trans. Variant B₃ was revealed to be associated with reduced C6:0, C11:0 level, and C20:0 level, but increased C17:0 iso, C24:0 and C10:1 index. Variant C₃ was associated with decreased C13:0 anteiso. The following genotypes; A₃A₃ (n = 73), A₃B₃ (n = 176) and A₃C₃ (n = 52) were examined for milk FA composition, whereas B₃B₃ (n = 11) and C₃C₃ (n = 3) were excluded from the analyses due to their low frequency of occurrence. In the genotype model, relative to the A₃A₃ genotype, the A₃B₃ genotype was associated with decreased levels of C8:0, C10:0, C11:0, C13:0 and grouped MCFA, but increased C24:0. Genotype A₃C₃ was associated with decrease grouped MCFA level and C10:0, C11:0 and C13:0 levels relative to the A₃A₃ genotype. These associations in NZ HF × J-cross cows suggest that variation in bovine MSTN and LEP could be explored for increasing the concentration of UFAs and decreasing the concentration of SFAs in milk.