Associations between gene variations and milk composition in New Zealand dairy cattle: A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Abstract

Milk solids (MS) of various types are an important export commodity for New Zealand (NZ). Of the MS, increased production of milk fat and protein, would increase farm and industry income, and accordingly the production traits of milk volume, fat percentage and protein percentage are included in the National Breeding Objective. This is underpinned by the Breeding Worth (BW) index, which is used to estimate an animal’s genetic merit for production. In addition to quantity, the compositions of the milk fat and protein are also important quality determinants for milk processing, milk products, and the nutritional value of milk. Gene-markers are useful tools for improving animal genetics and breeding. They have become an increasingly useful method for selecting superior dairy cattle, as the valued milk traits are dependent on multiple factors including genetics, breed, diet, and feeding system. In this context, this research set out to ascertain the effect of selected genes on milk traits in NZ pasture-grazed KiwicrossTM cows, and with the overall aim of establishing new gene-markers for cattle breeding. Five genes were studied, the diacylglycerol acyl-CoA acyltransferase 1 gene (DGAT1), the fatty acid binding protein 4 gene (FABP4), the stearoyl-CoA desaturase (Δ-9-desaturase) gene (SCD1), the perilipin-2 gene (PLIN2) and the lipin-1 gene (LPIN1). These genes were chosen because they had either been implicated in fatty acid (FA) metabolism in mammary gland cells, or potentially regulated milk fat synthesis. They were screened to find nucleotide sequence variation using a Polymerase Chain Reaction - Single Stranded Conformational Polymorphism (PCR-SSCP) approach, and then a modelling approach was used to ascertain whether the genetic variation (if detected) was associated with gross milk traits (including milk volume, fat percentage and protein percentage), and milk fat composition. At the gross milk trait level, variation in DGAT1 and FABP4 are associated with variation in milk volume, milk fat and milk protein content. Nucleotide sequence variation that has been reported previously is found in exon 8 of DGAT1 in the KiwicrossTM cows. If expressed, this variation results in the amino acid substitution p.K232A. The K variant of DGAT1 (frequency = 61.9% in 395 cows) is found to be associated with the production of less milk volume (KK cows: 22.441 ± 0.526 L/day), but high concentrations of milk fat (KK cows: 5.271 ± 0.067 %) and protein (KK cow: 4.073 ± 0.043 %), than for cows that were A (AA cows: 25.132 ± 0.609 L/day, 4.331 ± 0.077 % and 3.823 ± 0.049 % respectively) (P < 0.001). It has been reported previously that FABP4 has at least three haplotypes (haplotypes A, B and C), and that these are associated with gross milk traits. Variation in FABP4 and its association with milk fat composition are therefore investigated. Haplotype A, is associated with increased milk C22:0 (P = 0.001) and C24:0 FA (P < 0.001) levels, and the C10:0, C12:0, and C14:0 FA content and medium chain fatty acid (MCFA) content increased when haplotype B was present (P = 0.012, P = 0.009, P = 0.005, and P = 0.003 respectively). At the genotype level, AC cows produce more C22:0 (P = 0.021) and C24:0 (P = 0.030) FA, and the AB cows produce more C12:1 (P = 0.018) and C14:0 (P = 0.010) FA. In respect of milk fat composition, variation in DGAT1 and SCD1 are associated with variation in milk FAs. Cows with the DGAT1 p.232 K variant, produce more saturated FAs (P < 0.001) but less branched and unsaturated FAs (P < 0.001). The p.232 homozygous AA cows produce more (P < 0.001) CLA (1.070 ± 0.054 g/100 g) and C18:3 cis-9, 12, 15 FA (0.830 ± 0.021 g/100 g), but less C16:0 FA (35.739 ± 0.534 g/100 g) than the KK cows (0.864 ± 0.046 g/100 g, 0.751 ± 0.018 g/100 g and 38.437 ± 0.461 g/100 g respectively). For the SCD1 gene (SCD1), three genetic variations in exon 5 (c.702A>G, c.762T>C and c.878C>T) were found and the c.878C>T substitution would result in amino acid change p.A293V. One variation in intron 5 (c.880 + 105A>G) and four variations in the 3’UTR (c.*1783A>G, c.*1883C>T, c.*1984G>A and c.*2066T/C /G) were also found. The c.*1783A>G and c.*2066T/C/G substitutions produced three nucleotide sequence variants (a, b and c). There was linkage between the exon 5 variation and the 3’UTR variation, such that the sequence that would encode valine at position 293 of SCD1 is linked to 3’UTR variant a, and the sequence that would encode alanine, is linked to variants b and c. The VV cows produced less C10:1, C12:1 and C14:1 FA, but more C16:1 and C18:2 FA than the AA cows (P < 0.001). The presence of c is associated with decreased levels of C16:1 (P < 0.001), C17:1 (P = 0.001), C18:2 cis-9, trans-13 (P = 0.045), C18:2 cis-9, trans-12 (P = 0.018) FA and C16:1 FA index (P < 0.001). The presence of b is associated with increased levels of C13:0 iso FAs (P < 0.001), MUFA (P = 0.002), and C12:1 (P < 0.001). Variation in the 3’UTR of PLIN2 (c.*302T>C), which produce two nucleotide sequence variants (A5 and B5) was described. The B5B5 homozygous cows produced less palmitic acid (C16:0) (P = 0.048), but more medium chain fatty acids, than the A5A5 cows (P = 0.033). Overall, this study identified DGAT1 and FABP4 as good candidate genes for predicting gross milk trait variation in KiwicrossTM cows. Furthermore, DGAT1, FABP4, SCD1 and PLIN2 might be useful for predicting variation in milk fat composition. Variation was not found in the regions (exon 16, exon 17, part of intron 16 and part of intron 17) of LIPN1 that were investigated. To further this research, more dairy cattle from different dairy production systems will need to be studied to confirm that these genes might be useful markers for gross milk traits and milk fat composition.