Associations between Leptin Gene Polymorphisms and Somatic Cell Count in Milk of Jersey Cows

A total of 181 Jersey cows were used to investigate how leptin gene polymorphisms affect somatic cell count (SCC) in milk. Three single nucleotide polymorphisms were genotyped, namely the R4C polymorphism in exon 2, the Sau3AI polymorphism in intron 2 and the A59V polymorphism in exon 3. The genotype and allele frequencies for each polymorphism and the haplotype frequencies for all polymorphisms were estimated in the herd under study. Statistical analysis revealed that the R4C and Sau3AI polymorphisms significantly affected SCC (P ≤ 0.01) with C and T as a desirable allele, respectively. No associations were found between the A59V polymorphism and SCC in this study. However, all the genotype combinations (haplotypes) significantly affected this trait. The results indicate that selection for the R4C CC and Sau3AI TT animals might contribute to a reduction of SCC in Jersey cattle. Leptin, polymorphism, haplotype, somatic cell count, dairy cows, candidate gene, selection Leptin is a 16-kDa polypeptide hormone with a tertiary structure resembling that of some cytokines such as IL-6, IL-11, IL-12 and IL-15. It is produced primarily by fat cells but the presence of leptin was also demonstrated in the mammary gland tissue and in milk of different mammalian species (Zhang et al. 1994; Houseknecht et al. 1997; Estienne et al. 2000; Bonnet et al. 2002; McFadin et al. 2002). Leptin receptors are expressed in a variety of cells and tissues, including the immune cells. Leptin is mainly involved in maintaining the energy balance by controlling food intake and energy expenditure and it regulates the endocrine and immune functions (Houseknecht et al. 1998). It has been established that leptin plays a major role in both innate and adaptive immune responses. Leptin mediates proliferative and antiapoptotic activities in the monocytes and T lymphocytes and delays apoptosis of neutrophils (Bruno et al. 2005). Leptin was found to promote T helper responses (Matarese et al. 2005) and also to regulate phagocytic function in macrophages/monocytes and secretion of proinflammatory cytokines (TNF-α, IL-6, IL-12). Bernotiene et al. (2006) reported elevated circulating leptin level during infectious and inflammatory processes. Mastitis – inflammation of the mammary gland – is a major cause of economic losses to the dairy cattle industry as it reduces milk yield, alters milk composition and elevates treatment costs. Mastitis is accompanied by an increased somatic cell count (SCC) in milk and estimated genetic correlation between mastitis and SCC ranges between 0.53 and 0.77 (Carlén et al. 2004; Ødegård et al. 2004; Koivula et al. 2005). Therefore, SCC is a widely used indicator trait for mastitis, which in many countries is used as an indirect selection criterion for improving mastitis resistance (Interbull 2008). Monitoring and reducing SCC is the primary objective for dairy producers. However, it is necessary to keep within the physiological bounds. Many studies have been conducted recently to search for candidate genes associated with udder health and immune processes and some genes have been proposed, for example: TNF-α – tumour necrosis factor α, LTF – lactoferrin, DEF – defensin, CXCR – chemokine ACTA VET. BRNO 2010, 79: 237-242; doi:10.2754/avb201079020237 Address for correspondence: Dr. Hanna Kulig Department of Genetics and Animal Breeding, Westpomeranian University of Technology, Doktora Judyma 6, 71-466 Szczecin, Poland Phone/fax: +48 91 4541497 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm receptor, interleukin-8 (IL-8) receptor gene (Youngerman et al. 2004; Kamiński et al. 2006; Wojdak-Maksymiec et al. 2006). There have also been some reports on associations between leptin genotypes and SCC in dairy cattle (Buchanan et al. 2003). However, the majority of studies have been concerned with milk production, metabolic and/or reproductive traits in relation to leptin gene polymorphisms (Liefers et al. 2002; Liefers et al. 2005). The aim of this study was to determine leptin allele and genotype frequencies and to establish possible associations between leptin gene polymorphisms (R4C, A59V, Sau3AI, and their combinations) and SCC in Jersey cows. Materials and Methods The study included a total of 181 Jersey cows kept on a farm located in the western region of Poland. All the animals were born of 17 sires between 1994 and 2001 and kept under identical management conditions: standard feeding, seasonal pasture feeding, and water available ad libitum. The cows were milked twice a day with the use of a pipeline milking machine. Milk yield was evaluated with the A4 method in compliance with the recommendations of the International Committee for Animal Recording (ICAR). SCC data was recorded on the basis of monthly milking tests. SCC in the collected samples was determined with an instrumental method in compliance with the PN EN ISO / IEC 17025 standard, using Combifoss equipment (including Fossmatic 5000 apparatus, Foss, Hillerod, Denmark). All the analyses were carried out in a certified milk analysis laboratory. Blood from the external jugular vein was collected into tubes containing K3EDTA. DNA was isolated with MasterPure kit (Epicentre®) according to the manufacturer’s instructions. The analysed polymorphic sites are situated in: 1. the second exon; it is a T/C transition which results in R4C substitution in the protein; 2. the third exon; it is a C/T transition which results in A59V substitution in the protein; 3. the second intron; it is a C/T transition. Genotype analyses were performed using the PCR-RFLP method. Amplification of the desired leptin gene fragments was performed with appropriate primer pairs with the following nucleotide sequences: 5’-ATGCGCTGTGGACCCCTGTAT-3’ and 5’-TGGTG TCATCCTGGACCTTCC-3’, a 94 bp (base pair) long fragment (R4C polymorphism) (Buchanan et al. 2002); 5’-GGGAAGGGCAGAAAGATAG-3’ and 5’-TGGCAGACTGT TGAGGATC -3’, a 331 bp long fragment (A59V polymorphism) (Haegeman et al. 2000); 5’-GTCACCAGGATCAATGACAT-3’ and 5’-AGCCCAGGAATGAAGTCCAA-3’, a 1820 bp long fragment (Sau3AI polymorphism) (Pomp et al. 1997). The PCR was performed in a total volume of 20 ml containing 50-100 ng DNA, 20 mM Taq polymerase buffer, 2 mM MgCl2, 10 pmol each primer, 200 mM each dNTP, and 0.5 U Taq DNA polymerase. The PCR products were digested with appropriate restriction enzymes: the 94 bp fragment (R4C polymorphism) with Kpn2I at 55 oC, the 331 bp fragment (A59V polymorphism) with HphI at 37 oC and the 1820 bp fragment (Sau3AI polymorphism) with Sau3AI at 37 oC. The obtained restriction fragments were separated on 3% and 2% agarose gels with ethidium bromide (0.5 μg/ml) in the presence of an appropriate DNA standard, and described using the Vilber Lourmat software for photodocumentation of electrophoretic separation and image storage. The next stage involved a statistical analysis of associations between the leptin gene polymorphisms and SCC. Year/month of investigation, parity/month of lactation and cow (as a random factor nested within genotype) were also considered as sources of variability. SCC was transformed to the natural logarithm scale (ln SCC). SCC data was obtained from breeding documentation for 2001-2004. The statistical analysis of SCC in relation to leptin genotypes/haplotypes was carried out using the GLM (General Linear Model) multiple-factor mixed nested model (StatSoft 2006). The following linear model was applied: (ln SCC) yijkl = μ + ai + bj + ck + dl(ai) + eijkl where: yijkl – somatic cell count (ln SCC); μ – mean somatic cell count for herd (ln SCC); ai – effect of genotype/ haplotype (i = 1, 2, 3 for R4C, A59V and Sau3AI; i = 1, 2, ..., 6 for R4C/A59V; i = 1, 2, ..., 7 for R4C/Sau3AI and A59V/Sau3AI); bj – effect of study year/month (j = 1, 2, ..., 35); ck – effect of parity/month of lactation (k = 1, 2, ..., 68); dl(ai) – effect of cow, random factor nested within leptin genotype (l = 1, 2, ... , 181); eijkl – random error. The differences between mean SCCs were verified with Duncan multiple range test.