Polymorphism in FASN, SCD1 and ANXA9 Genes: A Preliminary Study of Markers Which May Serve As Genetic Predictors of the Fatty Acid Prole of Sheep Milk

In this study, single nucleotide polymorphisms (SNPs) in the ANXA9 (annexin 9), FASN (fatty acid synthase) and SCD1 (stearoyl-CoA desaturase 1) genes were analyzed as factors inuencing fatty acid proles in milk from Zošľachtená valaška sheep. SNP in selected genes was identied using polymerase chain reaction (PCR) and restriction fragment length polymorphism (PCR-RFLP). The long-chain fatty acids prole in sheep milk was identied by gas chromatography. Statistical analysis of the SCD1/Cfr13I polymorphism showed that the milk of the homozygous AA animals was characterized by a lower (P < 0.05) share of C4:0, C6:0, C8:0, C10:0, C12:0, C14:0 in comparison to the homozygous CC sheep. The milk of heterozygous sheep was characterized by a higher (P < 0.05) proportion of C13:0 acid compared to the milk of sheep with the homozygous AA type. A higher (P < 0.05) level of saturated fatty acids (SFA) was found in the milk of CC genotype sheep compared to the AA genotype. Our results lead to the conclusion that the greatest changes were observed for the SCD1/Cfr13I polymorphism and the least signicant ones for FASN/AciI. Moreover, it is the rst evidence that milk from sheep with SCD1/Cfr13I polymorphism and the homozygous AA genotype showed the most desirable fatty acids prole. C18:1n9t acid compared polymorphism in the ANXA9 gene acid was also found. Because presented results indicate the association of the analyzed genotypes with the fatty acid prole in Zošľachtená valaška, a larger prospective study will be continued to explore the effect of SNPs, also in the other genes showing effects on sheep milk. Moreover, results of our study can be useful for breeders, especially that Zošľachtená valaška sheep are included in the breeding program and sheep with homozygous AA (SCD1/Cfr13I) could be used during the breed improvement program.


Introduction
Globalization and the rapid development of food production have resulted in new consumer food trends.
Modern consumers are looking for products rich in valuable nutrients, vitamins and substances that have a positive effect on human health. In food, unsaturated fatty acids have pro-health properties, in particular, they contribute to reducing blood cholesterol 1 . The most desirable fatty acids in the human diet are conjugated linoleic acid (CLA), eicosapentaenoic acid (C20:5n-3, EPA) and docosahexaenoic acid (C22:6n-3, DHA), which play an important role in preventing cancer, cardiovascular, autoimmune, and psychological diseases 2 . In contrast, the SFA intake highly increases coronary disease risk, diabetes, obesity, atherosclerosis, and high low-density lipoprotein (LDL) levels 3 .
Sheep milk is a rich source of unsaturated fatty acids compared to cow and goat milk, which makes it desirable as a functional food 4 . The composition of milk, including the fatty acids pro le, is determined by genetic and environmental factors 5,6,7 . The composition of sheep milk is in uenced by both genetic and environmental factors. Breed, age and stage of lactation have a signi cant impact on changes in sheep's milk. The candidate gene approach is applied with regard to genes whose products might affect production traits. Among them, the most extensively studied genes are genes encoding milk proteins (e.g. caseins), hormones and their receptors. Other genes of interest in this area include genes encoding enzymes that participate in fatty acid metabolism as well as genes encoding fatty acid binding and transport proteins 8 .
Fatty acid synthase (FASN) encoded by the FASN gene is a multifunctional homodimeric enzyme that catalyzes the synthesis of fatty acids (FA), plays a key role in the synthesis of short-and medium-chain fatty acids in mammals 9,10 . Additionally, which is important in the adult life of mammals, it determines the energy homeostasis of the organism and is involved in the production of milk lipids during lactation involved in the transport across the cytoplasmic membrane, which is important in the mammary gland 12 . On the other hand, stearoyl-CoA desaturase 1 (SCD1) encoded by the SCD1 gene is the key and a ratelimiting enzyme involved in the metabolism of mammary lipids and is responsible for the conversion of SFA into monounsaturated fatty acids (MUFA). Also be a key enzyme in the production of the cis-9, trans-11 isomer of conjugated CLA. CLA can be found in ruminant milk and tissue fat and is considered a bene cial effect on human health 13,14 .
Dairy sheep farming systems vary from extensive to intensive according to economic relevance of the production chain and the speci c environmental and breed 15 . Only a few publications have comparative values of milk composition for different breeds of sheep and milk energy of sheep breeds, but milk components were not different among breeds 15 .
The available literature data have shown the effects of SNP on the basic composition and protein fractions in sheep milk, however, each variant of the polymorphism was considered separately for each of the FASN, ANXA9 and SCD1 genotypes 7,16,17 . Therefore, an attempt was made to determine which of the studied polymorphisms has a more signi cant impact on the proportion of fatty acids in sheep milk and could be the best candidate marker. Additionally, Zošľachtená valaška sheep are a local Slovak breed, which opens up prospects for future selection programs and animal protection strategies. For this purpose, this study analyzed SNP polymorphisms in the ANXA9, FASN and SCD1 genes as a factor in uencing the fatty acid pro le in the milk of Zošľachtená valaška sheep.

Animals and nutrition
The experiment was carried out in a ock of sheep of the Zošľachtená valaška a perspective breed, mainly for mountainous areas, which is included in the native Slovak breed. They are bred in the three Slovak regions (Spiš, Orava, and Liptov). The breed was generated by the intentionally combined crossing of Native Wallachian sheep with the rams of various imported breeds as, Cheviot, Hampshire, Lincoln, Texel and Leicester, therefore it is genetically interesting. As a new semi-coarse-wool breed, the Improved Wallachian sheep was recognized in 1982. At present, 128,930 pieces of this breed are kept in Slovakia 18 . Animals of this breed are characterized by good adaptation to di cult mountain conditions. Ewes weigh from 50 to 55 kg, they are seasonally polyoestrous during the fall season (October -November).
The composition of mammalian milk is in uenced by genetic and environmental factors. Breed, age and stage of lactation have a signi cant impact on changes in sheep's milk. Therefore, fty sheep were selected from a herd, taking into account age and lactation stage to eliminate the main variables in uencing milk parameters. After lamb weaning, the ewes produced 80-120 kg of milk during the 150 days of lactation. The material for the study was collected from 50 ewes in the similar phase of milking (25-30 days of lactation) and lactation (1st and 2nd lactation). In the lambing period, the sheep were kept in special buildings complying with the European Union Directive (No. 116, item 778, 2010) and were fed hay ad libitum, 250 g/ewe/day of wheat middlings, and 3 kg/ewe/day of haylage. To collect milk samples from ewes, their lambs were separated overnight. The milk was collected into sterile containers and transported to the laboratory at 4 °C. Besides, peripheral blood samples were collected from the external jugular vein (EJV) of ewes into test tubes containing anticoagulant (tripotassium ethylenediaminetetraacetic acid, K 3 EDTA) for DNA isolation and immediately transported to the laboratory, and frozen at −20 °C for subsequent analysis.
DNA isolation was performed using the MasterPure DNA Puri cation Kit for Blood, Version II (Lucigen, Middleton, WI, USA) according to the manufacturer's instructions. Animal genotyping was performed using PCR-RFLP. Three SNPs in the ANXA9 gene (intron 4, intron 5, GenBank: AY785286.1) and one in the FASN gene (exon 32, GenBank: GQ150557.1) and SCD1 (promoter region, GenBank: FJ513370.1) were analyzed. Table 1 shows the location of individual SNPs and the appropriate primers, designed using the Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/), enabling the ampli cation of selected fragments of the analyzed genes. In the case of the reverse primer for the FASN gene, a mismatched nucleotide was introduced to create a cleavage site for the enzyme (this nucleotide is underlined in the Table 1). The polymerase chain reaction (PCR) reaction was performed in a nal volume of 25 μl, using 2x PCR Master Mix (A&A Biotechnology, Gdynia, Poland),) containing 50 ng genomic DNA and 5 pmol of each primer.
DNA ampli cation was performed using an initial denaturation at 94°C for 5 min, followed by 30 cycles of 30 s denaturation at 94°C each, annealing at a temperature appropriate for each gene for 30 s and extension at 72°C for 30 s , ending with a nal extension of 8 minutes at 72°C (Table 1). After the ampli cation of selected fragments of SCD1, FASN and ANXA9 genes, the identi cation of polymorphic loci in these genes was carried out using appropriately selected restriction enzymes.
Individual PCR products were digested separately with restriction enzymes according to the manufacturer's recommendations. Next, the restriction fragments were separated on agarose gels with appropriately selected agarose concentration. The restriction enzymes and the obtained restriction fragments for individual genotypes are presented in Table 2. Fats were extracted from the milk samples using the Folch method [19]. Next, the obtained fat was converted to fatty acid methyl esters using the Christopherson and Glass procedure (1969) [20] with 2M KOH in methanol. The fatty acid pro le was determined, by using a gas chromatography method (Agilent Technologies 7890A, Agilent Technologies, Santa Clara, CA, USA), with a ame ionization detector and an HP-88 capillary column designed for the separation of fatty acid methyl esters (FAMEs) (100 m length, 25 mm i.d. × 0.20 μm). The initial oven temperature was 50 °C and was increased by 3 °C/min to 220 °C. The detector and dispenser temperatures were -270 °C and 270 respectively.
To analyse experimental chromatograms a comparative analysis of the retention times of the fatty acid methyl ester standards [Sigma-Aldrich] was performed using the ChemStation software (Agilent Technologies, USA).

Statistical analysis
The frequencies of genotypes and alleles and the Hardy-Weinberg equilibrium for individual SNPs were calculated using the POPGENE software 21 , the effective number of alleles (Ne) was evaluated according to Kimura and Crow (1964) 22 and expected heterozygosity (He) and the polymorphism information content (PIC) were evaluated according to Nei's methods 23 .
The statistical analysis of the in uence of selected SNPs on the fatty acid pro le in sheep milk was carried out in the Statistica 13.1 program (StatSoft Poland, Krakow, Poland). The results of the study were statistically analyzed using one-way ANOVA followed by a multiple comparisons Tukey Post-Hoc Test. Pearson correlation coe cient (r) with two-tailed test of signi cance was conducted to examine the relationship between certain parameters.

Results
The analysis of the obtained genotyping results suggested that for almost all polymorphic loci tested, the presence of all three possible genotypes was identi ed; only in the FASN gene, two out of three possible genotypes were identi ed. Table 3 shows the frequencies of genotypes and alleles and the expected heterozygosity, the effective number of alleles, PIC, and the value of χ2 calculated based on genotyping results.  24 indicates an average polymorphism (0.25 < PIC value < 0.5). The next analyzed parameter was the Hardy-Weinberg equilibrium value (HWE). The distribution of genotypes consistent with the HWE was at P > 0.05. The analysis of the results collected in Table 3 shows that the ANXA9/HinfI polymorphism was found to be incompatible with the Hardy-Weinberg equilibrium. We also assessed the effects of SNPs within the fatty acid synthase gene (FASN), however, we detected only two genotypes (CC, CT) out of 3 FASN/AciI polymorphism (without TT). Due to this, we did not calculate HWE for FASN/AciI.
The pro le of individual saturated fatty acids in sheep milk in relation to individual genotypes of the studied polymorphisms of the SCD1 and FASN genes is presented in Table 4. Statistical analysis for the SCD1/Cfr13I polymorphism showed that the milk of individuals with the homozygous AA genotype was characterized by a lower (P < 0.05) share of butanoic acid (C4:0), hexanoic acid (C6:0), octanoic acid (C8:0), decanoic acid (C10:0), dodecane (C12:0), tetradecanoate (C14:0) than the homozygous CC sheep. On the other hand, the milk of sheep with the heterozygous genotype was characterized by a higher (P < 0.05) share of tridecanoic acid (C13:0) as compared to the milk of sheep with the AA genotype. A higher (P < 0.05) total level of saturated fatty acids (SFA) was found in the milk of the homozygous CC sheep compared to those of the homozygous AA genotype.  Table 5 presents the share of unsaturated fatty acids in sheep milk in relation to the studied polymorphisms of the SCD1 and FASN genes. The analysis of the SCD1/Cfr13I polymorphism revealed that the proportion of (Z)-11-eicosenoic acid (C20:1) in milk was lower (P < 0.05) in the homozygous AA sheep in relation to the heterozygous and homozygous CC individuals. Higher (P < 0.05) levels of MUFA were noted in the homozygous AA individuals compared to the homozygous CC individuals. The relationship for all cis-5,8,11,14,17-eicosapentaenoic acid (C20:5n3) was reverse: in the milk of the homozygous AA sheep the level of this acid was lower (P < 0.05) compared to individuals with the CC genotype. The polymorphism in the FASN gene will not statistically affect the fatty acid pro le in sheep milk. For the remaining saturated and saturated fatty acids in milk, no statistical effect of polymorphisms for the SCD1 gene was observed. The analysis of the share of short-and long-chain saturated fatty acids with respect to the individual genotypes of the studied polymorphisms in the ANXA9 gene is presented in Table 6 (a, b, c). Statistical analysis for the ANXA9/HinfI polymorphism showed that the milk of individuals with the homozygous CC genotype was characterized by a higher (P < 0.05) content of pentadecanoic acid (C15:0) and eicosanoic acid (C20:0) than the milk of heterozygous sheep. In contrast, the milk of sheep with the CG genotype was characterized by a lower (P < 0.05) proportion of octadecanoic acid (C18:0) compared to the milk of sheep with the homozygous GG genotype. For the remaining saturated fatty acids in milk, no statistical effect of ANXA9 polymorphisms was observed.  a, b, c) shows the contribution of UFA in Zošľachtená valaška milk with respect to individual genotypes of the ANXA9 polymorphisms examined in this study. Analysis of the ANXA9/NlaIII polymorphism showed that the share of (all-Z) -5,8,11,14-eicosatetraenoic acid (20:4n6) in milk was lower (P < 0.01) in the sheep with the homozygous AA genotype in relation to individuals with heterozygous and homozygous GG genotypes. In the case of the ANXA9/HinfI polymorphism, it was shown that the milk of sheep with the homozygous GG genotype was characterized by a higher proportion of transoctadecenoic acid (C18:1n9t) compared to the homozygous CC (P < 0.01) and heterozygous (P < 0.05) sheep. Also, a higher (P < 0.05) share of CLA was found in the homozygous GG individuals compared to heterozygous ones. For the ANXA9/Tru1I polymorphism, it was found that animals with the homozygous AA genotype were characterized by a lower (P < 0.05) proportion of C18:1n9t acid in milk compared to heterozygous sheep and higher (P < 0.05) compared to the homozygous CC animals. The polymorphism in the ANXA9 gene did not statistically affect the share of other unsaturated fatty acids in sheep milk.   Furthermore, we estimated the genetic correlations among individual FAs, which were shown in (Table 8  and 9). For two individual SCFA (C4:0, C6:0), and one LCFA (C:14) negative genetic correlations were observed for SCD1/Cfr13I polymorphism and two negative correlation for two LCFA (C15:0, C20:0) over ANXA9/HinfI polymorphism. SCFA-short-chain fatty acids, LCFA -long-chain fatty acids, SFA -saturated fatty acids; (g/100g of fat). *Signi cance at P < 0.05 was marked by an asterisk.
Positive genetic correlations were observed between MUFA and UFA and SCD1/Cfr13I, although negative correlations for C20:5n3 was observed (Table 9). We also showed a positive correlation between C20:4n6 and ANXA9/NlaIII.  In the human diet, foods with a low n-6/n-3 share, ranging from 1:1 to 4:1, are desirable 33 . Maintaining a su ciently low n-6/n-3 ratio in the diet has a positive effect on the cognitive functions of the body and reduces the risk of depression 34 . Additionally, the high consumption rate of (n-3)/(n-6) PUFAs reduces the risk of neoplastic diseases and in ammations 2,33,35 .

Discussion
In the case of the SCD1/Cfr13I polymorphism, an increase in C20:5n3 was observed in milk of the sheep with homozygous CC genotypes, without changes in the level of n-6 acids, which may cause an increase in the value of (n-3)/(n-6). In the case of ANXA9/NlaIII polymorphism analysis for AA homozygous animals, a decrease in the C20:4n6 acid level was found, which can also be considered a favourable phenomenon. Supplementation of CLA in the diet of animals and humans improves the metabolism of glucose and lipids in the body 36 . In our research, an increase in the level of CLA in the milk of sheep with the GG ANXA9/HinfI genotype was observed.
According to studies by other authors, long-chain unsaturated fatty acids are important in the human diet 33,35,36 . Replacing SFA with PUFA in the human diet is bene cial for the cardiovascular system 37 . In the diet, saturated acids, such as C18:0, C14:0 and C12:0, adversely affect the increase of plasma lipoproteins 38 . On the other hand, the consumption of saturated fatty acids with a carbon chain from C12 to C16 increases the level of plasma LDL-C 37,39 . In our research, an increase in the level of C18:0 acid was noted in the ANXA9/HinfI polymorphism in the homozygous GG individuals, while in the homozygous AA sheep, in the case of the SCD1/Cfr13I polymorphism, a decrease in the level of C12:0, C13:0, C14:0 acids and an increase in MUFA level in milk was observed. Consuming MUFA has a positive effect on humans, reduces total cholesterol and LDL fraction in the blood 40 .
The share of individual fatty acids in the human diet determines both physical and mental health. The present study determined the relationship between the studied polymorphisms and the fatty acid pro le in milk and the results may be used in the selection program in sheep ocks: the choice of an appropriate genotype variant will ensure the desired fatty acid pro le in milk.

Conclusions
The research was aimed at searching for the best marker in uencing the fatty acid pro le in sheep milk. These preliminary ndings show that the greatest changes were observed in sheep with SCD1/Cfr13I polymorphism and the least signi cant ones in FASN/AciI. Milk obtained from the homozygous AA sheep (SCD1/Cfr13I) had the best fatty acid pro le. Slight changes in the milk of sheep were found for polymorphisms in the ANXA9 gene. For the ANXA9/NlaIII polymorphism (AA genotype animals) a favourable reduction in the level of C20:4n6 acid in the milk was noted. Changes were also found for the ANXA9/HinfI polymorphism (homozygous GG individuals). The milk of these sheep was characterized by an increase in the level of C18:1n9t and CLA acids, which is bene cial; unfortunately, an undesirable increase in the proportion of C18:0 acid was also found. Because presented results indicate the association of the analyzed genotypes with the fatty acid pro le in Zošľachtená valaška, a larger prospective study will be continued to explore the effect of SNPs, also in the other genes showing effects on sheep milk. Moreover, results of our study can be useful for breeders, especially that Zošľachtená valaška sheep are included in the breeding program and sheep with homozygous AA (SCD1/Cfr13I) could be used during the breed improvement program.