Towards Sustainable Sources of Omega-3 Long-Chain Polyunsaturated Fatty Acids in Northern Australian Tropical Crossbred Beef Steers through Single Nucleotide Polymorphisms in Lipogenic Genes for Meat Eating Quality

: This study aimed to identify single nucleotide polymorphisms (SNP) in lipogenic genes of northern Australian tropically adapted crossbred beef cattle and to evaluate associations with healthy lipid traits of the Longissimus dorsi (loin eye) muscle. The hypothesis tested was that there are signiﬁcant associations between SNP loci encoding for the fatty acid binding protein 4 (FABP4), stearoyl-CoA desaturase (SCD) and fatty acid synthase (FASN) genes and human health beneﬁcial omega-3 long-chain polyunsaturated fatty acids ( ω 3 LC-PUFA) within the loin eye muscle of northern Australian crossbred beef cattle. Brahman, Charbray, and Droughtmaster crossbred steers were fed on Rhodes grass hay augmented with desmanthus, lucerne, or both, for 140 days and the loin eye muscle sampled for intramuscular fat (IMF), fat melting point (FMP), and fatty acid composition. and FASN genes with signiﬁcant effects on lipid traits were identiﬁed with next-generation sequencing. The GG genotype at the FABP4 g.44677239C>G locus was associated with higher proportion of linoleic acid than the CC and CG genotypes ( p < 0.05). Multiple comparisons of genotypes at the SCD g.21266629G>T locus indicated that the TT genotype had signiﬁcantly higher eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids than GG genotype ( p < 0.05). Signiﬁcant correlations ( p < 0.05) between FASN SNP and IMF, saturated and monounsaturated fatty acids were observed. These results provide insights into the contribution of lipogenic genes to intramuscular fat deposition and SNP marker-assisted selection for improvement of meat-eating quality, with emphasis on alternate and sustainable sources of ω 3 LC-PUFA, in northern Australian tropical crossbred beef cattle, hence an acceptance of the tested hypothesis.


Introduction
According to the Food and Agriculture Organization of the United Nations (FAO), sustainable diets are protective and respectful of biodiversity and ecosystems, nutritionally

Loin Eye Muscle Sampling and Chemical Analysis
A minimally invasive biopsy technique was used to collect loin eye muscle samples from the 12th-13th rib interface of the steers transported to the feedlot after forage-feeding phase according to the protocol described earlier [49]. Samples from the steers slaughtered immediately after the forage-feeding phase were collected from the 12th-13th rib interface of the chilled carcasses 12 h after slaughter. The IMF of the biopsy and carcass samples was extracted as described by Flakemore et al., (2014) [50], and FMP was determined with the slip-point method [51]. The fatty acid composition was evaluated using a gas chromatography-mass spectrometry procedure [52].

Blood Sampling and Genomic DNA Extraction
Blood samples were collected into 10 mL EDTA-containing vacutainer tubes (BD, Sydney, Australia) via jugular venipuncture, transported in dry ice and stored at −80 • C until needed for laboratory analysis. Blood samples were thawed at room temperature and genomic DNA was extracted from a 2 mL aliquot using the NucleoSpin Blood Kit (Macherey-Nagel GmbH and Co. KG, Duren, Germany) according to the manufacturer's instructions. DNA yield and purity were determined with NanoDrop ND-1000 (Thermo Fisher Scientific Australia Pty Ltd., Scoresby, VIC, Australia).

Primer Design, Amplification of Target Genes, Clean-Up of PCR Products, Library Preparation, Sequencing and Data Analysis
The procedures were carried out as described previously [32] with slight modifications on the gene amplification conditions. The target genes were amplified using the primer sequences presented in Table S1 and the gel image of the amplification products is presented in Supplementary Figure S1. The amplification reactions were executed in a SimpliAmp Thermal Cycler (Thermofisher Scientific, Scoresby, VIC, Australia) in a total volume of 50 µL consisting of 25 µL of PCR master mix, 100 ng of DNA template, and 0.5 µM of each primer in a 3-step procedure: single initial denaturation at 98 • C for 1 min, 35 cycles of denaturation, annealing and extension at 98 • C for 15 s, 60 • C for 15 s, and 72 • C for 9 min, respectively, followed by a final extension at 72 • C for 9 min and a 4 • C hold. The FASN gene was amplified with PrimeSTAR GXL Master Mix (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) in a 2-step protocol. The amplification reaction mix consisted of 1. 25  sequences NC_ 037353.1, NC_ 037346.1, and NC_ 037341.1 obtained from the GenBank database were used as the SCD, FASN, and FABP4 reference sequences, respectively.

Calculations and Statistical Analysis
Data analyses and the plotting of figures were conducted with the R software v.4.0.2 (R Foundation for Statistical Computing, Vienna, Austria). The GDIcall online calculator (http: //www.msrcall.com/Gdicall.aspx (accessed on 14 January 2022)) was used to calculate SNP polymorphism information content (PIC). Hardy-Weinberg equilibrium (HWE) and expected heterozygosity (He) were calculated according to the methods described by Nei and Roychoudhury (1974) [53]. The HWE was tested for each identified SNP locus with the Chi-square test. Summary statistics including range, means, and standard deviations were computed and checked for data entry errors and outliers. The degree of linkage disequilibrium between each pair of loci was examined with distance-based hierarchical clustering of SNP loci [54] and the results presented as dendrograms and heatmaps. Linear correlations between genomic variants and muscle IMF, FMP, and fatty acid composition were estimated with Spearman's ρ correlations. Generalised least square procedure was used to fit linear models to investigate SNP associations with the loin eye muscle IMF, FMP, and fatty acid composition. Differences between means were compared using the Tukey-adjusted multiple comparisons test with a threshold for significance set at p < 0.05.

Associations between Single Nucleotide Polymorphisms, Intramuscular Fat, Fat Melting Point, and Fatty Acid Composition
Associations between FABP4 g.44677239C>G (rs110383592), SCD g.21266629G>T (novel), and FASN g.50783803G>A (novel) are presented in Table 2. No significant associations were observed between the FASN g.50783803G>A with IMF, FMP, or any fatty acid examined. However, FABP4 g.44677239C>G was significantly associated with linoleic acid (p = 0.03). Linoleic acid was lower for the CC than the GG genotypes at 45.8 ± 10.88 mg/100 g and 54.5 ± 7.3 mg/100 g, respectively (p = 0.02), but CG was not significantly different from the homozygotes (Figure 4). Significant associations between the SCD g.21266629G>T SNP with DPA, DHA, EPA+DHA, and EPA+DPA+DHA were observed (p ≤ 0.02). Multiple comparisons in Figure 5 indicate that EPA, DPA, and DHA were significantly higher for the TT compared to the GG genotypes (p ≤ 0.03). The DHA level was lower (p = 0.02) while EPA and DPA tended to be lower for the TT compared to the GT variants (p ≤ 0.08). No significant difference was observed for EPA, DPA, and DHA in GT compared to GG variants (p ≥ 0.47). The IMF and FMP levels were not associated with either FABP4 g.44677239C>G, SCD g.21266629G>T, or FASN g.50783803G>A (p ≥ 0.38). Table 2. Least Square Means ± SD of loin eye muscle IMF (%), FMP ( • C) and fatty acid concentrations (mg/100 g fresh muscle) by genotype at the FABP4 g.44677239C>G, SCD g.21266629G>T and FASN g.50783803G>A SNP loci.      ). Figure 4. Multiple comparisons of loin eye muscle linoleic acid content between genotype variants at the FABP4 g.44677239C>G SNP locus CC ( ), CG ( ), and GG ( ).

Discussion
Meat fatty acid composition influences meat shelf life, eating quality and consumers' health [55][56][57]. Although many studies have reported that diet modulation influences meat fatty acid composition, it is more difficult in ruminants compared to monogastric livestock due to microbial lipolysis [16,17] and biohydrogenation of unsaturated to saturated fatty acids in the rumen [18]. As a result, meat fatty acids are more saturated in ruminant than in monogastric animals [58,59]. On the other hand, studies have reported that fatty acid composition is heritable [21,27,60,61]. A recent study by Sakuma et al. [60] reported medium to high heritability estimates of 0.48 to 0.85 for six out of the eight fatty acids analysed. Therefore, there is an increased research interest in breeding, selecting and producing farm animals with desirable fatty acid composition [62].
Selection and breeding provide a long-term alternative to improving marbling level [63], and meat fatty acid composition [64,65]. Several SNP in genes encoding key enzymes and proteins involved in fatty acid metabolism have been reported as potential genetic markers for the improvement of IMF and fatty acid composition in different cattle breeds [33,35,41]. This study examined SNP in the FABP4, SCD, and FASN genes of northern Australian tropical crossbred beef cattle and identified SNP with significant influences on fatty acid composition of the loin eye muscle.

Fatty Acid Binding Protein 4 Gene Polymorphisms
The FABP4 gene is an important protein for long-chain fatty acid transport in mammals, and its polymorphism is associated with growth, fat deposition, and carcass traits in cattle [66][67][68]. Substitution of the G to C allele of the g.44677587 (rs723716479) locus was positively correlated with CLA, previously inversely linked with the risk of colorectal and breast cancer in some population-based studies [69]. The observed trend where the homozygous GG variant had the highest linoleic acid levels (almost 10 mg/100 g higher than homozygous CC in the g.44677239C>G loci) may indicate higher inflammatory eicosanoids synthesis. Linoleic acid is a building block in the synthesis of arachidonic acid, the precursor for prostaglandins and other inflammatory eicosanoids [70]. In contrast to findings of this study, variation in the g.44677959T>C (c.220) influenced palmitoleic acid in Japanese Black cattle [71]. This discrepancy may be due to epistatic interaction of the g.44677959T>C locus with polymorphisms at another locus in line with the observations of Xu et al., (2021) [72] on the effect of polymorphisms on FABP4 protein structure. They reported that the wild type protein with isoleucine in amino acid 74 had 58.33% sheet and 29.55% loop interactions that changed to 59.09% sheet and 28.79% loop when isoleucine was substituted with valine. This discrepancy may also be due to breed differences since the Japanese Black cattle are reported to be genetically predisposed to producing carcass lipids with higher concentration of MUFA, including palmitoleic acid, compared to other cattle breeds such as Japanese Brown, Holstein or Charolais steers, likely due to the activity of the delta 9 desaturase enzyme on palmitic acid [73,74].

Stearoyl-CoA Desaturase Gene Polymorphisms
For most diets, approximately 70% to 95% and 85% to 100% ω6 PUFA and ω3 PUFA, respectively, are hydrogenated in the rumen [75]. As a result, fatty acids are absorbed almost entirely as SFA and biohydrogenation intermediates comprising conjugated di-or trienoic fatty acids and trans-11 fatty acids, notably trans-vaccenic acid, due to chemical reduction of unsaturated fatty acids in the rumen by microorganisms in ruminants [75,76]. Therefore, the composition of fatty acids stored in the fat depots mirror the action of SCD on fatty acids substrates [77]. The enzyme SCD catalyses the desaturation of SFA and MUFA by inserting a cis-double bond in the delta (∆) 9 position of SFA substrates, with a higher preference for palmitic acid and stearic acid substrates transformed into palmitoleic acid and oleic acid, respectively [62,77,78]. Nucleotide substitution of C with T identified in the fifth exon of bovine SCD gene at the 878 CDS causes the replacement of the amino acid alanine with valine [37]. The replacement caused significantly higher MUFA and lower FMP in M. trapezius of CC compared to TT genotype cattle [37]. Similarly, Flekvieh bulls with the CC genotype had lower SFA and higher MUFA compared to the TT, but CC and the CT genotypes were similar [39]. The TT genotype of Chinese Simmental cattle were reported to have lower IMF compared to the CC genotype, but no difference was found between the heterozygous (CT) and either of the homozygous genotypes [79]. Additionally, the SNP had a significant association with stearic acid, oleic acid, SFA, and MUFA in Japanese black cattle with higher MUFA and lower SFA reported in animals with the CC variant [78]. In contrast, the SNP (g.21272422C>T) did not have significant effect on palmitic acid, stearic acid, palmitoleic acid, or oleic acid in the present study. However, findings of this study concur with a previous study that reported no effect of the SNP with palmitic acid, stearic acid, palmitoleic acid, or oleic acid in Canadian Angus and Charolaisbased commercial crossbred beef steers [40]. Moreover, Dujková et al., (2015) [80] found that the SNP did not influence fatty acid composition in Aberdeen Angus and Blonde d'Aquitaine cattle. Unsaturated fatty acids are synthesized through the activity of ∆5, ∆6 or ∆9 desaturases [81], hence the difference between studies may be due to the activity of other desaturases or other genes [76,82]. The SCD genotype was reported to explain only 4% of the MUFA composition in Japanese Black cattle [37], and 5% in MUFA and 4% oleic acid variation, respectively, in Wagyu × Limousin cattle [38]. The significant correlations between EPA, DPA, and DHA with at least 16 SCD SNP observed in this study corroborate the findings of a previous study in sheep that recorded significant correlations between two SCD SNP and ω3 long-chain PUFA [32]. The three ω3 long-chain PUFA are synthesized from alpha-linolenic acid through the activity of ∆6 desaturase and ∆5 desaturase among other enzymes, but not ∆9 desaturase since ALA already has a double bond between C9 and C10 [83,84]. Therefore, the correlation may be due to linkage disequilibrium between the SCD SNP and other loci responsible for the synthesis of ω3 long-chain PUFA. Nonetheless, the significant correlations of SCD SNP with the EPA, DPA and DHA with no influence on the SFA and MUFA observed in this study suggests that the SNP can be used as markers to select cattle for improved health beneficial ω3 long-chain PUFA with no negative influence on meat-eating quality denoted by the lack of correlation with oleic acid; the most abundant fatty acid in beef that is reported to improve fat softness and meat palatability [85].
Seafood sources including fish, crustaceans, and molluscs are recognized as the best dietary sources of long-chain ω3 oils [86]. However, sustainability of seafood as a source of ω3 LC-PUFA is threatened by the global decline in wild-harvest fish stocks [87], high cost of seafood [88], and low availability of seafood in many geographical locations [89]. On the other hand, beef contributes significantly to meat intake as it is the third most consumed meat in the world at 6.3 kg per capita [90]. Therefore, the significant correlations of SCD SNP with the EPA, DPA, and DHA suggests that marker assisted selection can be used to provide a sustainable source of dietary ω3 LC-PUFA in communities where beef constitutes a significant proportion of the diet.

Fatty Acid Synthase Gene Polymorphisms
The FASN gene is located in the BTA19 region where quantitative trait loci affecting milk fat content, meat fatty acid composition and related traits had been previously identified [36,91]. The enzyme FASN catalyses the de novo synthesis of palmitic acid, a substrate for palmitoleic acid synthesis through desaturation, and stearic acid through elongation [92][93][94]. Genome-wide association studies with varying breeds of cattle have reported significant effect of FASN SNP on intramuscular composition of SFA, MUFA, and linoleic acid [91,[95][96][97][98]. Previous studies had reported that FASN polymorphism significantly influenced the intramuscular composition of oleic acid, SFA and MUFA in Fleckvieh bulls [41], and palmitic acid, palmitoleic acid, oleic acid, SFA, and MUFA of the intramuscular adipose tissue in Japanese Black cattle [36,82,99]. Zhang et al., (2008) [20] reported an additive effect of the g.17924A>G SNP on fatty acid composition, where the G allele was associated with higher MUFA and lower SFA compared to the A allele in purebred Angus bulls. The SNP also influenced palmitoleic acid and oleic acid composition in commercial crossbred beef steers [40], and palmitic acid, palmitoleic acid, oleic acid, total MUFA, SFA, and marbling score in Korean cattle [100][101][102]. In addition, FASN polymorphisms influenced SFA, MUFA, and PUFA in Chinese Holstein cattle [98]. Oh et al., (2012) reported associations of five FASN exonic SNP with intramuscular fatty acid composition in Korean cattle. These findings align with this current study where FASN g.50784242C>T was positively correlated with IMF, palmitic acid, oleic acid, SFA, and MUFA, while g.50783803G>A was correlated with palmitic acid, stearic acid, oleic acid, SFA and MUFA. Majority of the previous studies suggested that polymorphisms influenced the tissue fatty acid composition through amino acids substitutions on the b-ketoacyl reductase domain and the thioesterase domain by changing the spatial structure of the substrate-binding site [36,100,102]. However, the g.50784242C>T was a synonymous mutation, while g.50783803G>A was in the intron, thus they did not influence the production of missense codons, but may have exerted their effect by changing the splicing regulatory sequences [33]. The effect of g.50784242C>T on palmitic acid, oleic acid, SFA and MUFA may be due to the differences in IMF content. A review by De Smet et al. [103] reported a linear increase in SFA and MUFA expressed in mg/100g muscle (r = 0.98) with IMF content.
Tissue CLA is primarily derived from endogenous synthesis from trans-11 C18:1 (vaccenic acid) by the SCD activity [104], and to a lesser extent, as an intermediate of microbial fatty acid biohydrogenation in the rumen [105,106]. In this study, SNP of the g.50787886A>G, g.50788691T>C, and g.50788956C>T loci were found to be correlated with high CLA levels. Although these polymorphisms were either synonymous or located in the intron, they may have influenced the function of FASN in palmitic acid synthesis, the substrate for trans-11 C18:1 and subsequently CLA synthesis. These findings suggest that these loci may be used to select cattle with high CLA composition, a fatty acid associated with lower risk for atherosclerosis, diabetes and cancer [69,106]. Put together, these findings indicate that polymorphisms on the FASN gene can be used to select individuals for improved IMF and fatty acid composition of northern Australian tropical crossbred beef cattle.

Conclusions
This study aimed to investigate the targeted identification of SNP in the FABP4, SCD, and FASN genes and their associations with fatty acid composition in the loin eye muscle of northern Australian tropical crossbred beef cattle. Single nucleotide polymorphisms on the FABP4 gene significantly influenced linoleic acid, SCD was associated with long-chain n3 PUFA and FASN impacted IMF, SFA, MUFA, CLA, and EPA compositions. These findings not only provide insights into the genetic role of SNP in fat deposition and lipid metabolism in tropical crossbred cattle of northern Australia, but also their potential use in markerassisted selection and breeding for improved meat-eating quality. The tested hypothesis of significant associations between SNP loci encoding for the fatty acid binding protein 4, stearoyl-CoA desaturase and fatty acid synthase genes and human health beneficial ω3 long-chain polyunsaturated fatty acids within the loin eye muscle of northern Australian crossbred beef cattle is therefore acceptable. This is the first study that demonstrates the presence of single nucleotide polymorphisms in lipogenic genes in northern Australian crossbred beef cattle which constitute over 50% of Australian beef production and exports.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/su14148409/s1, Table S1: Primer sequences for target gene amplification; Figure S1: Gel image of the amplification products of the three target genes visualised in 0.8% agarose gel.; Table S2: Genetic variants detected in the FABP4, SCD and FASN genes of tropically adapted crossbred steers; Figure S2: Correlation coefficients for all pairs of SNP loci of the FABP4 gene. The rectangles represent distance-based clustering of SNP loci; Figure S3: Correlation coefficients for all pairs of SNP loci of the SCD gene. The rectangles represent distance-based clustering of SNP loci; Figure S4: Correlation coefficients for all pairs of SNP loci of the FASN gene. The rectangles represent distance-based clustering of SNP loci.

Institutional Review Board Statement:
The study was conducted in accordance with the CSIRO Animal Ethics Committee approved guidelines (approval number 2019-38, issued on the 20 February 2020) and the Australian code of practice for the care and use of animals for scientific purposes.

Data Availability Statement:
The data presented in this study are available from the corresponding author on request.