Associations between genetic polymorphisms and dairy traits are of great interest in buffalo for explaining the mechanisms underlying their genetic variation and for providing useful tools for improving the accuracy and efficiency of traditional selection methods. Among dairy traits, of particular interest is the fatty acid (FA) composition of milk fat. The evaluation of the FA composition in milk from buffalo cows is of extreme importance to human health since high intakes of specific fatty acids such as C 18:2 cis 9- trans–11 and C18:1 trans-11 (vacenic) can help in the diseases prevention and help in maintaining and regulating of metabolism in humans. Variations in levels of unsaturated FA of milk fat of buffaloes are similar to those found in dairy cows from the partum to eighth week of lactation. However, higher concentrations of CLA in milk of buffalo cows bring greater benefits to human health due to higher amounts of fat in milk of these animals (Verdurico et al. Reference Verdurico, Gandra, Freitas, Barletta, Venturelli, Mingoti, Vendramini and Rennó2012). Candidate genes for fatty acid binding, transport, and metabolism in relation to production traits include FASN (fatty acid synthase), DGAT1 (diacylglycerol O-acyltransferase 1), SCD (stearoyl CoA desaturase), ACACA (acetyl-CoA carboxylase alpha), BLG (beta-lactoglobulin). Significant associations between polymorphisms in some of these genes and milk production or milk fat related traits have been reported in cattle (Zhang et al. Reference Zhang, Knight, Reecy, Wheeler, Shackelford, Cundiff and Beitz2009; Komisarek et al. Reference Komisarek, Michalak and Walendowska2011; Mao et al. Reference Mao, Chen, Chang, Chen, Ji, Wu, Shi, Wu, Zhang, Yang, Konig and Yang2012; Matsumoto et al. Reference Matsumoto, Inada, Kobayashi, Abe, Hasebe, Sasazaki, Oyama and Mannen2012; Molee et al. Reference Molee, Poompramun and Mernkrathoke2015), goat (Badaoui et al. Reference Badaoui, Serradilla, Tomàs, Urrutia, Ares, Carrizosa, Sànchez, Jordana and Amills2007; Dixit et al. Reference Dixit, Sivalingam, Tyagi, Saroha, Sharma and Nagda2015), sheep (Scatà et al. Reference Scatà, Napolitano, Casu, Carta, De Matteis, Signorelli, Annicchiarico, Catillo and Moioli2009; Moioli et al. Reference Moioli, Scatà, De Matteis, Annicchiarico, Catillo and Napolitano2013) and buffalo (Vohra et al. Reference Vohra, Bhattacharya, Dayal, Kumar and Sharma2006; Pauciullo et al. Reference Pauciullo, Cosenza, Steri, Coletta, La Battaglia, Di Berardino, Macciotta and Ramunno2012; Cardoso et al. Reference Cardoso, de Souza, Aspilcueta-Borquis, Araujo Neto, de Camargo, Hurtado-Lugo, Scalez, de Freitas, Albuquerque and Tonhati2015; de Freitas et al. Reference de Freitas, de Camargo, Stafuzza, Aspilcueta-Borquis, Venturini, Dias, Cardoso and Tonhati2016).
A possible candidate for milk fat composition could be also the oxytocin receptor encoding gene (OXTR). It acts as a receptor for oxytocin and the oxytocin-oxytocin receptor complex plays an important role in the uterus during calving. The OXTR belongs to the G protein-coupled receptor (GPCR) family and contains seven transmembrane (TM) domains, an extra-cellular N-terminus and an intracellular C-terminus, which are believed to be involved in receptor regulation (Bathgate et al. Reference Bathgate, Rust and Balvers1995). The conserved TM domains are involved in the activation and signal transduction of the G protein (Gimpl & Fahrenholz, Reference Gimpl and Fahrenholz2001), whereas the masking of G protein binding sites has been associated with changes in the relative orientation of TM domains 3 and 6 (Barberis et al. Reference Barberis, Mouillac and Durroux1998). The OXTR is differentially expressed in various tissues; it is up-regulated in the cows' uterus during oestrus and at the end of pregnancy, and it also increases in the mammary gland and during lactation (Gimpl & Fahrenholz, Reference Gimpl and Fahrenholz2001). OXTR gene maps on BTA22 where nine QTL for FA composition of meat have been found in a Charolais X Holstein cross population (Gutiérrez-Gil et al. Reference Gutiérrez-Gil, Wiener, Richardson, Wood and Williams2010). Five out of 9 have been found to influence the content of palmitic (C16:0), palmitoleic (C16:1), oleic (c9 C18:1) and conjugated linoleic (CLA) fatty acids and the sum of total fatty acid (SUMWFA) index. The other 4 QTL identified on this chromosome affected the stearic acid (C18:0) content, the SFA index and the palmitic : stearic ratio.
To date, the OXTR encoding sequences have been reported for mice (GenBank Acc. No. D86599), human (Acc. No. AY389507), swine (Acc. No. NM_214027), sheep (Acc. No. NM_001009752), cattle (Acc. No. AF101724) and water buffalo (Acc. No. FJ917398; XM_006070767). Cattle and buffalo OXTR cDNA contains 1176 nucleotides, encoding for 391 amino acids with a molecular weight of 43 kDa and an isoelectric point of 9·253 (Bathgate et al. Reference Bathgate, Rust and Balvers1995; Fleming et al. Reference Fleming, Spencer, Safe and Bazer2006; Arunmozhi et al. Reference Arunmozhi, Singh, Sarath, Agarwal, Doiphode and Shankar2014).
In human, the gene spans 17 kb and contains 3 introns and 4 exons. The genomic DNA for OXTR can be mainly divided into three segments. The segment 1 contains the 5′ non-coding region. The segment 2 starts upstream the ATG initiation codon and spans downstream encoding for most part of the receptor protein including the sixth transmembrane domain of the receptor. The segment 3, instead, contains the sequence encoding for the seventh transmembrane domain of the receptor, the C-terminal region and the entire 3′ noncoding region, including multiple polyadenylation signals. There are two introns between the three segments. The largest intron (about 12 kb in human and rat), interrupts the coding region between transmembrane domain 6 and 7. Compared to human, the structural organisation of the OXTR gene seems to be conserved also in the bovine species apart from only one exon upstream of that there is the ATG initiation codon (Feng, Reference Feng2000; Gimpl & Fahrenholz, Reference Gimpl and Fahrenholz2001).
Excluding these studies, to our knowledge, no information on genetic variability has been reported so far at the OXTR locus and, consequently, no association study with milk production traits has been carried out. This study aims to explore the genetic variability at Italian Mediterranean river buffalo OXTR locus and test possible associations between detected polymorphisms and milk fatty acids composition finalised to a genetic improvement of productive efficiency of the species.
Materials and methods
Sample collection
Individual blood and milk samples were collected from 306 Italian river buffaloes belonging to 14 farms, located in Salerno and Caserta province (Southern Italy), with a similar feeding management, based on 60% forage (silage and hay) and 40% concentrates. Sampling was carried out in collaboration with the Italian National Association of Buffalo Breeders (ANASB). Milk samples were collected over the whole lactation.
Fatty acid composition of milk samples was determined by gas chromatography (GC Turbo 3400 CX, Varian Inc., Palo Alto, CA, USA). Milk fat extraction was performed according to the Röse-Gottlieb method (AOAC, 1990) modified as described by Nudda et al. (Reference Nudda, Battacone, Bee, Boe, Castanares, Lovicu and Pulina2015). The fatty acid methyl esters (FAME) were prepared with a base-catalysed trans – esterification according to the International Dairy Federation standard procedure (1999). The FAME was separated in a capillary column (CP-select CB for Fame; 100 m × 0·32 mm i.d., 0·25-μm film thickness, Varian Inc., Palo Alto, CA). The injector and FID temperatures were 255 °C. The programmed temperature was 75 °C for 1 min. It was then raised to 165 °C at a rate of 8 °C/min, maintained at 165 °C for 35 min, increased to 210 °C at a rate of 5·5 °C/min, and finally increased to 240 °C at a rate of 15 °C/min. The split ratio was 1 : 100 with He at a pressure of 37 psi as the carrier gas. The fatty acids were identified by comparing retention times of peaks with those of methyl ester standards as reported by Nudda et al. (Reference Nudda, McGuire, Battacone, Manca, Boe and Pulina2011). In addition published isomeric profile were used to identify trans C18:1 and CLA isomers of interest (Griinari et al. Reference Griinari, Dwyer, McGuire, Bauman, Palmquist and Nurmela1998; Kramer et al. Reference Kramer, Cruz-Hernandez, Deng, Zhou, Jahreis and Dugan2004; respectively).
DNA isolation and PCR amplification conditions
DNA was isolated from leucocyte, using the procedure described by Goossens & Kan (Reference Goossens and Kan1981). DNA concentration and OD260/280 ratio of the samples were measured by the Nanodrop ND-2000C Spectrophotometer (Thermo Scientific).
The DNA regions of the OXTR gene spanning exons 1, 2 and 3 of ten individual samples, randomly chosen, were amplified by iCycler (BioRad) using primers designed on the complete bovine genome sequence (EMBL acc. no. NC_007320·5) (Table 1).
Table 1. Oligonucleotide primers sequence and positions
† Primers were designed by means of DNAsis-Pro (Hitachi Software Engineering Co., Japan).
PCR amplifications were performed according to the following chemical conditions: 100 ng of genomic DNA, 50 mm KCl, 10 mm Tris-HCl (pH 9·0), 0·1% Triton X-100, 3 mm MgCl2, 200 nmol of each primer, dNTPs each at 400 µm, 2·5 U Taq DNA Polymerase (Promega, Madison, WI), and 0·04% BSA in a final volume of 25 µl reaction mix. The thermal condition for the amplification of the exon 1 and 3 consisted of an initial denaturation at 97 °C for 2 min, annealing at 62 °C for 45 s and extension at 72 °C for 2 min, followed by 30 cycles at 94 °C for 45 s, 62 °C for 45 s and 72 °C for 2 min (except for the final extension of 10 min). For the amplification of the DNA region spanning the exon 2, the amplification conditions were the same except for the annealing temperature (67 °C).
All PCR products were analysed directly by electrophoresis in 1·5% TBE agarose gel (Bio-Rad, CA, USA) in 0·5X TBE buffer and stained with SYBR®green nucleic acid stain (Lonza Rockland, Inc, USA). PCR products were sequenced on both strands at CEINGE – Biotecnologie Avanzate (Naples, Italy).
Bioinformatic and statistical analysis
Allelic frequencies and Hardy-Weinberg equilibrium (χ 2 test) were calculated. Homology searches, comparison among nucleotide and amino acid sequences and multiple alignments were accomplished using DNAsis-Pro (Hitachi Software Engineering Co., Japan).
Phylogenetic analysis was performed using the evolutionary distances computed by the maximum composite likelihood method of MEGA 4 software (Tamura et al. Reference Tamura, Dudley, Nei and Kumar2007). The evolutionary distances were computed using the Poisson correction model and they are in the units of the number of amino acid substitutions per site. A bootstrap analysis based on 1000 iterations was used to evaluate the robustness of the tree. The amino acid sequences for the OXTR were obtained from the EMBL sequence database.
Associations between OXTR genotype, fatty acid composition traits, fatty acid classes and fat percentage were tested using the following mixed linear model:
$$\eqalign{y_{ijklmn} & = \mu + {\rm AGE}_i + {\rm DIM}_j + {\rm Month}_k + {\rm SNP}_l \cr & \quad + {\rm HERD}_m + e_{ijklmn}}$$
where: y ijklmn = Fatty acid considered in test; μ = Overall mean; AGE i = fixed effect of the age of the ith animal at calving expressed in years (6 classes: 1 = <4 years, 2 = 4 years, 3 = 5 years, 4 = 6 years, 7 = 5 years, 6 = >7 years); DIM j = fixed effect of the jth stage of lactation (10 levels of 30 d each); Month k = fixed effect of the kth month of calving (12 levels); SNP l = fixed effect of the lth SNP genotype; HERD m = Random effect of the mth herd; e ijklmn = Random residual.
Pairwise comparisons among different levels of fixed effects included in model were performed using a Bonferroni adjusted test.
The average gene substitution effect (α) was calculated as simple phenotypic regression upon the number of C alleles at the OXTR locus (0, 1, 2) weighted for the frequency of the phenotypes.
The variance component associated to the OXTR genotype (σ 2 OXTR ) was estimated by running a mixed linear model with the same structure of (1) but with the SNP effect treated as random. Then the contribution of the OXTR genotype (r 2 OXTR ) to the total phenotypic variance of the trait considered was calculated as:
$$r_{OXTR}^2 = \displaystyle{{\sigma _{OXTR}^2} \over {\sigma _{OXTR}^2 + \sigma _{HERD}^2 + \sigma _e^2}} $$
where (σ 2 HERD ) and (σ 2 e ) are herd and residual variance component estimated with model (1), respectively.
Results and discussion
Gene characterisation and polymorphism detection
The first exon (partial), the entire second exon and the third exon until the stop codon (TGA) of the OXTR gene of ten individual samples randomly chosen were amplified and sequenced. The sequences are available in EMBL with the following accession numbers: LT592263, LT592262, LT592264.
The Mediterranean river buffalo OXTR gene contains an uninterrupted ORF of 1176 nucleotides, corresponding to a predicted polypeptide length of 391 amino acids similar to those reported in cattle (Bathgate et al. Reference Bathgate, Rust and Balvers1995), sheep (Fleming et al. Reference Fleming, Spencer, Safe and Bazer2006) and Indian water buffalo (Arunmozhi et al. Reference Arunmozhi, Singh, Sarath, Agarwal, Doiphode and Shankar2014). The predicted molecular weight of the deduced amino acid sequence was 43 169 kDa with an isoelectric point of 9·41. The ORF is characterised by high CG content with an average value of 64·8%.
Exon 1
The DNA sequence spanning the last 631 nucleotides of the exon 1 showed a homology of about 97% with the corresponding bovine sequence (EMBL AF100633) and 99% with the non-annotated genomic bubaline sequence deposited in GeneBank with the access number KI418690·1 (from nt 1902413 to 1903043), which, therefore, we have identified as putative OXTR locus. The comparison of obtained sequences showed a total of 5 polymorphic sites: EMBL no. LT592263g.230G > T, g.522G > T, g.567C > G, g.577G > C, g.579G > T. Since this exon corresponds to the region 5′ UT, such mutations do not affect any triplet coding for the protein. The first SNP falls within a portion of exon 1 that appears to be differentially spliced in the bovine (exon 1a, Bathgate et al. Reference Bathgate, Rust and Balvers1995; Telgmann et al. Reference Telgmann, Bathgate, Jaeger, Tillmann and Ivell2003, EMBL AJ490937·1). The subsequent comparison with the buffalo genomic sequence (EMBL KI418690·1) allowed to identify a further nucleotide difference: g.476C > T (Fig. 1). The sequence analysis of the informative subjects showed only two haplotypes: (a) GGCGG and (b) TTCCT.
Fig. 1. Alignment of the assembled Italian Mediterranean buffalo OXTR exonic sequences (present study) with sequences available in EMBL related to the Indian buffalo OXTR cDNA (FJ917398), Italian Mediterranean buffalo OXTR cDNA (KI418690·1), non-annotated genomic bubaline sequence (NW_005785401·1) and genomic DNA (XM_006070767). Alignment was performed using DNAsis pro Software v2.0 (Hitachi). The sites of exon-exon junctions are underlined. In bold type the portion of exon 1 that appears to be differentially spliced in the bovine (exon 1a). Dashes indicate identical nucleotides. The stop codon is symbolised by *. Amino acid numbering is relative to the first aminoacid (Met) of the deduced encoded protein.
Exon 2
The DNA region including the second exon of the buffalo OXTR gene was sequenced for a total of 1084 bp (EMBL no. LT592262). The nt 157–159 correspond to the triplet ATG coding for the start codon of the translation (Methionine). The entire sequence showed a homology of 99% with the buffalo genome sequence (KI418690·1), as well as with the corresponding bovine sequence (EMBL AF100633).
A similar degree of homology was also observed with the partial sequence of the Indian buffalo OXTR cDNA (EMBL FJ917398) and with the partial Italian Mediterranean buffalo cDNA sequence (EMBL XM_006070767·1). In particular, the first comparison showed 6 SNP, three of which are responsible for amino acids changes: GTGVal > GCGArg, ATTIle > GTTVal, TGGTrp > CGGArg in position 60, 209 and 302, respectively. On contrary, only one silent SNP was detected by the latter comparison with the Italian Mediterranean buffalo cDNA (Fig. 1). No polymorphism was detected from the sequences alignment of the 10 subjects examined in this study.
Exon 3
The complete coding region (from the first nucleotide to the termination codon, TGA) of the third exon of OXTR gene was sequenced for a total of 248 bp (EMBL no. LT592264). The sequence alignment among the 10 investigated animals showed two SNP. The first (g.129C > T) is responsible for the amino acid replacement CGCArg > TGCCys in position 353, whereas the second (g.139C > AGly356) is a conservative mutation.
The following comparison with the sequences available in database showed that, excluding the species phylogenetical distant like cetaceans and Ailuropoda melanoleuca (Giant panda), the presence of a cysteine in position 353 does not characterise any other of the 13 mammalian species compared. However, it is interesting to point out that in primates, pinnipeds, felidae, canidae, chiroptera and talpidae, the thymine represents always the first nucleotide of the triplet, although in all these species it is replaced by TAC which encodes for the amino acid Tyr.
In regard of the conservative polymorphism, it is remarkable to notice that, with the exception of the Indian buffalo that is characterised by the presence of adenine, the other mammals considered in the present study are characterised by the presence of cytosine.
Additional polymorphisms were detected through the comparison of the sequences available in EMBL (XM_006070767 and NW_005785401·1) related to the cDNA and the genomic DNA of the Italian Mediterranean buffalo, respectively. In particular, the SNP g.222G > A and g.241C > T are responsible for two amino acid changes, Gly384 > Arg and Thr390 > Met, respectively (Fig. 1).
Analysis of the amino acid sequences and phylogenetic analysis
According to Feng et al. (Reference Feng, Bhav and Fairclough2000), the putative amino acid sequence derived from these exonic sequences confirmed the occurrence of seven hydrophobic transmembrane regions, which are characteristic of a G-protein coupled receptor. In the three extracellular loops (between transmembrane domains II and III, IV and V, VI and VII) little change was found among the species (Fig. 2). This finding leads to state that, throughout mammalian evolution, the primary amino acid sequence of OXTR was rather conserved to guarantee the further protein structures for the specific ligand-receptor binding properties. In general, few differences were found only at the edges (N- and C-terminus) and at the third intracellular loop (between transmembrane domains V and VI) which likely have less functionality.
Fig. 2. Comparison of protein-coding regions of the water buffalo OXTR with cattle, yak, sheep, bison, orca, minke whale, giant panda, dog, cat, pig, human, rat, flying fox, rabbit and cape golden mole. Accession numbers of sequences compared are shown in the figure. Dashes (–) indicate the identical amino acid residues. Amino acids full matching the buffalo sequence of the present work are shown as shaded. The protein portion corresponding to the 7 transmembrane receptor (rhodopsin family) is underlined, whereas the transmembrane (TM) domains are indicated by asterisks (*).
The multiple sequences alignment of the amino acid primary structures between the buffalo OXTR and the other considered species allowed the construction of the phylogenetic tree. Closely related mammalians were gathered in different clades. In particular, the phylogenetic analysis showed 3 main clades: A, B and C. The first one includes four embranchments: Minke Wale/Orca embranchment, both members of artiodactyl infraorder Cetacea, American bison/yak, Italian water buffalo (XP006070829)/Italian water buffalo (XM006070767), Indian water buffalo/Italian water buffalo (present work), cattle and sheep embranchment, all being members of cetartiodactyla. The clade B includes members of carnivore and the feral pig, a member of cetartiodactyla, with a Feral Pig/Giant Panda embranchment, whereas the Clade C includes members of Rodentia, Soricomorpha, Lagomorpha and Chiroptera with a rabbit/flying fox embranchment (Fig. 3).
Fig. 3. Phylogenetic relationship of the peptide sequence of Italian water buffalo (Bubalus bubalis) OXTR with other 14 peptide/protein sequences reported in different mammals. The MEGA v4.0 program was used to reconstruct a phylogenetic tree. Bootstrap values are indicated.
These data indicate a greater phylogenetic affinity among artiodactyla compared to that found with others orders. In particular, the Italian buffalo investigated herein showed a higher affinity with the Indian buffalo than the same Italian buffalo already present in database.
Fatty acids composition profile
The average fat content (%), the FA composition (%) and FA classes (%) are reported in the Table 2. Saturated FA (SFA) represented 71·6% of total FA ranging from 57·9 to 85·9% and C16:0 and C18:1 c9 were the most represented (34·8 and 19·1%, respectively). Monounsaturated FA (MUFA) were 25·17% of total milk FA ranging from 12·6 to 37·4% and the C18:1 c9 represents the majority (76% of total MUFA). The concentration of polyunsaturated FA (PUFA) was 3·21% of the total FA ranging from 1·39 to 5·11%, with the C18:2 n6 predominating (49% of total PUFA), while the C18:3 n3 was present in a lower amount 0·32% of total FA. Trans FA (TFA) represented 1·70% of total FA with the C18:1 t11 the most abundant (59% of total TFA). CLA isomers amounted to 0·76% of total milk FA and the isomer c9, t11 CLA represented the majority (55% of total CLA). The FA profile obtained in this study is typical of animals farmed in intensive systems, with a reduced occurrence of unsaturated fatty acids, compared to graze-based systems.
Table 2. Average fat content (%), fatty acid composition (%) and fatty acid classes (%) of 306 dairy buffaloes milk samples measured in 14 herd located in Southern Italy
Results of mixed model analysis did not find statistically significant effects of the age of the buffalo at calving on the fatty acid profile.
Month of calving significantly affected total CLA and TFA (P < 0·05), evidencing a difference between January and February (data not reported for brevity). DIM affected significantly all the group of FA analysed (P < 0·01) denoting a marked lactation curve effects.
Genotyping and association of OXTR polymorphism with milk FA composition traits
It is well known that the cysteine can be easily oxidised to form a dimer containing a disulfide bridge between two cysteines. Such dimer is known as cystine and this feature is very important for the analysis of the primary structure, for effects on changes in secondary structure and for stabilisation of the tertiary and quaternary structure of the proteins. Therefore, given the peculiarity of the mutation g.129C > T detected at the exon 3 and the resulting amino acid change (CGCArg > TGCCys in position 353), we decided to genotype the investigated buffalo population only for this SNP.
The genotyping of 306 buffalo DNA samples was performed at the KBiosciences (Herts, UK, http://www.kbioscience.co.uk) laboratory. The major allele had a relative frequency of 0·743 and the χ 2 value showed that there was no evidence of departure from the Hardy-Weinberg equilibrium (P ≤ 0·05). Genotype distribution of buffalo cows for the SNP g.129C > T is reported in Table 3.
Table 3. Genotyping data, allele frequency, relative frequencies of buffalo cows of the SNP g.129C > T at the exon 3 of the OXTR gene in Mediterranean river buffalo population
Results of model (1) showed statistically significant effects of the SNP g.129C > T at the OXTR locus on the milk FA composition traits. In particular, the CC genotype was associated with higher contents of odd branched-chain fatty acids (OBCFA) (P < 0·0006), polyunsaturated FA (PUFA n 3 and n 6) (P < 0·0032 and P < 0·0006, respectively), and Stearic acid (C18) (P < 0·02). In general, the estimated allele substitution effects (α) for these FAs indicate a positive percentage improvement with the increase of the number of C alleles. On the other hand, TT genotype animals showed higher level of Palmitic acid (C16) (P < 0·02), confirmed also by a negative α value (−0·732). Table 4 showed the simple statistics of the FA composition traits along with the genotype effects and allele substitution effects, including only significant associations. The contribution of the OXTR locus to the phenotypic variance of the considered traits was about 0·05 for the FA classes but lower for the single FA (Table 4).
Table 4. Least squares Means of the SNP g.129C > T genotypes at the exon 3 of the OXTR gene for some FA, estimation of average substitution effects (α) of a thymine for a cytosine, and contribution of the polymorphism to the phenotypic variance (r 2)
It is well known that n-3 FAs have important roles in the modulation and prevention of coronary heart disease. In fact, PUFA n-3 consumption lowers plasma triglycerides, resting heart rate, and blood pressure and might also improve myocardial filling and efficiency, lowers inflammation, and improves vascular function (Mozaffarian & Wu, Reference Mozaffarian and Wu2011). Similarly, PUFA n-6 regulates a wide variety of biological functions, which range from blood pressure and blood clotting to the correct development and functioning of the brain and nervous system (Wall et al. Reference Wall, Ross, Fitzgerald and Stanton2010). In addition, they have important roles in immune regulation and inflammation (Calder, Reference Calder2009). Regarding OBCFA, a potential antitumoral activity on human breast cancer cells has been reported (Wongtangtintharn et al. Reference Wongtangtintharn, Oku, Iwasaki and Toda2004). About Palmitic acids, which is the most abundant fatty acid of milk and dairy products, has been reported that replacing palmitic acid with oleic acid lowered total and LDL cholesterol concentrations, and the LDL : HDL ratio in humans (Kien et al. Reference Kien, Bunn, Stevens, Bain, Ikayeva, Crain, Koves and Muoio2014), even if its detrimental properties has been recently reappraised (Odia et al. Reference Odia, Ofori and Maduka2015).
Stearic acid has not been shown to elevate blood cholesterol and is rapidly converted to oleic acid in vivo (Bonamone & Grundy, Reference Bonamone and Grundy1988). In view of these observations, and of the results of this study we can suggest that the OXTR-CC genotype animals might be useful in selection toward increasing of OBCFA, polyunsaturated FA (PUFA n-3 and n-6), stearic acid and decreasing palmitic acid content in milk of Mediterranean river buffalo. Nevertheless, further studies are needed to confirm this.
Conclusions
In conclusion, the identified SNP is the first report to help in understanding the genetic structures of oxytocin receptor gene (OXTR) on fatty acid compositions in Mediterranean river buffalo. So far, polymorphisms of OXTR have been hypothesised to be correlated only with social behaviour related phenomena in humans and other mammals. The SNP g.129C > T at the buffalo OXTR locus was found to have a strong association with composition of several fatty acids of milk, in particular with higher contents of OBCFA, polyunsaturated FA (PUFA n 3 and n 6), Stearic acid (C18) and lower level of palmitic acid. All these variations might help in producing ‘healthy’ milk. Furthermore, with PUFA and stearic acid influencing meat traits, such as taste and texture, this SNP might even be associated with beef quality. The exact molecular and physiological mechanisms underlying the association of SNP with fatty acid content reported in this study are -so far- unknown and further studies need to deeply investigate this aspect. Nevertheless, the use of this polymorphism as genetic markers can be a useful tool for selection of animals and improve milk quality in Mediterranean river buffalo. This study provides useful genetic information regarding the relation between OXTR and fatty acid composition.
This work was financially supported by the Italian Ministry for Agriculture and Forestry Policy – MiPAAF (INNOVAGEN project)
Conflict of interest
None of the authors have any conflict of interest to declare.
