Animals and Sampling
Indigenous Holstein cows and purebred Mediterranean Buffaloes (Bubalus bubalis) imported from Italy in 2015 (by Guangxi HuaXu Buffalo Biotechnology co. LTD) were used for this study. All animals were kept under uniform feeding and management conditions. Milk samples were collected from 30 Buffalos or Holstein cows in their 1st or 2nd parity and 100~150 days in lactation. We collected 30 samples from each species in this study and each milk sample were collected in triplicate until the composition (lactose, fat, protein, total solid and solid not fat) was determined by a milk analyzer (MilkoScan FT120, Denmark) immediately after collection.
Milk Fat Extraction and Gas Chromatography Analysis
Milk fat extraction and gas chromatography analysis were performed following the method reported by Mele [41]. In brief, two grams of milk sample was mixed with 0.4 mL of ammonia 25%, 1 mL of ethyl alcohol 95%, and 5 mL of hexane, vortexed and centrifuged at 1600 × g at 4°C. The upper layer was collected, and a second extraction with 1 mL of ethyl alcohol 95% and 5 mL of hexane was performed. A third extraction was made by using 5 mL of hexane. The extracted fat was dried, weighed, and finally dissolved in hexane. FA composition was determined by gas chromatography using a Shimadzu GC-2014C (Kyoto, Japan) gas-chromatograph equipped with an FID and a capillary column (Agilent DB23, California, USA; 30m × 0.32 mm i.d.; film thickness 0.25μm). Injection ported 230 °C and detector 280 °C. The column was kept at 180 °C for 5min and heated up to 230 °C at 3 °C min-1. The carrier gas is kept in high-purity nitrogen and the injection volume is 1 μL. Individual FA methyl esters were identified by comparing them to a standard mixture of 37 Component FAME Mix (Supelco, Bellefonte, PA). The standards of PUFA-2, nonconjugated C18:2 isomer mixture, individuals cis-5,8,11,14,17 C20:5, cis-4,7,10,13,16,19 C22:6 (Supelco), cis-6,9,12 C18:3 and cis-9,12,15 C18:3 (Matreya Inc., Pleasant Gap, PA) were used to identify polyunsaturated FA. The identification of C18:1 isomer was based on commercial standard mixtures (Supelco) and published isomeric profiles (Wolff and Bayard, 1995). A nonadecanoic acid was used as an internal standard to increase the veracity of the peak normalization. For all studied FA, the coefficient of variation [(SD/mean) × 100] was < 3.5%, suggesting good repeatability of GC data. All milk FA compositions were expressed as g per 100 g of fat.
Total RNA Isolation
The total RNA in milk fat globules (MFG) was used for gene expression analysis as reported previously [42]. Milk samples were collected in the morning and mRNA was extracted immediately. The whole extraction process was performed at 4 ℃ (from the milk collection to the RNA extraction) and completed within two hours to improve the quality of mRNA. Milk samples were centrifuged at 2000 × g for 10 min at 4°C to isolate milk fat. The supernatant fat layer was separated and 500 μL fat was mixed with 1.5 mL of TRIzol LS solution (Invitrogen Life Technologies Inc., Carlsbad, CA). All the procedures were carried out at 4°C. RNA was dissolved in 30 μL of RNase-free water and stored at −80 °C. We further performed the agarose gel electrophoresis analysis and only the mRNA samples with a low 5s band were selected for the following experiment.
Single-Strand cDNA Synthesis
RNA purity was evaluated through absorbance readings (ratio of A260/A230 and A260/A280) by using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Before first-strand cDNA synthesis, contaminated genomic DNA was removed by DNase treatment. First-strand cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific, Waltham, MA). The single-strand cDNA obtained was stored at −20°C.
Primer Designing and qRT-PCR
Primers of the selected genes were designed by using the oligo 7 software (Table 5). The expressions of selected genes were quantified by using SYBR Green dye (4913914001, ROCHE) and fluorescence data were acquired using RT-PCR instrument (ABI 7500). A 20 μL mixture was performed in each run as follows: 1 μL cDNA, 8 μL H2O, 10 μL Faststart Universal SYBR Green Master (ROX), and 0.5 uM aliquots of both forward and reverse primers. The thermal cycling profile started with a 3-min dwell temperature of 94°C, followed by 40 cycles of 30 sec at 94°C, 30 sec at the primer specific annealing temperature (60°C), 30 sec at 72°C, and a final step during which fluorescence was acquired. After 40 cycles, a melting curve was generated by temperature increments of 0.1°C starting three 3 times, and relative gene expression was calculated using the 2-△△Ct method using GAPDH as a reference gene as reported previously [43].
Construction of Lentiviral Vector and Synthesis of siRNA
The SCD1 gene was cloned from buffalo mammary epithelial cells according to the SCD1 sequence available in GenBank (No. AY241933) and inserted into lentiviral vector. The siRNA targeting SCD1 gene were designed and synthesized by Gene Pharma (Shanghai, China) with a control sequence (Table 6). Lentiviral vectors containing the SCD1 gene and the siRNA with negative control (NC) were constructed by Gene Pharma (Shanghai, China). The lentiviral vectors were packaged and propagated in 293T cell line with the packaging plasmid (ΔNRF) and envelope plasmid encoding the vesicular stomatitis Virus-G glycoprotein (VSVG).
Cell Culture
The BMECs were cultured and purified as reported previously [44, 45]. Briefly, during lactation fresh tissue blocks from buffalo were obtained and washed 3 times and the acinus portion was extracted from mammary gland tissue and transferred into high-resistance PBS (containing 400 IU mL-1 of penicillin and 400 IU mL-1 of streptomycin). Then tissue pieces were placed in culture dishes on a clean bench, cut into 1 to 2 mm pieces and tiled on the bottom of the culture dish and cultured in the incubator for 4 h. Then they were inverted and cultured in the upright position overnight. The epithelial cells started to grow after about 12 days and epithelial cells were isolated by using trypsin digestion combined with a cell adherence speed method. The purification procedure was performed 3 times and the BMECs at 3 to 4 generation in the subculture were used for the following studies;
Infection and Transfection
Transfection of the lentiviral vectors was carried out by using the LipofectamineTM 2000 (Invitrogen, USA). 100 μM lentiviral vectors containing siRNA or SCD1 gene were used in each transfection and the transfected confluent cells were harvested for qRT-PCR analysis, 24h post transfection.
Statistical Analysis
All experiments were repeated three times. Results were expressed as mean ± standard error (SEM). Statistical analysis was performed by using Student’s t-test and analysis of variance (ANOVA) with DUNCAN’s Multiple Range Test (DMR) in SPSS 17.0 software (IBM Corp., Armonk, NY). Differences of P< 0.05 were considered to be significant.