Matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) has been applied to study sperm lipid profiles. Lipids are known to play a crucial role in sperm membrane physico-chemical behavior during cryopreservation. In this work, we show the results of characterization of sperm lipid profiles from 2 bulls with different capacities of in vitro embryo production by MALDI-MS direct analysis. The bull capacities judged by the rate of blastocyst formation after IVP with semen from seven different ejaculates per each animal were 19.1 and 35.3% for bulls 1 and 2, respectively (P < 0.05). For MALDI-MS analysis, frozen semen from each ejaculate was thawed in water at 25°C for 40 s. Sperm was washed 3 times by centrifugatin in 1 mL of PBS at 3000 × g for 10 min. Samples were stored at -20°C in 200 μL of methanol:PBS (vol/vol) solution until analysis. A Synapt HDMS mass spectrometer (Waters Corp., Milford, MA, USA) equipped with a MALDI was used. All spectra were collected for 45 s in the positive ion mode at the mass range of m/z 450 to 1200. The volume of 1 μL of the semen pellet was spotted in the target plate and allowed to dry. Afterward, 1 μL of 2,5-dihydroxybenzoic acid (DHB) was added as matrix. The 50 most intense monoisotopic ions were considered for principal component analysis (PCA). Values of m/z and relative ion intensities were processed using the software Pirouette v.3.11 (Infometrix, Woodinville, WA, USA). Direct MALDI-MS analysis of bulls 1 and 2 spermatozoa with no extraction provided informative spectra containing either [M + Na]+ or [M + H]+ ions characteristic of sphingomyelins, such as m/z 753.6 for SM 18:0, phosphocholines (m/z 780.6 for PC 34 : 2; 782.6 for PC 34 : 1; 806.6 for PC 36 : 6; 808.6 for 36 :2; 828.6 for PC38 : 6; and 830.6 for PC38 : 5), plasmalogens (m/z 790.6 for 1-palmitenyl-2-docosahexanoyl-GPC and 814.6 for 1-palmityl-2-docosaheaenoyl-GPC); and triacylglycerols (m/z 881.7 for sn-glycerol-palmitoleate-oleate-oleate). PCA showed clear separation between bulls 1 and 2 ejaculates, indicating that each bull presented a characteristic and reproducible (from different ejaculates) profiles. Differences in the relative intensities of the ions mentioned above contributed for bulls 1 and 2 differentiation by PCA. PC1 and PC2 explained 86.5% of the data variance. In conclusion, a fast sample preparation protocol followed by MALDI-MS appears to provide characteristic lipid fingerprints for crude spermatozoa (ejaculates) of bulls with different capacities of embryo in vitro production. Experiments involving a larger and more statically relevant set of samples are underway.
We thank the Brazilian research foundations FAPESP (2008/10756-7) and CNPq.
Establishment of regenerative callus, cell suspension system, and molecular characterization of Taxus wallichiana Zucc. for the in vitro production of Taxol
