Immediately after semen analysis, samples were centrifuged at 800 ×g for 10 min to separate the spermatozoa from the epididymal fluid. The volume of each sample contained an equivalent concentration of 1.5 million spermatozoa, by pooling the sample of four dogs, totalizing five pools of each segment of the epididymides (caput, corpus and cauda), according to previous works [28 (link), 29 (link)]. For the epididymal fluid analysis we used a fixed volume of 60 μL. Samples were stored at −20 °C until analysis.
For the fatty acids extraction, we utilized the transesterification method described by Lepage and Roy [30 (link)]. Samples were transferred to a glass cuvette, to which 10 μL of sodium chloride and triphenylphosphate (10 mg/mL; as internal standard) was added. Subsequently, 1 mL of the solution was combined to methanol:acetyl chloride, in a 100:5 proportion (3 mL:150 μL), and the cuvette was maintained at 100 °C for 60 min for fatty acids transmethylation [31 (link)].
After incubation, the cuvettes were maintained at room temperature for the addition of 1 mL of hexane, which allowed the solubilization of fatty acids, enabling the passage through gas chromatography. Samples were vortexed for 60 s and then centrifuged (640 ×g for 5 min). The supernatant was transferred to a glass jar and dried by N2 vapor. Sequentially, the sample was suspended in 50 μL of hexane, vortexed for 60 s and 1 μL of this solution was injected into the gas chromatograph (GC-17A®, Shimadzu, Kyoto, Japan).
The fatty acids were classified in accordance with the retention time of a pre-established curve, then sorted into saturated fatty acids (butyric, caproic, caprylic, capric, undecanoic, lauric, tridecanoic, myristic, pentadecanoic, palmitic, heptadecanoic, stearic, arachidic, heneicosanoic, behenic, tricosanoic and lignoceric), monounsaturated fatty acids (myristoleic, pentadecenoic, palmitoleic, elaidic, oleic, eicosenoic, erucic and nervonic) and polyunsaturated fatty acids (linoleic, linolelaidic, gamma-linolenic, alpha-linolenic, eicosadienoic, arachidonic, eicosatrienoic, eicosapentaenoic acid and docosahexaenoic).
For the fatty acids extraction, we utilized the transesterification method described by Lepage and Roy [30 (link)]. Samples were transferred to a glass cuvette, to which 10 μL of sodium chloride and triphenylphosphate (10 mg/mL; as internal standard) was added. Subsequently, 1 mL of the solution was combined to methanol:acetyl chloride, in a 100:5 proportion (3 mL:150 μL), and the cuvette was maintained at 100 °C for 60 min for fatty acids transmethylation [31 (link)].
After incubation, the cuvettes were maintained at room temperature for the addition of 1 mL of hexane, which allowed the solubilization of fatty acids, enabling the passage through gas chromatography. Samples were vortexed for 60 s and then centrifuged (640 ×g for 5 min). The supernatant was transferred to a glass jar and dried by N2 vapor. Sequentially, the sample was suspended in 50 μL of hexane, vortexed for 60 s and 1 μL of this solution was injected into the gas chromatograph (GC-17A®, Shimadzu, Kyoto, Japan).
The fatty acids were classified in accordance with the retention time of a pre-established curve, then sorted into saturated fatty acids (butyric, caproic, caprylic, capric, undecanoic, lauric, tridecanoic, myristic, pentadecanoic, palmitic, heptadecanoic, stearic, arachidic, heneicosanoic, behenic, tricosanoic and lignoceric), monounsaturated fatty acids (myristoleic, pentadecenoic, palmitoleic, elaidic, oleic, eicosenoic, erucic and nervonic) and polyunsaturated fatty acids (linoleic, linolelaidic, gamma-linolenic, alpha-linolenic, eicosadienoic, arachidonic, eicosatrienoic, eicosapentaenoic acid and docosahexaenoic).
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