The Fatty Acid Composition of Porcine

BIOLOGY OF REPRODUCTION 1, 330-334 (1969) The Fatty Acid Composition of Porcine Spermatozoa Phospholipids L. A. JOHNSON1, R. J. GERRITS1, AND E. P. YOUNG1 United States Department of Agriculture, and University of Maryland Received March 24, 1969 The fatty acid composition of three phospholipids of boar spermatozoa from 32 ejaculates was determined; choline, ethanolamine, and serine were the phospholipids in which 19 fatty acids and two aldehydes were identified. Thin-layer and gas-liquid chromatography were employed for separation of phospholipids and determination of fatty acids. Major saturated fatty acids present were palmitic (16:0), stearic (18:0), and myristic (14:0). Palmitic and stearic acid were present in highest concentrations, ranging from 8 to 19% and 4 to 36%, respectively, in the three s. Unsaturated fatty acids predominated in the choline and ethanolamine s, while saturated acids predominated in the serine s. Docosapentaenoic acid (22:5w6) was the major unsaturated acid at a high of 38.6%. Docosahexaenoic acid (22:6) was somewhat less at 25.2%. Oleic acid (18:1w9), linoleic acid (I8:2w6), and arachidonic acid (20:4w6) were present in lesser quantities. The fatty acid composition of boar spermatozoa phospholipid has not been elucidated. Numerous studies have been conducted to determine the fatty acids present in the semen of various other species. Patton and Flipse (1961) reported that myristic, palmitic, and stearic acid accounted for over 80% of the fatty acids in bovine spermatozoa lipid. Dietz et al. (1963) reported the presence of several fatty acids in whole bull semen lipid; palmitic acid predominated. Pursel and Graham (1967) reported the presence of 11 fatty acids in bovine spermatozoa phospholipid; docosahexaenoic acid was the predominant acid in the choline fraction. Ram semen phospholipid contains palmitic and arachidonic acid in large quantities (Gray, 1960). Hartree and Mann (1961) reported that ram semen contained six major fatty acids; palmitic and oleic acid were present in highest concentra- 1 Animal Husbandry Research Division, ARS, Beltsville, Maryland 20705. 2 Department of Animal Science, University of Maryland, College Park, Maryland 20740. tions and were implicated as possible sources of energy for the sperm cell. The purpose of this investigation was to determine the fatty acid composition of boar spermatozoa s. A preliminary report of this study has been published (Johnson et al., 1967). MATERIALS AND METHODS Semen Collection and Preparation Semen was collected from four crossbred littermate boars, 14 months of age. The boars were trained to mount a dummy sow and serve an artificial vagina. The semen was collected in a water-jacketed (37#{176}C) beaker covered with gauze to separate the gel particles from the liquid semen. Immediately after collection, semen was evaluated for quality by determining sperm motility under a light microscope at X100 magnification; by counting abnormal spermatozoa at x 430 and by determining spermatozoa concentration from the average of four hemocytometer counts. After collection, the spermatozoa were separated from the plasma by centrifugation at 30,000 g for 20 mm. Washing of spermatozoa after centrifugation was dispensed with because of the low level of lipid in the seminal plasma (Komarek et a!., 1965) in which only 0.29% lipid \ as present. This low level 330

PORCINE SEMEN FATTY ACIDS 331 of plasma lipid was confirmed by Johnson et at. (1969). Washing has been shown to cause a loss in phospholipid from the sperm cell (Hartree and Mann, 1959). The spermatozoa were stored in liquid nitrogen until analyzed. Spermatozoa from 32 ejaculates were analyzed. Extraction and Chromatography of Lipid The lipids were extracted and purified at room temperature with chloroform:methanol (2:1 v/v) according to the method of Folch et at. (1957). Separation of phospholipids was accomplished by thin-layer chromatography on silica gel H (Brinkmann Instruments, Westbury, N.Y.). Total lipid extract extended in chloroform was applied to the thinlayer with a 10O-el microsyringe. A total of 0.7-0.9 ml of extract was applied to the thinlayer in repetitive strokes as a streak 17 cm long. The plates were 20 X 20 cm with a 0.5-mm layer of silica gel H. The plates were developed in a solvent system containing chloroform: methanol: acetic acid: water (25:15:4:2 v/v/v/v) according to Skipski et at. (1964). Development time was 75 to 90 mm. This method of separation does not differentiate between the phosphatidyl and phosphatidal derivatives, hence the use of terminology. The phospholipid fractions were identified using pure standards (Applied Science Laboratory, State College, Pennsylvania) and cochromatography. Identification of the fractions on each plate was made by spraying the plate with a 0.04% solution of 2, 7 -dichlorofluorescein solution in methanol (Parker and Peterson, 1965) and viewing under ultraviolet light. Identified fractions were scraped from the plate into tubes for transesterificatjon. Fatty Acid Esterification Transesterification was performed by using a modification of the method of Bowyer et at. (1963). Four milliliters of 10% methanolic sulfuric acid (w/v) were added to the tube containing the phospholipid fraction (still adhered to the silica gel). Carborundum and hydroquinone were added and the mixture refluxed for 60 mm at 80#{176} C. After cooling, 1 ml of water was added and the methyl esters were extracted three times with 1-ml portions of hexane. The combined extracts were concentrated and injected into a gas chromatograph. Completeness of esterification was determined by thin-layer chromatography on silica gel G (Metcalfe et a!., 1966). Gas Chromatography A Perkin-Elmer Model 801 gas chromatograph, equipped with a hydrogen-flame ionization detector, was used. The columns were dual, glass, 1.83 meters long, 3.0 mm id. and packed with 10% ethylene glycol succinate silicone copolvmer (EGSS-X or EGSS-Y) on 100/120 mesh Gas Chrom P. The operating temperature was 140 to 200#{176}Cprogrammed at one degree per minute. The carrier gas was nitrogen at a flow rate of 50 ml per minute. The peak areas were computed with a disc integrator. Authentic methyl ester mixtures were used for instrument calibration, including H104 and NHI mixtures KA, KB, KC and KD (Applied Science Laboratory, State College, Pennsylvania). Fatty acid identification was conducted isothermally at 190#{176}C. Identification procedures were based on a comparison of relative retention times (to stearic acid) and separation factors (Ackman et at., 1963) to those of authentic methyl ester mixtures obtained from the Hormel Institute, Austin, Minnesota, and the National Heart Institute, NIH, Bethesda, Maryland. In addition, comparisons were made of relative retention times and separation factors to those determined for cod liver oil fatty acid methyl esters. RESULTS AND DISCUSSION The fatty acid composition of three s, choline, ethanolamine and serine, of boar spermatozoa are shown in Table 1. Palmitic acid (16:0) was the major saturated fatty acid in choline (CP) and ethanolamine (EP). However, in serine (SP) stearic acid (18:0) was predominant. The results are comparable to bull semen where palmitic acid and stearic acid were found to be predominant (Dietz et al., 1963; Pursel and Graham, 1967). Ram semen phospholipid was also characterized by a high palmitic acid content (Gray, 1960). Other saturated fatty acids present in the three s at greater than 1% of the total fatty acids were myristic (14:0), arachi - dic (20:0), and behenic (22:0). In addition to those acids listed in Table 1 as present in very small quantities (< 1%), pen tadecanoic acid (15:0) was present in some samples in trace quantities. The unsaturated fatty acids were very high in boar sperm s. CP and EP contained 72.8 and 64.1% unsaturated fatty acids, respectively, with docosapentaenoic acid (22: 5oj6) predominating. Docosapentaenoic acid is commonly found in erythrocyte phospholipid as well as in the phospholipid of vanous other tissues of humans (Dodge and Phil-

332 JOHNSON, GERRITS, AND YOUNG TABLE 1 FATTY Acm COMPOSITION OF THREE PIIOSPHATIDES IN PORCINE SPERMATOZOA Fatty acidb Serine Ethanolamine Choline or aldehyde 14:0 DMA 0.7 ± 0.1 2.6 ± 0.5 2.2 ± 0.4 14:0 1.7±0.1 2.7±0.4 5.2±0.4 16:0 DMA 1.3 ± 0.2 8.5 ± 0.9 4.8 ± 0.6 17:0 0.4±0.0 0.2±0.0 0.1±0.0 17:1 + 16:2 0.5 ± 0.2 0.3 ± 0.1 0.1 ±0.0 16:0 19.7 ± 0.6 15.1 ± 1.0 8.4 ± 0.6 16:1 0.3 ± 0.1 0.2 ± 0.0 <0.1-18:0 37.3 ± 1.2 5.5 ± 0.3 3.8 ± 0.6 18:lio9 4.6±0.4 2.0±0.2 2.9±0.3 18:2o6 2.8±0.5 1.3±0.1 0.6±0.0 20:0 2.6 ± 0.3 0.2 ± 0.1 0.4 ± 0.1 20:1 1.2 ± 0.4 0.3 ± 0.1 0.5 ± 0.1 20:2 0.7 ± 0.3 0.1 ± 0.0 0.5 ± 0.1 22:0 2.2 ± 0.2 0.8 ± 0.1 1.8 ± 0.1 20:4ffl6 + 22:1 7.6 ± 0.5 4.6 ± 0.2 1.2 ± 0.1? <0.1-0.3 ± 0.2 0.4 ± 0.1 20:5 0.4 ± 0.4 <0.1-0.2 ± 0.1 24:0 0.3 ± 0.1 0.1 ± 0.0 0.2 ± 0.1 24:1 + 22:4 0.9 ± 0.1 2.4 ± 0.1 2.1 ± 0.1 22:5o,6 11.3 ± 0.5 33.1 ± 0.8 38.8 ± 0.7 22:5o3 0.1 ± 0.0 0.4 ± 0.0 0.4 ± 0.1 22:6 3.5 ± 0.3 19.3 ± 0.7 25.1 ± 0.6 % Saturated 66.2 35.7 27.0 % Unsaturated 33.9 64.1 72.8 Mean values ± SE. b Nomenclature: Number of carbons:number of double bonds the w or terminal methyl group. DMA - Dimethylacetal. and position of first double bond relative to lips, 1967). High concentrations of 22:5w6 appear to be peculiar to spermatozoa. Davis et al. (1966) reported 22:5w6 to be present in rat epididymal sperm phospholipid (16. 4%). It was also present in rat testes phospholipid and triglyceride. The biosynthetic pathway of docosapentaenoic acid (22:5o6) was established in rat testes by Davis and Coniglio (1966). They determined that it was formed from arachidonic acid (20:4) which in turn had been formed from linoleic acid (18:2). Davis et al. (1966) found a significant increase in docosapentaenoic acid (22:5o6) of rat testes between the ages of 4 and 7 weeks. After further study, they concluded that the increase in 22:5w6 was associated with the appearance and maturation of the spermatids. Further studies by Davis and Coniglio (1967) support this contention. Their analysis of rat epididymal sperm mentioned above would also support these findings since the lipids of the spermatid would be expected to be a dominant factor in the lipid pattern of the testes. The high concentration of 22:5w6 in the boar sperm phospholipid (11.3 to 38.8%) determined in the present study might also indicate an association with sperm cell maturation. Docosahexaenoic acid (22:6) was present in significant quantities in CP and EP (25.1 and 18.8%). This acid has been reported previously in bull spermatozoa phospholipid by Pursel and Graham (1967). The two most unsaturated fatty acids (22: 5o6 and 22:6)

PORCINE SEMEN FATTY ACIDS 333 represent 52 to 64% of the total fatty acid complement. Aldehydes identified were palmitaldehyde (16:0 DMA) and myristaldehyde (14:0 DMA). The highest concentrations of aldehydes were found in EP with 8.5% palmitaldehyde. Reports of aldehydes in bull spermatozoa are widespread (Patton and Flipse, 1961; Dietz et al., 1963; Pursel and Graham, 1967). Several fatty acids in very minor amounts were also identified; each was below l/ of the total fatty acid complement. Complete separation of fatty acids was not achieved in two cases: therefore identity could not be established conclusively, hence they are listed together. In a third case arachidonic acid (20:4w6) elutes at the same time as erucic acid (22:1). It was determined, however, that arachidonic acid predominated and is so listed. One fatty acid present in minor amounts was not identified. The results indicate a similar fatty acid pattern for EP and CP. Senine contrasted sharply in that it was nearly as high in saturated acids (66.2%) as EP and CP were in unsaturated acids (64.1 and 72.8%). Earlier studies (Johnson et al., 1969) have shown that CP represents 43.6% of the total phospholipid whereas EP accounts for 25.6% and SP for only 5.4%. With these results in mind it is worthy to note that the s which are dominated by unsaturated fatty acids represent 69.2% of the total phospholipid (which itself constitutes 68% of the lipid, Johnson et a!., 1969) of boar spermatozoa. Therefore, whatever role played by fatty acids in boar sperm cell maturation or metabolism, it seems likely that the highly unsaturated acids may be of particular importance. ACKNOWLEDGMENTS Data presented are taken from a dissertation presented by the senior author in partial fulfillment of the requirements for the Ph.D. degree, University of Maryland, College Park. Gratitude is expressed to Mrs. M. R. Stallknecht for technical assistance and to Dr. R. Kifer for the cod liver oil fatty acid methyl esters. REFERENCES ACEMAN, R. G., BURGHER, R. D., AND JANGAARD, P. M. (1963). Systematic identification of fatty acids in the gas-liquid chromatography of fatty acid methyl esters: A preliminary survey of seal oil. Can. J. Biochem. Physiol. 41, 1627-1641. BowvER, D. E., LEAT, %V. M. F., HOWARD, A. F., AND GRESHAM, G. A. (1963). The determinaticn of the fatty acid composition of serum lipids separated by thin-layer chromatography; and a comparison with column chromatography. Biochim. Biophvs..4 cta 70, 423-43 1. DAVIS, J. T., BRIDGES, R. B., AND CGNIGLIO, J. G. (1966). Changes in lipid composition of the maturing rat testis. Biochem. J. 98, 342-346. DAVIS, J. T., AND C0NIGLI0, J. G. (1966). The biosynthesis of docosapentaenoic and other fatty acids by rat testes. I. Biol. Chen,. 241, 610-612. DAVIS, J. T., AND CONIGLIO, J. G. (1967). The effect of cryptorchidism, cadmium and anti-spermatogenic drugs on fatty acid composition of rat testes. J. Reprod. Fertility 14, 407-413. DIETS, R. W., PICICETT, B. %V., KOMAREK, R. J., AND JENsEN, R. G. (1963). Fatty acid composition of bovine semen. J. Dairy Sci. 46, 468-472. DODGE, J. T. AND PHILLIPS, G. B. (1967). Composition of phospholipids and of phospholipid fatty acids and aldehydes in human red cells. J. Lipid Res. 8, 667-6 75. FOLCII, J., LEES, M., AND SLOANE-STANLEY, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509. GRAY, G. M. (1960). The phospholipids of ox spleen with special reference to the fatty acid and fatty aldehyde composition of the lecithin and kephalin fractions. Biochem. J. 77, 82-91. HARTREE, E. F. AND MANN, T. (1959). Plasmalogen in ram semen and its role in sperm metabolism. Biochem. 1. 71, 423-434. HARTREE, E. F., AND MANN, T. (1961). Phospholipids in ram semen: metabolism of plasmalogen and fatty acids. Biochem. J. 80, 464-476. JOHNSON, L.A., GERRITS, R. J., AND YOUNG, E. P. (1967). The fatty acid composition of boar spermatozoa phospholipids. J. Animal Sci. 26, 1499 (abstr.). JOHNSON, L. A., GERRITS, R. J., AND YOUNG, E. P. (1969). Quantitative analysis of porcine spermatozoa and seminal plasma phospholipids as affected by frequency of ejaculation. J. Reprod. Fertility 19, 95-102. KOMAREX, R. J.,PICKETT, B. W., GIBSON, E. %V., AND JENSEN, R. G. (1965). Lipids of porcine sperma-

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