KIFER. SMITH, and YOUNG; EFFECT OF DIETARY FISH OIL 



Table 4. — Dressing percentage, lean-cut percentage, longistiimns dorsi area, and backfat thickness obtained from 



pigs fed various diets containing menliaden oil. 



petroleum ether (30° to 60° C boiling point), 

 poured into a separatory funnel, and washed 

 twice with a 20^^ solution of NaCl. The layer 

 of petroleum ether was evaporated in the rotary 

 evaporator, and the e.xtracted fat was trans- 

 ferred to containers in which it was protected 

 by nitrogen and was stored at — 20° C until 

 methyl esters were prepared from it for analysis. 



The methyl esters of the fatty acids were pre- 

 pared as follows: 



Five ml of anhydrous methanol and about 50 

 mg of freshly cut and shiny sodium were placed 

 into a small test tube. After the sodium had 

 reacted, six to eight drops of the extracted oil 

 were added and heated to reflux on a steam bath 

 for 2 min with agitation. The end point of the 

 reaction was signaled when the solution became 

 clear. 



The reaction solution was quenched with 5 ml 

 of distilled water and was transferred to a sep- 

 aratory funnel. The mixture was extracted with 

 two 10-ml portions of petroleum ether (30° to 

 60" C boiling point). The final water layer was 

 discarded, and the two petroleum ether extracts 

 were combined. The petroleum ether solution 

 was washed with 10 ml of 5 ""r aqueous HCl so- 

 lution. The acid wash was followed by succes- 

 sive washes with 15-ml and 10-ml aliquots of 

 20''r NaCl solution. The washing was com- 

 pleted when pH paper tested neutral. 



The ethereal solution of methyl esters was 

 dried over 3 g of anhydrous Na2S04, filtered, 

 and evaporated over a 60° C water bath, using 

 a vacuum rotary evaporator. 



To check for purity, we made a thin-layer 



chromatogram of the ester solution using silicic 

 acid paper. Methyl myristate was used as the 

 control. A solution of 90 parts petroleum ether, 

 10 parts ethyl ether, and 1 part formic acid was 

 used to elute the esters. The chromatogram was 

 develo])ed in iodine vapor. 



Methyl esters of pure fatty acids were used 

 as reference standards for the C14-24 saturated 

 acids, Cii; -:24 monoenoic acids, plus linoleic, lino- 

 lenic, ai'achidonic, eicosapentaenoic, and docosa- 

 hexaenoic acids. Also concentrates of 16: 2, 16:3, 

 16:4, and 18:4 methyl esters that were obtained 

 by fractional distillation and urea-inclusion com- 

 pound fractionalization were used as reference 

 standards." As a secondary reference mixture, 

 methyl esters from whole menhaden oil were also 

 analyzed. From a plot of the logarithms of the 

 retention times (relative to stearate) versus the 

 number of carbon atoms, nearly linear relations 

 were observed for homologous series (Farquhar, 

 Insull, Rosen, Stoff'el, and Ahrens, 1959). Iden- 

 tifications were further verified by applying the 

 graphical method of James (1960) for analyses 

 on columns jiacked with diethylene glycol succi- 

 nate polyester and Apiezon L. These plots pro- 

 vided the necessary reference data for identifi- 

 cation of the various tissue lipids analyzed." 



' Tlie staff of the National Marine Fisheries Service 

 Technological Laboratory, Seattle, Wash., made the 

 fractional distillations and urea-inclusion compound frac- 

 tionations. 



' The fatty acids of the oil fed and of the animal 

 tissues were identified initially in collaboration with the 

 staff of the National Marine Fisheries Service Techno- 

 logical Laboratory, Seattle, Wash. 



285 



