VITAMIN A in SELECTED. PALE-COLORED LIVERS of ALASKA FUR SEALS, 1948 Marine Biological L^h-it.tOfT LI 25 ». A- B. TT AUG 2 .1 1950 WOODS HOLE, MASS. SPECIAL SCIENTIFIC REPORT: FISHERIES No. 32 UNITED STATES DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE Explanatory Note The series embodies results of investigations, usually of restricted scope, intended to aid or direct management or utilization practices and as guides for administrative or legislative action. It is issued in limited quantities for the official use of Federal, State or cooperating agencies and in processed form. for econony and to avoid, delay in publication. Washington, D. C. July 1950 United States Department of the Interior Oscar Lo Chapnan, Secretary Fish and Wildlife Service Albert M<. Day, Director Special Scientific Report - Fisheries No. 32 VITAMIN A IN SELECTED, PALE-COLORED LIVERS OF ALASKA FUR SEALS, I9I48 By Victor B. Scheffer, Biologist, Neva Lo Karrick, Chemist, Fo Bruce Sanford, Chemist Branch of Commercial Fisheries Uo So Fish and Wildlife Service Seattle, Washington V Abstract Fifty-one livers selected for pale color were found, on the average, to wei^ 1,060 grams and to contain 2.k million "spec" units of vitamin A per poxmd and 3.9 percent oil. "Bie vitamin A potency of the liver oil ranged from 4,170 to 63^,000 units DjoOO x E (1 percent 1 cm, 3ii8 m ma, isopropanoljj per gram. The average potency of the liver oil was 137,000 "spec" units per gram. It appears from tiie present and from earlier work that paleness of liver is directly correlated with hi^ vitamin A potency and with higti oil content. It is apparently not associated with size of the seal or other readily ohservahle character- istics. The data on vitamin A potency of the oil do not resemble a normal distrihution. The data can he fitted "by the formula: f (X) = 23.k6 , 4,150 + X where X stands for the vitamin A potency of the oil and f (X) for the numher of individuals having X as a potency. Introduction In 1946 the livers of eight Alaska fur seals (Callorhinos ursinus) were collected on the Prihilof Islands and analyzed for oil and vitamin A in Seattle (Miyauchi and Sanford 1947). The vitamin A potency was hi^ enovi^ in certain of the livers to warrant commercial exploita- tion even in such a remote area as the Prihilof Islands. Consequently, a search was made for a means of segregating the livers of high from tiaose of low potency at the source. In 1947 it was found in a sample of 196 livers that the pale-colored ones were richer in vitamin A than the darker, maroon-colored ones (Sanford, Kenyon, and Scheffer 1949) . In 1948, 51 pale-colored livers were sampled selectively. The results of the analyses are given in this report. Collecting the Samples The fresh, warm livers from male fur seals approximately 3 to 4 years of age were collected. The carcasses of the seals had "been laid out "by the sealing ^ng on the killing fields of St. Paul Island, Alaska, on July 12 and 13, 1948. The livers of aibout 500 animals were examined, and the 51 among than that appeared to he palest were selected for testing. The color of the fur seal liver ranges from maroon (as in heef liver) to pale tan (as in cod liver) . By far the greatest numher of livers axe shades of maroon. In selecting a sanple of the palest 10 percent the color sense of the observer, rather than instruments, was relied upon. The 1948 sanples were handled in the same way as those collected in 1947, Ihat is, each liver was washed in cold water, placed in a waxed- ps5)er hag having a cellophane liner, and stored in a refrigerator at ap- proximately 20° F. until they were analyzed in the fall of 1948. As a separate operation, on July ISth, a stady was made of the relation between the size of a given seal and the paleness of its liver. This was done as follows: One hundred carcasses of known length were sajipled at random, (The "body length of every male fur seal killed on the Prihilof Islands is measured routinely to the nearest inch.) From these were selected the 10 having the palest livers. It was found that the mean length of the 10 seals having the pale livers was virtually the same as the mean length of the 90 seals having the darker livers (41.9 inches versus ^2.0.) Thus, in tiiis sainple there was no perceptible difference in size between seals having pale and those having dark livers. Analytical Procedure Each liver was ground in a meat chopper and a representative fraction was stored in a screw~c^ jar at -20° F. About ^ hours before analysis, the sample was thawed at 35° F. The sample was blended in a Waring Blendor, and about 10 grams of the resulting material was accurately weighed into a square shaking bottle of 180-millili ter capacity. A heaping teaspoon of powdered pumice, exactly 50 milliliters of ethyl ether, and two heaping teaspoons of anhydrous sodium sulfate were then added. In a shaking device, the bottles were subjected to lUU one-inch strokes a minute for one hour. They were then centrifuged and aliquots were removed for oil and vitamin A deteimnations. All saii5)les were run in duplicate. To estimate the oil corjcentration, a lO-milliliter aliquoti por- tion of the ether extract was pipetted into a tared, 50-milliliter beaker, and the ether was evaporated on an air bath. Three minutes after the dis- appearance of the ether, the beaker was removed and allowed to come to room temperature. The wei^it of the beaker and its contents then gave the remaining information necessary for the calculation of the oil concentra- tion in the liver. To determine the vitamin A potency, a 1-milliliter aliquot por- tion of the ether extract was diluted with isopropanol, and the optical density of the solution was measured at 328 m ma, by means of a Beckman spectrophotometer. The vitanan A potency was calculated as "spec" (spec- tropho to metric) -anits using the formula 2000 x E (l percent, 1 cm, 328 m mu, isopropanol) . It was recogoized that this gave only a gross estimate of vitamin A, but it was thou^t adequate for the present purpose. Results The results of the analyses are presented in Tables 1 and 2. On the average the livers weighed 2.^ pounds- and contained nearly h percent oil and 2.U million "spec" units of vitamin A per po\md. The range in potency was U,170 to 63^,000 "spec" units per gram of oil, and 0.lU6 million to 26.6 million "spec" units per liver. (The potency of 63^,000 "spec" units per gram of oil is the maximum thus fax encountered in our three-year study of for seal livers.) The richest liver contained 182 times as nuch vitamin A as the poorest. About one-half of the livers yielded an oil having a vitamin A potency of less than 50,000 "bpec" luiits per gram, and about one- third of the livers yielded an oil having a potency in excess of 100,000 "spec" units per gram. (The liver of the average 3-year-old male fur seal contrihutes ahout k percent of the weight of the "body and 5 percent of the wei^t of the skinned carcass.) Discussion The prime objective in 19^ was to find a means of identifying, in the fresh bsdies, the livers of potential value. In this we have partially succeeded. It ^jpears from the present and our earlier woik that paleness of liver is fairly indicative of high vitamin A potency. Thus, for the saii5)le of 51 pale livers taken in 19^ and reported here, the mean vitamin A potency of the liver oil was 137,000 "spec" units per gram of oil. For the random sample of 95 livers taken in 19^7, the mean potency was 57,^00 "spec" units. Althou^ the animals sampled were of the same age and sex, they were killed a year apart. It is barely possible that the observed dif- ference in vitamin A potency was dae to a difference in tiie 19^7 diet and the 191+8 diet of the study animals. Considering, however, the wide extent of the feeding range of the fur seal herd, the probability is great that the diet from one year to the next is fairly \mifom. Hence the difference in vitamin A potency probably stems from some source other than annual variation in food. Granted that paleness of the liver and high vitamin A potenisy are directly correlated, the next move is to find out what factors contribute to paleness. The li^t color is uniform throughout the liver and is not limited to the surface. It may, to a sli^t extent, be caused by an increase in fat content. Thus, in the 19^8 sanple of pale livers, the oil concen- tration was 3.91 percent, vftiile in the 19^7 random sanple of livers it was 2.99 percent. The livers having a hi^ vitamin A potency seem to contain more oil. If tile 51 livers are divided into two groups, the first (with 35 livers) having a potency of less than 100,000 "spec" units and the second (with 16 livers) having a potency of more than 100,000 "spec" units per gram of oil, it is found tiiat the mean concentration of oil in the liver of the first groi?) is 3.63 percent and in the second, h,55 percent. A similar relationship has been observed by Braekkan (19^) in whale liver oil. In fin, blue, and sperm whales he states that "tiie potency is usually hi^est in the sanples having the highest content of fat." Of all the livers available on the killing field, the palest one- tenth mi^t be used commercially. These are worth, at 16 cents a million units of vitamin A, about $0.88 each, f.o.b. Seattle. Furthermore, among the palest one- tenth there is a still smaller fraction, representing about three percent of all tiie livers, which are worth about $2.72 each. Farther observations of carcasses on the killing fields are planned in an attempt to segregate the livers of hi^ vitamin A potency. 3 Ttie wide variation in vitamin A content of fur seal liver oil poses an interesting problem. In outward appearance and "behavior the animals are alike. They are juvenile males, alx>ut 3 years of age, appear- iog on land at the same time. They are in a resting or sani-resting stage after a sojoiim of many months at sea. They are, as has "been shown, fairly unifonn in Ixidy length. Why, then, the vitamin A potency in their livers should vary so greatly is difficult to explain at the present time. That the jrariation is caused ty great differences in the diet of the individaal seal, is one of the more plausihle explanations.!/ Periiaps the variation depends upon the length of time "between the most recent meal of the seal and the time of sampling. That is, perhaps the vitamin A reserve tends to change as the male seal continues to fast on the "breeding grounds. It is known that the adult male seals may gp two months or more without food. How long the juvenile males, like those in our sanple, habitually fast in the summer season has not been determined. It can "be deduced, however, from study of the annuli or growth rings on the teeth that the deposition of calcium is interrupted, hence there is proTsably an annual fasting period for the subadult as well as the adult males. Theoretical Considerations The data on vitamin A potency (Table 1) do not resemble a normal distri"bution. When the o"bserved range is divided into ten equal classes, 62 percent of the values fall within the lowest class. When the individual values are arrayed in order, equidistant on the x-axis, while their loga- rithms are plotted on the y-axis, the plotted points fall nearly on a strai^t line. Dr. Z. W. Bimhaum, of the Laboratory of Statistical Research, University of Washington, has kindly examined our data. He states that he does not know of any model to which tiiey conform, although they can be quite well fitted by a distribution of the type f (X) = M L+ X where X stands for vitamin A potency and f (X) for the number of individuals having potency X. The parameters M and L will be positive constants which can be fitted by the method of least squares. The two constants can then be used to characterize the distribution. (Miss Elizabeth "^aughan, Statisti- cian, Alaska Fisheries Investigations, Fish and Wildlife Service, has kindly conpited the parameters for our data. She finds that L = k,150 and M = 23. U6.) Dr. Bimbaum suggests, further, that the distribution is bi- modal and the superinposition of two normal distributions. All evidence ■ from the field, however, points to a homogenous sanple. 1/ A tabulation by Victor B. Scheffer (in press) of the contents of 1,300 seal stomachs from the North Pacific Ocean and Bering Sea shows that the following items are of most frequent occurrence; squid, pollack, herring, Crustacea (chiefly from the fish stomachs), seal fish (Bathylasus) . sa.1mon, ailachon, and rockfish. Table 1. — Oil and Vitamin A in Selected, Pale-Colored Livers of Male For Seals, Summer of 19^3: Analyses of Individual Livers Sample Wei^t of liver Oil in liver Vitamin A potency of liver oil No. Grams Percent \iy weight "spec" units per gram of oil^/ 3U 909 3.66 4,170 38 905 2.98 5,600 6 9U6 3.85 5,650 hi 1101 1+.16 6.300 7 921 3.25 6,700 US 9ltS 3.29 10,300 37 1132 3.27 10,600 U5 126U 3.90 10,800 9 895 3.^7 11,100 11 1283 4.76 11,300 50 1363 3.13 11,300 23 969 3.88 13,400 1 138U 3.36 13,500 33 loiw 3.68 15,100 »^9 10U6 4.00 17,500 3 963 4.99 18,800 Z7 959 4.74 23,900 2U 998 3.'^ 25,800 39 92U 2.88 28,400 22 1260 3.95 29,500 20 1071 3.77 38,600 »^3 859 3.18 41,800 25 1150 2.76 42,500 Ho 1273 2.89 42,900 l46 10^46 4.38 46,400 47 981 3.26 47,000 26 1563 3.10 48,600 12 117^ 4.41 53,400 16 1018 3.16 53,800 31 860 3.34 58,500 U 878 3.14 59,800 29 1127 3.15 63,000 18 1209 4.38 86,200 28 1113 3.92 91,700 5 1157 3.42 92,900 51 827 3.67 137,000 ZL 1U99 3.92 i4o,ooo lU 99U 5.16 144,000 36 1031 3.17 182,000 17 1255 4.45 251,000 15 1200 4.90 318,000 8 1032 4.48 340,000 13 112lt 2.76 413,000 1/ 2000 X E (l percent 1 cm, 328 m ma, isopropanol) 5 Table 1. — Oil and Vitamin A in Selected, Pale-Colored Livers of Male For Seals, Summer of 19^8: (Continued) Analyses of Individual Livers Sample Wei^t of liver Oil in liver Vitamin A potency of liver oil No"i Grams Percent "by weight "spec" units per gram of oil—/ 32 822 5.31 Ul3,000 2 8^9 5.26 Ul9,000 19 1167 5.00 ^29,000 35, 756 1^.37 ^30,000 10 1266 U.85 lj-70,000 te 8l<-3 5.6U 508,000 30 8U7 5.13 628,000 ^ 892 U.70 63^^,000 Mean 1060 3.91 137,310 1/ 2000 X E (1 percent 1 cm, 328 m ma, isopropanol) Tal)le 2.— Summary of Data for 19U8 Arithmetic Variate Observed range mean Wei^t of liver, grams 756 to 1,563 1,060 Oil in liver, percent 2.76 to 5.6U 3.91 Vitamin A potency of liver oil, "spec" units per gram 4,170 to 634,000 ' 137,000 Literature Cited BEAEKKAN, OLAF R. 19^8. Vitamins in Whale Liver. Hvalrodets Skrifter (Oslo), no. 32, p. 17. MITAUCHI, DAVID T. and SAilTOED, P. BHJCE 19^7. Vitamin A Content of Far Seal Oils, Commercial Fisheries Review, vol. 9, no. 11, pp. 5-8, November 19U7. SMPOED, F. BHJCE, KENTON, KARL W. and SCHEFFER, VICTOR B. I9U9. Vitamin A in Liver of the Alaska Pur Seal. Commercial Fisheries Review, vol. 11, no. h, pp. 9-15, J^ril 19^9. 79651 6 Interior—Daplicating Section, Washington, D, C. Fur seal carcasses at byproducts plant, St. Paul Island, Alaska, July 15, 19U8. (Photo VBS 21; 09) Byproducts plant, St. Paul Island, Alaska, July 26, 19U8. (Photo VBS 2i4m) 7 Liver of bachelor fiir seal 5-5 years old, St. Paul Island, Alaska, June 30, 19Lt7. Weight of liver with gall bladder 1,581 grains (5,Ql4 pounds). (Photo VBS 2191) 79«S1 interior— Duplicating Section, Washington, D. C. iII^AmMP,' f-^fPrV.T.Serisls JMMllllllltllll l„„ 5 WHSE 01012