THE occurrence of blood spots in eggs

860 W. J. MUELLER, A. J. G. MAW AND E. G. BUSS fowl. Poultry Sci. 12: 69-82. Hunter, J. A., A. Van Wagenen and G. O. Hall, 1936. Seasonal changes in interior egg quality of Single Comb White Leghorn hens. Poultry Sci. IS: 115-118. Hutchinson, J. C. D., 1953. Effect of hot climate on egg weight. Poultry Sci. 32: 692-696. Jull, M. A., 1953. Egg quality declines as layers increase in age. World's Poultry Sci. J. 9: 273-274. Lamoureux, W. F., 1957. Albumen quality. The Kimberchik News, November. Lorenz, F. W., and H. J. Almquist, 1936. Seasonal variations in egg quality. Poultry Sci. 15: 14-18. Marble, D. R., 1943. Genetics of egg shape. Poultry Sci. 22: 61-71. Romanoff, A. L., and A. J. Romanoff, 1949. The Avian Egg. John Wiley and Sons, Inc., New York, N.Y. Blood Spot Incidence in Chicken Eggs and Vitamin A Level of the Diet 1 G. E. BEARSE, C. F. MCCLARY 2 AND H. C. SAXENA 3 Washington State University, Western Washington Experiment Station, Puyallup, THE occurrence of blood spots in eggs of the domestic fowl has been considered generally to be caused by genetic and environmental factors. A suggestion that diet might be associated with the incidence of blood spots in chicken eggs was noted at this Station (Bearse and Miller, 1940) when in experiments on vitamin A requirements of laying hens, diets with a low level of this vitamin produced eggs with numerous small blood spots. The occurrence of increased blood spots was attributed to a possible vitamin K deficiency because of low levels of alfalfa in the rations fed. Many nutritional factors have been studied to determine their effect on production of blood spots in eggs [Nalbandov and Card, 1944, 1947; Denton, 1947; Carver and Henderson, 1948; Sauter et al., 1952]. While some of these investigations 1 Scientific Paper No. 1895, Washington Agricultural Experiment Stations, Pullman. Project No. 801. 2 Present address: Heisdorf & Nelson Farms, Inc., P. O. Box 428, Kirkland, Wash. 3 Present address: Animal Nutrition Research Center, Institute of Agriculture, Anand, Bombay State, India. (Received for publication October 5, 1959) showed a beneficial effect from grass range, dried cereal grass, and fresh cut and dehydrated alfalfa, no significant reductions in blood spots were observed following vitamin supplementation. That vitamin A itself might be involved was indicated by Bearse et al. (1953). Subsequently, Scott et al. (1957) reported more blood spots in the eggs from hens fed 800 I.U. vitamin A per pound of ration than with hens fed higher levels. The data herein reported record the details of experiments supporting earlier reports from this Station of the influence of dietary vitamin A on blood spot incidence in eggs. EXPERIMENT 1 Washington Procedure. A group of 696 eight months old White Leghorn pullets were divided into 12 lots of 58 birds each, housed in litter floored pens and fed basal ration 1 of Table 1. The mash comprised 60 percent of the diet and the whole grain mixture 40 percent. These proportions were rigidly adhered to by frequent checking of mash consumption and adjustment of amounts

BLOOD SPOTS AND VITAMIN A 861 TABLE 1.Percent composition of basal rations fed Ingredients 1 1 2 3 4 5 Ground wheat Ground corn Ground barley Ground oats Wheat mixed feed Herring fish meal (70% pi Meat & bone scrap (45% rotein) protein) Soybean oil meal (45% protein) Dehydrated alfalfa (10 mg. carotene/100 g.) Whey, dried, whole Ground limestone Steamed bone meal Salt Whole oats Whole barley Whole wheat Whole corn 32.53 15.00 5.00 19.27 3.00 2.14 2.56.50 33.33 33.33 33.33 31.12. 21.34 2.87 4.07 50.0 Mash mixtures 28.12 21.34 3.00 2.87 4.07 Grain mixtures 50.0 28.34 19.80 5.00 20.50 2.26 3.50 50.0 8.00 40.00 6.00 14.00 5.00 2.40 4.00 75.0 1 The following supplements were added to each pound of mash: manganese sulfate, iodized, 113 mg. to rations 1, 2, 3 & 4 and 227 mg. to ration 5; sulfaquinoxaline, 38 mg.; vitamin D 3, 675 I.C.U.; vitamin B12 18 meg. to rations 1 & 4; riboflavin, 0.45 mg. to rations 1 and 0.90 mg. to rations 2, 3,4 & 5; vitamin A, 2,270 units to ration 5. as needed. A calcium supplement was fed ad libitum. Vitamin A was added to the basal mash in varying amounts. During the first 168 days all the levels fed were below the National Research Council (1954) requirement as determined by subsequent assay of the vitamin A supplements. This assay established the levels of vitamin A at 553, 758, 816, 962, 1.198 and 1,579 U.S.P. units of vitamin A per pound of ration. Blood spot observations were made on all eggs laid on two consecutive days after the birds had been on the experimental rations for 103 days and on all eggs laid on four consecutive days after 145 days on the rations. The eggs were broken out and examined on the day following oviposition, and the percentage having visible blood spots was determined. After 168 days on the above diets, changes were made in the vitamin A levels of the rations, and for 84 days quadruplicate lots received 1,115, 1,665, and 2,265 U.S.P. units of vitamin A per pound of ration. A third blood spot observation was made on the egg production of the last 5 days of the experiment. Results. Table 2 presents the results of the blood spot observations made in the first part of this experiment. At both the first and second observations, blood spot incidence tended to decrease as the level of vitamin A increased from 553 to 1,579 U.S.P. units per pound of diet, with only TABLE 2.Effect of level of vitamin A on incidence 1 of blood spots {experiment 1, part 1) U.S.P. units vitamin A per lb. ration 553 758 816 962 1,198 1,579 1st observation 2 % 39.6 22.7 22.4 19.2 20.3 16.1 2nd observation 3 % 35.5 30.5 26.8 23.7 22.1 15.2 1 Incidence refers to percent of eggs with blood spots of all sizes found in broken out eggs. 2 After 103 days on rations2 day's eggs. 3 After 145 days on rations4 day's eggs.

862 G. E. BEAESE, C. F. MCCLARY AND H. C. SAXENA TABLE 3.Effect of level of vitamin A on incidence 1 of blood spots {experiment 1, part 2) 1,115 23.2 1,665 14.6 2,265 13.5 1 Incidence refers to percent of eggs with blood spots of all sizes found in broken out eggs. 2 After 79 days on rations5 consecutive day's eggs. one exception. Statistical analysis of the data for the first observation showed differences which were at the.01 level of probability and application of Duncan's test (1955) showed the 553 U.S.P. unit vitamin A level to be significantly different from the rest with no statistically significant differences among the other levels. No significant differences were obtained by statistical treatment of the data of the second observation. The results of the second part of this experiment are shown in Table 3. These observations showed a higher percentage of blood spots for the lowest level, 1,115 units of vitamin A, than for the higher levels of 1,665 and 2,265 units. Duncan's test showed the lowest vitamin A level to be different (P < 0.05) from the two higher levels. EXPERIMENT 2 Procedure. A group of 910 six months old White Leghorns were randomized into 14 litter floored pens of 65 hens each. After 26 days on a vitamin A depletion ration, (ration 2, Table 1) the birds were fed a diet containing approximately 1,400 U.S.P. units of vitamin A per pound (ration 3, Table 1) for 112 days. Observations on blood spot incidence were made on all eggs laid during the last five days of this period. Following this observation the birds were fed another vitamin A deficient diet (ration 4, Table 1) for 19 days. Blood spot observations were made on all eggs laid during the last six days of this period. Diets 2 and 4 were estimated to contain approximately 50 U.S.P. units of vitamin A per pound. The total diet consisted of 60 percent mash and 40 percent whole grains. A calcium supplement was fed ad libitum. After the final depletion period, diets containing approximately 910, 2,270, 3,630 and 9,980 U.S.P. units of vitamin A per pound were each fed to triplicate lots of birds. The vitamin A supplement was a stabilized, gelatin coated product guaranteed to contain a minimum of 10,000 U.S.P. units of vitamin A per gram. Birds from two pens were used for liver storage determinations of vitamin A and the pens were removed from the experiment after the final 19 days depletion period. The supplemented rations were fed for four and one-half 28-day periods. All eggs laid during the last 14 days of the first period and six days near the end of each of the remaining periods were broken out and observed for presence of blood spots. When the feeding of the experimental rations was concluded the 12 pens were fed the regular Station laying ration for 61 days. Following this treatment the eggs produced during a period of seven days were observed for incidence of blood spots. Immediately before the final depletion period while the birds were still receiving ration 3, vitamin A content of the liver was determined on 12 birds from the two extra pens. This procedure was repeated after the birds had been fed ration 4 for 19 days. After 128 days on the various dietary levels of vitamin A, six birds in egg production were removed from each of the 12 pens and liver storage of vitamin A again determined. The assays for liver vitamin A were run by the method of Gallup and Hoefer (1946). Results. The birds to be fed the final depletion diet 4 were in a low state of vitamin A storage as a result of feeding diets 2 and

BXOOD SPOTS AND VITAMIN A 863 TABLE 4.Vitamin A assay of livers (experiment 2) Treatments 1 Before final vitamin A depletion After 19 days final depletion After 128 days on experimental rations 910 units vit. A/lb. of ration 2,270 units vit. A/lb. of ration 3,630 units vit. A/lb. of ration 9,980 units vit. A/lb. of ration 1 See narrative for details of treatments. U.S.P. units/ gram of liver 11.5 2.0 2.7 34.0 253.3 1,866.6 3. This is shown in Table 4 by the vitamin A assays of the livers which averaged 11.5 U.S.P. units per gram. Assays following the feeding of diet 4 for 19 days showed only 2.0 U.S.P. units per gram of liver (Table 4). Blood spot incidence increased between these two periods for each of the four groups of pens as shown in Table 5. The feeding of 910 U.S.P. units per pound of ration to hens depleted of vitamin A failed to stop this rise in percent blood spots immediately. An increased number of blood spot eggs were recorded for the first period observation after the birds had been on the 910 unit level of vitamin A from 14-28 days. Subsequently, there was a slight reduction in percent blood spots but the blood spot incidence never became lower than for the depletion period. Levels of 2,270, 3,630 and 9,980 U.S.P. units per pound caused a decrease in blood spot incidence over the depletion period Last 5 days before final 19- day depletion 10.84 8.12 8.68 8.38 observation. This decrease was evident at the end of period 1 for the two higher levels and at the end of period 2 for the 2,270 unit level. At the end of the 19 day depletion period, there was no significant difference in percent blood spots between the groups subsequently assigned to the four vitamin A levels. Analysis of the data on blood spot incidence for the period 4 observation showed the 908 unit level to be significantly (P <.01) higher than the three other levels of vitamin A. During the 61 days on the regular Station ration, containing approximately 8,650 U.S.P. units of vitamin A per pound, the eggs of birds which had been on the 910 unit level dropped from 19 percent blood spots to 3.47 percent, while the incidence on the other rations remained about the same as when the birds were fed the test diets. Table 4 shows that the birds on the 908 unit level failed to increase liver storage of vitamin A during the period of supplemental feeding. The storage of vitamin A in the livers of birds on the higher vitamin A levels increased; the higher the level the greater the storage. EXPERIMENT 3 Procedure. Six hundred and fifty White Leghorn hens about 15 months of age which had been randomized into four litter TABLE 5.Percent blood spots on several vitamin A levels (experiment 2) Last 6 days Vit. A 1 final 19-day units per depletion lb. of ration 15.02 13.05 13.79 15.28 910 2,270 3,630 9,980 1st period 23.07 13.14 10.59 9 2nd period 17.40 8.28 5.13 5.89 3rd period 16.70 4.20 4.30 3.90 4th period 19* 5.94 3.94 4.57 After 61 days 2 on commercial type ration (8,653 units) 3.47 5.66 5.96 3.98 * P<.01 compared to higher levels of vitamin A. 1 After depletion period. 2 Following 4th experimental period.

864 G. E. BEARSE, C. F. MCCLARY AND H. C. SAXENA TABLE 6.Effect of high vitamin A supplementation on incidence 1 of blood spots U.S.P. units U.S.P. units vitamin A % blood 2 spots vitamin A % blood 3 spots per lb. ration pre-exptl. pd. per lb. ration exptl. pd. pre-exptl. exptl. pd. 5,000 25.2 5,000 23.7 5,000 22.1 15,000 23.1 1 Incidence refers to percent of eggs with blood spots of all sizes found in broken out eggs. 2 Eggs from 25 days of 28-day period. 3 Eggs from last 3 days of 28-day period. floored pens 10 months previously and now consisted of approximately 163 birds each, were available for this experiment. These birds had been fed ration 5 (Table 1) containing about 5,000 U.S.P. units of vitamin A per pound. After observing blood spot incidence for 25 days, two pens were supplemented with 10,000 U.S.P. units of vitamin A per pound of feed. The other two pens were continued on the original diet (Ration 5). After 4 weeks all eggs laid on three consecutive days were broken and observed for blood spots. Results. Table 6 shows the results of this experiment. There is no indication from the data that supplementing a diet containing 5,000 U.S.P. units of vitamin A with an additional 10,000 units reduced blood spot incidence in the eggs. DISCUSSION The results of these experiments demonstrate a relationship between the vitamin A level of the diet of laying birds and incidence of total blood spots in their eggs. An increased incidence of blood spots was noted on rations containing less than approximately 1,100 U.S.P. units of vitamin A per pound of feed. The evidence presented does not indicate that the level for minimum blood spot incidence is higher than the National Research Council (1954) established requirement of 2,000 U.S.P. units of vitamin A per pound of feed. It was possible, however, to reduce the incidence of blood spots in birds depleted of vitamin A more rapidly with levels above 2,000 U.S.P. units per pound. These results were confirmed by Scott et al. (1957) who showed that low levels of vitamin A increase blood spot incidence in eggs and that minimum blood spot incidence is attained at 1,200 to 1,600 units per pound of feed. SUMMARY The results of three feeding experiments conducted with White Leghorn chickens have shown the following: 1. Blood spot incidence in eggs was increased at levels of vitamin A below 1,100 U.S.P. units per pound of feed. 2. Vitamin A levels in the range of 1,200 to 1,600 units per pound resulted in minimum blood spot incidence; no further reduction occurred with levels up to 15,000 units. 3. Birds depleted of vitamin A reserves decreased in percent blood spots more rapidly when fed diets containing 3,600 and 10,000 units of vitamin A per pound than when fed diets containing 2,300 units per pound. ACKNOWLEDGMENTS The authors gratefully acknowledge the assistance of V. L. Miller in vitamin A determinations of livers and the assistance and suggestions of L. R. Berg, J. G. Darroch and T. R. Russell in statistical analysis of the data. Furthermore, they are indebted to the Nopco Chemical Co. and Charles Pfizer and Co. for vitamin A supplements. REFERENCES Bearse, G. E., and M. W. Miller, 1940. Unpublished data of Western Washington Experiment Station, Puyallup. Bearse, G. E., C. F. McClary and H. C. Saxena, 1953. Blood spot incidence and the vitamin A level of the diet. Poultry Sci. 32: 888. Carver, J. S., and W. Henderson, 1948. The

BLOOD SPOTS AND VITAMIN A 865 effect of rutin and ascorbic acid and of alfalfa on blood and meat spots in hens' eggs. Poultry Sci. 27: 656. Denton, C. A., 1947. Observations on the incidence and characteristics of blood and meat spots in hens' eggs. Poultry Sci. 26: 272-276. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Gallup, W. D., and J. A. Hoefer, 1946. Determination of vitamin A in liver. Ind. Eng. Chem. (Anal, ed.) 18: 288-290. Nalbandov, A. V., and L. E. Card, 1944. The problem of blood clots and meat spots in chicken eggs. Poultry Sci. 23: 170-180. Nalbandov, A. V., and L. E. Card, 1947. The problem of blood clots and meat spots in HILE no research has been done on the effect of light on fowl spermatozoa, the effects of ultraviolet and "visible" light irradiation on the sperm and eggs of the sea urchin have been studied. Irradiation of the fertilized egg of the sea urchin with ultraviolet light inhibits the rate of cell division (Giese, 1938). Cleavage delay also occurs if the gametes are irradiated prior to fertilization (Giese, 1939; Blum et al., 1949; Marshak, 1949). Giese (1939) and Marshak (1949) both reported the spermatozoa to be more sensitive to the effects of ultraviolet radiation than were the eggs. The delay in cleavage induced with ultraviolet light could be reduced by treatment with intense "visible" light (Blum et al., 1949; Marshak, 1949; Wells and Giese, chicken eggs. II. Its importance in poultry flocks, and a study of the nutritional factors involved. Poultry Sci. 26: 400-409. National Research Council, 19S4. Nutrient requirements for domestic animals. No. 1 Nutrient requirements for poultry. Sauter, E. A., W. J. Stadelman and J. S. Carver, 1952. Factors affecting the incidence of blood spots and their detection in hens' eggs. Poultry Sci. 31: 1042-1049. Scott, M. L., F. W. Hill, L. C. Norris, G. F. Heuser, R. E. Reynolds, E. H. Parsons, Jr. and H. E. Butters, 1957. New information on the vitamin A requirements of chickens, ducks, and pheasants. Proceedings 1957 Cornell Nutrition Conference, pp. 132-136. The Effect of "Visible" Light on the Fertilizing Capacity of Fowl Spermatozoa* W. G. HUNSAKER AND J. R. AlTKEN Biochemistry and Nutrition Unit, Animal Research Institute, Central Experimental Farm, Ottawa, Canada (Received for publication October 5, 1959) 1950). Wells and Giese (1950) also reported, however, that "visible" light was inj'urious to sea urchin sperm, the inj'ury in this case being a reduction in motility and fertilizing capacity. Eggs fertilized with sperm irradiated with visible light also showed a delay in cleavage. During normal artificial insemination practices it is very unlikely that the spermatozoa would be subjected to ultraviolet irradiation sufficient to be inj'urious. Exposure to "visible" light, however, may be inj'urious to fowl sperm in a manner similar to that observed with sea urchin sperm. It seemed advisable, therefore, to design an experiment to study the effect of "visible" light on the fertlizing capacity of fowl spermatozoa. * Contribution No. 10, Animal Research Institute, Research Branch, Canada Department of Agriculture, Ottawa. PROCEDURE A group of 12 New Hampshire cockerels were ej'aculated under conditions of almost