A S S O C I A T I O N A F F A I R S

A B S T R A C T S O F P A P E R S P R E S E N T E D A T T H E E A S T E R N D I V I S I O N A L M E E T I N G O F T H E

A M E R I C A N D A I R Y S C I E N C E A S S O C I A T I O N

R U T G E R S - - T H E S T A T E U N I V E R S I T Y N E W B R U N S W I C K , N E W J E R S E Y

A U G U S T 3, 1966

Application of the disc theory for analysis of rennin and prorennin in vert ical slab acryl- amide gel electrophoresis. A. G. RAND, JR., Universi ty of Rhode Island, Kingston.

The disc theory of electrophoresis, developed by Ornstein and Davis, was applied to a ver- tical slab gel for the analysis of rennin and prorennin. Polyacrylamide gels of 11% con- centration were prepared in a vertical cell similar to the one designed by Raymond. A special slot form was utilized which produced openings in the top of the gel 19 mm deep with 0.19 ml capacity. A potential of 200 V was ap- plied for 30 minutes allowing the samples to migrate into the gel. The voltage was then increased to 800 V for a final period of 60 minutes.

Prorennin migrated slightly ahead of the rennin band, but the two proteins could not be completely separated. DEAE purified pro- rennin produced only one band, while crystal- line rennin showed one nmjor and two minor components.

Sources of variation affecting the relation- ship of milk protein determinations by Orange G and Kjeldahl methods. F. N. DicKinson, S. N. GAUI~T AND D. J. HANKINSON, Universi ty of Massachusetts, Amherst.

Protein determinations were made by both the Kjeldahl and Orange G Dye methods on 1150 two-milking composite samples of milk from cows of the five major dairy breeds sampled at varying stages of lactation over a period of four years. Orange G transmission readings were transformed to log~o prior to a statistical analysis which related the transmission readings to the Kjeldahls which were considered to be the true protein percentages. Linear mod- els were fitted to the two sets of data separately to estimate the effect of breed, month of lacta- tion and period of time. The periods corre- sponded to known changes in laboratory person- nel and equipment. All three sources of varia- tion had highly significant (P < .01) effects which corresponded closely in direction and relative magnitude on both estbnates of protein per cent. I t was then determined that the re- gresssion of log~0 transmission reading on Kjel- dahl was linear and did not differ significantly among breeds. A final model was fitted to the data regressing log~o transmission readings on Kjeldahl on a within breed, month of lactation, ~,~eriod basis. The resulting equation was solved

for values of protein per cent 2.00, 2.01, . . . . , 5.00, to produ?e a table which is used to con- vert transnfission readings directly to protein per cent.

Polyaerylamide-gel electrophoretic examina- t ion of the role of natural proteolytic milk enzymes in Cheddar cheese ripening. K. R. NAT]t: AND R. A. LEDFORD, Cornell University, Ithaca, New York.

Residual casein fractions of cheese and cheese milk were determined by polyacrylamide-gel electrophoresis ( P A E ) . Slight proteolytic changes in a~l- and /~-caseins were discernible with this technique. The facili ty to use si- multaneously casein standards enhanced identifi- cation of casein fractions changed by proteolysis. The appearance of a faint zone of greater mobility accompanied by a decrease of inten- si ty of the a~l-casein zone indicates the action of milk protease in milk preserved by toluene for 12 days. This change was not evident in pasteurized milk held under similar conditions. fl-casein appeared unchanged during toluene storage of both raw and pasteurized milk. a~l-Casein is degraded in raw and pasteurized cheese before fl-casein which appears resistant to proteolysis in cheddar ripening. Analysis of cultureless cheese, prepared by the addition of lactic acid indicates that rennet enzymes par- t ially hydrolyze a~-casein. Examination of the P A E patterns of raw and pasteurized cheese caseins indicates that milk protease is of little significance compared with the actions of rennet and bacterial enzymes in the proteotysis of casein in cheddar ripening.

Control of lactic streptococcal phage- associated lysins. D. R. TOURWLL~ A~D S. TO- KI~DA, The University of Vermont, Burlington.

This report concerns further studies of a lactic streptococcal phage-induced lysin (cl0- lysin) released from Streptococcu~' laetis strain C10 cells when infected with their homologous el0 phage. The cl0-1ysin has been purified approximately 600 fold by a multistep pro- cedure which included the use of the ultra- centrifuge, ammonium sulfate frationation, precipitat ion of nucleic acids with streptomycin sulfate, gel filtration and ion exchange chro- matography. The purified cl0-1ysin lost its activity when stored at room temperature and at 4 C or -- 60 C. The purified cl0-lysin was completely inactivated at 56 C for 20 minutes. The presence of reducing agents preserved, but

1572

JOURNAL OF DAIRY SCIENCE 1573

did not increase el0-1ysin activity. The cl0- lysin was inhibited by sulfhydryl binding com- pounds. This inhibition was reversed in the presence of excess 2-mercaptoethanol. The cl0- lysin was further characterized as having a molecular weight in excess of 200,000 as de- ternfined by gel filtration and a sedimentation rate of approximately 7S as determined by sucrose density gradient ultracentrifugation. Electrophoretieally, cl0-1ysin migrated as a single peak toward the cathode at pH 8.6. In- itial studies of another lactic streptococcal host-phage system revealed that a lysin (c2- lysin) is released when strain C2 cells are lysed by c2 phage. The c2-1ysin showed physico- chemical characteristics that were similar to cl0-1ysin; however, the pattern of e2 lysin activity differed from c10 lysin.

Effect of nitrogenous compounds during growth and subsequent storage on maintenance of activity of Streptococcus lactis. D. H. KING, ~ARY LEE HOLLAND AND J. A. KOBURGER, West ¥ i rg in ia University, Morgantown.

Stre'ptocoecus lactis grown in a lactose- tryptone-yeast extract medium, with and without added casein, maintained essentially the same activity during storage in phosphate buffer. The addition of casein to the storage buffer medium, however, greatly enhanced storage potential of both conditions. Cells, grown at various concentrations of lactose with increasing amounts of tryptone, were more active following storage when prior growth occurred at high nitrogen levels. Analyses of the spent buffers indicated that there was no lactate production; however, amino acids and ammonia were released by the cells during storage. To further test the hypothesis that nitrogenous compounds are im- portant for the maintenance of maximum ac- tivity, storage trials were conducted at 4°C and 22°C with various adjuncts. Nitrogenous com- pounds, notably casein, enhanced storage poten- tial while carbohydrates resulted in a more rapid loss of activity.

Determination of water content of butteroil by infrared spectrophotometry. J. ~V. SttER- BON AND J . V. ~¢[ELGAR, Department of Food Science, Cornell University, Ithaca, New York.

Up to 5% water in butteroil was found to follow a linear relation between absorbance and water content at 2.9 u and at 6.1 u, using a double beam infrared spectropho~ometer. Samples were prepared by melting butter, separathlg the phases, and drying the oil under vacuum at 100°C for three hours. Water was emulsified into samples of the dried oil to give nfixtures having 0 to 5% added water. Each sample was examined as a 10% solution, by volmne, in carbon tetraehloride. Sodium chlo- ride cells having a pathlength of 0.2 ram. were used. Each sample was run having a 10% solution of dry oil in carbon tetrachloride in the reference beam. Scattered light due to

large moisture droplets interfered with the test.

Rapid method for determining the copper content of milk. A. C. SMITH, University of Connecticut, Storrs.

The procedure involves treatment of 100 ml of nfilk in a 200 ml flask with 25 ml of 50% by weight of trichloroacetic acid, heating in boiling water for 15 rain and cooling. A 25 ml portion of the supernatant is aspirated off and transferred to a cream test bottle (extraction flask 1nay be used). The addition of 5 ml of 0.05% zinc dibenzyldithiocarbamate in toluene (carbon tetraehloride should be substituted for toluene with use of extraction flask), shaking for 10 rain, centrifuging for 5 rain, addition of deionized water to bring the toluene layer within the neck of the bottle and recentrifuging for 5 rain completes the extraction of copper. The toluene layer is poured into 1/2 in. test tubes, color intensity measured in a Spectronic 20 at 435 nm and ug of copper are obtained from a standard curve. The results are expressed as ppm after adjusting for a reagent blank, milk blank and volume of coagulated fat and protein.

The method agrees favorably with, and is not statistically different from, the dry-ashing" procedure. Analysis of 28 samples of milk to which 0.1 and 0.2 ppm of copper were added had recoveries varying from 82% to 113% and 85% to 105% with means of 99% and 97%, respectively.

Rapid method for copper analysis in milk. R. L. KI~G AND D. L. BASHORE, Department of Dairy Science, University of Maryland, College Park.

Analysis of milk for copper by a dry ashing procedure requires at least 2 days to complete. Losses due to overheating and contanfination due to long-time exposure are difficult to control. Direct analysis of TCA filtrate as reported by Olling [Neth. Milk Dairy J., 17:295 (1963)] has been evaluated and a procedure developed that is being used routinely to analyze about 20 samples of milk in duplicate in less than 6 hours by one person.

TCA (1 g/ml solution) is added to cold milk at the rate of 1 ml/]0 ml nfilk contained in a screw-top Erlemneyer flask. After thorough shaking the contents are heated for 30 minutes in a 95 ° C water bath with frequent nfixing during the first 10 minutes to mininfize clot formation. After cooling in ice water the con- tents are filtered through No. 40 Whatman paper into a 125 ml separatory funnel. The filtrate is treated in the same manner as the solution of milk ash in HC1 as described in the dry ashing procedure [J. Dairy Sci., 42:420 (!959) ].

Results obtained using this procedure are superior to the dry ashing procedure in terms of replicate analyses, recovery of added copper, control of contamination and time required.

J. DAIF~Y S('IEIqCE ~V~O~. 49. NO. 12

1574 ASSOCIATION AF~AIRS

Based on an average milk composition and recovery of added copper 107 g of TCA filtrate represents 100 g of milk.

Natural and supplemented tocopherol in the dairy ration and oxidized flavor. R. L. KtSG, H . H . TIKRITI AND MARIANNE OSKARSSON¢ De- p a r t m e n t of Dairy Science, Universi ty of Maryland, College Park.

The antioxidant properties of milk as in- fluenced by the ration are being studied using a herd of 22 Holstein cows selected as first-calf heifers. The control ration of alfalfa hay and grain produces milk uniformly susceptible to oxidized flavor induced by 1/10 p.p.m, added copper. Individual milks are evaluated weekly for their susceptibility to oxidized flavor by a flavor panel and by the TBA test. The milks are also analyzed for fat, copper, citric acid, as- corbic acid and tocopherol.

Supplementation of the basic ration with tocopherol acetate has been evaluated at several levels ranging from 1-]0 g/cow/day. An in- crease in resistance to oxidized flavor was ob- served at all levels and the magnitude was directly related to the quantity of tocopherol acetate in the ration. A direct relation was also observed between the tocopherol level in milk and the amount of added copper tolerated by the milk. Spontaneous oxidized flavor was eliminated at the lowest level supplemented. Green-chopped alfalfa and brome grass pasture produced a similar effect which appears to be directly related to the tocopherol content of the forage. When the source of tocopherol was removed from the ration the level in milk returned to that of the control in about 7 days. Tocopherol content of milk was increased over 5 fold at the highest level supplemented.

Responses of plasma calcium and parathy- roid hormone to prolonged E D T A infusion. C. F. I~AMBERG, JR., G. P. MAYER, D. S. KRON- FELD, L. M. SlqERVv~OOD, AND J . T. POTTS, JR., Universi ty of Pennsylvania, Kennet t Square, and National Hear t Institute, Bethesda, Md.

Continuous infusion of disodium EDTA at a rate of 57.5 or 40.0 m E q / h r produced a triphasic response in plasma Ca, which fell rapidly for 3-7 hr, then slowly for 4-56 hr and then rapidly again for 3-12 hr. The nlean rates of change in plasma Ca were 0.44, 0.05, and 0.20 m E q / L / h r in phases 1, 2, and 3 respec- tively in 6 experiments in which E D T A was infused at 57.5 mEq/hr . The infusions pro- duced a marked drop in milk production but the triphasic response was also seen in dry cows, ruling out decreased lactation as the sole mechanism. Plasma PTH, measured by ra- (lioimmummssay, averaged 0.92, 4.11, 3.73, and 3.90 m/xg/ml in pre-infusion samples, phase 1, 2, and 3, respectively. Plasma P T H appeared to depend on plasma Ca concentration and a scatterplot of P T H (Y,m/~g/ml) against plasma Ca(X,mEq/L) showed an inverse relationship

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(Y = 8.2 -- 1.3X, r = -- 0.52, P < 0.001). Sim- ilar lines were seen when the Ca was falling during the infusion (Y = 8.2-1.3X, r = -- 0.52, P < 0.001) and when the Ca was rising after the infusions (Y : 7.9-1.3X, r = -- 0.60, P < 0.02) indicating a graded response of P T H secretion. In a P T X cow, infused at 5.6 gm EDTA/hr , plasma P T H was not detectable and plasma Ca fell linearly without the appearance of the second phase. (Supported in p a ~ by N I H grant AM-08392 and AEC contract AT 30-1-3360).

Relation of concentrate fiber level and con- centrate urea level to ration digestibil ity and production performance in dairy cows. N . F . COLOVOS, J. B. HOLTE~, H. A. DAVIS AND W. E. U~BAN, Department of Animal Sciences and Biochemistry, Universi ty of New Hampshire, Durham.

Two experiments were carried out, each in- volving four Holstein cows in a latin square trial. In one trial the cows were in their first lactation (17 kg mean production) and in the other, mature cows (27 kg mean production) were used. Each trial commenced about one month postpartmn and continued 16 weeks. Concentrate fiber (5 vs. 8%) and urea (0 vs. 2.0% and 0 vs. 1.25 vs. 2.0 vs. 2.5%) were the treatment variables, ingredient and chemical composition of the concentrates being otherwise similar. Medium-quality, heat-dried grass hay, the sole forage, was fed at 2% of body weight. Nutrient digestibility, energy and nitrogen bal- ances, as well as rumeu and blood metabolite concentrations were studied.

Under the conditions of these experiments, milk production and butterfat percentage were not significantly changed, statistically, by the inclusion of as much as 2.5% urea in the con- centrate when the total ration was of high quality. Depression in the nutritive value of the rations containing urea was relatively small compared with the depression caused by high fiber level. Further experimentation on a large scale will be necessary to serve as a basis for practical feeding recommendations. Concen- trate and milk price economics should be inte- grated into such experiments.

Effect of stage of lactation on methylmal- onate isomerase activity in bovine liver. M.M. MAT~IAS AND J. M. ELLm~, Cornell University, Ithaca, New York.

Liver homogenates, with mitoehondria dis- rupted, were employed to study the rate of incorporation of label from propionate-1 or -22~C into methyhnalonate, succinate and other metabolic intermediates. Liver samples were obtained by biopsy f rom dairy cows represent- ing various stages of lactation and levels of milk production. Data summarized to date in- dicate great variation among cows in the rate of incorporation of label into intermediates beyond methyhnalonate. The rate was nega-

JOURNAL OF DAIRY SCIENCE ] 5 7 5

tively correlated (r : .60) with the level of milk production at the time of biopsy, and positively correlated (r---- .36) with liver vitamin B~ lev- els. The administration of vitamin B~.~ to cows appeared to increase the rate of incorporation. These findings are interpreted, tentatively, as evidence that methyhnalonyl-Co A isomerase activity is lower in the high-producing cow in early lactation than in later stages of lactation.

In vivo and in vitro monitoring of animal thyroids to evaluate environmental ~ I levels in milk. JESSE Y. HARRIS, Research Branch, DHEW, PHS, DRH, Roekville, Maryland.

I T M in thyroids of grazing animals can be measured in v i vo or .in v i t ro depending on the level present. Two lactating Holstein cows and nine lactating grade Toggenburg goats were orally dosed with carrier-free Na ~I . E:<creta ~:~I levels were determined by gamma spectro- scopy. Thyroid ~"~I levels were monitored with a portable C-M survey meter placed against the neck at the position of highest reading. One cow was sacrificed at the end of the experiment and an in v i t ro thyroid analysis made and correlated with the in v ivo measurement.

A simple model was developed to relate cattle and sheep or goat thyroid peak ~:'~I levels to peak milk levels following a single contami- nating event, namely: M ---- (K) (T) ; where (M) equals peak milk activity (pCi/L) , (T) equals peak thyroid level (pCi/g) and I( is a constant equal to 0.055 for cattle thyroids or .02 for sheep and goat thyroids. Cattle thyroid levels of approximately 2 /xCi (total) were detectable in v i vo and 1.0 pCi/g levels were

measurable i.n. vitq'o with a well crystal in con- junction with a single channel analyzer system.

In emergency situations, milk levels above 5,000 pCi/L should be predictable from simple field estimates, while in v i t ro thyroid analyses can be used to estimate milk levels of less than 0.1 pCi/L.

Goat hypothalamic electrode placement. A. W . ~'~AIIONEY, C. A. BAILE:, A~'D J. MAYER, Harvard University, Boston, Massachusetts.

Specific hypothalamie centers can be located routinely by this modified method. A goat is supported in a sling chute during surgery a~ld experimentation. After a portion of the skull is removed, a plexiglass plate, containing 61, 22-gauge stainless steel guide eannulas ar- ranged in 11 rostro-eaudal rows (5 rows of 5 and 6 rows of 6 eannulas) spaced over an area 20 mm wide by 15 mm long, is positioned on the skull so that the most rostral cannulas are 3 mm rostral to the bregma. The plate is secured with stainless steel screws and dental cement. The electrodes, 6 em long, are varnish- insulated 0.004 inch stainless steel wires with 28 gauge stainless steel tubing sheaths. After recovery and without anesthesia, electrodes are implanted 3 to 5 mm from the base of the brain. The goat's response to a 1.0 to 2.5 v, 50 cps impulse duration of 4 msee sthnulus can be immediately observed. The stimulation of eating by a satiated goat, for example, is used to locate the hypothalamic feeding center. Elec- trical activity in the hypothalamus will be discussed relative to hunger and satiety in goats.

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