Some Amino Acid Aspects of Bovine Semen

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Some Amino Acid Aspects of Bovine Semen

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D. ROUSSEL, PH.D.,* and 0. T. STALLCUP, PH.D.

A s EARLY As 1923, Steudel and Suzuki reported free amino acid in fish semen. Tyrosine was observed in 1941 by Wagner-Jauregg in human semen. The following amino acids: glycine, threonine, alanine, leucine, isoleucine, cystine, proline, tyrosine, phenylalanine, valine, lysine, arginine, aspartic acid, and glutamic acid-were observed in seminal plasma of man in the early 1950's by Jacobson and by Lundquist. Free amino acids have been identified in the semen of the normal bulP Flipse, in the bull, and Gregoire et al., in the human, bull, and rabbit, measured high quantities of seminal glutamic-oxalacetic transaminase (GOT), while Roussel and Stallcup 15 observed significant correlations between various semen characteristics, GOT, and glutamic-pyruvic transaminase (CPT). Free amino acids are formed after ejaculation in human semen as a result of the action of the endogenous proteolytic enzymes, 7 but Gassner and Hopwood3 observed no such increase in amino acids on incubation of bovine semen. In a recent investigation, Roussel and Stallcup16 observed high activity of GOT and CPT in the various reproductive organs of the mature bovine male. Glutamic acid, aspartic acid, serine, alanine, and glycine are the most abundant free amino acids in bull semen. Hopwood and Gassner observed that glutamic acid is contributed mainly by the testes and epididymis and is related to fertility. The investigation reported below was undertaken to gain additional knowledge on the quantitative aspect of amino acids in seminal plasma and spermatozoa of bovine origin. Correct interpretation of the fundamental aspects of various chemical constituents in semen is of the utmost importance in formulating procedures to maintain the survival of spermatozoa. Numerous investigators have observed an almost complete loss of motility when spermatozoa is separated from whole semen.

From the Department of Animal Sciences, University of Arkansas, Fayetteville, Ark., with the approval of the Arkansas Agricultural Experiment Station. The authors express their appreciation to Mrs. Ella Nora Griffon and Mr. Darrel B. Bragg for their assistance. *Present address: Delta Regional Primate Research Center, Covington, La.

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& STALL CUP

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& STERILITY

MATERIALS AND METHODS

By artificial vagina, semen samples were obtained from 13 healthy young Holstein-Friesian bulls, selected on the basis of their individual semen characteristics. The collection tube receiving the first ejaculate was stoppered, placed in a waterbath at 25° C., and thereafter placed in a Styrofoam carrier and maintained at 5° C. to prevent temperature shock. Samples were examined for volume, percentage of initial progressive motility, concentration of spermatozoa per milliliter, percentage of live spermatozoa, and percentage of abnormal spermatozoa, as described elsewhere.15 Thereafter, samples were centrifuged at 2000 g for 25 min. in a refrigerated centrifuge (5o C.). Seminal plasma then was removed from the centrifuged tube by means of a blood-volume pipette. A volume of phosphate buffer (pH 6.8) equal to that of the seminal plasma was added to the spermatozoa. The suspension of buffer and spermatozoa was utilized in determining the amount of amino acids present. These samples were frozen and assayed concurrently at a later date. Samples were hydrolyzed in a 1: 10 dilution of 10% hydrochloric acid in a sealed flask under vacuum for 1 hr. at 121 o C. at 15 psi. Thereafter, samples were evaporated and suspended in a buffer of pH 2 with an antioxidant ( thiodiglycol). Samples were assayed by an automated method* which is a modification of the Piez and Morris procedure. 14 Statistical analysis of these data was in accordance with methods outlined by Snedecor. 18 For the analysis, the data of percentages of motility and live and abnormal spermatozoa were transformed to arcsins. RESULTS

The ranges of values for semen characteristics were as follows: volume, 3.1-7.0 ml.; initial progressive motility, 5-70%; concentration of spermatozoa, 158-1900 X 106 cellsjml.; live spermatozoa, 16-83%; and abnormal spermatozoa, 4-69%. The means, standard deviations, and correlation coefficients are shown in Table 1. In spermatozoa, there were significant and negative correlation coefficients between volume of semen per ejaculate and concentrations of the following amino acids: aspartic acid, glycine, arginine; in seminal plasma, aspartic acid, glutamic acid, proline, isoleucine, leucine, tyrosine, phenylalanine, and arginine. The percentage of initial progressive motility was significantly and negatively correlated with threonine and proline concentration in seminal plasma. The activity of glycine, arginine, aspartic acid, and glutamic acid in spermatozoa were significantly * AutoAnalyzer, Technicon Instruments Corp., Chauncey, N. Y.

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TABLE 1. Coefficients of Correlations Between Semen Characteristics and Semen Amino Acid Activity

SpeciAmino acid

men*

Aspartic

e

n , *

y

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s SP

Threonine

-

a m f d d

333

SEMEN AMINO Acms

VoL. 18, No.3, 1967

s

SP Serine

s

Glutamic

s

SP SP

Proline

s

Glycine

s

SP SP

Alanine

s

SP Valine

s

SP Cystine

s

SP Methionine

s

SP Isoleucine

s

SP Leucine

s

SP Tyrosine

s

Phenylalanine

s

SP SP

Lysine

s

SP

Histidine

s

SP Arginine

s

SP MEANS S.D.

Volume (ml.)

Concentration Motility (X 1o• cella) ( o/o)

- .53t - .53t - .17 - .41 - .25 -.51 - .43 - .66t - .19 -LOOt - .53t -.50 - .14 -.51 - .28 - .41 - .10 -.50 - .07 - .48 - .13 - .53t - .21 - .53t - .08 - .63t -.11 -.7lt - .27 - .42 - .09 .00 - .64t - .68t 5.26 .96

.07 .17 .09 .85t .21 .26 .27 .20 .24 .53t .21 .27 .27 .22 .21 .10 .25 .13 .28 .23 .26 .18 .23 .27 .25 .35 .24 .15 .23 .07 .01 .18 .28 .31

34.76 13.31

.72t .05 .17 .10 .43 .09 .67t .00 .15 .07 .61t .04 .27 .15 .52 .02 .25 .05 .18 .15 .24 .20 .34 .06 .16 .10 .18 .19 .46 .05 .25 .10 .66t .08

789.58 462.98

Live cells ( o/o)

.03 .28 .12 - .34 - .21 .39 .18 .28 .25 - .7lt

-

.11 -

-

-

.38 .24 .34 .22 .20 .23 .28 .25 .32 .23 .32 .22 .40 .22 .42 .22 .26 .23 .22 .00 .30 .22 .46

44.76 14.54

Means S.D. (12 Abnormal samples, (mg./ cells mg./ml.) ml.) ( o/o)

.25 .22 .25 .47 .09 .24 .19 .20 .26 .39 .15 .18 .04 - .25 .13 .29 .02 .10 .02 .29 .02 .16 .04 .18 .02 .21 - .02 .38

.11 .30 - .17 - .15 - .05 - .32 24.96 14.46

*S indicates spermatozoa free of seminal plasma; SP, seminal plasma. tSignificant correlation (p 0.05). 0,01). +Significant correlation (p

< <

.387 .616 5.236 3.498 .275 .236 .384 .865 .422 .384 1.674 1.015 .408 .535 3.985 2.723 .417 .338 1.969 1.221 .144 .232 .982 1.496 .778 .440 .764 1.321 .142 .142 .639 1.046 .447 .206 .395 .613 .593 1.777 .577 .825 .266 .143 .260 .397 .545 .415 .859 1.515 .645 1.694 .761 .948 .602 .282 .432 .531 .489 .470 2.559 1.437 .264 .190 .508 .559 .298 .540 1.612 .800

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correlated with concentration of spermatozoa. A significant and negative correlation was observed between seminal plasma proline content and percentage of live spermatozoa. However, no significant correlations were found between percentage of abnormal spermatozoa and the amino acid contents of spermatozoa and seminal plasma. To facilitate a more equitable comparison of cellular amino acid activity between samples, the amino acid values were adjusted to a common basis of cell number ( 1000 X 106 cells jml.). Table 2 shows the relationships between adjusted amino acid values and the semen characteristics observed. The activities of arginine and aspartic acid in spermatozoa were significantly and negatively correlated with volume of semen, as were those of proline, tyrosine, and phenylalanine in seminal plasma. As observed previously in spermatozoa, proline and threonine ( -0.58 and -0.85, respectively) were significantly and negatively correlated with the percentage of initial progressive motility. There was a considerable improvement in the correlation between amino acids and concentration of spermatozoa. Extremely highly significant correlations were observed between threonine and proline ( -0.95 and --0.68, respectively), and percentage of live spermatozoa. No correlation was observed between amino acids and percentage of abnormal spermatozoa. Correlation coefficients were determined, to express the interrelationships of the amino acids activity observed in spermatozoa free of seminal plasma (Table 3) and in seminal plasma (Table 4). Generally speaking, most of the amino acids investigated in spermatozoa are interrelated, with the exception of aspartic acid, glutamic acid, threonine, proline, glycine, and arginine. Also, a majority of the amino acids in seminal plasma were significantly interrelated. DISCUSSION

It has been suggested that motile life span can be greatly extended simply by the addition of an amino acid to the suspending fluid, as well as by addition of certain proteins. Krampitz and Doepfmer reported that the total amino acid content of the seminal plasma from normal human semen was 1257 mg.j100 ml., as compared with 454 in azoospermic semen, 208 in aspermic semen, and only 35 mg. j100 ml. in normal blood plasma. These values are in general agreement with the data reported above. It has been reported 3 that the free amino acids of the castrated bull are depleted by castration and restored, in part, by parenteral administration of testosterone propionate. The activity of testosterone has been shown to vary among adult males. The presence of various free amino acids in rat

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SEMEN AMINO Acms

VoL. 18, No. 3, 1967

TABLE 2. Coefficients of Correlations Between Semen· Characteristics and Adjusted Semen Amino Acids Activity (Based on 1000 X 106 cells/mi.)

e

d

y s

s

Amino acid

-

Aspartic

e

Specimen*

s

Methionine

s

Isoleucine

s

Leucine

s

- .53t - .19 - .12 .36 - .27 - .17 - .44 - .23 - .14 - .72t - .39 - .21 - .13 - .15 - .32 - .01 - .14 .20 .07 .12 .13 .21 - .23

SP

s

-.11

Tyrosine

SP

e d t . e e

Threonine

s

Serine

s

Glutamic

s

Proline

s

Glycine

s

Alanine

s

s a f e d -

Valine

s

d s e n n e

e n o t

Volume (ml.)

SP SP SP SP SP SP SP

Cystine

s

SP SP SP

SP

Phenylalanine

s

Lysine

s

Histidine

s

Arginine

s

SP SP SP SP

- .09 - .66t - .12 - .65t - .30 - .04 - .10 .17 - .60t - .33

Concentration Motility (XlO' cells) ( o/o)

.11 -

-

.37 .13 .85t .22 .45 .33 .40 .23 .58t .12 .41 .28 .42 .20 .41 .21 .30 .27 .44 .28 .39 .25 .46 .27 .29 .27 .17 .24 .37 .05 .43 .22 .46

.22 -.55 - .13 - .89t - .01 - .57t - .22 - .57t - .17 - .63t - .35 - .54t .08 - .56t .02 - .62t .15 - .55t .16 - .61 .07 - .53t .03 - .61

.11

-

.28 .07 .40 .01 .62t .06 .47 .34 .6lt

Live cells ( o/o)

.09 .38 .20 .95t .17 .46 .31 - .39 .28 - .68t

-

.11 -

.42 .24 .43 .16 .40 .18 .34 .24 .44 .24 .41 .21 .47 .23 .35 .23 .24 .21 .39 .04 .47 .24 .49

Meam S.D. Abnormal (12 cells samples, (mg./ mg./ml.) ml.) ( o/o)

-

.08 .12 .17 .34 .04 .13 .10 .08 .15 .34 .08 .13 .00 .15 .07 .04 .05 .04 .00 .08 .00 .12 .05 .05 .02 .30 .03 .34 .02 .10 .26 .06 .31 .19

*S indicates spermatozoa free of seminal plasma; SP, seminal plasma. tSignificant correlation (p 0.05) . ;!:Significant correlation (p 0.01).

< <

.840 8.794 .329 1.403 .484 2.780 .729 6.912 .487 3.177 .332 2.475 .523 2.141 .179 1.779 .226 1.049 .602 1.466 .171 .645 .517 2.551 .723 1.389 .335 .865 .598 4.287 .233 .894 .754 2.584

.500 7.545 .419 .866 .393 2.221 .439 6.203 .655 2.521 .199 2.076 .748 1.624 .126 1.386 .438 .930 1.756 1.321 .258 .522 .516 1.976 1.656 1.346 .584 .953 .458 3.216 .262 .752 .387 1.765

TABLE 3. Coefficients of Correlations Between Amino Acids of Spermatozoa Free of Seminal Plasma Amino acid

GluThoronine Serine tamic

.18 Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine

.41 .34

Proline

.86* .22 .89* .06 -.03 -.07 .31

*Significant correlation (p

< 0.01).

Glycine

Alanine

Valine

Oyatine

.16 .21 .48 .30 .33 .31 .06 .97* .98* .96* .12 .94* -.25 -.06 -.29 .25 -.13 -.05 -.16 .19 -.15 -.09 .93* .99* .93*

.94*

Methio- I sonine leucine

Leucine

Hist-idine

Arginine

.19 .46 .21 1.34 .31 .88* .95* 1.00* -.21 .01 -.35 -.19 -.05 -.15 -.07 .18 -.18 .89* .96* .99* .87* .98* .89* .91* .94* .99* .89* .92* 1.00* .89* .95* 1.00* .89* .98* .98* .89* .92* 1.00* .89* .93* .86*

.91* .05 .36 .85* .12 .92* .16 .41

Tyro- Phenylsine alanine Lysine

.07 .31 .19 .08 .29 .31 .31 .30 .94* .99* .97* .94* -.38 -.15 -.28 .37 -.14 -.11 -.16 .16 -.22 .02 -.10 .22 .99* .99* .99* 1.00* .88* .96* .92* .89* .99* .98* .99* .99* .97* 1.00* .99* .99* .99* .97*

.11

.11 .04 .15 .27 .04 .08 .40 .17

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semen is due to the secretory function of the prostate. 13 On the other hand, Hess et al. extirpated the bovine seminal vesicles and observed that the seminal free amino acids were not altered significantly. However, fructose and citric acid values were extremely low. Amino acids are also controlled in part by such factors as age, nutrition, and amount of androgen. Numerous reports have shown that the percentage of initial progressive motility is the best single index for the prediction of fertility. This parameter was significantly and negatively correlated with seminal plasma threonine and proline. Also, the percentage of live cells was significantly correlated with seminal plasma proline. However, the free amino acid study by Gassner and Hill revealed that the highest semen grade is related to the highest free amino acid content. Their data further indicated that poor semen quality is related to amino acid imbalance. Similar observations were noted in this study. However, it was not possible to determine which amino acids were primarily responsible for the poor semen quality. Histidine was the least significant of the amino acids investigated. Free amino acid content of seminal plasma is significant and positively correlated with fertility, as shown by Hopwood and Gassner. Knoop and Krauss claimed that 1% glycine or proline improved the efficacy of egg yolk as an extender , for bull semen; more recently it has been reported21 that glycine increases the life span of fowl and bull spermatozoa diluted in saline and balanced solutions. However, Tyler and Tanabe believed that the beneficial effect of glycine in their experiments was because of toxic trace elements in the diluent. None of the 21 amino acids added to mammalian diluent by White had any significant beneficial action on ram, bull, or rabbit spermatozoa, and some were toxic. However, his method of evaluating the beneficial effects of glycine or arginine differed from that of the other investigators: he measured motility at hourly intervals over a 4-hr. period, whereas the marked effects of these added amino acids would be evident only after prolonged storage time and would be seen primarily in terms of extended longevity rather than as increased motility. Tosic and Walton reported that tyrosine, phenylalanine, and tryptophane are toxic to bull spermatozoa as shaking in air-an effect which they attributed to the formation of hydrogen peroxide. These relationships of free amino acids observed by other workers, 8 and the present data, strongly suggest the prepotency of semen samples. Hence, amino acids in seminal plasma do have a significant physiologic function in reproduction. There appears to be no advantage in the determination of the amino acids of sperm cell and seminal plasma as a means of measuring semen quality, with the exception of proline and threonine. It is likely

TABLE 4. Coefficients of Correlations Between Amino Acids of Seminal Plasma Amino acid

GluTheronine Serine tamic

.73* Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine

.93* .67t

*Significant correlation (p tSignificant correlation (p

.94* .6lt .82*

Proline

.88* .56t .90* .78*

< 0.01). < 0.05).

Glycine

.Ll.lanine

.97* .70* .93* .90* .89*

.96* .65t .97* .86* .89* .96*

Valine

.86* .59t .80* .72* .69* .86* .81*

Oys- Methio- I sonine leucine tine

.75* .68* .72* .67* .59t .71* .66* .79*

.95* .66* .93* .88* .87* .96* .92* .90* .74*

.95* .76* .92* .86* .85* .97* .94* .76* .76* .86*

Leucine

Tyrosine

.53t .97* .25 .71* .49 .92* .87* . .57t .40 .83* .57t .97* .49 .93* .52 .91* .37 .84* .55t .95* .42 .92* .56t

Histi- .Ll. rgiPhenylalanine Lysine dine nine

.85* .6lt .81* .82* .76* .85* .78* .85* .78* .92* .72* .88* .66*

.94* .66* .87* .85t .79* .91* .91* .78* .74* .87* .94* .92* .41 .75*

.56t .54t .74* .27 .64t .6lt .68* .67* .59t .62t .63t .67* .17 .50 .62t

.92* .57t .92* .82* .7Q* .94,. .92* .87* .73* .92* .86* .94* .74* .89* .86* .67*

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that the intracellular amino acid content of spermatozoa cells is rather stable, since all of the correlations between semen characteristics and amino acids activity of sperm cells are of a rather low order. No attempt was made to correlate the sundry amino acid groups (neutral amino acids, acidic amino acids, and basic amino acids) with semen characteristics. The amino acid levels in spermatozoa were considerably lower than those observed in seminal plasma. Also, a considerable amount of variation was observed among the concentration of amino acids. Aspartic acid was the most concentrated, followed by glutamic acid observed in seminal plasma. However, tyrosine was the most active amino acid in the spermatozoa, and aspartic acid and methionine had essentially the same activity. Leakage of intracellular spermatozoa amino acids is unlikely. Previous studies in this laboratory have shown that centrifugation over 2000 g resulted in no leakage of lactic dehydrogenase from the spermatozoa. Because enzyme molecules are larger than amino acid molecules, leakage of intracellular material from the spermatozoa is attributed to the exceptionally high permeability of the sperm cells and, the combined damage of cell structure during high-speed centrifugation. Hence, the acidic amino acids in spermatozoa as well as in seminal plasma were the most concentrated and the neutral amino acids the least, with the basic amino acids intermediate. Other compounds such as ammonia, N-amino butyric, and ornithine were present in both spermatozoa and seminal plasma. Other small peaks were observed on the chromatograms but were not identified. A phenomenon probably associated with enzymic degradation of proteins is the progressive accumulation of free ammonia, which occurs in whole semen and in seminal plasma on anaerobic, as well as aerobic, incubation. This occurrence has been observed in several species. 12 •17 The significant interrelationships shown in Tables 3 and 4 do suggest that most of the amino acids found in spermatozoa and seminal plasma are interdependent. That is, these amino acids do occur in a balanced ratio, and are more or less in a constant quantity. On the other hand, the amino acids not significantly related to each other suggest their presence in an unbalanced proportion. Perhaps other, uninvestigated factors may be responsible for their variation in spermatozoa and seminal plasma. Most of the significant correlations are extremely high and several are perfect (r=l.OO). The particular physiologic mechanism for each of the amino acids correlated with motility, survival or freezability, and fertilizing capacity of bovine semen is essentially unknown. Furthermore, the general physiologic functions of these amino acids are virtually unknown.

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SUMMARY

Assays were performed for 17 amino acids from the spermatozoa and seminal plasma of 13 healthy young Holstein-Friesian bulls. The amino acid levels were considerably lower in spermatozoa than in seminal plasma. The acidic amino acids were the most concentrated, followed by the basic amino acids, and the lowest values were for the neutral amino acids. Initial progressive motility and percentage of live spermatozoa were significantly and negatively correlated with proline and threonine activity in seminal plasma. The levels of the amino acids in spermatozoa as such are of little significance in the determination ol semen quality. Delta Regional Primate Research Station Covington, La.

REFERENCES 1. FLIPSE, R. J. Metabolism of bovine semen. IX. Glutamic-oxalacetic and glutamicpyruvic transaminase activities. 1 Dairy Sci 43:113, 1960. 2. GASSNER, F. X., and HILL, H. J. Correlation of fructose content of semen and rate of fructolysis to breeding efficiency of bulls. Proc II Internat Congr Physiol Path Animal Reprod July 7-11, 1952, p. 62. 3. GASSNER, F. X., and HoPWOOD, M. L. Seminal amino acid and carbohydrate pattern of bulls with normal and abnormal testes function. Proc Soc Exper Biol Med 81:31, 1952. 4. GREGOIRE, A. T., RAKOFF, A. E., and WARD, K. Glutamic-ox:alacetic transaminase in semen of human, bull and rabbit seminal plasma. lnternat 1 Fertil 6:13, 1961. 5. HEss, E. A., LUDWICK, T. M., MARTIG, R. C., and ELY, F. Influence of seminal vesiculectomy on certain physical and biochemical properties of bovine semen. 1 Dairy Sci 43:256, 1960. 6. HoPwooD, M. L., and GASSNER, F. X. The free amino acids of bovine semen. Fertil Steril 13:290, 1962. 7. JACOBSON, L. Free amino acids in human semen. Acta Physiol Scand 20:88, 1950. 8. JUNEJA, N. L., FAULKNER, L. C., and HoPWOOD, M. L. Biochemical aspects of semen in bovine seminal vesiculities. Fertil Steril16:361, 1965. 9. KNooP, C. E., and KRAuss, W. E. Storage of bovine spermatozoa in diluents containing certain amino acids. 1 Dairy Sci 27:657, 1944. 10. KRAMPITZ, G., and DoEPFMER, R. Determination of free amino-acids in human ejaculate by ion-exchange chromatography. Nature (Lond) 194:685, 1962. 11. LuNDQUIST, F. Biochemistry of human semen, amino acids and protolytic enzymes. Acta Physiol Scand 25:178, 1952. 12. MANN, T. On the presence and role of inositol and certain other substances in the seminal vesicle secretion of the boar. Proc Roy Soc B 142:21, 1954. 13. MARviN, H. N., and AwAPARA, J. Effect of androgen on concentration of certain amino acids in the rat prostate. Proc Soc Exper Biol Med 72:93, 1949. 14. PIEz, K. A., and MoRRIS, L. A modified procedure for the automatic analysis of amino acid. Anal Chern 1:187, 1960. 15. RoussEL, J. D., and STALLCUP, 0. T. Parallelism between semen characteristics and glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase activities. 1 Dairy Sci 48:1684, 1965. 16. RoussEL, J.D., and STALLCUP, 0. T. The distribution of lactic dehydrogenase and

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17. 18. 19. 20. 21. 22. 23.

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transaminase in the genital tissue of Holstein-Friesian bulls. I Dairy Sci (in press), 1967. SHERGIN, N. P. Acidity of semen. Trans Vet Path, Orenburg Vet Inst 1:51, 1933. SNEDECOR, G. W. Statistical Methods. (ed. 5). Iowa Press, Ames, Iowa, I956. ~TEUDEL, H., and SuzuKI, K. Amino acids in fish semen. Z Physiol Chem 127:1, 1923. TosiC, J., and WALTON, A. Metabolism of spermatozoa. Biochem I 47:199, 1950. TYLER, A., and TANABE, T. Y. Motile life of bovine spermatozoa in glycine and yolk-citrate diluents at high and low temperatures. Proc Soc Exper Biol Med 81: 367, 1952. WAGNER-}AUREGG, T. "Ober das vorkommendes Keptakosans immenschlichen Sperma. Z Physiol Chem 269:56, 1941. WmTE, I. G. The effect of some seminal constituents and related substances on diluted mammalian spermatozoa. Aust I Biol Sci 7:379, 1954.

Southern Seminar in Obstetrics and Gynecology The Southern Seminar in Obstetrics and Gynecology will be held at the Oak Grove Inn, Asheville, N. C., July 23-29, 1967. Visiting faculty wi1l include Donald G. McKay, M.D., John W. Greene, M.D., Raymond H. Kaufman, M.D., and Harry C. Shirkey, M.D. Reservations for hotel accommodations should be made directly to the Oak Grove Inn. To register for the seminar, please write to WILLIAM H. RoBERTSON, M.D., 2700 Tenth Ave. S., Birmingham, Ala. 35205, or to RoBERT R. FRANKLIN, M.D., Baylor University College of Medicine, Houston, Tex. 77025.