- Email: [email protected]
Fractionation of the Bovine Ejaculate1
TECHNICAL NOTES
represent daily milligram conjugate quantities (again in terms of equivalent MC-A-600), respectively, of 27, 2.8, 24, and 1.5. The concentration of 1-naphthol conjugates observed in the urine of a Holstein cow fed 450 ppm of Sevin was about 65 ppm calculated as 1-naphthol (3). REFEREI~CES
(1) GUTEN~IANN, W. H., HARDEE, D. D., HOLLAND, R. F., AND LISK, D..]-. Disappearance
of 4-(2,4-dichlorophenoxybutyric) Acid Herbicide in the Dairy Cow. J. Dairy Sci., 46: 991. 1963. (2) GUTENI~IANI~,W. H., AND LISK, D. J. Gas
FRACTIONATION
823
Chromatographic Residue Determination of Sevin as Brominated 1-Naphthyl Acetate. J. Agr. Food Chem. (In press.) 1964. (3) WHITEHURST,W. E., BISHOe, E. T., CRITCH~IELD, F. E., GYRISCO, G. G., HUDDLESTON, E. W., ARNOLD, H., AND LISK, D..]-. The
Metabolism of Sevin in Dairy Cows. J. Agr. Food Chem., 11: 167. 1963. W. H. GUTENYIANN D. G. WAGNER D. J. LISK Departments of Entomology and Animal Husbandry Cornell University Ithaca, New York
O F T H E B O V I N E E J A C U L A T E '1
The contribution of the accessory sex glands to the bovine ejaculate has been investigated (2, 4, 5) to determine the relationship of endocrine patterns to accessory sex gland function. The role of the accessory sex glands in reproduction is not well-defined. Alterations in the function of these glands may or may not be related to fertility. Separating the ejaculate into well-defined components would enhance our knowledge of accessory gland function. The bull has presented a problem in this respect, since the bovine ejaculatory process is said to be instantaneous (6). Lutwak-Mann and Rowson (6) were able to collect two well-defined fractions of bull semen using electric stimulation, but felt that it was not feasible to collect separate fractions by means of the artificial vagina. This study was conducted to determine the possibility of fractionating bovine semen during collections with the artificial vagina and to compare these to fractions collected by electroejaculation. MATERIALS AND ]~ETt{ODS
Semen was collected by both methods from three mature dairy bulls. Eighteen ejaculates were obtained with the artificial vagina (AV) and nine with the electroejaculator ( E E ) . Twelve AV samples were collected before and six were collected following the 9 EE samples. The collection interval was 1 wk. During AV collections each bull was subjected to intensive teasing before allowing a mount. During this period a presperm fraction was collected with a tong-handled device equipped with a director cone and collection vial. The sperm-rich fraction was obtained with a standard artificial vagina. A post-sperm fraction was collected with the long-handled Published with the approval of the Director of the Colorado Agricultural Experiment Station as Scientific Series Paper No. 948. This investigation was supported by a PHS Research Grant, GM 07120.
device as the bull dismounted. An electronic ejaculator (Marden) with a rectal electrode was used to obtain the EE samples. The presperm fraction was collected as long as it remained clear. When tile ejaculate was distinctly milky in appearance, the sperm-rich fraction was collected separately. A post-sperm fraction was collected as the sperm density decreased. All samples were frozen in dry ice within 5 rain of collection. Sperm cell comlts were determined in a hemocytometer. The colorimetric method of Roe (9) as adapted by Mann (7) was used for fructose determinations. The nitrogen content of whole fractions was analyzed by Kjeldahl's method. Free amino acids were measured by the method of Hopwood and Gassner (5). The p H was measured with a Beckman p H meter, using a single-drop hydrogen electrode. RESULTS AND DISCUSSIOI~ The data in Table 1 for the presperm fractions collected by both methods were similar to those of Lutwak-Mann and Rowson (6), in that the concentrations of cells, nitrogen, and fructose were low. These values and the free amino acid concentrations markedly increased in the sperm-rich fluid. Values for the postsperm collections obtained with the AV were intermediate between the two other fractions, but more closely resembled the presperm fluid. The hydrogen ion activities of AV samples did not differ from those collected by EE in the presperm and post-sperm fractions. Spermrich samples collected by E E were more alkaline than those collected by the AV method (P < 0.01). This finding and significant differences between the two methods of collection of the three fractions for sperm count, fructose, and nitrogen indicate an unnatural admixing of seminal fluids during EE collections. The three fractions were more distinctly separated by the AV method. I t appears that only the sperm-rich fraction
JOURNAL
824
DAIRY
OF
TABLE
SCIENCE
1
Characteristics of three fractions of bull semen collected by artificial vagina (AV) or by electroej aculation ( E E )
Presperm
Sperm-rlch AV
Post-sperm
AV
EE
EE
AV
EE
4÷ 2 18
8± 4 9
],062 ± 124 16
572 ± 80 ~ 9
19 ± 8 5
3± 2 18
76 ± 38* 9
602 ± 50 16
444 ± 77 9
139 ± 64 5
503 ~- 85 ~* 9
11 ± 3 18
27 ± 6 ~ 9
59 ± 13 15
48 ± 10 9
38 ± 13 4
49 -~ 11 9
47.6 ± 7.0 4 7.80 ~ 0.05 5
50.4 ± 13.1 9 7.63 ± 0.16 9
Sperm count
(miltions/ml)
No. samples Fructose
(mg/lO0 ml)
No. samples Nitrogen
(~g/ml)
No. samples
132 ± 65 9
Free amino acids
(mg/l~)O ml) 17.4 ~- 6.9 27.2 No. samples 14 pit 8.45 ± 0.10 7.97 No. samples 18 * Significant from AV collection ** Significant from AV collection ± Refers to standard deviation.
± 9 ± 9 at at
8.5
86.0 ± 11.1 12 0.45 6.73 ± 0.09 16 5% level. 1% level.
would be deposited in the female g e n i t a l t r a c t d u r i n g n a t u r a l service. I t was possible to collect large volumes of p r e s p e r m fluid with the A V method. I t was n o t possible to p r o l o n g collection of the p r e s p e r m fluid d u r i n g E E collections, because these fluids were p r o g r e s sively c o n t a n l i n a t e d b y other, s p e r m - c o n t a i n i n g fluids. No a t t e m p t s were made to subdivide the pre- o r p o s t - s p e r m f r a c t i o n s d u r i n g electroejaculation. The t e c h n i q u e of o b t a i n i n g p o s t - s p e r m fractions a t t e n d a n t to A V collection was difficult, since the p e n i s was r a p i d l y r e t r a c t e d a n d posit i o n i n g of the collection device was a problem. F l u i d s successfully collected h a d c o n c e n t r a t i o n s of cells, n i t r o g e n , a n d a m i n o acids two to five tinms g r e a t e r in the p o s t - s p e r m t h a n in the p r e s p e r m fractions. M a n n (8) m e n t i o n e d t h a t L u t w a k - M a n n a n d Rowson (6) h a d o b t a i n e d a p o s t - s p e r m f r a c t i o n which r e p r e s e n t e d a n a h n o s t p u r e secretion o f the v e s i c u l a r glands, with a c h a r a c t e r i s t i c a l l y h i g h c o n t e n t of fructose, citrate, a n d 5-nucleotidase. O u r results i n d i c a t e t h a t the vesicular g l a n d s actively cont r i b u t e to the p o s t - s p e r m h ' a c t i o n b u t do n o t g r e a t l y influence the p r e s p e r m f r a c t i o n . These results e x p l a i n p r e v i o u s l y r e p o r t e d discrepancies between A V a n d E E collections (1, 4 ) . I t h a d b e e n observed t h a t v o l u m e a n d p H were g r e a t e r in samples collected by E E , whereas cell c o n c e n t r a t i o n a n d f r u c t o s e levels were g r e a t e r in A V collections. D u r i n g A V collections the bull s e p a r a t e s the fractions. On the o t h e r h a n d , the various sex g l a n d s are s t i n m l a t e d more or less s i n m l t a n e o u s l y d u r i n g E E colleetions~ m a k i n g i t difficult to o b t a i n the same degree of f r a c t i o n a t i o n . H i l l et al. (3), r e p o r t i n g on the s p e r m - r i c h fraction~ f o u n d only a slight difference in p H , b u t the
67.6 --~ 13.3 9 7.30 ± 0.11 ~ ' 9
volume of t h e i r selected f r a c t i o n was less t h a n the volume of the same f r a c t i o n collected with the A V . I t is a p p a r e n t t h a t the f r a c t i o n a t i o n of bovine semen with the A V is s u p e r i o r to f r a c t i o n s o b t a i n e d b y electrostinmlation. LLOYD C. FAULK~ER Animal Reproduction Laboratory AND J. F. MASKEN AI~D M. L. HOPWOOD E n d o c r i n e Section Colorado S t a t e U n i v e r s i t y F o r t Collins REFERENCES (1) AUSTIN, ,]'. W., HUPP, E. W., AND MURPHREE, 1~. L. Comparison of Quality of Bull Semen Collected in the Artificial Vagina and by Eleetroejaculation. J. Dairy Sei., 44: 2292. 1961. (2) HESS, E. A., LUDWICK, T. M., MA•TIG, R. C., AND ELY, F. Influence of Seminal Vesiculectomy on Certain Physical and Biochemical Properties of Bovine Semen. J. Dairy Sci., 43: 256. 1960. (3) HILL, H. J., SCOTT, F. S., HOI~IA]'~, N., AND GASSNEE, F. X. Eleetroejaculation i~l the Bull. J.A.V.M.A., 128: 375. 1956. (4) I-IoPwOOD, M. L., FAULI~NEI~, L. C., AND GASSNER~ F. X. Effect of Exhaustive Ejaculation on Composition of Bovine Semem J. Dairy Sci., 46: 1409. 1963. (5) HOPWOOD, M. L., AND GASSNER, F. X. The Free Amino Acids of Bovine Semen. Ferti]. & Steril., 13: 290. 1962. (6) LUTWAK-MANN, C., AND ROV~SON, L. E. A. The Chemical Composition of the Pre-sperm Fraction of Bull Ejaculate Obtained by
825
T E C H N I C A L NOTES
Electrical Stimulation. J. Agr. Sci., 43: 131. 1953. (7) MA~N, T. Fructose Content and Fructolysis in Semen. Practical Application in the Evaluation of Semen Quality. J. Agr. Sci., 38: 323. 1948.
PRICE
(8) MAN~, T. The Biochemistry of Semen. John Wiley & Sons, Inc., New York. 1954. (9) ROE, J. H. A Colorimetric Method for Deterruination of Fructose in Blood and Urine. J. Biol. Chem., 107:15. 1934.
SCHEDULE FOR REPRINTS OF PAPERS THAT THE JOURNAL OF DAIRY SCIENCE
APPEAR
IN
C. J. C~VSE~ Treasurer Box 327, ~ontieello, Illinois 61856 The Executive Board, at the time of the Annual Meeting of the American Dairy Science Association at the University of Wisconsin, June, 1961, increased the price of reprints 25%, effective J u l y 1, 1961. The new reprint schedule follows :
ceived within 30 days after the papers are published in the JOURNAL; otherwise, the type will have been destroyed. In case the original type has been destroyed, it is possible to supply reprints by a special photographic process, and their cost will be
Number of pages No. of reprints 50 100 200 300 400 590 600 700 800 900 1,000
2 17.50 20.00 22.50 [email protected] 30.00 33.75 37.50 41.25 45.00 ¢8..75 57.25
4 20.00 22.50 28.75 33.75 40.00 45.00 51.25 56.25 62~.50 6,7.59 73.75
8 36.95 41.25 51.25 62.50 72.50 83.75 93.75 105.00 115.0'0 126.25 136.25
12
16
20
(Cost ~n dollars) 51.95 67.50 78.75 51.25 77.50 92.50 7 6 . 2 5 9 7 . 5 0 117.50 9 1 . 2 5 117.50 143.75 1 0 7 . 5 0 13.7.50 1 7 0 . 0 0 1 2 2 . 5 0 157.50 195.0'0 137.50 177.50 221.25 153.75 197.50 246.25 168.75 218.75 272.50 1 8 5 . 0 0 2~8.75 2 9 8 . 7 5 2 0 0 . 0 0 2 5 8 . 7 5 323.75
I f covers for reprints are desired, the cost of 50 covers will be $12.18, and for each additional 100 covers, the cost will be $8.75. Back copies of the JOURNAL will cost $2 each. The reprints are made from standing type within 30 days after the papers appear in the JOVR~AL. Requests for a few reprints of a pa pe r should be sent to the authors, whose names and addresses appear with the title. The Secretary and the Editor's office do not keep supplies of the various reprints. Orders f o r large numbers of reprints should be sent to The Garrard Press, 510 North Hickory Street, Champaign, Illinois. These orders must be re-
24
28
3~
97.50 112.50 143.75 173.75 205.0.0 236.25 266.25 297.50 328.75 358.75; 390.00
115.00 13~.59 162.75: 205.00 241.25 277~25 313.75 350.0'01 3186.25 422'.50 498.75
125.00 145.00 185.00 226.25 266.25 306.25 346.25 387.50 427.50 467.50 507.50
50% more than the regular ones. F o r example, 100 reprints of 32 pages will cost $217.50. It is hoped that the publication of this reprint schedule will make it easier for interested people to obtain reprints in any number desired and, at the same time, aid in disseminating useful information to the dairy and related industries. The JOUR~AT. OF DAIRY SCIEI~'CE is copyrighted. Reproduction of complete papers from this JOURNAL by any organization is not permitted. The reproduction of graphs, tables, and illustrations for books and other periodicals may be authorized by the Editor-in-Chief.
represent daily milligram conjugate quantities (again in terms of equivalent MC-A-600), respectively, of 27, 2.8, 24, and 1.5. The concentration of 1-naphthol conjugates observed in the urine of a Holstein cow fed 450 ppm of Sevin was about 65 ppm calculated as 1-naphthol (3). REFEREI~CES
(1) GUTEN~IANN, W. H., HARDEE, D. D., HOLLAND, R. F., AND LISK, D..]-. Disappearance
of 4-(2,4-dichlorophenoxybutyric) Acid Herbicide in the Dairy Cow. J. Dairy Sci., 46: 991. 1963. (2) GUTENI~IANI~,W. H., AND LISK, D. J. Gas
FRACTIONATION
823
Chromatographic Residue Determination of Sevin as Brominated 1-Naphthyl Acetate. J. Agr. Food Chem. (In press.) 1964. (3) WHITEHURST,W. E., BISHOe, E. T., CRITCH~IELD, F. E., GYRISCO, G. G., HUDDLESTON, E. W., ARNOLD, H., AND LISK, D..]-. The
Metabolism of Sevin in Dairy Cows. J. Agr. Food Chem., 11: 167. 1963. W. H. GUTENYIANN D. G. WAGNER D. J. LISK Departments of Entomology and Animal Husbandry Cornell University Ithaca, New York
O F T H E B O V I N E E J A C U L A T E '1
The contribution of the accessory sex glands to the bovine ejaculate has been investigated (2, 4, 5) to determine the relationship of endocrine patterns to accessory sex gland function. The role of the accessory sex glands in reproduction is not well-defined. Alterations in the function of these glands may or may not be related to fertility. Separating the ejaculate into well-defined components would enhance our knowledge of accessory gland function. The bull has presented a problem in this respect, since the bovine ejaculatory process is said to be instantaneous (6). Lutwak-Mann and Rowson (6) were able to collect two well-defined fractions of bull semen using electric stimulation, but felt that it was not feasible to collect separate fractions by means of the artificial vagina. This study was conducted to determine the possibility of fractionating bovine semen during collections with the artificial vagina and to compare these to fractions collected by electroejaculation. MATERIALS AND ]~ETt{ODS
Semen was collected by both methods from three mature dairy bulls. Eighteen ejaculates were obtained with the artificial vagina (AV) and nine with the electroejaculator ( E E ) . Twelve AV samples were collected before and six were collected following the 9 EE samples. The collection interval was 1 wk. During AV collections each bull was subjected to intensive teasing before allowing a mount. During this period a presperm fraction was collected with a tong-handled device equipped with a director cone and collection vial. The sperm-rich fraction was obtained with a standard artificial vagina. A post-sperm fraction was collected with the long-handled Published with the approval of the Director of the Colorado Agricultural Experiment Station as Scientific Series Paper No. 948. This investigation was supported by a PHS Research Grant, GM 07120.
device as the bull dismounted. An electronic ejaculator (Marden) with a rectal electrode was used to obtain the EE samples. The presperm fraction was collected as long as it remained clear. When tile ejaculate was distinctly milky in appearance, the sperm-rich fraction was collected separately. A post-sperm fraction was collected as the sperm density decreased. All samples were frozen in dry ice within 5 rain of collection. Sperm cell comlts were determined in a hemocytometer. The colorimetric method of Roe (9) as adapted by Mann (7) was used for fructose determinations. The nitrogen content of whole fractions was analyzed by Kjeldahl's method. Free amino acids were measured by the method of Hopwood and Gassner (5). The p H was measured with a Beckman p H meter, using a single-drop hydrogen electrode. RESULTS AND DISCUSSIOI~ The data in Table 1 for the presperm fractions collected by both methods were similar to those of Lutwak-Mann and Rowson (6), in that the concentrations of cells, nitrogen, and fructose were low. These values and the free amino acid concentrations markedly increased in the sperm-rich fluid. Values for the postsperm collections obtained with the AV were intermediate between the two other fractions, but more closely resembled the presperm fluid. The hydrogen ion activities of AV samples did not differ from those collected by EE in the presperm and post-sperm fractions. Spermrich samples collected by E E were more alkaline than those collected by the AV method (P < 0.01). This finding and significant differences between the two methods of collection of the three fractions for sperm count, fructose, and nitrogen indicate an unnatural admixing of seminal fluids during EE collections. The three fractions were more distinctly separated by the AV method. I t appears that only the sperm-rich fraction
JOURNAL
824
DAIRY
OF
TABLE
SCIENCE
1
Characteristics of three fractions of bull semen collected by artificial vagina (AV) or by electroej aculation ( E E )
Presperm
Sperm-rlch AV
Post-sperm
AV
EE
EE
AV
EE
4÷ 2 18
8± 4 9
],062 ± 124 16
572 ± 80 ~ 9
19 ± 8 5
3± 2 18
76 ± 38* 9
602 ± 50 16
444 ± 77 9
139 ± 64 5
503 ~- 85 ~* 9
11 ± 3 18
27 ± 6 ~ 9
59 ± 13 15
48 ± 10 9
38 ± 13 4
49 -~ 11 9
47.6 ± 7.0 4 7.80 ~ 0.05 5
50.4 ± 13.1 9 7.63 ± 0.16 9
Sperm count
(miltions/ml)
No. samples Fructose
(mg/lO0 ml)
No. samples Nitrogen
(~g/ml)
No. samples
132 ± 65 9
Free amino acids
(mg/l~)O ml) 17.4 ~- 6.9 27.2 No. samples 14 pit 8.45 ± 0.10 7.97 No. samples 18 * Significant from AV collection ** Significant from AV collection ± Refers to standard deviation.
± 9 ± 9 at at
8.5
86.0 ± 11.1 12 0.45 6.73 ± 0.09 16 5% level. 1% level.
would be deposited in the female g e n i t a l t r a c t d u r i n g n a t u r a l service. I t was possible to collect large volumes of p r e s p e r m fluid with the A V method. I t was n o t possible to p r o l o n g collection of the p r e s p e r m fluid d u r i n g E E collections, because these fluids were p r o g r e s sively c o n t a n l i n a t e d b y other, s p e r m - c o n t a i n i n g fluids. No a t t e m p t s were made to subdivide the pre- o r p o s t - s p e r m f r a c t i o n s d u r i n g electroejaculation. The t e c h n i q u e of o b t a i n i n g p o s t - s p e r m fractions a t t e n d a n t to A V collection was difficult, since the p e n i s was r a p i d l y r e t r a c t e d a n d posit i o n i n g of the collection device was a problem. F l u i d s successfully collected h a d c o n c e n t r a t i o n s of cells, n i t r o g e n , a n d a m i n o acids two to five tinms g r e a t e r in the p o s t - s p e r m t h a n in the p r e s p e r m fractions. M a n n (8) m e n t i o n e d t h a t L u t w a k - M a n n a n d Rowson (6) h a d o b t a i n e d a p o s t - s p e r m f r a c t i o n which r e p r e s e n t e d a n a h n o s t p u r e secretion o f the v e s i c u l a r glands, with a c h a r a c t e r i s t i c a l l y h i g h c o n t e n t of fructose, citrate, a n d 5-nucleotidase. O u r results i n d i c a t e t h a t the vesicular g l a n d s actively cont r i b u t e to the p o s t - s p e r m h ' a c t i o n b u t do n o t g r e a t l y influence the p r e s p e r m f r a c t i o n . These results e x p l a i n p r e v i o u s l y r e p o r t e d discrepancies between A V a n d E E collections (1, 4 ) . I t h a d b e e n observed t h a t v o l u m e a n d p H were g r e a t e r in samples collected by E E , whereas cell c o n c e n t r a t i o n a n d f r u c t o s e levels were g r e a t e r in A V collections. D u r i n g A V collections the bull s e p a r a t e s the fractions. On the o t h e r h a n d , the various sex g l a n d s are s t i n m l a t e d more or less s i n m l t a n e o u s l y d u r i n g E E colleetions~ m a k i n g i t difficult to o b t a i n the same degree of f r a c t i o n a t i o n . H i l l et al. (3), r e p o r t i n g on the s p e r m - r i c h fraction~ f o u n d only a slight difference in p H , b u t the
67.6 --~ 13.3 9 7.30 ± 0.11 ~ ' 9
volume of t h e i r selected f r a c t i o n was less t h a n the volume of the same f r a c t i o n collected with the A V . I t is a p p a r e n t t h a t the f r a c t i o n a t i o n of bovine semen with the A V is s u p e r i o r to f r a c t i o n s o b t a i n e d b y electrostinmlation. LLOYD C. FAULK~ER Animal Reproduction Laboratory AND J. F. MASKEN AI~D M. L. HOPWOOD E n d o c r i n e Section Colorado S t a t e U n i v e r s i t y F o r t Collins REFERENCES (1) AUSTIN, ,]'. W., HUPP, E. W., AND MURPHREE, 1~. L. Comparison of Quality of Bull Semen Collected in the Artificial Vagina and by Eleetroejaculation. J. Dairy Sei., 44: 2292. 1961. (2) HESS, E. A., LUDWICK, T. M., MA•TIG, R. C., AND ELY, F. Influence of Seminal Vesiculectomy on Certain Physical and Biochemical Properties of Bovine Semen. J. Dairy Sci., 43: 256. 1960. (3) HILL, H. J., SCOTT, F. S., HOI~IA]'~, N., AND GASSNEE, F. X. Eleetroejaculation i~l the Bull. J.A.V.M.A., 128: 375. 1956. (4) I-IoPwOOD, M. L., FAULI~NEI~, L. C., AND GASSNER~ F. X. Effect of Exhaustive Ejaculation on Composition of Bovine Semem J. Dairy Sci., 46: 1409. 1963. (5) HOPWOOD, M. L., AND GASSNER, F. X. The Free Amino Acids of Bovine Semen. Ferti]. & Steril., 13: 290. 1962. (6) LUTWAK-MANN, C., AND ROV~SON, L. E. A. The Chemical Composition of the Pre-sperm Fraction of Bull Ejaculate Obtained by
825
T E C H N I C A L NOTES
Electrical Stimulation. J. Agr. Sci., 43: 131. 1953. (7) MA~N, T. Fructose Content and Fructolysis in Semen. Practical Application in the Evaluation of Semen Quality. J. Agr. Sci., 38: 323. 1948.
PRICE
(8) MAN~, T. The Biochemistry of Semen. John Wiley & Sons, Inc., New York. 1954. (9) ROE, J. H. A Colorimetric Method for Deterruination of Fructose in Blood and Urine. J. Biol. Chem., 107:15. 1934.
SCHEDULE FOR REPRINTS OF PAPERS THAT THE JOURNAL OF DAIRY SCIENCE
APPEAR
IN
C. J. C~VSE~ Treasurer Box 327, ~ontieello, Illinois 61856 The Executive Board, at the time of the Annual Meeting of the American Dairy Science Association at the University of Wisconsin, June, 1961, increased the price of reprints 25%, effective J u l y 1, 1961. The new reprint schedule follows :
ceived within 30 days after the papers are published in the JOURNAL; otherwise, the type will have been destroyed. In case the original type has been destroyed, it is possible to supply reprints by a special photographic process, and their cost will be
Number of pages No. of reprints 50 100 200 300 400 590 600 700 800 900 1,000
2 17.50 20.00 22.50 [email protected] 30.00 33.75 37.50 41.25 45.00 ¢8..75 57.25
4 20.00 22.50 28.75 33.75 40.00 45.00 51.25 56.25 62~.50 6,7.59 73.75
8 36.95 41.25 51.25 62.50 72.50 83.75 93.75 105.00 115.0'0 126.25 136.25
12
16
20
(Cost ~n dollars) 51.95 67.50 78.75 51.25 77.50 92.50 7 6 . 2 5 9 7 . 5 0 117.50 9 1 . 2 5 117.50 143.75 1 0 7 . 5 0 13.7.50 1 7 0 . 0 0 1 2 2 . 5 0 157.50 195.0'0 137.50 177.50 221.25 153.75 197.50 246.25 168.75 218.75 272.50 1 8 5 . 0 0 2~8.75 2 9 8 . 7 5 2 0 0 . 0 0 2 5 8 . 7 5 323.75
I f covers for reprints are desired, the cost of 50 covers will be $12.18, and for each additional 100 covers, the cost will be $8.75. Back copies of the JOURNAL will cost $2 each. The reprints are made from standing type within 30 days after the papers appear in the JOVR~AL. Requests for a few reprints of a pa pe r should be sent to the authors, whose names and addresses appear with the title. The Secretary and the Editor's office do not keep supplies of the various reprints. Orders f o r large numbers of reprints should be sent to The Garrard Press, 510 North Hickory Street, Champaign, Illinois. These orders must be re-
24
28
3~
97.50 112.50 143.75 173.75 205.0.0 236.25 266.25 297.50 328.75 358.75; 390.00
115.00 13~.59 162.75: 205.00 241.25 277~25 313.75 350.0'01 3186.25 422'.50 498.75
125.00 145.00 185.00 226.25 266.25 306.25 346.25 387.50 427.50 467.50 507.50
50% more than the regular ones. F o r example, 100 reprints of 32 pages will cost $217.50. It is hoped that the publication of this reprint schedule will make it easier for interested people to obtain reprints in any number desired and, at the same time, aid in disseminating useful information to the dairy and related industries. The JOUR~AT. OF DAIRY SCIEI~'CE is copyrighted. Reproduction of complete papers from this JOURNAL by any organization is not permitted. The reproduction of graphs, tables, and illustrations for books and other periodicals may be authorized by the Editor-in-Chief.