Survival of Bull Sperm in Milk and Yolk Extenders with Added Catalase

SURVIVAL OF BULL SPEt~M IN MILK AND YOLK EXTENDERS ADDED

WITH

CATALASE

R. It. FOOTE Department of Animal Husbandry, Corne]l University, Ithaca, New York SUMMARY

After 12 days of storage at 5 C the percentage of motile sperm in CUE containing 0, 1, 10, 100, and 200 ~g of catalase per milliliter of extender was 28, 37, 44, 44, and 45, in CU-16 was 35, 43, 47, 52, and 50, in CU-16 plus chlorpromazine hydroehloride was 29, 35, 35, 41, and 43, and in 20% yolk-citrate was 24, 34, 38, 37, and 34, respectively. All levels of catalase improved sperm survival significantly (P < .01), but little additional response was observed with catalase levels higher than 10 vg/ml of extender. The concentration of catalase required to produce a maximum response was much greater than that theoretically required to eliminate any hydrogen peroxide produced. No benefieial effect of adding 20 vg of catalase per ml of skimmilk or skimmilk-glycerol extender was found. The percentage of motile sperm after 12 days of storage at 5 C averaged 47 for CU-16 extender with catalase, 11 for skimnfilk, and 12 for skimmilk with catalase. The reason for the differential response to catalase in egg yolk versus milk has not been elucidated.

Bull semen has been reported to contain little catalase (7). Tosic and Walton (12) showed that bull sperm produced H oO=, and that added eatalase was helpful in preventing I-I~O~ from depressing motility of sperm. Also, catalase had been shown to partially protect sperm cells from the harmful effects of agitation, oxygen, and exposure to light (2, 5, 9, 10, 14). But VanDemark et al. (13) found no effect of catalase on fertility of semen used principally within two days of collection. Present trends of retaining some semen in the field for longer periods of time before use could result in H~O~, causing appreciable damage to sperm cells. The studies reported here were undertaken to establish levels of catalase optimum for sperm survival in egg yolk and milk extenders. Since these studies were completed catalase has been reported by t t a f s (8) to improve fertility of bull sperm stored in homogenized whole milk containing glycerol, but not to improve fertility of sperm in egg yolk. No studies with varying levels of catalase were reported. EXPERI]~ENTAL PROCEDURE Semen obtained from bulls in the regular stud of the New York Artificial Breeders' Cooperative, Inc. was rapidly evaluated for sperm motility and concentration. Sufficient semen was Received for publication May 19, 1962.

added to the experimental extenders to give ten million motile sperm per milliliter of extender. On the average, the semen was extended at a rate greater than 1:100. Composition and preparation of the CUE and CU-16 extenders used has been described previously by Foote et al. (4, 6). Skinnnilk was prepared according to the method described by Almquist et al. (1). The final concentration of glycerol in the glycerolated skimmilk was 10% by volume. All extenders contained 1,000 units of penicillin and 1,000 t~g of dihydrostreptomycin per milliliter. The catalase used throughout was a twicecrystallized lyophilized beef liver eatalase obtained from Nutritional Biochemicals Corporation. The standard method for assaying this product changed during the investigations reported here. The same product originally listed as containing 100 units/nag later was listed as containing 3,000 units/rag. To avoid confusion, all data here are listed in terms of micrograms (tLg), rather than units of catalase. Two milliliters of experimentally extended semen was stored at 5 C in corked 2.5-ml plastic tubes. At regular intervals these tubes were mixed, subsampled, and the percentage of motile sperm determined microscopically at 430× in a stage incubator set at 37 C. The pH's of the extenders were determined with a Beckman Model G p H meter. 907

908

i~. H.

FOOTE

TABLE 1 Sperm survival in yolk extenders with added catalase (Average of ten ejaculates) CUE

CU-16

Days stored at 5 G

0

1.0

10

100

0

1.0

10

100

0

1.0

10

100

1 4 8 12

62 49 46 31

62 54 45 38

62 54 ~6 39

63 56 51 48

63 52 45 29

62 52 52 36

63 55 48 40

62 52 48 39

60 49 37 24

61 46 44 30

62 5~ 43 32

61 48 45 36

47

50

50

54

~7

50

52

50

42

45

~7

48

Average

Catalase, ~g/ml

50%EYC ~

Catalase, ~g/ml

Catalase, ~g/ml

This 50% yolk-citrate extender was the type used in earlier studies (13, 14). The statistical analyses were carried out as outlined by Steel and Torrie (11). Bulls were considered to be a random variable, and catalase levels, types of extenders, and other variables were considered to be fixed effects. RESULTS

In the first experiment, levels of catalase were chosen which might bracket some of the levels used before purified preparations were readily available commercially (12, 14). Catalase was added to CUE and CU-16, as well as a 50 % egg yolk-citrate extender used in earlier studies (13, 14), at levels of 0, 2.5, 5.0, and 10 ~g/ml of extender. Catalase significantly improved survival of sperm in ten ejaculates of semen stored for 12 days at 5 C (P < .01). The highest motility was observed in the extenders containing the most catalase. Since the upper lbnit of response did not appear to have been .established, the experiment was repeated, using a greater range of catalase concentrations. The results of this second experiment are shown in Table 1. Again eatalase improved sperm survival at all levels (P < .01). Increasing the level of catalase above 10 tLg/ml seemed to be beneficial in CUE but not in the other extenders. The 50% egg yolk-citrate extender gave significantly poorer results ( P < .01), but the motility estimations were more difficult to make in this extender. The p H of all extenders was within the range from 6.6 to 6.8. The results of a third experiment are shown in Table 2. In this experiment a higher level of catalase was added, a 20% yolk-citrate extender was included, and chlorpromazine hydroehloride was added to one extender. The latter compound was included because of the possibility of its affecting cell permeability (4). Again, catalase improved sperm survival (P < .01). The highest level of catalase was not superior to the 100 t~g/ml level, and most of the improvement was achieved by including

10 t~g/ml. Clumping of sperm was observed occasionally in the extenders containing high levels of eatalase. Sperm sm~/ival in the 20% yolk-citrate was inferior to the other extenders (P < .01). Subsequent studies revealed that catalase at 20 tLg/ml gave a uniformly good response in yolk-extenders without the clumping observed at higher levels. The effect of adding this level of catalase to skimmilk and to skimmilk-glyeerol extcnders in two experiments is shown in Tables 3 and 4. No response to catalase was observed in the skimmilk, or in the skimmilkglycerol extender. Sperm survival in the CU-16 was significantly higher than in skimmitk extender (P ( . 0 1 ) , and glycerol improved sperm survival in skimmilk (P < .01). DISCUSSIOI~

The addition of from 1 to 100 t~g of catalase per milliliter of yolk extender substantially improved the smwival of bull sperm stored at 5 C. Additional amounts of eatalase did not improve sperm survival and tended to increase clumping of sperm. The beneficial effect of catalase in sperm survival is presumably accomplished through the decomposition of H20~ by eatalase. Desjardins and t I af s (3) have reported that egg yolk contains small quantities of eatalase. The consistent response reported here with added catalase indicates that the amount present in egg yolk is insufficient for optimmn sperm survival. The amount of catalase required for maximum response is far more than should be required to decompose the I-I~O~ produced (12). Perhaps a high extracellular concentration is required to obtain a small quantity of intracellular catalase, where H..O~ production might damage the cell. However, it is not certain that catalase will penetrate the sperm cell membrane. The inclusion of ehlorpromazine hydrochloride, as a possible means of altering perme-

909

SURVIVAL OF BBULL SPERM SURVIVAL O:~ ULL SPERM

TABLE T ABLE 3

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TABLE 4 T A B L E 4~

:g ~

Sperm survival i~ skimmilk-glyeeroI with added added Sperm in skimmilk-glycerol catalase eatalase (Average of ten ejaculates) ejaculates)

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ability, increased ability, pprovided r o v i d e d no evidence for f o r increased ff ec t'Iveness of t ratIOns ' low concentrations of catalase. eeffectiveness 0f owI concen 0 f catalase. The ineffectiveness of catalase catalase ~n in the skim.The ineffecti:,en~ss of the skimmilk extender is difficult to explall. explain. The The necmIlk extender IS dIfficult essary present in milk from which essary substrate substrate is present in milk f r o m which sperm produce tt~O., H.O. in the same s p e r m cells could could produce in the same manner Hafs has rreported m a n n e r as in in egg yolk. H a f s (8) (8) has eported that catalase homogenized whole milk milk that catalase added added to homogenized containing glycerol resulted in a sizable sizable and and containing resulted in t a t'IS t'Ica11y Signi . 'fi can t improvement . t'in S llnprovemen III ffertility, er t'l't 1 1 y, statistically significant but catalase had no no effect on fertility when but catalase had f e r t i l i t y when added yolk extender, Whether added to yolk extender. W h e t h e r glycerol interacts in way with with catalase teracts in any any way catalase to improve improve fertility is unknown. I t did did not unknown. It not interact interact with with motility, The glycerol to affect motility. The beneficial effects of may be be because because glycerol can utiof glycerol may can be utilized by s p e r m to produce fructose, which in by sperm produce fructose, which in by glycolysis. glycolysis. tturn u r n sperm sperm can can utilize by The experiments experiments reported clearly demThe reported here here clearly onstrate that catalase prolongs survival bull onstrate that catalase prolongs survival of of bull sperm yolk extenders but not not in s p e r m in yolk extenders but in skimmilk. skimmilk. The reasons for this differential response, as The reasons f o r this differential response, as the reasons reasons for the apparent well as the f o r the a p p a r e n t excess of of catalase required f o r optimum optimum response in egg catalase required for response in 'dated. have not not been been elUCI yolk, have elucidated.

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Skimmilkglycerol

Skimmi]k Skimmilk

Days Days stored stored at at 5 0C

0 catalase eatalase

1 4 8 12 12

61 54 32 12 12

60 50 33 16 16

59 56 44 32 32

59 54 49 34 34

40

40

48

49

20 ~g/ml p.g/ml catalase catalase

0 catalase catalase

20 ~g/ml p.g/ml catalase catalase

(% motile rnotile sperm) sperrn)

~

~

55 49 30 12 12 3,6 36

p

't>

"Qi'

55 48 31 ]1 11 36

~

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<;-;

(% motile rnotUe sperm) sperrn)

~

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r--.?

00 II-!

55 57 57 53 47 47 53

Skimmilk

+'

lei <0

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1 4 8 12 12 Average

Skimmilk plus 20 Izg/m] p.g/ml eatalase catalase

OJ)

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btl ,...;

~

Days Days stored stored at 5,5 0C at

CU-16 plus 20 t~g/m] p.g/ml cata]ase catalase

00

Average

R.

910

H.

FOOTE

ACKNOWLEDGMENTS

The author is grateful to Miss Linda Gray, Mrs. Nina Schales, Mrs. Sally Shipman, and Mrs. Norton Roarke for assistance in obtaining and summarizing the data, and to the New York Artificial Breeders' Cooperative, Inc., for the supply of semen. I~EFERElqCES (1) ALRIQUIST, J. O., FLIPSE, R. J-., AND TItAOKER, D. L. Diluters for Bovine Semen. IV. Fertility of Bovine Spermatozoa in Heated Homogenized Milk and_ Skimmilk. J. Dairy Sci., 37: 13'03. 1954. (2) AN])EaS]~, H., A~D ROTTE~NST~V, K. The Effect of Glucose, Fructose, Catalase, and Peroxidase in the Extender on the Viability of Bull Spermatozoa. (In Danish.) Ann. Rept. Sterility Research Inst., Roy. Vet. Agr. Coll., Copenhagen, 33. 1961. (3) DI~SJARDINS, C., AND HAPS, •. D. Enzymatic Degradation of Hydrogen Peroxide in Egg Yolk. J. Dairy Sci., 44: 1183. 1961. (4) F o o ~ , R. H., AND GRAY, L. C. Effect of Promazine Hydrochloride and Chlorpromazinc Itydrokhloride on the Motility and Fertility of Bovine Semen. J. Dairy Sci., 43: 1499. 1960. (3) F o o ~ , R. H., AND GravY, L. C. Effect of Dyes, Colored Tubes, and Catalase on the Survival of Bovine Spermatozoa Agitated and Exposed to Light at 5°C. J. Dairy Sei., 43: 1889. 1960. (6) FOOT~, R. H., GarY, L. C., YouNo, D. C., AND DUNN, H. O. Fertility of Bull Semen

(7)

(8)

(9)

(10)

(11)

(]2)

(]3)

(]4)

Stored Up to Four Days at 5°C. in 20% E g g Yolk Extenders. J. Dairy Sci., 43: 1330. 1960. F o o ~ , R. H., VomT, V. T., AND SClZAL~S, N. Catalase Content of Rabbit, Ram, and Bull Semen. (Abstr.) ft. Animal Ski., 19: 1218. 1960. HAYS, H. D. Fertility of Bull Sperm with Added Catalase. J. Dairy Sci., 44: 1529. 1961. NOl~lyIAI~, C., AND GOL~)BE~, E. Effect of Light on Motility, Life-Span, and Respiration of Bovine Spermatozoa. Science, 130: 624. 1959. PI%INC~,, P. W., Alq-D ALMQUIST, J. O. The Effect of Agitation upon the Livability of Bovine Spermatozoa. J. Dairy Sei., 31: 839. 194,8. STEEL, R. G. D., AND To~m~ J. H. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. 1960. TOSlO, J., AND WALTON, A. Metabolism of Spermatozoa. The Formation and Elimination of Hydrogen Peroxide by Spermatozoa and Effects on Motility and Survival. Biochem. J . , 4~7 : 199. 1950. VAND~I~IAI~K, N. L., BI~kVTON, R. W., AND FOO~'~, R. H. The Fertility of Bovine Semen in Citrate-Yolk Extendcrs Containing Added Catalase. 3-. Dairy Ski., 33: 661. 1950. VANDE~ARK, N. L., SALISBURY, G. W., AN]) Bt~ATrI~ON, R. W. Oxygen Damage to Bull Spermatozoa and Its Prevention by Cata]ase. J. Dairy Sci., 32: 353. ]949.