Investigations on Thawing Frozen Bovine Spermatozoa

Investigations on Thawing Frozen Bovine Spermatozoa B. W. PICKETT, PH.D., R. C. HALL, E. W. GIBSON, M.S.

JR.,

M.S.,

J. J.

LUCAS, PH.D.,* and

is one of the many factors significantly affecting the survival of frozen bovine spermatozoa. 3 , 4.13, 16. 23 Blackshaw, Bruemmer et al., and Hafs and Elliott reported that thawing bovine semen in water maintained at 38 to 40° C. resulted in superior sperm survival, as compared to thawing at lower temperatures. According to a review by Smith this is the case with most animal cells, as would be expected theoretically. However, VanDemark et al. observed significantly greater motility in samples thawed at 5° C. as compared to those thawed at 38°; Brugman et al., O'Dell and Almquist, and Rowson found no difference in survival between spermatozoa water-thawed at 38 to 40° and those thawed at 5° C. Undoubtedly, numerous factors contribute to the lack of agreement among investigators. For example, results of work at this station 1G comparing thawing, freezing and storage methods, and extenders, showed that in only one of eight possible motility comparisons was semen thawed in ice water (2°C.) significantly better than semen thawed at 40°. Although definite proof was lacking, it appeared that within the ranges studied, there was an inverse relationship between rate of cooling and rate of rewarming. Fertility is the ultimate test of superiority of a thawing method, but unfortunately the data are limited. Arnott conducted two studies on water thawing, comparing 30° to 15° and 15° to 4°; he found a significant dif-

RATE OF REWARMING OR THAWING

From the Department of Animal Industries, University of Connecticut, Storrs, Conn. Scientific contrihution No. 110, Agricultural Experimt'nt Station. This work was supported in part hy grants-in-aid from the Chas. H. Hood Dairy Foundation, the National Association of Animal Bret'clers, and the Northeastern Council of Artificial Brt'eding Cooperatives. ·We are grateful to the New England Selective Breeding Association, especially to Mr. Orville Curtis, lahoratory manager, for the use of their facilities, and to Union Carbide Corporation, Linde Division, for lending us the Model BF-I Freezer and for supplying the liquid nitrogen. *Storrs Agricultural Experiment Station Biometrician.

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ference in 60- to 90-day per cent nonreturn (% NR) in favor of 15° C. in the first study, and a 2% NR difference in favor of thawing at 4 ° C. in the latter study. Dunn et al. observed no difference in % NR between semen thawed at 40° as compared to semen thawed at 5°. In contrast, in a limited field trial conducted by Hafs and Elliott, thawing at 40° was superior to 5° and both were better than thawing at 20°. With the increasing use of frozen semen by commercial artificial-breeding organizations, the search for a reliable method to predict fertility has been intensifiedY' 15, 18, 19 In studies conducted in this laboratory,I5 significant correlations (up to .79) between motility of frozen semen after exposure to various stress tests and 60- to 90-day % NR were demonstrated. Kelly and Hurst incubated semen at 38 ° C. after thawing in ice water, and estimated motility hourly up to 24 hr. A correlation of .582 between mean motility X hours survived and fertility was obtained. Roussel et al. 18 , 19 confirmed the relationship between fertility and motility after incubation at 38° c., as evidenced by a correlation of .79 after incubation for only 5 hr. tI The experiments reported herein were conducted in an effort to define more clearly the parameters of thawing frozen semen and to investigate the possibility of employing some of the thawing methods to predict the potential fertility of frozen semen. MATERIALS AND METHODS Experiment 1

Two ejaculates from each of 5 Holstein bulls were collected over a period of 5 weeks. Immediately after collection, sperm concentration was determined photometrically and per cent progressive motility (% PM) estimated microscopically. Heated, whole homogenized milk and egg yolk-citrate (EYC) extenders were prepared as previously outlined. 2 A sufficiently large number of cells were added to the nonglycerol-containing fraction (one-half of the total volume) of each extender at 32° C., to provide a final concentration before freezing of 20 million motile spermatozoa per milliliter. The partially extended semen was cooled to 5° C. over a 3to 3.5-hr. period and the remaining one-half of the extender, containing glycerol, added in four equal parts at I5-min. intervals. The extended semen was equilibrated at 5° C. for 15 hr., hermetically sealed in glass ampules, and placed on six-ampule canes for freezing. The canes were randomly divided for freezing by (a) manual addition of crushed dry ice to an isopropyl alcohol bath (DIF) and (b) a liquid-nitrogen biological

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FERTILITY

&

STERILITY

freezer. * An attempt was made to decrease the freezing chamber temperatures at 1° per minute from +5 to -15° C. and at 4° per minute from -15 to -79° C. One-half of the ampules from each extender and each freezing method was placed in dry ice-alcohol (DI) storage of -79° C. and the other one-half in liquid nitrogen (LN) at -196° C. Two ampules from each treatment were thawed in ice water (mean temperature 1 ° c., range 0 to 2°) and two ampules in tap water (mean 15° c., range 14 to 16°); the contents were poured into 3-ml. test tubes and an aliquot taken for microscopic evaluation by two observers. Time allowed for thawing in ice and tap water was 12 and 5 min., respectively. Following the post-thaw estimate the tubes were stoppered, incubated for 1 hr. at approximately 32 0 , and motility estimated again. The ampules of semen had been held at their respective storage temperatures for approximately 12 months before evaluation to permit motility to reach a plateau before study. In the analysis of variance of the data,22 bulls were considered as random variables, treatments as fixed, and bulls X treatments as error to test the effect of treatment. In addition, orthogonal comparisons were made among treatments. 22 Experiment 2

Six technicians employed by the New England Selective Breeding Association (NESBA, Woodbridge, Conn.) were divided into 2 groups. Half of the technicians inseminated cows during August 1962 with semen thawed in tap water, while the other group inseminated cows with semen thawed in ice water. The following month the treatments were reversed. Semen from all bulls of all breeds in routine use by the association was used in the study. Technicians using tap water were requested to thaw semen in an insulated container with at least 1 pint of water (maintained at 15 to 18 ° C.), allow 3 to 5 min. for thawing, and use it within 15 min. Those using ice water were instructed to keep the container well iced, allow 8 to 10 min. for thawing, and use the semen within 1 hr. Fertility data, based on 60- to 90-day % NR's from first-service inseminations, were obtained for each technician for each treatment, and only data on those ejaculates used in both treatments were accepted. A weighted ttest was used to determine the level of significance between results of the treatments. 22 *Model BF-I, Linde Co., New York, N. Y.

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Experiment 3

A single ejaculate was obtained each collection day from a Holstein bull in routine use at NESBA until a total of 10 bulls had been used. Semen was extended in EYC and processed as outlined in Experiment 1, except that a dilution of 1: 4 (semen: extender) was used for the initial cooling from 32 to 5° C.,14 and a final concentration of 37.5 million progressively motile cells per milliliter of extended semen was employed. Freezing was accomplished in a liquid nitrogen semen freezer, * with a programmed freezing rate as previously described. 6 When -79° C. was reached in the freezing cycle, 12 ampules were quickly removed and placed in DI storage, then the temperature of the remaining ampules was lowered at approximately 13° per minute to -196° C. and transferred to LN storage. Approximately 24 hr. later, two ampules from each storage method were thawed in (a) ice water (mean temperature 1° c., range 0 to 2°); (b) tap water (mean 15° C., range 13 to 16); (c) alcohol (mean 1 ° C., range -1 to 4 0); (d) air (mean 20° c., range 14 to 25° ); and (e) warm water (mean 40° C., range 38 to 40°). The time allowed for thawing was 12, 5, 40, 30, and 1.33 min., respectively. Immediately after thawing 2 ampules from each treatment were poured into 3-ml. test tubes and motility determined (0 hr.). The tubes were stoppered, placed in a water bath maintained at approximately 32°, and motility estimates made every hour for 7 hr. or until a reading of 0 was ob· tained for 2 consecutive hours. Frozen semen from each ejaculate not used in the laboratory study was shipped to technicians, thawed according to our previously described method,14 and used for breeding cows. The 60- to 90-day % NR was obtained on each ejaculate from November 1962 through June 1963. Any ejaculate used to breed fewer than 30 first-service cows was eliminated from the analysis. 8 Under these conditions, one ejaculate (from Bull G) had to be discarded; consequently the motility data for that bull were also eliminated when fertility was correlated with motility. The motility data were subjected to an analysis of variance essentially as described previously. Simple correlations, weighted on the basis of the number of first services per bull, were calculated on each thawing and storage procedure at each incubation time. 22 Duncan's Multiple Range Test was used to test the differences among thawing means under each storage condition. 22 *Model 3005, Frozen Semen Products, Division of Cryo-therm, Inc., Breiningsville, Pa.

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FERTILITY

PICKETT ET AL.

& STERILITY

RESULTS AND DISCUSSION Experiment 1

A significant difference in the mean per cent progressive motility (Table 1) was found between extenders (P < 0.01), storage temperatures, and freezing and thawing methods, and significant interactions between extenders and methods of freezing and between extenders and methods of storage (P < 0.001). Although survival in EYC was superior to milk when averaged for all treatments, the principal differences were due to the poorer survival of semen in milk frozen in LN, particularly after I-hr. incubation. Thus, motility of spermatozoa frozen in milk is equal to that of semen frozen in EYC when frozen at the rate used in the DI freezing and when evaluated immediately after thawing. The observation that freezing in DI was superior (P < 0.001) to LN freezing is in agreement with the data of Macpherson and Bean et al. However, more recentl y6 no difference between LN and DI freezing was found when proper freezing rates were employed. It has been shown previously that -196° C. storage results in higher revival rates than -79° C. storage and that incubation for 1 hr. at 32° C. significantly reduces motility.2, 16, 17 Thawing semen in tap water resulted in lower motilities than thawing in ice water (P < 0.001). If tap water is used to thaw frozen semen because of convenience and availability, 1 the semen should be used as soon as possible after thawing. TABLE 1. Effect of Freezing and Thawing Procedures on Progressive Motility of Spermatozoa Incubated at 32° C. for 1 hr. (Mean % for 10 Ejaculates) Storage at _79°0.

Stm'age at -196°0. DI freezing LN freezing Time after - - - - - - thawing Thawing Thawing Thawing Thawing (hr.) at 15°0. at ro. at 15°0. at ro.

LN freezing Thawing at 15°0.

DI freezing

Thawing at ro.

Thawing at 15°0.

Thawing at 1°0.

14 4

26 12

31 10

38 19

21

28

28

4

10

8

34 14

MILK EXTENDER

o 1

38 8

39 14

51 30

46 27 Eye

o

43

1

26

48 29

48 30

EXTENDER

50 34

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Experiment 2

The 60- to gO-day % NR's by thawing methods by technician are presented in Table 2. Although there was a weighted mean difference of 1.26% in favor of thawing in ice water, this difference was not significant (P > 0.05). Thus, these results are in general agreement with the 2% NR difference obtained by Arnott, between semen thawed at 4 and 15° C. and the 1.6% found by Hafs and Elliott between semen thawed at 5 and 20° C. Experiment 3

The effect of the thawing method and storage temperature on motility of spermatozoa at various hours of incubation at 32° C. is shown in Table 3. Although semen had been stored only 24 hr. in DI, sufficient deterioration had occurred to cause a significant difference (P < 0.01) in the motility when compared with that of semen stored in LN. Regardless of this difference, the linear rates of decrease in motility with incubation time did not differ between storage temperatures. On the basis of Duncan's Multiple Range Test, thawing in water at 400 or 1 ° was better (P < 0.05) than thawing in 15° water or 1 ° alcohol regardless of storage temperature, while thawing in air resulted in the lowest motilities observed (P < 0.01). The motility data are essentially in agreement with those obtained by other investigators, i.e., there was no difference between thawing in 38 0 to 40° water and in ice water, and either appears to be better than thawing at an intermediate temperature. 1. 13 The reasonably good survival obtained by the slowest thawing method, 1 ° alcohol, was surprising in view of the suggestion by Smith that slower rates of thawing reduce surTABLE 2. Effect of Thawing Temperature on Fertility of Frozen Bovine Semen Thawing at 15°0. Technician

1 2 3 4 5 6 TOTALS

Thawing at JOO.

No. of first services

60-90 0/0 NR*

No. of first services

60-90 0/0 NR*

Difference

WMDt

101 57 68 54 52 74 406

72.3 75.4 76.5 64.8 73.1 71.6 72.4

92 88 47 40 82 84 433

72.8 76.1 74.5 80.0 70.7 69.0 73.2

-0.5 -0.7 +2.0 -15.2 +2.4 +2.6 -0.8

-1.26

*0/0 NR indicates percentage of nonreturn. tWeighted mean difference.

FERTILITY & STERILITY

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648

TABLE 3. Effect of Thawing Media and Temperature and Storage Procedure on Survival of Bovine Spermatozoa During Incubation at 32° C. (Mean Progressive Motility of 10 Ejaculates) Incubation time (hr.) Storage Thawing temperature procedure*

0

1

2

.1

4

5

6

'7

])fean

WATER

40°C.

LN DI

15°C.

LN DI

1°C.

LN DI

51 51

48 45

43 40

39 38

35 34

30 29

25 22

19 15

36 34

48 45

42 36

40 35

36 29

30 25

28 16

18 13

14 10

32 26

49 45

46 44

42 40

40 37

36 34

30 29

26 22

16 14

36 33

36 32

30 30

28 20

18 12

12 8

31 27

32 26

24 20

17 13

12 6

8 4

26 22

ALCOHOL

1°C.

LN DI

48 42

40 38

38 36 AIR

20°C.

LN DI

*LN indicates at -79°C.

42 41

37 34

37 30

storage in liquid nitrogen at

_196°C.;

DI indicates storage in dry ice-alcohol

vival. Failure of these results to agree with those obtained by Blackshaw, Hafs and Elliott, and VanDemark et al. may be due to differences in freezing rates,4, 16 extenders,3 and/or dilution ratios. Table 5 shows weighted correlations between the motility of the frozen and thawed semen (Table 3) and the fertility of the bull ejaculates (Table 4) in terms of % NR. Data on Bull G are excluded. The highest correlations were obtained on semen used immediately after thawing from LN storage regardless of media, and the magnitudes of the correlations were affected by the thawing media employed. Water, regardless of temperature, gave best results, followed by air and finally alcohol. The correlation coefficients of .37 to .59 obtained at 0 hr. are in close agreement with the coefficients of .383 to .582 reported by Kelly and Hurst, and the coefficient of .505 obtained by Roussel et al./ 8 but considerably lower than the .79 obtained by others.15,19 The methods used in the above studies are rather time consuming and laborious; as shown in Table 5, similar correlations between motility and fertility can be obtained immediately after thawing with at least three of

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SPERMATOZOA THAWING

TABLE 4. Fertility of a Single Ejaculate from Each of 9 Holstein Bulls No. of return inseminations

No. of first services

Bull

182 84 121 43 60 72 79 190 136 967

A

B

C D E F H I

J TOTALS

60-90% NR*

45 28 31 8 20 23 19 47 43 264

75.3 66.7 74.4 81.4 66.7 68.0 75.9 75.3 68.4 72.8

*Percentage of non return.

TABLE 5. Weighted Correlations Between Motility and Fertility Incubation time (hr.) Thawing temperature

Storage procedure

0

2

1

5

3

-4

6

.25 .24 .38 .05 .3.5 .37

.37 .38 .34 .08 .30 .38

.19 .21 .16 -.05 .36 .29

.15 .05 .06 -.13 .16 .00

.10 .00 -.06 -.05 -.01 -.12

.44 .32

.40 .28

.33 .24

.33 .06

.10 -.05

.03 -.07

.32 .33

.34 .17

.01

.00 -.06

-.05 -.21

-.05 -.31

7

WATER

40°C. 15°C. 1°C.

LN DI LN DI LN DI

.59 .45 .59 .50 .58 .54

.37 .43 .46 .41 .54 .45

.40 .42 .29 .18 .45 .30

ALCOHOL

1°C.

LN DI

.42 .37

.39 .23 AIR

20°C. P

< 0.05 =

LN DI .60; P

< 0.10 =

.52 .42

.46 .26

.11

.52.

the thawing procedures, and nothing is to be gained (in fact, there was a loss of predictability) by prolonging the tests beyond 0 hr. lt is difficult to explain the correlation coefficients of .58 and .54 obtained at 0 hr. with ice water thawing when values of! .19 and .09 were previously obtained 17 under similar conditions, and they were in good agreement with

650

PICKETT ET AL.

FERTILITY & STERILITY

immediate post-thaw correlations reported by others.ll, 11, 14 It appears that in addition to the difficulties pointed out by Roussel et al. IIl of obtaining correlations approaching 1.0, there are factors involved in the processing procedures that affect the magnitude of the correlations.

SUMMARY 1. On the basis of motility estimates, thawing semen in ice water (1 ° C.) was found to be superior to thawing in water maintained at 15°. 2. Although differences were not statistically significant, fertility was highest when spermatozoa were thawed in ice water. 3. There was no significant difference in motility between semen thawed in water at 40° C. and semen thawed in ice water (1 ° C. ), and both methods were better than any of the others used, regardless of storage temperature. 4. Correlations of 0.4 to 0.6 were obtained between motility estimated immediately after thawing and fertility. Department of Animal Industries University of Connecticut Storrs, Conn.

REFERENCES 1. ARNOTT, W. J. Problems of artificial breeding of cattle. Australian Vet. I. 37:140, 1961. 2. BEAN, B. H., PICKETT, B. W., and MARTIG, R. C. Influence of freezing methods, extenders and storage temperatures on motility and pH of frozen bovine semen. I. Dairy Sc. 46:145, 1963. 3. BLACKSHAW, A. W. Factors affecting the revival of bull and ram spermatozoa after freezing to -79 C. Australian Vet. I. 31:238, 1955. 4. BRUEMMER, J. H., EDDY, R. W., and DURYEA, W. J. Temperature control in the low temperature preservation of spermatozoa. I. Cell. & Compo Physiol. 62:113, 1963. 5. BRUGMAN, H. H., POORE, M. E., and SCHMIDT, R. P. Investigation of motility of sperm thawed at different temperatures and time intervals. J. Dairy Sc. 41 :737, 1958. 6. CLEGG, E. D., PICKETT, B. W., and GIBSON, E. W. Non-mechanical nitrogen vapor freezing of bull semen. Storrs (Connecticut) Agri. Exper. Sta. Res. Rep. 5: 1965. In press. 7. DUNN, H. 0., HAFS, H. D., and YOUNG, G. F. Laboratory and field studies with frozen semen. I. Animal Sc. 12:893, 1953. 8. ERB, R. E., EHLERS, M. H., MIKOTA, L., and SCHWARZ, E. The relation of simple semen quality tests to fertilizing capacity of bull semen. Washington Agri. Exper. Sta. Tech. Bull. No.2, 1950. 9. ERICKSON, W. E., and GRAHAM, E. F. Factors affecting the fertility of frozen bovine spermatozoa. I. Dairy Sc. 42:520, 1959. 10. HAFS, H. D., and ELLIOTT, F. 1. Effect of thawing temperature and extender composition on the fertility of frozen bull semen. J. Animal Sc. 13:9,58, 19.54. 0

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11. KELLY, J. W., and HURST, V. Relationship between certain laboratory criteria and fertility of frozen bovine semen. Am.]. Vet. Res. 24: 136, 1963. 12. MACPHERSON, J. W. Some observations of procedures for freezing and handling bovine semen. The 25th Anniversary Issue of the Istituto Sperimentale Italiano "L. Spallanzani" Per La Fecondazione Artificiale, 1962, p. 372. 13. O'DELL, W. T., and ALMQUIST, J. O. Techniques for freezing bull spermatozoa in heated milk and preliminary breeding results. ]. Dairy Sc. 37:652, 1954. 14. PICKETT, B. W., HALL, R. C., JR., LUCAS, J. J., and GIBSON, E. W. Influence of sperm number on fertility of frozen bovine semen. J. Dairy Sc. 47:916, 1964. 15. PICKETT, B. W., MACDoNALD, W. A., GOSSLEE, D. G., and COWAN, W. A. Correlation between certain laboratory stress tests and fertility of frozen bovine spermatozoa. ]. Dairy Sc. 44: 1134, 1961. 16. PICKETT, B. W., MARTIG, R. C., and BEAN, B. H. Effect of various methods of thawing on motility of frozen bovine spermatozoa. ]. Dairy Sc. 45: 1578, 1962. 17. PICKETT, B. W., MARTIG, R. C., and COWAN, W. A. Preservation of bovine spermatozoa at -79 and -196° C. J. Diary Sc. 44:2089, 1961. 18. ROUSSEL, J. D., PATRICK, T. E., and KELLGREN, H. C. Parallelism between laboratory stress test and fertility. A. I. Digest 12(9):8,1964. 19. ROUSSEL, J. D., PATRICK, T. E., KELLGREN, H. C., and SHELWICK, J. O. Influence of nitrogen and argon gases on post-thawing motility, laboratory stress tests, and fertility of frozen bovine spermatozoa. ]. Dairy Sc. 46: 1278, 1963. 20. ROWSON, L. E. A. The storage of bull semen at low temperatures. Vet. Rec. 65:559, 1953. 21. SMITH, A. U. "Effects of Low Temperatures on Living Cells and Tissues." In Biological Applications of Freezing and Drying. Ed. by Harris, R. J. C. Acad. Press, New York, 1954. 22. SNEDECOR, G. W. Statistical Methods (ed. 5). Iowa State College Press, Ames, Iowa, 1956. 23. VANDEMARK, N. L., MILLER, W. J., KINNEY, \;Y. C. JR., RODRIQUEZ, c., and FRIEDMAN, M. E. Preservation of bull semen at sub-zero temperatures. Illinois Agri. Exper. Sta. Bull. 621, 1957.