EFFECT OF RATES OF FREEZING, THAWING AND LEVEL OF GLYCEROL ON THE SURVIVAL OF BOVINE SPERMATOZOA IN STRAWS 1

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1 EFFECT OF RATES OF FREEZING, THAWING AND LEVEL OF GLYCEROL ON THE SURVIVAL OF BOVINE SPERMATOZOA IN STRAWS 1 O. L. Rodriguez z, W. E. Berndtson, B. D. Ennen and B. W. Pickett Colorado State University, Fort Collins SUMMARY Extended semen from six Hereford bulls was placed in.25-ml Continental straws and frozen in the vertical position. Treatments were arranged factorially with glycerol levels of 5, 7, 9 or 11%; semen cooled from 5 to -130 C in 3.5 (fast), 20 (moderate) or 40 min (slow); and thawed in water at 5 C for 3 min, 35 C for 12 sec, 55 C for 8 sec, 75 C for 7 sec or 90 C for 5 seconds. Fast freezing resulted in greater postthaw motility than moderate (P <.05) or slow rates (P <.01), regardless of thawing method. Glycerol levels of 7 to 11% provided optimal survival when averaged over rates of freezing and thawing (P <.05). Post-thaw motility improved as the temperature of the thawing bath was increased from 5 to 55 C (P <.01). Further increases in thawing bath temperature to 90 C did not enhance survival. The post-thaw motility of spermatozoa frozen rapidly in straws and thawed at 55 to 90 C exceeded that for ampules from split-ejaculates frozen in 1.0-ml ampules (P <.01). Semen from one Angus, two Hereford and three Charolais bulls was frozen in a second study at the fast, moderate and slow rates in straws maintained in the horizontal or vertical position. The final extended semen contained 5, 7, 9 or 11% glycerol; and all semen was thawed in 75 C water for 7 seconds. Fast freezing resulted in post-thaw motility equal to 1 Animal Reproduction Laboratory, Department of Physiology and Biophysics. The advice and suggestions of Mr. George Covington, Cryenco, Denver, CO 80216, for the design of the freezing apparatus used in this study are greatly appreciated. Supported in part by grants-in-aid from Cryenco and Noba, Inc. through the National Association of Animal Breeders. 2Present address: lnstituto Nacional de lnvestigaciones Pecuarias, Km. 15 1/2 Carretera Mexico- Toluca, Palo Alto, D. F., Mexico. 3Continental Plastics, Inc., P. O. Box 336, Darien, WI or greater than that with the slower methods when the spermatozoa were suspended in 5 to 9% glycerol, but the slower rates of freezing were superior when 11% glycerol was used (P <.01). Glycerol levels of 7 and 9"% were superior to 11% (P <.05), when averaged over all other factors. Higher post-thaw motility was obtained when spermatozoa were frozen in straws placed horizontally as opposed to vertically (P<.01). (Key Words: Bovine Spermatozoa, Freezing, Semen, Glycerol, Straws.) INTRODUCTION Frozen semen in plastic straws can be stored more efficiently than that in 1.0-ml glass ampules (Pickett and Berndtson, 1974). In addition, the fertility of frozen bovine spermatozoa packaged in plastic straws has compared favorably to that of spermatozoa in glass ampules (Macpherson and King, 1966; Rajamannan, 1970; Bean, 1972; H. C. Kellgren, u~published data). The adopt.ion of straws has been accompanied by changes in semen processing, including seminal dilution rate and the rates of freezing and thawing. Interactions have been demonstrated among rates of freezing and thawing, and the type and level of cryopreservative with a variety of tissues and cells (Mazur and Schmidt, 1968; Mazur et al., 1969; Leibo et al., 1970), including bovine spermatozoa (Nagase et al., 1968). If optimal results are to be achieved, semen processing and handling techniques must be reevaluated for semen frozen in straws. These studies were conducted to establish optimal combinations of rates of freezing and thawing and level of glycerol for bovine spermatozoa in.25-ml Continental straws 3 and to compare the survival of spermatozoa frozen at similar rates in straws maintained horizontally or vertically. 129 JOURNAL OF ANIMAL SCIENCE, Vol. 41, No. 1,1975

2 130 RODRIGUEZ ET AL. EXPERIMENTAL PROCEDURE Ejaculates from six Hereford bulls with at least 50% progressively motile spermatozoa were utilized on a split-sample basis in Experiment 1. After initial evaluation of the spermatozoal concentration, percentage of progressively motile spermatozoa and total seminal volume, semen from each bull was diluted 1:4 (one part semen plus four parts extender) with a 20% egg yolk-2.9% sodium citrate extender and cooled slowly to 5 C over 4 hr (Benson et al., 1968). After cooling, additional extender was added to provide one-half the final volume. The extended semen was then divided into four portions to permit final dilutions resulting in 5, 7, 9 or 11% glycerol (v/v). Extender containing glycerol was added in four equal parts at 15-rain intervals to provide a final concentration of 20 x 106` progressively motile spermatozoa per milliliter. After hr of equilibration (Emmens and Martin, 1956, 1961; Blackshaw et al., 1957; Martig and Almquist, 1966), semen was packaged in 1.0-ml glass ampules or.25-ml straws. Semen in ampules was frozen in the liquidnitrogen vapor of a Linde 640 C storage unit (Clegg et al., 1965). To permit controlled freezing of. semen in straws, a resistor was placed in the bottom of a MVE CBF-21 vapor-freezing unit and attached to a variac power supply. The m6uth of the tank was fitted with a lid with. a single opening to accommodate a basket 7.6 cm square and 6.35 cm deep, designed to contain approximately 100 straws. The sides of the basket were of stainless steel, while a wire screen formed the bottom. This screen allowed nitrogen vapor to flow freely through the basket and also supported the straws. Two stainless steel plates with.48 cm holes spaced.79 cm apart (center to center) were used to support individual straws in the vertical position and permitted uniform spacing. Plastic tubing (.6 cm I.D.) attached to an aspirator was placed in the vapor above the liquid nitrogen. Freezing rates (table 1) were regulated by co~atrolling the rate of nitrogen "boil-off" with the variac power supply and/or by diverting the flow of vapor away from the straws by applying vacuum to the tubing. After freezing, all semen was stored at -196 C for 2 to 3 weeks. Semen in ampules was thawed in iced water for 10 minutes. Semen in straws was thawed by total immersion in iced water (approximately 5 C) for 3 min, or in water at 35 C for 12 sec, 55 C for 8 sec, 75 C for 7 sec or 90 C for 5 seconds. Post-thaw motility was evaluated in duplicate (two samples per treatment) at 37 C immediately following thawing (0 hr) by each of two independent observers without knowledge of the treatments. Samples from each treatment were also examined after 1 and 3 hr of incubation in iced water to permit accentuation of differences due to treatment, and because correlations between fertility and the TABLE 1. MEAN TIME RATES (+, SE) OF THREE DIFFERENT METHODS FOR FREEZING SEMEN IN STRAWS a Mean time Freezing rate Freezing method (min) (C/min) Slow from 5 C to -15 C from -15Cto-79C 16.0+, from -79Cto-130C ,.4 Moderate from5cto0c 4.4+, ,.1 fromocto-10c 5.7+, ,.3 from -10 C to C , ,.4 Fast from 5 C to--130c acooling rates were measured with a Leeds & Northrup Speedomax W/L Recorder using a fine copper-constantan thermocouple positioned in the center of a straw located at the center of each basket (mean of 12 freezes).

3 FACTORS AFFECTING SURVIVAL OF BOVINE SPERMATOZOA 131 post-thaw motility of spermatozoa frozen in ampules are higher after incubation than immediately after thawing (Pickett et al., 1961). A second study was conducted to compare the post-thaw motility of spermatozoa frozen in.25-ml Continental straws maintained in horizontal or vertical positions. Semen was obtained from one Angus, two Hereford and three Charolais bulls, evaluated and processed as in Experiment 1. A rack was fitted to the top of the freezing unit which permitted individual straws to be positioned horizontally with.625 cm spacing (center to center). Fast, moderate and slow freezing rates were obtained by the methods used in Experiment 1. Frozen semen was stored in liquid nitrogen for 2 to 3 weeks, thawed in 75 C water for 7 sec, and evaluated for motility immediately after thawing. Each freezing and thawing rate combination for straws in Experiment 1 was considered as a single treatment, and ampules formed one additional treatment. Thus, the data for straws and ampules formed a factorial with 16 treatments, 4 glycerol levels, 3 post-thaw incubation times, 2 observers and 6 bulls. Results with straws were also analyzed separately as a 4 x 32 x 5 x 2 x 6 factorial with 4 glycerol levels, 3 freezing rates, 3 post-thaw incubation times, 5 thawing rates and 2 observers replicated with 6 bulls. Experiment 2 was treated as a 3 x 22 x 4 x 6 factorial with 3 freezing rates, 2 freezing methods (horizontal and vertical), 2 observers, 4 glycerol levels and 6 bulls. For all analyses of variance, bulls were considered random, and all other factors were treated as fixed effects. When significant treatment effects were observed, differences among individual treatment means were further compared by the Studentized range (hsd) test (Snedecor and Cochran, 1967). Due to limitations in the number of means which can be compared with the hsd procedure, an lsd procedure was utilized to permit a comparison among the individual treatment means involved in the treatment (straws and ampules) x glycerol level interaction in Experiment 1. R ESU LTS The freezing rates in Experiment 1 for straws maintained vertically were monitored at the center (from end to end) of a straw near the center of the freezing basket. Variation between the freezing rate in the center versus that in the outer row of the basket was less than the variation among freezes. The outer row was always filled with straws containing extender only, to reduce variation in freezing rates among individual straws within the same basket. In spite of this precaution, there were large variations in the rate of freezing within each straw (figure 1). Since nitrogen vapor flowed past the straws from bottom to top, the bottom of the straws cooled more quickly. However, each time it became necessary to reduce the rate of cooling by diverting the flow of nitrogen vapor, the temperature within the straws tended to approach equilibrium, i.e., the bottoms of the straws rewarmed while the tops continued to cool. The extent to which the bottoms of the straws rewarmed was dependent upon the length of time vacuum was applied. The vacuum was never sufficient to completely prevent the flow of cold nitrogen vapor past the straws. Thus, the bulk of the semen within the straws continued to cool throughout the freezing process, and the rewarming which occurred reflected the establishment of temperature equilibrium within individual straws. The post-thaw motility of spermatozoa frozen in straws in Experiment 1 is shown in table 2. Since no difference in post-thaw,io- -Io- i.3o- -~o- M~UT~ FROU START OF Fr~-EZ~ Figure 1. Typical range of freezing rates within individual straws frozen at fast, moderate and slow rates in.25-ml Continental straws maintained in the vertical position. The temperature was regulated as necessary based upon the temperature at the center (from end to end) of a straw placed in the center of the freezing basket. The curves were obtained by monitoring the temperature at the center of a straw and at points 5 mm from each end (bottom and top) of two adjacent straws. (Note: Simultaneous measurement of the temperature at each of three locations in the same straw was not possible since leakage occurred when thermocouples were placed in each end of the straw.)

4 132 RODRIGUEZ ET AL. r,-.~ r t"q eq r Z 0,. t"q,~..~ r r t'q r ~t.-, ~m ~z z ~--~,,...~ eq t'~l eqeq ~lcq ~,'q eq t"q Z ~ v t'xl r-.i r t"q r t"q ~,r r r ~',1 r r t"q r r eqeq eqcq r eq d 0 9 t~ r,1 t"-"o ~',~,~'-,re~ t'~:p t'-t"q 9..~ ~--I r eq r t'~l t"q eq t"q r,,... Ox 1'~1 t"q eqcq r r t"q t~ t"-.i eq t"q r r ~e~ r r ~r t"q M~ <~ [.-, a. Z 0 t'q r eqcq r r t'~l r r E 0 e.~ '-d

5 FACTORS AFFECTING SURVIVAL OF BOVINE SPERMATOZOA 133 motility was found between the two observers, the results were pooled. Post-thaw motility of spermatozoa in straws averaged 26, 23 and 17% immediately following thawing for the fast, moderate and slow freezing rate, respectively. The fast rate of freezing was superior to the moderate rate (P <.01), and both were better than the slow rate of freezing (P <.0IL The higher survival at the more rapid rates of freezing was independent of rate of thawing, but was dependent upon the level of glycerol. Cryoprotection was greatest (P <.05) when semen was frozen in 7 to 11% glycerol at the slow rate or 9% glycerol at the moderate rate (P <.05); while 7 to 9% glycerol was optimal during fast freezing (P <.01). Thus, the optimal glycerol level for spermatozoa frozen in straws tended to decrease as the rate of freezing was increased. Increasing the thawing bath temperature from 5 to 55 C resulted in higher post-thaw motility (P <.01) while further increases to 90 C did not improve spermatozoal recovery (table 2). The improved post-thaw motility in samples thawed at 55 to 90 C was generally independent of the rate of freezing or level of glycerol. However, thawing at 5 C was especially deleterious in the presence of higher glycerol levels, particularly in conjunction with the slow rate of freezing. Post-thaw motility did not decrease significantly within 1 hr of post-thaw incubation in iced water; thus, only values following 0 and 3 hr of incubation are presented (table 2). Postthaw motility declined (P <.05) within 3 hr of post-thaw incubation. The decrease in motility was not influenced appreciably by freezing rate, thawing rate Or glycerol level. No evidence of cold-shock was noted when spermatozoa were thawed in warm water and immediately placed in iced water and incubated for up to 3 hours. The post-thaw motility of spermatozoa frozen in ampules averaged 20, 24, 28 and 19% for 5, 7, 9 and 11% glycerol levels, respectively, at 0 hr after thawing. After 3 hr of post-thaw incubation, these values were 17, 20, 23 and 18%, respectively. This represented an average reduction in post-thaw motility of 3.4 percentage points over the 3-hr incubation period, compared to a corresponding reduction of 2.5 percentage points for spermatozoa in straws. When averaged across all other treatments, the mean post-thaw motility of spermatozoa frozen in straws by the fast method and thawed in 55 to 90 C water was greater (P <.01)than that of spermatozoa in ampules. This relationship also existed when the same comparison included the optimal glycerol level for each treatment. Straws were placed horizontally or vertically for freezing in Experiment 2. Cooling was faster at the ends of straws frozen horizontally. However, the differences in temperatures were small and rewarming did not occur when nitrogen vapor was diverted. The results of Experiment 2 are summarized in table 3. Since there was no-significant difference between the two observers, the results were pooled. When averaged over all other treatments, the post-thaw motility of spermatozoa frozen to -130 C in 20 rain was similar to that for spermatozoa frozen in 3.5 TARLE 3. THE INFLUENCE OF GLYCEROL LEVEL AND RATES AND METHODS OF FREEZING ON THE POST-THAW MOTILITY OF SPERMATOZOA IN EXPERIMENT 2 Freezing rate a and method b 3.5 rain 20 rain 40 rain Glycerol level (%) Vert. Horiz. Vert. Horiz. Vert. Horiz. Mean Mean atime to cool semen from +5 to C. brefers to straws positioned horizontally or vertically during freezing.

6 134 RODRIGUEZ ET AL. min (P >.05), but greater (P <.01) than for spermatozoa frozen in 40 minutes. The horizontal method of freezing was superior to the vertical method (P <~.01), regardless of the rate of freezing. There were no differences in the post-thaw motility of spermatozoa frozen in 5, 7 or 9% glycerol (P >.05), but glycerol levels of 7 and 9% provided greater recovery than that with 11% (P <.05). Significant interactions between freezing rate and glycerol level were observed. The optimal glycerol levels for spermatozoa frozen by the fast method were 5 to 9%, while this increased to levels of 7 to 11% for spermatozoa frozen at the two slower rates. When averaged over all other treatments, the highest mean post-thaw motility was obtained when spermatozoa were frozen by the fast method in the presence of 7% glycerol. Discussion Freezing rate was confounded by intermittent periods of rewarming in Experiment 1 (figure 1). Therefore, the survival of spermatozoa was compared after freezing by techniques in which rewarming did (vertical) or did not occur (horizontal). Although the horizontal freezing method produced slight improvements in the post-thaw motility of spermatozoa frozen at the two slower rates, a similar improvement also occurred with the fast freezing method, despite the fact that rewarming did not occur during fast freezing by the vertical method. Thus, the poorer survival following slow freezing in both experiments cannot be attributed to rewarming. Since rewarming during freezing was not a serious problem and since spermatozoa in straws can apparently be frozen at widely differing rates without noticeable effects upon post-thaw motility, it is difficult to explain why survival was greater for spermatozoa in straws frozen horizontally. However, it should be emphasized that the advantage, averaged over all other treatments, was only 1.4 percentage points. Almquist and Wiggin (1973a, b) and Robbins et al. (1973) have concluded that freezing rates are relatively unimportant in affecting post-thaw motility or acrosomal retention of spermatozoa frozen in straws if the semen is thawed rapidly. In contrast, the slow rate of freezing in the present investigations resulted in reduced spermatozoal survival, regardless of how the semen was thawed. The lack of conformity between the present findings and those of other investigators presumably stems from differences in the range of freezing rates studied. Although a significant difference was noted between the fast and moderate rates of freezing in Experiment 1, this difference was small and was not observed in the second study. Since temperature decreased from 5 to -130 C in 3.5 min with the fast freezing rate, compared to 20 rain for the moderate freezing rate, it is apparent that bovine spermatozoa in straws can tolerate a wide range of freezing rates without appreciable effects upon post-thaw motility. These findings are similar to those of Clegg et al. (1965) in which no difference in post-thaw motility was obtained when spermatozoa in ampules were frozen to -120 C in approximately 8 to 42 minutes. In contrast, the slow rate of freezing, which approximated the conventional rates of freezing widely employed for semen in glass ampules (Clegg et al, 1965), gave lower postthaw motility for spermatozoa in straws in both experiments. Based upon the present findings it would appear that such freezing rates should be avoided for spermatozoa packaged in straws. A level of 7% glycerol has been optimal for spermatozoa frozen in ampules (Miller and VanDemark, 1954; Cragle et al., 1955; Martin, 1965). However, from data of Berndtson and Foote (1972b) and Robbins et al. (1973) it appeared that higher glycerol levels might be advantageous for freezing spermatozoa in straws. In support of this concept, levels of up to 11% glycerol were satisfactory when spermatozoa in straws were frozen slowly. However, the optimal glycerol level tended to decrease as freezing rate increased. These findings are consistent with the observation that the optimal glycerol level for spermatozoa frozen rapidly in pellets was lower than that for spermatozoa frozen slowly in ampules (Nagase et al., 1964; Berndtson and Foote, 1972a). Clearly, the optimal glycerol level must be determined for each individual method of freezing, but levels of 7 to 9% glycerol appear optimal for spermatozoa diluted in egg yolk-citrate extender and frozen over a wide range of rates in straws. Current procedures for thawing spermatozoa frozen in straws include: (1)air thawing (Bean, 1972), (2) thawing in the cow (Davidovid et al.,

7 FACTORS AFFECTING SURVIVAL OF BOVINE SPERMATOZOA ), (3) rolling the straw between the palms of the hands (Macpherson and Penner, 1972), and (4) thawing in warm water (Aamdal and Andersen, 1968; Robbins et al., 1972, 1973; Almquist and Wiggin, 1973a). Best results have usually been obtained with rapid thawing (Pickett and Berndtson, 1974), and spermatozoal survival was enhanced by increasing the temperature of thawing from 5 to 55 C. Almquist and Wiggin (1973b) also obtained higher mean post-thaw motility when the thawing bath temperature for spermatozoa in.3-ml Continental straws was increased from 5 to 35 C. Further improvement was noted when the temperature was increased from 35 to 75 C, but an additional increase to 95 C was not beneficial. During warm water thawing care must be exercised to prevent excessive warming during thawing or cold shock may result on subsequent exposure to ambient temperature. At the time of thawing an individual straw was grasped with forceps and quickly removed from the liquid nitrogen and placed in the thawing bath. The exposure of each straw to a given water bath was timed to permit thawing without allowing the temperature of the semen to rise above approximately 5 C. No evidence of cold shock was observed when spermatozoa were thawed by these methods and subsequently placed in iced water. It should be emphasized that the temperature of the vapor in the neck of an average liquid nitrogen field unit is much higher than that of liquid nitrogen (Saacke, 1974). Thus, if straws are exposed to temperatures substantially above -196 C prior to removal for thawing, the terminal temperature following thawing in warm water might be higher, and the potential for excessive heating and/or cold shock would be increased. For this reason thawing times in warm water should be kept as brief as possible while still permitting complete thawing. LITERATURE CITED Aamdal, J. and K. Andersen Fast thawing of bull semen frozen in straws. VI Inter. Congr. Animal Reprod. Artif. Insem. II:973. Almquist, J. O. and H. B. Wiggin. 1973a. Survival of bull spermatozoa frozen and thawed by different methods in plastic straws. A. I. Digest 21(7):12. Almquist, J. O. and H. B. Wiggin. 1973b. Effect of different combinations of freezing and thawing rates upon survival of bull spermatozoa in U. S. plastic straws. A. I. Digest 21(9):10. Bean, B Laboratory procedures and field trial results with straws. Proc. Fourth N.A.A.B. Tech. Conf. Artif. lnsem, and Reprod. p. 77. Benson, R. W., T. J. Sexton, B. W. Pickett, J. J. Lucas and M. R. Gebauer Influence of processing techniques and dilution rates on survival of frozen bovine spermatozoa. Univ. Conn. (Storrs) Agr. Exp. Sta. Res. Pep. 28. Berndtson, W. E. and R. H. Foote. 1972a. The freezability of spermatozoa after minimal prefreezing exposure to glycerol or lactose. Cryobiology 9:57. Bcrndtson, W. E. and R. H. Foote. 1972b. Bull sperm survival following freezing in ampules, pellets and straws. VII Inter. Cong. Animal Reprod. Artif. Insem. II:1353. Blackshaw, A. W., C. W. Emmens, I. Martin and J. Heyting The preparation of deep frozen semen. A. I. Digest 5(1):6. Clegg, E. D., B. W. Pickett and E. W. Gibson Non-mechanical nitrogen vapor freezing of bull semen. Univ. Conn. (Storrs) Agr. Exp. Sta. Res. Rep. 5. Cragle, R. G., R. M. Myers, R. K. Waugh, J. S. Hunter and R. L. Anderson The effects of various levels of sodium citrate, glycerol, and equilibration time on survival of bovine spermatozoa after storage at -79 C. J. Dairy Sci. 38:508. Davidovic', A., S. Borjanovic, B. Cvetkov and D. Knezevic' Some characters of deep frozen semen and new possibilities of its use. Vet. Glasn. 25:447. (Animal Breed. Abstr. 40. No. 406.) Emmens, C. W. and 1. Martin The controlled introduction of deep-freeze techniques into artificial insemination. Ill. Inter. Congr. Animal Reprod. Artif. Insem. III:18. Emmens, C. W. and I. C. A. Martin The effects of equilibration period and sugar content of the diluent on the survival and fertility of bull spermatozoa deep-frozen to -79 C. IV Inter. Congr. Animal Reprod. Artif. Insem. IV:964. Leibo, S. P., J. Farrant, P. Mazur, M. G. Hanna, Jr. and L. H. Smith Effects of freezing on marrow stern cell suspensions: Interactions of cooling and warming rates in the presence of PVP, sucrose, or glycerol. Cryobiology 6:315. Macpherson, J. W. and G. J. King Plastic straws for frozen semen (A field evaluation). Canad. J. Comp. Med. Vet. Sci. 30:109. Macpherson, J. W. and P. Penner A modified straw technique for frozen semen. A. I. Digest 20(2):6. Martig, R. C. and J. O. Almquist Effect of glycerol equilibration time on fertility of bull spermatozoa frozen in homogenized milk-fructose diluent. A. I. Digest 14(5):8. Martin, I. C. A Effects of rate of cooling to 5 C, storage at 5 C, glycerol concentration, sodium chloride, fructose and glycine on the revival of deep-frozen bull spermatozoa. J. Agr. Sci. 64:425. Mazur, P., J. Farrant, S. P. Leibo and E. H. V. Chu Survival of hamster tissue culture cells after

8 136 RODRIGUEZ ET AL. freezing and thawing. Interactions between protective solutes and cooling and warming rates. Cryobiology 6:1. Mazur, P. and J. J. Schmidt Interactions of cooling velocity, temperature, and warming velocity on the survival of frozen and thawed yeast. Cryobiology 5:1. Miller, W. J. and N. L. VanDemark The influence of glycerol level, various temperature aspects and certain other factors on the survival of bull spermatozoa at sub-zero temperatures. J. Dairy Sci. 37:45. Nagase, H., E. F. Graham and T. Niwa Pelleted semen: The effect of glycerol level and composition of thawing solution on fertility of bovine spermatozoa. V Inter. Congr. Animal Reprod. Artif. Insem. IV:404. Nagase, H., S. Yamashita and S. Irie Protective effects of sugars against freezing injury of bull spermatozoa. VI Inter. Congr. Animal Reprod. Artif. Insem. II:llll. Pickett, B. W. and W. E. t3erndtson The preservation of bovine spermatozoa by freezing in straws. A review. J. Dairy Sci. 57:1287. Pickett, B. W., W. A. MacDonald, D. G. Gosslee and W. A. Cowan Correlation between certain laboratory stress tests and fertility of frozen bovine spermatozoa. J. Dairy Sci. 44:1134. Rajamannan, A. H. J A method of packaging semen - the straw. Proc. Third N.A.A.B. Tech. Conf. Artif. lnsem. Reprod. p. 49. Robbins, R. K., L. E. Gerber and R. G. Saacke Influence of thaw rate on maintenance of the acrosomal cap. J. Anim. Sci. 35:253. (Abstr.). Robbins, R. K., M. L. O'Connor, P. T. Chandler and R. G. Saacke Freezing of bovine semen in "French straws." J. Anim. Sci. 37:327. (Abstr.). Saaeke, R. G Concepts in semen packaging and use. Proc. Eighth Conf. Artif. Insem. Beef Cattle, p. 11. Snedecor, G. W. and W. G. Cochran Statistical methods. Iowa State University Press, Ames.