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Effect of Thawing Time on Fertility of Bovine Spermatozoa in French Straws1
Effect of Thawing Time on Fertility of Bovine Spermatozoa
in F r e n c h S t r a w s 1
J. O. ALMQUIST, = K. E. GRUBE, a and J. L. ROSENBERGER 4 The Pennsylvania State University University Park 16802
ABSTRACT
Thawing times of 9 and 40 s were compared in a 6-mo fertility experiment involving 18,057 first service inseminations to 23 Holstein bulls. Semen extended in Tris-yolk and packaged i n . 5-ml French straws was thawed in warm water baths (32 to 35°C) for either 9 or 40 s on alternate days. Based on 75-day nonreturn rates in log-linear models, fertility was significantly higher when semen was thawed for 40 s (66.3%) than for 9 s (64.4%). The improvement with longer thawing was not affected significantly by month (January through June). Thus, warming of semen in straws to a temperature approaching body temperature provided a slight increase in fertility even during cold weather. INTRODUCTION
There is agreement that rapid rates of thawing are beneficial to postthaw motility, acrosomal retention, and fertility of bovine spermatozoa packaged in plastic straws. However, there is lack of agreement when warm water is used as to whether thawing should be timed to prevent seminal temperature from rising above 5°C (5, 11) or to allow seminal temperature to rise well above 5°C (2, 12). There is concern that cold shock injury (1, 13) could occur under field conditions when semen in straws is thawed rapidly in warm water for 30 s or longer. Thus, fertility data involving different thawing times during cold weather are needed. Spermatozoal integrity is reduced by
Received February 2, 1981. l Journal Series No. 6177, Pennsylvania Agricultural Experiment Station. 2Dairy Breeding Research Center, Department of Dairy and Animal Science. 3Sire Power, Inc., R. D. 2, Tunkhannock, PA 18657. 4 Department of Statistics. 1982 J Dairy Sci 65:824--827
sudden postthaw warming of semen at 5°C (5, 13), supporting the need to determine the effect of thawing duration in warm water on fertility. In (2), fertility was slightly higher when seminal temperature in straws was raised to about 30°C by thawing for 30 s rather than 12 s in warm water even during cold weather. The experiment involved semen which was extended in milk and packaged in .3-ml Continental straws. The present experiment to extend these observations included a wider range of thawing times, a different extender, different size and kind of straw, and different seminal processing and freezing procedures. EXPERIMENTAL PROCEDURE
The experiment was from January through June, 1979, with semen used routinely by Sire Power, Inc. The neat semen was treated with antibiotics (6) and then extended all at once at 37°C in Tris-yolk-glycerol (8) containing 1 mg dihydrostreptomycin, 1,000 units penicillin, and 500 units polymyxin B/ml, packaged in .5-ml French straws at 21°C and cooled to 5°C during 1.5 to 2 h. The final concentration of extended semen was commonly 40 x 1 0 6 with a range of 30 to 60 x 106 motile sperm/ml. After a 4 -+ .5 h equilibration period at 5°C, semen was frozen in straws held in 10-straw goblets (10) on canes (400- to 700-straw load) on horizontal racks in static nitrogen vapor at - 1 5 0 ° C . The semen was cooled from 5°C to crystallization at a rate of from 10 to 12°C/rain and from crystallization to - 1 1 0 ° C at 8 to 10°C/rain. Straws were plunged into liquid nitrogen 20 min after being placed in static vapor. Twenty-three technicians in Pennsylvania and Maryland used thawing times of 9 and 40 s throughout the experiment. During April, May, and June, two technicians in New Jersey were added. Each technician was instructed to thaw semen for either 9 or 40 s on alternate days; 9 s was the routine thawing time used before the experiment. A wrist watch with second hand was used for timing. Semen was thawed by 824
THAWING SEMEN IN STRAWS AND FERTILITY immersing individual straws in warm water (32 to 35°C) contained in a .3-liter insulated bottle equipped with a small, dial-type thermometer. Technicians were instructed to check the bath temperature before removing the straw from the liquid nitrogen refrigerator and to raise the water temperature to 35°C whenever it was at or below 32°C. Based on trials with a microthermocouple centered within a straw containing Tris-yolk extender (unpublished data), postthaw seminal temperatures were about - 4 ° C after 9 s and 35°C after 40 s thawing in a 35°C water bath. Technicians were requested to dry straws thoroughly with clean, absorbent tissue after removal from the water bath and to use the semen for insemination as soon as possible after thawing. To reduce the possibility of cold shock after thawing, straws were carried inside the technician's outer garment to the inseminating site either wrapped in a paper towel or placed inside the inseminating device, the end of which was wrapped with a paper towel. Although semen from a large number of dairy and beef bulls was involved and all breeding receipts were marked with the code letter for thawing time, breeding receipt information was recorded on computer cards for only 23 Holstein bulls used during each month. All second services were matched to first services by ear tag or registration number. For statistical analysis, differences among ejaculates within bulls were ignored and 75-day nonreturn percentages were determined for each of the 23 bulls by two treatment classes (thawing times) on the basis of first services. As described (2), the log-linear model approach to contingency table analysis (3) was used to test for treatment differences in models which also accounted for differences among bulls, months, and inseminators. For this experiment, a three-factor table was utilized to classify the 18,057 inseminations with factors R (the dichotomous response factor indicating success or failure of the insemination), T (the treatment factor indicating either 9 or 40 s thawing time), and B (the bull factor). Tests for important two- and threefactor interactions in four-factor tables were constructed by the difference in chi-square goodness of fit statistics obtained by fitting the model without and with the interaction of interest. These tests were used to determine the
825
importance of month (M), inseminator (I), and the interactions of M and I with response rate by treatment. The 18,057 inseminations were classified in four-way tables with factor M, along with factors R, T, and B ; similar four-way tables were constructed for factor I. Whether the treatment effect changed across months or across inseminators was determined by Chisquare test on three-way interactions, RTM, or RTI, respectively.
RESULTS AND DISCUSSION
The test for a three-way interaction (RTB) in the full three-factor classification [X2(22)= 26.1, .3>P>.2] indicated that the treatment effect was consistent among bulls and that the model (RT, RB, TB) adequately represented the data. The hypothesis of no interaction of treatment by response (RT) was tested, and the chi-square statistic Ix2(1) = 6.0, P<.05] showed a significant treatment effect on response between the 9 and 40 s thawing times in favor of 40 s thawing (Table 1). The hypothesis of no interaction of bull by response (RB) was tested, and the chi-square statistic [X2(22) = 135.4, P<.01] indicated significant variation in response rate among bulls regardless of thawing time. This increase in nonreturn rate of 1.9 percentage points in favor of a longer thawing time, which elevated the seminal temperature to about 32°C, confirms the increase o f 1.9 percentage points we reported (2). In a trial (2) involving 21,424 first services, fertility was higher (P<.01) when semen was thawed for 30 s (72.0%) than for 12 s (70.1%). The earlier experiment (2) involved milk-diluted semen, stepwise addition of glycerol at 5°C to the partially diluted semen, packaging in .3-ml Continental straws at 5°C, and seminal freezing in five-straw goblets on canes in a mechanical forced vapor freezer. In contrast, the present experiment involved complete Tris-yolk-glycerol extender, packaging in .5-ml French straws at 21°C, and seminal freezing in 10-straw goblets on canes in static nitrogen vapor. Thus, even though two distinctly different systems were used, a similar increase in fertility in favor of longer thawing was obtained in two large experiments. Fertility in limited trials (4, 7, 9) also favored longer thawing. The difference in fertility favoring 40 s Journal of Dairy Science Vol. 65, No. 5, 1982
826
ALMQUIST ET AL.
TABLE 1. Effect of thawing time in warm water and month of year on fertility of spermatozoa frozen in French straws (23 bulls). Month of year
No. first services 9 sa
40 sa
Jan Feb Mar Apr May Jun Overall
1526 1415 1694 1493 1464 1472 9064
1550 1451 1718 1444 1431 1399 8993
Percent 75
Percentage points in favor of 40 s thawing
62.0 62.3 65.9 65.3 65.2 65.8 64.4
3.7 2.3 .9 1.3 3.6 -.8 1.9"
65.7 64.6 66.8 66.6 68.8 65.0 66.3
aTime in 32 to 35°C water bath. bservices weighted equally, *P<.05.
thawing could be related to greater acrosomal maintenance when the semen was warmed to a temperature approaching body temperature. Laboratory studies (5, 13) showed that acrosomal retention and motility of spermatozoa were damaged when semen at or near 5°C was warmed subsequently. Thus, warming of cold semen (about - 4 ° C after 9 s thawing) during and after insemination might have resulted in greater postthaw losses of acrosomes than when warm semen (about 32°C after 40 s thawing) was used. Based on the findings that in vitro warming damages spermatozoal integrity (5, 13) and that fertility is lower when cold semen is inseminated (2), Senger (12) suggested that semen in straws should be allowed to reach body temperature (thawing at 35°C for 30 to 60 s) during thawing. Use of this procedure should be accompanied by efforts to guard against cold shock. Nonreturn rate varied (P<.05) by month (RM). Of greater importance, however, the increase in fertility in favor of 40 s thawing (Table 1) did not change significantly from January through June, i.e., the test for a response by treatment by month interaction (RTM) was not significant (P>.5). The improvement of fertility with longer thawing was not affected by cold weather, which confirms (2). In both of our experiments, technicians were instructed to protect thawed semen by wrapping the straw or the end of the loaded inseminating device with a paper Journal of Dairy Science Vol. 65, No. 5, 1982
towel. In addition, the wrapped straw or inseminating device was carried inside the technician's outer garment to the inseminating site. Improvement in fertility with longer thawing might not be achieved if not accompanied by these precautions against cold shock. Bulls varied (P<.01) in nonreturn rate. There was wider variation among bulls for treatment response in this experiment than for (2). Fertility favored 40 s thawing time for 17 of 23, or 74%, of the bulls. Fertility also varied (P<.01) among inseminators; 75-day nonreturn rates ranged from 55 to 71% for 9 s thawing and from 61 to 77% for 40 s thawing. Although the three-way interaction (RTI) was significant (P<.05), fertility favored 40 s thawing for 15 of 25, or 60%, of the inseminators. Result was the same when months were included in the four-factor model, RTMI. Although technicians differed, they used each thawing time treatment on alternate days, and selection of the bull was without regard to treatment that day. Our experiment involved extended semen containing from 15 to 30 × 106 progressively motile spermatozoa per straw prior to freezing; most semen was extended at 20 × 106. The effect of lower numbers of motile spermatozoa per unit on the difference in fertility between the 9 and 40 s thawing times, especially during cold weather, is not known. The small increase in fertility in the present experiment and four other trials (2, 4, 7, 9)
THAWING SEMEN IN STRAWS AND F E R T I L I T Y supports use of a rapid thawing method which will r a i s e t h e t e m p e r a t u r e o f s e m e n in s t r a w s well above 5°C even during cold weather. Nevertheless, care should be taken to minimize exposure of thawed semen and inseminating devices to cold.
6
7
ACKNOWLEDGMENT
8
T h e a u t h o r s w i s h t o t h a n k t h e s t a f f o f Sire Power, Inc., Tunkhannock, PA, f o r t h e i r assistance and cooperation. 9
REFERENCES 1 Almquist, J. O. 1976. Effect of cold shock after thawing on acrosomal m a i n t e n a n c e and motility of bovine spermatozoa frozen in plastic straws. J. Dairy Sci. 59:1825. 2 Almquist, J. O., J. L. Rosenberger, and R. J. Branas. 1979. Effect of thawing time in warm water on fertility of bovine spermatozoa in plastic straws. J. Dairy Sci. 62:772. 3 Bishop, Y.M.M., S. E. Feinberg, and P. W. Holland. 1975. Discrete multivariate analysis. The MIT Press, Cambridge, MA. 4 Chandler, J. E., R. L. Nebel, A. Baham, and R. W. Adkinson. 1980. Effect of thawing temperature and time on fertility of bovine semen packaged in •3 ml straws. J. Dairy Sci. 63 (Suppl. 1 ) :95. (Abstr.) 5 De Abreu, R. M., W. E. Berndtson, R. L. Smith, and B. W. Pickett. 1979. Effect of post-thaw warming on viability of bovine spermatozoa
10
11
12
13
827
thawed at different rates in French straws. J. Dairy Sci. 62:1449. Elliott, F. 1., D. M. Murphy. D. E. Bartlett, and R. A. Kubista. 1962. The use of p o l y m y x i n B sulfate with dihydrostreptomycin and penicillin for the control of 'Vibrio Fetus' in a frozen semen process. A. I. Digest 10(2):10. Forde, B., and K. Gravir. 1973. A u n i f o r m m e t h o d of thawing frozen semen. Norwegian Vet. 85:146. (Anim. Breed• Abstr. 42:432. 1974.) Gilbert, G. R., and J. O. Almquist. 1978. Effects of processing procedures on post-thaw acrosomal retention and motility of bovine spermatozoa packaged in .3-ml straws at r o o m temperature. J. Anim. Sci. 46:225. Jondet, R., and Y. Rabadeux. 1977. D~gel des paillettes de 0.25 ml dans un barn d eau at +35 C: Influence compar6e de deux t e m p s dimmersion sur ie pouvior f & o n d a n t des spermatozoides. Elevage Insem. 159:18. Landa, C. A., and J. O. Almquist. 1979. Effect of freezing large n u m b e r s of straws of bovine spermatozoa in an automatic freezer on post-thaw motility and acrosomal retention. J. Anim. Sci. 4 9 : 1 1 9 0 . Rodriguez, O. L., W. E. Berndtson, B. D. Ennen, and B. W. Pickett. 1975. Effects of rates of freezing, thawing and level of glycerol on the survival of bovine spermatozoa in straws. J. Anim. Sci. 41:129. Senger, P. L. 1980. Handling frozen bovine semen - factors which influence viability and fertility. Theriogenology 13 : 51. Senger, P. L., W. C. Becker, and J. K. Hillers. 1976. Effect of thawing rate and post-thaw temperature on motility and acrosomal maintenance in bovine semen frozen in plastic straws. J. Anita. Sci. 42:932.
Journal of Dairy Science Vol. 65, No. 5, 1982
in F r e n c h S t r a w s 1
J. O. ALMQUIST, = K. E. GRUBE, a and J. L. ROSENBERGER 4 The Pennsylvania State University University Park 16802
ABSTRACT
Thawing times of 9 and 40 s were compared in a 6-mo fertility experiment involving 18,057 first service inseminations to 23 Holstein bulls. Semen extended in Tris-yolk and packaged i n . 5-ml French straws was thawed in warm water baths (32 to 35°C) for either 9 or 40 s on alternate days. Based on 75-day nonreturn rates in log-linear models, fertility was significantly higher when semen was thawed for 40 s (66.3%) than for 9 s (64.4%). The improvement with longer thawing was not affected significantly by month (January through June). Thus, warming of semen in straws to a temperature approaching body temperature provided a slight increase in fertility even during cold weather. INTRODUCTION
There is agreement that rapid rates of thawing are beneficial to postthaw motility, acrosomal retention, and fertility of bovine spermatozoa packaged in plastic straws. However, there is lack of agreement when warm water is used as to whether thawing should be timed to prevent seminal temperature from rising above 5°C (5, 11) or to allow seminal temperature to rise well above 5°C (2, 12). There is concern that cold shock injury (1, 13) could occur under field conditions when semen in straws is thawed rapidly in warm water for 30 s or longer. Thus, fertility data involving different thawing times during cold weather are needed. Spermatozoal integrity is reduced by
Received February 2, 1981. l Journal Series No. 6177, Pennsylvania Agricultural Experiment Station. 2Dairy Breeding Research Center, Department of Dairy and Animal Science. 3Sire Power, Inc., R. D. 2, Tunkhannock, PA 18657. 4 Department of Statistics. 1982 J Dairy Sci 65:824--827
sudden postthaw warming of semen at 5°C (5, 13), supporting the need to determine the effect of thawing duration in warm water on fertility. In (2), fertility was slightly higher when seminal temperature in straws was raised to about 30°C by thawing for 30 s rather than 12 s in warm water even during cold weather. The experiment involved semen which was extended in milk and packaged in .3-ml Continental straws. The present experiment to extend these observations included a wider range of thawing times, a different extender, different size and kind of straw, and different seminal processing and freezing procedures. EXPERIMENTAL PROCEDURE
The experiment was from January through June, 1979, with semen used routinely by Sire Power, Inc. The neat semen was treated with antibiotics (6) and then extended all at once at 37°C in Tris-yolk-glycerol (8) containing 1 mg dihydrostreptomycin, 1,000 units penicillin, and 500 units polymyxin B/ml, packaged in .5-ml French straws at 21°C and cooled to 5°C during 1.5 to 2 h. The final concentration of extended semen was commonly 40 x 1 0 6 with a range of 30 to 60 x 106 motile sperm/ml. After a 4 -+ .5 h equilibration period at 5°C, semen was frozen in straws held in 10-straw goblets (10) on canes (400- to 700-straw load) on horizontal racks in static nitrogen vapor at - 1 5 0 ° C . The semen was cooled from 5°C to crystallization at a rate of from 10 to 12°C/rain and from crystallization to - 1 1 0 ° C at 8 to 10°C/rain. Straws were plunged into liquid nitrogen 20 min after being placed in static vapor. Twenty-three technicians in Pennsylvania and Maryland used thawing times of 9 and 40 s throughout the experiment. During April, May, and June, two technicians in New Jersey were added. Each technician was instructed to thaw semen for either 9 or 40 s on alternate days; 9 s was the routine thawing time used before the experiment. A wrist watch with second hand was used for timing. Semen was thawed by 824
THAWING SEMEN IN STRAWS AND FERTILITY immersing individual straws in warm water (32 to 35°C) contained in a .3-liter insulated bottle equipped with a small, dial-type thermometer. Technicians were instructed to check the bath temperature before removing the straw from the liquid nitrogen refrigerator and to raise the water temperature to 35°C whenever it was at or below 32°C. Based on trials with a microthermocouple centered within a straw containing Tris-yolk extender (unpublished data), postthaw seminal temperatures were about - 4 ° C after 9 s and 35°C after 40 s thawing in a 35°C water bath. Technicians were requested to dry straws thoroughly with clean, absorbent tissue after removal from the water bath and to use the semen for insemination as soon as possible after thawing. To reduce the possibility of cold shock after thawing, straws were carried inside the technician's outer garment to the inseminating site either wrapped in a paper towel or placed inside the inseminating device, the end of which was wrapped with a paper towel. Although semen from a large number of dairy and beef bulls was involved and all breeding receipts were marked with the code letter for thawing time, breeding receipt information was recorded on computer cards for only 23 Holstein bulls used during each month. All second services were matched to first services by ear tag or registration number. For statistical analysis, differences among ejaculates within bulls were ignored and 75-day nonreturn percentages were determined for each of the 23 bulls by two treatment classes (thawing times) on the basis of first services. As described (2), the log-linear model approach to contingency table analysis (3) was used to test for treatment differences in models which also accounted for differences among bulls, months, and inseminators. For this experiment, a three-factor table was utilized to classify the 18,057 inseminations with factors R (the dichotomous response factor indicating success or failure of the insemination), T (the treatment factor indicating either 9 or 40 s thawing time), and B (the bull factor). Tests for important two- and threefactor interactions in four-factor tables were constructed by the difference in chi-square goodness of fit statistics obtained by fitting the model without and with the interaction of interest. These tests were used to determine the
825
importance of month (M), inseminator (I), and the interactions of M and I with response rate by treatment. The 18,057 inseminations were classified in four-way tables with factor M, along with factors R, T, and B ; similar four-way tables were constructed for factor I. Whether the treatment effect changed across months or across inseminators was determined by Chisquare test on three-way interactions, RTM, or RTI, respectively.
RESULTS AND DISCUSSION
The test for a three-way interaction (RTB) in the full three-factor classification [X2(22)= 26.1, .3>P>.2] indicated that the treatment effect was consistent among bulls and that the model (RT, RB, TB) adequately represented the data. The hypothesis of no interaction of treatment by response (RT) was tested, and the chi-square statistic Ix2(1) = 6.0, P<.05] showed a significant treatment effect on response between the 9 and 40 s thawing times in favor of 40 s thawing (Table 1). The hypothesis of no interaction of bull by response (RB) was tested, and the chi-square statistic [X2(22) = 135.4, P<.01] indicated significant variation in response rate among bulls regardless of thawing time. This increase in nonreturn rate of 1.9 percentage points in favor of a longer thawing time, which elevated the seminal temperature to about 32°C, confirms the increase o f 1.9 percentage points we reported (2). In a trial (2) involving 21,424 first services, fertility was higher (P<.01) when semen was thawed for 30 s (72.0%) than for 12 s (70.1%). The earlier experiment (2) involved milk-diluted semen, stepwise addition of glycerol at 5°C to the partially diluted semen, packaging in .3-ml Continental straws at 5°C, and seminal freezing in five-straw goblets on canes in a mechanical forced vapor freezer. In contrast, the present experiment involved complete Tris-yolk-glycerol extender, packaging in .5-ml French straws at 21°C, and seminal freezing in 10-straw goblets on canes in static nitrogen vapor. Thus, even though two distinctly different systems were used, a similar increase in fertility in favor of longer thawing was obtained in two large experiments. Fertility in limited trials (4, 7, 9) also favored longer thawing. The difference in fertility favoring 40 s Journal of Dairy Science Vol. 65, No. 5, 1982
826
ALMQUIST ET AL.
TABLE 1. Effect of thawing time in warm water and month of year on fertility of spermatozoa frozen in French straws (23 bulls). Month of year
No. first services 9 sa
40 sa
Jan Feb Mar Apr May Jun Overall
1526 1415 1694 1493 1464 1472 9064
1550 1451 1718 1444 1431 1399 8993
Percent 75
Percentage points in favor of 40 s thawing
62.0 62.3 65.9 65.3 65.2 65.8 64.4
3.7 2.3 .9 1.3 3.6 -.8 1.9"
65.7 64.6 66.8 66.6 68.8 65.0 66.3
aTime in 32 to 35°C water bath. bservices weighted equally, *P<.05.
thawing could be related to greater acrosomal maintenance when the semen was warmed to a temperature approaching body temperature. Laboratory studies (5, 13) showed that acrosomal retention and motility of spermatozoa were damaged when semen at or near 5°C was warmed subsequently. Thus, warming of cold semen (about - 4 ° C after 9 s thawing) during and after insemination might have resulted in greater postthaw losses of acrosomes than when warm semen (about 32°C after 40 s thawing) was used. Based on the findings that in vitro warming damages spermatozoal integrity (5, 13) and that fertility is lower when cold semen is inseminated (2), Senger (12) suggested that semen in straws should be allowed to reach body temperature (thawing at 35°C for 30 to 60 s) during thawing. Use of this procedure should be accompanied by efforts to guard against cold shock. Nonreturn rate varied (P<.05) by month (RM). Of greater importance, however, the increase in fertility in favor of 40 s thawing (Table 1) did not change significantly from January through June, i.e., the test for a response by treatment by month interaction (RTM) was not significant (P>.5). The improvement of fertility with longer thawing was not affected by cold weather, which confirms (2). In both of our experiments, technicians were instructed to protect thawed semen by wrapping the straw or the end of the loaded inseminating device with a paper Journal of Dairy Science Vol. 65, No. 5, 1982
towel. In addition, the wrapped straw or inseminating device was carried inside the technician's outer garment to the inseminating site. Improvement in fertility with longer thawing might not be achieved if not accompanied by these precautions against cold shock. Bulls varied (P<.01) in nonreturn rate. There was wider variation among bulls for treatment response in this experiment than for (2). Fertility favored 40 s thawing time for 17 of 23, or 74%, of the bulls. Fertility also varied (P<.01) among inseminators; 75-day nonreturn rates ranged from 55 to 71% for 9 s thawing and from 61 to 77% for 40 s thawing. Although the three-way interaction (RTI) was significant (P<.05), fertility favored 40 s thawing for 15 of 25, or 60%, of the inseminators. Result was the same when months were included in the four-factor model, RTMI. Although technicians differed, they used each thawing time treatment on alternate days, and selection of the bull was without regard to treatment that day. Our experiment involved extended semen containing from 15 to 30 × 106 progressively motile spermatozoa per straw prior to freezing; most semen was extended at 20 × 106. The effect of lower numbers of motile spermatozoa per unit on the difference in fertility between the 9 and 40 s thawing times, especially during cold weather, is not known. The small increase in fertility in the present experiment and four other trials (2, 4, 7, 9)
THAWING SEMEN IN STRAWS AND F E R T I L I T Y supports use of a rapid thawing method which will r a i s e t h e t e m p e r a t u r e o f s e m e n in s t r a w s well above 5°C even during cold weather. Nevertheless, care should be taken to minimize exposure of thawed semen and inseminating devices to cold.
6
7
ACKNOWLEDGMENT
8
T h e a u t h o r s w i s h t o t h a n k t h e s t a f f o f Sire Power, Inc., Tunkhannock, PA, f o r t h e i r assistance and cooperation. 9
REFERENCES 1 Almquist, J. O. 1976. Effect of cold shock after thawing on acrosomal m a i n t e n a n c e and motility of bovine spermatozoa frozen in plastic straws. J. Dairy Sci. 59:1825. 2 Almquist, J. O., J. L. Rosenberger, and R. J. Branas. 1979. Effect of thawing time in warm water on fertility of bovine spermatozoa in plastic straws. J. Dairy Sci. 62:772. 3 Bishop, Y.M.M., S. E. Feinberg, and P. W. Holland. 1975. Discrete multivariate analysis. The MIT Press, Cambridge, MA. 4 Chandler, J. E., R. L. Nebel, A. Baham, and R. W. Adkinson. 1980. Effect of thawing temperature and time on fertility of bovine semen packaged in •3 ml straws. J. Dairy Sci. 63 (Suppl. 1 ) :95. (Abstr.) 5 De Abreu, R. M., W. E. Berndtson, R. L. Smith, and B. W. Pickett. 1979. Effect of post-thaw warming on viability of bovine spermatozoa
10
11
12
13
827
thawed at different rates in French straws. J. Dairy Sci. 62:1449. Elliott, F. 1., D. M. Murphy. D. E. Bartlett, and R. A. Kubista. 1962. The use of p o l y m y x i n B sulfate with dihydrostreptomycin and penicillin for the control of 'Vibrio Fetus' in a frozen semen process. A. I. Digest 10(2):10. Forde, B., and K. Gravir. 1973. A u n i f o r m m e t h o d of thawing frozen semen. Norwegian Vet. 85:146. (Anim. Breed• Abstr. 42:432. 1974.) Gilbert, G. R., and J. O. Almquist. 1978. Effects of processing procedures on post-thaw acrosomal retention and motility of bovine spermatozoa packaged in .3-ml straws at r o o m temperature. J. Anim. Sci. 46:225. Jondet, R., and Y. Rabadeux. 1977. D~gel des paillettes de 0.25 ml dans un barn d eau at +35 C: Influence compar6e de deux t e m p s dimmersion sur ie pouvior f & o n d a n t des spermatozoides. Elevage Insem. 159:18. Landa, C. A., and J. O. Almquist. 1979. Effect of freezing large n u m b e r s of straws of bovine spermatozoa in an automatic freezer on post-thaw motility and acrosomal retention. J. Anim. Sci. 4 9 : 1 1 9 0 . Rodriguez, O. L., W. E. Berndtson, B. D. Ennen, and B. W. Pickett. 1975. Effects of rates of freezing, thawing and level of glycerol on the survival of bovine spermatozoa in straws. J. Anim. Sci. 41:129. Senger, P. L. 1980. Handling frozen bovine semen - factors which influence viability and fertility. Theriogenology 13 : 51. Senger, P. L., W. C. Becker, and J. K. Hillers. 1976. Effect of thawing rate and post-thaw temperature on motility and acrosomal maintenance in bovine semen frozen in plastic straws. J. Anita. Sci. 42:932.
Journal of Dairy Science Vol. 65, No. 5, 1982