Preservation of Bull Semen at Sub-Zero Temperatures

THAWING

The importance of carefully controlled cooling and storage has been emphasized in the foregoing sections. The need for controlling thawing rates and the temperature of thawing was not clearly defined in the early work on freezing bull semen. The British used a thawing temperature of 40° C., which was satisfactory. If there is a need to hold the semen for a time after thawing, then a lower thawing temperature might be more desirable so that cooling again will not be necessary.Comparison of thawing temperatures of 5° C. and 38° C. The effects of thawing at temperatures of 38° (body temperature) and 5° C. (refrigerator temperature) were investigated. The first trial involved thawing as rapidly as possible by dropping glass ampules of frozen semen into water baths at the two temperatures. The frozen semen samples contained glycerol levels of 4, 6, 8, and 10 percent. The mean percentages of motile sperm found after thawing thirteen diluted semen samples treated in this manner are shown in Figure 6.

The 5° C. thawing temperature resulted in a higher percentage of sperm survival at all the glycerol levels than 38° C., with the difference in favor of 5° C. becoming greater as the glycerol level increased. The reason for the interaction between glycerol level and thawing temperature is not known. It may be that the presence of the higher levels of glycerol at 38° C. brought about harmful metabolic activity. The difference in survival of sperm in semen thawed at 5° C. and at 38° C. continued during storage at 5° C. (Table 15). It was also evident that the interaction between glycerol level and thawing temperature continued during storage (Fig. 7).

Fig. 7

Effect of thawing temperature on sperm motility during storage at 5°
C. following freezing and thawing	(Fig. 7)

Table 15.—Effect of Glycerol Level, Thawing Temperature,
and Storage at 5° C. After Thawing on Sperm Motility

(Average of 13 ejaculates)

Thawing temperature (° C.)	Glycerol level (percent)	Spermmotility(percent)
		Post- thawing	Afterstorageat5°C.		Average
		Post- thawing	1 day	3 days	Average
38	4	28.5	17.3	5.1	17.0
	6	31.5	22.4	9.2	21.1
	8	33.1	15.0	4.6	17.6
	10	19.5	3.6	0.8	8.0
	Average	28.2	14.6	4.9	12.2
5	4	29.2	21.7	19.8	23.9
	6	37.7	33.8	23.5	31.7
	8	41.5	33.1	17.3	30.6
	10	33.1	18.5	6.0	19.2
	Average	35.4	26.8	16.6	20.6

It is obvious that motility falls off rapidly after the semen is thawed. In a field trial in which the initial intent was to test the effect of glycerol levels on fertility of frozen semen, the semen was thawed in the morning and used during the same day. Survival of the sperm with 4 percent glycerol was so poor that only a few breedings were made with these samples. Even at 7 and 10 percent, the fertility results were much lower than with semen that had not been subjected to freezing. At that time it was felt that thawing the samples and using them throughout the day may have caused the low fertility results. Since then, a large-scale experiment by Cornell University investigators, in cooperation with the New York Artificial Breeders’ Cooperative, has shown definitely that thawing should be delayed until a few minutes prior to breeding.[11] If the semen is used immediately, a thawing temperature of either 5° or 38° C. appears to be suitable. However, there is less danger of cold shock due to recooling if 5° C is used.Thawing rate in plastic and in glass. Glass ampules transmit cold or heat more readily than plastic ones. The temperature rise is rapid in both glass and plastic when samples are taken from the storage box at -79° C. and placed in water at 5° C. However, complete thawing occurs more rapidly in glass than in plastic ampules. The changes in temperature that occurred when glass and plastic ampules were thawed in a water bath at 5° C. are shown in Figure 8. The initial temperature rise for the first minute or two was about the same, then the rate of warming in the plastic slowed and actual melting of the frozen sample occurred a little over a minute later in the plastic than it did in the glass. Both were thawed in less than four minutes.

Fig. 8

Warming rates of diluted semen samples in plastic vials and in glass
ampules	(Fig. 8)

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Table 15.—Effect of Glycerol Level, Thawing Temperature, and Storage at 5° C. After Thawing on Sperm Motility

Table 15.—Effect of Glycerol Level, Thawing Temperature,
and Storage at 5° C. After Thawing on Sperm Motility