Preservation of Bull Semen at Sub-Zero Temperatures

DILUTION RATES

The first trials by the British at freezing bull semen were made with samples containing many millions of sperm cells. In routine artificial breeding, it is common to add extenders to semen so that one milliliter of diluted semen may contain only 10 million living sperm cells. (This number still insures optimal fertility.) Frequently the addition of 100 or more parts of the yolk extender to each part of the original semen sample is possible without reducing the sperm numbers below 10 million per milliliter. No one knew if this process of dilution would affect the resistance of bull sperm to freezing. The effect of various rates of dilution on the freezability of bull sperm was tested with 10 semen samples. The results, presented in Table 6, show that the numbers of sperm between 10 and 90 million per milliliter did not influence the percentage of sperm that survived freezing.

In a later trial it was found that sperm survival was slightly better at lower dilution rates than in the same samples frozen following dilution to 15 million sperm per milliliter. However, field trials with frozen semen carried out by others, using sperm numbers as low as 15 million per milliliter of semen inseminated or even lower, have been highly satisfactory.[11], [12]

During the early studies in the Illinois laboratory, the effects of glycerol level were also tested.[13] These effects are discussed in the section on glycerol additions beginning on page 17.Effect of further dilution and refreezing after the initial freezing. Under some circumstances it might be advantageous to freeze semen with a high concentration of sperm cells and then extend it further after thawing. With such a procedure less storage space is needed than when dilution is carried to the maximum before freezing. Two experiments were conducted to test the effects of dilution and storage at 5° C. and dilution and refreezing following an initial freezing of concentrated samples.

Table 6.—Effect of Sperm Numbers and Glycerol Level
in Final Mixture on Freezability of Bull Sperm at -79° C.

(Average of 10 ejaculates)

Glycerollevel (percent)	Post-thawingmotility(percent)[F]
	Numberofsperm(millions/ml.)			Average
	90	30	10	Average
5	36.0	34.0	36.0	35.0
10	22.0	24.0	23.0	23.0
15	3.2	0.9	0.2	1.4
Average	20.3	19.8	19.9	20.0

[F] Mean initial motility of sperm before freezing was 55 percent.

Four semen samples were split and extended at rates of 1:1 (semen to extender) and 1:10. These were frozen, then thawed and halved. One half was further extended to a level of 15 million sperm per milliliter; the sperm numbers in the other remained unchanged. Each of these halves was split again, and one portion of each was stored at 5° C. for 3 to 7 hours. The other two portions were refrozen.

Table 7.—Effect of Further Dilution and Refreezing on Sperm Motility
After the Initial Freezing of Bull Semen

Dilutionof semen (semen: extender)	Pre- freezing motility (percent)	Post-thawing motility
		After first freezing	Afterstorage[G]		Afterrefreezing[H]
		After first freezing	No further dilution	Diluted to15 million/ml	No further dilution	Diluted to15 million/ml
		Firsttrial:4samples
1:1	60	49	46	34	31	6
1:10	53	45	36	30	25	5
		Secondtrial:7samples
1:9	67	47	41	35	28	11
15million/ml	67	30	32	..	18	..

[G] Stored at 5° C. for 3 to 7 hours after first thawing.[H] Refrozen following first thawing.

Table 8.—Effect of Glycerol Level and Storage at 5° C. on Motility
of Sperm in Yolk-Citrate Extender

Glycerollevel (percent)		Sperm motility
		Post- thawing			After storage at 5° C.									Average
		Post- thawing			1 day			3 days			7 days			Average
		percent	rate		percent	rate		percent	rate		percent	rate		percent	rate
Control[I]		56	2.5		55	1.9		46	1.8		38	1.4		48	1.90
	0	54	2.4		44	1.9		46	1.8		36	1.4		45	1.87
	5	52	2.2		50	1.9		46	1.7		32	1.4		45	1.80
1	0	52	2.3		46	1.8		42	1.7		28	1.6		42	1.85
2	0	52	2.1		50	1.7		44	1.6		38	1.1		46	1.62
3	0	50	0.7		44	0.5		42	0.4		30	0.4		42	0.51
Average		53	2.0	3	47	1.6	2	44	1.5	0	34	1.2	2	..	....

[I] The control differed from the 0-glycerol treatment in that no additional citrate or glycerol solution was added.

A similar trial was carried out with seven samples; one portion was diluted 1:9; the other was extended at the outset to 15 million sperm per milliliter. Results for both tests are summarized in Table 7.

From Table 7 it can be seen that refreezing following an initial freezing further reduced the number of surviving sperm. The second freezing was more detrimental to the portion of the samples extended to 15 million sperm per milliliter than to the portion that was refrozen at a higher sperm concentration. The percentage of motile sperm remained fairly high in the portions that were diluted to 15 million sperm and stored at 5° C. However, in all cases, survival was best in the samples at the lower dilution levels.

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Table 6.—Effect of Sperm Numbers and Glycerol Level in Final Mixture on Freezability of Bull Sperm at -79° C.

Table 7.—Effect of Further Dilution and Refreezing on Sperm Motility After the Initial Freezing of Bull Semen

Table 8.—Effect of Glycerol Level and Storage at 5° C. on Motility of Sperm in Yolk-Citrate Extender

Table 6.—Effect of Sperm Numbers and Glycerol Level
in Final Mixture on Freezability of Bull Sperm at -79° C.

Table 7.—Effect of Further Dilution and Refreezing on Sperm Motility
After the Initial Freezing of Bull Semen

Table 8.—Effect of Glycerol Level and Storage at 5° C. on Motility
of Sperm in Yolk-Citrate Extender