Temperature control in the low temperature preservation of spermatozoa

Abstract

Polge ('53), Erickson et al. ('54), and Van Demark and Kinney ('54) recognized the necessity of controlled slow cooling for the low temperature preservation of bull semen. They suggested that cooling semen at rates between 1 " to 3°C per minute over the range -I-5" to about -15°C was optimal for recovery of viable and fertile bovine spermatozoa. Recently, more rapid cooling was shown not to be detrimental to motility of spermatozoa (Luyet and Keane, '55; Sialy and Smith, '57; Kennelly et al., '60).With the development of cryogenic equipment in our laboratory to control accurately the rate of cooling over specific temperature ranges (Cowley et al., '62), we were able to examine critically the contribution of thermal history to the quality of preserved semen. Bull semen extended in egg yolk-citrate-glycerol solution was cooled at rates from 0.2" to over 200°C per minute and stored in liquid nitrogen refrigerators. The data reported below indicate that controlling the rate of cooling between 4" to 20°C per minute over the range -10" to -330°C permits optimal recovery of viable spermatozoa from lowtemperature-stored bovine semen. METHODS AND MATERIALSSemen' from each of five bulls in service at the New York Artificial Breeders Cooperative was extended to contain about 20 million motile sperm per ml before freezing. The final concentrations of the components of the extender were 20% v/v egg yolk, 3% w/v sodium citrate dihydrate, and 7% w/v glycerol. After 18-hour equilibration at about 4"C, 1-ml aliquots of the extended semen in glass semen vials were cooled with cold gas or by direct immersion in liquid nitrogen (LN) and stored in the Linde liquid nitrogenUnless otherwise noted, samples were stored for two to ten days before being thawed in ice water and evaluated for viability.Average rates of cooling over specific temperature ranges were calculated from temperature changes recorded with a potentiometer (Varian G 11A) connected to a copper-constantan thermocouple centrally located in one of the semen samples. Controlled rate cooling was achieved with cold nitrogen gas in a prototype of the Linde Biological Freezer-Model 3 (BF-3) or with LN by immersion of samples in metal, plastic and glass vials.Viability of semen samples was evaluated by the dye exclusion technique of Mayer et al. ('51) for differentiating live and dead sperm. No difficulty was experienced with the Eosin B-Fast Green stain when live sperm were counted immediately upon addition of the stain. Penetration of Eosin into sperm suspended in glycerolated semen was observed when counting was delayed five or ten minutes. In practice not more than a few minutes elapsed from staining to completion of the counting. Four slides were made of each sample; a total of 100 sperm were evaluated on each slide, The average viability of semen before freezing was 85% live sperm. RESULTSTwo parameters associated with the preservation of bovine spermatozoa were investigated: cooling rates and warming rates.

Influence of Diluents and Processing Times After Ejaculation on the Survival of Deep-Frozen Ram Spermatozoa

“…From Polge's (1957) studies a freezing rate of 4 degOjmin might be expected to decrease the survival of spermatozoa which had been equlibrated for a short period, as in experiment 2. However, more recent work by Bruemmer, Eddy, and Duryea (1963) demonstrated that between 0 and -10°0 and -10 and -30°0, bull spermatozoa tolerate average cooling rates of 1-6 and 4-12 degOjmin equally well, and better than rates either faster or slower than these.…”

“…The analyses of variance were computed (Claringbold 1957) using the sets of orthogonal polynomial coefficients tabulated by Fisher and Yates (1957) to partition levels of each factor. However, in experiment 3 and 4 (Tables 5 and 8) other sets of orthogonal coefficients, each contrast having a single degree of freedom, were used to partition components of one or two factors in order to test the validity of the hypotheses proposed in the experimental design.…”

Fertility of Chickens Inseminated Intraperitoneally with Semen Preserved in Liquid Nitrogen

“…The cooling rates within various temperature ranges for optimum survival of bull spermatozoa have been studied using liquid nitrogen. Bruemmer et al (1963) reported that bull spermatozoa survival was highest with cooling rates of 1°C. to 5°C.…”

Viabilities of Frozen Mammalian Cells Following Slow Thawing.

“…Bruemmer et aZ (6) have also noted a correlation between cooling rate and viability of bovine spermatozoa following slow thawing. Dimethylsulfoxide generally ing, although good viability wits sometimes afforded better protection than glycerol obtained with glycerol as well.…”