CRYOSURVIVAL OF RAM SPERMATOZOA IN HYPERTONIC AND ISOTONIC DILUENTS

CRYOSURVIVAL OF RAM SPERMATOZOA IN HYPERTONIC AND ISOTONIC DILUENTSAnimal Research Centre, Agriculture Canada, Ottawa, Ontario KIA 0C6. Con- tribution no. 1049, received 29 Dec. 1981, accepted 18 Mar. 1982.
Frsen, P. S., ArNswonrH, L. AND Frrnrull, R. W. 1982. Cryosurvival of ram spermatozoa in hypertonic and isotonic diluents. Can. J. Anim. Sci. 62: 425-428.
The cryosurvival (percentage of motile spermatozoa and rate of foward progfes- sion) of ram spermatozoa after freezing in hypertonic and isotonic diluents containing a buffered dextran-sugar combination or bufferdd dextran-sugar plus egg yolk was compared immediately after thawing and after incubation at 39" for 60 min. Cryosurvival of spermatozoa was comparable in both diluents. However, the cryosurvival of spermatozoa was increased significantly in hyper- tonic diluents compared with that in isotonic diluents, regardless of their com- position.
La cryosurvie (pourcentage de spermatozoides motiles et taux de progression) de spermatozoides de b6lier aprbs cong6lation dans des diluants hypertoniques et isotoniques contenant un m6lange de dextrane-sucre tamponn6 ou le mOme m6lange plus du jaune d'oeuf a 6t6 comparde juste aprds d6cong6lation et incubation e 39'C pendant 60 min. La cryosurvie est comparable pour les deux diluants, mais elle s'accroit significativement selon qu'il s'agit des diluants hypertoniques plut6t qu'isotoniques, peu importe leur composition.
Key words: Spermatozoa,
As a part of a research program directed towards increasing the cryosurvival and fertilizing ability of frozen-thawed ram spermatozoa, attention has been drawn to the beneficial effects of some high molec- ular weight compounds on cryosurvival of several different cell types (Damjanovic and Thomas 1974;' Lionetti et al. 1976; McGann 1978; Pribor and Pribor 1973) to replace or complement the egg yolk and milk portions of diluents used for freezing ram semen. In our laboratory, several high molecular weight compounds have been tested in glycerolated diluents for freezing ram spermatozoa, with dextran and hy- droxyethyl starch resulting in the best cry- osurvival. Also, previous studies have shown that freezing of ram spermatozoa in moderately hypertonic skim milk di-
Can. J. Animal Sci. 62: 425-428 (June 1982)
sheep, cryosurvival, diluents
luents resulted in acceptable fertility and improved cryosurvival (Colas 1979; Fiser et al. 1981) when compared with isotonic diluents.
This study was carried out to compare the cryosurvival of ram spermatozoa after freezing in hypertonic and isotonic diluents containing a buffered dextran-sugar com- bination or buffered dextran-sugar, egg yolk combination. Pooled semen was used in order to eliminate the great variation among rams with respect to sperm survival after freezing.
MATERIALS AND METHODS Semen was collected, using an artificial vagina, from l0 adult rams of the synthetic strains being developed at the Animal Research Centre in Ottawa (Heaney et al. 1980). Rams were
0008-3984 I 821 6202-042s 52.00 e 1982 Agricultural Institute of Canada
425
426 CANADIAN JOURNAI- OF ANIMAT, SCIENCE
housed in windowless rooms maintained at ap- proximately 16'C throughout the year. Light of 200 lx intensity at 1 m above ground level was provided by incandescent lamps and op- erated under a day-length pattern coffesponding to latitude 45'N, with changes to the control- ling time clock being made weekly. Ejaculates were collected once a week for 6 wk beginning in early November. During this period the day- length changed from 10 to 8.5 h of light. The semen volume, spermatozoa concentration and spermatozoa motility were recorded immedi- ately after collection. Spermatozoa concentra- tion was determined spectrophotometrically and spermatozoa motility was assessed microscop- ically, as percentage of motile spermatozoa (0-10070) estimated to the nearest 57o and as
the rate of forward progression (motility rating) on a scale of 0 5. Ejaculates collected at each collection period were pooled, divided into two equal portions and each portion was extended at 30'C with two volumes of one of the fol- lowing diluents:
Diluent A: 80Vc (vo1/vol) lactose (321 mM); 20Vo (.vollvol) egg yolk; (osmolality 330 mOs/ kg water);
Diluent B:47.2 g dextran (mol. wt 150.000- 200.000); 8.2 g sodium citrate dihydrate; 1.9 g TES; 4.3 g glycine; 31.6 g lactose; 14.1 g raffinose; and 6.0 g fructose per litre of so-
lution; (osmolality 320 mOs/kg water). This resulted in an average spermatozoa concentra- tion of 1360 x 106/mL. Extended semen sam- ples were cooled at O.3'Clmin to 5'C. Each sample was then subdivided into two aliquots and extended further in the ratio 3:2 (diluted semen:glycerolated diluent) using one of the following glycerolated diluents:
lsotonic diluent C: 88.157o (volivol) diluent B; 1l.257o (vol/vol) glycerol. Hypertonic di- luent D: 49.6 g dextran; 8.6 g sodium citrate dihydrate; 2.0 g TES; 4.5 g glycine; 64.1 g
lactose; 119.8 g raffinose; and 6.3 g fructose per litre of solution. The osmolality of 600 mOs/kg was determined before addition of glycerol. The final solution contained 11.257a (vol/vol) glycerol. The glycerolated diluents were added drop-wise over 30 45 min to the extended semen samples maintained at 5"C and agitated slowly with a magnetic bar stirrer. After equilibration for 2 h the extended semen was transferred in 0.5-mL portions into five plastic straws (observations) per treatment in six replicates (weeks) and frozen at a linear
rate of l5"C/min to -100"C, using a program- able freezing system (Fiser et al. 1981), before storage in liquid nitrogen. After storage for 7
days the straws were thawed in a water bath at 39'C for 30 sec and resuspended in isotonic skim milk diluent. The spermatozoa motility was estimated microscopically as described above after thawing and after incubation at
39'C for 60 min. These procedures resulted in four diluent combinations, isotonic (AC) and (BC), and hypertonic (AD) and (BD); diluents AC and AD included egg yolk lactose in the first dilution, whereas diluents BC and BD used clear buffered dextran-sugar solution in both dilutions. The final average sperm con- centration was 810 x 10b/ml-.
Data were analyzed by analysis of variance (ANOVA) using linear contrasts to compare differences between diluents on cryosurvival (Steel and Torrie 1960).
RESULTS AND DISCUSSION The effect of different extender combina- tions on cryosurvival of ram spermatozoa is presented in Fig. I as post-thaw motility percent and rating of spermatozoa forward progression immediately after thawing and after 60 min incubation at 39'C. The post- thaw spermatozoa motility percent and rat- ing in both hypertonic diluents (AD,BD) were significantly higher (P<0.01 and P<0.05, respectively) than those after freezing in isotonic diluents (AC, BC). The percent of motile spermatozoa and motility rating after freezing and thawing did not differ significantly (P>0.05) be- tween either the two hypertonic diluents (AD vs. BD) or the two isotonic diluents (AC vs. BC) (Table 1). Although the pro- portional decrease in sperm motility was slightly higher in hypertonic diluents, the resulting motilities were still greater than those observed in isotonic diluents. Im- mediately after thawing, motility percent and rating averaged 43.87o and 3.4, and 4l .6Vo and 3.1 for hypertonic diluents AD and BD, respectively. After 60 min in- cubation, these values decreased to 35.jVo and 2.8, and 34.'7 and 2.7, respectively, compared with 27.2Vo and 2.6, and 25.3Vo and 2.5 observed in semen incubated for
C an
. J . A
ni m
. S ci
. D ow
nl oa
de d
fr om
c dn
sc ie
nc ep
ub .c
om b
y 17
1. 24
3. 0.
16 1
on 0
3/ 06
/2 3
F or
p er
so na
y.
60 min after freezing and thawing in re- spective isotonic diluents AC and BC. Pace and Graham (1974) have reported that glycerol provided little protection dur- ing freezing and thawing of bovine sper-
Fig. 1. The effect of diluents and post-thaw incubation on motility percent (upper graph) and motility rating (lower graph). Open col- umns indicate immediate post-thaw motility in isotonic diluents AC and BC and hvoertonic diluents AD and BDI F indicates the motility of fresh pooled semen. Shaded columns indi- cate the motility of semen after 60 min of incubation at 39"C (means + SD).
427
matozoa without the presence of egg yolk, and have suggested that high molecular weight lipoproteins in egg yolk (Watson 1976) and low molecular weight glycerol may interact with each other or with the spermatozoa membrane to yield a syner- gistic effect during freezing and thawing to improve cryosurvival (Jeyendran and Graham 1980). A similar protective mech- anism may be involved when dextran-sugar combinations are substituted for egg yolk. From their experiments, Williams and Hanis (1977) indicate that dextran is sur- face active and may possibly distribute into the lipids of the cell membrane and thus it may provide protection against the cold shock. The replacement of egg yolk by a dextran-sugar combination in ram semen diluents resulted in comparable cry- osurvival. Spermatozoa cryosurvival was significantly increased in moderately hy- pertonic diluents as compared with the is- otonic diluents, regardless of their com- position. This agrees with a previous report on the beneficial effect of moderately hy- pertonic diluents on ram spermatozoa cry- osurvival (Fiser et al. 1981). The exposure of spermatozoa to moderately hypertonic conditions before freezing results in the partial removal of freezable water from the cell through exoosmosis and, therefore, reduces the volume of intracellular ice for- mation. Glycerol, used at the same con- centration in all extenders, provided cry- opreservation by lowering the freezing point, by lowering the vapor pressure of cell suspensions and by reducing further the amount of intracellular ice, and kinet- ically by increasing the viscosity of extra- and intracellular solutions (Mervman et al. 1977).
COLAS, G. 1979. Fertility in the ewe after artificial insemination with fresh and frozen semen at the induced estrus, and influence of the photoperiod on the semen quality of the ram. Livest. Prod. Sci. 6: 153-166. DAMJANOVIC, V. and THOMAS, D. 1974. The use of polyvinylpyrrolidone as a cryopro-
FISER ET AI-. -
Table 1. Analysis of variance of ram spermatozoa cryosurvival
Mean squares
Source Motility
df percentage Motility rating
Diluent (AC, BC, AD, BD) AC, BC vs. AD, BD AC vs. BC AD vs. BD Incubation time Diluent x incubation time Replicates/treatment Observations/repl icate s
40 r92
2402** 6934+*
10.2 1 ** 0.90 0.58 0.12
*,+*P<0.05 and P<0.01, respectively.
tectectant in the freezing of human lympho- cytes. Cryobiology 1l: 312 316. FISER, P. S., AINSWORTH, L. and LANG- FORD, G. A. 1981. Effect of osmolality of skim milk diluents and thawing rate on cry- osurvival of ram spermatozoa. Cryobiology lE: 399-443. HEANEY, D. P., AINSWORTH, L., BATRA, T. R., FISER, P. S., HACKETT, A. J., LANGFORD, G. A. and LEE, A. J. 1980. Research for an intensive total confinement sheep production system. Agric. Can., Anim. Res. Inst. Tech. Bull. No. 2, 56 pp. JEYENDRAN, R. S. and GRAHAM, E. F. 1980. An evaluation of cryoprotective com- pounds on bovine spermatozoa. Cryobiology I7: 458-464. LIONETTI, F. J., HUNT, S. M. and LIN, P. S. 1976. Improved method for the cryopres- ervation of human red cells in liquid nitrogen with hydroxyethyl starch. Cryobiology 13: 489-499. McGANN, L. 1978. Differing action of pen- etrating and non-penetrating cryoprotective agents. Cryobiology 15: 382-390.
MERYMAN. H. T., WILIAMS, R. J. and
DOUGLAS, St. M. 1977. Freezing injury frorn "solution effects" and its prevention by natural and artificial cryopreservation. Cryobiology 14:
28'7 302. PACE. M. M. and GRAHAM, E. F. 1974. Components in egg yolk which protect bovine spermatozoa during freezing. J. Anim. Sci. 39:
rt44-1149. PRIBOR, D. B. and PRIBOR, H. C. 19'73.
Studies with Dextran 40 in cryopreservation of blood. Cryobiology 10: 9T103. STEEL, R. G. D. and TORRIE, J. H. 1960.
Principles and procedures of statistics. McGraw- Hill Book Company Inc., New York. WATSON, P. F. 1976. The protection of ram and bull spermatozoa by the low density li- poprotein fraction of egg yolk during storage at 5'C and deep-freezing. J. Therm. Biol. l: 137 -l4l .
WILLIAMS, R. J. and HARRIS, D. 1977.The distribution of cryoprotective agents into lipid interfaces. Cryobiology 14: 670-680.
C an
. J . A
ni m
. S ci
. D ow
nl oa
de d
fr om
c dn
sc ie
nc ep
ub .c
om b
y 17
1. 24
3. 0.
16 1
on 0
3/ 06
/2 3
F or
p er
so na