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The effects of nonpenetrating cryoprotectants added to TEST-yolk-glycerol extender on the post-thaw motility of ram spermatozoa
CRYOBIOLOGY
23, 512-517 (1986)
The Effects of Nonpenetrating Cryoprotectants TEST-Yolk-Glycerol Extender on the Post-thaw Ram Spermatozoa1 M. K. SCHMEHL, Animal Physiology,
Deprrrtmcnt
I. A. VAZQUEZ,
of Animd
Science, University
AND E. F. of Minnesota,
Added to Motility of
GRAHAM St. Paul, Minnesota
55108
The effects of adding five different concentrations of 17 polymeric compounds to TEST-yolk-glycerol extender on ram spermatozoa survival was studied. These were Aquacide (I. 11, and III); dextran (0.8-1.6, 1.9, 15-20, 70, and 200-300 kDa); three types of Dri-Sweet; hydroxyethyl starch: methylcellulose, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, and Supercol 912. All the compounds tested except the Dri-Sweet compounds and hydroxyethyl starch significantly (P 4 0.05) decreased percentages of motile cells in unfrozen samples. The use of dextran (O.g- 1.6 kDa; hydrolyzed dextran separ-ated by ethanol) and Aquacide II significantly (p < 0.05) increased post-thaw motility of spermatozoa frozen in pellets. Dextrdn (15-20 kDa), dextran (0.8- I.6 kDa), Aquacide II, and hydroxyethyl starch significantly (P < 0.05) increased the percentages of post-thaw motility of ram spermatozoa frozen in the presence of glycerol and egg yolk. 0 1986Academic Press, Inc.
The medium used to suspend cells during cryopreservation greatly affects ice crystal formation, crystallization temperature, and cryosurvival. The addition of colloids in aqueous solutions during freezing modifies the shape and growth of crystal formations (12). The formation of different ice crystal patterns is dependent upon temperature, concentration, and the specific compound used (13). The temperature of recrystallization during thaw is also dependent on the specific compound used (12). Many types of compounds provide cryoprotection to cells during the freezing process (3). Penetrating cryoprotectants, such as glycerol and dimethyl sulfoxide (DMSO), reduce cell dehydration by preventing the conccntration of the extracellular media (14) and are more effective at lower cooling rates (19). Nonpenetrating cryoprotectants, such as sucrose, dextran, and polyvinyl pyrrolidone, induce formation of ice crystal lattices or clathrates as external shields around the cell membrane (10) or these Received May 12, 1986; accepted September 5, 1986. i Scientific Journal Series, Paper No. 14,924, Minnesota Agricultural Experiment Station.
agents may enable the cell membranes to leak reversibly with osmotic stress (14). These types of agents function more effectively at high cooling rates (17, 19). Roy and Djerassi (18) reported that a combination of penetrating (DMSO) and nonpenetrating (sucrose) cryoprotectants had a synergistic effect for freezing of platelets. Combinations of penetrating agents with sugars have been used to cryopreserve spermatozoa from many species (9). Platov er al. (16) reported that extender with gum arabic and glycerol added, increased the percentage of ewes lambing after insemination with frozen ram semen. However, there are few other reports on polymeric compounds used in combination with penetrating cryoprotectants for the preservation of spermatozoa. The objective of this study was to examine the cryoprotective properties of nonpenetrating polymeric compounds used in combination with glycerol and egg yolk for the preservation of ram spermatozoa. MATERIALS
Extender preparation. In preliminary studies, 85 polymeric compounds were 512
001 l-2240/86 $3.00 CopyrIght All right,
0 1986 by Academic Press. Inc. of rrpruduction in any fwm rcsavcd
AND METHODS
CRYOPROTECTANTS
AND POST-THAW MOTILITY
screened for solubility in our extender system and for toxicity to spermatozoa. Insoluble compounds or compounds that did not support >30% motile spermatozoa after 24 hr at 5°C were eliminated. Saturated solutions of 17 polymeric compounds were prepared in TEST extender, 320 mosmol/kg osmotic pressure, pH 7.15 (7). Measured amounts of each compound were slowly added to 100 ml of TEST until saturated. The solutions were heated to dissolve the remaining material. The temperature used differed with each compound and was determined by the appearance of small bubbles in the solutions, prior to boiling. The temperature was then decreased approximately 2°C to prevent boiling and excess loss of water during the heatingistirring process. Osmotic pressure was adjusted to 325-330 mosmol/kg and the final volume of each solution was used to calculate the concentration of each substance. The compounds used, source, and concentrations are in Table I. The final extenders consisted of 20% yolk (v/v), 80, 64, 32, or 16% (v/v) of each compound, with the remaining percentage composed of TEST extender. The extenders were centrifuged at 15,OOOgfor 20 min and filtered through No. 612 filter paper, and either 0 or 4% glycerol (v/v) was added. TEST-20% yolk with 0 and 4% glycerol (v/v) was used as the control extender. Due to the processing time required, the extenders were tested in three groups (I -5, 6- 10, 1I- 17), each with different pooled semen samples and each with two samples of a TEST-yolk controls, with and without glycerol. Each group of extenders was tested with three different semen samples. Semen. Semen was collected from 1 to 3 rams, assessed for >65% motile spermatozoa, pooled, and extended 1:25 with the extender solutions. The semen was cooled at -0.2”Cimin to 5°C in a beaker of water and frozen in 0.1-m] pellets on dry ice at 4 hr. Percentages of progressively motile spermatozoa with normal head rota-
OF SPERMATOZOA
513
TABLE I Compounds, Source, and Concentrations of Additives Compound I. Methyl cellulose 2. Polyethylene glycol, 6 kDa 3. Dextran, 200-300 kDa 4. Dextran. 70 kDa 5. Dextran, 15-20 kDa 6. Hydrolyzed dextran 0.8-1.6 kDa 7. Aquacide I 8. Aquacide II 9. Aquacide III 10. Polyvinyl pyrrolidone Type NP-KI5 1I. Poly(vinyl alcohol) 12. Dri-Sweet 13. OK Dri-Sweet 14. OK Dri-Sweet 36 IS. Hydroxyethyl starch 16. Hydrolyzed dextran. I .9 kDa 17. Supercol 912
Source”
g/l00 ml
A
0.50
B C D C
2.26 9.76 8.47 14.39
E F F F
4.95 0.50 0.25 8.47
G B H H H I
8.69 1.68 5.86 8.35 8.71 3.92
D J
6.67 0.76
u A, Fisher Scientific, Springfield, N.J. M-280; B, J. T. Baker Chemical Co., Phillipsburg, N.J. (2) U219. (I I) U229. C, ICN. Nutritional Biochemicals, Cleveland. Ohio. D, Pharmacia Fine Chemicals, Piscataway, N.J. (4) T70. (16) PD8116. E, Prepared from our laboratory by Pharmacia Fine Chemicals. F, Calbiothem, San Diego, Calif. (7) 1785. (8) 17851. (9) 17852. G, General Aniline and Film Corp., New York, N.Y. H, The Hubinger Co., Keokuk. Iowa: I. Sigma Chemicals. St. Louis. MO. H-6382. J, General Mills. Minneapolis. Minn.
tion were estimated from nonfrozen samples just prior to freezing and after 24 hr storage at 5°C. Three pellets from coded samples were thawed individually in an aluminum thaw block at 37°C. The percentages of motile spermatozoa were estimated and mean values were determined. The thaw block consisted of a 50-mm-wide x 75-mm-long x lPmm-thick block with 20 wells, 6 mm diameter x 13 mm deep. The semen samples were analyzed immediately after thawing. All motility estimates were made using a closed circuit television system on a microscope. The study was repeated three times.
514
SCHMEHL,
VAZQUEZ,
Each group of extenders was analyzed statistically with their respective TESTyolk controls using a factorial design (3 replicates x 5 or 7 extenders x 2 percentages of glycerol) using the general linear model (GLM, (6), a least-squares procedure). Mean differences were determined by Duncan’s multiple range test (6) at the P > 0.05 level of significance. Control samples were compared with percentage polymeric stock solution for individual added compounds using the contrast option in GLM
AND GRAHAM
erol (31.7, 27.5, and 28.0% for control Groups 1, 2, and 3, respectively). DISCUSSION
Methyl cellulose, dextran, peptone, and polyvinyl pyrrolidone (PVP) gave little protection without glycerol during cryopreservation of boar spermatozoa but Salamon et ul. (21) suggested that these compounds, used in conjunction with other cryoprotective agents, may provide additional protection. Glucose, fructose, lactose, and raf(6). finose, as well as sugar alcohols, are frequently used in semen extenders to improve cryopreservation (8, 9). Platov et RESULTS al. (15) tested gum arabic, cherry resin, There were no significant improvements apricot resin, and inulin as cryoprotectants over the respective control in nonfrozen for ram semen. Semen preserved with the semen storage for any extender at 4 hr resins and inulin had decreased post-thaw (prefreeze) or 24 hr at 5°C (Table 2). How- fertility while semen with gum arabic had ever, dextran 0.8- 1.6 kDa and Aquacide II slightly increased fertility over the control. (Group 2) were significantly better than the In later studies (16), ram spermatozoa prearabic-cicontrol for post-thaw samples. There were served in lactose-yolk-gum trate-glycerol extender yielded a 79.6% no significant differences between the control and methyl cellulose or polyethylene lambing rate compared to 65.1% for a lacacid-yolk extender. Dexglycol (Group l), the control and Aquacide tose-Tris-citric I (Group 2), and the control and Dri-Sweet, tran and HES with glycerol have also been hydroxyethyl starch (HES), or dextran, I .9 used to increase the cryosurvival of ram kDa (Group 3) for post-thaw motility. Post- spermatozoa (4). These data suggest a synthaw samples in all extenders with 4% glyc- ergism of cryoprotective effects. In our erol were significantly higher than samples study, both dextran and HES provided without glycerol, reagardless of polymeric good post-thaw recovery of motile spermacompound added. Mean percentages of tozoa. However, gum arabic in TEST exmotile spermatozoa were 42.2, 39.8, and tender was eliminated during the prelimi26.4% with 0% glycerol and 45.4, 42.4, and nary screening of compounds. Possibly, 39.9% with 4% glycerol for 4 and 24 hr there is difference in the protective effects nonfrozen storage and post-thaw samples, of gum arabic when used with different exrespectively. Dextran, 15-20 kDa with 16% tender systems. The data in our study also suggest a synstock solution, dextran, 0.8-1.6 kDa with 48% stock solution, Aquacide 11, with 32% ergism of cryoprotective effects for combistock solution, and hydroxyethyl starch nations of penetrating with nonpenetrating with 16% stock solution (Table 3) signifi- agents for certain compounds and concencantly improved post-thaw recovery of trations of compounds. Hydrolyzed dexspermatozoa frozen with 4% glycerol. tran (0.8-I .6 kDa) and Aquacide II both However, none of the polymeric com- yielded significantly higher percentages of pounds tested without glycerol significantly post-thaw motility. Although the hydroincreased post-thaw recovery of sperma- lyzed dextran with the lowest molecular tozoa above control samples without glyc- weight (0.8- 1.6 KDa) provided more pro-
CRYOPROTECTANTS
AND POST-THAW MOTILITY
OF SPERMATOZOA
515
TABLE 2 Motility of Ram Spermatozoa in TEST-Yolk-Glycerol Extender with Different Polymeric Compounds % Motile spermatozoa Fresh semen Treatment compound Group 1 Control If Methyl cellulose Polyethylene glycol, 6KDa Dextran 200-300 KDa Dextran 70 KDa Dextran IS-20 KDa Group 2 Control 2f Dextran, OX- I .6 KDag Aquacide 1 Aquacide II Aquacide III Polyvinyl pyrrolidone Group 3 Control Y Poly (vinyl alcohol) Dri-Sweet OK Dri-Sweet OK Dri-Sweet 36 Hydroxyethyl starch Dextran, I .9 kDag Supercol 9 I2
Prefreeze
Frozen semen 24 hr
Post-thaw
52._sa,e 46X6 44.0’ 43.7’ 4J.Sb 46.2b,’
41,9o.b.e
42.3h 42.36 45.00 4l.Y
3s.9a,c 37.6” 35. IO 24.6b 28.76 2J.9b
54.2” 49.2’ 48.8’ 43.Sd 49.4c
48.50 44.7b 46.0b 46.2b 37.9< 4S.Ob
33.6” 40.2a 3J.9”,b 39.6” 21.2’ 33.36
42.9Q 34.2c.d 40.2”,b 41.7”,h 39.w 41 ,W 3JSb.’ 3 I .8d
40.4” 36.3< 39.7a,b 40.3” 37.5”.h,’ 40.0” 36.8b,c 33.3”
3S.h”J,c
51.9b
41.Ob
21.3d 35.6”,b,c 33.36,< 32.6’ 39.3” 38.6”,b 32.F
u-d Means in the same column and within group not followed by the same letter are significantly different (P < 0.05). p Controls N = 12; Samples N = 30. fTEST-yolk-glycerol extender without additives. X Hydrolyzed dextran, separated by ethanol precipitation.
tection than the higher molecular weight dextran (15-20, 70, 200-300 kDa), the reverse was true for the Aquacides. Aquacide I is described as a sodium salt of carboxymethylcellulose, 70 kDa, and Aquacide II is a sodium salt of carboxymethylcellulose, 500 kDa. Here, the higher molecule weight compound provided the most protection. Aquacide III is a flake polyethylene glycol (20 kDa) which provided less protection during freezing than the control while polyethylene glycol (6 kDa) resulted in motility which was not different (P > 0.05) than its control. Apparently, neither the basic chemical skeleton nor the molecular weight alone determines the protective properties
of polymeric materials but possibly a combination of the two factors. All the polymeric compounds studied with the exception of the Dri-Sweet compounds and HES significantly depressed percentages of motile spermatozoa in fresh semen. However, the Dri-Sweet compounds (corn starch products) did not increase post-thaw recovery of motile cells but the lowest concentration of HES tested provided significantly higher percentages of motile spermatozoa post-thaw than the control sample. A combination of DMSO and HES yields higher post-thaw recovery of hemopoietic origin cells than does DMSO alone (11, 22). However, DMSO
516
SCHMEHL,
VAZQUEZ,
AND GRAHAM
TABLE 3 Percentage Post-thaw Motility of Ram Spermatozoa Frozen in TEST-Yolk-Glycerol Polymeric Compounds
Extender with Different
% Stock solution Treatment/compound
0
Control I” Methyl cellulose Polyethylene glycol, 6 kDa Dextran 200-300 kDa Dextran 70 kDa Dextran IS-20 kDa Control 2” Dextran, 0.8- 1.6 KDac Aquacide I Aquacide II Aquacide 111 Polyvinyl pyrrolidone Control 3” Poly (vinyl alcohol) Dri-Sweet OK Dri-Sweet OK Dri-Sweet 36 Hydroxyethyl starch Dextran, I .9 kDac Supercol912
43.6
16
32
48
64
80
46.1 37.2 36.7 37.8 61.7’
34.4 39.4 25.0 27.8 35.6
45.0 41.7 41.1 46.2 37.8
41.1 42.2 30.0 42.6 27.2
45.6 36.1 15.0 27.8 22.2
41.1 51.7 51.1 46.1 51.1
54.4 48.9 55.0* 38.3 43.3
60.0” 45.0 46.1 27.8 28.9
46.7 47.8 42.2 16.1 30.0
44.4 39.4 52.8 13.3 25.0
40.6 39.4 41.1 35.6 51.7* 44.4 33.9
36.1 49.4 40.6 32.5 47.8 47.2 40.6
38.3 48.3 40.6 42.8 47.2 45.6 36.7
31.1 41.1 36.1 39.4 41.7 46.1 33.9
23.3 34.4 31.7 40.6 44.4 56. I 35.0
38.6
43.1
D TEST-yolk-glycerol extender without additives. b Controls N = 6; samples N = 3. c Hydrolyzed dextran separated by ethanol precipitation. * Significantly (P < 0.05) higher than the respective control value.
was not used in this study because it provides less cryoprotection to ram spermatozoa than glycerol (2, 20). The combination of HES and PVP with 15% glycerol has been used to cryopreserve several cell culture lines (1). However, glycerol concentrations over 8% have detrimental effects on frozen ram semen (5). The concentrations of polymeric compounds in our study are lower than the concentrations used by the above researchers (11, 22) because the solutions were adjusted for osmotic pressure and also many of the compounds we tested were not very soluble in TEST extender. TEST extender has a low ionic strength which probably decreases the solubility of some of the compounds used. Generally, the higher concentrations used in this study showed a decrease in percentages of motile
spermatozoa post-thaw, The increase in viscosity associated with higher concentrations of polymeric compounds may be detrimental to spermatozoa. The improved post-thaw motility in this study with the addition of lower weight hydrolyzed dextrans (0.8- 1.6 kDa) along with the established protective properties of sugars (3, 14, 19) such as glucose (180 Da), sucrose (340 Da), and lactose (360 Da) suggests that direct comparisons need to be made between these compounds. Also, the optimal cryoprotection may be afforded by polysaccharides with molecular weights between 0.36 and 0.8-1.6 kDa. Additionally, the polymeric compounds that supported the highest percentages of post-thaw motility need to be evaluated by fertility studies with frozen semen. Motility estimation is one of several laboratory evalua-
CRYOPROTECTANTS
AND POST-THAW MOTILITY
tions that are commonly used to evaluate semen quality but no laboratory assay can, presently, be used to accurately predict fertility of frozen-thawed semen (9). However, we have found that motility estimates used with multiple replications can be used to evaluate semen quality in comparative treatment studies utilizing split ejaculate techniques. This enables the researcher to eliminate treatments prior to the costly procedure of fertility trials. REFERENCES 1. Conscience, J. F., and Fischer, F. An improved preservation technique for cells with hemopoietic origin. Cr~ohiolog~ 22, 495-498 (1985). 2. Curiel, G. B., and Mendez. J. V. Action of cryoprotective agents dimethyl sulfoxide and glycerol on the acrosome of the ram sperm during freezing. Vet. Mu. 12, 211-216 (1981). 3. Doebbler, G. F. Cryoprotective compounds: Review and discussion of structure and function. Ctyohiolog?~ 3, 2-11 (1966). 4. Fiser, P. S., Ainsworth, L., and Fairfull, R. W. Cryosurvival of ram spermatozoa in hypertonic and isotonic diluents. C~ncld. J. Anirn. Sc,i. 62, 425-428 (1982). 5. Fiser, P. S., and Fait-full, R. W. The effect of glycerol concentration and cooling velocity on cryosurvival of ram spermatozoa frozen in straws. Cryobiology 21, 542-551 (1984). 6. Goodnight, J. H. GLMiintroduction: GLMireference. In “SAS User’s Guide,” pp. 237-265. SAS Institute Inc., Cary, N.C. (1979). 7. Graham, E. F., Crabo, B. G., and Brown. K. I. Effect of some zwitter ion buffers on the freezing and storage of spermatozoa. 1. Bull. 1. Dairy Sci. 55, 372-378 (1972).
8. Graham, E. F., Crabo, B. G., and M. M. Pace. Current status of semen preservation in the ram, boar and stallion. J. Anirn. Sci. 47 (Suppl. II), 80-119 (1978). 9. Graham, E. F., Schmehl, M. L., and Deyo, R. C. M. Cryopreservation and fertility of fish, poultry and mammalian spermatozoa. In “Proceedings 10th Tech. Conf. A.I. Reprod.,
OF SPERMATOZOA
517
NAAB,” pp. 4-29 (1984). 10. Karow, A. M.. and Webb, W. R. Tissue freezing: A theory for injury and survival. Cryobiology 2, 99-108 (1965). 11. Luscinskas, F. W., Lionetti, F. J., Melaragno, A. J., and Valeri, C. R. Long term cryopreservation of dog granulocytes. Cryobiology 20, l-6 (1983). 12. Luyet, B. J. Phase transitions encountered in the rapid freezing of aqueous solutions. Ann. N. Y. Acud. SC;. 125, 502-521 (1965). 13. Luyet. B., and Rapatz, G. Patterns of ice formation in some aqueous solutions. Biodynrrmicrc 8, l-68 (1958). 14. Meryman, H. T. Cryoprotective agents. Cryobiology 8, 173-183 (1971). 15. Platov, E. M., Avanov, N. Y., and Balaskov, N. G. Freezing ram semen in diluents containing polysaccharides. Ovtsevodswo Mask. 10, 38-39 (1980). 16. Platov, E. M.. Karol, V. K.. and Bashkatov, L. P. An experiment on the use of deep frozen ram semen. Zhivot. No. 3, 41-42 (1983). 17. Rapatz, G., and Luyet, B. Combined effects of freezing rates and of various protective agents on the preservation of human erythrocytes. C/yobio/ogy
4, 215-222 (1968).
18. Roy, A. J., and Djerassi, I. Prevention of freezing injury by simultaneous use of agents with different modes of action. Cryabiologp 1 (Suppl. 1). 20 (1965). 19. Rowe, A. W. Biochemical aspects of cryoprotective agents in freezing and thawing. Cryohiology 3, 12-18 (1966). 20. Salamon, S. Deep freezing of ram semen: Recovery of spermatozoa after pelleting and comparison with other methods of freezing. Ausr. J. Biol. Sc,i. 21, 351-360 (1968). 21. Salamon, S., Wilmut, I., and Polge, C. Deep freezing of boar semen. I. Effects of diluent composition, protective agents, and method of thawing on survival of spermatozoa. Aust. J. Bid. SC;. 26, 219-230 (1973).
22. Stiff, P. J., Murgo, A. J., Zaroulis, C. G.. DeRisi, M. F., and Clarkson, B. D. Unfractionated human marrow cell cryopreservation using dimethyl sulfoxide and hydroxyethyl starch. Cryobio1og.v 20, 17-24 (1983).
23, 512-517 (1986)
The Effects of Nonpenetrating Cryoprotectants TEST-Yolk-Glycerol Extender on the Post-thaw Ram Spermatozoa1 M. K. SCHMEHL, Animal Physiology,
Deprrrtmcnt
I. A. VAZQUEZ,
of Animd
Science, University
AND E. F. of Minnesota,
Added to Motility of
GRAHAM St. Paul, Minnesota
55108
The effects of adding five different concentrations of 17 polymeric compounds to TEST-yolk-glycerol extender on ram spermatozoa survival was studied. These were Aquacide (I. 11, and III); dextran (0.8-1.6, 1.9, 15-20, 70, and 200-300 kDa); three types of Dri-Sweet; hydroxyethyl starch: methylcellulose, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, and Supercol 912. All the compounds tested except the Dri-Sweet compounds and hydroxyethyl starch significantly (P 4 0.05) decreased percentages of motile cells in unfrozen samples. The use of dextran (O.g- 1.6 kDa; hydrolyzed dextran separ-ated by ethanol) and Aquacide II significantly (p < 0.05) increased post-thaw motility of spermatozoa frozen in pellets. Dextrdn (15-20 kDa), dextran (0.8- I.6 kDa), Aquacide II, and hydroxyethyl starch significantly (P < 0.05) increased the percentages of post-thaw motility of ram spermatozoa frozen in the presence of glycerol and egg yolk. 0 1986Academic Press, Inc.
The medium used to suspend cells during cryopreservation greatly affects ice crystal formation, crystallization temperature, and cryosurvival. The addition of colloids in aqueous solutions during freezing modifies the shape and growth of crystal formations (12). The formation of different ice crystal patterns is dependent upon temperature, concentration, and the specific compound used (13). The temperature of recrystallization during thaw is also dependent on the specific compound used (12). Many types of compounds provide cryoprotection to cells during the freezing process (3). Penetrating cryoprotectants, such as glycerol and dimethyl sulfoxide (DMSO), reduce cell dehydration by preventing the conccntration of the extracellular media (14) and are more effective at lower cooling rates (19). Nonpenetrating cryoprotectants, such as sucrose, dextran, and polyvinyl pyrrolidone, induce formation of ice crystal lattices or clathrates as external shields around the cell membrane (10) or these Received May 12, 1986; accepted September 5, 1986. i Scientific Journal Series, Paper No. 14,924, Minnesota Agricultural Experiment Station.
agents may enable the cell membranes to leak reversibly with osmotic stress (14). These types of agents function more effectively at high cooling rates (17, 19). Roy and Djerassi (18) reported that a combination of penetrating (DMSO) and nonpenetrating (sucrose) cryoprotectants had a synergistic effect for freezing of platelets. Combinations of penetrating agents with sugars have been used to cryopreserve spermatozoa from many species (9). Platov er al. (16) reported that extender with gum arabic and glycerol added, increased the percentage of ewes lambing after insemination with frozen ram semen. However, there are few other reports on polymeric compounds used in combination with penetrating cryoprotectants for the preservation of spermatozoa. The objective of this study was to examine the cryoprotective properties of nonpenetrating polymeric compounds used in combination with glycerol and egg yolk for the preservation of ram spermatozoa. MATERIALS
Extender preparation. In preliminary studies, 85 polymeric compounds were 512
001 l-2240/86 $3.00 CopyrIght All right,
0 1986 by Academic Press. Inc. of rrpruduction in any fwm rcsavcd
AND METHODS
CRYOPROTECTANTS
AND POST-THAW MOTILITY
screened for solubility in our extender system and for toxicity to spermatozoa. Insoluble compounds or compounds that did not support >30% motile spermatozoa after 24 hr at 5°C were eliminated. Saturated solutions of 17 polymeric compounds were prepared in TEST extender, 320 mosmol/kg osmotic pressure, pH 7.15 (7). Measured amounts of each compound were slowly added to 100 ml of TEST until saturated. The solutions were heated to dissolve the remaining material. The temperature used differed with each compound and was determined by the appearance of small bubbles in the solutions, prior to boiling. The temperature was then decreased approximately 2°C to prevent boiling and excess loss of water during the heatingistirring process. Osmotic pressure was adjusted to 325-330 mosmol/kg and the final volume of each solution was used to calculate the concentration of each substance. The compounds used, source, and concentrations are in Table I. The final extenders consisted of 20% yolk (v/v), 80, 64, 32, or 16% (v/v) of each compound, with the remaining percentage composed of TEST extender. The extenders were centrifuged at 15,OOOgfor 20 min and filtered through No. 612 filter paper, and either 0 or 4% glycerol (v/v) was added. TEST-20% yolk with 0 and 4% glycerol (v/v) was used as the control extender. Due to the processing time required, the extenders were tested in three groups (I -5, 6- 10, 1I- 17), each with different pooled semen samples and each with two samples of a TEST-yolk controls, with and without glycerol. Each group of extenders was tested with three different semen samples. Semen. Semen was collected from 1 to 3 rams, assessed for >65% motile spermatozoa, pooled, and extended 1:25 with the extender solutions. The semen was cooled at -0.2”Cimin to 5°C in a beaker of water and frozen in 0.1-m] pellets on dry ice at 4 hr. Percentages of progressively motile spermatozoa with normal head rota-
OF SPERMATOZOA
513
TABLE I Compounds, Source, and Concentrations of Additives Compound I. Methyl cellulose 2. Polyethylene glycol, 6 kDa 3. Dextran, 200-300 kDa 4. Dextran. 70 kDa 5. Dextran, 15-20 kDa 6. Hydrolyzed dextran 0.8-1.6 kDa 7. Aquacide I 8. Aquacide II 9. Aquacide III 10. Polyvinyl pyrrolidone Type NP-KI5 1I. Poly(vinyl alcohol) 12. Dri-Sweet 13. OK Dri-Sweet 14. OK Dri-Sweet 36 IS. Hydroxyethyl starch 16. Hydrolyzed dextran. I .9 kDa 17. Supercol 912
Source”
g/l00 ml
A
0.50
B C D C
2.26 9.76 8.47 14.39
E F F F
4.95 0.50 0.25 8.47
G B H H H I
8.69 1.68 5.86 8.35 8.71 3.92
D J
6.67 0.76
u A, Fisher Scientific, Springfield, N.J. M-280; B, J. T. Baker Chemical Co., Phillipsburg, N.J. (2) U219. (I I) U229. C, ICN. Nutritional Biochemicals, Cleveland. Ohio. D, Pharmacia Fine Chemicals, Piscataway, N.J. (4) T70. (16) PD8116. E, Prepared from our laboratory by Pharmacia Fine Chemicals. F, Calbiothem, San Diego, Calif. (7) 1785. (8) 17851. (9) 17852. G, General Aniline and Film Corp., New York, N.Y. H, The Hubinger Co., Keokuk. Iowa: I. Sigma Chemicals. St. Louis. MO. H-6382. J, General Mills. Minneapolis. Minn.
tion were estimated from nonfrozen samples just prior to freezing and after 24 hr storage at 5°C. Three pellets from coded samples were thawed individually in an aluminum thaw block at 37°C. The percentages of motile spermatozoa were estimated and mean values were determined. The thaw block consisted of a 50-mm-wide x 75-mm-long x lPmm-thick block with 20 wells, 6 mm diameter x 13 mm deep. The semen samples were analyzed immediately after thawing. All motility estimates were made using a closed circuit television system on a microscope. The study was repeated three times.
514
SCHMEHL,
VAZQUEZ,
Each group of extenders was analyzed statistically with their respective TESTyolk controls using a factorial design (3 replicates x 5 or 7 extenders x 2 percentages of glycerol) using the general linear model (GLM, (6), a least-squares procedure). Mean differences were determined by Duncan’s multiple range test (6) at the P > 0.05 level of significance. Control samples were compared with percentage polymeric stock solution for individual added compounds using the contrast option in GLM
AND GRAHAM
erol (31.7, 27.5, and 28.0% for control Groups 1, 2, and 3, respectively). DISCUSSION
Methyl cellulose, dextran, peptone, and polyvinyl pyrrolidone (PVP) gave little protection without glycerol during cryopreservation of boar spermatozoa but Salamon et ul. (21) suggested that these compounds, used in conjunction with other cryoprotective agents, may provide additional protection. Glucose, fructose, lactose, and raf(6). finose, as well as sugar alcohols, are frequently used in semen extenders to improve cryopreservation (8, 9). Platov et RESULTS al. (15) tested gum arabic, cherry resin, There were no significant improvements apricot resin, and inulin as cryoprotectants over the respective control in nonfrozen for ram semen. Semen preserved with the semen storage for any extender at 4 hr resins and inulin had decreased post-thaw (prefreeze) or 24 hr at 5°C (Table 2). How- fertility while semen with gum arabic had ever, dextran 0.8- 1.6 kDa and Aquacide II slightly increased fertility over the control. (Group 2) were significantly better than the In later studies (16), ram spermatozoa prearabic-cicontrol for post-thaw samples. There were served in lactose-yolk-gum trate-glycerol extender yielded a 79.6% no significant differences between the control and methyl cellulose or polyethylene lambing rate compared to 65.1% for a lacacid-yolk extender. Dexglycol (Group l), the control and Aquacide tose-Tris-citric I (Group 2), and the control and Dri-Sweet, tran and HES with glycerol have also been hydroxyethyl starch (HES), or dextran, I .9 used to increase the cryosurvival of ram kDa (Group 3) for post-thaw motility. Post- spermatozoa (4). These data suggest a synthaw samples in all extenders with 4% glyc- ergism of cryoprotective effects. In our erol were significantly higher than samples study, both dextran and HES provided without glycerol, reagardless of polymeric good post-thaw recovery of motile spermacompound added. Mean percentages of tozoa. However, gum arabic in TEST exmotile spermatozoa were 42.2, 39.8, and tender was eliminated during the prelimi26.4% with 0% glycerol and 45.4, 42.4, and nary screening of compounds. Possibly, 39.9% with 4% glycerol for 4 and 24 hr there is difference in the protective effects nonfrozen storage and post-thaw samples, of gum arabic when used with different exrespectively. Dextran, 15-20 kDa with 16% tender systems. The data in our study also suggest a synstock solution, dextran, 0.8-1.6 kDa with 48% stock solution, Aquacide 11, with 32% ergism of cryoprotective effects for combistock solution, and hydroxyethyl starch nations of penetrating with nonpenetrating with 16% stock solution (Table 3) signifi- agents for certain compounds and concencantly improved post-thaw recovery of trations of compounds. Hydrolyzed dexspermatozoa frozen with 4% glycerol. tran (0.8-I .6 kDa) and Aquacide II both However, none of the polymeric com- yielded significantly higher percentages of pounds tested without glycerol significantly post-thaw motility. Although the hydroincreased post-thaw recovery of sperma- lyzed dextran with the lowest molecular tozoa above control samples without glyc- weight (0.8- 1.6 KDa) provided more pro-
CRYOPROTECTANTS
AND POST-THAW MOTILITY
OF SPERMATOZOA
515
TABLE 2 Motility of Ram Spermatozoa in TEST-Yolk-Glycerol Extender with Different Polymeric Compounds % Motile spermatozoa Fresh semen Treatment compound Group 1 Control If Methyl cellulose Polyethylene glycol, 6KDa Dextran 200-300 KDa Dextran 70 KDa Dextran IS-20 KDa Group 2 Control 2f Dextran, OX- I .6 KDag Aquacide 1 Aquacide II Aquacide III Polyvinyl pyrrolidone Group 3 Control Y Poly (vinyl alcohol) Dri-Sweet OK Dri-Sweet OK Dri-Sweet 36 Hydroxyethyl starch Dextran, I .9 kDag Supercol 9 I2
Prefreeze
Frozen semen 24 hr
Post-thaw
52._sa,e 46X6 44.0’ 43.7’ 4J.Sb 46.2b,’
41,9o.b.e
42.3h 42.36 45.00 4l.Y
3s.9a,c 37.6” 35. IO 24.6b 28.76 2J.9b
54.2” 49.2’ 48.8’ 43.Sd 49.4c
48.50 44.7b 46.0b 46.2b 37.9< 4S.Ob
33.6” 40.2a 3J.9”,b 39.6” 21.2’ 33.36
42.9Q 34.2c.d 40.2”,b 41.7”,h 39.w 41 ,W 3JSb.’ 3 I .8d
40.4” 36.3< 39.7a,b 40.3” 37.5”.h,’ 40.0” 36.8b,c 33.3”
3S.h”J,c
51.9b
41.Ob
21.3d 35.6”,b,c 33.36,< 32.6’ 39.3” 38.6”,b 32.F
u-d Means in the same column and within group not followed by the same letter are significantly different (P < 0.05). p Controls N = 12; Samples N = 30. fTEST-yolk-glycerol extender without additives. X Hydrolyzed dextran, separated by ethanol precipitation.
tection than the higher molecular weight dextran (15-20, 70, 200-300 kDa), the reverse was true for the Aquacides. Aquacide I is described as a sodium salt of carboxymethylcellulose, 70 kDa, and Aquacide II is a sodium salt of carboxymethylcellulose, 500 kDa. Here, the higher molecule weight compound provided the most protection. Aquacide III is a flake polyethylene glycol (20 kDa) which provided less protection during freezing than the control while polyethylene glycol (6 kDa) resulted in motility which was not different (P > 0.05) than its control. Apparently, neither the basic chemical skeleton nor the molecular weight alone determines the protective properties
of polymeric materials but possibly a combination of the two factors. All the polymeric compounds studied with the exception of the Dri-Sweet compounds and HES significantly depressed percentages of motile spermatozoa in fresh semen. However, the Dri-Sweet compounds (corn starch products) did not increase post-thaw recovery of motile cells but the lowest concentration of HES tested provided significantly higher percentages of motile spermatozoa post-thaw than the control sample. A combination of DMSO and HES yields higher post-thaw recovery of hemopoietic origin cells than does DMSO alone (11, 22). However, DMSO
516
SCHMEHL,
VAZQUEZ,
AND GRAHAM
TABLE 3 Percentage Post-thaw Motility of Ram Spermatozoa Frozen in TEST-Yolk-Glycerol Polymeric Compounds
Extender with Different
% Stock solution Treatment/compound
0
Control I” Methyl cellulose Polyethylene glycol, 6 kDa Dextran 200-300 kDa Dextran 70 kDa Dextran IS-20 kDa Control 2” Dextran, 0.8- 1.6 KDac Aquacide I Aquacide II Aquacide 111 Polyvinyl pyrrolidone Control 3” Poly (vinyl alcohol) Dri-Sweet OK Dri-Sweet OK Dri-Sweet 36 Hydroxyethyl starch Dextran, I .9 kDac Supercol912
43.6
16
32
48
64
80
46.1 37.2 36.7 37.8 61.7’
34.4 39.4 25.0 27.8 35.6
45.0 41.7 41.1 46.2 37.8
41.1 42.2 30.0 42.6 27.2
45.6 36.1 15.0 27.8 22.2
41.1 51.7 51.1 46.1 51.1
54.4 48.9 55.0* 38.3 43.3
60.0” 45.0 46.1 27.8 28.9
46.7 47.8 42.2 16.1 30.0
44.4 39.4 52.8 13.3 25.0
40.6 39.4 41.1 35.6 51.7* 44.4 33.9
36.1 49.4 40.6 32.5 47.8 47.2 40.6
38.3 48.3 40.6 42.8 47.2 45.6 36.7
31.1 41.1 36.1 39.4 41.7 46.1 33.9
23.3 34.4 31.7 40.6 44.4 56. I 35.0
38.6
43.1
D TEST-yolk-glycerol extender without additives. b Controls N = 6; samples N = 3. c Hydrolyzed dextran separated by ethanol precipitation. * Significantly (P < 0.05) higher than the respective control value.
was not used in this study because it provides less cryoprotection to ram spermatozoa than glycerol (2, 20). The combination of HES and PVP with 15% glycerol has been used to cryopreserve several cell culture lines (1). However, glycerol concentrations over 8% have detrimental effects on frozen ram semen (5). The concentrations of polymeric compounds in our study are lower than the concentrations used by the above researchers (11, 22) because the solutions were adjusted for osmotic pressure and also many of the compounds we tested were not very soluble in TEST extender. TEST extender has a low ionic strength which probably decreases the solubility of some of the compounds used. Generally, the higher concentrations used in this study showed a decrease in percentages of motile
spermatozoa post-thaw, The increase in viscosity associated with higher concentrations of polymeric compounds may be detrimental to spermatozoa. The improved post-thaw motility in this study with the addition of lower weight hydrolyzed dextrans (0.8- 1.6 kDa) along with the established protective properties of sugars (3, 14, 19) such as glucose (180 Da), sucrose (340 Da), and lactose (360 Da) suggests that direct comparisons need to be made between these compounds. Also, the optimal cryoprotection may be afforded by polysaccharides with molecular weights between 0.36 and 0.8-1.6 kDa. Additionally, the polymeric compounds that supported the highest percentages of post-thaw motility need to be evaluated by fertility studies with frozen semen. Motility estimation is one of several laboratory evalua-
CRYOPROTECTANTS
AND POST-THAW MOTILITY
tions that are commonly used to evaluate semen quality but no laboratory assay can, presently, be used to accurately predict fertility of frozen-thawed semen (9). However, we have found that motility estimates used with multiple replications can be used to evaluate semen quality in comparative treatment studies utilizing split ejaculate techniques. This enables the researcher to eliminate treatments prior to the costly procedure of fertility trials. REFERENCES 1. Conscience, J. F., and Fischer, F. An improved preservation technique for cells with hemopoietic origin. Cr~ohiolog~ 22, 495-498 (1985). 2. Curiel, G. B., and Mendez. J. V. Action of cryoprotective agents dimethyl sulfoxide and glycerol on the acrosome of the ram sperm during freezing. Vet. Mu. 12, 211-216 (1981). 3. Doebbler, G. F. Cryoprotective compounds: Review and discussion of structure and function. Ctyohiolog?~ 3, 2-11 (1966). 4. Fiser, P. S., Ainsworth, L., and Fairfull, R. W. Cryosurvival of ram spermatozoa in hypertonic and isotonic diluents. C~ncld. J. Anirn. Sc,i. 62, 425-428 (1982). 5. Fiser, P. S., and Fait-full, R. W. The effect of glycerol concentration and cooling velocity on cryosurvival of ram spermatozoa frozen in straws. Cryobiology 21, 542-551 (1984). 6. Goodnight, J. H. GLMiintroduction: GLMireference. In “SAS User’s Guide,” pp. 237-265. SAS Institute Inc., Cary, N.C. (1979). 7. Graham, E. F., Crabo, B. G., and Brown. K. I. Effect of some zwitter ion buffers on the freezing and storage of spermatozoa. 1. Bull. 1. Dairy Sci. 55, 372-378 (1972).
8. Graham, E. F., Crabo, B. G., and M. M. Pace. Current status of semen preservation in the ram, boar and stallion. J. Anirn. Sci. 47 (Suppl. II), 80-119 (1978). 9. Graham, E. F., Schmehl, M. L., and Deyo, R. C. M. Cryopreservation and fertility of fish, poultry and mammalian spermatozoa. In “Proceedings 10th Tech. Conf. A.I. Reprod.,
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NAAB,” pp. 4-29 (1984). 10. Karow, A. M.. and Webb, W. R. Tissue freezing: A theory for injury and survival. Cryobiology 2, 99-108 (1965). 11. Luscinskas, F. W., Lionetti, F. J., Melaragno, A. J., and Valeri, C. R. Long term cryopreservation of dog granulocytes. Cryobiology 20, l-6 (1983). 12. Luyet, B. J. Phase transitions encountered in the rapid freezing of aqueous solutions. Ann. N. Y. Acud. SC;. 125, 502-521 (1965). 13. Luyet. B., and Rapatz, G. Patterns of ice formation in some aqueous solutions. Biodynrrmicrc 8, l-68 (1958). 14. Meryman, H. T. Cryoprotective agents. Cryobiology 8, 173-183 (1971). 15. Platov, E. M., Avanov, N. Y., and Balaskov, N. G. Freezing ram semen in diluents containing polysaccharides. Ovtsevodswo Mask. 10, 38-39 (1980). 16. Platov, E. M.. Karol, V. K.. and Bashkatov, L. P. An experiment on the use of deep frozen ram semen. Zhivot. No. 3, 41-42 (1983). 17. Rapatz, G., and Luyet, B. Combined effects of freezing rates and of various protective agents on the preservation of human erythrocytes. C/yobio/ogy
4, 215-222 (1968).
18. Roy, A. J., and Djerassi, I. Prevention of freezing injury by simultaneous use of agents with different modes of action. Cryabiologp 1 (Suppl. 1). 20 (1965). 19. Rowe, A. W. Biochemical aspects of cryoprotective agents in freezing and thawing. Cryohiology 3, 12-18 (1966). 20. Salamon, S. Deep freezing of ram semen: Recovery of spermatozoa after pelleting and comparison with other methods of freezing. Ausr. J. Biol. Sc,i. 21, 351-360 (1968). 21. Salamon, S., Wilmut, I., and Polge, C. Deep freezing of boar semen. I. Effects of diluent composition, protective agents, and method of thawing on survival of spermatozoa. Aust. J. Bid. SC;. 26, 219-230 (1973).
22. Stiff, P. J., Murgo, A. J., Zaroulis, C. G.. DeRisi, M. F., and Clarkson, B. D. Unfractionated human marrow cell cryopreservation using dimethyl sulfoxide and hydroxyethyl starch. Cryobio1og.v 20, 17-24 (1983).