Effect of glycerol concentration on frozen boar sperm

THERIOGENOLOGY

EFFECT OF GLYCEROL CONCENTRATIONON FROZEN BOAR SPERM V. G. Pursel, L. L. Schulman and L. A. Johnson U. S. Dept. of Agriculture Animal Physiology and Genetics Institute AgriculturalResearch Center Beltsville,Maryland 20705 Received for Publication:Nov. 9, 1977 ABSTRACT In experiment1, Beltsville F3 extender containing0 to 7% glycerol was addetdto boar sperm. Glycerol was either retained during freezing or removsedby centrifugationbefore freezing. When glycerolwas retained, there was a significantnegative linear relationshipbetween the percentage of sperm acrosomeswith a normal apical ridge (NAR) and the percentage of glycerol. When glycerolwas removed before freezing,the percentageof NAR acrosomes did not differ among samples. The percentageof motile sperm and the percentageof glycerol in the original extender were linearly related regardlessof whether glycerolwas retained or removed before freezing. In experiment2, four concentrationsof glycerol, three cooling times and two dilution rates were comparedwhen semen was frozen in BeltsvilleF5 extender. The post-thaw results for percentages of NAR acrosomes and sperm motility were optimum with 1% glycerol and a 1:4 dilution rate. Cooling time had a minor effect on the freezing results. In experiment3, the competitivefertilizing capacityof boar sperm frozen with 1% glycerol was compared with that frozen without glycerol. The number of ova fertilizedby sperm frozen with 0 or 1% glycerol, 52 and 72 ova, respectively,were nearing statisticaldifferencefrom a SO:50 ratio (Pc.07). These results indicated that under the conditionsof this study glycerolwas of some positive value as a cryoprotectantfor boar sperm. INTRODUCTION The cryoprotectiveproperties of glycerol were discoveredby Polge et al. (1). Since then glycerol has been used extensivelyin freeze prese
The authors express their appreciationto Dr. A. B. Borko. vet, Insect ChemosterilantsLaboratory,BARC, for kindly supplying the TEPA.

w:DGMENTS

Mention of products or companies in this report does not constituteendorsement by the U. S. Dept. of Agricultureto the exclusion of others not mentioned.

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Glycerol has a beneficial effect on the fertilizing capacity of stored semen of cattle and a detrimental effect on semen of swine (2,3), fowl (4,5,6) and horses (7). Wilmut and Polge (8) found that the fertilizing capacity of boar semen was reduced when the semen was stored in 5 or 10% glycerol at 20' C but not at 5' C. However, the detrimental effects of glycerol appear to be magnified during freezing or thawing because semen that was frozen with 4 to 8% glycerol in the extender lost its fertilizing capacity (2,9,10,11) whereas semen that "as frozen with less than 3% glycerol in the extender retained its fertilizing capacity (3,12,13). The purpose of the present study was to determine the effects of glycerol concentration during freezing on acrosome morphology, motility and fertilizing capacity of boar sperm. MATERIALS AND METHODS Boar semen was collected by the gloved-hand technique into a 250-ml insulated vacuum bottle; the opening was covered with a milk filter disc to separate the gel particles. The semen "as fractionated visually into sperm-rich and sperm-poor portions at collection; only the sperm-rich portion "as used. Experiment 1. This experiment "as conducted to determine (a) the effect of glycerol concentration on post-thaw acrosome morphology and motility and (b) whether removal of glycerol immediately before freezing would be beneficial. Treatments were assigned in a 2 x 5 factorial arrangement. Semen from two ejaculates from each of three boars was used, and duplicate samples were processed for each treatment. Three-milliliter aliquots of semen from each ejaculate were centrifuged at room temperature for 10 min at 300 g and the seminal plasma was removed by aspiration. The extender used in this experiment was Beltsville F3 (BF3) composed of 4% lactose hydrate, 2% casein, 2% Tris (hydroxymethyl) aminomethane, 1% citric acid monohydrate and .5% fructose (w/v). BF3 extender containing 0, 1, 3, 5 or 7% glycerol (v/v) was added until the original semen volume was attained, and the sperm were resuspended by gentle mixing. The extended semen was cooled to So C over a 6-hr period. At the end of the 6-hr cooling period, half of the semen (treatment A) "as frozen on Dry Ice into .15-ml pellets. The other half of the semen (treatment B) "as centrifuged at 5' C for 10 min at 800 g, the supernatant "as removed by aspiration, BF3 extender (5' C) without glycerol was added to bring the final volume to 3 ml, the spermatozoa were resuspended, and the semen was frozen on Dry Ice into .15-ml pellets. The pellets were thawed in a Teflon-coated pan in a 37' C waterbath. Treatment B samples contained 0, .14, .44, .6 and .82% glycerol, respectively, during freezing and thawing according to osmolarity determinations. Experiment 2. This experiment was designed to compare four concentrations of glycerol, three cooling times and two dilution rates. The extender used in this experiment "as Beltsville F5 (BFS) composed of 3.2% glucose, 1.2% Tes-N-Tris (hydroxymethyl) methyl 2 aminoethane sulfonic acid, .2% Tris (hydroxymethyl) aminomethane, .5% Orvus ES Paste (Proctor and Gamble, Cincinnati, Ohio) and 20% egg yolk. The extender was centrifuged at 12,000 g for 10 min and the extender decanted (14). Semen from four boars "as used. Semen was placed into test tubes in .5- or 2-ml aliquots within 30 min after collection and held at 22 to 24" C. Tubes with 2 ml of semen were centrifuged

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at 300 g for 10 min, 1.5 ml of seminal plasma was removed by aspiration and the sperm were resuspended in the remaining seminal plasma. The tubes containing .5 ml of semen were not centrifuged. At 2 hr after semen collection, .5 ml of BF5 extender was added to each tube, the semen and extender were gently mixed, the tubes were divided into three groups and gradually cooled to 5' C in 1, 2 or 4 hours. At the end of the cooling period, 1 ml of BF5 containing 0, 2, 4, or 6% glycerol (v/v) was added to each tube. The final dilution rates were 1:l (original volume:final volume) for the 2-ml samples and 1:4 for the .5-ml samples. The semen and extender were gently mixed (final glycerol concentration was 0, 1, 2 or 3%), and the semen was frozen on Dry Ice into .2-ml pellets. After freezing, the pellets were transferred to liquid nitrogen and stored for 24 to 72 hours. Six pellets per sample were thawed in 2.5 ml of Beltsville thawing solution (BTS) that was preheated to 50° c (14). mriment 3. This experiment was designed to compare the competitive fertilizing capacity of boar sperm frozen with 1% glycerol with that frozen without glycerol. The competitive fertility test is an alternative means of assessing fertility which is conducted by mixing equal numbers of sperm from two treatments before insemination. The two sperm populations then compete for fertilizing ova within the female. This test eliminates many sources of variation, such as inseminator efficiency, level of female fertility, and death or abortion in females, that are inherent in usual fertility tests. The greater efficiency of the competitive fertility test as compared to the usual fertility test has been demonstrated in cattle (15,16), swine (17), rabbits 1:18,19) and chickens (17,20). Bedford and Overstreet (18) indicated this technique seems particularly suitable for detecting differences in semen treatments which may not become apparent in tests involving separate insemination into different females. One of the requirements for conducting a competitive fertility test is that the identity of the fertilizing sperm must be detectable in the zygote. In this experiment one sperm population was marked with the chemosterilant Ova that are fertilized by a TEPA (Tris(l-aziridinyl) phosphine oxide). and can be distinguished from TEPA-marked sperm have retarded cleavage normally developing ova when recovered 5 days after ovulation. TEPA marking does not alter the ability of the sperm to compete with unmarked sperm for fertilization of ova (21). Semen from two or three boars was pooled and redivided into aliquots containing 3 x log sperm. S erm in one-half of the aliquots were marked (T) with 2 mg of TEPA per 10g sperm for 10 min using the procedure described earlier (21); the other half of the aliquots were unmarked (C). The marked and unmarked aliquots were frozen in BF5 extender containing 0 or 1% final glycerol concentration; a 2-hr cooling time, a 1:l dilution rate and the freezing process described in Experiment 2 were used. Eac'h sample of 3 x log sperm was thawed in 20 ml of BTS that had been preheated to 50' C. The two treatment samples of 3 x lo9 sperm each were combined just before insemination, i.e., T with 0% glycerol and C with 1% Nineteen gilts were glycerol or T with 1% glycerol and C with 0% glycerol. catheter was used to insemininseminated with the mixed semen. A spiral-ti ate gilts twice per estrous period with 6 x 10 s sperm per insemination.

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Ova were recovered about 162 hr after the onset of estrus, fixed in acetic ethanol for at least 24 hr, stained with aceto-orcein and examined as whole mounts for cleavage and nuclear development. Ova that contained pronuclei to four nuclei were considered to have been fertilized by a TEPAmarked sperm (21). Ova that had developed into morula or blastocysts were considered to have been fertilized by an unmarked sperm. Sperm Evaluation. Immediately after the pellets were thawed, two l-ml sub-samples were removed. One sub-sample was held at 37' C for 30 min, after which the percentage of motile sperm was estimated at 37' C by phase-contrast microscopy at 250X. The other sub-sample was examined by phase contrast microscopy as described by Purse1 -et al. (22). Two slides were examined for each sample; 100 acrosomes were assessed per slide, and the percentage of sperm with a normal apical ridge (NAR) was determined. All samples were coded before evaluation so that sample identity was not known to the evaluator. Statistical Analysis. The percentages of NAR and motility were subjected to arcsin transformation before analysis of variance. In experiments 1 and 2, orthogonal comparisons were performed. In experiment 3, the proportions of ova fertilized were analyzed by chi-square. RESULTS Experiment 1. When glycerol was not removed before freezing (treatment A), the percentages of NAR acrosomes and motile sperm were significantly different among treatments (Pc.005 for both, Table 1). The percentage of NAR acrosomes decreased linearly as the percentage of glycerol increased (Pc.005). However, there was a positive linear relationship between the percentage of motile sperm and the percentage of glycerol in the extender (PC.005). When glycerol was removed by centrifugation before freezing (treatment B), the percentage of NAR acrosomes was similar for all treatments (P>.lO). However, the positive linear relationship remained between the percentage of motile sperm and the percentage of glycerol in the original extender (Pc.01). Experiment 2. The percentage of NAR acrosomes decreased linearly as the percentage of glycerol increased (Pc.005, Table 2). The percentage of motile spermatozoa was significantly higher for those samples frozen with glycerol (0 -vs 1 + 2 + 3, Pc.005). Cooling times of 1, 2 and 4 hr did not affect the percentage of NAR acrosomes (P>.lO); however, the percentage of motile sperm was higher for samples cooled 1 hr than for samples cooled 2 and 4 hr (Pc.025). The percentage of NAR acrosomes was significantly higher in the semen diluted 1:4 than in the semen diluted 1:l (Pc.005). The dilution rate did not affect the percentage of motile sperm. There were no significant interactions among glycerol concentrations, cooling times and dilution rates for either evaluation criterion.

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Q'eriment 3. Fertilized ova were recovered from 14 of the 19 gilts inseminated with the sperm mixtures (Table 3). A total of 149 ova were recovered from the 14 gilts; 23 ova were unfertilized, 62 ova were cleaving normally (16 or more nuclei), 63 ova had delayed cleavage caused by fertilization with a TEPA-treated sperm (4 nuclei or less) and 1 ova was unclassifiable (11 nuclei).

The number of ova fertilized by sperm frozen with 0 or 1% glycerol 52 and 73 (ova, respectively, were nearing statistical difference from a 50:50 ratio (:?<.07, Table 3). The percentages of NAB acrosomes for the two sperm populations used for insemination were not different, but the percentage of motile sperm was significantly higher for 1% glycerol than for 0% glycerol (PC.01). TEPA-marked sperm fertilized 50.4% of the fertilized ova that were recovered. This result confirms our earlier observation that TEPA-marked sperm compete equally well with unmarked sperm (21). DISCUSSION In experiment 1, the presence of 3 to 7% glycerol in the BF3 extender during freezing and thawing (treatment A) had a deleterious effect on the acrosome morphology, a result that was contrary to the beneficial effect of glycerol on post-thaw motility. These observations confirm those reported earlier (8). Much of the adverse effect of glycerol on the acrosome morphology could be counteracted by removal of most of the glycerol by centrifugation before freezing (treatment B) without adversely affecting post-thaw motility. The results of experiment 2 indicated that 1% glycerol in BF5 extender was the optimum concentration for maintenance of NAE acrosomes and sperm motility during freezing and thawing for the various dilution rates and cooling times that were tested. Cooling time had a very minor effect on post-thaw survival, and a dilution rate of 1:4 was slightly superior to 1:l The four over all treatments as far as acrosome morphology was concerned. percentage point advantage for the 1:4 dilution rate is a sinor consideration in comparison with the practical advantages offered by freezing in the more concentrated form (14). Th.e results of experiment 3 indicated that sperm frozen with 1% glycerol had a slight advantage over sperm frozen without glycerol when the two sperm populations were placed in direct competition for fertilizing the ova. Glycerol was much more effective in protecting sperm motility during freezing than it Even though was in protecting fertilizing capacity or acrosome morphology. glycerol was of some positive value as a cryoprotectant of boar sperm fertility, Efforts to identify a more we greatly need a more effective substance. effective cryoprotectant for boar sperm have thus far been unsuccessful (23, 24). REFERENCES

1.

Polge, C., Smith, A. U. and Parkes, A. S. Revival of spermatozoa after Nature (London) vitrification and dehydration at low temperatures. 164:166 (1949).

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2.

Polge,

3.

Graham, E. F., Rajammanan, A. H., Schmehl, M. K. L., Maki-Laurila, M. and Bower, R. E. Preliminary reports on procedure and rationale for freezing boar semen. A. I. Digest 19:12-14 (1971).

4.

Polge, C. Functional survival of fowl spermatozoa after freezing at -79O c. Nature (London) 167-949-950 (1951).

5.

Neville, W. J., Macpherson, J. W. and Reinhart, B. The contraceptive action of glycerol in chickens. Poult. Sci. 50:1411-1415 (1971).

6.

Sexton, T. J. Effects of various cryoprotective agents on the viability and reproductive efficiency of chicken spermatozoa. Poult. Sci. 52: 1353-1357 (1973).

7.

Demick, D. S., Voss, J. L. and Pickett, B. W. Effect of cooling, storage, glycerolization and spermatozoa1 numbers on equine fertility. J. &rim. Sci. 43:633-637 (1976).

a.

Wilmut, I. and Polge, C. The fertilizing capacity of boar semen stored in the presence of glycerol at 20, 5 and -79' C. J. Reprod. Fert. 38:105-113 (1974).

9.

Settergren, I. Experiments on the deep-freezing of boar semen at -76' C. Nord. Vet. Motet (Helsingfors) Sekt D. Rapp. 26 (1958).

C.

Artificial

insemination

in pigs.

Vet. Rec.

68:62-76

(1956).

10.

King, G. J. and Macpherson, J. W. Boar semen studies II. Laboratory fertility results of a method for deep freezing. Canadian J. Comp. Med 6 Vet Sci. 31:46-47 (1967).

and

11.

Dalrymple, J. R. and Macpherson, J. W. Low temperature preservation boar spermatozoa. Canadian J. Anim. Sci. 49:45-49 (1969).

of

12.

Carbo, B. and Einarsson, S. Fertility in deep frozen boar spermatozoa. Acta. Vet. Stand. 12:125-127 (1971).

13.

Pursel, V. G. and Johnson, L. A. Procedure for the preservation of boar spermatozoa by freezing. U. S. Department of Agriculture, ARS 44-227 (1971).

14.

Pursel, V. G. and Johnson, L. A. Freezing of boar spermatozoa: Fertilizing capacity with concentrated semen and a new thawing procedure. J. Anim. Sci. 40:99-102 (1975).

15.

Beatty, R. A., Bennett, G. H., Hall, J. G., Hancock, D. L. An experiment with heterospermic insemination Reprod. Fert. 19:491-502 (1969).

16.

Stewart, D. L., Spooner, R. L., Bennett, G. H., Beatty, R. A. and Hancock, J. L. A second experiment with heterospermic insemination in cattle. J. Reprod. Fert. 36:107-116 (1974).

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17. Martin, P. A. and Dziuk, P. J. Assessment of relative fertilityof males (cockerelsand boars) by competitivemating. J. Reprod. Fert. 49:323-329 (1977). 18. Bedford, J. M. and Overstreet,J. W. A method for objective evaluation. of the fertilizingability of spermatozoairrespectiveof genetic character. J. Reprod. Fert. 31:407-414 (1972). 19. O'Reilly,P. J., Graves, C. N. and Dziuk, P. J. Heterospermicinsemination of rabbit semen as a means of evaluatingtechniquesof semen handling. J. Reprod. Fert. 29:49-56 (1972). 20. Martin, P. A. and Dziuk, P. J. The effect of storage on spermatozoa in vitro and the compositionof the extender on the proportionof off--spring from heterospermicinseminationin the chicken. J. Reprod. Fert. 59:297-300 (1977). 21. Pursel, V. G., Johnson, L. A. and Borkovec,A. B. Effects of -in vitro treatmentof boar spermatozoawith TEPA on the fertilizationand developmentof pig eggs. J. Reprod. Fert. 45:549-552 (1975). 22. Pursel, V. G., Johnson, L. A. and Rampacek, G. B. Acrosome morphology of boar spermatozoaincubatedbefore cold shock. J. Anim. Sci. 34: 278-283 (1972). 23. W:lmut, I. and Polge, C. The low temperaturepreservationof boar spermatozoa. I. The motility and morphology of boar spermatozoafrozen and thawed in the presence of permeatingprotective agents. Cryobiology 14:471-478 (1977). 24. Graham, E. F. and Crabo, B. G. Some factors influencingthe freezing o:Zboar spermatozoa. Proc. VIIth Int. Congr. Anim. Reprod.,Munich II, 1627 (1972).

Table 11. Effect of Glycerol on Post-thaw Acrosome Morphology and Sperm Motility. TreatmentAa Glycerol level in NAR Motility origin,31 extender (%) S.E. (X) S.E. 0% 1% 3% 5% 7%

34b 21 15 8 4

1.6 2.1 1.5 1.3 0.4

13 25 30 28 34

2.4 3.4 3.4 3.0 5.0

Treatment Ba Motility NAR (X) S.E. (%) S.E. 40 40 37 32 34

2.2 2.6 1.9 1.9 3.2

22 25 31 40 37

3.0 3.1 3.8 3.3 3.3

aTreatmentA: extender and glycerol not removed before freezing;Treatment B: extender and glycerol removed and replaced with BF3 extender without glycerol before freezing. b Mean for 12 replications.

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Table 2.

Effect of Dilution Fate, Cooling Time, and Glycerol Concentration on Post-thaw Acrosome Morphology and Sperm Motility.

Dilution

Cooling time (hours) 1 2 4

1:l

Mean 1:4

1 2 4 Mean

Percent glycerol 0 1 2 3 Mean --------------Percent NAR-----------60a 62 50 40 53 62 64 56 44 56 58 56 50 43 52 60

61

52

42

54

64 62 64

64 65 64

60 56 62

43 50 41

58 58 58

63

64

59

45

58

-_________---percent

1:l

1 2 4 Mean

1:4

1 2 4 Mean

Motility__--_----

11 6 4

22 16 18

22 18 15

20 8 16

19 12 13

7

19

18

15

15

10 14 13

16 19 18

19 18 12

14 16 15

15 17 14

12

18

16

15

15

aMean for four replicates.

****** Table 3.

*****

*****

Fertilizing Capacity, Acrosome Morphology and Motility of Boar Spermatozoa Frozen with or without 1% Glycerol.

Itema

Number of gilts insem. wlfert. ova

Glycerol 0% 1% -No. ova fert.-

Sperm mixture T/l% + C/O% C/l% + T/O% Total

10 9 19

7 7 14 >

40 (T) 33 (0

29 (0 23 (T)

73 (58.4)

52 (41.6)

---Percent---Semen evaluation NAP acrosomes Motility

64.1 45.3

65.9 26.1

aT = TEPA, C = control, 0 and 1% = glycerol.

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