Sperm membrane functional integrity and response of frozen-thawed bovine spermatozoa during the hypoosmotic swelling test incubation at varying temperatures

ELSEVIER

SPERM MEMBRANE FUNCTIONAL INTEGRITY AND RESPONSE OF FROZENTHAWED BOVINE SPERMATOZOA DURING THE HYPOOSMOTIC SWELLING TEST INCUBATION AT VARYING TEMPERATURES J.R. Correa,’ G. Heersche, Jr’,* and P.M. Zavos’ ‘Department of Animal Sciences and ‘Cooperative Extension Service, University of Kentucky Lexington, KY, 40546, USA Received for publication: Accepted:

April October

3,

1996 15,

1996

ABSTRACT The objective of this study was to assess the sperm membrane integrity and permeability of frozen-thawed bovine spermatozoa, processed at varying temperatures during and abler thawing, by exposing the spermatozoa to standardized hypoosmotic conditions. The hypoosmotic swelling (HOS) test was employed to measure changes in sperm membrane fUnctional status and permeability. Frozen specimens (from 5 bulls) were thawed at 37°C for 10 set and transferred to a water bath at 37 (Aliquot l), 21 (Aliquot 2) or 5°C (Aliquot 3) to complete thawing (1 to 2 min). The specimens were maintained and processed at these temperatures for additional 5 to 10 min. Specimens were slowly diluted 1: 1 (v/v) and washed with Ham’s F-10 media containing 3% (w/v) BSA. The HOS test was performed by adding 0.1 ml of the sperm specimen to 1.O ml of a 100 mOsm& HOS diluent. The following treatments were performed: 1) Aliquot 1 (control), specimens were incubated in HOS solutions at 37°C for 5 min; 2) Aliquot 2, specimens were incubated in HOS solutions at 21 or 37°C for 5 min; and 3) Aliquot 3, specimens were incubated in HOS solutions at 5 or 37°C for 5 min. Samples were obtained from the sperm specimen-HOS diluent mixtures at 1 min intervals (during the 5 min incubation period), fixed and assessed for sperm swelling patterns. The sperm response to the HOS test for specimens processed at temperatures below 37°C was higher when samples were incubated in HOS diluents at 37°C. This finding indicates that the potential for sperm swelling (measurement of sperm membrane functional status) can be maintained when spermatozoa are processed at temperatures below 37°C. The highest response to the HOS test was observed in spermatozoa processed at 21 “C and incubated in a HOS solution at 37°C. The response to the HOS test was superior to the one observed in specimens maintained and processed at 37°C throughout. Thawing of spermatozoa at 37°C followed by processing at 21°C seems to reduce the negative effects associated with osmotic shock and results in the preservation of the sperm membrane functional status during the in vitro handling of frozen-thawed bovine spermatozoa. 0 1997 by Elsevier Science Inc. Key words: spermatozoa, thawing, processing, membrane integrity Acknowledgments The authors wish to express their appreciation to the American Breeders Service for providing frozen semen specimens for this research effort. This study was presented at the 2 1th Annual Meeting of the American Society of Andrology, Minneapolis, Minnesota, April 25-29, 1996. Theriogenology 47:715-721, 0 1997 by Elsewer Science

1997 Inc.

0093-691X/97/$17.00 PII SOO93-691X(97)0002%0

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Theriogenology

INTRODUCTION Cryopreserved spermatozoa are very susceptible to changing osmotic conditions encountered during freezing, thawing and processing (6,7,15,18). Frozen spermatozoa encounter hypertonic conditions during freezing-thawing, and isotonic conditions during in vitro sperm wash procedures or when placed in the female reproductive tract fluids during the performance of AI (3,4,9-l 1). Abrupt changes in osmotic pressure result in occurrence of osmotic shock, reduced sperm viability and sperm membrane damage (3-5). Osmotic shock refers to the cellular changes (coiling of the sperm tail) that occur during the exposure of spermatozoa to isotonic conditions after exposure to hypertonic conditions. The recommended thawing procedure for frozen bovine spermatozoa used for AI in the field involves thawing at 37°C for a minimum of 30 set (2,13). Reductions in osmotic stress and maintenance of post-thaw bovine sperm membrane integrity can be obtained via thawing and processing at various temperatures (5). Thawing at 37°C for 10 set, followed by transfer to a water bath at 21 “C (1 min) to complete thawing, and subsequent processing at 21 “C seems to allow for a nonabrupt transition between hypertonic and isotonic conditions during sperm processing (5). Spermatozoa thawed and processed in this manner are then rewarmed to 37°C and used for AI and IVF or for in vitro evaluation trials (5). Spermatozoa thawed and processed in this manner showed improved viabiity and maintenance of sperm membrane integrity during short-term incubation (postprocessing) at 37°C for 2 h (5). However, the sperm membrane behavior to changing temperatures during thawing and processing have not been assessed. The status of the sperm membrane can be assessed by evaluating the response of the processed spermatozoa to hypoosmotic conditions (4,12). The hypoosmotic swelling (HOS) test evaluates whether an intact membrane is fimctionally active (4,12). It was suggested by Jeyendran et al. that the ability of spermatozoa to swell in the presence of a hypoosmotic diluent reflects normal water transport across the sperm membrane, which is a sign of normal membrane integrity and functional activity (12). The functionally active spermatozoa exposed to hypoosmotic stress (I-IOS test) undergoes swelling, due to the influx of water, and subsequently increases in volume to establish an equilibrium between the fluid compartment within the spermatozoon and the extracellular environment (12). Spermatozoa with damaged or inactive membranes are unable to support osmotic swelling as shown by others (12). Thus, the HOS test may prove useful in assessing changes in the sperm membrane fimctional integrity during thawingprocessing procedures (3). The objective of this study was to assess the frozen-thawed bovine sperm membrane integrity status and its behavior (i.e., maintenance, reversibility or irreversibility of the sperm swelling response) during the HOS test incubation at varying temperatures. MATERIALS AND METHODS Frozen semen from 5 bulls, packaged in 0.5 ml French straws, was provided by the American Breeders Service (ABS Global, Inc., DeForest, WI, USA). The cryopreservation medium used was modified 2.9% (w/v) sodium citrate containing 20% (v/v) chicken egg yolk and 7% (v/v) glycerol (13). The media used for post-thaw sperm processing and incubation was Ham’s F- 10 containing 3% (w/v) BSA (SpermPrepm medium; ZBL, Inc., Lexington, KY, USA). A diagrammatic representation of the experimental procedures is depicted in Figure 1. Semen specimens were thawed at 37°C for 10 set and transferred to a water bath at 37 (Aliquot l), 21 (Aliquot 2) or 5°C (Aliquot 3) to

717

Frozen Specimen I v Thaw at 37°C for 10 to 15 set and transfer to a water bath at 37,21 or 5°C for 1 to 2 min to complete thawing I ‘I v Ahquot 2 (21°C)

‘I Aliquot 1 (37°C)

v Aliquot 3 PC)

Dilute 1: 1 (v/v) with Ham’s F- 10 I v Sperm wash: filter semen specimens and reconstitute to original volume I v Assess”

i Hypoosmotic swelling (HOS) test incubation at varying temperatures

v Aliquot 1

‘I 37°C

I v v Aliquot 2

v Aliquot 3

I Split

I Split I v

! 21°C

37°C

5°C

37°C

i Incubate for 5 min (at varying temperatures) and assess at 1 min intervals for percentage of swollen spermatozoa Figure 1. Diagrammatic representation of the experimental procedures. ‘Sperm specimens were assessed for motility characteristics and percentage of spermatozoa with coiled tails (as a consequence of osmotic shock) prior to the HOS test incubation.

Theriogenology

complete thawing (1 to 2 min; 5). The specimens were maintained and processed at these temperatures for additional 5 to 10 min. Processing consisted of slow dilution, sperm wash and resuspension to 0.5 ml (4). Spermatozoa were washed by filtering the cryopreservation medium through a 0.22~pm Millipore tilter unit (Millipore Co., Bedford, MA, USA). The sperm specimen was aspirated into a 1.O ml tuberculin syringe. The filtering unit was attached to the syringe and a 22-g needle was attached to the filtering unit to facilitate aspiration of the Ham’s F-10. The previously diluted cryopreservation medium was filtered through the filtering unit and the spermatozoa remained in the filter matrix. Fresh Ham’s F-10 (0.5 ml) was the aspirated into the syringe. The flow created by the aspiration of media resulted in the removal and recovery of spermatozoa into the syringe. This procedure was repeated 2 more times and the specimen was transferred to a 12x75mm culture tube (Fisher Scientific, Inc., Pittsburgh, PA, USA). Sperm samples (pre-dilution and post-resuspension) were evaluated for percentage and grade of motility (8,19), and percentage of spermatozoa with coiled tails (3,4,17). Spermatozoa were exposed to a HOS test diluent (100 mOsm/L) at varying temperatures following processing (3,4). Hypoosmotic Swelling (HOS) Test Incubation The HOS test was performed by adding 0.1 ml of the sperm specimen to 1.0 ml of a 100 mOsm/L HOS diluent (3). The sperm specimen-HOS diluent mixture was incubated for 5 min. The following treatments were performed: 1) Aliquot 1 (Control), specimens were incubated in the 100 mOsm/L HOS diluent at 37°C; 2);Aliquot 2, specimens were split and incubated at 21 or 37°C; and 3) Aliquot 3, specimens were split and incubated at 5 or 37°C. Samples (10.0 to 20.0 ~1) were obtained from the sperm specimen-HOS diluent mixtures at 1 min intervals, fixed (wet mount) and assessed for sperm swelling patterns (12). The fixed specimens were assessed under a phase-contrast microscope at x 400 and a total of 200 spermatozoa per specimen was assessed (3,12). The results were reported as means f SD, and evaluated by ANOVA. The Least Significant Difference method was used to determine significance among the various means (16). RESULTS The results obtained in this study are summarized in Tables 1 and 2. The percentage and grade of motility was similar among spermatozoa processed at varying temperatures. The occurrence of osmotic shock was less for spermatozoa processed at 5 and 21°C than at 37°C. Effects between bulls (in terms of response to the HOS test) were not significantly different @Q-0.05). The percentage of swollen spermatozoa increased during the HOS test incubation (for all treatments evaluated) and most of the sperm swelling response occurred between 0 to 1 min of incubation. The swelling response of specimens processed at 5 or 2 1°C was higher, regardless of incubation interval, when samples from these specimens were incubated in the HOS diluent at 37°C vs incubation at 5 or 21°C. However, the swelling response for spermatozoa processed at 5°C and incubated in the HOS diluent at 37 vs 5°C was not significantly different at the end of incubation (P>O.OS). The highest response to the HOS test (of all treatments assessed) was observed in spermatozoa processed at 21 “C and incubated at 37°C in the HOS diluent (P
75.5 f 1.1 74.0 f 1.4 71.5 f 1.4

37

21

5

54.0 f 0.6’

58.0 f 2.0’

51.0* 1.5c

54.0 f 0.6’

58.0 f 1.58

58.0 l 2.3*

67.0 f 1.5b

61.0 f 2.3’

4

5 5 46.0 f 3.8’ 50.0 f l.od 51.0 f l.2c 51.0 f 0.6’ B.b~c.dValues with different superscripts within a column are significantly different (P
50.0 f 2. lb

37

5

55.0 f l.OC

65.0 i 1.2b

60.0~22.1”

3

65.0 f 1.2b

60.0 f 2.0’

2

54.0 f l.oc

50.0 f 3.sb

21

21

65.0 f 2.6b

59.0 f 0.6’

1

Incubation time (minutes)

52.0 f 1.0’

55.0 f 0.6’

60.0 f 3.8’

67.0 f 5.3b

61.0 f 2.3’

5

swelling (HOS) test incubation at varying

7.4 f 0.5

3.2hO.l

bovine spermatozoa during hypoosmotic

6.0 *0.7

9.2 f 0.4

W)

Spermatozoa with coiled tails*

3.5 f 0.1

3.6kO.l

Grade (0 to 4)

54.0 f l.oc

60.0 rt 2.3’

37

21

47.0 i 3 .gBvb

0

37

HOS diluent

37

Processing

Temperature (“C)

Table 2. Percentage of swollen frozen-thawed temperatures (means f SD)

‘Spermatozoa with coiled tails undergoing osmotic shock.

W)

Motility

Processing temperature (“C)

Sperm characteristics assessed

Table 1. Sperm characteristics of frozen-thawed bovine spermatozoa processed at various temperatures prior to the hypoosmotic swelling (HOS) test (means f SD)

51

2 6 %

9

0 g.

Theriogenology DISCUSSION In a previous study the authors determined that thawing frozen bovine spermatozoa at 37°C for 10 to 15 set, followed by transfer to a water bath at lower temperatures (21 and S’C) to complete thawing and processing, decreased the occurrence of osmotic shock and resulted in maintenance of the sperm membrane Iimctional integrity (5). The spermatozoa were maintained and processed (sperm wash) at a given temperature (37,21 or 5°C) to remove the cryopreservation medium (hypertonic) and return the spermatozoa to isotonic conditions, followed by rewarming to 37°C. The objective of the current study was to assess the sperm swelling potential and behavior of spermatozoa processed in a manner similar to the procedures described above and exposed to a HOS diluent at the temperature used for processing and also at 37°C. The results obtained in this study suggest that the potential for sperm swelling can be maintained following processing of cozen-thawed bovine spermatozoa at temperatures below 37°C. The highest response to the HOS test was noted with spermatozoa processed at 21 “C and incubated at 37°C during the HOS test, which confirms results reported previously (5). These findings indicate that the sperm membrane fluidity and permeability decreases at temperatures below 37°C but increases following rewarming at 37°C (reversible effect). Thus, processing of spermatozoa at temperatures below 37°C is possible without the deleterious effects of osmotic shock and loss of sperm membrane functional integrity. The thawing and processing approaches evaluated in this study could enable the in vitro handling of frozen-thawed spermatozoa with minimum damage to the sperm membrane integrity and maintenance of viability following processing procedures. REFERENCES 1. Almquist JO. Effect of cold shock after thawing on acrosomal maintenance and motility of bovine spermatozoa frozen in plastic straws. J Dairy Sci 1976;59: 1825- 1829. 2. Bemdtson WE, Pickett BW. Techniques for the cryopreservation and field handling of bovine spermatozoa. In: The National Research Council (eds), The Integrity of Frozen Spermatozoa. Washington, DC: National Academy of Sciences, 1978;53-77. 3. Correa JR, Zavos PM. The hypoosmotic swelling test: its employment as an assay to evaluate the fUnctional integrity of the frozen-thawed bovine sperm membrane. Theriogenology 1994;42:351-360. 4. Correa JR, Zavos PM. Frozen-thawed bovine spermatozoa diluted via slow or rapid dilution method: measurements on occurrence of osmotic shock and sperm viability. Theriogenology 1995;44:963-971. 5. Correa, JR, Rodriguez MC, Patterson DJ, Zavos PM. Thawing and processing of cryopreserved bovine spermatozoa at various temperatures and their subsequent effects on sperm viability, osmotic shock and sperm membrane finctional integrity. Theriogenology 1996;46:413-420. 6. Fiser PS, FairIirll RW. The effect of glycerol concentration and cooling velocity on cryosurvival of ram spermatozoa frozen in straws. Cryobiology 1984;21:542-55 1. 7. Foote RH. Freezing bull spermatozoa: a review. Cryobiology 1978;15:350-351. 8. Graham EF, Crab0 BG, Brown RI. Effect of zwitter ion buffers on the freezing and storage of spermatozoa. I. Bull. J Dairy Sci 1972:55:372-378.

Theriogenology

9. Hammerstedt RH, Graham JR, Nolan P. Ctyopreservation of mammalian sperm: what we ask them to survive. J Androl 1990;11:73-88. 10. Jeyendran RS, Van der Ven HH, Perez-Pelaez MM, Zaneveld LJD. Effect of glycerol and cryopresetvation on oocyte penetration by human spermatozoa. Andrologia 1985; 17124l-248. 11. Jeyendran RS, Van der Ven HH, Perez-Pelaez MM, Zaneveld LJD. Non-beneficial effects of glycerol on the oocyte penetrating capacity of cryopreserved and incubated human spermatozoa. Cryobiology 1985;22:434-437. 12. Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crab0 BG, Zaneveld LJD. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984;70:219-225. 13. Pace MM, Sullivan JJ, Elliot FI, Graham EF, Coulter GH. Effects of thawing temperature, number of spermatozoa and spermatozoa1 quality on fertility of bovine spermatozoa packaged in 0.5ml trench straws. J Anim Sci 1981;53:693-701. 14. Pickett BW. Factors affecting the utilization of frozen bovine semen for maximum reproductive efficiency. AI Digest 1971;19:8-23. 15. Salisbury GW, Van Demark NL, Lodge JR. Principles and techniques of freezing spermatozoa. In: Salisbury GA (ed), Physiology of Reproduction and Artificial Insemination of Cattle. San Francisco: WH Freeman Co., 1978;494-554. 16. SAS User’s Guide: Statistics. Cary, NC: SAS Institute, Inc., 1989. 17. Zavos PM. Opisthosmotic shock of frozen-thawed human spermatozoa. Infertility 1982;5:247255. 18. Zavos PM. Principles of cryopreservation of human spermatozoa: state-of-the-art. Infertility 1991;13:239-246. 19. Zavos PM Correa JR, Zarmakoupis PN. Improvements and short-term viability of mouse epididymal spermatozoa recovered through the SpermPrep TMliltration method. Theriogenology 1994;42:1035-1042.