Effects of ouabain on the response to osmotic changes in dog and boar spermatozoa

ELSEVIER

EFFECTS

OF OUABAIN ON THE RESPONSE TO OSMOTIC CHANGES DOG AND BOAR SPERMATOZOA Joan E. Rodriguez-Gil

IN

and Teresa Rigau

Unit of Reproduction of Animal Pathology and Production School of Veterinary Medicine, Autonomous University of Barcelona E-08193 Bellaterra, Spain Department

Received

for publication: December 6, 1994 Accepted: June 30, 1995 ABSTRACT

Although there is much information on the response of spermatozoa from different species to osmotic changes, little has been reported about the mechanism/s by which spermatozoa react to similar changes in the osmotic pressure of the medium. In this study we examine the effect of inhibition of Na'/K+, ouabainsensitive ATP-ase on the response of canine and porcine when spermatozoa they are incubated in hypoosmotic and hyperosmotic media. The presence of ouabain slightly decreased the percentages of total and progressive motility, and increased the percentages of altered acrosomes (from 13.0 k 0.3% to 17.2 f 0.4% in the presence of 10e4 M ouabain) and, specially, swollen tails (from 0.6 f 0.1% to 5.9 + 0.2% in the presence of 10m4 M of ouabain) in fresh dog semen, although it did not affect these parameters in boar semen samples. Moreover, ouabain increased the percentage of both altered acrosomes and swollen tails in canine spermatozoa incubated in 100 mOsm and in 900 mOsm media at concentrations higher than 10.' M and 10.' M, respectively. The percentage of viability of canine spermatozoa was not modified by ouabain after incubation in 100, 300 or 900 mOsm media. did significantly affect Furthermore, ouabain not boar spermatozoa incubated in 100, 300 or 900 mOsm media. Although ouabain induced a significant decrease in L-lactate production in canine spermatozoa in an isoosmotic medium (from 4.7 + 0.4 pmol/mg protein x 60 min to 2.6 f 0.3 pmol/mg protein x 60 min in the presence of 1O-4 M ouabain), there was no significant effect on L-lactate production in boar spermatozoa. These results indicate that while dog spermatozoa acted against changes in the osmotic pressure by a mechanism(s) related to Na'/K', ouabainsensitive ATP-ase, boar spermatozoa reacted to some mechanism(s) not related to ionic pumps. Key words:

dog, boar, spermatozoa, hyperosmotic medium

ouabain,

hypoosmotic

Acknowledgments The authors thank Alejandro Peria for technical Chuck Simmons for editorial assistance.

Theriogenology 45:673-668, 1996 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

medium,

assistance

and

0093-691X/96/$15.00 PII sOO93-691x(96)ooo16-3

INTRODUCTION

In recent years, there has been an increasing number of reports on the ability of spermatozoa from different species to react to changes in osmotic pressure hypoosmotic swelling test (HOS TestJtn)hzf ,",",9,meudslk?'wT~~ satisfactory results in semen analysis of humans (22) as well as in the dog (9,10,15), ram (21), bull (3) and mouse (19). This test is based on a tail swelling reaction of the spermatozoa incubated in a hypoosmotic medium, with the percentage of swollen tails being correlated with semen quality in these species. response to a hypoosmotic medium has However, in the boar,sperm required a different test, the osmotic resistance test (ORT), which measures the percentage of altered acrosomes not swollen tails for indicating semen quality (18,201. These data suggest that boar spermatozoa have some functional and/or structural differences which could explain their response to hypoosmotic conditions. The adaptability of a cell to changes in the osmotic conditions of the external medium is greatly dependent on the activity of its membrane osmotic pumps. In eucaryotic cells, one ionic pumps is the Na+/K+, ouabainof the most important sensitive ATP-ase observed that the (5,ll). It has been inhibition of this pump by ouabain induced the swelling of many types of eucariotic cells when they were exposed to a hypoosmotic medium (11, indicating that this pump is important in regulating response to a decrease of 71. On the other hand, Na'/K' ATP-ase activity is also important in regulating the response of cells to an increase of 71 (1). This point is essential when spermatozoa are processed for freezing, since the changes between a hyperosmotic and an isoosmotic 'TI induced in the freezing-thawing mechanisms form part of the basis of most of the spermatozoa1 injuries observed during the freezing-thawing process (12,13). Thus, it is possible that Na'/K' ATP-ase plays an important role in the ability of spermatozoa to resist osmotic changes during freezing. The main objective of this study was to evaluate the effect that ouabain, as inhibitor of the Na'/K' ATP-ase, causes in the response that both canine and porcine spermatozoa show to the variations of 7~ in the medium. Ouabain increases a swelling reaction in dog but not boar spermatozoa incubated in a hypoosmotic medium. Under hyperosmotic conditions, the presence of ouabain does not induce any significant changes in the percentages of viability and altered acrosomes in either dog or boar semen samples. These results indicate that Na'/K', ouabainsensitive ATP-ase plays an important role in the response to osmotic changes in canine but not porcine sperm cells, at least This difference could be an under hypoosmotic conditions. important one since the insensitivity of boar spermatozoa to ouabain could in part explain the differences in behavior between porcine and canine spermatozoa when they were subjected to in changes in osmotic conditions. Moreover, this difference response to ouabain could also carry over to the freezing/thawing

Theriogenology process

875

in the different

species.

MATERIALS AND METHODS Animals,

and Sample Collection

Semen was obtained from 11 purebred Beagle dogs ranging from 1 to 5 yr of age. The dogs were kept in outdoor kennels, exercised twice daily and fed a balanced diet with free access to water. Semen was collected once or twice weekly without using a female by manual stimulation into warmed (37 OC) sterile glass or plastic funnels. Only the sperm-rich fraction of the ejaculates was used. Samples were maintained at 37 OC in a water bath for as long as 10 min before being used, without addition of diluents. Semen was also obtained from 11 healthy boars ranging from 1 to 4 yr of agethatbelonged to a commercial herd. The breeds of swine represented were Large White (3), Landrace (3), Belgium Landrace (3) and Pietrain (2). The ejaculates were manually collected and immediately diluted at 37 OC in a thermoregulated water bath. The dilution of semen was carried out to a final concentration of lo7 sperm/ml in a commercial extender for semen (MR-A extender. Kubus S.A. Majadahonda, refrigerated Spain) ; the diluted semen was placed into 100-mL plastic bottles ready for use in artificial insemination. One bottle was kept at 15 to 17 OC for about 1 h (the time that was spent in reaching the laboratory from the location of sample collection), then S-mL aliquots from this bottle were kept in a 37 OC water bath for as long as 10 min before being used. To observe if dilution of boar semen in a commercial extender had any effect on osmotic response, a preliminary study was performed that compared the percentages of viability, swollen tails and altered acrosomes after incubation in a hypoosmotic medium in both diluted and undiluted samples. After 25 experiments, no significant difference between the 2 groups (data not shown) could be observed. The following experiments were performed on the diluted samples; it was assumed that extender did not affect osmotic response of boar spermatozoa. Evaluation

of Spermatozoa

The percentage of viable spermatozoa, and the percentages tails and morphological of altered acrosomes, swollen abnormalities were observed by using the dual staining method (61, as modified by Rodriguez-Gil et al. (15). These percentages were established after counting 200 to 250 sperm/slide, at x 1000 magnification. Curled or coiled tails were considered as swollen, in both dog and boar sperm samples. Straight or slightly curved tails were considered as normal. Other tail appearances were considered independently of the osmotic conditions, and were grouped with the morphological abnormalities category. Proximal and distal cytoplasmic droplets were included with the neck and mid-piece

Theriogenology abnormalities. Head abnormalities did not include lost acrosomes, which were separately counted acrosomes.

detached or as altered

Total and progressive motilities were determined by visual estimation at x 400 magnification in a phase-contrast microscope on a warmed stage (37 "C). Total motility was defined as the percentage of motile spermatozoa, whereas progressive motility was defined as the percentage of motile spermatozoa that were moving forward. Hypo- and Hyperosmotic

Incubations

Hypoosmotic incubations were performed as described previously (15). Briefly, 100-PL aliquots of semen were added to 900 ,uL of the appropriate medium, which was previously warmed at 37 oc. Incubation was maintained at 37 OC until the time of analysis, and at that time, 25 to 30 FL were taken from the incubation mixture to carry out the analysis. The isoosmotic medium (n=301 + 5 mOsm) contained 3.7% (w/v) dihydrate sodium citrate in distilled water. The 20, 50, 70, 100, 500, 700 and 900 mOsm of medium, respectively, contained 0.26, 0.65, 0.91, 1.30, 6.17, 8.63 and 11.10% sodium citrate dihydrate; the measured osmolarities for these media were 20 f 1, 51 f 3, 72 + 1, 102 jr 3, 505 * 7, 709 f 11 and 912 + 12 mOsm, respectively. Incubation

of Spermatozoa

with Ouabain

To study the effects of ouabain, aliquots of 300 to 400 PL of semen were preincubated for 30 min at 37 OC in a water bath with 15 PL of the appropriate aqueous solution of ouabain, until a final concentration of ouabain ranging from lo-' to 1O‘4 M was obtained. At this time, 25 to 30-PL aliquots were taken for the appropriate analysis. In the 0 M ouabain groups, semen was added to 15 ,LlLof twice-distilled water. After this, 50 PL of the ouabain-treated spermatozoa were added to 450 FL of the previously warmed (37 OC) hypo-, iso-, and hyper-osmotic solutions indicated above. To avoid a possible reversal of the ouabain effect, the osmotic solutions were added with ouabain, or, in the 0 M ouabain group, with distilled water. In all cases, the final concentration of ouabain in the mixtures of osmotic solutions and semen was equal to that in the preincubated semen samples. These mixtures were incubated for 45 min at 37 OC in a water bath, and afterwards aliquots were taken to carry out the analysis. In all cases, samples were coded using a double blind system to avoid bias of the results. Determination

of L-Lactate

Production

production was determined as The rhythm of L-lactate previously described (16). Aliquots of 100 PL of semen were added to 900 FL of an adjusted (pH 7.4) isoosmotic solution containing 2.94% (w/v) dihydrate sodium citrate and 5.4% (w/v) fructose (7~ = 303 f 6 mOsm), previously warmed at 37 OC. The solutions were incubated for 60 min at 37 OC. Both semen and the isoosmotic

Theriogenology

877

solution were treated with different solutions of ouabain or with distilled water, as indicated above. The mixture was then centrifuged at 3000 x g for 20 min, and L-lactate concentration was determined in the supernatants. Pellets were resuspended in 100 PL (final volume) of distilled water, and homogenized by ultrasound; the protein content of the pellets was then determined. The L-lactate concentration was determined enzymatically (14). Protein concentration was evaluated by the Bradford method (2), using the Bio-Rad reactive mixture (Bio-Rad Laboratories GmbH. Miinchen, Germany). Statistical

Methods

Putative differences between groups were analyzed by the Student's t-test for paired data and, when comparing non paired results, by two-way ANOVA. In this latter analytical procedure the effects of time, 71and the combined effects of time x A were calculated. For the boar data, a first experimental design was performed to evaluate the possibility that difference of breeds exerted some influence on the results. Analysis showed that the influence of breed on the results was negligible. Therefore, analyses were performed obviating this point. All data are expressed as means + SEM. Suppliers Ouabain was obtained from Boehringer Mannheim (Mannheim, Germany). Sodium citrate dihydrate came from Merck (Darmstaad, Germany). All the other reagents were of analytical grade. RESULTS The general characteristics in Table 1.

of dog and boar semen are shown

Effects of Hypo- and Hyper-osmotic Media on Viability, Acrosomes and Swollen Tails in Canine and Porcine Spermatozoa Before initiating our study on the effects of ouabain on dog and boar sperm cell response to changes in the r of the medium, we incubated dog and boar spermatozoa in several hypo- and hyperosmotic media, and then calculated the percentages of viability, altered acrosomes and swollen tails (only in hypoosmotic media, since it is not logical to suspect that a hyperosmotic medium would cause tail swelling). in an isoosmotic medium Incubation of dog spermatozoa induced a decrease in viability, which was time-dependent. Thus, the percentage of viability after 120 min of incubation, significantly decreased from about 90 to about 70% (P s 0.01 for time x K after an ANOVA analysis; data not shown). Similar effects were observed when dog spermatozoa were incubated in a 100 mOsm hypoosmotic medium (data not shown). However, when the media had progressively decreasing values of k. the effects on viability were more accentuated. Thus, in 70-mOsm medium,

Theriogenology

878 Table 1.

Generalcharacteristics offreshsperm-rich of dog and boar semen samples Dog

Viability

(%)

Concentration Total motility Progressive

(106/mL) (%I

motility

Tail abnormalities

(%)

(%)

Neck and mid-piece abnormalities (%) Head abnormalities Altered

acrosomes

Swollen

tails

(%I

(%) (%I

fractions Boar

90.1 + 0.2

84.8 k 0.4

805 & 11

797 f 6

94 * 1

89 + 1

93 + 1

77 f 1

4.1 f 0.1

9.9 + 0.3

2.2 * 0.2

1.9 r 0.2

2.2 f 0.1

0.5 * 0.1

11.4 f 0.2

16.5 * 0.4

0.2 * 0.1

0.2 * 0.1

Results are expressed as means f SEM. Data were obtained from 23 different samples from 11 dogs, and from 19 different samples from 11 boars.

viability decreased about 60% after 120 min of incubation. At 50 mOsm, it decreased about 40%; in 20-mOsm medium, it decreased about 30%; and in distilled water (7~= 0 mOsm), viability was nearly 0% after only 20 min of incubation (data not shown). Boar spermatozoa responded to osmolarity changes in a similar manner as dog spermatozoa when they were incubated in the same media. In an isoosmotic medium, a progressive, and was significant, decrease in the percentage of viability observed, with initial values of about 90% decreasing to those of about 70% after 120 min (P s 0.01 for time x 71after an ANOVA analysis; data not shown). Similar results were observed in the spermatozoa incubated in 100-mOsm medium (data not showy/. HS the r of the medium further decreased, the effects on viability were more pronounced. In this way, viability was reduced to about 50% after 120 min in 70- mOsm medium, to about 40% in 50-mOsm medium and to about 25% in 20-mOsm medium (data not shown). When boar spermatozoa were incubated in distilled water, viability was practically 0% after 20 min of incubation. Thus, viability was greatly dependent on the values of 71 of the media, both in dog and in boar spermatozoa. The effects of the hypoosmotic media on the percentage of on the altered acrosomes were inverse to those observed percentage of viability. In both cases, the increase of the percentage of altered acrosomes was significant, ranging from

Theriogenology

879

about 20% after 5 min of incubation to about 30 to 35% after 120 min (P s 0.05 for time x 'TIafter an ANOVA analysis; data not shown). As the 7rof the media decreased, the rate of increase of the percentage of altered acrosomes concomitantly increased. In 100-mOsm medium, the percentage of altered acrosomes after 12~ min of incubation was about 40% in both species; at 70 mosm, about 50%; at 50 mOsm, about 60%; and at 20 m&am, about 75% (data not shown). In distilled water, the percentage cf altered acrosomes in both dog and boar spermatozoa was higher than 90% after 10 min of incubation (data not shown). The percentage of swollen tails was also dependent on the K of the media. In an isoosmotic medium, neither dog nor boar spermatozoa showed a significant percentage of swollen tails over 120 min of incubation (Figures 1A and 1B). In 100-mOsm medium, dog spermatozoa showed a fast and significant increase in the percentage of swollen tails, reaching values of about 75% after 20 to 30 min of incubation (P s 10m6 for time x 71 after an ANOVA analysis; Figure 1A). Boar spermatozoa also showed an increase in the percentage of swollen tails, although this incidence reached values of only about 30% after 30 to 45 min of incubation (P 5 10m6 for time x 71 after an ANOVA analysis; Figure 1B). The percentage of swollen tails in 70- and 50-mOsm media was not significantly different from that observed in a 100-mOsm medium in dog spermatozoa (Figure lA), although the values in SO-mOsm medium were slightly higher in boar than dog samples (Figure 1B). In 20-m&m medium the percentage of swollen tails in dog spermatozoa was lower than that in IOO-mOsm medium; in boar slightly higher, although no samples this percentage was significant difference was observed in either dog or in boar water, spermatozoa (Figures 1A and 1B). In distilled the percentage of swollen tails was very high in dog spermatozoa, reaching values of about 95% after 10 to 20 min of incubation (Figure 1A). However, in boar spermatozoa this percentage was no higher than about 30% (Figure 1B). It is worth noting that in tails was distilled water the morphology of the swollen characteristic of and slightly different from those observed in the other hypoosmotic media, both in dog and boar semen samples (data not shown). Hyperosmotic media had little effect on the percentage of viability of either dog or boar spermatozoa. When samples were incubated in 500, 700 and 900 mOsm media, viability decreased from about 80% after 5 min of incubation to about 60 to 70% after 120 min (P 5 0.05 after an ANOVA analysis; data not shown). Similar results were observed when samples were incubated in an isoosmotic medium (data not shown). Thus, hyperosmotic media did not greatly affect the sperm viability under the conditions of our study. Similar results were observed when the percentage of altered different with the acrosomes was analyzed. Incubation hyperosmotic media (at 500, 700 and 900 mOsm) did not greatly modify the percentage of altered acrosomes in isoosmotic medium, either in dog or in boar spermatozoa (data not shown).

880

20

0

0

10

20

30

40

50

60

70

80

90

100

110

120

TIME (minutes) 100

B)

00

10

20

30

40

50

60

70

80

90

100

110

120

TIME (minutes)

Figure 1.

Time-dependent effects of hypoosmotic media on the percentages of swollen tails in dog and boar spermatozoa. Dog (A) and boar (B) spermatozoa were incubated in isoosmotic Cm), 0 (v), 20 (01, 50 X , 70 (A) and 100 mOsm (*) media. At the indicated of times, aliquots were taken, and percentages swollen tails were calculated. Results are expressed as means f SEM for 7 separate experiments.

Theriogenology

881

Effects of Ouabain on Dog and Boar Sperm Characteristics Incubation for 30 min with ouabain induced significant changes in some parameters of semen quality in dog semen. As shown in Table 2, ouabain at concentrations of 10.' and 10m4 M induced a slight but significant decrease in :he percentage of viability and in total and progressive motilities. Ouabain also significantly increased the percentage of altered acrosomes at concentrations of 10.' and 1O‘4 M. However, the primary effect of ouabain was on the percentage of swollen sperm tails, with a significant increase observed at a concentration of lo-' M (Table LI

.

By contrast, no significant effect of ouabain was observed on samples of boar semen (Table 2). Effects Tails

of Ouabain

on Sperm

Viability,

Acrosomes

and

Swollen

Ouabain did not significantly affect the percentage of viability of dog and boar spermatozoa, that had been preincubated with the ouabain at concentrations ranging from 10m7 to 10e4 M, and then incubated again for 45 min in an isoosmotic, hypocsmotic (100 mOsm) or hyperosmotic (900 mOsm) medium containing ouabain (Table 3). Similarly, ouabain did not significantly affect the percentage of altered acrosomes in the iso-, hypo- or hyperosmotic medium in either species (Table 3). However, although ouabain did not modify the percentage of swollen tails in boar spermatozoa incubated in the iso- (Figure 2A) and the hypoosmotic media (Figure 2B), the presence of this inhibitor at increased the concentrations as low as 10.' M significantly percentage of swollen tails in the dog samples, both in the isoosmotic (from P 5 0.01 at 10.'M ouabain to P B lo-" at 1O‘4 M ouabain; Figure 2A) and in hypoosmotic media (from P < 0.05 at 10~'M ouabain to P 5 10m6 at 10e4M ouabain; Figure 2B). In all cases, the putative significance of the differences was tested the by using Student's t-test for paired data, observing differences of each ouabain concentration against the values of their paired "no ouabain" samples. Effects of Ouabain Spermatozoa

on the L-Lactate

Production

in Dog and Boar

The production of L-lactate in an isoosmotic medium was not modified by ouabain in boar spermatozoa (Table 4). However, in the dog samples, ouabain induced a significant decrease of Llactate production that was evident at concentrations as low as 10 -’ M (Table 4). DISCUSSION The results of this study suggest that Na'/K', ouabainsensitive ATP-ase, is related to the swelling reaction observed in dog spermatozoa incubated under hypoosmotic conditions. Although a significant increase in the percentage of swollen

(%)

(%)

(%)

(%)

(%I

(%)

(%)

0.5kO.l

0.5kO.l 0.6kO.l

0.2*0.1

0.3kO.l

21.6kO.6

21.9tO.6

20.4kO.6

20.8kO.6

85 f 1

79.OkO.5

* 84.OkO.4 * 89 + 1 * 88 f 1 ** 17.2kO.4 ***** 5.9kO.2

21.OkO.7

78 + 1

81 + 1

82 + 1

86 f 1

*

-4

82 f 1

87 + 1

85 f 1

87 f 1

79.0f0.6

84.7kO.4 * 89 + 1 * 89 f 1 * 16.3kO.4 ***** 3.6fO.l

10

80 f 1

81.3kO.5

13.9kO.4 l *** 2.8kO.l

90 * 1

92 * 1

80.0+0.6

13.6kO.3 **** 2.OkO.l

90 + 1

91 + 1

87.2+0.4

79.8kO.6

0.6kO.l

13.OkO.3

92 f 1

93 + 1

86.4kO.3

88.OkO.3

10-S

* PsO.01 vs no ouabain. ** PsO.001 vs no ouabain. ***** P~10.~ vs no ouabain.

**** P=~10-~vs no ouabain.

Samples were treated with ouabain, as indicated in Materials and Methods. Results are expressed as of 16 different samples from dog and 14 different samples from boar. Putative means f SEM significance of differences was obtained by using the Student's t-test for paired data.

Swollen tails

Altered acrosomes

Progressive motility

Total motility

Viability

Boar

Swollen tails

(%I

motility

(%)

Altered acrosomes

Progressive

Total motility

(%)

10-6

on sperm quality 10.'

with ouabain

0

Effects of incubation

(MI

Viability

Ouabain

Table 2.

2. g 3 Q 0 B

2

100 mOsm

900 mOsm

Altered acrosomes

Altered

14.7kO.6

14.6kO.9

32.9+2.7

33.1k2.6

900 mOsm

Results are significance

32.2k2.6

30.852.2

Putative

27.3k2.0

30.2k3.1

25.3kl.O

71.6k2.3

69.1k2.4

75.6kO.8

17.9k2.6

17.4k1.7

14.1kO.4

83.Ok1.5

83.OkO.6

84.050.4

1o‘4

in dog and boar

expressed as means k SEM of 16 (dog) and 14 (boar) different samples. of differences was obtained by using the Student's t-test for paired data.

acrosomes

Altered

25.1r1.4 25.6k1.4

28.9k3.1

27.124.5

100 mOsm

Altered acrosomes

24.6kl.O

24.2kO.9

25.8kO.9

25.OkO.9

300 mOsm

Altered acrosomes

72.Ok1.5

69.2i2.2

68.922.0

7i.Ok2.i

900 mOsm

Viability

77.7kO.8

18.7+2.8

16.OkO.8

15.2kO.5

83.3+1.3

84.OkO.7

84.4kO.4

1om5

71.7k1.7

68.9k2.4

70.3k2.4

100 mOsm

Viability

77.7kO.8

15.5kl.O

16.1-10.7

15.6kO.3

85.2kO.5

83.9kO.8

84.3kO.4

10-6

71.7*3 .o

76.8kO.8

76.720.7

mOsm

300

BOAR SPERMATOZOA

16.OkO.S

14.2kO.5

Viability

acrosomes

300 mOsm

Altered acrosomes

84.5kO.6 15.1kO.5

84.5~0.6

900

Viability

83.OkO.6

84.2kO.3

14.OkO.4

83.7kO.4

100 mOsm

Viability

mOsm

10~?

DOG SPERMATOZOA 84.9kO.3

0

300 mOsm

CM)

Effects of ouabain on the percentages of viability and altered acrosomes spermatozoa incubated in; iso-, hypo- and hyper-osmotic media

Viability

Ouabain

Table 3.

3

B

2 3.

884

Theriogenology 20 . A)

15 -

5 T

I

Of O

0.0000001

0.000001

I0.00001

17

0.0001

log [OUABAIN] (M)

80 -

T

B)

( 60 -=

40 t

0

’

0

I

0.0000001

I

I

0.000001

0.00001

0.0001

log[OUABAIN] (M) Figure 2.

Effects of ouabain on the percentages of swollen tails in dog and boar spermatozoa incubated in isoand hypo-osmotic media. (~1 Dog (0) and boar spermatozoa were incubated for 45 minutes in isoosmotic (A) or 100 mOsm (B) media in the presence of the indicated concentrations of ouabain, as indicated in Materials and Methods. Results are expressed as means + SEM for 8 separate experiments.

885

Theriogenology Table 4.

Effects of ouabain on L-lactate production boar spermatozoa 0

10-7

Dog

4.7 k 0.4

** 3.7 + 0.3

3.7 k 0.4

Boar

4.9 + 0.1

5.2 + 0.4

4.9 * 0.4

OUABAIN

10-6

in dog and

10‘5 l *

***

3.1 * 0.4 4.8 * 0.4

1O-4 ****

2.6 + 0.3 5.3 * 0.3

Data are expressed as means + SEM for 7 separate experiments. Putative significance of differences was obtained by using the Student's t-test for paired data, comparing each treatment of ouabain with its paired "no ouabain" value. ** PsO.001 vs no ouabain. *** PslO-“ vs no ouabain. ***i p*10-6 vs no ouabain.

tails after ouabain incubation in an isoosmotic medium has also been observed, the increase was not great, suggesting that other ionic pumps (not ouabain-sensitive) may also be involved in the swelling reaction in dog spermatozoa. Moreover, the additive effect of ouabain on the increase in the percentage of swollen tails under hypoosmotic conditions in our study further upholds this suggestion. Under the conditions of our laboratory, boar spermatozoa were not sensitive to ouabain action in either isoosmotic or hypoosmotic medium, as demonstrated by the low percentage of swollen tails observed after hypoosmotic incubation. At least 2 possible explanations can be offered: first, boar spermatozoa contain a very low percentage of Na'/K', ouabain-sensitive ATPase activity; second, the overall composition of the ionic pumps from that of dog in boar sperm membranes is different spermatozoa. This could be an important point since the Nat/K', ouabain-inhibitive activity has been described in a wide variety of eucaryotic cells (l,S,ll), and it is not fully understood why boar spermatozoa lack significant activity of these pumps, while dog sperm cells show this activity, at least when its effects on tail membranes are examined. It is possible that both boar and ATP-ase show some Na'/K', ouabain-sensitive dog spermatozoa activity in other zones, as in the acrosomal membrane, that are not reflected in changes in tail morphology or in L-lactate production. Thus, differences in the response to hypoosmotic environments between dog and boar spermatozoa could be explained, ATP-ase in Na'/K', ouabain-sensitive in part, by differences activity. The successful conservation of spermatozoa requires that the cells be maintained under the best osmotic conditions. Since ionic pumps are cellular structures that regulate the osmotic (5), it is logical to assume that cellular environment of the ionic pumps would affect differences in composition

886

Theriogenology

differencesin sperm preservation. It has been described that the storage of canine sperm-rich fraction at 4 to 5 OC for 24 h without addition of any preservative solution does not modify (9,101, or only slightly decreases important (15), some such as the percentages parameters of semen quality, of viability, motility and response to the HOS test. However, the storage of fresh, undiluted boar semen at 4 to 5 OC for 24 h greatly decreases semen quality, as observed by the percentages motility and response to the ORT measure of viability, (unpublished data). Thus, the different compositions of ionic pumps in dog and boar spermatozoa might be related to differences in sperm quality following storage, without the use of additives, at 4 to 5 OC. Under our conditions, ouabain did not affect the percentages and altered acrosomes in either dog or boar of viability spermatozoa under a moderate hyperosmotic environment. The lack of these effects could be due to the fact that the osmotic tolerance of both types of spermatozoa fully counteracts the hyperosmotic stress caused by media with 71 as high as 900 mOsm. In fact, it has been described that incubation in media with 1~ between 300 and 1000 mOsm only slightly affects the percentage of unlysed cells in human spermatozoa (7). Similar effects have also been described in ram spermatozoa (4,8), although in the latter a significant decrease in motility (4) and in the percentage of intact spermatozoa (8) in media with 7~greater than 600 mOsm were reported. However, cellular damage has been shown when human and ram spermatozoa were incubated in hyperosmotic media and were then returned to isoosmotic conditions (4,7,8). However, n was not the only reason for these effects, since it has been stated that the presence of different components, like glycerol, fructose or NaCl in the hyperosmotic media also cause some variations in effects of high values of 71 on the cellular is possible that the integrity of human spermatozoa (7). It composition of hyperosmotic media used in our study negated these effects. It is necessary to further study the effects observed at higher K along with different compositions of hyperosmotic media to fully evaluate the role of the Na'/K', ouabain-sensitive ATP-ase in hyperosmotic stress. REFERENCES 1.

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