Effect of sperm diluents on the acrosome reaction in canine sperm

ELSEVIER

EFFECT OF SPERM DILUENTS ON THE ACROSOME REACTION IN CANINE SPERM S. Sirivaidyapong,la F.P.Cheng,* A. Marks,’ W. F. Voorhout,’ M. M. Bevers’ and B. Colenbrander’ ’ Department of Farm Animal Health ’ Institute of Biomembranes, Department of Biochemistry and Cell Biology Faculty of Veterinary Medicine Utrecht University, Yalelaan 7, 3584 CL, Utrecht, The Netherlands Received for publication: March 8, 1999 Accepted: October 12, 1999 ABSTRACT In this study we investigated the influence of sperm diluting media and temperature on the incidence of the acrosome reaction in dog sperm. Ejaculates were collected from 5 dogs, diluted with six different media and then incubated at 37°C and 20°C. Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) and ethidium homodimer as a vital stain were used in combination to determine the acrosomal status of viable spermatozoa, the technique was validated using electron microscopy. The outer acrosomal membrane of dog spermatozoa was shown to be the specific binding site for FITC-PNA. After 6 h of incubation, ejaculates diluted in media with a high Ca2+ concentration showed a significantly higher percentage (mean&SD) of acrosome reacted spermatozoa [64*7 and 58*9 in sperm capacitation medium with (SP-TALP-1) and without BSA (SP-TALP-2), respectively] than those diluted in media with a low Ca2+concentration[36&5, 39*4, 18*2 and 20*4 in Canine Capacitation Medium (CCM), Egg Yolk Tris dog semen extender (EXT-I), Modified Egg Yolk Tris extender (EXT-2) and Modified CCM (MCCM), respectively]. The increase in the percentage of acrosome reaction (AR) was slower at 20°C than at 37°C. In addition, the percentage of viable acrosome reacted spermatozoa increased significantly from 19*5 and 22*3 in non-bound sperm to 27*4 and 3M6 in zona pellucida bound sperm (diluted in EXT-2 and MCCM, respectively). We conclude that the composition of the spermatozoa diluent has a marked effect on the incidence of the acrosome reaction. Therefore, both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation. Q 2000 by Elsevier Science Inc.

Key words: acrosome, zona pellucida, dog spermatozoa Acknowledgements The authors thank Chulalongkom University and Thai government for providing a grant to Dr.S.Sirivaidyapong and Dr.T.Stout for reading of manuscript. a Correspondence and reprint requests:Fax.31-30-2531054, s.sirivaidvanong(ii),vet.uu.nl Theriogenology 53:769-602, Q 2000 by Elsevier Science

2000 Inc.

0093-691WOO/$-see front matter PII SOO93-691X(99)00274-5

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Theriogenology INTRODUCTION Although, freshly ejaculated spermatozoa are both viable and motile they are not yet capable of fertilizing an oocyte and, indeed, they require a species-specific time- span for the physiological changes necessary for the development of fertilizing ability. This process is known as capacitation (2, 5, 8) and has also been defined as the acquisition of the ability to undergo the acrosome reaction (AR) (30, 32). The AR is an important process for fertilization in mammals (42) because it is essential for zona pellucida penetration by the spermatozoa. The acrosomal status needs to be safeguarded when sperm is handled prior to or during artificial insemination or IVF procedures because acrosome reacted sperm can no longer penetrate the zona. Hence, the media used for sperm processing and sperm storage should prevent acrosome alterations, but preserve the ability of spermatozoa to undergo a physiological acrosome reaction when required. Several factors are known to induce the AR both in vivo and in vitro and these include zona pellucida proteins (ZP3) and progesterone (6, 10, 23, 26, 28, 30, 39). Each of these two factors can activate at least two types of Ca2+ channels. One channel mediates a rapid Ca2’ influx via a direct route and the other regulates Ca2+ influx indirectly, via a cell-signaling cascade, such that the influx is delayed (18, 24, 29, 38). During fertilization, the AR in mammalian spermatozoa is Ca2’ dependent and only millimolar concentrations are required for a maximal response (6, 20). In fact, the Ca2+ is considered an essential component of a medium for in vitro acrosome reaction (43). A relatively high concentration of Ca” in the incubation medium will cause a rapid influx of calcium ions while high intracellular Ca” levels will trigger the AR (11, 44). Both the AR and capacitation are Ca2’ dependent processes and, while the minor capacitationrelated changes in Ca2’ influx take place over a relatively long time- span, the AR occurs rapidly and appears to be triggered by a relatively large influx of Ca2+ (21). The effect of CaZfon the AR can be reduced by magnesium, as was observed for guinea pig and hamster spermatozoa (36). Most sperm incubation media also contain serum albumin which efficiently binds cholesterol and thus causes a decrease in the cholesterol : phospholipid (C : PL) ratio of the sperm plasma membrane. Furthermore, it has been suggested that a lower C : PL ratio decreases the membrane microviscosity, relaxes the packing of phospholipids in the membrane and permits greater calcium influx. All of which lead through unspecified intermediate steps to the fusion of the plasma and outer acrosomal membranes (13). A recent study on the AR of mouse spermatozoa indicated that BSA triggers the AR (25) while glucose has been reported to favor the occurrence of the AR of dog (27) and hamster spermatozoa (4). Taken together, the above indicates clearly that Ca” concentrations and other components of the media used for sperm processing can affect sperm acrosomal status. Sperm plasma membrane fusogenity is also affected by cooling, a procedure often employed when sperm is processed for IVF or artificial insemination. Cooling of sperm induces morphological changes of the plasma membrane, such as lateral phase separation of lipids and the clustering of proteins (7, 14) resulting, for example, in an

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increased percentage of AR in porcine sperm (40). Thus, when studying the acrosomal status of sperm, the consequences of handling sperm at room temperature instead of at body temperature should be taken into account. The aims of this study were to investigate the effects of different sperm diluting media and temperature on the acrosomal status of dog sperm over time. The media investigated were a widely used canine semen extender (E g Yolk Tris Extender: 12, 15, 33) and a number of sperm capacitation media in which CaQ+concentrations were adapted (9, 17, 32). Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) was used for staining the acrosome of dog spermatozoa, after the technique had been validated. Zona pellucida was used as a physiological inducer of the acrosome reaction of sperm diluted in media which did not induce the AR during incubation. Motility and viability were assessedas standard sperm quality parameters. MATERIALS AND METHODS Sperm preparation The sperm-rich fraction of the ejaculate was collected from 4 fertile Greyhound and 1 Beagle dogs, once a week for 3 weeks. Immediately after collection, ejaculates were diluted (1: 1 at 20°C) in one of six different media and then centrifuged at 300 x g for 10 min. The following media were used: An Egg Yolk Tris dog semen extender (Veterinary faculty, Utrecht University, The Netherlands : 12, 15, 33) which contained glucose and fructose but not Ca”. A Modified Egf Yolk Tris extender with Ca”. A sperm capacitation medium with a relatively high Ca +concentration with (32) or without BSA. A Canine Capacitation Medium (26, 27) with a low Ca2+concentrationwhich also contained glucose and BSA. A Modified CCM (MCCM) to which magnesium and fructose had been added. The concentrations of some of the specific components of the various media are given in Table 1. The diluted ejaculates were centrifuged at 300 x g for 10 min and, after the removal of the supematant, the pellets were resupended in the medium used for the initial dilution. The ejaculates were diluted to a final concentration of lo7 sperm/ml and preincubated for 6 h either at 37” C or 20°C in 5% CO2 in humidified air. Motility assessment The motility of the spermatozoa was assessed after semen collection, after washing (centrifugation) and after incubation for 0, 2, 4 and 6 h, using a phase contrast microscope at a magnification of x 100 by an experienced technician (3 1). Assessmentof sperm viability Sperm viability was assessedas described by Althouse and Hopkins (1) using the LIVE/DEAD@ kit, calcein-AM in combination with ethidium homodimer (Calcein-AM EthD-1 : ViabilityQtotoxicity, Molecular Probes Inc. Eugene, OR, USA). For the

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792 Table 1 .The composition

of the six media used to dilute dog ejaculates Media

Media Component ImMol unless stated btherwise) Calcium chloride Potassium

chloride

Magnesium chloride Sodium chloride Sodium bicarbonate Sodium phosphate Potassmm phosphate Sodium pyruvate Sodium lactate Glucose Fructose BSA (g/L) Hepes Tris Gentamycin S (g/L) Penicilhn G (IU/L) Streptomycin S (g/L)

Phenol red (g/L) Egg Yolk (g/L) Citric acid Glycme PH Osmolarity

(mOSM)

SP-TALP-I

-

a

2.0 3.1 0.4 100 25 0.3 0 1 21.6 0 0 10 10 0 0.25 0 0 0 0 0 0 7.21 303

SP-TALP-2b

CCMC

EXT-ld

EXT-2e

MCCMf

0.4 2.0 1.7 0 1.7 4.8 3.1 4.8 0 0 0 0 0.4 0.4 0.4 11.5 11.5 83.5 100 83.5 11.9 11.9 37.6 25 37.6 0 0 0 0.3 0 1.2 0 1.2 0 0 1 0.3 0 0 0.3 0 21.6 21.6 21.6 0 27.7 11.1 2.8 0 2.8 27.7 27.7 27.7 0 0 2.0 2.0 0 2.0 0 0 0 0 10 0 0 0 133.7 133.7 0 0.25 0 0 0 0 0 100,000 200,000 200,000 200,000 0.05 2 2 0.1 0 0 0.02 0 0 0.005 0 0 0 0.25 0 42.9 42.9 0 0 0 0 0 83.4 83.4 0 7.14 7.3 7.66 6.8 7.55 285 291 298 309 280

quantitative assessment of sperm viability, 200 sperm cells From a stained sample were assessed in randomly selected fields using an epifluorescence microscope (x 500) equipped with a DMU set of filters (BH2-RFCA; Olympus, Tokyo, Japan). Spermatozoa with accumulated green calcein in all cellular compartments were classified as viable, whereas spermatozoa stained red (EthD-1) and spermatozoa with accumulated green a Sperm capacitation

medium with a relatively

high Ca2+concentration

b

with BSA.

Sperm capacitation medium with a relatively high C$+concentration without BSA. c Canine Capacitation Medium with a low Ca’+concentration which also contained glucose and BSA. d Egg Yolk Tris extender which is commonly used as an extender for semen preservation. g Modified Egg Yolk Tris extender with Ca2’. Modified

CCM to which magnesium

and fructose had been added.

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calcein in the acrosome only and displaying were classified as non-viable. The viability incubation. Localization

of the FITC-PNA

binding

red EthD-1 in the post-acrosomal region, was assessed after 0, 2, 4 and 6 h of

site on dog spermatozoa

The dog spermatozoa were processed for immuno-electromnicroscopy to verify the location of the specific binding site of the PNA lectin. Spermatozoa were washed by centrifuging and resuspending in PBS at a concentration of 10’ cells/ml and they were then mixed with an equal volume of 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and fixed overnight at 4°C. The fixed sperm samples were resuspended in 10% (w/v) gelatine in 0.1 M phosphate buffer (pH 7.4) at 38°C. After pelletization and solidification at 4’C, the sperm pellet was fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Small blocks of approximately 1 mm3 were cut and infiltrated with 2.3 M sucrose overnight at 4°C and then frozen in liquid nitrogen. Ultrathin cryosections were cut at -12O’C using a Reichert Ultracut S/FCS ultramicrotome (Leica Aktiengesellschaft, Wien, Austria) with a cryo-diamond knife (Drukker International, Cuyk, The Netherlands) and transferred to formvar carbon-coated nickel grids. Thawed ultrathin cryosections were floated on 2% gelatine in 0.1 M phosphate buffer (pH 7.4) for 10 min to block non-specific binding sites, on 0.02% glycine/PBS for 10 min to block remaining free aldehyde groups, and finally for 30 minutes on block buffer containing 5% BSA and 0.1% cold water fish gelatine (BSA-C; Aurion, Wageningen, The Netherlands) in PBS. Sections were incubated with FITC-PNA, diluted 1:500 in 0.1% BSA-C in PBS for 1 h, rinsed three times for 10 min, and incubated for 1 h with rabbit anti-FITC (Molecular Probes, Inc., Eugene, OR, USA) diluted 1: 1,000 in BSA-C/PBS. The sections were rinsed three times for 10 min with BSA-C/PBS and immunoreactivity was visualised by incubating them with 10 m-n protein A-gold particles, prepared by the tamric acid-citrate method (35). Finally, immunolabeled sections were rinsed four times for 5 min with distilled water, stained on a drop of 2% uranyl acetate-oxalate (pH 7) for 5 min, and embedded in 1.8% methylcellulose containing 0.3% uranyl acetate. Immunogold-labeled cryosections were examined and photographed using a Philips CM10 electron microscope (Philips, Eindhoven, the Netherlands) at 80 kV (9). Assessment of acrosomal

status

An aliquot of each sperm sample (200 ~1) of each sperm sample was mixed with 200 ul of 4 uM EthD-1 in PBS and incubated for 5 min at 37°C. Subsequently, 20 ~1 of a solution containing 1 mg/ml DNA (Sigma, St.Louis, MO, USA) in PBS was added for 3 min (37) to bind the excess EthD-1. Next, 200 pl of 1% Paraformaldehyde in PBS was added and mixed with the sperm sample and the mixture was kept in the dark for 10 min. Then the sperm suspension was centrifuged at 300 x g for 4 min, the supematant was removed and the pellet was resuspended in 1 ml PBS before being centrifuged again at 300 x g for 4 min, to remove remaining paraformadehyde. After removal of the supematant the second time, the pellet was resuspended gently in 100 ul PBS. Subsequently, 100 ul of 0.1% Nonidet-P40 (Fluka, Buchs, Germany) was added and the

Theriogenology suspension was incubated in the dark for 5 min to permeabilize the sperm plasma membrane. Then, 1 ml PBS was added and the sample was centrifuged at 300 x g for 4 min to remove any remaining Nonidet-P40. The washing procedure was repeated and the sperm pellet was resuspended in 100 ul of PBS. To stain the sperm acrosome 100 ,ul of 100 pgiml FITC labeled peanut agglutinin (FITC-PNA; E-Y.Lab. Inc., San Mateo, CA, USA) in PBS was added to this suspension and incubated at 37’ C in 5% CO* in humidified air for 30 min. Subsequently, 1 ml of PBS was added and the sperm suspension was centrifuged at 300 x g for 4 min to remove the excess of FITC-PNA. The washing procedure was repeated and, finally, the sperm pellet was resuspended in 100 ul of PBS. Five pl of this suspension was transferred to a glass slide, mounted with 5 u1 antifade solution as described by Johnson and Gloria (22), and covered with a coverslip. Slides were stored in the dark until examination. To estimate the proportion of acrosome reacted spermatozoa, 200 spermatozoa were assessed in randomly selected fields, using the epifluorescence microscope. The acrosomal status and viability were assessed simultaneously at 0, 2, 4 and 6 h of incubation. Spermatozoa were considered to have an intact acrosome if the FITC-PNA staining was distributed over the entire acrosome. Spermatozoa with either slight fluorescence on the equatorial segment or no FITC-PNA staining, had completed the AR and had shed the outer acrosome membrane (9). Oocytes and preparation

of hemizonae

Canine ovaries were obtained following ovariectomy. The ovaries were transported in physiological saline solution at room temperature and cumulus-oocyte complexes (COCs) were recovered by placing the ovaries in a petri dish containing PBS and slicing them into small pieces (approximately 1x1 mm). COCs were recovered using a finely pulled Pasteur pipette and they were vortexed to remove cumulus cells. The denuded oocytes were washed with PBS and stored at 4 “C in a salt solution containing 2 M (NH& SO4 and 10% dextran (w/v) (17). On the day of the experiment, salt stored oocytes were washed 5 times in PBS. Each oocyte was transferred to a 20 ul droplet of PBS in 1 of 4 wells of a 35 mm plastic 4-well petri dish (Greiner B.V., Alphen A.D. Rijn, The Netherlands). After transfer, the oocytes settled down and attached themselves to the bottom of the well. Oocytes were gently bisected using a MTB-05 microblade (Micromanipulator Microscope, Carson city, NV, USA) connected to a Narishige micromanipulator (Narishige, Tokyo, Japan) fixed to a CK2 inverted, phase-contrast microscope (Olympus, Tokyo, Japan: x 200). The microblade was lowered slowly until it contacted the oocyte and the position of the blade was then adjusted to ensure a mid-line cut of the zona pellucida. Subsequently the microblade was lowered and the oocyte and zona were divided into two equal halves. The ooplasm inside each hemizona was then dislodged by scrapping the inner side of hemizona with the microblade and each hemizona was placed in a separate 50 ul droplet of either EXT-2 or MCCM in a 35 mm plastic petri dish, covered with mineral oil and kept at room temperature until used for the sperm-hemizona binding.

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Assessmentof sperm acrosomal status after zona pellucida binding Semen samples diluted with EXT-2 or MCCM (concentration 10’ sperm/ml) were used in this experiment because a preliminary study indicated that a low percentage of AR occurred in semen diluted with EXT-2 and MCCM at 37” C. After preincubating the diluted sperm samples for 6 h, the samples were diluted further to a concentration of 6~10~motile sperm/ml. A 50 u1 droplet of diluted sperm suspension was then added to each hemizona droplet, to which 2 uM of EthD-1 had already been added. The spermhemizona droplets were incubated at 37’ C in 5% CO2 in humidified air, for 5 min. Subsequently, the sperm-hemizona complexes were rinsed 5 times with PBS using a finely pulled Pasteur pipette, to remove loosely attached spermatozoa, and then washed 3 times with PBS containing 0.025 mg/ml DNA to remove the excess EthD-1. Next, the complexes were fixed for 10 min with 1 % paraformaldehyde in PBS. The spermhemizona complexes were then washed three times with PBS and transferred to a droplet of 0.05% Nonidet-P40 for 1 min to permeabilize the sperm plasma membranes, and then fixed for 10 min with 1% paraformaldehyde in PBS. After a further wash with PBS, the complexes were incubated in 50 ul droplets of 0.02 M glycine in PBS for 30 min at room temperature, to block any remaining free aldehyde groups. Finally, the sperm-hemizona complexes were incubated in 50 ~1 droplets of 20 &ml FITC-PNA, washed three time with PBS and processed for microscopical examination. Samples of sperm diluted either in EXT-2 or MCCM and obtained after 6 h of incubation were also processed for the assessment of acrosomal status, as described previously. Preparation of sperm-hemizona complexes for microscopical examination Each sperm-hemizona complex was transferred to a 20 ~1 droplet of SlowFadeTMLight antifade suspension (Molecular probes, Eugene, Oregon, USA) placed on a Poly-LLysine (Sigma) coated cover slip (20 x 20 mm). After attachment of the sperm hemizona complexes to the cover slip, the cover slip was inverted on to a microscope slide. To avoid pressure on the sperm-hemizona complex, the microscopic slide was supported at the comers with droplets of a mixture of paraffin and Vaseline. The space between the cover slip and the microscope slide was filled with antifade and sealed with nail polish. The samples were examined under an epifluorescene microscope (at a magnification of x 500) equipped with a set of DMU filters. One ejaculate from each of 5 dogs was split and diluted with either EXT-2 or MCCM and used in this experiment. Statistical Analysis Data are expressed as means+SD. The differences on percentage of acrosome reacted spermatozoa between diluted ejaculates incubated at 20°C and 37’C were tested using Chi-square analysis. Data were subjected to one way ANOVA and Levene’s test to

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determine whether the different media affected the acrosomal status and viability of spermatozoa. Differences were considered significant when P< 0.05. RESULTS Immunogold localization of bound FITC-PNA to ultrathin cryosections of fixed dog spermatozoa showed that FITC-PNA binding was limited to the acrosomal cap of spermatozoa and, indeed, was confined to the outer acrosomal membrane. FITC-PNA did not bind to the plasma membrane (Figure 1).

Figure 1. Immunogold localization of bound FITC-PNA on ultrathin cryosections of fixed dog spermatozoa. The bound FITC-PNA was detected with a polyclonal antibody against FITC and 10 nm protein A-gold. FITC-PNA bound only to the outer acrosome membrane and did not bind to the sperm plasma membrane. The percentage of non-viable sperm, both acrosome intact and acrosome reacted increased gradually from an average of 4*3 tolkt10 (mean&D) during the 6 h of incubation. There was no effect of the media on the percentage of non-viable sperm and the percentage of viable AR spermatozoa in fresh ejaculates was < 2. Following incubation at 37°C this percentage increased gradually during the 6 h of incubation to

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64*21, 58+17, 36*6, 39*18, 18*5 or 20*4 in SP-TALP-1, SP-TALP-2, CCM, EXT-1, EXT-2 and MCCM, respectively (Figure 2A). Thus, the ejaculates diluted in SP-TALP-1 and SP-TALP-2 and incubated at 37’C showed after 4 and 6 h a significantly higher percentage of AR than those diluted in the other media. The percentage of viable AR spermatozoa in the diluted samples incubated at 37°C was significantly higher than that in the diluted samples incubated at 20°C (Figure 2B). The percentage of non-viable sperm, either acrosome intact or acrosome reacted increased from 4+3 to 817 after incubation at 20°C. There was no significant difference in the percentage of acrosome reacted spermatozoa between sperm samples incubated at 20°C for up to 6 h in the various media. At 6 h, the percentage of acrosome reacted spermatozoa was significantly higher in SP-TALP- 1 (20*2), SP-TALP-2 (2 1k3) and CCM (1854) than in EXT- 1 (12k 4), EXT-2 (1 U-2) and MCCM (1 l&4) (Figure 2B).

80

60

0 0 : @%ISP--rALP-1

2

hour I

SP-TALP-2 -_____

l-lows c¶ CCM

4

hours

I

EXT-

6 1

ca EXT-2

hours I

Mczcprl,

Figure 2. The effect of the six different media on the acrosomal status of spermatozoa incubated at 37’C (A) and 20°C (B). a, b and c indicate significant differences at a given time point for a specific temperature. Mean&SD of 15 ejaculates from 5 fertile dogs are shown.

798

Theriogenology The procedure of washing sperm by centrifugation and resuspension did not affect motility significantly. However, the motility of spermatozoa decreased during incubation at 37°C (0 to 6 h) from 87*4 to 52*10,82*7 to 20&g, 83*4 to 32*8,83&8 to 55*8,78*6 to 2&S and 77%5 to 20*9 in the samples diluted with SP-TALP-1, SP-TALP-2, CCM, EXT-1, EXT-2 and MCCM, respectively. The samples incubated at 20°C showed a similar decline in the percentage of motile sperm over time as those incubated at 37°C when samples diluted in the same media were compared. The percentage of viable acrosome reacted spermatozoa was significantly higher (27*4 and 30*6 for EXT-2 and MCCM, respectively) after binding to zona pellucida than before (19+5 and 2213 for EXT-2 and MCCM, respectively). DISCUSSION The acrosomal status of dog spermatozoa can be visualized using FITC-PNA and we have used fluorescence microscopy to demonstrate that FITC-PNA binding is limited to the outer acrosomal membrane. No staining of any other membrane of the spermatozoon was observed. In fact, the sperm plasma membrane did not stain at all, an observation that contradicts that made by Bain et al. (3). However we were able to verify the outer acrosomal membrane as the specific binding site for PNA using electron microscopy. Hence, this lectin is a useful tool for studying the acrosomal status of canine spermatozoa, as it is for porcine (17) and equine (9) spermatozoa. The percentage of viable and motile AR sperm increased during incubation, with the increase being greatest for sperm samples diluted in either SP-TALP-1 or SP-TALP2. This effect may have resulted from the high Ca” concentrations in these media causing a rapid influx of calcium ions since the high intracellular calcium levels would then trigger the AR (11, 44). In this respect, sperm samples diluted in media with low Ca*+ concentration showed a lower % of AR. That Ca*’ is required in the capacitation medium for proper induction of the capacitation and the AR has been shown in both the hamster (41) and the mouse (19). On the other hand, the capacitation and the AR of guinea pig spermatozoa are not dependent on Ca2’ being present in the medium (34). In preliminary experiments, we were able to demonstrate that the addition of EDTA, a Ca2’ chelator, to SP-TALP, prevented the AR in dog spermatozoa. In addition, the percentage of AR was reduced by adding magnesium to the CCM incubation medium. Magnesium probably inhibits the AR in dog sperm as has been also observed for guinea pig and hamster spermatozoa (36). By contrast, glucose has been reported to favor the occurrence of the AR in dog spermatozoa (27) and hamster spermatozoa (4) and, our study which compared EXT-1, which had a relatively high glucose concentration, to EXT-2 and MCCM which had lower glucose concentrations, supports this view, especially when it is remembered that both of these last two extenders contained Ca2+. Lipids and protein in egg yolk may also affect the AR while BSA has been shown to trigger the AR of spermatozoa of some species e.g. the mouse (25) probably by affecting the cholesterol : phospholipids ratio in the sperm plasma membrane and thus influencing Ca ‘+ influx (13). That there was no

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difference in AR between sperm diluted in SP-TALP-1 and SP-TALP-2 indicated that, in this instance at least, BSA did not affect the AR. However, the high Ca” concentration in these media may have masked any effect of albumin on the acrosome reaction. Interestingly, the sperm samples incubated at 20°C showed the same trend in percentage of the AR except that the changes occurred more slowly than at 37°C. With regard to temperature, cooling porcine sperm to 15’C affects the lipid composition and fluidity of the sperm plasma membrane. As the temperature decreases,movement of phospholipids becomes restricted (40) and this may induce changes in intracellular Ca2’ that affect the AR. However, cooling sperm also reduces cell metabolism (40) and thus could delay the AR. For dog spermatozoa, as for that of other species, the zona pellucida can induce AR (23). In the present study, the acrosomal status of dog spermatozoa bound to the zona pellucida was compared to that of non-bound spermatozoa using an incubation medium that preserved acrosomal status (EXT-2 or MCCM). The use of matching pairs of hemizonae eliminated the variation between oocytes in their binding capacity and AR inducing capacity (16). The percentage of AR was increased significantly for zona bound spermatozoa. In some species,such as the pig (17), only AR intact spermatozoa bind to the zona pellucida and our results indicate that spermatozoa diluted in media that preserve acrosomal status are still sensitive to AR induction by the zona pellucida. Moreover, this indicates that even when the media have a low Ca2’ concentration, the sperm are able to capacitate and can readily undergo the AR. We conclude that the AR of dog spermatozoa is affected greatly by the Ca” concentration of the chosen sperm diluent. High Ca2’ concentrations enhance the AR while low Ca2’ concentrations delay the AR. However, other components of the media also play important roles in the incidence of AR, while handling sperm at 20°C rather than at 37°C decreasesthe rate of AR induction. Therefore, the media that are used to dilute dog semen and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation. REFERENCES 1. 2. 3. 4. 5.

Althouse GC, Hopkins SM. Assessment of boar sperm viability using a combination of two fluorophores. Theriogenology 1995;43: 595-603. Austin, CR. The capacitation of mammalian sperm. Nature 1952; 170: 326. Bain HK, Pabst MA, Bawa SR. Changes in the lectin binding sites on the testicular, epididymal, vas, and ejaculated spermatozoon surface of dog. Andrologia 1993; 25: 19-24. Bavister BD, Yanagimachi R. The effects of sperm extracts and energy sourceson the motility and acrosome reaction of hamster spermatozoa in vitro. Biol Reprod 1977; 16: 228-237. Bedford JM. Significance of need for sperm capacitation before fertilization in eutherian mammals. Biol Reprod 1983; 28: 108-120.

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