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Effects of spermatozoal concentration and post-thaw dilution rate on survival after thawing of dog spermatozoa
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EFFECTS OF SPERMATOZOAL CONCENTRATION AND POST-THAW DILUTION RATE ON SURVIVAL AFTER THAWING OF DOG SPERMATOZOA A. Peiia a and C. Linde-Forsberg b Department of Obstetrics and Gynecology, Center for Reproductive Biology, Box 7039 Swedish University of Agricultural Sciences,SE-750 07 Uppsala, Sweden Received for publication: December 82, 1999 Accepted:
March 23, 2000
ABSTRACT The objectives of this study were to evaluate the effects and interactions of freezing dog semen using 4 different sperm concentrations (50 x 106, 100 x 106, 200 x 106 and 400 x 106 spermatozoa/ml) in OS-mL straws and diluting the thawed semen at 4 different rates (1 :O, 1:1, 1:2 and 1:4) on post-thaw survival and longevity of dog spermatozoa during incubation at 38OC.Fifteen ejaculates were collected from 12 dogs and pooled. The semenpool was divided into 4 aliquots containing respectively 4200 x 106, 2100 x 106, 1050 x 106 and 525 x 106 spermatozoa, which were centrifuged. Sperm pellets were rediluted with TRIS-glucose-egg yolk extender containing 5% glycerol and 0.5% of Equex STM Paste to obtain the designated sperm concentrations. The semen was frozen in 0.5~mL straws 4 cm above liquid nitrogen (LN2). The straws were thawed at 70°C for 8 set and the contents of each straw were divided into 4 aliquots and diluted with TRIS buffer at 38OC at rates of 1:O, 1:l, 1:2 and I:4 (semen:buffer), respectively, making a total of 16 treatments. Sperm motility was subjectively evaluated after thawing and at l-h intervals during 8 h of incubation at 38’JC. Plasma membrane integrity and acrosomal status were evaluated at 1, 3,6, 12 and 18 h post-thaw using a triple-staining procedure and flow cytometry. For data pooled across the post-thaw dilution rate, motility was higher (P
Key words: dog semen,cryopreservation, sperm concentration, dilution rate, flow cytometry Acknowledgments: The authors thank the Department of Small Animal Sciences, SLU, for allowing semen collection from some of their dogs. a Present address:Area of Reproduction and Obstetrics, Department of Animal Pathology, Faculty of Veterinary Medicine, University of Santiago de Compostela, 27.002, Lugo, Spain. bAuthor to whom reprint requestsshould be addressed. Thenogenology 54:703-718,200O 0 20M1 Ebevier Science Inc.
0093-691WOO/$-eee front matter PII: SO093-691 X(00)003848
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Several protocols have been developed for cryopreservation of dog spermatozoa, using different packaging systems (straws, pellets, ampules, aluminum tubes) (2, 12, 15, 29, 30, 34) and insemination doses of varying volumes and spermatozoa1numbers. Dog spermatozoaare, however, most commonly packaged in 0.5-mL straws at concentrations varying for different cryopreservation protocols and for different authors, such as 40 x 106 spermatozoa/ml (20); 44 x 106 spermatozoa/ml (7); 50 x 106 spermatozoa/ml (28); 66 x 106 spermatozoa/ml (11); 100 x 106 spermatozoa/ml (33, 34, 37); 150 x 106 spermatozoa/ml (9); and 200 x 106 spermatozoa/ml (3 l/32). The minimum sperm number needed to obtain maximum pregnancy rates after artificial insemination (AI) with frozen-thawed semen in the canine species has not been established. Farstad and Andersen Berg (9) suggestedthat 150 to 200 x 106 spermatozoa per insemination, with a post-thaw survival rate of at least 50%, gives acceptable conception rates and litter size. To obtain an insemination dose with at least 100 x 106 progressive motile spermatozoa,usually one to five OS-mL straws are necessary,depending on the sperm number per straw and the post-thaw survival rate. However, a potential effect of spermatozoa1 concentration on the post-thaw sperm survival when the semenis frozen in 0.5-mL straws has not been investigated in this species,and this may be of importance to maximize the number of live spermatozoarecovered after cryopreservation. One method of thawing dog semen consists of emptying the content of each thawed straw into a volume of isotonic medium at 37 to 38OCand keeping the sperm suspension at that temperature for 5 min before assessing the post-thaw semen quality and using it for AI or research purposes (18, 27, 28, 33). This procedure allows the volume of the insemination dose to be increased when the sperm concentration is high, and, because the thawing medium usually contains additional metabolizable substrate and buffering capacity (27, 28) it also reduces the toxic effects of, for example, glycerol, egg yolk and Equex by dilution. However, the exposure of frozen-thawed spermatozoato isotonic conditions after a period of hypertonic exposure may cause an osmotic shock due to an abrupt reduction of glycerol concentration in the extracellular medium (13). The degree of osmotic stress imposed on the spermatozoa by post-thaw dilution may vary with the dilution rate and be dependent on whether the dilution is made quickly or slowly, becausethe speedof glycerol removal from the spermatozoaseemsto influence the severity of cell damage(4, 5,6, 19). Such osmotic shock is thought to occur also in the female reproductive tract fluids during AI (13). Whether post-thaw dilution of dog semen is beneficial for sperm survival and, if so, which dilution rate would be the optimal to maximize the spermatozoa1longevity in vitro, has not been investigated. The objectives of this study were to evaluate the effects and interactions of 1) freezing dog semen in a TRIS-egg yolk-5% glycerol-0.5% Equex extender at 4 different sperm concentrations (50 x 106, 100 x 106, 200 x 106 and 400 x 106 spermatozoa/ml) in 0.5-mL straws, and 2) sperm dilution immediately post-thaw with TRIS buffer at 4 different dilution
Theriogenology rates (1 :O, 1:1, I:2 and 1:4) on the post-thaw sperm viability and membrane integrity during incubation at 38OC. MATERIALS AND METHODS Animals Four privately owned and 8 research dogs were used in this study: 2 Bavarian Schweisshunds, 6 Beagles, 1 Briard, 1 German Shepherd and 2 crossbreds,ranging between 10 mo and 8 yr of age. Collection and Evaluation of Ejaculates One ejaculate was collected from each of 9 dogs and 2 ejaculates were collected from each of the other 3 with an interval of around 1 h between the first and second collections, to obtain a total number of spermatozoaof 8.3 x 109 from the 15 ejaculates, which were obtained over a 2.5-h period. The sperm-rich fraction was collected by digital manipulation in a calibrated plastic vial (17) and analyzed to determine its volume, sperm concentration and motility. The percentage of motile spermatozoa was estimated by subjective microscopic examination at a magnification of x 400 using a phase contrast microscope, and the sperm concentration was determined using a photometer (SpermaCue, Minittib, Tiefenbach, Germany). Ejaculate characteristics varied between dogs, with means and standard deviations for volume, concentration, Bnd motility being 1.8~tO.9mL, 290.8&l 02.8 x 106 spermatozoa/ml and 92*3%, respectively. SemenProcessing All the ejaculates were pooled, and 1 aliquot was removed to evaluate the motility and the sperm morphology of the pool. The motility of the pool was 90%; the proportion of normal spermatozoa,as evaluated in formol-saline (l), was 83.5%; and the proportion of sperm-head defects, as evaluated in the carbol-fuchsine stained smears(36), was 7.6%. The pool of ejaculates was divided into 4 aliquots containing, respectively, 4200 x 106, 2100 x 106, 1050 x 106 and 525 x 106 spermatozoa.The 4 aliquots were centrifuged twice (due to the high sperm concentration) at 700 x g for 8 min, whereupon the seminal plasma was discarded and each sperm pellet was rediluted with 5.25 to 5.5 mL of Extender 1 (Ext-1C ; 28) to equal1the volume of the 4 aliquots. The addition of Ext-1 resulted in sperm concentrations of approximately 800 x 106,400 x 106,200 x 106and 100 x 106 spermatozoa/ml, respectively. The 4 semen samples were allowed to equilibrate for 1 h to 4’JCin a cooler. After equilibration, 5.25 mL of Extender 2 (Ext-2-E; 28) at 4OC was added to each sample, resulting in CExt-1 : 2.4 g TRIS, 1.4 g citric acid, 0.8 g glucose, 0.06 g Na-Benzylpenicillin, 0.1 g Streptomycin sulphate, 20 mL egg yolk, 3 mL glycerol and distilled water to 100 mL @H 6.4, 843 mOsm).
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concentrations of approximately 400 x 106, 200 x 106, 100 x 106 and 50 x lo6 spermatozoa/ml, respectively. The composition of Ext-2-E was the same as that of Ext-I except that it contained 7% glycerol and 1% (v/v) of Equex STM Paste (Nova Chemical Sales Inc., Scituate, MA, USA), pH 6.4, 1685 mOsm, and the final extender concentration was, thus, 5% glycerol and 0.5% Equex. The extended semen was packaged in OS-mL straws, which were frozen on a rack (dimensions: 21 x 14 cm) placed 4 cm above the surface of LN2 contained in a stainless steel box (dimensions: 28 x 15 x 8.5 cm) for 10 min. The steel box was placed into a closed Styrofoam box (dimensions: 36 x 36 x 23 cm). The freezing rate obtained with this method was measured by placing a type-K copper thermocouple, connected to a temperature recorder (ChesselRmodel 4001, Chessel Ltd, West Sussex, England), in the center of a filled straw: it was -320C/min from +5OCto -1 lOC, followed by -2.80C/min from -1lOC to -15OC, -270C/min from -15OC to -5OOC,and -lOOC/min from -5OOCto -1OOOC.The straws were immersed in the LN2. The thawing of the first 2 straws (replicate 1, specified below) started approximately after I8 h of storage in LN2 and the subsequentreplicates were thawed on the following 15 days. Twenty straws were obtained for each of the 4 sperm concentrations. The thawing was done by immersing the straws in a water bath at 7OOCfor 8 sec. The content of each straw was emptied in a 3-mL plastic tube, and immediately 100~pL aliquots of thawed semen were taken using a micropipette and diluted with 100 pL (1: 1 dilution rate), 200 pL (1:2 dilution rate), and 400 pL (1:4 dilution rate) of TRIS buffed (28) previously placed in 3-mL tubes and kept at 38OC.The remaining volume of thawed semenwas left as undiluted control (1:O dilution rate). Sperm motility was assessedin 5 replicates using 2 straws for each replicate to ensure the necessaryvolume of semen (i.e., 10 straws of each concentration were used for the assessment of motility), and plasma and acrosomal membrane integrity of spermatozoa was assessedin another 5 replicates using another 2 straws for each one (i.e., 10 straws of each concentration were used for the assessmentof membrane integrity). The 2 straws corresponding to each replicate were thawed and diluted separately as specified above, so that the aliquots from each straw were diluted immediately after thawing without delay, and then subsamplesfrom each of 2 straws with the samedilution rate were mixed and evaluated as a single replicate. Post-Thaw Spermatozoa1Evaluation Motility. Spermatozoa1motility was evaluated immediately post-thaw (0 h) and at l-h intervals during 8 h of incubation at 38OC.The proportion of progressively motile spermatozoa from 8 randomly selected fields in each sample was evaluated subjectively in a Makler chamber (Israel Electrooptical Industry, Rehovot, Israel) at 37OC using a phase contrast microscope at x 320 magnification.
d TRIS buffer: 2.4 g TRIS, 1.4 g citric acid, 0.8 g glucose, 0.06 g Na-Benzylpenicillin, 0.1 g Streptomycin sulphate in 100 mL of distilled water (pH 6.51,249 mOsm).
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Plasma membrane intearitv and acrosomal status. Post-thaw sperm plasma membrane integrity and acrosomal status in samples containing 100 x 106, 200 x 106 and 400 x 106 spermatozoa/ml were assessedby flow cytometry after 0, 3, 6, 12 and 18 h of incubation at 38OC.Samples containing 50 x 106 spermatozoa/n& were evaluated only at 0, 3 and 6 h postthaw. The straws containing 50 x 106 spermatozoa/ml were the first group to be analyzed by flow cytometry. After the 6-h evaluation of these samples,it was seen that high percentagesof spermatozoa still had intact plasma and acrosomal membranes and that there were no differences between subsamplesdiluted after thawing at different dilution rates. Therefore, for the other sperm concentrations left to be evaluated, the incubation period was extended as long as it was needed to detect possible differences between the treatments, which proved to be 18 h. A staining procedure previously described (24) for the simultaneous assessment of plasma and acrosomal membrane integrity of dog spermatozoa was used. Briefly, a lo-mM stock solution of Carboxy-SNARF-1 (SNARF) (Molecular Probes, Inc., Eugene, OR, USA) in DMSO was prepared and stored frozen in small aliquots. The stock solution was thawed just before use and diluted in PBS to a final concentration of 100 pM. A 1S-mM stock solution of propidium iodide (PI), (Sigma, St. Louis, MO, USA) in PBS and a 2yM stock solution of fluorescein isothiocyanate (FITC) conjugated Pisum sativum agglutinin (PSA) (Sigma, St. Louis, MO, USA) in PBS were prepared and stored frozen. These solutions were added to lOOnL aliquots of thawed semen to make the following concentrations: SNARF=25 FM, PI=50 PM and FITC-PSA=O.l pM (or a 0.39 PM concentration of FITC). Semen samples were incubated at 38W in the dark for 30 min. After incubation, the stained sperm suspension was further diluted with 500 pL of TRIS buffer and analyzed by flow cytometry. Flow cytometric analysis was performed on a Facstar Plus flow cytometer (Becton Dickinson, San Jose, CA, USA) as described previously (24). Cytogram Analysis Dot plot cytograms were obtained for FL1 (fluorescence sensor that collects wavelengths between 500 and 560 nm) and FL3 (fluorescence sensor that collects wavelengths higher than 640 nm). The SNARF and PI positive cells were both detected in the FL3 sensor; the two populations were easily identified becausethe PI fluorescence intensity was higher than that of SNARF. The FITC fluorescence was detected in the FL1 sensor. Four populations with different fluorescences were quantified: 1) one population with a low intensity of red and green fluorescence (live cells with intact acrosomes,stained only by SNARF); 2) another population with a low intensity of red fluorescence and a high intensity of green fluorescence (live cells with reacted acrosomes,stained by SNARF and FITC-PSA); 3) a third population with a high intensity of red fluorescence and a low intensity of green fluorescence (dead cells stained only by PI); and 4) a population with high intensities of both red and green fluorescence (dead cells with damagedacrosomes,stained by PI and FITC-PSA).
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Flow cytometry data on the integrity of sperm plasma and acrosomal membranes are presented, respectively, as percentages of total live spermatozoa (Populations 1 and 2) and percentagesof acrosome-reactedlive spermatozoa(Population 2), becausethe acrosomal status of only the live spermatozoa was considered to be more relevant than that of the total sperm population. Statistical Analysis Data on post-thaw progressive motility, sperm plasma membrane integrity and acrosomal status were analyzed using the general lineal model procedure (GLM, Minitab Statistical software, State College, PA, USA) at each evaluation time. Effects included in the models were the concentration of spermatozoa (50 x 106, 100 x 106, 200 x 106 and 400 x 106 spermatozoa/ml) in 0.5-mL straws, the post-thaw dilution rate (l:O, l:l, 1:2 and 1:4) and the interaction between them. When significant effects were detected in the models, differences between treatments were analyzed with Tukey’s Studentized Range (HSD) Test using the SAS program (Cary, North Carolina, USA) and were considered statistically significant at PcO.05 level. RESULTS Effect of Spermatozoa1Concentration For data pooled across post-thaw dilution rates, the spermatozoa1concentration had a significant effect (P
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In samples with 200 or 400 x lo6 spermatozoa/ml, the decline in the proportion of total live spermatozoa during 6 h of incubation was not significant. In contrast, semen samples frozen with 50 x 106 or 100 x 106 spermatozoa/ml had a lower (PcO.05) percentage of total live spermatozoa after 3 h of incubation than immediately after thawing, but the decline between 3 and 6 h was not significant. Table 1. Progressive motility percentages(mea&SD) of dog semen samples frozen in OS-mL straws with different spermatozoal concentrations, for all dilution rates, during incubation post-thaw at 38eC. Spermatozoal concentration (spermatozoa/mL) Incubation time so x 106 loo x 106 200 x 106 400 x 106 Oh 54.5+9.7 b 49.7k7.7 b 65Sk5.6 a 51.2k4.2 b lh 54.5k8.2 b 50.7+8.1 b 64.m7.0 a 54.5+5.1 b 2h 50.5k8.6 b 48.7+8.1 b 60.7h7.3 a 50.5+3.9 b 3h 44.5*9.0 b 44.2rt14.6 b 60.5b8.6 a 47.0+4.4 b 4h 36.a8.4 C 38.5kl4.0 C 56.7h6.9 a 46.5+5.4 b 5h 21.5h9.7 d 32.Ohl3.3 C 41.2klO.7 b 50.5h6.9 a 6h 9.2h9.2 cl 23.7kl5.3 C 43.5hl4.0 a 34.7k17.8 b 7h 3.1Zt5.9c 14.&12.1 b 31.Oh18.0a 29.7*18.3 a 8h 1.1*2.6b 6.5~k8.4b 21.2kl6.3 a 20.0k17.9 a a b, CDifferent superscript letters in the samerow indicate significant differences (P
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concentrations evaluated. Between 3 and 6 h of incubation the proportions of acrosome-reacted live spermatozoa were higher (P
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Table 4. Progressive motility percentages(mean&SD) of frozen-thawed dog semen samples diluted immediately after thawing with TRIS buffer at different dilution rates, for all sperm concentrations, during incubation post-thaw at 38cC. Post-thaw dilution rate Incubation time 1: 0 1: 1 1:2 1: 4 54.S7.2 ab Oh 58.h7.3 a* 57.5*7.7a* 50.5&12.2b* lh 54.Ozt5.0a 56.5h7.1 a 58.7*7.6a 54.5k12.8 a 2h 51.755.4 a 53.7+7.0a 53.5k8.3 a 51.5*12.2a 3h 44.7hl2.3 a 51.758.0 a 52.5+9.1 a 47.2*15.3 a 4h 39.5*11.9a 46.7k10.7a 47.7k8.7 a 43.7*15.7 a 5h 26.5+13.4 b** 38.2k13.0 a** 42.2~Hl.l a** 38.2h17.5 a** 6h 9.7h10.6 b** 31.7*18.6a** 37.Oh15.3a** 32.7h19.2 a** 7h 0.7+1.8 b** 21.5~t15.2a** 28.5h16.9 a** 27.U19.0 a** 8h o.ozto.0b** 6.5h6.3 b** 21.7+16.2a** 20.6h17.8 a** % b Different superscript letters in the same row indicate significant differences (*= PcO.05; **=P
The percentageof live spermatozoawith reacted acrosomes(Table 6) was not influenced by the dilution rate during the first 6 h of incubation at 38cC. After 12 h of incubation a tendency (P=O.O71)was seen for undiluted samples to have lower proportions of acrosome reactions. After 18 h, a significant (P
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Table 6. Percentagesof acrosome-reactedlive spermatozoa(mean+SD) in frozen-thawed dog semen samples diluted immediately after thawing with TRIS buffer at different dilution rates, for all sperm concentrations, during incubation post-thaw at 38OC. Post-thaw dilution rate Incubation time 1: 0 1:4 1: 1 1: 2 Oh o.oko.0 a o.oko.0 a 0.0~0.0 a o.o+o.o a 3h 1.2+1.9 a 0.9k2.3 a 1.9h3.4 a 1.0*2.2 a 6h .5.0+3.2a 3.1h3.2 a 3.714.2 a 3.313.4 a 12h 6.9k3.5 a 11.7*8.4 a 11.115.9a 11.613.6 a 18h 3.9+2.4 a 8.8+6.2 b 18.919.0 d 14.5k7.4 c a~bc~dDifferent superscript letters in the samerow indicate significant differences (P
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contrast, the highest proportion of live spermatozoa with reacted acrosomes after 6 h of incubation was observed with the concentration of 400 x 106 spermatozoa/ml and post-thaw dilution rates of 1:2 (14.0%*7.8%) and 1:4 (13.0%*2.9%). However, after 18 h of incubation, the lowest percentage of acrosome reactions (PcO.05) was observed with the concentration of 400 x 106 spermatozoa/ml and a post-thaw dilution rate of 1:4, for which only 26.6%9.6% of the live spermatozoa had reacted acrosomes.In contrast, the highest percentage (PcO.05) was observed with the concentration of 200 x 106 spermatozoa/ml and a post-thaw dilution rate of 1:2, for which 60.2Y&9.4% of the live spermatozoahad reacted acrosomes. DISCUSSION There is a lack of information concerning the effects of sperm concentration for freezing of dog semen. Studies in other species, especially in the equine, suggest that this factor may have an influence on the post-thaw sperm survival. Palacios et al. (21) reported better motility post-thaw when equine spermatozoawere frozen at 800 x 106 spermatozoa/mL than at 100 x 106 spermatozoa/ml, and Parlevliet et al. (22) observed an interaction between sperm concentration and freezing method. For high sperm concentrations (1000 x 106 spermatozoa/ml), post-thaw motility was better when a fast cooling rate was used during the freezing, whereas for low sperm concentrations (200 x 106 spermatozoa/ml) the post-thaw motility was better when a slow cooling rate was used. Leipold et al. (16) found that post-thaw motility was similar for equine spermatozoafrozen at 1600 x 106 spermatozoa/ml and at 400 x 1Oe spermatozoa/ml, but pregnancy rates tended to be higher for mares inseminated with spermatozoa frozen at r600 x 106 spermatozoa/ml than at 400 x 106 spermatozoa/ml. Furthermore, when a low and a high number of spermatozoaper insemination dose (320 x 106 vs. 800 x 106 progressively motile spermatozoa) were compared, there tended to be a higher pregnancy rate using the lower spermatozoa1number if spermatozoa were packaged at the higher concentration. Heitland et al. (14), however, reported better post-thaw motility when stallion spermatozoawere frozen in OS-mL straws at sperm concentrations of 20,200 or 400 x 106 spermatozoa/ml than at 800 x 106 spermatozoa/ml or higher. In addition, Colas (3) obtained better fertility results when ram semen was diluted to a constant concentration of 900 x 106 spermatozoa/ml (34% of 91 ewes lambed) than to a constant rate (1:4, semen volume: extender volume) where the sperm concentration would be variable (25% of 101 ewes lambed). This author suggested that the effect was related to the final glycerol concentration in the spermatozoa. The results of the present study show that dog spermatozoa, under the cryopreservation conditions described here, had a higher thermoresistance when they were frozen at concentrations of 200 x 106 and 400 x 106 spermatozoa/ml than at lower sperm concentrations. This might suggest that latent damage of sperm membranes after thawing is less severewhen freezing is done at higher sperm concentrations. The cause of such an effect is unknown, but it is not likely to have been due to a lower glycerol concentration in the sperm cells, becausedog spermatozoaseem to be rather tolerant to a range of glycerol levels (8, 12, 20, 23, 27). The effect, instead, might be due to an influence of the sperm concentration on factors determining the cryoinjury of spermatozoa, such as the “solution effect” and the formation of ice crystals. However, Watson and Duncan (35) observed that cell concentrations
Theriogenology in the range of 3 to 12 x10* spermatozoa/ml did not influence the effects of either the salt concentration or the unfrozen water fraction during the crystallization phase of the freezing on the survival of ram spermatozoa. In the present study, the proportions of acrosomereactions during the first period of postthaw incubation seemedto increase with increasing numbers of spermatozoaper straw. There is not an obvious explanation for such an effect, which has also been observed in equine spermatozoa(21), but acrosomal enzymes and toxic products released from dead spermatozoa might have contributed to the destabilization of acrosomal membranesin live spermatozoa,and to larger extent at higher sperm concentrations. At the end of the incubation period, this effect was no longer apparent, but it might have been obscured by the effects of the prolonged incubation as such. The highest post-thaw dilution rate evaluated in the present study exerted a negative effect on the progressive motility of spermatozoa immediately after the dilution; however, it resulted in a higher sperm longevity. There is some evidence that the thawing injury of sperm cells is due to the fact that increasingly hyperosmotic conditions during freezing are reversed during thawing, and cells are progressively subjected to hypoosmotic conditions when compared with the highest hyperosmotic environment the cells were exposed to during the freezing as the crystallization of the extracellular water progressed (6). This osmotic stress should be expected to be more pronounced when thawed spermatozoaare further diluted in a glycerol-free medium, becauseit induces an abrupt removal of glycerol from the spermatozoa to reach the equilibrium on both sides of the plasma membrane and, therefore, cells have to accommodate new volume changes in addition to those suffered during the thawing. Furthermore, at higher post-thaw dilution rates with a glycerol-free medium, the osmotic stress imposed on the cells is supposedto be higher due to a bigger difference between osmolarities. On the other hand, a quick dilution after thawing in a single step was found to be detrimental for bull and mouse spermatozoa, whereas a slow dilution was not (4, 19). Fiser and Fairfull (lo), however, reported that the rate of glycerol removal after thawing did not affect the percentage of motile spermatozoa and intact acrosomes in ram spermatozoa. In the present study, the dilution post-thaw was done immediately after thawing in a single step, and samples diluted at a rate of 1:4 showed the lowest progressive motility immediately post-thaw and the highest plasma membrane integrity, presumably due to a more severe osmotic stress, which impaired the straight movement of spermatozoa while maintaining intact sperm membranes. However, after 8 and 12 h of incubation, respectively, sperm motility and viability were higher at increasing dilution rates. These results indicate that, after the osmotic stress imposed on spermatozoa by a rapid dilution post-thaw, spermatozoa were able to adjust to the new extracellular osmotic conditions, maintaining motility and the integrity of their membranes. The improved longevity at increasing dilution rates is thought to have been caused not only by the additional source of energy and buffering capacity, but also by reducing the glycerol concentration in spermatozoaand its toxic effects at high temperature.
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In this study, the integrity of sperm plasma membranes,in general for all treatments and particularly for those with the higher dilution rates, was maintained in a high percentage of sperm cells for a longer incubation period in comparison with that observed in previous studies. It indicates that the cryopreservation protocol used here provided adequate stabilizing conditions for the sperm membranes at high temperature. The freezing and thawing methods used in this study were found to be the least detrimental for sperm survival among several other treatments evaluated in a previous study (25). Motility, on the other hand, was affected considerably more by the incubation than membrane integrity over the same periods. It is not known whether this population of non-motile but apparently viable spermatozoa could regain motility within the female reproductive tract; however, the addition of caffeine had no such effect (data not shown). The decline in sperm motility after incubation periods when there were still high numbers of viable spermatozoadoes not seemto have been causedby the depletion of available substrate for the spermatozoa. If that were the case, the decline in sperm motility should be expected to occur earlier in semen sampleswith the higher sperm concentrations due to the faster depletion of substrate by the presenceof more sperm, but the opposite was found (Table 1). Furthermore, in the semen samples diluted at a rate of 1:4, which had the best longevity, the same phenomenon was observed. One explanation might be that the premature loss of motility in live cells is due to latent damagecausedby cryopreservation. The post-thaw dilution rate had no significant effect on the proportion of acrosome reactions detected during most of the incubation period, although, at the end, increasing proportions of reacted acrosomes were seen at increasing dilution rates. However, the ratio between the percentage of acrosome-reactedlive spermatozoaand the percentage of live cells was rather similar for the different dilution rates. Thus, it does not appear that post-thaw dilution of spermatozoa,at the rates evaluated in this study, increasesthe acrosomal damageof spermatozoa. In conclusion, the results of the present study suggest that dog spermatozoa frozen in a TRIS-egg yolk-5% glycerol-0.5% Equex extender at concentrations of 200 x 106 spermatozoa/ml and diluted immediately post-thaw with a plain TRIS buffer at rates of 1:4 or 1:2, survive better than when frozen with lower or higher sperm concentrations and/or not being diluted after thawing. Further studies are needed to investigate whether motility may be induced again in the populations of non-motile spermatozoa with intact plasma membranes, and whether it could be reflected in an increase of fertility rates after AI with frozen semen in this species. REFERENCES 1. Bane A. Acrosomal abnormality associatedwith sterility in boar. Proc 4th Int Congr Anim Reprod (ICAR) 1961;810-817. 2. Battista M, Parks J, Concannon P. Canine sperm post-thaw survival following freezing in straws orpellets using PIPES, Lactose, Tris or Test extenders. Proc 1lfh Int Congr Anim Reprod (ICAR) 1988;3:229-231.
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3. Colas G. Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-frozen ram semen. J Reprod Fertil 1975;42:277285. 4. Correa JR, Rodriguez MC, Patterson DJ, Zavos PM. Thawing and processed of
cryopreserved bovine spermatozoa at various temperatures and their effects on sperm viability, osmotic shock and sperm membrane functional integrity. Theriogenology 1996; 46: 413-420. 5. Correa JR, Zavos PM. Frozen-thawed bovine spermatozoa diluted by slow or rapid
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8. 9. 10. 11.
dilution method: measurements on occurrence of osmotic shock and sperm viability. Theriogenology 1995;44:963-971. Curry MR, Watson PF. Osmotic effects on ram and human sperm membranesin relation to thawing injury. Cryobiology 1994;31:39-46. Dobrinski I, Lulai C, Barth AD, Post K. Effects of four different extenders and three different freezing rates on post-thaw viability of dog semen. J Reprod Fertil 1993;47(Suppl):291-296. England GCW. Cryopreservation of dog semen: a review. J Reprod Fertil 1993;47(Suppl):243-255. Farstad W, Andersen-Berg K. Factors influencing the success rate of artificial insemination with frozen semenin the dog. J Reprod Fertil 1989;39(Suppl):289-292. Fiser PS, Fairfull RW. The effect of glycerol related osmotic changes on post-thaw motility and acrosomal integrity of ram spermatozoa.Cryobiology 1989:26:64-69. Fontbonne A, Badinand F. Canine artificial insemination with frozen semen: comparison of intravaginal and-intrauterine deposition of semen.J Reprod Fertil 1993;47(Suppl):325327.
12. Foote RH. Extenders for freezing dog semen.Am J Vet Res 1964;25:37-40 13. Hammerstedt RH, Graham JK, Nolan JP. Cryopreservation of mammalian sperm: What we ask them to survive? J Androl 1990;11:73-88. 14. Heitland AV, Jasko DJ, Squires EL, Graham JK, Pickett BW, Hamilton C. Factors affecting motion characteristics of frozen-thawed stallion spermatozoa. Equine Vet J 1996;28:47-53. 15. Ivanova-Kicheva MG, Bobadov N, Somlev B. Cryopreservation of canine semen in pellets and in 5-ml aluminium tubes using three extenders. Theriogenology 1997;48:13431349. 16. Leipold SD, Graham JK, Squires PM, McCue PM, Brinsko SP, Venderwall DK. Effect of spermatozoa1 concentration and number on fertility of frozen equine semen. Theriogenology 1998;49:1537-1543. 17. Linde-Forsberg C. Achieving canine pregnancy by using frozen or chilled extended semen.Vet Clin No Am: Sm Anim Prac 1991;21:467-485. 18. Linde-Forsberg C, Strom Holst B, Govette G. Comparison of fertility data from vaginal vs. intrauterine insemination of frozen-thawed dog semen: a retrospective study. Theriogenology 1999;52:1l-23. 19. Nakagata N, Takeshima T. High fertilizing ability of mouse spermatozoa diluted slowly after cryopreservation. Theriogenology 1992;37:1283-1291.
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20. Olar TT, Bowen RA, Pickett BW. Influence of extender, cryopreservative and seminal processing procedures on post-thaw motility of canine spermatozoa frozen in straws. Theriogenology 1989;31:451-461. 21. Palacios A, Valencia J, Zarco L. Efecto de1 sistema de envasado y la concentration espermaticasobre el daho acrosomal y la motilidad postdescongelacion de1semen equino. Vet Mex 1992;4:315-318. 22. Parlevliet J, Malmgren L, Boyle M, Wiickener a, Bader H, Colenbrander B. Influence of conservation method on the motility and morphology of stallion semen (an international project). ActaVet Stand 1992;88:153-162. 23. Peiia AI, Barrio F, Quintela LA, Herraddn PG. Effect of different glycerol treatments on frozen-thawed dog sperm longevity and acrosomal integrity. Theriogenology 1998;50:163-174. 24. PeAa A, Johannisson A, Linde-Forsberg C. Post-thaw evaluation of dog spermatozoa using new triple staining and flow cytometry. Theriogenology 1999;52:965-980. 25. Petia A, Linde-Forsberg C. Effects of Equex, one- of two-step dilution, and two freezing and thawing rates on post-thaw survival of dog spermatozoa.Theriogenology, 2000;54:859-875. 26. Rota A, Iguer-Ouada M, Verstegen J, Linde-Forsberg C. Fertility after vaginal of uterine deposition of dog semen frozen in a Tris extender with or without Equex STM paste. Theriogenology 1999;51:1045-1058. 27. Rota A, Linde-Forsberg C, Vannozzi J, Romagnoli S, Rodriguez-Martinez H. Cryosurvival of dog spermatozoa at different glycerol concentrations and freezing/thawing rates. Reprod Dom Anim 1998;33:355-361. 28. Rota A, Strom B, Linde-Forsberg C, Rodriguez-Martinez H. Effects of Equex STM Paste on viability of frozen-thawed dog spermatozoa during in vitro incubation at 38OC. Theriogenology 1997;47:1093-1101. 29. Seager SWJ, Fletcher WS. Progress on the use of frozen semen in the dog. Vet Record 1973;92:6-10. 30. Seager SWJ, Platz C, Fletcher WS. Conception rates and related data using frozen dog semen.J Reprod Fertil 1975;45:189-192. 3 1. Silva LDM, Gnclin K, Lejeune B, Verstegen JP. Comparisons of intravaginal and intrauterine insemination of bitches with fresh or frozen semen. Vet Ret 1996; 138: 154157. 32. Silva LDM, Verstegen JP. Comparisons between three different extenders for canine intrauterine insemination with frozen-thawed spermatozoa. Theriogenology 1995; 44: 571-579. 33. Strom B, Rota A, Linde-Forsberg C. In vitro characteristics of canine spermatozoa subjected to two methods of cryopreservation. Theriogenology 1997;48:247-256. 34. Thomas PGA, Larsen RE, Hahn CN. A comparison of three packaging techniques using two extenders for the cryopreservation of canine semen. Theriogenology 1993;40:11991205. 35. Watson PF, Duncan AE. Effect of salt concentration and unfrozen water fraction on the viability of slowly frozen ram spermatozoa.Cryobiology 1988;25: 131- 142.
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36. Williams WW. Technique of collecting semen for laboratory examination with a review of several diseasedbulls. Cornell Vet 1920;10:87-94. 37. Wilson MS. Non-surgical intrauterine artificial insemination in bitches using frozen semen.J Reprod Fertil 1993;47(Suppl):307-311.
EFFECTS OF SPERMATOZOAL CONCENTRATION AND POST-THAW DILUTION RATE ON SURVIVAL AFTER THAWING OF DOG SPERMATOZOA A. Peiia a and C. Linde-Forsberg b Department of Obstetrics and Gynecology, Center for Reproductive Biology, Box 7039 Swedish University of Agricultural Sciences,SE-750 07 Uppsala, Sweden Received for publication: December 82, 1999 Accepted:
March 23, 2000
ABSTRACT The objectives of this study were to evaluate the effects and interactions of freezing dog semen using 4 different sperm concentrations (50 x 106, 100 x 106, 200 x 106 and 400 x 106 spermatozoa/ml) in OS-mL straws and diluting the thawed semen at 4 different rates (1 :O, 1:1, 1:2 and 1:4) on post-thaw survival and longevity of dog spermatozoa during incubation at 38OC.Fifteen ejaculates were collected from 12 dogs and pooled. The semenpool was divided into 4 aliquots containing respectively 4200 x 106, 2100 x 106, 1050 x 106 and 525 x 106 spermatozoa, which were centrifuged. Sperm pellets were rediluted with TRIS-glucose-egg yolk extender containing 5% glycerol and 0.5% of Equex STM Paste to obtain the designated sperm concentrations. The semen was frozen in 0.5~mL straws 4 cm above liquid nitrogen (LN2). The straws were thawed at 70°C for 8 set and the contents of each straw were divided into 4 aliquots and diluted with TRIS buffer at 38OC at rates of 1:O, 1:l, 1:2 and I:4 (semen:buffer), respectively, making a total of 16 treatments. Sperm motility was subjectively evaluated after thawing and at l-h intervals during 8 h of incubation at 38’JC. Plasma membrane integrity and acrosomal status were evaluated at 1, 3,6, 12 and 18 h post-thaw using a triple-staining procedure and flow cytometry. For data pooled across the post-thaw dilution rate, motility was higher (P
Key words: dog semen,cryopreservation, sperm concentration, dilution rate, flow cytometry Acknowledgments: The authors thank the Department of Small Animal Sciences, SLU, for allowing semen collection from some of their dogs. a Present address:Area of Reproduction and Obstetrics, Department of Animal Pathology, Faculty of Veterinary Medicine, University of Santiago de Compostela, 27.002, Lugo, Spain. bAuthor to whom reprint requestsshould be addressed. Thenogenology 54:703-718,200O 0 20M1 Ebevier Science Inc.
0093-691WOO/$-eee front matter PII: SO093-691 X(00)003848
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Several protocols have been developed for cryopreservation of dog spermatozoa, using different packaging systems (straws, pellets, ampules, aluminum tubes) (2, 12, 15, 29, 30, 34) and insemination doses of varying volumes and spermatozoa1numbers. Dog spermatozoaare, however, most commonly packaged in 0.5-mL straws at concentrations varying for different cryopreservation protocols and for different authors, such as 40 x 106 spermatozoa/ml (20); 44 x 106 spermatozoa/ml (7); 50 x 106 spermatozoa/ml (28); 66 x 106 spermatozoa/ml (11); 100 x 106 spermatozoa/ml (33, 34, 37); 150 x 106 spermatozoa/ml (9); and 200 x 106 spermatozoa/ml (3 l/32). The minimum sperm number needed to obtain maximum pregnancy rates after artificial insemination (AI) with frozen-thawed semen in the canine species has not been established. Farstad and Andersen Berg (9) suggestedthat 150 to 200 x 106 spermatozoa per insemination, with a post-thaw survival rate of at least 50%, gives acceptable conception rates and litter size. To obtain an insemination dose with at least 100 x 106 progressive motile spermatozoa,usually one to five OS-mL straws are necessary,depending on the sperm number per straw and the post-thaw survival rate. However, a potential effect of spermatozoa1 concentration on the post-thaw sperm survival when the semenis frozen in 0.5-mL straws has not been investigated in this species,and this may be of importance to maximize the number of live spermatozoarecovered after cryopreservation. One method of thawing dog semen consists of emptying the content of each thawed straw into a volume of isotonic medium at 37 to 38OCand keeping the sperm suspension at that temperature for 5 min before assessing the post-thaw semen quality and using it for AI or research purposes (18, 27, 28, 33). This procedure allows the volume of the insemination dose to be increased when the sperm concentration is high, and, because the thawing medium usually contains additional metabolizable substrate and buffering capacity (27, 28) it also reduces the toxic effects of, for example, glycerol, egg yolk and Equex by dilution. However, the exposure of frozen-thawed spermatozoato isotonic conditions after a period of hypertonic exposure may cause an osmotic shock due to an abrupt reduction of glycerol concentration in the extracellular medium (13). The degree of osmotic stress imposed on the spermatozoa by post-thaw dilution may vary with the dilution rate and be dependent on whether the dilution is made quickly or slowly, becausethe speedof glycerol removal from the spermatozoaseemsto influence the severity of cell damage(4, 5,6, 19). Such osmotic shock is thought to occur also in the female reproductive tract fluids during AI (13). Whether post-thaw dilution of dog semen is beneficial for sperm survival and, if so, which dilution rate would be the optimal to maximize the spermatozoa1longevity in vitro, has not been investigated. The objectives of this study were to evaluate the effects and interactions of 1) freezing dog semen in a TRIS-egg yolk-5% glycerol-0.5% Equex extender at 4 different sperm concentrations (50 x 106, 100 x 106, 200 x 106 and 400 x 106 spermatozoa/ml) in 0.5-mL straws, and 2) sperm dilution immediately post-thaw with TRIS buffer at 4 different dilution
Theriogenology rates (1 :O, 1:1, I:2 and 1:4) on the post-thaw sperm viability and membrane integrity during incubation at 38OC. MATERIALS AND METHODS Animals Four privately owned and 8 research dogs were used in this study: 2 Bavarian Schweisshunds, 6 Beagles, 1 Briard, 1 German Shepherd and 2 crossbreds,ranging between 10 mo and 8 yr of age. Collection and Evaluation of Ejaculates One ejaculate was collected from each of 9 dogs and 2 ejaculates were collected from each of the other 3 with an interval of around 1 h between the first and second collections, to obtain a total number of spermatozoaof 8.3 x 109 from the 15 ejaculates, which were obtained over a 2.5-h period. The sperm-rich fraction was collected by digital manipulation in a calibrated plastic vial (17) and analyzed to determine its volume, sperm concentration and motility. The percentage of motile spermatozoa was estimated by subjective microscopic examination at a magnification of x 400 using a phase contrast microscope, and the sperm concentration was determined using a photometer (SpermaCue, Minittib, Tiefenbach, Germany). Ejaculate characteristics varied between dogs, with means and standard deviations for volume, concentration, Bnd motility being 1.8~tO.9mL, 290.8&l 02.8 x 106 spermatozoa/ml and 92*3%, respectively. SemenProcessing All the ejaculates were pooled, and 1 aliquot was removed to evaluate the motility and the sperm morphology of the pool. The motility of the pool was 90%; the proportion of normal spermatozoa,as evaluated in formol-saline (l), was 83.5%; and the proportion of sperm-head defects, as evaluated in the carbol-fuchsine stained smears(36), was 7.6%. The pool of ejaculates was divided into 4 aliquots containing, respectively, 4200 x 106, 2100 x 106, 1050 x 106 and 525 x 106 spermatozoa.The 4 aliquots were centrifuged twice (due to the high sperm concentration) at 700 x g for 8 min, whereupon the seminal plasma was discarded and each sperm pellet was rediluted with 5.25 to 5.5 mL of Extender 1 (Ext-1C ; 28) to equal1the volume of the 4 aliquots. The addition of Ext-1 resulted in sperm concentrations of approximately 800 x 106,400 x 106,200 x 106and 100 x 106 spermatozoa/ml, respectively. The 4 semen samples were allowed to equilibrate for 1 h to 4’JCin a cooler. After equilibration, 5.25 mL of Extender 2 (Ext-2-E; 28) at 4OC was added to each sample, resulting in CExt-1 : 2.4 g TRIS, 1.4 g citric acid, 0.8 g glucose, 0.06 g Na-Benzylpenicillin, 0.1 g Streptomycin sulphate, 20 mL egg yolk, 3 mL glycerol and distilled water to 100 mL @H 6.4, 843 mOsm).
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concentrations of approximately 400 x 106, 200 x 106, 100 x 106 and 50 x lo6 spermatozoa/ml, respectively. The composition of Ext-2-E was the same as that of Ext-I except that it contained 7% glycerol and 1% (v/v) of Equex STM Paste (Nova Chemical Sales Inc., Scituate, MA, USA), pH 6.4, 1685 mOsm, and the final extender concentration was, thus, 5% glycerol and 0.5% Equex. The extended semen was packaged in OS-mL straws, which were frozen on a rack (dimensions: 21 x 14 cm) placed 4 cm above the surface of LN2 contained in a stainless steel box (dimensions: 28 x 15 x 8.5 cm) for 10 min. The steel box was placed into a closed Styrofoam box (dimensions: 36 x 36 x 23 cm). The freezing rate obtained with this method was measured by placing a type-K copper thermocouple, connected to a temperature recorder (ChesselRmodel 4001, Chessel Ltd, West Sussex, England), in the center of a filled straw: it was -320C/min from +5OCto -1 lOC, followed by -2.80C/min from -1lOC to -15OC, -270C/min from -15OC to -5OOC,and -lOOC/min from -5OOCto -1OOOC.The straws were immersed in the LN2. The thawing of the first 2 straws (replicate 1, specified below) started approximately after I8 h of storage in LN2 and the subsequentreplicates were thawed on the following 15 days. Twenty straws were obtained for each of the 4 sperm concentrations. The thawing was done by immersing the straws in a water bath at 7OOCfor 8 sec. The content of each straw was emptied in a 3-mL plastic tube, and immediately 100~pL aliquots of thawed semen were taken using a micropipette and diluted with 100 pL (1: 1 dilution rate), 200 pL (1:2 dilution rate), and 400 pL (1:4 dilution rate) of TRIS buffed (28) previously placed in 3-mL tubes and kept at 38OC.The remaining volume of thawed semenwas left as undiluted control (1:O dilution rate). Sperm motility was assessedin 5 replicates using 2 straws for each replicate to ensure the necessaryvolume of semen (i.e., 10 straws of each concentration were used for the assessment of motility), and plasma and acrosomal membrane integrity of spermatozoa was assessedin another 5 replicates using another 2 straws for each one (i.e., 10 straws of each concentration were used for the assessmentof membrane integrity). The 2 straws corresponding to each replicate were thawed and diluted separately as specified above, so that the aliquots from each straw were diluted immediately after thawing without delay, and then subsamplesfrom each of 2 straws with the samedilution rate were mixed and evaluated as a single replicate. Post-Thaw Spermatozoa1Evaluation Motility. Spermatozoa1motility was evaluated immediately post-thaw (0 h) and at l-h intervals during 8 h of incubation at 38OC.The proportion of progressively motile spermatozoa from 8 randomly selected fields in each sample was evaluated subjectively in a Makler chamber (Israel Electrooptical Industry, Rehovot, Israel) at 37OC using a phase contrast microscope at x 320 magnification.
d TRIS buffer: 2.4 g TRIS, 1.4 g citric acid, 0.8 g glucose, 0.06 g Na-Benzylpenicillin, 0.1 g Streptomycin sulphate in 100 mL of distilled water (pH 6.51,249 mOsm).
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Plasma membrane intearitv and acrosomal status. Post-thaw sperm plasma membrane integrity and acrosomal status in samples containing 100 x 106, 200 x 106 and 400 x 106 spermatozoa/ml were assessedby flow cytometry after 0, 3, 6, 12 and 18 h of incubation at 38OC.Samples containing 50 x 106 spermatozoa/n& were evaluated only at 0, 3 and 6 h postthaw. The straws containing 50 x 106 spermatozoa/ml were the first group to be analyzed by flow cytometry. After the 6-h evaluation of these samples,it was seen that high percentagesof spermatozoa still had intact plasma and acrosomal membranes and that there were no differences between subsamplesdiluted after thawing at different dilution rates. Therefore, for the other sperm concentrations left to be evaluated, the incubation period was extended as long as it was needed to detect possible differences between the treatments, which proved to be 18 h. A staining procedure previously described (24) for the simultaneous assessment of plasma and acrosomal membrane integrity of dog spermatozoa was used. Briefly, a lo-mM stock solution of Carboxy-SNARF-1 (SNARF) (Molecular Probes, Inc., Eugene, OR, USA) in DMSO was prepared and stored frozen in small aliquots. The stock solution was thawed just before use and diluted in PBS to a final concentration of 100 pM. A 1S-mM stock solution of propidium iodide (PI), (Sigma, St. Louis, MO, USA) in PBS and a 2yM stock solution of fluorescein isothiocyanate (FITC) conjugated Pisum sativum agglutinin (PSA) (Sigma, St. Louis, MO, USA) in PBS were prepared and stored frozen. These solutions were added to lOOnL aliquots of thawed semen to make the following concentrations: SNARF=25 FM, PI=50 PM and FITC-PSA=O.l pM (or a 0.39 PM concentration of FITC). Semen samples were incubated at 38W in the dark for 30 min. After incubation, the stained sperm suspension was further diluted with 500 pL of TRIS buffer and analyzed by flow cytometry. Flow cytometric analysis was performed on a Facstar Plus flow cytometer (Becton Dickinson, San Jose, CA, USA) as described previously (24). Cytogram Analysis Dot plot cytograms were obtained for FL1 (fluorescence sensor that collects wavelengths between 500 and 560 nm) and FL3 (fluorescence sensor that collects wavelengths higher than 640 nm). The SNARF and PI positive cells were both detected in the FL3 sensor; the two populations were easily identified becausethe PI fluorescence intensity was higher than that of SNARF. The FITC fluorescence was detected in the FL1 sensor. Four populations with different fluorescences were quantified: 1) one population with a low intensity of red and green fluorescence (live cells with intact acrosomes,stained only by SNARF); 2) another population with a low intensity of red fluorescence and a high intensity of green fluorescence (live cells with reacted acrosomes,stained by SNARF and FITC-PSA); 3) a third population with a high intensity of red fluorescence and a low intensity of green fluorescence (dead cells stained only by PI); and 4) a population with high intensities of both red and green fluorescence (dead cells with damagedacrosomes,stained by PI and FITC-PSA).
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Flow cytometry data on the integrity of sperm plasma and acrosomal membranes are presented, respectively, as percentages of total live spermatozoa (Populations 1 and 2) and percentagesof acrosome-reactedlive spermatozoa(Population 2), becausethe acrosomal status of only the live spermatozoa was considered to be more relevant than that of the total sperm population. Statistical Analysis Data on post-thaw progressive motility, sperm plasma membrane integrity and acrosomal status were analyzed using the general lineal model procedure (GLM, Minitab Statistical software, State College, PA, USA) at each evaluation time. Effects included in the models were the concentration of spermatozoa (50 x 106, 100 x 106, 200 x 106 and 400 x 106 spermatozoa/ml) in 0.5-mL straws, the post-thaw dilution rate (l:O, l:l, 1:2 and 1:4) and the interaction between them. When significant effects were detected in the models, differences between treatments were analyzed with Tukey’s Studentized Range (HSD) Test using the SAS program (Cary, North Carolina, USA) and were considered statistically significant at PcO.05 level. RESULTS Effect of Spermatozoa1Concentration For data pooled across post-thaw dilution rates, the spermatozoa1concentration had a significant effect (P
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In samples with 200 or 400 x lo6 spermatozoa/ml, the decline in the proportion of total live spermatozoa during 6 h of incubation was not significant. In contrast, semen samples frozen with 50 x 106 or 100 x 106 spermatozoa/ml had a lower (PcO.05) percentage of total live spermatozoa after 3 h of incubation than immediately after thawing, but the decline between 3 and 6 h was not significant. Table 1. Progressive motility percentages(mea&SD) of dog semen samples frozen in OS-mL straws with different spermatozoal concentrations, for all dilution rates, during incubation post-thaw at 38eC. Spermatozoal concentration (spermatozoa/mL) Incubation time so x 106 loo x 106 200 x 106 400 x 106 Oh 54.5+9.7 b 49.7k7.7 b 65Sk5.6 a 51.2k4.2 b lh 54.5k8.2 b 50.7+8.1 b 64.m7.0 a 54.5+5.1 b 2h 50.5k8.6 b 48.7+8.1 b 60.7h7.3 a 50.5+3.9 b 3h 44.5*9.0 b 44.2rt14.6 b 60.5b8.6 a 47.0+4.4 b 4h 36.a8.4 C 38.5kl4.0 C 56.7h6.9 a 46.5+5.4 b 5h 21.5h9.7 d 32.Ohl3.3 C 41.2klO.7 b 50.5h6.9 a 6h 9.2h9.2 cl 23.7kl5.3 C 43.5hl4.0 a 34.7k17.8 b 7h 3.1Zt5.9c 14.&12.1 b 31.Oh18.0a 29.7*18.3 a 8h 1.1*2.6b 6.5~k8.4b 21.2kl6.3 a 20.0k17.9 a a b, CDifferent superscript letters in the samerow indicate significant differences (P
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concentrations evaluated. Between 3 and 6 h of incubation the proportions of acrosome-reacted live spermatozoa were higher (P
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Table 4. Progressive motility percentages(mean&SD) of frozen-thawed dog semen samples diluted immediately after thawing with TRIS buffer at different dilution rates, for all sperm concentrations, during incubation post-thaw at 38cC. Post-thaw dilution rate Incubation time 1: 0 1: 1 1:2 1: 4 54.S7.2 ab Oh 58.h7.3 a* 57.5*7.7a* 50.5&12.2b* lh 54.Ozt5.0a 56.5h7.1 a 58.7*7.6a 54.5k12.8 a 2h 51.755.4 a 53.7+7.0a 53.5k8.3 a 51.5*12.2a 3h 44.7hl2.3 a 51.758.0 a 52.5+9.1 a 47.2*15.3 a 4h 39.5*11.9a 46.7k10.7a 47.7k8.7 a 43.7*15.7 a 5h 26.5+13.4 b** 38.2k13.0 a** 42.2~Hl.l a** 38.2h17.5 a** 6h 9.7h10.6 b** 31.7*18.6a** 37.Oh15.3a** 32.7h19.2 a** 7h 0.7+1.8 b** 21.5~t15.2a** 28.5h16.9 a** 27.U19.0 a** 8h o.ozto.0b** 6.5h6.3 b** 21.7+16.2a** 20.6h17.8 a** % b Different superscript letters in the same row indicate significant differences (*= PcO.05; **=P
The percentageof live spermatozoawith reacted acrosomes(Table 6) was not influenced by the dilution rate during the first 6 h of incubation at 38cC. After 12 h of incubation a tendency (P=O.O71)was seen for undiluted samples to have lower proportions of acrosome reactions. After 18 h, a significant (P
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Table 6. Percentagesof acrosome-reactedlive spermatozoa(mean+SD) in frozen-thawed dog semen samples diluted immediately after thawing with TRIS buffer at different dilution rates, for all sperm concentrations, during incubation post-thaw at 38OC. Post-thaw dilution rate Incubation time 1: 0 1:4 1: 1 1: 2 Oh o.oko.0 a o.oko.0 a 0.0~0.0 a o.o+o.o a 3h 1.2+1.9 a 0.9k2.3 a 1.9h3.4 a 1.0*2.2 a 6h .5.0+3.2a 3.1h3.2 a 3.714.2 a 3.313.4 a 12h 6.9k3.5 a 11.7*8.4 a 11.115.9a 11.613.6 a 18h 3.9+2.4 a 8.8+6.2 b 18.919.0 d 14.5k7.4 c a~bc~dDifferent superscript letters in the samerow indicate significant differences (P
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contrast, the highest proportion of live spermatozoa with reacted acrosomes after 6 h of incubation was observed with the concentration of 400 x 106 spermatozoa/ml and post-thaw dilution rates of 1:2 (14.0%*7.8%) and 1:4 (13.0%*2.9%). However, after 18 h of incubation, the lowest percentage of acrosome reactions (PcO.05) was observed with the concentration of 400 x 106 spermatozoa/ml and a post-thaw dilution rate of 1:4, for which only 26.6%9.6% of the live spermatozoa had reacted acrosomes.In contrast, the highest percentage (PcO.05) was observed with the concentration of 200 x 106 spermatozoa/ml and a post-thaw dilution rate of 1:2, for which 60.2Y&9.4% of the live spermatozoahad reacted acrosomes. DISCUSSION There is a lack of information concerning the effects of sperm concentration for freezing of dog semen. Studies in other species, especially in the equine, suggest that this factor may have an influence on the post-thaw sperm survival. Palacios et al. (21) reported better motility post-thaw when equine spermatozoawere frozen at 800 x 106 spermatozoa/mL than at 100 x 106 spermatozoa/ml, and Parlevliet et al. (22) observed an interaction between sperm concentration and freezing method. For high sperm concentrations (1000 x 106 spermatozoa/ml), post-thaw motility was better when a fast cooling rate was used during the freezing, whereas for low sperm concentrations (200 x 106 spermatozoa/ml) the post-thaw motility was better when a slow cooling rate was used. Leipold et al. (16) found that post-thaw motility was similar for equine spermatozoafrozen at 1600 x 106 spermatozoa/ml and at 400 x 1Oe spermatozoa/ml, but pregnancy rates tended to be higher for mares inseminated with spermatozoa frozen at r600 x 106 spermatozoa/ml than at 400 x 106 spermatozoa/ml. Furthermore, when a low and a high number of spermatozoaper insemination dose (320 x 106 vs. 800 x 106 progressively motile spermatozoa) were compared, there tended to be a higher pregnancy rate using the lower spermatozoa1number if spermatozoa were packaged at the higher concentration. Heitland et al. (14), however, reported better post-thaw motility when stallion spermatozoawere frozen in OS-mL straws at sperm concentrations of 20,200 or 400 x 106 spermatozoa/ml than at 800 x 106 spermatozoa/ml or higher. In addition, Colas (3) obtained better fertility results when ram semen was diluted to a constant concentration of 900 x 106 spermatozoa/ml (34% of 91 ewes lambed) than to a constant rate (1:4, semen volume: extender volume) where the sperm concentration would be variable (25% of 101 ewes lambed). This author suggested that the effect was related to the final glycerol concentration in the spermatozoa. The results of the present study show that dog spermatozoa, under the cryopreservation conditions described here, had a higher thermoresistance when they were frozen at concentrations of 200 x 106 and 400 x 106 spermatozoa/ml than at lower sperm concentrations. This might suggest that latent damage of sperm membranes after thawing is less severewhen freezing is done at higher sperm concentrations. The cause of such an effect is unknown, but it is not likely to have been due to a lower glycerol concentration in the sperm cells, becausedog spermatozoaseem to be rather tolerant to a range of glycerol levels (8, 12, 20, 23, 27). The effect, instead, might be due to an influence of the sperm concentration on factors determining the cryoinjury of spermatozoa, such as the “solution effect” and the formation of ice crystals. However, Watson and Duncan (35) observed that cell concentrations
Theriogenology in the range of 3 to 12 x10* spermatozoa/ml did not influence the effects of either the salt concentration or the unfrozen water fraction during the crystallization phase of the freezing on the survival of ram spermatozoa. In the present study, the proportions of acrosomereactions during the first period of postthaw incubation seemedto increase with increasing numbers of spermatozoaper straw. There is not an obvious explanation for such an effect, which has also been observed in equine spermatozoa(21), but acrosomal enzymes and toxic products released from dead spermatozoa might have contributed to the destabilization of acrosomal membranesin live spermatozoa,and to larger extent at higher sperm concentrations. At the end of the incubation period, this effect was no longer apparent, but it might have been obscured by the effects of the prolonged incubation as such. The highest post-thaw dilution rate evaluated in the present study exerted a negative effect on the progressive motility of spermatozoa immediately after the dilution; however, it resulted in a higher sperm longevity. There is some evidence that the thawing injury of sperm cells is due to the fact that increasingly hyperosmotic conditions during freezing are reversed during thawing, and cells are progressively subjected to hypoosmotic conditions when compared with the highest hyperosmotic environment the cells were exposed to during the freezing as the crystallization of the extracellular water progressed (6). This osmotic stress should be expected to be more pronounced when thawed spermatozoaare further diluted in a glycerol-free medium, becauseit induces an abrupt removal of glycerol from the spermatozoa to reach the equilibrium on both sides of the plasma membrane and, therefore, cells have to accommodate new volume changes in addition to those suffered during the thawing. Furthermore, at higher post-thaw dilution rates with a glycerol-free medium, the osmotic stress imposed on the cells is supposedto be higher due to a bigger difference between osmolarities. On the other hand, a quick dilution after thawing in a single step was found to be detrimental for bull and mouse spermatozoa, whereas a slow dilution was not (4, 19). Fiser and Fairfull (lo), however, reported that the rate of glycerol removal after thawing did not affect the percentage of motile spermatozoa and intact acrosomes in ram spermatozoa. In the present study, the dilution post-thaw was done immediately after thawing in a single step, and samples diluted at a rate of 1:4 showed the lowest progressive motility immediately post-thaw and the highest plasma membrane integrity, presumably due to a more severe osmotic stress, which impaired the straight movement of spermatozoa while maintaining intact sperm membranes. However, after 8 and 12 h of incubation, respectively, sperm motility and viability were higher at increasing dilution rates. These results indicate that, after the osmotic stress imposed on spermatozoa by a rapid dilution post-thaw, spermatozoa were able to adjust to the new extracellular osmotic conditions, maintaining motility and the integrity of their membranes. The improved longevity at increasing dilution rates is thought to have been caused not only by the additional source of energy and buffering capacity, but also by reducing the glycerol concentration in spermatozoaand its toxic effects at high temperature.
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In this study, the integrity of sperm plasma membranes,in general for all treatments and particularly for those with the higher dilution rates, was maintained in a high percentage of sperm cells for a longer incubation period in comparison with that observed in previous studies. It indicates that the cryopreservation protocol used here provided adequate stabilizing conditions for the sperm membranes at high temperature. The freezing and thawing methods used in this study were found to be the least detrimental for sperm survival among several other treatments evaluated in a previous study (25). Motility, on the other hand, was affected considerably more by the incubation than membrane integrity over the same periods. It is not known whether this population of non-motile but apparently viable spermatozoa could regain motility within the female reproductive tract; however, the addition of caffeine had no such effect (data not shown). The decline in sperm motility after incubation periods when there were still high numbers of viable spermatozoadoes not seemto have been causedby the depletion of available substrate for the spermatozoa. If that were the case, the decline in sperm motility should be expected to occur earlier in semen sampleswith the higher sperm concentrations due to the faster depletion of substrate by the presenceof more sperm, but the opposite was found (Table 1). Furthermore, in the semen samples diluted at a rate of 1:4, which had the best longevity, the same phenomenon was observed. One explanation might be that the premature loss of motility in live cells is due to latent damagecausedby cryopreservation. The post-thaw dilution rate had no significant effect on the proportion of acrosome reactions detected during most of the incubation period, although, at the end, increasing proportions of reacted acrosomes were seen at increasing dilution rates. However, the ratio between the percentage of acrosome-reactedlive spermatozoaand the percentage of live cells was rather similar for the different dilution rates. Thus, it does not appear that post-thaw dilution of spermatozoa,at the rates evaluated in this study, increasesthe acrosomal damageof spermatozoa. In conclusion, the results of the present study suggest that dog spermatozoa frozen in a TRIS-egg yolk-5% glycerol-0.5% Equex extender at concentrations of 200 x 106 spermatozoa/ml and diluted immediately post-thaw with a plain TRIS buffer at rates of 1:4 or 1:2, survive better than when frozen with lower or higher sperm concentrations and/or not being diluted after thawing. Further studies are needed to investigate whether motility may be induced again in the populations of non-motile spermatozoa with intact plasma membranes, and whether it could be reflected in an increase of fertility rates after AI with frozen semen in this species. REFERENCES 1. Bane A. Acrosomal abnormality associatedwith sterility in boar. Proc 4th Int Congr Anim Reprod (ICAR) 1961;810-817. 2. Battista M, Parks J, Concannon P. Canine sperm post-thaw survival following freezing in straws orpellets using PIPES, Lactose, Tris or Test extenders. Proc 1lfh Int Congr Anim Reprod (ICAR) 1988;3:229-231.
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