The effect of Equex STM paste and sperm morphology on post-thaw survival of cat epididymal spermatozoa

Cryopreservation of epididymal spermatozoa is a potentially valuable tool for preserving genetic material from individuals of endangered species that die accidentally. Improvement of sperm-freezing protocols would increase the efficacy of gene banking from endangered felids, and the domestic cat can be used as a model for the wild felids. Addition of the detergent Equex STM paste to semen freezing extenders has been found to improve post-thaw survival and longevity of spermatozoa from various species but has never been tested for cat spermatozoa. Spermatozoa from cats with a high percentage of morphologically abnormal spermatozoa are more susceptible for cold injury and osmotic stress than spermatozoa from normozoospermic cats. Therefore, the aims of this study were to investigate: (a) if addition of Equex STM paste to a semen freezing extender would improve post-thaw sperm survival, and (b) if there is a relation between the percentage of morphologically normal spermatozoa and cryopreservation induced damage in cat epididymal spermatozoa. Spermatozoa harvested from epididymides of 10 male cats were frozen in a Tris egg yolk extender with or without the addition of Equex STM paste (0.5%, v/v). Sperm motility, membrane integrity and acrosomal status were evaluated immediately after harvesting, and at 0, 2, 4 and 6 h post-thaw. Sperm membrane integrity and acrosomal status were also evaluated after cooling to 4 degrees C, just before freezing. Cooling did not cause significant damage to the spermatozoa, whereas freezing damaged sperm membranes and acrosomes. Addition of Equex to the freezing extender had a significant positive effect on the percentage of intact acrosomes immediately after thawing (P > 0.05), but had a negative effect on the longevity of the spermatozoa; the percentages of membrane intact and motile spermatozoa being significantly lower in the presence of Equex than in the controls at 6h after thawing. The percentage of morphologically normal spermatozoa was not found to be correlated with either cryopreservation induced acrosome or plasma membrane damage, or with post-thaw motility (P > 0.05). The results clearly show that addition of Equex STM paste in the freezing extender protects the acrosomes of cat epididymal spermatozoa during the freezing--thawing process, but reduces the sperm longevity during in vitro incubation at 38 degrees C. Our results also indicate that the percentage of morphologically normal epididymal spermatozoa is not correlated with cryopreservation induced sperm damage using the described freezing protocol.     

Cryopreservation of dog semen: the effects of Equex STM paste on plasma membrane fluidity and the control of intracellular free calcium

Understanding cryoinjury of dog spermatozoa is crucial to preserving fertilizing ability. This study examined flow cytometric indicators of sperm function to explore the reported benefits of Equex STM paste. The motility of cryopreserved spermatozoa immediately and 1h after thawing was higher in the extender containing 0.5% Equex; no significant differences between the two extenders were observed regarding viability, acrosomal integrity and intracellular Ca(2+) concentration. The proportion of spermatozoa having high membrane fluidity increased significantly post-thawing. The interaction between time after thawing and treatment was significant for plasma membrane fluidity. Dilution in a commercial diluent for transport before processing caused a significant increase in intracellular Ca(2+), which may affect functional survival. No significant difference with or without Equex was detected in plasma membrane fluidity. However, a significant interaction between Equex and dogs was detected. A significant decrease in intracellular Ca(2+) was detected in the live cell population both after dilution in Andersen's buffer and again after cooling and equilibration. One hour post-thaw, the proportion of live spermatozoa with high calcium concentration increased to a similar proportion as that seen in diluted semen; the interaction between diluent and dog was significant. The results suggest that Equex in the diluent benefited motility after cryopreservation. Live spermatozoa with high intracellular Ca(2+) after cryopreservation seem to have a favoured survival in the first hour after thawing. Nevertheless, survival after cryopreservation was severely compromised, explaining the relatively poor fertility of cryopreserved dog semen.     

Effects of Equex STM Paste on the quality of frozen-thawed epididymal dog spermatozoa

This study was carried out to investigate the cryoprotective efficacy of Equex STM Paste on the quality of canine post-thaw epididymal spermatozoa. Following castration, spermatozoa were flushed from the cauda epididymides. Epididymal spermatozoa from 13 of 16 dogs with a sperm motility of >70% were frozen in an egg yolk-Tris extender, supplemented with Equex STM Paste (0.5%, v/v); the extender free of Equex STM Paste served as a control cryoprotective diluent. The quality of spermatozoa, judged by its motility, plasma membrane integrity and acrosome integrity, was evaluated on four occasions, immediately after collection, after equilibration and at 0 and 2h post-thaw. Reducing the temperature to 4 degrees C for 2h prior to freezing decreased sperm motility (P=0.001), but had no effects on membrane integrity or acrosome integrity. Immediately after thawing, the percentage of acrosome-intact spermatozoa significantly decreased in samples frozen without Equex STM Paste compared to freshly collected or Equex-treated samples. After incubation at 37 degrees C for 2h post-thaw, a greater percentage of motile spermatozoa (P=0.018) and spermatozoa with intact acrosomes (P=0.001) were observed in Equex-treated samples compared with the control. The percentage of membrane-intact spermatozoa did not differ significantly between Equex-treated and control samples at any time. Supplementation with Equex STM Paste in the semen extender was effective for freezing canine epididymal spermatozoa because it protected acrosome integrity against damage induced by cryopreservation and it prolonged post-thaw sperm motility during in vitro incubation at 37 degrees C.     

Quality and fertilizing ability of electroejaculated cat spermatozoa frozen with or without Equex STM Paste

An optimal protocol for cat semen cryopreservation has not yet been defined. Addition of Equex STM Paste has been tested for epididymal cat spermatozoa but not for ejaculated cat spermatozoa. Furthermore, the effect of Equex STM Paste on fertilizing ability of cryopreserved semen has never been evaluated in that species. Therefore, the aims of the current study were to investigate if addition of Equex STM Paste to a freezing extender for electroejaculated cat (Felis catus) semen would improve postthaw sperm quality and if sperm fertilizing ability after cryopreservation with or without Equex STM Paste was preserved. Semen was collected by electroejaculation and frozen in a Tris-glucose-citrate egg yolk extender supplemented with (0.5% vol/vol) or without Equex STM Paste. In Experiment 1, sperm motility, membrane integrity, and acrosomal status were determined immediately after collection and at 0, 3, and 6 h postthaw. In Experiment 2, frozen semen from the two groups was used for in vitro fertilization (IVF) of in vitro-matured cat oocytes. Cleavage rate was recorded 30 h after IVF, and embryo development was evaluated on Days 6 and 7 of culture. In Experiment 1, the rate of motile spermatozoa after freezing-thawing was higher when Equex STM Paste was added to the freezing extender, but progressive motility score was not influenced (P > 0.05). Sperm membrane integrity was positively affected (P < 0.05) by the addition of the detergent. Intact acrosomes after thawing were similar (P > 0.05) between groups. Even if the decreasing rates of motility and membrane integrity were more rapid in presence of Equex than those in controls, total motility and sperm viability were similar at 3 and 6 h after thawing (P > 0.05). In Experiment 2, there was no difference in fertilizing ability and embryo development between the two groups (P > 0.05). The results of this study demonstrate that the addition of Equex STM Paste in the freezing extender avoids the loss of motile spermatozoa and maintains fertilizing ability of frozen-thawed spermatozoa.     

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.     

Fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without Equex STM paste

Twenty-five bitches were artificially inseminated with semen that was frozen-thawed using an egg yolk-Tris-glucose-citrate extender containing 5% glycerol with, or without the addition of 0.5% Equex STM Paste. Semen was collected on 2 occasions from 11 dogs, pooled, and evaluated for sperm motility, morphology and plasma membrane integrity. Each pool was then divided in 2 parts, diluted with 1 of the 2 extenders, and frozen in 0.5-mL straws. In the bitches, plasma progesterone was assayed daily during late proestrus and estrus. Artificial insemination (AI) was performed twice on Days 3 and 5 after the estimated LH peak. For each insemination, 200x10(6) spermatozoa were used. Ten bitches were inseminated with semen frozen without Equex: In 5 females, semen was deposited transcervically into the uterus with the aid of a fiberoptic endoscope and a urethral catheter, while the remaining 5 bitches were inseminated in the cranial vagina using a Norwegian catheter. Fifteen bitches were inseminated with semen frozen-thawed with Equex: Two groups of 5 bitches were inseminated according to the techniques described above, while 5 bitches were inseminated vaginally using the Osiris catheter. Pregnancy was diagnosed and the number of fetuses counted by ultrasound examination. Post-thaw, spermatozoa frozen with Equex tended to have higher total and progressive motility and to survive longer in vitro than when the extender without Equex was used. Spermatozoal concentration, age of the bitches, duration of heat and estrus, and progesterone concentration at LH peak and at the first and second AI did not differ among the 5 groups. The overall pregnancy rate of 84% (21/25) was close to what can be expected from well controlled natural matings. For both freezing extenders tested, 5/5 bitches were pregnant after uterine deposition of semen and 4/5 were pregnant when semen was deposited in the anterior vagina using the Norwegian catheter. With the Osiris catheter, 3/5 inseminations resulted in a pregnancy. No significant differences in pregnancy rate or number of fetuses were found between groups, site of deposition or freezing extender.     

Effect of post-thaw dilution with autologous prostatic fluid on dog semen motility and sperm acrosome status

The aim of this study was to evaluate the effect of post-thaw dilution with autologous prostatic fluid on motility parameters, longevity and acrosome status of frozen-thawed dog spermatozoa. After semen collection, seminal plasma was separated by centrifugation and stored frozen until use. Sperm pellets were diluted in two steps with an egg yolk-Tris extender to a final concentration of 5% glycerol and 0.5% Equex STM Paste. After thawing, semen was diluted 1:2 either with Tris buffer or with the autologous prostatic fluid. Motility was evaluated using a phase contrast microscope and a computer-assisted motility analyser system immediately after thawing and at hourly intervals up for 4h at 38 degrees C. The status of acrosomes was assessed with Spermac stain at thawing and after 2 h of incubation. Motility and straight line velocity were initially higher in prostatic fluid-diluted samples (0 h and 0 and 1h, respectively), but decreased to values similar to those of Tris-diluted samples in a time-dependent manner. In contrast, both the curvilinear velocity and amplitude of lateral head displacement were lower in prostatic fluid-diluted samples (1 and 3 h and 0, 1 and 3 h, respectively). The dilution did not have any significant effect on the percentage of acrosome-intact spermatozoa at either thawing or after 2 h. The pattern of motility of prostatic fluid-diluted samples suggests a reduction in hyperactivated motility with time, even though prostatic fluid neither prolonged spermatozoa longevity nor had any effect on the status of spermatozoa acrosomes.     

Effects of Equex, one- or two-step dilution, and two freezing and thawing rates on post-thaw survival of dog spermatozoa

The objectives of the present study were to evaluate the effects of adding Equex to a TRIS-extender, diluting the semen in 1 or 2 steps, freezing according to 2 methods, thawing at 2 rates, and the interactions between these treatments, on the post-thaw survival of dog spermatozoa at 38 degrees C. Ten ejaculates were obtained from 8 dogs. Each ejaculate was centrifuged, and the seminal plasma was discarded. Each sperm pellet was diluted with 2 mL of a TRIS-glucose-egg yolk extender containing 3% glycerol (Extender 1 [Ext-1]). Ejaculates were then pooled (9 x 10(9) spermatozoa), and Ext-1 was added to obtain 200 x 10(6) spermatozoa/mL. The semen pool was carefully mixed and divided into aliquots, and processed according to a 2 x 2 x 2 x 2 factorial design to evaluate the effects of 1) adding the same volume of a second TRIS-glucose-egg yolk extender with 7% glycerol that contained (Ext-2-E) or didn't contain (Ext-2) 1% of Equex STM Paste (final concentration of spermatozoa 100 x 10(6) spermatozoa/mL, glycerol 5%, Equex 0% [Ext-2] or 0.5% [Ext-2-E]); 2) diluting the semen in 1 step (adding Ext-2 or Ext-2-E before equilibration) or in 2 steps (adding Ext-2 or Ext-2-E after equilibration, just before the freezing operation); 3) freezing the straws horizontally in a styrofoam box 4 cm above liquid nitrogen (LN2) or by lowering them vertically into a LN2 tank in 3 steps; and 4) thawing at 70 degrees C for 8 sec or at 37 degrees C for 15 sec. A total of 16 treatment combinations were evaluated. Sperm motility was evaluated after thawing and at 1-h intervals during 7 h of incubation at 38 degrees C by subjective examination and by using a CASA-system. Plasma membrane integrity and acrosomal status were evaluated simultaneously at 1, 3 and 6 h post-thaw using a triple fluorescent staining procedure and flow cytometry. The best post-thaw survival and thermoresistance of spermatozoa was obtained when Equex was present in the extender (P<0.0001); the semen dilution was performed in 2 steps instead of 1 (P<0.0001); the freezing was carried out using the box instead of the tank (P<0.05); and the straws were thawed at 70 degrees C for 8 sec instead of at 37 degrees C for 15 sec (P<0.0001).     

Effects of Equex from different sources on post-thaw survival,longevity and intracellular Ca2+ concentration of dog spermatozoa

The aims of the present study were to compare the effects of two commercial preparations (Equex STM Paste or Equex Pasta), whose active ingredient is sodium dodecyl sulphate (SDS), added to a Tris-egg yolk-based extender, on post-thaw sperm survival and longevity, as well as on the intracellular Ca(2+) concentration of dog spermatozoa during incubation at 38 degrees C. One ejaculate was collected from each of eight dogs. Each ejaculate was centrifuged, the semen plasma discarded, and the sperm pellet rediluted with a Tris-glucose-egg yolk extender containing 3% glycerol (Ext-1) at a sperm concentration of 200 x 10(6) spermatozoa (spz)/ml. The diluted semen was divided in three aliquots of equal volume and allowed to equilibrate for 1h at 4 degrees C. After equilibration, the same volume of three different second extenders was added, respectively, to each of the three aliquots: (A) Ext-2A (same composition as Ext-1 except that it contained 7% glycerol and 1% Equex STM Paste), (B) Ext-2B (same composition as that of Ext-1 except that it contained 7% glycerol and 1% Equex Pasta), and (C) Ext-2 (Control: same composition as that of Ext-1 except that it contained 7% glycerol). Semen samples were packed in 0.5 ml straws and frozen on a rack 4 cm above liquid nitrogen (LN(2)) in a styrofoam box. Thawing was at 70 degrees C for 8s. Sperm motility was evaluated after thawing and at 1 h intervals for 5h at 38 degrees C by subjective examination and by using a CASA system. Plasma membrane integrity and acrosomal status were evaluated at 1, 4 and 7h post-thaw using a triple staining procedure and flow cytometry. Intracellular Ca(2+) concentration of live spermatozoa was evaluated by flow cytometry at 1, 4 and 7h post-thaw after co-loading the sperm cells with the Ca(2+) indicators Fluo 3 AM and Fura Red AM, and with PI. Post-thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better (P<0.005) when Ext-2A (containing Equex STM Paste) was used. There was no difference between Ext-2B (containing Equex Pasta) and Ext-2 (Control). The mean intracellular Ca(2+) concentration (arbitrary units) of cryopreserved spermatozoa (range: 0.23+/-0.12 to 1.26+/-0.46) was higher than that of fresh spermatozoa (0.13+/-0.06). When using Ext-2A, the live spermatozoa frequently (P=0.012) appeared divided in two subpopulations, with high (1.26+/-0.46) and low (0.27+/-0.09) intracellular Ca(2+) content, respectively. When using Ext-2B or Ext-2, the live spermatozoa were more frequently seen in a single population with low intracellular Ca(2+) concentration (0.30+/-0.35 and 0.23+/-0.12, for Ext-2B and Ext-2, respectively).     

Evaluation of chilled and frozen-thawed canine spermatozoa using a zona pellucida binding assay

Zona pellucida binding assays provide information about the fertilizing ability of spermatozoa. A zona-binding assay for canine spermatozoa using intact, denuded homologous oocytes has not been evaluated previously. In the present study, an assay using canine oocytes derived from frozen-thawed ovaries was evaluated using three types of semen: fresh untreated; killed; and a 50:50 mixture of untreated and killed spermatozoa. The assays were performed on 3 x 20 oocytes for each sperm treatment, using semen from pooled ejaculates (0.5 x 10(6) spermatozoa in each 50 microliter droplet containing five oocytes). There was a significant difference (P < 0. 001) between all treatments. Thereafter, the same procedure was used to evaluate methods of chilling and freeze-thawing of canine semen. There was a trend (P = 0.067) for more sperm binding after 1 day of chilling compared with after 4 days of chilling. Semen samples frozen using an extender (with or without the addition of Equex STM paste) were evaluated. Equex had a significant (P = 0.034) positive effect on the capacity of the spermatozoa to bind to the zona pellucida. In conclusion, the addition of a zona pellucida binding assay to established in vitro tests should give a better estimate of the damage caused by the various procedures when developing new techniques for chilling and freeze-thawing. Furthermore, the present study showed that chilling for 4 days tended to reduce the zona-binding capacity of the spermatozoon, and that Equex STM paste had a beneficial effect on the capacity of the frozen-thawed spermatozoon to bind to the zona pellucida.     

The effect of homologous prostatic fluid on motility and morphology of dog epididymal spermatozoa extended and frozen in Biladyl with Equex STM paste or Andromed

Although dog prostatic fluid decreases the longevity of ejaculated dog spermatozoa, it also increases their rate of motility and their fertility after vaginal insemination, as well as the fertility of epididymal spermatozoa after uterine insemination. These findings indicate a need to further characterize the effects of prostatic fluid on dog spermatozoa. This study was done to determine the effects (P<0.05) of homologous prostatic fluid added prior to cooling, after thawing, or at both times to epididymal spermatozoa from 21 dogs. The effects of two extenders were also determined. The one extender was Biladyl(*) with Equex STM paste(**) (BilEq) and the other Andromed(*) (Minitüb, Tiefenbach, Germany (*); Nova Chemical Sales, Scituate, MA, USA (**)). The response variables were percentage progressively motile spermatozoa (Prog) and morphology after thawing. Prog was measured at various times until 8h after extension (unfrozen spermatozoa) or until 2h after thawing. Prog after thawing was higher with BilEq than Andromed, when no prostatic fluid was added prior to cooling, and when prostatic fluid was added after thawing. BilEq resulted in a higher mean percentage of spermatozoa with bent principle pieces than Andromed and the addition of prostatic fluid prior to cooling resulted in lower mean percentages of cytoplasmic droplets and bent principle pieces than when none was added. The optimal combination was BilEq with prostatic fluid added prior to cooling (in order to inhibit the development of bent principle pieces) and after thawing (to achieve higher motility until 1h after thawing). This study shows that BilEq is more suitable for the freezing of epididymal spermatozoa than Andromed and that prostatic fluid improves the freezability and post-thaw longevity of epididymal spermatozoa frozen in BilEq.     

Evaluation of dog semen quality after slow (biological freezer) or rapid (nitrogen vapours) freezing

Three ejaculates were collected from each of five dogs. After initial evaluation, the sperm-rich fractions were diluted to 100 x 10(6) spermatozoa x mL(-1) in two steps with an egg yolk-TRIS extender containing a final concentration of 5% glycerol and 0.5% Equex STM paste. Half of the 0.5 mL straws obtained from each ejaculate were frozen on nitrogen vapours (4 cm above the liquid surface) ("rapid freezing"), while the other half was frozen in a biological freezer at a rate of 0.5 degrees C x min(-1) between 5 degrees C and -10 degrees C and of 8 degrees C x min(-1) between -10 degrees C and -60 degrees C, followed by immersion in liquid nitrogen ("slow freezing"). After an average storage of 30 days, the straws were thawed in a water-bath at 37 degrees C for 1 min. Progressive motility was subjectively estimated hourly for 8 h on semen incubated at 38 degrees C. Immediately after thawing and after 2 h of incubation, motility parameters were also measured by a motility analyser. Sperm membrane function and chromatin stability were assessed immediately post-thaw, using the hypo-osmotic swelling test and acridine orange staining, respectively. Slow freezing significantly improved total post-thaw motility, which showed a slower decline over time, although spermatozoal average path and straight line velocity were lower compared to the fast rate. Also the number of intact membrane spermatozoa was significantly higher in slow-frozen samples while the proportion of spermatozoa with single-stranded DNA was minimal after both freezing procedures.     

Effects of spermatozoal concentration and post-thaw dilution rate on survival after thawing of dog spermatozoa

The objectives of this study were to evaluate the effects and interactions of freezing dog semen using 4 different sperm concentrations (50 x 10(6), 100 x 10(6), 200 x 10(6) and 400 x 10(6) spermatozoa/mL) in 0.5-mL straws and diluting the thawed semen at 4 different rates (1:0, 1:1, 1:2 and 1:4) on post-thaw survival and longevity of dog spermatozoa during incubation at 38 degrees C. Fifteen ejaculates were collected from 12 dogs and pooled. The semen pool was divided into 4 aliquots containing respectively 4,200 x 10(6), 2,100 x 10(6), 1,050 x 10(6) and 525 x 10(6) 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 degrees C for 8 sec and the contents of each straw were divided into 4 aliquots and diluted with TRIS buffer at 38 degrees C at rates of 1:0, 1:1, 1:2 and 1:4 (semen:buffer), respectively, making a total of 16 treatments. Sperm motility was subjectively evaluated after thawing and at 1-h intervals during 8 h of incubation at 38 degrees C. 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< 0.001) in samples frozen with 200 x 10(6) spermatozoa/mu. The integrity of sperm plasma membranes after 18 h incubation was higher (P<0.05) in samples frozen with 200 x 10(6) and 400 x 10(6) spermatozoa/mL. For data pooled across sperm concentration, samples diluted at a rate of 1:2 or 1:4 had better (P<0.001) motilities after 8 h of incubation than undiluted samples or those diluted at 1:1. The integrity of the sperm plasma membranes was higher (P<0.001) at increasing dilution rates. When the 16 treatments were compared, the best longevity was obtained when semen packaged at a concentration of 200 x 10(6) spermatozoa/mL was diluted immediately after thawing at 1:4 dilution rate.     

Thawing dog spermatozoa in just-boiled water: submersion time and effect on sperm quality compared to thawing in water at 70 degrees C

Dog spermatozoa have better quality after thawing in water at 70-75 degrees C instead of 35-38 degrees C. The aim of Experiment 1 was to determine the time needed to thaw 0.5 mL straws in just-boiled (98 degrees C) water and that of Experiment 2 to determine whether thawing frozen dog spermatozoa in just-boiled water will result in better quality than thawing in water at 70 degrees C. Prior to freezing the straws of Experiment 1, a Type J thermocouple with wire diameters of 0.08 mm (Osiris Technical Systems, Centurion, South Africa) was placed in the center of each of ninety-three 0.5 mL straws (IMV Technologies, L'Aigle, France) filled with extender (Biladyl* with 0.5%, v/v of Equex STM paste**) and 54 filled with extender plus 200 x 10(6)spermatozoa/mL (Minitüb, Germany (*) and Nova Chemical Sales, MA (**)). Thirty straws with extender were thawed in water at 70 degrees C and the others in just-boiled water. Temperatures inside straws were recorded 10 times/s during warming. Two ejaculates were then collected from each of eight dogs and one from each of three others. Extended ejaculates from the same dog were pooled, frozen 8 cm above liquid nitrogen, and 2 straws from each of the 11 batches thawed in water at 70 degrees C for 8s and 2 in just-boiled water for 6.5s. Sperm morphology and viability were assessed on eosin-nigrosin smears made after thawing and the percentage progressively motile spermatozoa was estimated immediately, 1, 2 and 3h after thawing. The optimal submersion time in just-boiled water was 6.5s for both sperm concentrations, resulting in average temperatures of 23.6+/-1.5 degrees C (+/-S.E.M.) and 24.9+/-1.6 degrees C inside straws with extender or extender plus spermatozoa (P=0.6). The temperature inside straws thawed in water at 70 degrees C was 13.6+/-1.7 degrees C after 8s. Apart from a 1.5% higher (P<0.05) mean percentage motile sperm 2h after thawing, thawing dog spermatozoa in just-boiled (98 degrees C) water holds no benefit over thawing in water at 70 degrees C, which is easier to do.     

A two-step dilution tris-egg yolk extender containing Equex STM significantly improves sperm cryopreservation in the African wild dog (Lycaon pictus)

Conservation management of endangered African wild dogs (AWD; Lycaon pictus) can benefit greatly from development of sperm freezing and artificial insemination. Previous freezing attempts yielded nearly 0% motile sperm within 2 h of thawing. In this study, two canine freezing protocols were tested: Protocol 1: a one-step dilution in TRIS-20% egg yolk containing 8% glycerol; and Protocol 2: a two-step dilution in TRIS-20% egg yolk containing a final extender concentration of 5% glycerol and 0.5% Equex STM, coupled with a TRIS-citrate-fructose thawing solution. Semen was collected by electroejaculation from n = 24 AWDs, of which eight ejaculates of sufficient quality (four good quality with initial sperm motility of 75.0 ± 4.4% and four poor quality; showing rapid decrease in sperm motility to 3.3 ± 3.3% prior to freezing) were frozen. For good quality samples, motility and sperm motility index persisted for up to 8 h for Protocol 2, and was higher between 2 and 6 h after thawing with a decrease from 4 h of incubation. Motility dropped to nearly 0% after 2 h incubation for Protocol 1. Viability was higher for Protocol 2 throughout the 8 h of incubation, with a decrease after 6 h, compared to 4 h for Protocol 1. Acrosome integrity was higher for Protocol 2 throughout post-thaw incubation, with a decrease after 2 h for both protocols. Protocols did not differ in normal sperm morphology or DNA integrity. Poor quality samples yielded similar results, except for acrosome integrity, which declined for Protocol 2. In conclusion, a two-step dilution in TRIS-egg yolk-glycerol extender containing Equex STM yields significantly improved post-thaw quality and longevity of AWD spermatozoa, making it suitable for sperm banking and artificial insemination initiatives.     

Effect of glycerol concentration, Equex STM® supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen-thawed epididymal alpaca (Vicugna pacos) sperm

Two experiments were conducted to determine the effects of glycerol concentration and Equex STM® paste on the post-thaw motility and acrosome integrity of epididymal alpaca sperm. In Experiment 1, epididymal sperm were harvested from male alpacas, diluted, and cooled to 4 °C in a Lactose cooling extender, and pellet-frozen in a Lactose cryodiluent containing final glycerol concentrations of 2, 3, or 4%. In Experiment 2, epididymal sperm were diluted in Biladyl®, cooled to 4 °C, stored at that temperature for 18-24 h, and further diluted with Biladyl® without or with Equex STM® paste (final concentration 1% v:v) before pellet freezing. In Experiment 1, sperm motility was not affected by glycerol concentration immediately (2%: 16.1 ± 4.6%; 3%: 20.5 ± 5.9% and 4%: 18.5 ± 6.6%; P > 0.05) or 3h post thaw (< 5% for all groups; P > 0.05). Post-thaw acrosome integrity was similar for sperm frozen in 2% (83.6 ± 1.6%), 3% (81.3 ± 2.0%) and 4% glycerol (84.8 ± 2.0%; P > 0.05) but was higher 3h post-thaw for sperm frozen in 3% (75.7 ± 3.8%) and 4% (77.2 ± 4.1%) than 2% glycerol (66.9 ± 2.7%; P < 0.05). In Experiment 2, sperm motility was higher immediately after thawing for sperm frozen in the presence of Equex STM® (Equex®: 21.5 ± 3.5%; control: 14.4 ± 2.1%; P < 0.05) but was similar at 3h post-thaw (P > 0.05). Acrosome integrity was similar for sperm frozen with or without Equex STM® paste immediately (control: 89.6 ± 1.2%; Equex®: 91.1 ± 1.4%; P > 0.05) and 3 h post-thaw (control: 69.3 ± 3.7%; Equex®: 59.9 ± 5.8%; P > 0.05). Sperm cryopreserved in medium containing 3-4% glycerol and 1% Equex STM® retained the best motility and acrosome integrity, even after liquid storage for 18-24 h at 4 °C prior to cryopreservation.     

In vitro characteristics of canine spermatozoa subjected to two methods of cryopreservation

The in vitro viability of canine spermatozoa was evaluated after freezing-thawing using the Andersen method, and the commercial CLONE method. These methods differ in the extenders used, number of dilution steps, and equilibration times as well as in both freezing and thawing techniques and rates. Insemination with semen frozen-thawed by either method gives high whelping rates in practice, implying that dog spermatozoa can retain their fertilizing ability after being subjected to widely different preservation methods. The in vitro viability of spermatozoa processed by these methods has not been previously evaluated in detail. Three ejaculates were collected from each of 5 fertile dogs. Each ejaculate was divided into 2 parts and frozen in medium straws according to the 2 methods. Two straws were thawed and examined from each freezing batch. Sperm motility was assessed in the undiluted semen, and in frozen-thawed semen immediately after thawing, and after storage for 3, 6 and 24 h at room temperature (Straw 1) or 1, 2 and 3 h at 37 degrees C (Straw 2, thermoresistance test). The integrity of the sperm plasma membrane was evaluated in undiluted, in equilibrated (diluted and chilled), and in frozen-thawed spermatozoa using fluorophore probes. The acrosome morphology of frozen-thawed spermatozoa was assessed using a commercial stain (Spermac). Motility immediately after thawing was significantly higher with the CLONE method (75.3% [SD = 4.0] for Straw 1 and 73.7% [SD = 3.2] for Straw 2) than with the Andersen method (70.0% [SD = 5.1] and 69.7% [SD = 3.2]). Motility decreased during storage after thawing. Spermatozoa frozen-thawed using the CLONE method showed a significantly lower thermoresistance. The proportion of spermatozoa with intact plasma membrane was not affected by the equilibration procedure used with either method but was significantly decreased (P < 0.001) after thawing with both methods. The percentage of spermatozoa exhibiting changes thought to represent different stages of acrosomal degradation, was 45.7% (SD = 5.3) using the Andersen method and 44.1% (SD = 9,4) using the CLONE method. Both cryopreservation methods thus resulted in high initial post-thaw sperm motility and membrane integrity but low thermoresistance, and under both methods a large proportion of sperm cells were undergoing acrosomal degradation. The methods differed significantly in terms of their effect on sperm motility but not on plasma membrane integrity or acrosomal morphology.     

Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry

A new triple fluorescent staining method was developed to evaluate frozen-thawed dog spermatozoa. This method was used to compare functional parameters of canine spermatozoa cryopreserved using 2 different freezing-thawing protocols. One ejaculate from each of 10 dogs was split into 2 aliquots and processed using the Andersen method or the CLONE method. Semen samples were evaluated immediately after thawing and after 3 h of incubation at 37 degrees C. Plasma membrane integrity and acrosomal status of the spermatozoa were evaluated simultaneously by flow cytometry using a combination of 3 fluorescent dyes: Carboxy-SNARF-1 (SNARF), to identify the live spermatozoa; propidium iodide (PI), which only stains dead cells or cells with damaged membranes; and fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA), which binds to the acrosomal content of spermatozoa with damaged plasma and outer acrosomal membranes. The accuracy of this new staining method in quantifying the proportions of live and dead spermatozoa by flow cytometry was evaluated by comparing it with the staining technique using carboxyfluorescein diacetate and propidium iodide (CFDA-PI), which yielded high correlation coefficients. The triple-stained sperm samples were also analyzed by epifluorescence microscopy, and both methods proved to be highly correlated. Post-thaw progressive motility and plasma membrane integrity were similar for the 2 freezing procedures, but the proportion of damaged acrosomes after thawing was lower using the Andersen method and the spermatozoa had a higher thermoresistance. This new triple staining method for assessing canine sperm viability and acrosomal integrity provides an efficient procedure for evaluating frozen-thawed dog semen samples either by flow cytometry or fluorescence microscopy.     

Effect of seminal plasma on the cryopreservation of equine spermatozoa

Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80% seminal plasma for 15 min, prior to being diluted to a final concentration of 50 x 10(6)sperm/mL in a Lactose-EDTA freezing diluent and cryopreserved. Sperm motility was analyzed at 10 and 90 min after thawing, while sperm viability and acrosomal integrity were analyzed 20 min after thawing. Seminal plasma did not affect sperm motility, viability or acrosomal integrity (P>0.05). Experiment 2 tested the main affects of seminal plasma level (5 or 20%), incubation temperature (5 or 20 degrees C) and incubation time (2, 4 or 6 h) prior to cryopreservation. In this experiment, spermatozoa were incubated with 5 or 20% seminal plasma for up to 6h at either 5 or 20 degrees C prior to cryopreservation in a skim milk, egg yolk freezing extender. Samples cooled immediately to 5 degrees C, prior to freezing had higher percentages of progressively motile spermatozoa than treatments incubated at 20 degrees C (31 versus 25%, respectively; P<0.05), when analyzed 10 min after thawing. At 90 min post-thaw, total motility was higher for samples incubated at 5 degrees C (42%) compared to 20 degrees C (35%; P<0.05). In addition, samples containing 5% seminal plasma had higher percentages of total and progressively motile spermatozoa (45 and 15%) than samples exposed to 20% seminal plasma (33 and 9%; P<0.05). In conclusion, although the short-term exposure of sperm to seminal plasma had no significant effect on the motility of cryopreserved equine spermatozoa, prolonged exposure to seminal plasma, prior to cryopreservation, was deleterious.