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.     

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.     

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 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).     

Flow cytometric assessment of fresh and frozen-thawed Canada goose (Branta canadensis) semen

The present study was conducted to investigate spermatozoal membrane integrity, acrosome integrity, mitochondrial activity, and chromatin structure in fresh and frozen-thawed Canada goose (Branta canadensis) semen with the use of the flow cytometry. The experiment was carried out on ten, 2-year-old, Canada goose ganders. The semen was collected twice a week, by a dorso-abdominal massage method, then pooled and subjected to cryopreservation in straws, in a programmable freezing unit with the use of dimethyloformamide (DMF) as a cryoprotectant. Frozen samples were thawed in a water bath at 60 °C. The freezing procedure was performed ten times. For the cytometric analysis the fresh and the frozen-thawed semen was extended with EK extender to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with SYBR-14 and propidium iodide (PI), acrosomal damage was evaluated with the use of PNA-Alexa Fluor^®488 conjugate, mitochondrial activity was estimated with Rhodamine 123 (R123), and spermatozoal DNA integrity was measured by the sperm chromatin structure assay (SCSA). The cryopreservation of Canada goose semen significantly decreased the percentage of live cells, from 76.3 to 50.4% (P < 0.01). Moreover, we observed the significant decrease in the percentage of live spermatozoa with intact acrosomes (P < 0.01), but we did not detect significant changes in the percentage of live spermatozoa with ruptured acrosomes. However, after thawing 50% of Canada goose live spermatozoa retained intact acrosomes. Furthermore, the percentage of live spermatozoa with active mitochondria was significantly lower in the frozen-thawed semen than in the fresh semen (P < 0.05). Nevertheless, after thawing the mitochondria remained active in almost 50% of live cells. In the present study, we observed no changes in the percentage of sperm with fragmented DNA after freezing-thawing of Canada goose semen. In conclusion, the present study indicates that even the fresh Branta canadensis semen might have poor quality, the cryopreservation of its semen did not provoke spermatozoal DNA defragmentation and half of the spermatozoa retained intact acrosomes and active mitochondria after freezing-thawing.     

Cytoskeletal proteins F-actin and β-dystrobrevin are altered by the cryopreservation process in bull sperm

The cryopreservation process has an important impact on sperm structure and physiology. The negative effects have been mainly observed on the plasma membrane, which is directly stabilized by the cytoskeleton. Since cytoskeleton proteins are osmosensitive and thermosensitive, the aim of this study was to evaluate the damage caused to the bull sperm cytoskeleton by cryopreservation (freezing-thawing). Fresh and frozen-thawed bull semen samples were exposed to a treatment with the neutral detergent Brij 36-T. Electron microscopy evidenced important damages at the sperm perinuclear theca after the protein extraction protocol; the perinuclear theca was partially solubilized, the perinuclear theca substructure disappeared in the cryopreserved samples. Furthermore, the sperm head's shape was significantly altered on the cryopreserved samples. Fluorescence analysis showed a decrease of the intensity of actin and dystrobrevin on the frozen-thawed samples. Western blot assays revealed a stronger signal for actin and β-dystrobrevin in the frozen-thawed sperm samples than in the fresh ones. Our results suggest that the cryopreservation process highly alters the sperm cytoskeleton stability, causing its proteins to become more fragile and therefore more susceptible to be extracted.     

Comparing ethylene glycol with glycerol for cryopreservation of canine semen in egg-yolk TRIS extenders

The objective of this work was to evaluate the possibility of substituting glycerol (G) for ethylene glycol (EG) when cryopreserving dog semen. A total of 15 ejaculates from 13 dogs was pooled into five samples and frozen in egg-yolk Tris extenders with variable ethylene glycol and glycerol concentrations, with or without Equex STM Paste. Two widely used glycerol extenders (Uppsala Equex II and Norwegian) were utilized as controls. Semen quality parameters assessed after thawing were total subjective motility (TSM), computer assisted sperm analysis (CASA), eosin-nigrosin staining, and flow cytometry (FC) after staining with the PI/Fitc-PSA (fluorescein isotiocianate conjugated with the agglutinin of Pisum sativum, PSA) fluorochromes. No advantages were seen in using EG to replace G when freezing dog semen or combining EG and G in the freezing medium. The Uppsala Equex II provided the best overall post-thaw parameters, followed by the egg-yolk Tris experimental extender with 5% EG and Equex STM Paste. The extender with 4% EG produced similar results to the Norwegian extender. High correlations (r>0.98) were obtained between eosin-nigrosin staining and FC, as well as between subjective and computerized motility assessment (r>0.90).     

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.     

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.     

Practical application of seminal plasma

Natural mating deposits a large number of sperm in a high volume of seminal plasma. Semen processed for AI has the seminal plasma greatly diluted, and processing of boar sperm for cryopreservation removes all seminal plasma. Boar sperm were evaluated for the impact of seminal plasma on the functional status of boar sperm. Seminal plasma prevented or reversed capacitation in frozen-thawed boar sperm. However, supplementing frozen-thawed semen doses with 10% seminal plasma did not significantly affect the number of sperm found in the utero-tubal sperm reservoir, nor did it affect farrowing rates.     

Evaluation of fresh and frozen-thawed fowl semen by flow cytometry

The aim of this study was to assess the spermatozoal viability, acrosome integrity, mitochondrial activity, and DNA status in the frozen-thawed fowl semen with the use of flow cytometry. The experiment was carried out on 10 sexually adult roosters of meat type line Flex. The semen was collected three times a week by dorso-abdominal massage method, then pooled and subjected to cryopreservation using “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. For cytometric analysis the fresh and frozen-thawed semen was extended with EK diluent to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with dual fluorescent probes SYBR-14 and propidium iodide (PI). Acrosomal damages were evaluated using phycoerythrin-conjugated lectin PNA from Arachis hypogaea. The percentage of live spermatozoa with functional mitochondria was estimated using Rhodamine 123 (R123) and PI. The spermatozoal DNA integrity was measured by sperm chromatin structure assay (SCSA). The freezing-thawing process decreased the viability, mitochondrial activity in the chicken sperm and increased the percentage of dead cells with ruptured and intact acrosomes, and also the percentage of spermatozoa with fragmented DNA. In conclusion, the present study indicates that fluorescent staining and flow cytometry may be useful for assessment of the changes of fowl semen quality caused by cryopreservation process. This technique allows precise examination of spermatozoa functional characteristic in a very short time.     

Detection of lipid peroxidation in frozen-thawed avian spermatozoa using C(11)-BODIPY(581/591)

The aim of this study was to perform flow cytometric analysis of C11-BODIPY581/591 oxidation in fowl and geese sperm as a marker for membrane lipid peroxidation (LPO) and to establish if the cryopreservation process would make sperm membranes more susceptible to oxidative stress. The experiment was carried out on 10 meat type line Flex roosters and 10 White Koluda® geese. The semen was collected two times a week, by dorso-abdominal massage method and pooled from 10 individuals of each species. Fowl semen samples were subjected to cryopreservation using the “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. Geese semen samples were cryopreserved in plastic straws in a programmable freezing unit with Dimethyloformamide (DMF) as the cryoprotectant. A fluorescent lipid probe C11-BODIPY581/591 provided with two double bonds that are oxidized during their contact with ROS, was used for the purpose of the assessment of the LPO in freshly diluted semen samples and frozen-thawed semen samples. This probe changes its color according to its state (non peroxidized: red; peroxidized: green). Flow cytometric analysis was used to monitor these changes. The White Koluda® geese fresh semen had a higher level of LPO than the Flex fresh semen (P > 0.01). The cryopreservation of fowl semen significantly (P > 0.01) increased the percentage of live and dead spermatozoa with lipid peroxidation. In frozen-thawed semen of White Koluda® geese the percentage of live spermatozoa with LPO significantly decreased (P > 0.05) whereas significantly (P > 0.01) higher level of dead cells with LPO was observed. There were significant differences between the two studied species. After thawing, the percentage of live and dead spermatozoa with lipid peroxidation was higher in fowl semen than in geese semen (P > 0.01). In conclusion, our data clearly indicate the existence of species specific differences in susceptibility of spermatozoa to the oxidation of PUFAs in the cell membranes, where such oxidation is caused by cryopreservation. This study shows that avian spermatozoa are vulnerable to radicals and frozenthawed sperm have higher level of LPO than fresh sperm. According to our observation, fowl semen is more susceptible to LPO than geese semen.     

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen

The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor^®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.     

Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution

The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs–Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3 h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1 M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in mKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm.     

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.     

Dialysis of boar semen prior to freezing-thawing: its effects on post-thaw sperm characteristics

Low-molecular weight components of the seminal plasma have a detrimental effect on sperm function. The present study was undertaken to evaluate the effect of the removal of low-molecular weight components by dialysis on sperm characteristics prior to and after freezing. Semen, collected from 5 boars, was extended in Kortowo-3 extender (K-3, Poland) and cooled for 3h (control non-dialysis) or dialyzed for 5h in semi-permeable dialysis bags of 12-14kDa molecular weight cut-off prior to freezing. The semen samples were diluted in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or lactose-lyophilized lipoprotein fractions-glycerol extender (lactose-LPFo-G), packaged into aluminum tubes and frozen in a controlled programmable freezer. Pre-frozen and frozen-thawed spermatozoa were evaluated for motility, plasma membrane (SYBR-14 and propidium iodide) and acrosome integrity, mitochondrial function (Rhodamine 123) and ATP content. The results of the study showed that dialysis significantly improved the sperm characteristics prior to freezing. Dialysis enhanced (P<0.05) post-thaw sperm motility, plasma membrane integrity and mitochondrial function, but had no significant effect (P>0.05) on recovery of spermatozoa with intact acrosomes. Furthermore, dialyzed spermatozoa exhibited higher (P<0.05) ATP content compared with the control after freezing-thawing. Consistent inter-boar variability was detected mainly in dialyzed semen following freezing-thawing. These results indicated that the improvement in sperm quality characteristics prior to freezing and the post-thaw sperm recovery were due to the removal of low-molecular weight components from the seminal plasma. It can be suggested that dialysis is effective in improving the post-thaw quality of boar spermatozoa and has also great practical importance in improving the protocols for cryopreservation of semen. Dialysis may also contribute to a better understanding of different mechanisms underlying cryo-induced damage to boar spermatozoa.     

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 cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm

Evaluating cryoinjury of canine spermatozoa is crucial to improving the probability of fertilization. Recently, studies on sperm ROS production, phospholipid scrambling, and DNA damage induced by cryopreservation have been reported. However, the consequences of cryopreservation on these crucial factors are lacking with respect to canine semen. Therefore, the current study was designed to investigate the effects of the freezing-thawing procedure on these factors in canine semen. Ejaculates from five dogs were cryopreserved and thawed. Spermatozoa before and after a freezing-thawing process were assessed for phosphatidylserine (PS) translocation (Annexin V [AN]/propidium iodide [PI] assay), intracellular H₂O₂ level (dichlorofluorescein [DCF]/PI assay), DNA integrity (sperm chromatin structure assay), and conventional sperm parameters. The freezing-thawing process decreased motility, viability, normal morphology, and membrane integrity in canine sperm (P < 0.05). The frozen-thawed semen also showed a decrease in AN−/PI− sperm (%) and an increase in the PS translocation index, the intracellular H₂O₂ level in the viable sperm fraction, and the DNA fragmentation compared with that of fresh semen (P < 0.05). In conclusion, the freezing-thawing procedure significantly affects PS translocation, the intracellular H₂O₂ level, and DNA integrity in canine semen, which may explain the lower fertilization rate and in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) outcome when frozen-thawed spermatozoa are used. It is therefore recommended that these parameters be used as an additional parameter for the assessment of sperm quality after freeze-thawing in canine semen.     

Changes in sperm membrane and ROS following cryopreservation of liquid boar semen stored at 15 °C

Boar semen is occasionally transferred to different locations in liquid form at 15 °C for cryopreservation. However, the use of frozen boar semen is limited due to the high susceptibility of boar sperm to cold shock. The aim of this study was to help improve the quality of frozen boar semen by determining the changes in sperm membrane and ROS during the cryopreservation processes of 15 °C-stored boar semen. Semen was collected from ten Duroc boars and transferred to our laboratory in liquid form stored at 15 °C. After cooling to 5 °C and freezing-thawing, conventional sperm parameters (total motility, progressive motility, and normal morphology), plasma membrane integrity, acrosomal membrane status, and intracellular ROS were evaluated. Sperm function, as assessed by conventional parameters, was unaffected by cooling but was decreased by freezing-thawing (P<0.05). However, the cooling and freezing-thawing processes led to damages in the sperm plasma membrane, and the cooling process caused increase in mean PNA (peanut agglutinin)-fluorescence intensity in viable acrosome-intact sperm (P<0.05). In ROS evaluation, the cooling process decreased intracellular (·)O(2) and H(2)O(2) in viable sperm (P<0.05), while the freezing-thawing process increased intracellular H(2)O(2) (P<0.05) without change in intracellular (·)O(2) in viable sperm. Our results suggest that, in liquid boar semen stored at 15 °C, cooling may be primarily responsible for the destabilization of sperm membranes in viable sperm, while freezing-thawing may induce reductions in sperm function with increase in membrane damage and H(2)O(2).     

Dimethylformamide is no better than glycerol for cryopreservation of canine semen

The objective of this study was to compare the effects of dimethylformamide (DMF) and glycerol in canine (Canis lupus familiaris) semen cryopreservation based on postthaw motility and velocity evaluated by computer-assisted sperm analysis (CASA) and the effects on subjective progressive motility, percentage of live sperm, and plasma membrane functional integrity. The semen was diluted in two steps with an egg-yolk Tris extender containing 6% glycerol or DMF, frozen in 0.25-mL straws, and stored in liquid nitrogen. Immediately after thawing, samples were accessed for subjective sperm motility, sperm membrane functional integrity, percentage of live sperm, and evaluation by CASA. There were differences (P < 0.05) between glycerol and DMF with regard to subjective progressive motility (43.1% vs. 21.5%), objective progressive motility (11.8% vs. 6.2%), velocity average pathway (31.1 vs. 23.1 μm/sec), and amplitude of lateral head (3.3 vs. 3.9 μm), which confirmed the efficiency of glycerol. In conclusion, objective analysis performed by CASA confirmed that no benefits were derived by using DMF to replace glycerol for cryopreservation of canine semen.