DNA integrity of fresh and frozen canine epididymal spermatozoa

The aims of this study were to evaluate: (1) the effect of cryopreservation on DNA fragmentation of canine epididymal spermatozoa, and (2) the potential protective effect of melatonin on post-thaw sperm quality (motility, morphology, acrosomal and DNA integrity). Epididymal spermatozoa were collected after orchiectomy of ten dogs. Sperm samples were frozen in the presence or absence of melatonin (1 mM). DNA fragmentation index (percentage of spermatozoa with fragmented DNA) was similar in fresh samples (3.3 ± 3.6) and samples frozen with (4.2 ± 3.8) or without (3.6 ± 3.7) melatonin. Sperm motility was significantly (p < 0.0001) higher in fresh compared to frozen samples. The presence of melatonin in the freezing extender did not affect the sperm motility. Proportions of spermatozoa with normal morphology were similar in fresh and frozen samples, irrespective of the presence of melatonin in the extender. Acrosome integrity was significantly decreased (p < 0.01) by cryopreservation, and melatonin did not exert any beneficial effects. In conclusion, DNA fragmentation of canine epididymal spermatozoa was not affected by the freezing procedure, and the presence of melatonin did not preserve motility and acrosome integrity which were adversely affected by cryopreservation. The evaluation of DNA status of thawed gametes is particularly relevant for epididymal spermatozoa since these spermatozoa are usually stored and used in assisted reproductive techniques.     

Recovery and cryopreservation of epididymal sperm from domestic cat using powdered coconut water (ACP-117c) and TRIS extenders

Cryopreservation of cats epididymal spermatozoa allows the conservation of the genetic material and the study of the cryogenic effect applied to the gametes of other felines. However, this biotechnique still presents variable results, being necessary the investigation of alternative extenders. Powdered coconut water (ACP-117c) has been efficient in the sperm freezing of several species and in the cat sperm refrigeration. Therefore, we aimed to evaluate the effect of the freezing stages and the quality of the cats’ epididymal spermatozoa after thawing, using ACP-117c. Epididymides (n = 36) from 18 cats were processed using TRIS (n = 18) or ACP-117c (n = 18) for sperm recovery. The sperm were immediately evaluated. Then, this was cooled, glycerolized, frozen and thawed, and re-evaluated at each stage for sperm kinetics by Computer Assisted Semen Analysis, viability, functionality (HOST), mitochondrial activity (DAB) and morphology. There was a reduction in total motility and progressive motility after thawing in both groups, and TRIS was superior to ACP-117c. The curvilinear velocity reduced after thawing with ACP-117c. Viability decreased after glycerolization in TRIS. Although it also reduced after thawing in both groups, it was higher in TRIS. There was no change on HOST. Mitochondrial activity decreased during the cryopreservation steps for both extenders. Nevertheless, TRIS presented a higher percentage of spermatozoa from DAB class I and II after thawing. Morphology did not differ between extenders. Therefore, ACP-117c is an alternative for the recovery of cat epididymal spermatozoa; however, it is not efficient for freezing. Glycerolization and thawing are the most critical stages, regardless of the extender.     

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.     

Effect of initial freezing temperature on the semen characteristics of frozen-thawed ram spermatozoa in a semi-arid tropical environment

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including initial freezing temperature. The present study was conducted to observe the effect of initial freezing temperature on post-thawing motility of ram spermatozoa of native and crossbred rams maintained in a semi-arid tropical environment. Good quality semen obtained from native Malpura and crossbred Bharat Merino rams were pooled within breed and diluted at a rate of 1000 million spermatozoa per milliliter in TEST—yolk–glycerol extender. Diluted semen samples were loaded in 0.25 ml straws and cooled to −25, −75 or −125 °C freezing temperature at the rate of −25 °C/min under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50 °C in a water bath for 10 s and motility characteristics of the frozen-thawed spermatozoa were assessed by a computer-assisted spermatozoa analysis technique. Initial freezing temperature significantly affected the post-thawing motility of sperm in both the breeds. The post-thawing % motility and rapid motile spermatozoa were significantly higher at initial freezing temperature of −125 °C and lower at −25 or −75 °C. The percentage medium motile sperm were similar at all three initial freezing temperatures. The percentage of slow motile and linearity of sperm varied (P<0.01) between the different freezing temperatures. The curvilinear velocity, average path velocity and straight line velocity of spermatozoa were higher (P<0.01) at −125 °C than −25 or −75 °C. Although the lateral head displacement of spermatozoa did not vary significantly between the different initial freezing temperatures, the stroke frequency was significantly lower at −25 °C than −75 or −125 °C. Except for % linearity, the average path velocity and straight line velocity, other spermatozoa characteristics were not significantly different between breeds. The interaction between freezing temperature and breed was significant only for the % motility and linearity of the spermatozoa. The study indicates that initial freezing temperature has a significant effect on spermatozoa motility and velocity following post-thawing. The best motile spermatozoa following thawing were achieved at −125 °C freezing temperature.     

Cryopreservation effects on domestic cat epididymal versus electroejaculated spermatozoa

Frozen-thawed epididymal spermatozoa have already been successfully used in artificial insemination in the domestic cat, proving to be a valuable resource for the reproduction of felid species, which are threatened with extinction. The aim of this study was to compare the effects of freezing and thawing on domestic cat semen collected by electroejaculation (EL) and from the epididymides (EP) and vasa deferentia. Ten adult cats were anesthetized, electroejaculated and immediately thereafter, orchiectomized. Epididymal spermatozoa were collected through the compression of caudae epididymidis and vasa deferentia. Spermatozoa were frozen-thawed following a single protocol. Sperm motility, sperm progressive status (0-5), plasma membrane integrity and morphology (light and transmission electron microscope) were assessed on two occasions, immediately after collection and after freezing and thawing. There were no significant differences between the electroejaculated and epididymal fresh or frozen-thawed spermatozoa for any of the variables. However, the incidence of acrosome defects after freezing and thawing increased by 19% based on light microscopy, whereas ultrastructural images revealed acrosome damages in most sperm cells. Since these acrosomal changes are known to affect sperm fertilising capacity, further studies are needed to optimize cryopreservation techniques for epididymal as well as electroejaculated domestic cat spermatozoa.     

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.     

Freeze–thaw induced genotoxicity in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) spermatozoa in relation to total antioxidant status

Use of cryopreserved semen has become an important tool in assisted reproduction but freezing and thawing cause sub-lethal damage to spermatozoa. This is detrimental to sperm because of the membrane damage including permeability and integrity. An excess generation of reactive oxygen species (ROS) creates oxidative stress due to reduced antioxidant status of the cryopreserved spermatozoa. In the present study fresh buffalo semen was collected and divided into two aliquots. One aliquot was used for fresh semen analysis and the other was cryopreserved in Tris-egg yolk-citrate extender. The semen samples were used to study different sperm quality parameters like motility, viability, membrane integrity and total antioxidant status. The DNA integrity in fresh and cryopreserved spermatozoa was also studied using comet assay. The sperm quality parameters like post-thaw sperm motility, viability, membrane integrity and total antioxidant status of cryopreserved spermatozoa were significantly lowered (P < 0.05) compared to fresh spermatozoa. The DNA fragmentation in cryopreserved spermatozoa was significantly higher (P < 0.01) as compared to fresh spermatozoa. The results show that the irreversible DNA damage occurs in spermatozoa during cryopreservation.     

Ginger and echinacea extracts improve the quality and fertility potential of frozen-thawed ram epididymal spermatozoa

The current study examined the impact of the supplementation of ginger and echinacea extract, as natural antioxidant agents, in freezing extender on the quality and fertility potential of ram epididymal spermatozoa after cryopreservation. Epididymal spermatozoa isolated from Forty testicles, obtained from 20 rams, with motility >80% and total morphological abnormalities <10% were pooled, divided into 7 aliquots and used for cryopreservation. The semen samples were re-suspended with basic Tris egg yolk diluent containing ginger and echinacea extracts (5, 10 and 20 mg/l). The control diluent comprised of only extender and lacked any antioxidant agent. For the determination of sperm quality, frozen straws were thawed after 7–10 days, and then the sperm characteristics were assessed. The supplementation of ginger at a concentration of 10 mg/l, as well as the addition of 10 and 20 mg/l echinacea extract significantly improved total motility and velocity parameters. The status of acrosome integrity and lipid peroxidation significantly improved in spermatozoa when supplemented with 10 mg/l ginger and 20 mg/l echinacea extract. Also, 5 mg/l ginger extract and 20 mg/l echinacea extract significantly improved mitochondrial activity. The highest ratio of the dispersion of sperm chromatin was observed in spermatozoa treated with 10 mg/l ginger extract. The cleavage rate was markedly higher in matured oocytes that were fertilized with frozen spermatozoa treated with 20 mg/l ginger extract and 10 mg/l echinacea. The application of ginger and echinacea extract resulted in improvement in the quality and fertility of frozen-thawed spermatozoa. However, future studies are wanted to elucidate how the active components in these extracts prevent cryo-damages in spermatozoa.     

Computer-assisted sperm analysis of fresh epididymal cat spermatozoa and the impact of cool storage (4 °C) on sperm quality

Epididymal cat sperm is commonly used for in vitro fertilization. Because of the high variability in preparation protocols and methods of evaluation, sperm quality may vary considerably between experiments and laboratories. The aims of the present study were (1) to describe an epididymal sperm preparation protocol to produce clean, highly motile samples using density gradient centrifugation, (2) to provide reference values of computer-assisted semen analysis (CASA) parameters of fresh epididymal cat sperm after density gradient centrifugation and (3) to investigate the effect of cool storage on various spermatozoa characteristics. After slicing the epididymides, viable and motile sperm cells were isolated using Percoll^® centrifugation. Sperm motility parameters were subsequently assessed using CASA in experiment 1. In experiment 2, fresh (day 0) sperm samples were evaluated for motility parameters (HTR) and stained for assessment of acrosomal status (FITC-PSA), morphology (eosin/nigrosin (E/N)), membrane integrity (E/N and SYBR^®14-PI) and DNA fragmentation (TUNEL). After addition of a Tris–glucose-citrate diluent containing 20% egg yolk, samples were cooled to 4 °C and reassessed on d1, d3, d5, d7 and d10. Cool storage impaired most motility and velocity parameters: MOT, PMOT, VAP, VSL, VCL, BCF, RAPID and the percentage of normal spermatozoa showed a decrease over time (P < 0.05) as compared to fresh samples. In contrast, STR, ALH, membrane integrity, DNA fragmentation and the percentage of acrosome intact spermatozoa were not affected by cool storage. However, the influence of cool storage of cat spermatozoa on subsequent in vitro embryo development and quality after IVF requires further investigation.     

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.     

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5–ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate.     

Caffeic acid improves microscopic sperm parameters and antioxidant status of buffalo (Bubalus bubalis) bull semen following freeze-thawing process

This study investigates the effects of cryopreservation and supplementation of buffalo's semen with Caffeic acid. It studies the effects of different Caffeic acid concentrations on cryopreservation capacity of the buffalo and evaluates their influence on various sperm parameters like motility, viability, progressive motility, sperm plasma membrane integrity, and antioxidant status. Twenty-four semen samples were collected with an artificial vaginal from three adult water buffalos. The semen samples were evaluated and the qualified ejaculates were separated and were diluted in a Tris-based extender. The resulting samples were classified into 5 groups: No antioxidant (control), Control sham (NaOH), Caffeic acid 50 μM, Caffeic acid 100 μM, and Caffeic acid 200 μM. The semen samples encountered cryodamage and the quality was deteriorating during the cryopreservation (P < 0.05). The semen evaluation after thawing showed that the groups of samples receiving 100 μM Caffeic acid had higher viability, total motility, and lower abnormal sperm and better linearity (LIN), curvilinear velocity (VCL), straight-line velocity (VSL) and path velocity and higher intact plasma membrane (P < 0.05) compared to other groups. It is notable that adding 100 μM Caffeic acid to freezing extenders enhances the CAT, GPx, SOD, and GSH and also ameliorates total antioxidant capacity of spermatozoa after thawing. It is notable that the addition of 100 μM Caffeic acid decreases the amount of Malondialdehyde. These reactions lead us to conclude that 100 μM Caffeic acid enhances the semen quality of water buffalo after thawing.     

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction

Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe²⁺) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n = 13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe²⁺, EE-CAT plus Fe²⁺, EE-GPx plus Fe²⁺ and EE-SOD plus Fe²⁺). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6 h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6 h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2 h after thawing (P < 0.05). Catalase supplementation, however, improved DNA integrity at 4 h (P < 0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6 h, linear motility at 6 h, mitochondrial activity at 6 h, membrane integrity at 2 and 6 h, and DNA integrity at 4 h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2 h after thawing (P < 0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2 h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe²⁺ negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P < 0.05). After thawing, there were, however, no significant differences between the control plus Fe²⁺ and the antioxidative enzymes supplementation plus Fe²⁺ groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.     

Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47 ± 0.46 nmol/10⁸ cells. Following semen cryopreservation, GSH decreased to 1.62 ± 0.13 nmol/10⁸ cells, a 64% reduction (p < 0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p < 0.01). Addition of 1 mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5 mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze–thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.     

Comparison among different cryoprotectants for cryopreservation of epididymal sperm from agouti (Dasyprocta leporina)

We verify the effects of different cryoprotectants on the cryopreservation of agouti (Dasyprocta leporina) epididymal sperm. We used 16 pairs of testes–epididymis complexes of sexually mature animals. We immediately evaluated epididymal sperm obtained by retrograde flushing for concentration, motility, vigor, viability, osmotic response, and morphology. Samples were extended in a coconut water extender plus 20% egg yolk, containing glycerol, ethylene glycol, dimethylsulfoxide – DMSO, or dimethylformamide. Finally, samples were stored in 0.25 mL straws, frozen in liquid nitrogen, and thawed after one week, being reevaluated and assessed for membrane integrity using fluorescent probes. The higher values for postthawing sperm motility, vigor, and membrane integrity were achieved by the usage of glycerol, when compared to ethylene glycol and dimethylformamide (P < 0.05); however, no differences were found between glycerol and DMSO (P > 0.05). All cryoprotectants provided a similar effect on the preservation of sperm morphology, osmotic response, and viability (P > 0.05). Therefore, here onwards, there was testing of glycerol and DMSO at 3 and 6% concentrations using the same freezing–thawing protocol reported previously. As the main result, DMSO at 6% concentration provided a decrease in sperm parameters, as well as in the chromatin integrity and in the binding capability of sperm. In conclusion, glycerol 3 or 6% and DMSO 3% can be used as alternative cryoprotectants for agouti epididymal sperm cryopreservation.     

Comparisons between three different extenders for canine intrauterine insemination with frozen-thawed spermatozoa

Ejaculates from 7 dogs were obtained on the same day and were pooled. This pooled semen was separated into 3 equal fractions and processed simultaneously, the only difference being in the extender used for freezing. The extenders were laiciphos (containing laiciphos, egg yolk, distilled water and glycerol- Group 1); Tes/Tris (containing Tes/Tris, egg yolk, distilled water and glycerol- Group 2); and biociphos (containing biociphos with glycerol in it, egg yolk and distilled water- Group 3). Spermatozoa were conditioned in 0.5ml French straws and presented normal characteristics before freezing and after thawing. The sperm concentration of the pooled was 683 x 10(6) sperm/ml; sperm motility was above 95%, the percentage of live spermatozoa was above 95% and was of good quality and mobility. Characteristics of the spermatozoa after thawing were the same for spermatozoa frozen with laiciphos and Tes/Tris. Mean sperm concentration was 201.5 +/- 4.95 x 10(6) sperm/ml, sperm motility was 65%, the percentage of live spermatozoa was 80% and the quality of motility.was good. Spermtozoa frozen with biociphos had the following post-thaw characteristics: sperm concentration was 201 x 10(6) sperm/ml, sperm motility was 50%, the percentage of live spermatozoa was 78% and the quality of mobility was medium. Abnormalities were less than 15% for all spermatozoa after thawing. Intrauterine artificial inseminations were performed by laparoscopic intrauterine insemination twice at Days 3 and 5 after the estimated LH peak in 15 normally cyclic Beagle bitches (5 per group) presenting normal hormonal profiles. There were no differences between groups. The females were inseminated with 1.0 ml of spermoatozoa (concentration of 200 x 10(6) sperm/ml) diluted with 1.0 ml of extender. A 60% pregnancy rate was obtained in bitches inseminated with frozen-thawed spermatozoa extended with laiciphos or Tes/Tris and 100% in bitches inseminated with spermatozoa extended with biociphos. Females inseminated with laiciphos, Tes/Tris and biociphos had a mean litter size of 5 +/- 2.6, 3 +/- 1 and 3.4 +/- 1.3 pups, respectively. This study demonstrated that post-thaw assessment of sperm characteristics is not the best technique for evaluating sperm fertility after freezing or for assessing different semen extenders.     

Computer-assisted sperm analysis of fresh epididymal cat spermatozoa and the impact of cool storage (4 degrees C) on sperm quality

Epididymal cat sperm is commonly used for in vitro fertilization. Because of the high variability in preparation protocols and methods of evaluation, sperm quality may vary considerably between experiments and laboratories. The aims of the present study were (1) to describe an epididymal sperm preparation protocol to produce clean, highly motile samples using density gradient centrifugation, (2) to provide reference values of computer-assisted semen analysis (CASA) parameters of fresh epididymal cat sperm after density gradient centrifugation and (3) to investigate the effect of cool storage on various spermatozoa characteristics. After slicing the epididymides, viable and motile sperm cells were isolated using Percoll^® centrifugation. Sperm motility parameters were subsequently assessed using CASA in experiment 1. In experiment 2, fresh (day 0) sperm samples were evaluated for motility parameters (HTR) and stained for assessment of acrosomal status (FITC-PSA), morphology (eosin/nigrosin (E/N)), membrane integrity (E/N and SYBR^®14-PI) and DNA fragmentation (TUNEL). After addition of a Tris–glucose-citrate diluent containing 20% egg yolk, samples were cooled to 4 °C and reassessed on d1, d3, d5, d7 and d10. Cool storage impaired most motility and velocity parameters: MOT, PMOT, VAP, VSL, VCL, BCF, RAPID and the percentage of normal spermatozoa showed a decrease over time (P < 0.05) as compared to fresh samples. In contrast, STR, ALH, membrane integrity, DNA fragmentation and the percentage of acrosome intact spermatozoa were not affected by cool storage. However, the influence of cool storage of cat spermatozoa on subsequent in vitro embryo development and quality after IVF requires further investigation.     

Assessment of the efficacy of Sephadex G-15 filtration of bovine spermatozoa for cryopreservation

Semen from five dairy AI bulls was split-filtered through a Sephadex G-15 filter and frozen in a Tris-citric acid buffer egg yolk-based extender. The effect of filtration was studied morphologically for individual sperm abnormalities. Computer-assisted sperm analysis (CASA) was used for motility and sperm motion assessment. Flow cytometry was used to disclose sperm viability (SYBR-14/PI), mitochondrial membrane potential (Mitotracker Deep Red/SYBR 14), acrosome integrity (SYBR 14/PE-PNA/PI), plasma membrane stability (Merocyanine 540/YO-PRO 1/Hoechst 333342), and chromatin stability (acridine orange staining). Filtration significantly reduced the concentration of recovered spermatozoa (P < 0.01), but improved semen quality, reducing the number of spermatozoa with various forms of morphological defects. Filtration also affected percentages of sperm motility after equilibration and after freezing/thawing. Sperm motion characteristics were, however, not significantly affected by filtration at any stage of the cryopreservation protocol, including post-extension, equilibration, or freezing/thawing. Filtration enhanced sperm viability after thawing (P < 0.05), but had no significant effect (P > 0.05) on recovery of spermatozoa with high mitochondrial potential, intact acrosomes, or preserved sperm chromatin structure. Sperm plasma membrane stability was also not affected by the filtration method used (P > 0.05). It can be concluded that filtration effectively separates weaken or abnormal spermatozoa in pre-freezing semen samples and therefore the procedure could be recommended to improve post-thaw sperm viability of selected, fertile sires.     

Effect of preincubation of cryopreserved porcine epididymal sperm

Preincubation of spermatozoa is important for capacitation and successful fertilization in vitro. The effects of preincubation time on frozen-thawed boar epididymal spermatozoa as measured by sperm motility, acrosomal integrity and fertilization ability in vitro were examined. Epididymal spermatozoa were collected from three Large White boars and frozen. The thawed spermatozoa were preincubated for 0, 15, 30, 60 and 120 min. Their motility was evaluated by a sperm motility analyzer and then the sperm motility indexes (SMIs) were calculated. The status of their acrosomal integrity was evaluated by triple-staining. Then, their fertilization ability was examined by in vitro fertilization (IVF) using porcine oocytes matured in vitro. SMIs of spermatozoa and the incidences of acrosome-intact live spermatozoa from the three boars were high (21-39 for SMI and 50-61% for acrosome-intact live spermatozoa) just after thawing, but both decreased as the duration of preincubation was prolonged (2-10 and 23-40%, respectively). The incidences of sperm penetration were high (61-89% of inseminated oocytes) when the sperm were preincubated for 0-60 min. However, sperm penetration decreased as the preincubation period was prolonged to 120 min. The degree of this decrease differed depending upon the boar from which the spermatozoa were obtained (10-72%). When the two parameters, sperm motility and acrosomal integrity, were analyzed statistically, the latter parameter rather than the former one showed a significant effect on penetration ability in vitro after each duration of preincubation. These results suggest that preincubation of frozen-thawed boar epididymal spermatozoa is not required for IVF and also that the maintenance of acrosomal integrity in unreacted status, rather than the maintenance of sperm motility, is important for fertilization ability after thawing and during preincubation of boar epididymal spermatozoa.     

Comparison between glycerol and ethylene glycol for dog semen cryopreservation

The aim of this study was to compare the effect of ethylene glycol versus glycerol for dog semen freezing, on post-thaw longevity, motility and motility parameters, and on plasma membrane functional integrity. Semen was diluted in two steps with an egg yolk TRIS extender containing a final concentration of either 5% glycerol or 5% ethylene glycol, and frozen in 0.5 ml straws, with 100 x 10(6) spermatozoa/ml, over nitrogen vapours. Semen motility was evaluated both under a light microscope and with a Computer Assisted Motility Analyser System, immediately after thawing and then hourly till 4h of incubation. Sperm membrane functional integrity was assessed with the hypoosmotic swelling test (60 mOsm fructose solution) applied at thawing and then hourly, for 4 h, on incubated samples. Motility (light microscope) and total and progressive motility (analyser) were significantly higher in ethylene glycol frozen samples at thawing (P < 0.01); from hour 1 onwards the effect of the cryoprotectant became not significant. Semen frozen with ethylene glycol showed higher path velocity and higher straight line velocity till 3 h after thawing; however, ethylene glycol semen samples also showed higher curvilinear velocity and higher lateral head displacement, which may indicate a capacitation-like condition affecting sperm membranes and possibly reducing post-thaw longevity. Functional integrity of plasma membrane was similar in glycerol and ethylene glycol samples till 3 h after thawing, then ethylene glycol samples showed a higher decline. The strong though short-lived positive effect of ethylene glycol is worth being evaluated further.