Recovery and cryopreservation of sika deer (Cervus nippon) spermatozoa from epididymides stored at 4 degrees C

The aim of this study is to clarify influence of cold storage of deer epididymides on sperm quality and suitability for cryopreservation. The epididymides were obtained postmortem from sika deer during the breeding season. When epididymides were removed 8-12h postmortem and stored at 4 degrees C for 1-4 days, the collected spermatozoa showed low motility (6.4%). When spermatozoa were collected from epididymides removed within 4h postmortem, sperm motility and viability were 71.8 and 82.4%, respectively. Sperm motility decreased as prolongation of the storage period of the epididymides continued up to 7 days, but sperm viability was not affected. Pyknosis of the epithelial cells and their release into the lumen were observed in the stored epididymides. Epididymal spermatozoa frozen on Day 0 showed 58.1% motility and 83.2% viability. Motility of the frozen-thawed spermatozoa from epididymides stored at 4 degrees C for 1 day (41.9%) was similar to that of nonfrozen spermatozoa from epididymides stored for 4 days (41.8%). These results suggest that refrigeration of deer epididymides or cryopreservation of spermatozoa from refrigerated epididymides can be used for assisted reproductive techniques when epididymal spermatozoa cannot be collected immediately.     

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.     

Storage of red deer epididymides for four days at 5 degrees C: effects on sperm motility,viability, and morphological integrity

The purpose of this study was to assess the sperm motility, the plasma membrane integrity and the morphology of red deer spermatozoa when maintained within epididymides stored for 4 days at 5 degrees C, and to evaluate whether such stored spermatozoa are able to withstand a refrigeration process. Thirty pairs of testes, with attached epididymides, were collected from 30 hunter-killed mature stags (Cervus elaphus hispanicus), and spermatozoa from each one of the pairs were immediately collected in Triladyl medium, evaluated and refrigerated (Control Group). The remaining testes and epididymides were gradually cooled to 5 degrees C and stored for 1, 2, 3, and 4 days (Experimental Groups), after which spermatozoa were processed as described previously for the control group. The effects on spermatozoa that had been stored within epididymides for various times were determined by assaying sperm motility index (SMI), plasma membrane integrity and sperm morphology (SM). In the same way, SMI and SM were assessed after spermatozoa refrigeration at 5 degrees C for 3 hours in different groups (SMI-R, SM-R). There was no significant decrease in plasma membrane integrity of spermatozoa recovered from epididymides stored at 5 degrees C for 4 days. Similarly, the percentage of morphologically normal spermatozoa remained unaffected during the first 3 days of storage. In contrast, during storage sperm motility evaluation revealed significantly (P<0.05) lower SMI values for samples from epididymides stored 2, 3, and 4 days (47.7+/-3.6, 45.5+/-4.4, 44.1+/-5.2) than that of the control group (57.6+/-1.6). Similar results were obtained after refrigeration of spermatozoa in Triladyl at 5 degrees C. These data suggest that it might be possible to recover functional spermatozoa from red deer epididymides stored at 5 degrees C during several days when epididymal spermatozoa cannot be collected and cryopreserved immediately.     

Characteristics of Iberian red deer (Cervus elaphus hispanicus) spermatozoa cryopreserved after storage at 5 degrees C in the epididymis for several days

The aim of this study was to assess the influence of prolonged cold storage of Iberian red deer epididymides on post-thaw sperm characteristics. Thirty-seven pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control group). The remaining epididymides were cooled to 5 degrees C and stored for 12, 24, 48, 72 and 96 h (experimental groups), after which spermatozoa were collected and frozen as in the control group. After thawing, sperm motility, membrane and acrosome integrities, mitochondrial function and DNA damage were evaluated. The motility of spermatozoa stored in the epididymis for up to 96 h did not decrease significantly (P>0.05) but, after cryopreservation, a decline in sperm motility was seen in spermatozoa stored for 48 h, or later. A slower decrease in sperm membrane and acrosome integrities after cryopreservation were seen as storage time progressed. Some differences were seen when different methods were used to assess the same sperm parameter although changes followed similar patterns. This was the case for acrosome integrity (phase contrast microscopy versus fluorescent lectin) or membrane integrity (hypo-osmotic swelling test or nigrosin-eosin stain versus propidium iodide). We conclude that frozen-thawed spermatozoa of Iberian red deer recovered from epididymides stored at 5 degrees C have a good sperm quality (including motility) during less than 48 h of storage for most of the sperm parameters assessed.     

Effects of long-term chilled storage of red deer epididymides on DNA integrity and motility of thawed spermatozoa

We have carried out a study on the influence of prolonged cold storage (5 degrees C) of Iberian red deer epididymides on post-thaw sperm motility and DNA integrity. Twenty-nine pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control). The remaining epididymides were cooled to 5 degrees C and stored for 24, 96 and 192 h (experimental groups), after which spermatozoa were collected and frozen. Samples were evaluated before freezing, after thawing, and after a 2-h period of incubation at 37 degrees C. Motility was evaluated by means of a CASA system and chromatin stability was assessed following the Sperm Chromatin Structure Assay (SCSA). Our results showed that, during the first 96 h, the motility (total and progressive) did not significantly decline when assessed after cryopreservation, although there was a significant decline when epididymides had been stored for 192 h at 5 degrees C (P<0.001). The present study demonstrates that motility and DNA status of thawed spermatozoa collected from refrigerated epididymes, at least 96 h post-mortem, were good enough to consider their eventual use. Most importantly, sperm DNA integrity after thawing was apparently not affected by storage time, even after 192 h.     

Freezing of epididymal spermatozoa from dogs after cool storage for 2 or 4 days

An experiment was conducted to investigate the freezing ability of canine epididymal spermatozoa after cool storage at 5 degrees C for 2 or 4 days. Spermatozoa were collected from the caudae epididymidis from 16 dogs. Total motility, plasma membrane integrity and acrosome integrity were evaluated immediately on harvesting, and after 2 and 4 days of storage at 5 degrees C, and at 0 and 2 h post-thaw at 37 degrees C. Sperm motility decreased significantly during cold storage, compared to freshly harvested spermatozoa (P < 0.001). Although there was no significant effect of pre-freeze storage time on post-thaw motility, there was a tendency towards decreased motility in spermatozoa that had been stored for 4 days, compared to spermatozoa that were frozen immediately after collection (P = 0.09). The number of post-thaw spermatozoa with an intact plasma membrane was decreased in spermatozoa cold-stored for 4 days (P < 0.001). There was no significant effect of pre-freeze storage time on the acrosomal status of post-thaw spermatozoa. In conclusion, canine epididymal spermatozoa were stored at 5 degrees C for up to 4 days without a clear detrimental effect on post-thaw motility and acrosome integrity, but storage may have decreased post-thaw motility. Results were, however, generally low.     

Influence of storage time on functional capacity of flow cytometrically sex-sorted boar spermatozoa

Sex-sorting of boar spermatozoa is an emerging biotechnology, still considered suboptimal owing to the slowness of the process, which requires long sorting periods to obtain an adequate number of spermatozoa to perform a non-surgical insemination. This period involves storage of sorted cells that could impair their functional capacity. Here, we have studied how the storage of sex-sorted boar spermatozoa affects their functional capacity. Sorted spermatozoa were assessed at various times (0, 2, 5h or 10h) during storage after sorting and compared with diluted and unsorted spermatozoa for sperm motility patterns, plasma membrane and acrosomal integrity and their ability to penetrate homologous IVM oocytes. Sex-sorted sperm motility and membrane integrity only decreased significantly (p<0.05) by the end of the storage period (10h) compared to unsorted spermatozoa. Sperm velocity, ALH and Dance increased significantly (p<0.05), immediately post-sorting, returning to unsorted sperm values during storage. Acrosome integrity was not seriously affected by the sorting process, but decreased (p<0.05) during storage after sorting. Sorted spermatozoa stored 2h after sorting did not differ from unsorted in penetration rates and numbers of spermatozoa per oocyte, reaching the highest (p<0.05) penetration rates and sperm numbers per oocyte, when co-cultured for 6 or more hours. Non-storage or storage for 5h or 10h negatively (p<0.05) affected sperm penetration ability. In conclusion, although flow cytometrically sex-sorted spermatozoa are able to maintain motility, viability and acrosomal integrity at optimal levels until 10h of storage after sorting, fertilizing ability is maintained only over shorter storage times (<5h).     

Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro

The effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona-free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid-stored (LIS, stored at 18°C for 3 days), and frozen (FRO, frozen by pellet method and stored at ?196°C for 3 days). A highly motile sperm population was isolated by the swim-up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39°C in a Tris-buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39°C for 3 hr in Hams F-10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome-reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P < .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P < .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.     

Motility and fertility of alginate encapsulated boar spermatozoa

Ejaculated boar spermatozoa are vulnerable to cold shock. Prolonged storage of boar spermatozoa at low temperatures reduces survival rate, resulting in a bottleneck for the extension of artificial insemination in pig husbandry. This study evaluated whether alginate microencapsulization processing can improve the longevity of boar spermatozoa stored at 5 degrees C and the fertility of microencapsulated spermatozoa in vivo. Sperm-rich fraction semen from three purebred boars were concentrated and microencapsulated using alginate at 16-18 degrees C, and then were stored at 5 degrees C. Following storage for 1, 3 and 7 days, the microcapsule was taken out to assess sperm release under 37 degrees C incubation with or without 110 rpm stirring. The percentage of sperm released from microcapsules with 110 rpm stirring was higher than without stirring (81 versus 60%) after 24h of incubation. In another experiment, semen was also microencapsulated to evaluate the sperm motility. The motility of spermatozoa was assessed at 10 min, 8, 24, 32, 48, 56 and 72 h following incubation at 37 degrees C for nine consecutive days. The fertility of the free and microencapsulated semen was assessed by inseminating sows, and the reproductive traits (conception rate, farrowing rate, and litter size) were recorded. The motility of encapsulated spermatozoa was significantly higher than that of free semen after 8h incubation at 37 degrees C after storing for over three days (P<0.05). No significant difference existed in conception rate, farrowing rate, and litter size between the microencapsulated and non-encapsulated semen after four days of storage. In conclusion, microencapsulation can increase the longevity of boar spermatozoa and may sustain in vivo ova fertilization ability.     

Freezing of stored, chilled dog spermatozoa

The aims of this study were to find out if dog spermatozoa can be stored chilled for 1 or 2 days prior to freezing without a deterioration in post-thaw vitality and longevity, and to compare two extenders; the Uppsala Equex-2 (UE-2) and a TRIS egg yolk extender (EYT). Pooled dog semen was frozen immediately after collection, or was extended and stored at 4 degrees C for 1 or 2 days before freezing. Sperm motility and acrosome integrity were evaluated before freezing and for 6h post thaw at 38 degrees C, while sperm plasma membrane integrity was evaluated post thaw. There were no effects of pre-freeze storage time or extender on post-thaw motility or plasma membrane integrity, but a significant effect of extender (P < 0.0153) on post-thaw acrosomal integrity was found, UE-2 being better than EYT. There was a significant (P < 0.0001) negative effect of post-thaw storage time on acrosome integrity, but this was not influenced by pre-freeze storage time or extender. In conclusion, we found that dog spermatozoa can be frozen after 1 or 2 days of cold storage without significant deterioration in post-thaw motility, acrosome integrity or sperm plasma membrane integrity compared to when frozen immediately after collection. The UE-2 extender was superior to the EYT extender for freezing of cold stored dog spermatozoa.     

Apoptotic-like changes in the spermatozoa of fresh and stored boar semen and the quality of embryos produced in vivo

The aim of this study was to determine the apoptotic-like changes in the spermatozoa of fresh and stored boar semen and to investigate the relationship between this phenomenon and the quality of embryos produced in vivo. The experiments were divided into two series. In the first series, ten ejaculates were collected from five boars, which were crossbreeds of the Polish Landrace and Large White breeds. The semen was stored as a liquid until Day A (the day on which sperm motility decreased to 30%). Three fluorescence methods were used to evaluate semen quality: an assay to assess the early changes in sperm membrane integrity using the fluorophore YO-PRO-1, an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled annexin-V and the mitochondrial-specific probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) for measuring changes in mitochondrial membrane potential. Our results showed that liquid preservation of boar semen causes apoptotic-like changes in the sperm, and a significant increase in both: apoptotic sperm (YO-PRO-1(+)/PI(-)) and early apoptotic sperm (annexin-V(+)/PI(-)) were observed between Day 0 (fresh semen) and Day A only in semen from three of the five boars. In the second series of experiments, the semen from boar nos. 1, 2, and 3 was selected for insemination of superovulated gilts. The fertilizing capacity of fresh and stored semen with different levels of apoptotic spermatozoa was measured based on the morphology and the number of cells of embryos that were obtained after insemination with this semen. Our studies indicated no significant differences in the fertilization rate of gilts after insemination with fresh and stored semen with increased levels of apoptotic spermatozoa. After insemination with stored semen, a significantly greater number of degenerated embryos were observed, but the morphologically normal blastocysts obtained after insemination with either fresh or stored semen had a similar number of nuclei.     

Cryopreservation of epididymal bovine spermatozoa from dead animals and its uses in vitro embryo production

The present study aimed to evaluate viability and in vitro fertilizing ability of cryopreserved epididymal spermatozoa obtained from dead animals. To collect spermatozoa, epididymides from three males (Bulls A1, A2 and A3) were collected at a local slaughterhouse. As a reference ejaculate from a bull with known in vitro fertility, was used. Sperm characteristics (motility, chromatin and acrosome integrity) were evaluated before and after cryopreservation. Then, frozen spermatozoa from all animals were used for in vitro fertilization. Cleavage and blastocyst rates at 48 h (day 2) and 168 h (day 7) post in vitro insemination, for bull A1 (82.1 and 38.6%) and A2 (80.7 and 33.8%) were similar (P>0.05) to the reference bull (88.9 and 57.2%). Bull A3 had the lesser cleavage (42.0%) and blastocyst (26.1%) rates. The results showed that epididymal spermatozoa from dead animals can be successfully cryopreserved and used in vitro production of embryos.     

Comparing sugar type supplementation for cryopreservation of boar semen in egg yolk based extender

Cryopreservation of boar semen is still considered suboptimal due to lower fertility when compared to fresh semen. The aim of this study was to evaluate the effects of the addition of different sugars (lactose, trehalose and glucose) on boar spermatozoa cryopreserved in an egg yolk based extender. Ejaculates were collected from a boar previously selected and semen samples were processed using the straw freezing procedure. In experiment 1, subsamples of semen were frozen in three different extenders: recommended lactose egg yolk extender (LEY); trehalose egg yolk extender (TEY) and glucose egg yolk extender (GEY). Sperm quality was assessed for motility, viability, acrosome integrity and hypoosmotic swelling test response upon collection, after freezing and thawing and then every hour for 3 h. Results showed that total motility at 1 and 3 h, progressive motility at 3 h, positive hypoosmotic response at 2 and 3 h and acrosome integrity at all times were significantly improved when trehalose was added to the extender. In experiment 2, sugar influence was also demonstrated in vitro fertilization. A total of 1691 oocytes were in vitro matured and inseminated with frozen-thawed sperm at 2000:1 sperm:oocyte ratio and coincubated for 6 h. Presumptive zygotes were cultured in NCSU-23 medium to assess fertilization parameters and embryo development. Both penetration and monospermy rates were significantly higher for trehalose frozen semen. A significant increase was observed in efficiency and blastocyst formation rates from TEY to the other groups. Our results demonstrated that trehalose extender enhances spermatozoa viability and its in vitro fertilization parameters in boar ejaculates with good sperm freezability. Further studies are necessary to assess the impact of sugars on the entire population.     

Short-term nonfrozen storage of mouse epididymal spermatozoa

Six simple methods for short-term (up to 8 d), nonfrozen (5 to 20 degrees C) storage of mouse epididymides were compared with respect to the motility and fertility of spermatozoa. A high percentage of progressively motile spermatozoa was obtained from epididymis stored for 8 d at 5 degrees C in mineral oil (78.3%), covered with body fat (80.0%), or stored in the intact body of the euthanized donor animal (77.5%). Fertilized eggs (6.4% fertilization rate) were obtained by IVF using spermatozoa that had been stored in mineral oil at 5 degrees C for at least 8 d, and offspring were obtained from 77.5% of transferred eggs that were fertilized by spermatozoa stored for 2 d. These methods inhibited moisture loss from the preserved epididymal spermatozoa, thereby allowing spermatozoa to be stored for a few days without loss of either motility or fertility. These methods make possible such wide-ranging applications as the long-distance transport of epididymis spermatozoa. While in storage at 5 degrees C, the tail of each recovered spermatozoon was bent midway along the tail, possibly owing to damage to the plasma membranes and due to the spermatozoa's hardening in the phospholipid by exposure to the low temperature.     

Birth of mice after in vitro fertilization using C57BL/6 sperm transported within epididymides at refrigerated temperatures

The transportation of cryopreserved spermatozoa is an economical, efficient, and safe method for the distribution of mouse strains from one facility to another. However, spermatozoa from some strains, including C57BL/6 (B6), are very sensitive to freezing and thawing and frequently fail to fertilize eggs by conventional in vitro fertilization methods at the recipient mouse facility. Since many genetically engineered mice have the B6 genetic background, this sensitivity poses a major obstacle to studies of mouse genetics. We investigated the feasibility of transporting spermatozoa within epididymides under non-freezing conditions. First, we examined the interval that B6 and B6D2F1 (BDF1) spermatozoa retained their ability to fertilize when stored within epididymides at low temperatures (5 degrees C or 7 degrees C). Fertilization rates were >50%, irrespective of the spermatozoa used, when epididymides were stored for 3d at 7 degrees C. B6 spermatozoa, but not BDF1 sperm, had better retention of fertilizing ability at 7 degrees C versus 5 degrees C. We then transported freshly collected B6 and BDF1 epididymides from a sender colony to a recipient colony using a common package delivery service, during which the temperature was maintained at 5 degrees C or 7 degrees C for 2d. Sufficiently high fertilization rates (68.0-77.5%) were obtained for all experimental groups, except for B6 spermatozoa transported at 5 degrees C. These spermatozoa were successfully cryopreserved at the recipient facility and, yielded post-thaw fertilization rates of 27.6-66.4%. When embryos derived from the B6 spermatozoa that were transported at 7 degrees C were transferred into recipient females, 52.7% (38/72) developed to term. In conclusion, transportation of epididymides at refrigerated temperatures is a practical method for the exchange of mouse genetic resources between facilities, especially when these facilities do not specialize in sperm cryopreservation. For the B6 mouse strain, the transportation of epididymides at 7 degrees C rather than 5 degrees C, is recommended.     

Recovery of motile,membrane-intact spermatozoa from canine epididymides stored for 8 days at 4 degrees C

The purpose of this study was to determine how long canine spermatozoa remain motile and with intact membranes when maintained within epididymides stored at 4 degrees C, and to determine whether such stored spermatozoa are able to bind to canine zonae pellucidae. Testes with attached epididymides, obtained from 32 dogs (26 purebred; six mixed breeds) at orchiectomy, were refrigerated at 4 degrees C, and spermatozoa were collected from caudae epididymides at nine time intervals ranging from 5 to 192 h. The effects on spermatozoa that had been refrigerated within epididymides for various times were determined by assaying sperm motility, integrity of plasma membranes and of acrosomes, and measuring binding of membrane-intact spermatozoa to canine zonae pellucidae. Membrane integrity was assessed using a double fluorescent dye, and acrosome integrity by staining with Pisum sativum agglutinin. For the zona-binding assay at various refrigeration time points, duplicate sets of six oocytes each, isolated from ovaries retrieved at elective ovariohysterectomy, were placed into 100 microl droplets of sperm capacitation medium containing 5 x 10(6) spermatozoa/ml. One minute later, oocytes were rinsed vigorously by pipetting, and then incubated for 1 h at 38.5 degrees C in a humidified atmosphere of 5% CO2 in air; the number of membrane-intact spermatozoa bound to zonae were counted. There was no significant decrease in membrane integrity and acrosome integrity of spermatozoa recovered from epididymides stored at 4 degrees C within the first 48 h of refrigeration. In contrast, sperm motility decreased significantly within the first 5 h of refrigeration (P < 0.05), but then declined more gradually thereafter. Some spermatozoa recovered from epididymides that had been refrigerated for 192 h retained their capability to bind to zonae pellucidae, although the mean number of refrigerated spermatozoa (0.4) bound to zonae was less than that of fresh samples (9.0). Membrane integrity of spermatozoa recovered from epididymides refrigerated for various times was highly correlated (r = 0.88) with sperm motility. Even after storage for 192 h (8 days) at 4 degrees C, motile spermatozoa could be recovered from the epididymides, and such refrigerated spermatozoa were capable of binding to zonae. We interpreted these data to indicate that it might be possible to recover functional spermatozoa from postmortem specimens of domestic and nondomestic canids.     

Assessment of sperm viability,mitochondrial activity,capacitation and acrosome intactness in extended boar semen during long-term storage

The purpose of this study was to assess sperm quality in extended boar semen during in vitro storage in order to determine which extender should be used and how long boar semen can be stored. Freshly ejaculated boar semen was diluted with equal volumes of Beltsville thaw solution (BTS), Androhep, KIEV or Zorlesco extenders and stored at 17 degrees C for up to 15 days. Sperm quality was evaluated by examining viability using SYBR-14/PI and Hoechst 33258 staining, mitochondrial activity using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining, acrosome intactness by Coomassie blue staining, and capacitation status by chlortetracycline (CTC) staining. There were over 50% viable spermatozoa in boar semen extended with Zorlesco and Androhep extenders on Day 13 of storage. The percentage of JC-1-stained spermatozoa was 53.8 +/- 2.1% for Zorlesco and 57.7 +/- 1.60% for Androhep extenders on Day 13 of storage. The percentage of acrosome-intact spermatozoa detected by Coomassie blue staining was higher than that in the SYBR-14PI-, Hoechst 33258-, and JC-1-stained samples in our study. The results from SYBR-14/PI, Hoechst 33258, JC-1, and Coomassie blue staining were highly correlated (r > or = 0.9461). There were less than 15% capacitated spermatozoa in the semen extended with BTS, Androhep and Zorlesco extenders during 9 days of storage. However, most viable boar spermatozoa became capacitated by Day 13 of storage. The rank order of four extenders for maintaining sperm viability and mitochondrial activity was as follows: Androhep, Zorlesco, BTS, KIEV.     

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.     

The use of ejaculated boar semen after freezing in 2 or 6% glucerol for in vitro fertilization of porcine oocytes matured in vitro

Two concentrations of glycerol in a freezing diluent were tested with respect to the in vitro fertilizing capacity of frozen-thawed boar spermatozoa which, before exposure to oocytes, were subjected to 3 methods of fractionation. These were 1) the upper fraction, 2) the swim-up and 3) percoll gradinet-centrifugation. The highest proportions of motile spermatozoa were obtained by the swim-up procedure, while acrosomal integrity was best preserved by the upper fraction procedure. Raising the glycerol concentration from 2 to 6% (v/v) during freezing decreased the proportion of spermatozoa with a normal apical ridge. Spermatozoa separated by the upper fraction method showed the greatest penetration of oocytes and produced the highest indidence of polyspermy. The glycerol level affected penetration and polyspermy only with spermatozoa separated in a percoll gradient, where the higher level of glycerol increased oocytes penetration and polyspermy. Pronuclei formation was influenced by the separation procedure and by the glycerol concentration in the freezing diluent. The results indicate that frozen boar semen can be used for in vitro fertilization more successfully than fresh semen since penetration by frozen upper fraction spermatozoa was similar to, the degree of polyspermy was lower, and the formation of two pronuclei was greater (P<0.01) than in oocytes exposed to fresh semen.     

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.