Comparison of assessment of fowl sperm viability by eosin-nigrosin and dual fluorescence (SYBR-14/PI)

The kinetics of fowl sperm viability/mortality following short-term and long-term in vitro storage were studied using 2 different staining methods: eosin/nigrosin (observed under light microscopy) and SYBR-14/PI (dual fluorescence). Based on data obtained at 0, 30 min and at 2, 4 and 24 h (T0, T30, T2, T4, and T24) after in vitro storage (4 degrees C, agitated) of fresh or frozen-thawed semen, the dual association SYBR-14/PI was more effective than eosin/nigrosin (P < 0.05) staining for the detection of sperm viability/mortality at early stages (30 min) in nonfrozen ejaculates stored above 0 degree C. In cryopreserved preparations, the 2 techniques were comparable for assessing viable spermatozoa immediately after thawing, but higher percentages (P < 0.05) of nonviable spermatozoa were detected by the SYBR-14/PI procedure for up to 4 h of in vitro storage post thawing (4 degrees C, agitated). Finally, comparable results were observed between the 2 techniques 24 h after beginning in vitro storage post thawing. It is concluded that the dual association SYBR-14/PI procedure is more effective (or, at least, more rapid) than eosin/nigrosin staining for the assessment of sperm viability/mortality in both fresh and cryopreserved samples of fowl semen. However, in the latter case, the thawing stage needs to be followed by a period of in vitro storage lasting at least 4 h to allow for easier discrimination between viable and nonviable populations of spermatozoa.     

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.     

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.     

Influence of thawing method on motility, plasma membrane integrity and morphology of frozen-thawed stallion spermatozoa

Different thawing methods are used for stallion semen, however, it is unclear which method is the optimal one. To determine if the thawing temperature has an effect on semen quality, we compared 2 thawing temperatures, 75 degrees C and 37 degrees C. The following parameters were used to measure sperm quality: sperm motility, sperm viability, plasma membrane integrity and sperm morphology. Twenty-three ejaculates from 10 Dutch Warmblood stallions were thawed either at 37 degrees C for 30 sec or at 75 degrees C for 7 sec. Sperm motility was evaluated by a Hamilton Thorn Motility Analyser. Plasma membrane integrity and sperm viability were evaluated by using a live/dead fluorescein stain containing a calcein AM probe and ethidium homodimer-1 probe. The eosinaniline blue staining method was used to evaluate the percentage of live and dead cells, as well as sperm morphology. There was no significant difference (P = 0.84) between sperm motility after thawing at 37 degrees C and 75 degrees C. There was also no significant difference (P = 0.053) between the percentage of live spermatozoa using the calcein AM/ethidium homodimer stain after thawing at 37 degrees C and 75 degrees C. There was, however, a significant difference (P = 0.032) between the percentage of live spermatozoa using the eosin-aniline blue stain after thawing at 37 degrees C compared with that at 75 degrees C. In conclusion, our laboratory results indicated that stud farms using frozen semen should thaw the straws at 37 degrees C instead of 75 degrees C. The lower temperature is easier to work with, as thawing at the higher temperature requires special equipment and has to be timed very carefully to avoid damage to the spermatozoa.     

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

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

Cryopreservation of turbot (Scophthalmus maximus) spermatozoa

The aim of this study was to develop a method for cryopreserving turbot semen and to compare sperm motility characteristics, metabolic status and fertilization capacity of frozenthawed and fresh semen. The best results were obtained when spermatozoa were diluted at a 1:2 ratio with a modified Mounib extender, supplemented with 10% BSA and 10% DMSO. For freezing sperm samples, straws were placed at 6.5 cm above the surface of liquid nitrogen (LN) and plunged in LN. The straws were thawed in water bath at 30 degrees C for 5 sec. Use of this simple method resulted in a 60 to 80% reactivation rate of the thawed spermatozoa. Although the percentage of motile spermatozoa in the frozen-thawed semen samples was significantly lower than in fresh semen, spermatozoa velocity and respiratory rate remained unchanged. The process of cryopreservation significantly decreased intracellular ATP content. The fertilization rate of frozen-thawed spermatozoa was significantly lower than that of fresh spermatozoa, but it increased with sperm concentration.     

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 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).     

Effect of staining and sorting on boar sperm membrane integrity, mitochondrial activity and in vitro blastocyst development

The objective of this study was to determine the effects of staining with Hoechst 33342 and of the entire sorting procedure on boar sperm membrane integrity (using Annexin-V/PI), mitochondrial activity (using JC-1/SYBR/PI) and blastocyst development in vitro; the effect of storage at 17 degrees C for 24h prior to Hoechst staining and sorting was also investigated. The Hoechst staining and the whole sorting procedure reduced the percent of live spermatozoa in both fresh (day 0) and stored (day 1) semen, as determined by both assays; nevertheless, there was no increase in live sperm cells showing signs of early damage (Annexin-V positive, propidium negative), whose percentages remained nearly zero. The majority of Annexin-V positive cells were propidium positive, therefore dead. JC-1 staining evidenced a correlation between mitochondrial activity and viability. However, a significant difference between viable sperm cells and sperm cells with active mitochondria was detected in control and stained sperm, whereas almost all viable sorted spermatozoa had active mitochondria. No significant differences in the in vitro produced blastocysts both on day 0 and 1 were observed. In conclusion, despite the damages induced by sorting procedures, semen sorted as fresh or after storage at 17 degrees C can be successfully used for in vitro production of pig embryos.     

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.     

Roles of lipid peroxidation and cytoplasmic droplets on in vitro fertilization capacity of sperm collected from bovine epididymides stored at 4 and 34 degrees C

Sperm recovery from the cauda epididymis can be very advantageous, for example, in case of the unexpected death of a genetically highly valuable animal, for preserving endangered species, or when the collection of sperm by other means becomes impossible. Studies indicate that epididymides stored at cooler temperatures result in better quality sperm. One of the factors that could negatively affect sperm viability during storage is lipid peroxidation, in which the sperm membrane's ability to resist attacks by reactive oxygen species (ROS) plays an important role. Another factor is the presence of cytoplasmic droplets, which appear in high numbers in epididymal sperm, and are known to influence oxidative stress. The objectives of this study were: to determine whether the post-slaughter storage temperature of the epididymis would effect the sperm membrane's resistance to lipid peroxidation and/or the sperm cell's fertilizing capacity in vitro and to elucidate the role played by the cytoplasmic droplets. Forty-eight testicles with epididymides (24 bulls) were collected following slaughter, and divided into two groups. One testicle from each pair was stored at 4 degrees C, and the other at 34 degrees C, for 2h, after which sperm was collected from the caudae epididymides. Sperm concentration was measured, and an aliquot containing 10(8)sperm was subjected to induced lipid peroxidation with ferrous sulphate and ascorbate (37 degrees C, 2h). Subsequently, thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation, were measured. A second aliquot of the same sample was used in a routine in vitro fertilization performed in duplicate. Sperm from caudae epididymides stored at 34 degrees C resulted in lower rates of total blastocyst formation and had a higher percentage of distal droplets, when compared to sperm from epididymides stored at 4 degrees C (21.2+/-2.42 and 71.8+/-4.7% versus 33.5+/-1.8 and 23.7+/-4.7%, respectively, P<0.05). Storage temperature had no effect on TBARS levels. For samples stored at 4 degrees C, TBARS were negatively correlated with distal droplets (r=-0.63, P<0.05) and positively correlated with proximal droplets (r=0.42, P<0.05). In conclusion, our results show that short-term storage of epididymides at 4 degrees C provided sperm of higher quality and in vitro fertilizing capacity than storage at 34 degrees C. Although resistance to oxidative stress could not be shown to directly influence these results, distal sperm droplets that appeared in high numbers in the cooled epididymal sperm samples, may have exerted an antioxidant effect. We hypothesize that this protection against ROS is one of the functions of distal sperm droplets in the epididymis.     

Membrane status of boar spermatozoa after cooling or cryopreservation

This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 degrees C, 4 h), cooling (to 15 or 5 degrees C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was unaffected by freezing and thawing if held at 15 degrees C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 degrees C and 5 degrees C and at thawing compared with that of the unheld controls. The results of this study suggest that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 degrees C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 degrees C during cooling.     

Evaluation on sperm quality of freshly ejaculated boar semen during in vitro storage under different temperatures

The purpose of this study was to assess the sperm quality of fresh ejaculated boar semen stored under different temperatures for up to 48 h in order to use the fresh semen efficiently. Spermatozoa were evaluated by 4 methods: Using trypan blue staining, the viability of spermatozoa stored at 39, 20, 15 and 4 degrees C for 48 h were 1.6, 46.9, 42.0 and 31.0%, respectively. Employing the hypoosmotic swelling test (HOST) showed 1.7%(39 degrees C), 28.7%(20 degrees C), 24.1%(15 degrees C), and 20.1%(4 degrees C) coiled-tail spermatozoa following 48 h storage. With Coomassie blue staining, the rates of acrosome-intact spermatozoa stored for 48 h were 4.5%(39 degrees C), 35.3%(20 degrees C), 55.7%(15 degrees C) and 22.8%(4 degrees C). Using fluorescein isothiocyanate-peanut agglutinin (FITC-PNA), the percentages of acrosome-intact spermatozoa stored for 48 h were 4.3%(39 degrees C), 43.2%(20 degrees C), 17.3%(15 degrees C) and 14.8%(4 degrees C), respectively. The cytoplasmic droplets were found in 18.66% of the spermatozoa in fresh semen and were gradually shed during storage. The results of these 4 methods were highly correlated and could be used to characterized sperm-cell quality effectively. These findings indicated that both membrane integrity and viability of spermatozoa could be preserved well during in vitro storage at 20 degrees C and 15 degrees C for 24 to 48 h.     

Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores

The objectives of this study were to determine the effect of cryoprotectants on sperm viability and develop a freezing protocol for long-term storage of P. monodon spermatophores. Spermatophores suspended for 30 min in calcium-free saline (Ca-F saline) containing the cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propylene glycol (PG), formamide, and methanol at concentrations of 5, 10, 15, or 20% were studied using a modified eosin-nigrosin staining technique. The smallest reductions in apparent sperm viability occurred with DMSO; therefore, a freezing protocol was developed using Ca-F saline containing 5% DMSO. Spermatophores were cryopreserved using three protocols; cooling to a final temperature of -30, -80 or -80 degrees C and immediately stored in liquid nitrogen (cooling rates of -2, -4, -6, -8, -10, -12, -14 or -16 degrees C/min). Frozen spermatophores were thawed (2 min) at 30, 60, 70, or 90 degrees C. Successful cryopreservation of spermatophores in liquid nitrogen was achieved by a one-step cooling rate of -2 degrees C/min between 25 and -80 degrees C before storing in liquid nitrogen. Optimal thawing was in a 30 degrees C water bath for 2 min; this yielded live sperm after storage in liquid nitrogen for 210 days. Average sperm viability for fresh (97.8+/-2.9%) and cryopreserved spermatophores held for less than 60 days (87.3+/-4.1%) did not differ (P>0.05); however, that for spermatophores stored in liquid nitrogen between 90 and 210 days were lower (P<0.05) and varied from 27.3+/-3.4 to 53.3+/-4.3%. Thawed spermatophores previously held in liquid nitrogen for less than 62 days fertilized eggs (fertilization and hatching rates of 71.6-72.2% and 63.6-64.1%, respectively) at rates comparable to fresh spermatophores (70.8-78.2% and 66.3-67.8%, respectively). In conclusion, sperm within cryopreserved spermatophores stored in liquid nitrogen retained their viability for up to 210 days.     

Cryopreservation of sperm in common carp Cyprinus carpio: sperm motility and hatching success of embryos

In this study, fish sperm cryopreservation methods were elaborated upon for ex situ conservation of nine strains of Bohemian common carp. Common carp sperm were diluted in Kurokura medium and chilled to 4 degrees C and dimethyl sulfoxide was added. Cryotubes of sperm with media were then cooled from +4 to -9 degrees C at a rate of 4 degrees C min(-1) and then from -9 to -80 degrees C at a rate of 11 degrees C min(-1), held for 6 min at -80 degrees C, and finally transferred into liquid N(2). The spermatozoa were thawed in a water bath at 35 degrees C for 110 s and checked for fertilization yield, hatching yield of embryos, and larval malformations. Fresh and frozen/thawed sperm were evaluated for the percentage and for the velocity of motile sperm from video frames using image analysis. The percentage and velocity of sperm motility at 15 s after activation of frozen/thawed sperm was significantly lower than that of fresh sperm (nine males). ANOVA showed a significant influence of fresh vs frozen/thawed sperm on fertilization rate (P < 0.0001), but differences in hatching rate and in larval malformation (0-6.8%) were not significant, and different males had a significant influence on fertilization and hatching rate (P < 0.003 and P < 0.007, respectively). Multiple range analysis (LSD) showed significant differences between fresh and frozen/thawed sperm regarding fertilization rate (68 +/- 11 and 56 +/- 10%, respectively) and insignificant differences between fresh and frozen/thawed sperm on the hatching rate (50 +/- 18 and 52 +/- 9%, respectively). The percentage and velocity of fresh sperm motility were correlated, respectively, with the fertilization yield of frozen/thawed sperm at the levels r = 0.51 and r = 0.54.     

Effect of storage on sperm membrane integrity and other functional characteristics of canine spermatozoa: In vitro bioassay for canine semen

The effect of storage of canine semen on sperm membrane integrity, as determined by the hypoosmotic swelling test, and on other functional characteristics of the canine spermatozoa was evaluated by established procedures. The results of this study indicated that storage of canine semen at a chilling temperature of 5 degrees C for 24 h did not significantly impair the physical and functional characteristics of the canine spermatozoa. The overall mean percentage of motility, hypo-osmotic swelling response, which assessed sperm membrane integrity, acrosome-reacted spermatozoa, acrosomal defects, and the percentage of live spermatozoa, did not significantly differ between the fresh and chilled semen samples. However, storage altered the rate of motility and acrosome reaction. The percentage of acrosome reaction in the canine capacitating medium peaked earlier in chilled than in fresh semen. It is probable that storing semen at 5 degrees C initiated/triggered the acrosome reaction. This did not amount to impairment of functional properties. Significant correlations were observed between hypo-osmotic swelling vs motility (r=0.98, P<0.002); hypo-osmotic swelling vs acrosome reaction (r=0.83, P<0.08); and acrosome reaction vs motility (R=0.89, P<0.04) in the fresh semen, and between hypo-osmotic swelling vs motility (r=0.87, P<0.05) and hypo-osmotic swelling vs acrosome reaction (r=0.56, P<0.05) in the chilled semen. It was concluded: that 1) storage of canine semen at 5 degrees C for 24 h did not significantly impair the physical and functional integrity of the spermatozoa; 2) the significant association between motility or acrosome reaction vs hypo-osmotic swelling indicates their value in assessing sperm viability; and 3) the hypo-osmotic swelling assay could have predictive value in screening out subfertile males with apparently normal spermiograms.     

The effect of prolonged cold storage of eland (Taurotragus oryx) cauda epididymides on the spermatozoa: possible implications for the conservation of biodiversity

The objectives of this study were to investigate the effects of prolonged storage of cauda epididymides at 4 degrees C on spermatozoa, and to determine the practicality of utilising epididymal sperm, harvested from testes collected during routine culling of game animals, in assisted reproductive technologies. Testes from eland (Taurotragus oryx) were collected and epididymides removed and maintained at 4 degrees C. Sperm motility, viability, morphology and membrane integrity were examined at 12 h intervals for 108 h. Sperm motility and viability were significantly lower at the end of the experiment than at the start (P < 0.05) and there was individual variation in the rate at which motility and viability declined. The total number of normal sperm decreased significantly with prolonged storage at 4 degrees C. Midpiece defects were the most common and head and tail abnormalities were rare. A significant decrease in acrosomal and nuclear membrane integrity was observed with prolonged cold storage but there was no significant change in cell membrane integrity. However, about 30% of epididymal sperm survived for 3 days at 4 degrees C and more than 10% survived for 4 days, and it should be possible to use sperm from culled animals in some assisted reproductive technologies.     

Evaluation of fresh and frozen-thawed semen of individual ganders by assessment of spermatozoa motility and morphology

Individual differences in gander Anser anser L. reaction to semen collection procedure, quality and quantity of fresh semen and its susceptibility to the freezing process are discussed. Semen was collected individually by dorso-abdominal massage, from 1-year old White Koluda ganders (n = 12) every 2-3 days. Ganders' reactions to massage were observed during the entire reproductive cycle (from 11 February to 13 June, from every male 40 semen collections were performed). For individual evaluation and freezing purpose semen was collected 13 times from every male. In the fresh semen, the following parameters were evaluated: ejaculate volume, color, density, blood or fecal contamination, motility, concentration and morphology of spermatozoa. Motility and spermatozoa morphology were evaluated in the frozen-thawed semen. Semen diluted in 2:1 ratio with EK diluent was frozen with 6% of dimethyl-formamide (DMF) to -140 degrees C at a rate 60 degrees C/min. Semen was thawed by placing the straws in a 60 degrees C water-bath for 4-5 s. Ten out of 12 ganders had from 67.5 to 100.0% positive reactions resulting in semen ejaculation. Significant (P < or = 0.01) differences in fresh semen quality of particular ganders were observed for all evaluated traits. In 1-year-old gander semen morphologically intact spermatozoa constitute only 27.8-45.2% of all cells. Therefore, the sperm quality factor (SQF), proposed by the authors, which includes ejaculate volume, sperm concentration and the percentage of live normal spermatozoa, seems to be a good predictor of gander semen fertilizing ability. The SQF of individual ganders varied from 7.7 to 11.5. The percentage of live normal spermatozoa in the frozen-thawed semen depended mainly on fresh semen quality. In relation to the fresh semen average from 57.2 to 63.2% of spermatozoa survived freezing process and from 23.9 to 38.5% remained morphologically intact.     

Preservation of boar semen at 18 degrees C induces lipid peroxidation and apoptosis like changes in spermatozoa

Boar sperm functions, lipid peroxidation status, mitochondrial membrane potential (DeltaPsi(m)) and membrane permeability (apoptosis like features) were assessed during liquid preservation. Four ejaculates each from four Hampshire boars were extended with Beltsville Thawing Solution and preserved at 18 degrees C. At 0, 24, 48, 72 and 96 h of storage, each ejaculate was examined for sperm functions, lipid peroxidation, DeltaPsi(m), and membrane permeability. The lipid peroxidation status of the sperm was assessed based on the malonaldehyde (MDA) levels. Detection of DeltaPsi(m) was done using 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)]/propidium iodide (PI) assay and Yo-pro-1/PI assay was used to detect change in plasma membrane permeability. The sperm motility, viability and acrosomal integrity declined significantly (p<0.05) from 0 to 96 h of preservation. At the start of the preservation, the MDA levels (nM/10(9) sperm) were low in sperm (99.83+/-2.69) and seminal plasma (191.98+/-11.58), which gradually increased up to the 96 h of storage. Highest negative correlation (r value) was observed between MDA levels and sperm motility (-0.97), live percent (-0.97), acrosomal integrity (-0.97) and hypo-osmotic sperm swelling test (HOSST) positive sperm percentage (-0.98). Strong positive correlation was observed between HOSST positive sperm percentage and intact acrosome percentage (r=0.98). There was a significant (p<0.05) increase in the sperm cells with low DeltaPsi(m) from 0 to 96 h of preservation. Before preservation, 14.85+/-4.66% of sperm cells of the ejaculate showed low mitochondrial membrane potential, whereas after 96 h of preservation, this proposition of cells increased up to 32.00+/-6.25%. The apoptotic sperm population was 8.33+/-2.31% in fresh semen, while this population was 25.19+/-4.25% at 96 h of preservation and the difference was significant (p<0.05). The findings of the present study revealed that liquid preservation of boar semen at 18 degrees C induces lipid peroxidation, decrease mitochondrial membrane potential and increase the plasma membrane permeability.     

Characteristics and in vitro fertilizing ability of giant panda (Ailuropoda melanoleuca) frozen‐thawed epididymal spermatozoa obtained 4 hours postmortem: A case report

When Chu‐Lin, a male giant panda (studbook #249), died at Madrid Zoo, his reproductive tract was removed 4 hr postmortem and the epididymal spermatozoa were collected. Extended sperm were kept at 5°C for 4 hr, loaded into straws, and frozen for 7 min in liquid nitrogen vapor before the straws were plunged into liquid nitrogen. Two straws were thawed and evaluated. Sperm motility was assessed in fresh, refrigerated, and thawed spermatozoa (75%, 60%, 35%, respectively). Sperm viability and acrosome status were estimated using a triple‐stain technique (TST). The results showed 33% live sperm with intact acrosomes after thawing. A hypoosmotic swelling (HOS) test demonstrated the retention of membrane integrity in 72% of thawed sperm. To evaluate the in vitro fertilizing ability of thawed sperm, a sperm penetration assay (SPA) was performed. The values obtained for the percentage of penetration and the penetration index were 62% and 1.78 sperm/oocyte, respectively. The results obtained demonstrate that epididymal sperm recovered from a giant panda postmortem can be successfully cryopreserved. The sperm fertilizing ability demonstrated in vitro after thawing may provide a final opportunity for this male to contribute to the currently small germplasm reserves of this endangered species, and to reproduce in the future through assisted reproductive technology. Zoo Biol 23:279–285, 2004. © 2004 Wiley‐Liss, Inc.