长牡蛎精子超低温冷冻后超微结构损伤研究

采用程序降温仪分步降温冷冻保存长牡蛎（Crassostrea gigas）精液,并用扫描电镜、透射电镜研究了精子的超微结构损伤。超低温冷冻保存后长牡蛎精子的运动率、受精率及孵化率与鲜精无显著差异。鲜精中84.5%的精子形态结构正常,冻精中73%的精子形态结构正常。形态结构正常的精子表现为顶体、质膜、线粒体与鞭毛结构完整、染色质形状规则,顶体、线粒体及中心粒结构正常,鞭毛形态完整、微管结构清晰;形态结构异常的精子表现为顶体脱落、解体,精子头部质膜膨胀、破裂、染色质肿胀、破裂、解体,线粒体移位、脱落、膨胀,嵴退化或消失,鞭毛弯折、断裂,微管解聚。结果显示,以10% DMSO为抗冻保护剂,HBSS溶液为稀释液,1:4的稀释比例,添加海藻糖,采用分步降温法冷冻保存,对长牡蛎精子具有较好的抗冻保护作用,合适的冻存方法可以有效的保护太平洋牡蛎精子冷冻过程中结构损伤。研究有助于长牡蛎种质资源的收集保存及应用。     

Cryopreservation of flounder (Paralichthys olivaceus) sperm with a practical methodology

A simple and convenient protocol for the cryopreservation of the flounder (Paralichthys olivaceus) sperm was established for "on the spot" cryopreservation of large quantities of semen. The use of three cryoprotectants, dimethyl sulphoxide (DMSO), glycerol (Gly) and methanol was tested in the method. The percentage of motile sperm present in semen after it had been frozen and thawed in the presence of DMSO, Gly or methanol was 60.5+/-3.6, 79.17+/-4.5 and 13.25+/-4.7%, respectively. The fertilization rates of this sperm were 67.06+/-15.1, 76.20+/-10.0 and 44.93+/-22.6%, while the hatching rates of eggs fertilized with this sperm were 37.40+/-8.3, 48.18+/-25.7 and 23.35+/-10.8%, respectively. It was found that Gly and DMSO were better cryoprotectants than methanol, with Gly giving the best overall results. Under scanning electron microscopy, it could be seen that while the majority of the frozen-thawed sperm remained morphologically normal, some exhibited lost or dilated mitochondria, swollen mid-pieces, broken tails, or damaged cell membrane, which probably caused the decrease in motility and fertility of the frozen-thawed sperm.     

Successful fertilization of Varicorhinus macrolepis eggs with sperm subjected to two freeze-thaw cycles

Although sperm from several fish species have been successfully cryopreserved, few studies have been done in small and/or endangered species. The aim of the present work was to develop a method of freezing and refreezing Varicorhinus macrolepis semen in 1.8 mL cryovials. The effect of extenders and cryoprotectants on the motility of post-thaw sperm was examined. The motility of frozen-thawed sperm in extender D-15 was higher than that in MPRS and fish Ringer solution (P<0.05). Dimethyl sulfoxide (DMSO) and glycerol provided greater protection to sperm than methanol during freezing and thawing; the most effective concentration of DMSO and glycerol was 10%. The fertilization rate of frozen-thawed sperm was not significantly different from that of fresh sperm. Furthermore, mean (+/-S.D.) hatching rate did not differ significantly between frozen-thawed (82.7+/-12.4%) and fresh sperm (90.7+/-4.5%). Although frozen-thawed sperm that was immediately refrozen had 0% post-thaw motility, frozen semen that was refrozen after dilution with D-15 (containing DMSO at a ratio of 1:2) had post-thaw motility of 38.3+/-2.9%. Motility was lower for refrozen than for frozen sperm (P<0.05). Furthermore, fertilization and hatching rates of refrozen sperm were 42.9+/-6.7 and 34.1+/-10.5%, respectively, which were lower than that of fresh sperm (P<0.05).     

Cryopreservation of bull spermatozoa alters the perinuclear theca

The perinuclear theca (PT) is involved in several important sperm functions leading to fertilization. The objective of this study was to investigate the effect of cryopreservation of bull spermatozoa on the integrity of the PT and the relationship between PT integrity and semen characteristics. Semen from seven bulls was evaluated before and after cryopreservation, comparing the integrity of the plasma membrane (hypo-osmotic test), percentage of live and dead spermatozoa (triple stain), acrosome integrity (triple stain) and the integrity of the PT (negative stain by electron microscopy). Cryopreservation of bull semen caused substantial damage to the PT; the proportion of spermatozoa with a damaged PT was 15.2% versus 52.5% (P<0.05) in fresh versus frozen-thawed spermatozoa, respectively. Furthermore, on average, 67.4% (range, 64-72%) of fresh spermatozoa were live, compared to 53.1% (range, 49-58%) for frozen-thawed spermatozoa; there was an inverse correlation between the percentage of live spermatozoa and the percentage with damage to the PT. Although 59.1% of frozen-thawed spermatozoa had an intact acrosome, only 43.7% of them still remained alive. In frozen-thawed semen, there was a high correlation (r=0.69) between live spermatozoa with an intact acrosome and spermatozoa that maintained an intact PT. In conclusion, freezing/thawing of bull spermatozoa altered the PT and maintaining PT integrity may be necessary to maintain acrosome integrity.     

In vitro fertilization of porcine oocytes with fresh and frozen-thawed ejaculated or frozen-thawed epididymal semen obtained from identical boars

The objective of this study was to compare the in vitro fertilizing capacity of porcine spermatozoa from fresh and frozen-thawed semen and frozen-thawed epididymal spermatozoa obtained from identical boars. Prior to IVF, fresh spermatozoa were capacitated in TCM 199. Frozen semen samples were stored in 0.25-ml plastic straws using a lactose/glycerol/orvus-es-paste extender. Cumulus-oocyte-complexes (COC) obtained from superovulated prepuberal gilts were fertilized in vitro within 2 h after aspiration with one of the semen samples. After final dilution for IVF, frozen-thawed epididymal semen samples showed motility rates (72.2 +/- 5.6%) similar to those of spermatozoa in fresh semen (76.4 +/- 4.5%), while sperm motility decreased in frozen-thawed ejaculated semen (40.2 +/- 9.4%). Considerable individual differences in sperm motility between boars were observed for ejaculated semen but not for epididymal semen. Enhanced fertilizing capacity of frozen-thawed epididymal spermatozoa was confirmed by pronucleus formation and cleavage rates, with significantly more embryos developing to the 2- and 4-cell stages compared with the groups fertilized with fresh or with frozen-thawed ejaculated semen (59.7 vs 14.6 and 16%). In conclusion, consistent in vitro fertilization rates with minimal semen variability are obtained using frozen-thawed epididymal semen. Following a modified freezing protocol, epididymal spermatozoa can easily be frozen in small containers for IVF, with higher resultant motility and fertilization rates than with ejaculated semen.     

Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African catfish (Clarias gariepinus)

A new integrated approach including computer-assisted sperm analysis (CASA), viability staining and fertilization was used to study the quality of cryodiluents used in fish sperm cryopreservation. As an example the sperm quality of an African catfish, Clarias gariepinus (Burchell, 1822), was assessed by its fertilizing ability, motility and viability at day 0 (fresh), after 2 days' storage at 4 degreesC and after 2 days, 5 months and 10 months frozen at -196 degreesC using solutions containing dimethyl sulphoxide (DMSO) or glycerol as permeating cryoprotectants. Four of the best freezing solutions were used, namely, Steyn's extender (S1, S4) and Mounib's extender (M3, M4) associating 10% hen's egg yolk. Progressive sperm movement measured by CASA and expressed by the straight line velocity (VSL), the average path velocity (VAP) and the curvilinear velocity (VCL) was highly correlated with hatching rates obtained from fertilization using minimal sperm:egg ratios. After 2 days, the motility of spermatozoa frozen with DMSO and 10% egg yolk had deteriorated less than that of spermatozoa stored at 4 degreesC. Post-thaw hatching rates reflected the post-thaw sperm viability, which was cryodiluent dependent: 14.9+/-2.0% (S4), 17.0+/-4.2% (S1), 25.9+/-3.7% (M4) and 52.1+/-3.4% (M3) after 5 months of cryopreservation. The percent motility of 10-months-frozen spermatozoa was high in M3 (70.7+/-11.4%) and M4 (64.0+/-2.0%) cryoprotected sperm when measured between 5 and 20 sec after activation, but decreased rapidly to 24.3+/-8.3% (M3) and 23.0+/-9.0% (M4) between 21 and 35 sec after activation. Mounib's extender (M3, M4) provided the best cryoprotection to the spermatozoa for all post-thaw sperm quality measurements and at all freezing durations. Sperm motility was positively related to fertility. Our method will make it possible to develop even better extenders and cryoprotectants.     

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

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

Viability and fertilizing capacity of cryopreserved sperm from three North American acipenseriform species: a retrospective study

Populations of sturgeon across the globe are threatened due to unregulated harvest and habitat loss, and the status varies among species across North America. Ready access to viable and functional sperm would contribute to recovery programmes for these species. In this study, we examined the motility, viability (cell membrane integrity) of cryopreserved sperm from three North American acipenseriform species and fertilizing capacity. Milt samples were collected from captive shortnose sturgeon (Acipenser brevirostrum), wild paddlefish (Polyodon spathula) and pallid sturgeon (Scaphirhynchus albus) and cryopreserved using combinations of Modified Tsvetkova’s (MT) extender, Original Tsvetkova’s extender, and modified Hanks’ balanced salt solution, along with the cryoprotectants methanol (MeOH) or dimethyl sulfoxide (DMSO). A dual‐staining technique using the fluorescent stains SYBR‐14 and propidium iodide was employed with flow cytometry to determine the percentages of spermatozoa that were viable by virtue of having intact membranes. The percentage of viable spermatozoa ranged from 5% to 12% in shortnose sturgeon, 30–59% in paddlefish, and 44–58% in pallid sturgeon. In the first experiment with shortnose sturgeon sperm, methanol allowed for higher values for dependent variables than did DMSO, and sperm viability generally correlated with post‐thaw motility. However, fertilization rate, neurulation, or hatching rates were independent from these factors. In the second experiment with shortnose sturgeon, 5% MeOH combined with MT yielded higher values for all parameters tested than the other combinations: viability was correlated with motility, fertilization rate, and hatching rate. Overall, viability and post‐thaw motility was not affected by the use of hyperosmotic extenders (OT) or cryoprotectants (DMSO), but their use decreased fertilization percentages. For paddlefish sperm (experiment 3), MT combined with 10% MeOH was clearly a good choice for cryopreservation; viability and motility results were correlated, but independent of fertilization. For pallid sturgeon sperm (experiment 4), MT with 5–10% MeOH showed significantly higher sperm quality and fertilization parameters. Membrane integrity can be used as a predictor of fertilization by cryopreserved sperm, however additional sperm quality parameters, supplementary to motility and membrane integrity, would be useful in the refining and optimizing cryopreservation protocols with acipenseriform sperm.     

Ubiquitination and its influence in boar sperm physiology and cryopreservation

Recent reports document the potential use of the ubiquitin protein as an indicator of mammalian sperm quality or fertility, based on poor morphology, sperm count, and other cellular qualities. However, its influence on cellular physiologic mechanisms and boar sperm cryopreservation are unknown. The objective of this research was to determine the influence of boar sperm ubiquitination (n=12 boars) on motility (using CASA), and flow cytometry and fluorescent probes (in parentheses) to evaluate mitochondrial activity (JC-1), plasma and acrosomal membrane integrity (PI and FITC-PNA), membrane fluidity (M540), and chromatin stability (TUNEL) for fresh and frozen-thawed samples. The effects of ubiquitination (determined flow cytometrically) on the ability of frozen-thawed boar sperm to capacitate (FLUO-3AM) and acrosome react (FITC-PNA) were also investigated using flow cytometry. Cryopreservation induced a decrease in the percentage of sperm that were ubiquitinated from 29 to 20% (P<0.0001), but no significant effects of ubiquitin on sperm quality (motility, membrane integrities and organization) were detected. The ability of sperm to capacitate and acrosome react was influenced by ubiquitination. Samples with more ubiquitinated boar sperm were able to maintain plasma membrane integrity (PMI) better and have fewer live acrosome-reacted cells over 120min of induced capacitation (P<0.05). In conclusion, frozen-thawed ubiquitinated boar sperm were better able to survive the physical stresses of induced capacitation, yet were still capable of capacitating and acrosome reacting, which may enable use of this assay for in the vitro evaluation of the quality of boar sperm.     

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

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

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

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

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.     

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

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

DNA fragmentation in chicken spermatozoa during cryopreservation

Semen cryopreservation is fundamental both for the practice of artificial insemination, and for the conservation of genetic resources in cryobanks; nevertheless, there is still not an efficient standard freezing procedure assuring a steady and suitable level of fertility in fowl, and consequently there is no systematic use of frozen semen in the poultry industry. This study examined changes in motility (CASA), cell membrane integrity (Ethidium Bromide (EtBr) exclusion procedure and stress test) and DNA fragmentation (neutral comet assay) in fowl spermatozoa before, during and after cryopreservation and storage at −196 °C. An optimized comet assay for chicken semen was studied and applied to the analyses. Semen collected from 18 Mericanel della Brianza (local Italian breed) male chicken breeders was frozen in pellets and thawed in a water bath at 60 °C. Measurements were performed on fresh semen soon after dilution, after equilibration with 6% dimethylacetamide at 4 °C (processed semen) and after thawing. Sperm DNA damage occurred during cryopreservation of chicken semen and the proportion of spermatozoa with damaged DNA significantly increased from 6.2% in fresh and 6.4% in processed semen to 19.8% in frozen-thawed semen. The proportion of DNA in the comet tail of damaged spermatozoa was also significantly affected by cryopreservation, with an increase found from fresh (26.3%) to frozen-thawed (30.9%) sperm, whereas processed semen (30.1%) didn't show significant differences. The proportion of total membrane damaged spermatozoa (EtBr exclusion procedure) did not increase by 4 °C equilibration time, and greatly and significantly increased by cryopreservation; the values recorded in fresh, processed and frozen semen were 2.9, 5.6, and 66.7% respectively. As regards the proportion of damaged cells in the stress test, all values differed significantly (7.1% fresh semen, 11.7% processed semen, 63.7% frozen semen). Total motility was not affected by equilibration (52.1% fresh semen, 51.9% processed semen), whereas it decreased significantly after cryopreservation (19.8%). These results suggest a low sensitivity of frozen-thawed chicken spermatozoa to DNA fragmentation, therefore it should not be considered as a major cause of sperm injuries during cryopreservation.     

Fluorescent staining as a method of assessing membrane damage and post-thaw survival of ram spermatozoa

We studied the relationship between motility and membrane damage, as assessed by fluorescent staining, in fresh and in frozen-thawed ram spermatozoa. Semen from Merino rams was incubated with 6-carboxyfluorescein diacetate and propidium iodide. In both fresh and frozen-thawed samples, the percentage of intact spermatozoa was lower than the motility rate, thus indicating the presence of damaged but motile spermatozoa. Freezing and thawing resulted in a marked loss of membrane integrity, whereas motility decreased to a lesser extent. There was a positive relationship (r=0.64; P<0.001) between membrane integrity immediately after thawing and motility after 8 h of incubation at 37 degrees C. These results demonstrate the usefulness of the fluorescent staining method for the prediction of ram sperm quality and post-thaw survival.     

Long-term cryopreservation of sperm from Mandarin fish Siniperca chuatsi

In order to develop cryopreservation techniques for long-term preserving the sperm of Mandarin fish Siniperca chuatsi, we examined the effects of various extender and cryopreservation on post-thaw motility. We found the optimal freezing procedures for the Mandarin fish sperm is diluting the semen in D-15 extender, chilling it to 4 degrees C, adding ME2SO to a final concentration of 10% (v/v), then transferring the semen in cryotubes, holding the cryotubes for 10 min at 6 cm (about -180 degrees C) above the surface of liquid nitrogen, for 5 min on the surface of liquid nitrogen, and finally plunged into liquid nitrogen. After thawed at 37 degrees C for 60s, the sperm had the highest post-thaw motility (96.00+/-1.73%). The optimal fertilization procedures for the frozen sperm is mixing the eggs with sperm, then adding 1 ml of swimming medium (SM=45 mM NaCl+5 mM KCl+20mM Tris-HCl, pH 8.0) immediately. At the sperm/egg ratio of 100,000:1, the fertilization rate and the hatching rate of the frozen sperm cryopreserved for 1 week or 1 year in liquid nitrogen (66.01+/-5.14% and 54.76+/-4.40% & 62.97+/-14.28% and 52.58+/-11.17%) were similar to that of fresh sperm (69.42+/-8.11% and 59.82+/-5.27%) (p>0.05). This is the first report that the Mandarin fish (S. chuatsi) sperm can successfully fertilized eggs after long-term cryopreservation.     

In vitro characteristics of frozen-thawed, sex-sorted bull sperm after refreezing or incubation at 15 or 37 °C

The objective was to determine the in vitro characteristics of frozen-thawed dairy bull sperm after sex-sorting and refreezing and thawing (0, 2, and 4 h post-thaw; 37 °C) or post-sort incubation at 15 or 37 °C for 30 and 24 h, respectively. These sperm were compared with nonsorted frozen-thawed sperm (control) and with nonsorted sperm undergoing two cryopreservation procedures (FF; 0, 2, and 4 h). Frozen-thawed sex-sorted (FS) sperm maintained at 15 or 37 °C had higher (P < 0.001) progressive motility (PM), velocity, mitochondrial function, viability, and acrosome integrity than that of control sperm but similar total motility at 0 and 2 h of incubation. Frozen-thawed sex-sorted sperm incubated at 15 °C maintained high levels of motility (66.5 ± 1.6%) and viability/acrosome integrity (64.9 ± 1.2%) at 24 h incubation and, after rewarming and further 6 h incubation at 37 °C, acceptable levels of motility (35.8 ± 1.6%) and viability/acrosome integrity (51.2 ± 1.2%) were maintained. Frozen-thawed sex-sorted sperm maintained at 37 °C had lower levels of motility, integrity, mitochondrial respiration, and velocity from 4 h of incubation onward than that of those incubated at 15 °C. However, when frozen-thawed sex-sorted sperm were refrozen (FSF), motility and velocity were depressed at all hours compared with levels exhibited by control sperm, but membrane viability/acrosome integrity and mitochondrial respiration were similar at 0 and 2 h post-thaw. Acrosome integrity of sperm in all groups undergoing sorting was exceptionally high at 0 h (≥90%), even after re-cryopreservation and 4 h of incubation (77.5 ± 1.3%). Double frozen-thawed nonsorted sperm (FF) had similar motility to FSF sperm at 0 and 2 h post-thaw but at all time points had the lowest (P < 0.001) levels of acrosome intact/viable sperm and mitochondrial respiration and the lowest velocity at 0 h. In conclusion, whereas sex-sorting improved the functionality of frozen-thawed sperm, refreezing depressed motility, viability, and velocity but not acrosome integrity after extended incubation compared with that of control sperm. Furthermore, frozen-thawed, sex-sorted sperm may be stored for transport at 15 °C for up 24 h without detrimental effects on in vitro sperm characteristics.     

Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa using flow cytometry: the effects of extender and cryoprotectant

Although the development of semen cryopreservation in the African elephants (Loxodonta africana) has been accomplished, effective procedures for cryopreservation of Asian elephant (Elephas maximus) spermatozoa have not been established. In the present study, we investigate the freezing methods for conservation of Asian elephant spermatozoa under field conditions and identify the most suitable freezing protocols which provide acceptable post-thaw semen quality. Semen was collected from two Asian elephant bulls (EM1 and EM2, 10 ejaculates from each bull) by manual manipulation and were assessed for volume, pH, sperm cell concentration, and progressive motility. Eight out of 20 ejaculates were of acceptable quality (progressive motility >/= 60%), and were used for cryopreservation studies. Semen were frozen in TEST + glycerol, TEST + DMSO, HEPT + glycerol, or HEPT + DMSO. The post-thaw progressive sperm motilities were assessed, and sperm cells were stained with PI and FITC-PNA for membrane and acrosomal integrity assessment using flow cytometry. Post-thaw progressive motility of spermatozoa (EM1: 42.0 +/- 4.3%; EM2: 26.0 +/- 17.3%) and the percentage of membrane and acrosome intact spermatozoa (EM1: 55.5 +/- 8.1%; EM2: 46.3 +/- 6.4%) cryopreserved in TEST + glycerol were significantly higher than (P < 0.05) those frozen in the other medium investigated choices for cryopreservation of Asian elephant spermatozoa. The data support the use of TEST + glycerol as an acceptable cryopreservation media of Asian elephant semen for the establishment of sperm banks.     

Effect of cryopreservation of zona-binding capacity of canine spermatozoa in vitro

The hemizona assay (HZA) was used as a functional test for zona pellucida binding capacity of fresh and frozen-thawed canine spermatozoa. We investigated 30 ejaculates from 3 dogs with sperm motility > 70% and sperm concentration > 5.10(8) cells per ejaculate with up to 20% abnormal and dead spermatozoa. Fifteen ejaculates were each divided into 2 portions: one portion was used for analysis of fresh semen, the other for cryopreserved semen. On the day of the experiments, in vitro-matured canine oocytes were bisected into 2 equal hemizonae. One half of the hemizonae were coincubated with fresh capacitated (control) spermatozoa, the other half of the hemizonae were coincubated with frozen-thawed (tested) spermatozoa at final concentration of 1 to 2 x 10(6) cells/mL in 200 microL droplets of BSA-supplemented Toyoda, Yokojama and Hoshi (TYH) medium at 37 degrees C, 5%, CO2 for 1 h. Sperm suspensions were examined kinesigraphically for post capacitation type of movement. The Student's t-test was used to compare differences between semen parameters. The data on HZA binding activity of fresh and frozen-thawed canine semen were analyzed by ANOVA and then by the Newman-Keuls multiple range method. The results showed no differences in the initial semen quality parameters among the 3 dogs. After thawing, the semen from Dog 1 and Dog 2 demonstrated relatively uniform sperm parameters, while in Dog 3 sperm motility, and viability and the percentage of morphologically normal spermatozoa were significantly decreased. The binding activity of frozen-thawed spermatozoa from the 3 dogs was significantly reduced (29.40 +/- 9.02, 18.60 +/- 3.30, 8.20 +/- 4.49) compared with that (107.20 +/- 19.22, 109.80 +/- 20.75, 78.20 +/- 12.47; P < 0.01) of fresh spermatozoa. The results showed that semen samples with similar sperm parameters prior to cryopreservation displayed different sperm zona-binding capacity after freezing. The HZI (value of sperm binding capacity of frozen-thawed vs fresh semen samples) was higher in Dog 1 (27.43) than in Dog 2 (16.90) or Dog 3 (10.40), and thus confirmed the variation of zona binding activity after thawing between dogs. The freezability of individual dog semen is discussed. In conclusion HZA may be a valuable tool for evaluating the post-thaw fertilizing ability of canine spermatozoa.     

Effects of media,fertilization technique,extender, straw volume,and sperm to egg ratio on hatchability of cyprinid embryos,using cryopreserved semen

To enable cryopreservation of fish semen to become an efficient, routine technique, much more detailed information is required. Therefore, the present study was conducted to investigate various fertilization techniques and media, straw volumes as well as optimal semen volume for cryopreservation. The bleak (Chalcalburnus chalcalburnus) was used as the main model for investigation. Using frozen-thawed semen the fertilization rate was similar up to the morula stage, independent of the fertilization technique. Thereafter, in egg batches fertilized using the wet technique most embryo development stopped. In egg batches fertilized using the dry technique, embryonic development proceeded normally. For cryopreserved semen full activation of sperm motility was obtained at ratios of fertilization media (hatchery water and all tested types of saline solutions) to semen of 10:1. Sperm motility rate was much higher in the saline solutions than in water. In contrast hatching rates were higher when water was used as fertilization medium. Therefore, the requirements necessary for optimal sperm motility and optimal sperm-egg contact were different and so for these parameters optimal levels could not be achieved. When adjusting the freezing and thawing conditions 0.5ml straws as well as larger straws (1.2ml) proved suitable for cryopreservation of cyprinid semen. The highest fertilization rates were obtained with sperm to egg ratios of (1.3-2.5) x 10(6):1 and were 77-92% of fresh semen control. This was also similar for Ch. nasus, R. meidingerii, B. barbatus and C. carpio and suggests that the cryopreservation requirements of spermatozoa are not species specific.