Sperm cryopreservation of green swordtail Xiphophorus helleri, a fish with internal fertilization

Sperm cryopreservation for fishes with internal fertilization is essentially unexplored although many species of these fishes are valuable biomedical research models. To explore methods for sperm cryopreservation within the live-bearing genus Xiphophorus, this study used X. helleri to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio. Sperm motility and survival duration after thawing showed significant differences among different cryoprotectants with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as the cryoprotectant, the highest motility after thawing was observed with Hanks' balanced salt solution (HBSS) at 300 mOsmol/kg. Samples cooled from 5 to -80 degrees C at 20 degrees C/min yielded the highest post-thaw motility although no significant difference was found in the first 4h after thawing for cooling rates across the range of 20-35 degrees C/min. Evaluation of equilibration time revealed no significant difference between 20 min and 2h, but the highest motility at 10 min after thawing was found with a 20-min equilibration. Dilution ratios of sperm-to-extender at 1:20, 1:60, and 1:120 showed no significant differences in motility and survival duration after thawing, but the dilution of sperm solutions with HBSS (320 mOsmol/kg) immediately after thawing reduced the decline of sperm motility, and significantly prolonged the survival duration. Based on these findings, the highest average sperm motility (77%) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsmol/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS-glycerol of 1:20, equilibrated for 10 min, cooled at 20 degrees C/min from 5 to -80 degrees C before being plunged in liquid nitrogen, and thawed in a 40 degrees C water bath for 7s. If diluted immediately after thawing, sperm frozen by the protocol above retained continuous motility after thawing for more than 8 days when stored at 4 degrees C.     

Refrigerated storage and cryopreservation of black drum (Pogonias cromis) spermatozoa

Procedures were developed for the collection, refrigerated storage and cryopreservation of black drum spermatozoa. Sperm samples were collected by removing and slicing the testis, and suspending the spermatozoa in Hanks' balanced salt solution (HBSS) at 200 mOsm/kg. Threshold activation (10%) of black drum spermatozoa occurred at 370 mOsm/kg, and complete activation occurred at 580 mOsm/kg in HBSS. Sperm cells activated in artificial seawater had higher motility than those activated in HBSS at osmolalities from 350 to 500 mOsm/kg. Spermatozoa stored at 4 degrees C in HBSS or artificial seawater at osmolalities from 202 to 290 mOsm/kg retained motility longer than did those stored at other osmolalities Dilution rate had no effect on sperm storage time at 4 degrees C. Four chemicals were evaluated as cryoprotectants: dimethyl sulfoxide (DMSO), n,n-dimethyl acetamide (DMA), methanol, and glycerol. Glycerol and DMA at concentrations of 10% significantly reduced motility within 52 min. Spermatozoa were cryopreserved at 3 freezing rates (-27, -30, or -45 degrees C/min) in a nitrogen vapor shipping dewar or a computer-controlled freezer. Spermatozoa frozen using 10% DMSO had the highest post-thaw motility at a freezing rate of -27 or -30 degrees C/min. Spermatozoa frozen using 5% glycerol, 5% DMSO, or 10% DMSO had the highest post-thaw motility at a freezing rate of -45 degrees C/min.     

Initial studies on sperm cryopreservation of a live-bearing fish, the green swordtail Xiphophorus helleri

Swordtails and platyfish of the genus Xiphophorus are valuable models for biomedical research and are also commercially raised as ornamental fish valued by aquarists. While research use and commercial interest increases yearly in these fish, cryopreservation of sperm is unexplored in this genus. Xiphophorus are live-bearing fishes characterized by small body sizes, limited sperm volumes, and internal fertilization, an atypical reproductive mode for fish. These attributes make research involving cryopreservation of Xiphophorus germplasm challenging. To explore methods for sperm cryopreservation, this study evaluated the effect of different loading volumes of sperm suspension in 0.25-ml French straws, different dilution ratios of sperm to extender, an osmolality range of extender without cryoprotectant and with dimethyl sulfoxide (DMSO) as cryoprotectant, and short-term storage at room temperature and 4 degrees C after thawing. No significant difference in sperm motility due to straw loading volume was observed after thawing. Sperm motility was observed to decrease with increasing dilution. The osmolality of Hanks' balanced salt solution (HBSS) without cryoprotectant in which the highest sperm motility (67%) was observed was 320 +/- 3 mOsm/kg, which was also the osmolality of X. helleri blood plasma. When cryopreserved with 10% DMSO, however, the highest motilities within 10 min after thawing were observed with HBSS in the range of 240-300 mOsm/kg. Sperm suspended in HBSS at 320 mOsm/kg with a dilution factor of 100 maintained motility for 24h at room temperature, but persisted for 10 days when stored at 4 degrees C. These results provided the first evidence that cryopreservation may be applied to conservation of genetic resources in live-bearing fishes.     

Effects of centrifugation, glycerol level, cooling to 5 degrees C, freezing rate and thawing rate on the post-thaw motility of equine sperm

Five experiments evaluated the effects of processing, freezing and thawing techniques on post-thaw motility of equine sperm. Post-thaw motility was similar for sperm frozen using two cooling rates. Inclusion of 4% glycerol extender was superior to 2 or 6%. Thawing in 75 degrees C water for 7 sec was superior to thawing in 37 degrees C water for 30 sec. The best procedure for concentrating sperm, based on sperm motility, was diluting semen to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium at 20 degrees C and centrifuging at 400 x g for 15 min. There was no difference in sperm motility between semen cooled slowly in extender with or without glycerol to 5 degrees C prior to freezing to -120 degrees C and semen cooled continuously from 20 degrees C to -120 degrees C. From these experiments, a new procedure for processing, freezing and thawing semen evolved. The new procedure involved dilution of semen to 50 x 10(6) sperm/ml in centrifugation medium and centrifugation at 400 x g for 15 min, resuspension of sperm in lactose-EDTA-egg yolk extender containing 4% glycerol, packaging in 0.5-ml polyvinyl chloride straws, freezing at 10 degrees C/min from 20 degrees C to -15 degrees C and 25 degrees C/min from -15 degrees C to -120 degrees C, storage at -196 degrees C, and thawing at 75 degrees C for 7 sec. Post-thaw motility of sperm averaged 34% for the new method as compared to 22% for the old method (P<0.01).     

The use of cryomicroscopy in guppy sperm freezing

The present study employed cryomicroscopy to derive an optimal sperm freezing protocol for guppy (Poecilia reticulata) sperm. Evaluation criteria during the freezing-thawing process were assessed for nucleation temperature (Tn), temperature when more than 50% of sperm display bending mid-piece (Tb), temperature when more than 80% of sperm stop moving (Tm), thawing temperature (Tt), and post-thaw motility. We compared four different cryoprotectants: 5% N-dimethyl formamide (DMF), 6% methanol (MEOH), 10% dimethyl sulfoxide (DMSO), and 14% glycerol, as well as glycerol at different concentrations of 7-50%; cooling and rewarming rates ranged from 5 to 100 °C/min. The protocol that yielded the highest post-thaw motility was samples suspended in 14% glycerol, cooled at 25 °C/min, and thawed at 100 °C/min, which was in complete agreement with our previous findings derived from a controlled-rate freezer. In addition, Tb and Tm were found to be negatively correlated with post-thaw motility, suggesting their possible role in predicting freezing success. The present study for the first time demonstrated the usefulness of cryomicroscopy in deriving an optimal sperm freezing protocol for aquatic species.     

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of egg yolk, glycerol and cooling rate

Two experiments were conducted to determine the effects of egg yolk (EY), glycerol, and cooling rate on the cryosurvival of red deer epididymal spermatozoa. The aim of Experiment 1 was to examine the effects of two EY types (clarified EY, CE, prepared by centrifugation, and whole EY, WE), and four EY concentrations (0, 5, 10 and 20%) on cryosurvival of red deer epididymal spermatozoa. Sperm samples were diluted to a final sperm concentration of approximately 200 x 10(6)spermatozoa/ml with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility, viability and of plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Cryopreservation of red deer epididymal spermatozoa frozen in a clarified EY extender, and with a 20% EY resulted in more vigorous post-thaw and post-incubation motilities (P<0.0001). Moreover, our results showed that regardless of the egg yolk concentration tested, the best sperm quality was obtained with the use of CE. Therefore, the objective of Experiment 2 was to explore the post-thaw effects of four clarified egg yolk concentrations (0, 5, 10 and 20%), two final glycerol concentrations (3 and 6%), and two cooling rates from 22 to 5 degrees C (slow: 0.23 degrees C/min; rapid: 4.2 degrees C/min) on red deer epididymal spermatozoa. At thawing, the effects of CE and glycerol concentrations, and cooling rate, all independently affected post-thaw sperm quality, while there were no effects of interactions on post-thawing sperm quality. Therefore, we studied each variable separately. Differences (P<0.05) for most of the semen parameters evaluated were found between the two final glycerol concentrations tested, with the high values after thawing found with the use of 6% glycerol (58.8+/-1.4 versus 46.2+/-1.4, for sperm motility). Moreover, the cooling rate did not have an effect on the semen characteristics, except for NAR (P<0.05), with the high values after thawing found with the use of the rapid protocol (64.5+/-1.4 versus 59.9+/-1.4). In conclusion, the use of 20% CE and 6% glycerol in combination with a rapid cooling rate, significantly improved red deer epididymal spermatozoa freezability.     

Rhesus monkey sperm cryopreservation with TEST-yolk extender in the absence of permeable cryoprotectant

Recently, there has been increased interest in ultra-rapid freezing with mammalian spermatozoa, especially for vitrification in the absence of cryoprotectants. Sperm cryopreservation in non-human primates has been successful, but the use of frozen-thawed sperm in standard artificial insemination (AI) remains difficult, and removal of permeable cryoprotectant may offer opportunities for increased AI success. The present study intended to explore the possibility of freezing rhesus monkey sperm in the absence of permeable cryoprotectants. Specifically, we evaluated various factors such as presence or absence of egg yolk, the percentage of egg yolk in the extenders, and the effect of cooling and thawing rate on the success of freezing without permeable cryoprotectants. Findings revealed that freezing with TEST in the absence of egg yolk offers little protection (<15% post-thaw motility). Egg yolk of 40% or more in TEST resulted in decreased motility, while egg yolk in the range of 20-30% yielded the most motile sperm. Cooling at a slow rate (29 °C/min) reduced post-thaw motility significantly for samples frozen with TEST-yolk alone, but had no effect for controls in the presence of glycerol. Similarly, slow thawing in room temperature air is detrimental for freezing without permeable cryoprotectant (<2% motility). In addition to motility, the ability of sperm to capacitate based on an increase in intracellular calcium levels upon activation with cAMP and caffeine suggested no difference between fresh and frozen-thawed motile sperm, regardless of treatment. In summary, the present study demonstrates that ejaculated and epididymal sperm from rhesus monkeys can be cryopreserved with TEST-yolk (20%) in the absence of permeable cryoprotectant when samples were loaded in a standard 0.25-mL straw, cooled rapidly in liquid nitrogen vapor at 220 °C/min, and thawed rapidly in a 37 °C water bath. This study also represents the first success of freezing without permeable cryoprotectant in non-human primates.     

Centrifugation and addition of glycerol at 22 degres C instead of 4 degrees C improve post-thaw motility and fertility of stallion spermatozoa

The aims of this study were to evaluate the effects of cooling rate to 4 degrees C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 degrees C (14 ejaculates), a moderate cooling rate (37 degrees C to 4 degrees C in I h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 degrees C to 4 degrees C in 4 h) (39%; P<0.05). When centrifugation/glycerol-addition was performed at 22 degrees C (37 degrees C to 22 degrees C in 10 min) (10 ejaculates), post-thaw motility was lower when spermatozoa were frozen directly from 22 degrees C (23%) than when spermatozoa were cooled to 4 degrees C (22 degrees C to 4 degrees C in 1 h) before freezing (47%; P<0.0001). When centrifugation/glycerol-addition was performed at 22 degrees C (before cooling at a moderate rate), as opposed to 4 degrees C (after cooling at a moderate rate), a significant improvement of 1) recovery of spermatozoa after centrifugation (P<0,0001), 2) post-thaw motility of spermatozoa at thawing (40% vs 36% (n < or = 291 ejaculates/group), P<0.0001) and 3) per-cycle fertility (56% vs 42% (n > or = 190 cycles/group), P<0.01) was observed. In conclusion, centrifugation/glycerol-addition at 22 degrees C followed by cooling to 4 degrees C at a moderate rate results in an improvement of post-thaw motility, spermatozoa recovery rate and per cycle fertility.     

Post-thaw functional status of boar spermatozoa cryopreserved using three controlled rate freezers: a comparison

This study compared variation in the quality of cryopreserved boar spermatozoa and the control and accuracy of cooling rates between three semen freezers (CryoLogic Freeze Control CL3000, Planer Products Kryo Save Compact KS1.7/Kryo 10 Control module and a controlled rate 'Watson' freezing machine developed within our laboratory). Five ejaculates were collected from each of 15 boars (five boars from each of three breeds). Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% v/v glycerol) and placed into 0.5 ml straws. Three straws per treatment, from each ejaculate were cooled to -5 degrees C at 6 degrees C/min, held at -5 degrees C for 30s while ice crystal formation was induced, then further cooled from -5 to 80 degrees C at either 40 degrees C/min (Kryo Save Compact KS1.7 and Watson) or 6 degrees C/min (Freeze Control CL3000). Precise measurements of temperature fluctuations during the programmed cooling curves were made by inserting thermocouples into the semen filled straws. Semen was assessed for %motile cells, motility characteristics using computer-assisted semen analysis (CASA), plasma membrane integrity (%SYBR-14 positive stained spermatozoa) and acrosome integrity (%FITC-PNA positive stained spermatozoa). Spermatozoa cryopreserved using the Freeze Control CL3000 system (maximum rate of 6 degrees C/min) exhibited reduced post-thaw viability (14.2+/-2.8% mean plasma membrane intact spermatozoa) when compared to both the KS1.7 and Watson freezers (optimal rate of 40 degrees C/min) (18.4+/-3.2 and 25.7+/-3.7% mean plasma membrane intact spermatozoa, respectively). Differences in motility characteristics were observed between spermatozoa cryopreserved at 40 degrees C/min with the Watson apparatus preserving a larger proportion of sperm with progressive motility. Cooling curves in the CL3000 and KS1.7 were interrupted by a pronounced increase in temperature at -5 degrees C that corresponded with the latent heat of fusion released with ice crystal formation. This temperature change was significantly reduced in the cooling curves produced by the Watson freezer. These findings suggest that preserving spermatozoa using the Watson freezer improved post-thaw semen quality, with regard to sperm motility characteristics. Furthermore, that post-thaw semen viability was enhanced by minimising temperature fluctuations resulting from the release of the latent heat of fusion at ice crystal formation.     

Sensitivity of domestic cat (Felis catus) sperm from normospermic versus teratospermic donors to cold-induced acrosomal damage.

Freeze-thawing cat sperm in cryoprotectant results in extensive membrane damage. To determine whether cooling alone influences sperm structure and viability, we compared the effect of cooling rate on sperm from normospermic (N; > 60% normal sperm per ejaculate) and teratospermic (T; < 40% normal sperm per ejaculate) domestic cats. Electroejaculates were divided into raw or washed (Ham's F-10 + 5% fetal calf serum) aliquots, with the latter resuspended in Ham's F-10 medium or Platz Diluent Variant Filtered without glycerol (20% egg yolk, 11% lactose). Aliquots were 1) maintained at 25 degrees C (no cooling; control), 2) cooled to 5 degrees C in a commercial refrigerator for 30 min (rapid cooling; approximately 4 degrees C/min), 3) placed in an ice slush at 0 degrees C for 10 min (ultrarapid cooling; approximately 14 degrees C/min), or 4) cooled to 0 degrees C at 0.5 degrees C/min in a programmable alcohol bath (slow cooling); and aliquots were removed every 4 degrees C. All samples then were warmed to 25 degrees C and evaluated for percentage sperm motility and the proportion of intact acrosomes using a fluorescein-conjugated peanut agglutinin stain. In both cat populations, sperm percentage motility remained unaffected (p > 0.05) immediately after exposure to low temperatures and after warming to 25 degrees C. However, the proportion of spermatozoa with intact acrosomes declined (p < 0.05) after rapid cooling ( approximately 4 degrees C/min) to 5 degrees C (N, 65.6%; T, 27.5%) or ultrarapid cooling ( approximately 14 degrees C/min) to 0 degrees C (N, 62.1%; T, 23.0%) in comparison to the control value (N, 81.5%; T, 77.5%). Transmission electron microscopy of cooled sperm revealed extensive damage to acrosomal membranes. In contrast, slow cooling (0.5 degrees C/min) to 5 degrees C maintained (p > 0.05) a high proportion of spermatozoa with intact acrosomes (N, 75.5%; T, 68.3%), which also remained similar (p > 0.05) between cat populations (N, 64.7%; T, 56.8%) through continued cooling to 0 degrees C. Results demonstrate that 1) rapid cooling of domestic cat sperm induces significant acrosomal damage without altering sperm motility, 2) spermatozoa from teratospermic males are more susceptible to cold-induced acrosomal damage than normospermic counterparts, and 3) reducing the rate of initial cooling markedly decreases sperm structural damage.     

Effects of cryopreservation methods on post-thaw motility of spermatozoa from the Japanese pearl oyster, Pinctada fucata martensii

In order to develop cryopreservation techniques for Japanese pearl oyster spermatozoa, the effects of various cryopreservation conditions on post-thaw motility were examined. Spermatozoa cryopreserved with 10% methanol (MET), dimethylformamide or dimethylacetamide plus 90% diluent comprising 80% seawater and 20% fetal bovine serum (FBS) showed higher percentages of post-thaw motility than those cryopreserved with 10% dimethylsulfoxide or glycerol. When spermatozoa were cryopreserved with various concentrations (0-20%) of MET and 100-80% diluent, 10% MET showed the highest percentages of post-thaw motility. When spermatozoa were cryopreserved with 10% MET and 90% diluent comprising various concentrations (0-100%) of FBS or Ringer solution mixed with seawater, the percentages of post-thaw motility peaked at 20% FBS or Ringer solution, and were significantly higher for 20% FBS than for 20% Ringer solution. The percentages of post-thaw motility increased with increasing dilution ratios from 2.5- to 50-fold. Spermatozoa cooled to -50 degrees C and then immersed in liquid nitrogen (LN) showed higher post-thaw motility than those cooled to -30 degrees C or -40 degrees C. When spermatozoa were cryopreserved to -50 degrees C at various cooling rates by changing the sample height above the LN surface, the post-thaw motilities of spermatozoa cooled at 10 cm (cooling rate: -21.3 degrees C/min) and 12.5 cm (-15.6 degrees C/min) from the LN surface were higher than those at 5, 7.5 or 15 cm. These results indicate that 10% MET plus 90% diluent comprising 80% seawater and 20% FBS is a suitable extender for cryopreservation of Japanese pearl oyster spermatozoa and that samples should be cooled to -50 degrees C at a cooling rate between -15 and -20 degrees C/min for efficient storage.     

Semen cryopreservation in Bactrian camel (Camelus bactrianus) using SHOTOR diluent: effects of cooling rates and glycerol concentrations

Experiments were conducted with a final goal of providing a suitable protocol for cryopreservation of Bactrian camel semen. In Experiment I, the effect of average cooling rate (slow cooling: 0.14 versus fast cooling: 0.55 degrees C/min) on the viability of chilled semen was evaluated. In Experiment II, the effect of different concentrations of glycerol (4, 6 and 8%) on the post-thaw viability of frozen sperm was investigated. In Experiment III, the efficiency of SHOTOR diluent was compared with IMV buffers for the cryopreservation of camel semen. Viability parameters including progressive forward motility (PFM), plasma membrane integrity and percentage of live spermatozoa were assessed. Progressive forward motility of sperm cooled at the faster rate was superior after incubating for 24h at 4 degrees C compared to that cooled at the slower rate (P<0.05). Post-thaw viability of Bactrian camel sperm was better using a final glycerol concentration of 6% compared to 4 and 8% (P<0.05). Progressive forward motility of frozen-thawed sperm was greater using SHOTOR diluent (29.9%) compared to IMV buffers (4.2%, P<0.05). In conclusion, semen cryopreservation in Bactrian camel is feasible when it is extended in SHOTOR diluent, cooled within 1h (average cooling rate: 0.55 degrees C/min) to 4 degrees C, and then exposed to glycerol, at the final concentration of 6%.     

Effects of cooling and warming rate to and from -70 degrees C, and effect of further cooling from -70 to -196 degrees C on the motility of mouse spermatozoa

We have previously reported high survival in mouse sperm frozen at 21 degrees C/min to -70 degrees C in a solution containing 18% raffinose in 0.25 x PBS (400 mOsm) and then warmed rapidly at approximately 2000 degrees C/min, especially under lowered oxygen tensions induced by Oxyrase, a bacterial membrane preparation. The best survival rates were obtained in the absence of glycerol. The first concern of the present study was to determine the effects of the cooling rate on the survival of sperm suspended in this medium. The sperm were cooled to -70 degrees C at rates ranging from 0.3 to 530 degrees C/min. The survival curve was an inverted "U" shape, with the highest motility occurring between 27 and 130 degrees C/min. Survival decreased precipitously at higher cooling rates. Decreasing the warming rate, however, decreased survivals at all cooling rates. The motility depression with slow warming was especially evident in sperm cooled at the optimal rates. This fact is consistent with our current view that the frozen medium surrounding sperm cells is in a metastable state, perhaps partly vitrified as a result of the high concentrations of sugar. The decimation of sperm cooled more rapidly than optimum (>130 degrees C/min), even with rapid warming, is consistent with the induction of considerable quantities of intracellular ice at these rates. When glycerol was added to the above medium, motilities were also dependent on the cooling rate, but they tended to be substantially lower than those obtained in the absence of glycerol. The minimum temperature in the above experiments was -70 degrees C. When sperm were frozen to -70 degrees C at optimum rates, lowering the temperature to -196 degrees C had no adverse effect.     

Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: (1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me₂SO), N,N-dimethylacetamide (DMA), N,N-dimethyl formamide (DMF), and glycerol with concentrations of 5%, 10%, and 15% for 60 min of incubation at 4 °C; (2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and (3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5% and 10%) did not change the sperm motility after 30 min; Me₂SO, DMA, and DMF (10% and 15%) and glycerol (5%, 10% and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 to 25 °C/min) and were compared to Me₂SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ? 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ?10% which was significantly lower than that of methanol and ME. With Me₂SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ? 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.     

Semen cryopreservation of small abalone (Haliotis diversicolor supertexa)

Methods for cryopreserving spermatozoa and maximizing fertilization rate in Taiwan small abalone, Haliotis diversicolor supertexa, were developed. The gametes (spermatozoa and eggs) of small abalone were viable 3 h post-spawning, with fertilization, and development rate decreasing with time. A minimum of 10(2) cell/ml sperm concentration and a contact time of 2 min between gametes is recommended for artificial insemination of small abalone eggs. Eight cryoprotectants, dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), ethylene glycol (EG), propylene glycol (PG), butylene glycol (BG), polyethylene glycol, glycerol and methanol, were tested at concentrations between 5 and 25% to evaluate their effect on motility of spermatozoa exposed to cryoprotectant for up to 60 min at 25 degrees C before freezing. The least toxic cryoprotectant, 10% DMSO, was added to artificial seawater (ASW) to formulate the extender for freezing. Semen was diluted 1:1 with the extender, inserted into 1.5 ml microtubes and frozen using a cooling rate between -3.5 and -20 degrees C/min to various transition temperatures (0, -30, -60, -90 and -120 degrees C), followed by transfer and storage in liquid nitrogen (-196 degrees C). The microtubes were thawed from +45 to +145 degrees C/min. Spermatozoa, cooled to -90 degrees C at a cooling rate of -12 or -15 degrees C/min and then immersed in liquid nitrogen, had the best post-thaw motility. Post-thaw sperm motility was markedly reduced compared to fresh sperm. More frozen-thawed spermatozoa are required to achieve fertilization rates comparable to those achieved using fresh spermatozoa.     

Freeze-induced membrane damage in ram spermatozoa is manifested after thawing: observations with experimental cryomicroscopy.

Cryoinjury in ram sperm was investigated by direct observation, using cryomicroscopy, to validate model hypotheses of freezing injury in such a specialized cell. Fluorescein diacetate was used to determine when during the freeze-thaw cycle the sperm membrane became permeable. In noncryoprotected sperm plasma membrane, integrity was maintained throughout the cooling and freezing process, but fluorescein leakage occurred during rewarming. The temperature of post-thaw permeabilization varied in relation to the minimum temperature reached during freezing; cells cooled to -10 degrees C retained fluorescence into the post-thaw temperature range of 9-24 degrees C (mean +/- SEM; 13.25 +/- 0.91 degrees C), whereas cells cooled to -20 degrees C lost fluorescence shortly after thawing (mean +/- SEM; 2.62 +/- 0.91 degrees C). Sperm cooled to 5 degrees C, but not frozen, retained fluorescence during rewarming up to 20-30 degrees C. The inclusion of glycerol and egg yolk in the freezing medium significantly and independently increased the post-thaw permeabilization temperature. Maintenance of fluorescence was also correlated with ability to resume motility after thawing. Sperm reactivation experiments were undertaken to examine deleterious effects of freezing upon the flagellar microtubular assembly. No direct evidence for such effects was obtained. Instead, a highly significant correlation between minimum freezing temperature and post-thaw temperature of initial reactivation was detected.     

Effect of osmotic immobilization on refrigerated storage and cryopreservation of sperm from a viviparous fish, the green swordtail Xiphophorus helleri

In this study, refrigerated storage and cryopreservation of sperm from the green swordtail Xiphophorus helleri were investigated. Previous cryopreservation research in this species utilized motile sperm because unlike in most fish species, Xiphophorus sperm can remain continuously motile after collection for a week with refrigerated storage. However, this species reproduces by internal fertilization, and given the significant requirements for motility within the female reproductive tract and potential limitations on sperm energetic capacities, immobilization of sperm prior to insemination could be used to improve fertilization success. Thus, the goal in this study was to use osmotic pressure to inhibit the motility of sperm after collection from X. helleri, and to test the effect of immobilization on refrigerated storage and cryopreservation. The objectives were to: (1) estimate the motility of sperm at different osmotic pressures, and determine an osmotic pressure suitable for immobilization; (2) cryopreserve the immobilized sperm, and estimate the motility after thawing with or without dilution, and (3) compare motility of non-immobilized and immobilized sperm after thawing, centrifugation, and washing to remove cryoprotectant. Motility was determined when sperm were suspended in 11 different osmotic pressures (24-500 mOsmol/kg) of Hanks' balanced salt solution (HBSS). Motility was observed between 116 and 425 mOsmol/kg. Sperm were not motile when the osmolality was lower than 116 or higher than 425 mOsmol/kg. Motility of the immobilized (non-motile) sperm could be activated by changing the osmotic pressure to 291-316 mOsmol/kg, and motility of immobilized sperm from hypertonic HBSS (425 mOsmol/kg) was significantly higher than that from hypotonic HBSS (145 mOsmol/kg) after 48 h of storage. At an osmolality of 500 mOsmol/kg, HBSS was used as extender to maintain immobilized sperm during cryopreservation with glycerol as the cryoprotectant. High motility (approximately 55%) was obtained in sperm after thawing when cryopreserved with 10-15% glycerol, and dilution of thawed sperm in fresh HBSS (1:4; V:V) was found to decrease the motility significantly. No difference was found in the motility of thawed sperm cryopreserved with 14% glycerol and extended in 310 and 500 mOsmol/kg HBSS. Washing by centrifugation prolonged the motility of thawed sperm from 24 to 72 h in HBSS at 310 and 500 mOsmol/kg. This study showed that sperm from X. helleri could be immobilized by use of specific osmotic pressures, and that the immobilization did not affect sperm motility after thawing. The immobilization of sperm by osmotic pressure could minimize reduction of the energetic capacities necessary for insemination, traversal, and residence within the female reproductive tract, and fertilization.     

Effects of low-molecular-weight fractions (LMWF) from milk, egg yolk, and seminal plasma on freezability of bovine spermatozoa

The effects of the dialyzable fractions from bovine seminal plasma, egg yolk, and milk and of two buffer systems (TEST and sodium citrate) on post-thaw sperm motility were studied. Each basic salt solution was used in the experimental design. These solutions were used as extender systems in combination with egg yolk and glycerol. After collection, semen samples were extended (1:20), cooled to 5 degrees C in 1.5 hr, and frozen in 0.5-cc French straws after 3 hr of equilibration. Post-thaw samples were assayed for percentage of motile cells immediately after thawing and after 4 hr of incubation at room temperature (22 degrees C). Egg yolk (25%) provided the same protection as did the combination of colloidal material present in the skim milk-yolk extenders. The use of TEST as a buffer provided significantly higher (P less than 0.01) sperm post-thaw motility than milk salts or Na citrate. Sperm survival in extenders containing high concentrations of seminal plasma and/or egg yolk salts was significantly lower (P less than 0.01). Spermatozoa frozen in the presence of 6% glycerol resulted in sperm motility significantly (P less than 0.05) higher than that of spermatozoa frozen with 3% glycerol. However, no difference was observed between these two concentrations when TEST solution was used.     

Systematic factor optimization for sperm cryopreservation of tetraploid Pacific oysters, Crassostrea gigas

The availability of tetraploid Pacific oysters provides a unique opportunity for comparative studies of sperm cryopreservation between diploids and tetraploids. In parallel to studies with sperm from diploid oysters, this study reports systematic factor optimization for sperm cryopreservation of tetraploid oysters. Specifically, this study evaluated the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), and straw size. Similar to sperm from diploids, the optimal cooling rate was 5 degrees C/min to -30 degrees C, followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations showed that a combination of the cryoprotectants 6% polyethylene glycol/4% propylene glycol and 6% polyethylene glycol/4% dimethyl sulfoxide yielded consistently high post-thaw motility. A long equilibration (60 min) yielded higher percent fertilization, and confirmed that extended equilibration could be beneficial when low concentrations of cryoprotectant are used. There was no significant difference in post-thaw motility between straw sizes of 0.25 and 0.5 mL. Despite low post-thaw fertilization (<10%) in general for sperm from tetraploids, optimized protocols in the present study effectively retained post-thaw motility for sperm from tetraploid oysters. This study confirmed that sperm from tetraploid Pacific oysters were more negatively affected by cryopreservation than were those of diploids. One possible explanation is that sperm from these two ploidies are different in their plasma membrane properties (e.g., structure, permeability, and elasticity), and the plasma membrane of sperm from tetraploids is more sensitive to cryopreservation effects. The fact that combinations of non-permeating and permeating cryoprotectants improved post-thaw motility in sperm from tetraploids provided presumptive evidence for this interpretation.     

Effects of extender composition,cooling rate,and freezing on the motility of sea bass (Dicentrarchus labrax,L.) spermatozoa after thawing

A successful cryopreservation procedure for sperm must guarantee recovery of the morphological and functional characteristics of the cells following thawing so that preserved semen can to be used comparably with non-preserved semen. The aim of this work was to identify a species-specific freezing protocol for sea bass (Dicentrarchus labrax) spermatozoa by optimising all the stages in the cryopreservation procedure. In the first stage of the experiments, the cryoprotectants and the relative concentrations that had the least toxic effect on motility at room temperature were selected. The capacity of the selected cryoprotectant substances was then assessed in freezing tests as follows: dimethyl sulfoxide (Me(2)SO) 5% and 7%, ethylene glycol (EG) 7% and 10%, propylene glycol (PG) 7% and 10%. The cryoprotectant that gave the best results in this second stage of the experiments was EG 10%, and this was then used for the optimisation of the different stages in the freezing procedure: two different times of adaptation to the cryoprotectant were tested (15min and 6h), as well as the effects of adding an energy substrate (1.25mM sodium pyruvate) to assess its possible use as an energy source. Lastly, using the extender (diluent+Na-pyruvate+EG10%) and the adaptation procedure (6h at 0-2 degrees C) that had given the best results in the preceding stages of the experiments, four cooling rates were tested: 10, 12, 15, 24 degrees C/min. It was shown that the semen that was diluted immediately after collection in extender that contained the cryoprotectant (EG 10%), was equilibrated for 6h at 0-2 degrees C and then cooled at a rate of 15 degrees C/min, showed motility on thawing comparable to that of fresh semen (P=0.045).