Cryopreservation of seabream (Sparus aurata) spermatozoa

The aim of this research was to optimize protocols for freezing spermatozoa of seabream (Sparus aurata). All the phases of the cryopreservation procedure (sampling, choosing the cryoprotective extender, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa under examination, so as to be able to restore on thawing the morphological and physiological characteristics of fresh semen. Seabream spermatozoa were collected by stripping and transported to the laboratory chilled (0-2 degrees C). Five cryoprotectants, dimethyl sulfoxide (Me(2)SO), ethylene glycol (EG), 1,2-propylene glycol (PG), glycerol, and methanol, were tested at concentrations between 5 and 15% by volume to evaluate their effect on the motility of semen exposed for up to 30 min at 26 degrees C. The less toxic cryoprotectants, 10% EG, 10% PG, and 5% Me(2)SO, respectively, were added to 1% NaCl to formulate the extenders for freezing. The semen was diluted 1:6 with the extender, inserted into 0.25-ml plastic straws by Pasteur pipette, and frozen using a cooling rate of either 10 or 15 degrees C/min to -150 degrees C followed by transfer and storage in liquid nitrogen (-196 degrees C). The straws were thawed at 15 degrees C/s. On thawing, the best motility was obtained with 5% Me(2)SO, although both 10% PG and EG showed good results; no differences were found between the two freezing gradients, although semen frozen with the 10 degrees C/min gradient showed a slightly higher and more prolonged motility.     

Effects of extender composition, cooling rate and freezing on the fertilisation viability of spermatozoa of the Pacific oyster (Crassostrea gigas)

The aim of this research was to optimise protocols for freezing spermatozoa of the Pacific oyster. All the phases of the cryopreservation procedure (choice of cryoprotectant, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa to restore on thawing the morphological and physiological characteristics of fresh semen. The choice of type and concentration of cryoprotectant in which semen is incubated before freezing is fundamental for a successful cryopreservation: the cryoprotectants (dimethylsulfoxide--Me(2)SO, ethylene glycol--EG, propylene glycol-PG, and glycerol in concentrations between 5 and 15%) were tested for their toxicity on the semen exposed up to 30 min at +26 degrees C (room temperature) by evaluating its ability to fertilise and the embryo development to the regular D larval stage. The best cryoprotectants, Me(2)SO, EG, and PG 5, 10, and 15% respectively, were used for the pre-cooling (adaptation/cooling) tests. Two different adaptation/cooling procedures were tested: (A) from +26 degrees C to 0-2 degrees C (2.6 degrees C/min) and (B) at +26 degrees C for 15 min. Lastly, using the cryoprotectants and the adaptation procedure (B) that had given the best results in the preceding stages of the experiment, four cooling rates were tested: 6, 11, 16, and 21 degrees C/min. It was seen that the semen that was incubated with EG 10%, adapted at +26 degrees C for 15 min, and then cooled at a rate of 6 degrees C/min showed a percentage of regular D larvae on thawing comparable to that of fresh semen (p > 0.05).     

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).     

Sperm cryopreservation of a live-bearing fish, the platyfish Xiphophorus couchianus

The objectives of this study were to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio, as well as somatic relationships of body length, body weight, and testis weight to sperm density in the platyfish Xiphophorus couchianus. Sperm motility and survival duration after thawing were significantly different between cryopreservation with dimethyl sulfoxide (DMSO) and glycerol, with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as cryoprotectant, the highest motility after thawing was observed with Hanks' balanced salt solution (HBSS) across a range of 240-300 mOsm/kg. Samples cooled from 5 to -80 degrees C at 25 degrees C/min yielded the highest post-thaw motility, although no significant difference was found for cooling rates across the range of 20-30 degrees C/min. In addition, the highest motility after thawing was found in samples equilibrated from 10 to 30 min with 14% glycerol and cooled at 25 degrees C/min. The post-thaw motility declined rapidly with use of 10% glycerol and cooling at 5 degrees C/min across the equilibration range of 10 min to 2h. Sperm motility with a dilution ratio of sperm to extender of 1:10 was not different at 10 min after thawing with those samples at greater dilutions, but declined significantly from Day 1 after thawing and showed lower survival duration when stored at 4 degrees C. However, the additional dilution of sperm solutions with HBSS (300 mOsm/kg) immediately after thawing significantly slowed the decline of motility and prolonged the duration of survival. Based on the above findings, the highest average sperm motility (78+/-3 %) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsm/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS-glycerol of 1:20, equilibrated for 10 min, cooled at 25 degrees C/min from 5 to -80 degrees C before plunging into liquid nitrogen, and thawed at 40 degrees C in a water bath for 7 s. If diluted within 5 h after thawing, sperm frozen by the above protocol retained continuous motility for 15 days when stored at 4 degrees C.     

Semen characteristics, cryopreservation, and successful artificial insemination in the Blue rock pigeon (Columba livia)

The present study was undertaken in the Blue rock pigeon (Columba livia) to evaluate the annual semen characteristics, to identify a suitable extender for semen short-term storage, to determine a protocol for cryopreservation of semen and finally to check whether intracloacal insemination would lead to the birth of a chick. Semen characteristics such as semen volume, sperm concentration, sperm motility, and percentage of normal spermatozoa were maximum during the monsoon season. TALP was observed to be the most suitable semen extender and the sperm survived best at 37 degrees C at a dilution of 1:100 in TALP. Further, cryopreservation studies on pigeon semen indicated that 8% DMSO with or without egg yolk (20%) proved to be a better cryoprotectant compared to glycerol and polyethylene glycol. In addition, the slow freezing protocol was better than the fast-freezing protocol and about 40% of the cryopreserved spermatozoa were motile following thawing. Computer-aided semen analysis indicated that pigeon spermatozoa were extremely active immediately after dilution in TALP and exhibited linear trajectories persisting up to 9h. But, with time there was a time-dependent decrease in the velocity parameters (VAP, VSL, and VCL). Cryopreserved spermatozoa following thawing also exhibited linear trajectories but had reduced velocity as evident from the significant decrease in VAP, VSL, and VCL. Further, artificial inseminations using fresh semen resulted in 45% fertilization and birth of a live chick.     

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.     

Comparison between glycerol and ethylene glycol for the cryopreservation of equine spermatozoa: semen quality assessment with standard analyses and with the hypoosmotic swelling test

The aims of this study were to compare glycerol (G) at customary concentrations and ethylene glycol (EG) as cryoprotectants for stallion semen in a skimmed milk (SM) extender, to test different EG concentrations and to compare the results of manual and computerized analysis with the hypoosmotic swelling (HOS) test. Ejaculates from two stallions were collected over 3 weeks (6 ejaculates per stallion), diluted in a SM based extender, divided into 4 fractions, centrifuged and diluted again to a concentration of 100 x 10(6) mL(-1) progressive motile spermatozoa (PMS) in addition with the cryoprotectant (3% G, 3% EG, 6% EG, 9% EG). Sperm motility was assessed both by microscopy (in raw and frozen-thawed semen immediately after thawing) and with an HTM-IVOS analyzer (Hamilton-Thorne Research, MA, USA), at 0, 1, 4, 6, and 12 h after thawing and storage at 21 degrees C. Raw and frozen-thawed (0 h) semen samples for G and EG at 3% were also submitted to the HOS test with a 100 mOsm sucrose solution and were evaluated to detect the presence of swollen tails. The higher EG concentrations (i.e. 6% EG and 9% EG) significantly reduced the percentage of motile and PMS, immediately after thawing. At the same concentration, i.e. 3%, G resulted in a higher percentage of PMS than EG (36.2 vs. 30%, P < 0.05), but at 12 h after thawing and storage at 21 degrees C, no significant differences were detected between G and EG at 3%. The correlations between progressive motility (assessed by direct microscope observation or measured through the HTM analyzer) and the HOS test results for 3%G and EG were r = 0.61 and r = 0.35, respectively. The HOS test confirmed its suitability as a complementary method of analysis for stallion semen. We conclude that with the SM extender used, EG could substitute G as the cryoprotectant for stallion semen if used at the same or lower concentration.     

Effects of cooling and freezing on the motility of Ostrea edulis (L., 1758) spermatozoa after thawing

The aim of this work was to evaluate the effects of temperature, cryoprotectant agents and freezing curves on sperm motility of Ostrea edulis. All phases of cryopreservation were studied (evaluation of semen motility pattern, choice of cryoprotectants and freezing rates) to restore after thawing the motility characteristics distinctive of fresh semen.To assess the temperature effects on sperm motility, semen was activated using four different temperatures (25, 18, 10 and 3 °C). Sperm aliquots were maintained inactive at these temperatures for 1 and 3 h, then activated with FSW at same temperature of conservation. Sperm was activated and incubated to 3 °C with dimethylsulfoxide (Me₂SO), ethylene glycol (EG), 1–2 propylene glycol (PG) (5%, 7%, 10% and 15% final concentrations), glycerol (GlOH; 5%, 10% and 15% final concentrations) and methanol (MetOH; 4% and 10% final concentrations) for 10, 20 and 30 min. A first evaluation of freezing rates was made by testing four freezing curves: −1, −3, −6 and −10 °C/min. Then, an optimization was made by testing four freezing curves: −2.5, −3.0, −3.5 and −4 °C/min.The selected temperature for short term conservation has been 3 °C, because only this temperature has allowed good sperm motility conservation after 3 h of dry-storage; this is a time sufficient to conduct cryopreservation procedures. The sperm showed a particular sensitivity to GlOH and PG to all tested concentrations and to 15% Me₂SO. EG and MetOH to all concentrations and Me₂SO to concentrations lower than 15% have not shown significant toxic effects. The freezing rate −3 °C/min using 15% EG has shown an highest percentage of RVF (rapid, vigorous and forward) spermatozoa (class 3, about 75% of fresh semen) and an highest sperm motility duration.     

Cryopreservation of canine semen using a coconut water extender with egg yolk and three different glycerol concentrations

Semen was collected by digital manipulation from six adult dogs. The second fraction of the ejaculate was used in this study. The semen was assessed by macroscopic and microscopic criteria 1 min after collection, first dilution, cooling, glycerol addition and thawing. Experiments were conducted to compare the effect of three different concentrations of glycerol in coconut water extender. The freezing method employed was that one described for caprine semen with slight changes. Semen was thawed at 37 degrees C for 1 min. Spermatozoal motility after thawing was 49.2+/-26, 44.2+/-18.3 and 35.8+/-26.8% for groups with 4, 6 and 8% glycerol, respectively. The vigor after thawing was 2.6+/-1.1, 2.7+/-1.0 and 2.1+/-1.2 for these groups, respectively. There was no difference among groups in motility and vigor. However, a smaller percentage of total and secondary abnormalities was observed using 6% glycerol in coconut water extender. In conclusion, the three glycerol concentrations (4, 6 or 8%) can be used successfully in cryopreservation of canine semen using a coconut water extender.     

Influence of extender, freezing rate, and thawing rate on post-thaw motility, viability and morphology of coyote (Canis latrans) spermatozoa

The objective of this study was to examine the post-thaw effects of three cryoprotective extenders (Tris-fructose-citric acid extender, Tris-glucose-citric acid extender, and lactose extender), three linear freezing rates (-1, -6, and -20 degrees C/min), and three thawing rates (37 degrees C water bath for 120s, 60 degrees C water bath for 30s, and 70 degrees C water bath for 8s) on coyote spermatozoa. After thawing, the findings supported that cryopreservation of coyote (Canis latrans) spermatozoa frozen at a moderate freezing rate (-6 degrees C/min), in either a Tris-fructose or Tris-glucose extender, and thawed at a slow rate (37 degrees C water bath for 120s) or moderate rate (60 degrees C water bath for 30s), resulted in a more vigorous post-thaw motility (range, 57.5-44.0%) and viability (range, 64-49.6%) with the least amount of morphological and acrosomal abnormalities.     

Effect of glycerolization procedure and removal of seminal plasma on post-thaw survival and got-release from Boer goat spermatozoa

Forty ejaculates (20 for each of 2 experiments) were collected from 4 Boer goat bucks at weekly intervals to study the effect of glycerolization procedure and removal of seminal plasma on progressive motility, percent live spermatozoa and release of glutamic oxaloacetic transaminase (GOT) before and after the freezing of semen. Stepwise glycerolization at 37 degrees C gave higher progressive motility and percentage of live spermatozoa both before freezing and after thawing than onestep glyceroliza-tion at 37 degrees C or stepwise extension with glycerol being added after cooling to 5 degrees C. The GOT-release was reduced before freezing and after thawing of semen with stepwise glycerolization (P < 0.05). Progressive motility and the percentage of live spermatozoa were higher (P < 0.05) after the freezing of whole semen than in washed spermatozoa. The concentration of GOT in the extra-cellular fluid was lower in washed spermatozoa prior to freezing (P < 0,05); but after thawing, the washed spermatozoa released more GOT than spermatozoa in whole semen. Removal of seminal plasma prior to freezing spermatozoa in an extender containing egg yolk had an unfavorable effect on their post-thaw motility and integrity.     

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.     

Effect of seminal plasma on the cryopreservation of equine spermatozoa

Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80% seminal plasma for 15 min, prior to being diluted to a final concentration of 50 x 10(6)sperm/mL in a Lactose-EDTA freezing diluent and cryopreserved. Sperm motility was analyzed at 10 and 90 min after thawing, while sperm viability and acrosomal integrity were analyzed 20 min after thawing. Seminal plasma did not affect sperm motility, viability or acrosomal integrity (P>0.05). Experiment 2 tested the main affects of seminal plasma level (5 or 20%), incubation temperature (5 or 20 degrees C) and incubation time (2, 4 or 6 h) prior to cryopreservation. In this experiment, spermatozoa were incubated with 5 or 20% seminal plasma for up to 6h at either 5 or 20 degrees C prior to cryopreservation in a skim milk, egg yolk freezing extender. Samples cooled immediately to 5 degrees C, prior to freezing had higher percentages of progressively motile spermatozoa than treatments incubated at 20 degrees C (31 versus 25%, respectively; P<0.05), when analyzed 10 min after thawing. At 90 min post-thaw, total motility was higher for samples incubated at 5 degrees C (42%) compared to 20 degrees C (35%; P<0.05). In addition, samples containing 5% seminal plasma had higher percentages of total and progressively motile spermatozoa (45 and 15%) than samples exposed to 20% seminal plasma (33 and 9%; P<0.05). In conclusion, although the short-term exposure of sperm to seminal plasma had no significant effect on the motility of cryopreserved equine spermatozoa, prolonged exposure to seminal plasma, prior to cryopreservation, was deleterious.     

Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integrity, plasma membrane functionality and sperm longevity. The fertility of semen frozen in the presence of sorbitol was also tested by artificial insemination. Sperm quality was significantly decreased following freezing and thawing (P < 0.05). Fructose was inferior for protecting sperm during cryopreservation when compared to sorbitol and glucose (P < 0.05). Although the viability, motility and acrosome integrity of sperm cryopreserved with a glucose-containing extender did not significantly differ from sperm frozen in the sorbitol-based extender when examined at 2 and 4 h post-thaw, all of these parameters plus plasma membrane functionality were improved for sperm frozen in the sorbitol extender than in the glucose extender when examined 10 min post-thaw. Two of four mares (50%) inseminated with semen frozen with a sorbitol-containing freezing extender became pregnant. It is concluded that different sugars have different abilities to protect against cryoinjury during freezing and thawing of stallion sperm. This study demonstrated that an extender containing sorbitol as primary sugar can be used to successfully cryopreserve equine sperm; moreover, the quality of frozen-thawed sperm appeared to be better than when glucose or fructose was the principle sugar in the freezing extender.     

Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification

In this study, the cryoprotectant ethylene glycol (EG) was tested for its ability to improve and facilitate the cryopreservation of in vitro produced (IVP) bovine embryos. Embryos were cryopreserved in EG solutions supplemented with either newborn calf serum (NBCS) or polyvinyl alcohol (PVA). To assess EG toxicity, the embryos were equilibrated in EG concentrations from 1.8 to 8.9 M at room temperature for 10 min and then cultured for 72 h on a cumulus cell monolayer. The hatching rate was highest for day 7 blastocysts frozen in 3.6 M EG (98%) and was not different from the control group (85%). The controlled freezing (0.3 degrees C/min to -35 degrees C) of expanded day 7 blastocysts resulted in a hatching rate of 81%, which was similar to that of the nonfrozen controls (76%). Differential staining revealed only very few degenerate blastomeres attributed to freezing and thawing. Upon direct nonsurgical transfer of day 7 expanded blastocysts frozen in 3.6 M EG, a pregnancy rate of 43% was achieved, while the pregnancy rate after transfer of other developmental stages was significantly lower (22% with expanded day 8 blastocysts). When bovine IVP embryos were incubated at room temperature in 7.2 M EG preceded by preequilibration in 3.6 M EG, the hatching rate of day 7 expanded blastocysts reached 93%. Upon vitrification of IVP day 7 and day 8 blastocysts and expanded blastocysts in 7.2 M EG, the latter showed a higher hatching rate (42%) than blastocysts (12%). Overall, PVA as supplement to the basic freezing solution instead of NBCS had deleterious effects on survival after controlled freezing or vitrification. The simple cryopreservation protocol employed in this study and the low toxicity of ethylene glycol highlight the usefulness of this approach for controlled freezing of IVP embryos. However, further experiments are needed to improve the pregnancy rate following embryo transfer and to enhance survival after vitrification.     

Cryopreservation of spermatozoa in cyprinid fishes

The present study investigated semen cryopreservation in cyprinid fish using computer-assisted sperm motility analysis for viability control. Spermatozoa of the bleak, Chalcalbumus chalcoides, were used as a basic model to describe the toxic and cryoprotective effects of internal and external cryoprotectants, their most effective concentrations and combinations, the freezing and thawing conditions, and the effects of equilibration. We also used these data to develop a cryopreservation protocol for Barbus barbus, Chondrostoma nasus, Ctenopharyngodon idella, Cyprinus cario, Hypohtalmichthys molitrix, Leuciscus cephalus, Rutilus meidingerii, and Vimba vimba. For all investigated species the optimal extender composition was a buffered physiological sperm motility-inhibiting saline solution containing 10% DMSO and 0.5% glycin. The optimal sperm equilibration period in the extender was < or = 5 min. Freezing was performed in an insulated box in liquid nitrogen vapor and it was optimal at 4 to 5 cm above the surface of the liquid, depending on the species. Thawing was optimal in a 25 degrees C water bath whereby the thawing time ranged depending on species from 15 to 45 sec. This cryopreservation protocol resulted in frozen-thawed semen with 35 to 65% motile and 5 to 25% locally motile spermatozoa depending on the quality of fresh semen.     

Influence of extender, cryoperservative and seminal processing procedures on postthaw motility of canine spermatozoa frozen in straws

Experiments were conducted to evaluate two extenders (egg-yolk Tris and egg-yolk lactose), varying concentrations of two cryopreservatives (glycerol and dimethyl sulfoxide), and rates for cooling to 5 degrees C, cooling from 5 to -100 degrees C, and warming for canine spermatozoa packaged in 0.5-ml French straws. At optimal concentrations of glycerol, egg-yolk Tris extender was superior to egg-yolk lactose in preserving spermatozoal motility. Addition of dimethyl sulfoxide, alone or in combination with glycerol in either extender, was not beneficial to spermatozoal survival after thawing. Canine spermatozoa withstood a range of cooling and equilibration times with no detrimental effect on spermatozoal motility prior to freezing. However, there were differences in spermatozoal motility immediately after thawing; these differences were variable, resulting in a cooling time by equilibration time interaction. Spermatozoal motility after thawing was best preserved by freezing in egg-yolk Tris extender containing 2-4% glycerol, using a moderate rate of cooling from 5 to -100 degrees C (-5 degrees C/min from 5 to -15 degrees C, then -20 degrees C/min from -15 to -100 degrees C). Three of 12 bitches inseminated intravaginally with semen frozen using this protocol became pregnant.     

The effect of thawing velocity on survival and acrosomal integrity of ram spermatozoa frozen at optimal and suboptimal rates in straws

The effect of various thawing velocities on the motility and acrosomal maintenance of ram spermatozoa frozen at 20 degrees C/min (optimal) or 2 degrees C/min (suboptimal) was studied. The freeze-thaw motility and the percentage of intact acrosomes of spermatozoa frozen at 20 degrees C/min increased progressively with the thawing velocity. In semen frozen at 2 degrees C/min, motility of spermatozoa and the percentage of intact acrosomes declined drastically when the thawing velocity obtained in air at 20 degrees C was increased by thawing in water at 20 degrees C. Thawing at higher temperatures markedly increased both motility and acrosomal preservation, but the best results with semen frozen at 2 degrees C/min were lower than those obtained with semen frozen at 20 degrees C/min. The optimal freeze-thaw conditions for semen protected by 4% glycerol were freezing at 20 degrees C/min and thawing in water at 60 or 80 degrees C for 8 or 5 sec, respectively. Semen collected from rams exposed to a decreasing photoperiod exhibited higher motility after freezing and thawing than those exposed to an increasing photoperiod. However, there was no effect on acrosomal preservation after freezing at 20 degrees C/min.     

Effect of differential addition of glycerol and pyruvate to extender on cryopreservation of Mediterranean buffalo (B.bubalis) spermatozoa

The composition of the extender in which semen is diluted before freezing plays a major role in successful cryopreservation of spermatozoa. Substances of high osmolarity, like glycerol, protect sperm cells during the freezing process and energy-rich compounds, like pyruvate provide extra energy during capacitation and fertilization. Since cryopreservation procedures for Buffalo spermatozoa have not been adequately defined, the aim of the study was to improve the survival rate of buffalo (Bubalus bubalis) spermatozoa after cryopreservation by optimizing the timing for adding glycerol and by enriching the cryoprotectant extender with an energy source substrate. Semen was collected with an artificial vagina from 5 bulls and the ejaculates were immediately evaluated for motility, forward progressive motility and for viability, pooled and held at room temperature (28 degrees C) for 1 h. Then aliquots of pooled semen were subjected to dilution and equilibration in triplicate as follows: Experiment 1. Glycerol (3%) in a commercial extender was added to the semen at 28 degrees C and cooled to 5 degrees C for 1 h; then extender with 11% glycerol was added before further equilibration (initial glycerol addition; IGA) and the samples held at 5 degrees C for 1, 3 or 5 additional hours (IGA 1, n = 24; IGA 3, n = 24; IGA 5, n = 24) before freezing. Experiment 2. Glycerol (3%) was added and the mixture brought to 5 degrees C as described above. Then extender with 11% glycerol was added (late glycerol addition; LGA) and after equilibration for 1, 3 and 5 h (LGA 1, n= 24; LGA 3, n = 24; LGA 5, n = 24) the samples were frozen. In Experiments 3 and 4 Na pyruvate (1.25 mM) was added to the extender as described for IGA and LGA above (IPA and LPA samples). The effect of addition time (initial vs late) of glycerol and pyruvate was evaluated by measuring sperm motility, progressively forward motility and viability. After freezing-thawing the percentage of motile spermatozoa was significantly higher (0.001相似文献   

Ultramicroscopic and biochemical changes in ram spermatozoa cryopreserved with trehalose-based hypertonic extenders

The ability of a range of extenders to cryopreserve ram spermatozoa was tested. The extenders were modified by the inclusion of citrate, Tris buffer, trehalose, and EDTA. Ejaculates from three Pampinta rams were evaluated and pooled at 30 degrees C. The semen was diluted to contain 1 x 10(9) cells/mL, cooled to 5 degrees C, loaded into 0.25-mL straws, frozen and stored in liquid nitrogen. Evaluation was based on the hypoosmotic swelling test (HOS test), electron microscopy, and biochemical parameters such as lipid peroxidation and reduced and total glutathione levels, all measured after thawing. The HOS test indicated that the percentage of intact plasma membranes after freezing and thawing was significantly higher for the hypertonic extender containing trehalose (T), compared with an extender containing trehalose+EDTA (TE) or an isotonic Tris extender (B) (p < 0.05). Membrane evaluation by ultramicroscopy also indicated better sperm cryopreservation in extender T compared with the others, and there was a significant reduction in the number of damaged membranes (27%, p < 0.0002). The level of reduced glutathione was significantly higher after sperm cryopreservation in either hypertonic diluent (T and TE) with respect to the isotonic extender B, immediately after thawing (12%) and after a 3-h post-thawing thermotolerance test at 37 degrees C (17%, p = 0.007). Total glutathione levels did not show statistical differences among the extenders. After 3h post-thawing incubation at 37 degrees C, lipid peroxide levels in spermatozoa were statistically lower for T than TE (35%) or isotonic extender B (44%) (p = 0.002). Taken together these results indicate a reduction in the oxidative stress provoked by freezing and thawing when semen is cryopreserved in extender T. The antioxidant properties of extender T may be related to its effectiveness in membrane cryopreservation.