Effect of cholesterol-loaded cyclodextrin on the cryosurvival of bull sperm

Bull sperm were treated with several levels of cholesterol-loaded cyclodextrin (CLC) and frozen in egg yolk diluents containing either Tris or sodium citrate, to determine the CLC concentration that best benefits bull sperm cryosurvival. After thawing, higher percentages of motile (60%) and viable (55%) sperm were obtained when 1.5mg/ml CLC was added to sperm prior to freezing, than for sperm frozen in egg yolk Tris alone (42 and 46%, respectively; P < 0.05). Increasing concentration of CLCs, maintained higher percentages of viable sperm up to addition of 6.0mg/ml CLC when the percentages of viable sperm began to decline (50%; P < 0.05). Addition of 1.5mg/ml CLC to sperm frozen in sodium citrate diluent resulted in 53% motile sperm compared to 37% for control, although these were not different (P > 0.05). The beneficial effects of CLC addition were observed regardless of whether sperm incubated with CLC at 22 or 37 degrees C (P > 0.05) and maximum effects were observed when sperm incubated with CLC for 15min. Longer incubation times, up to 60min, resulted in similar results (P > 0.05). The amount of cholesterol that incorporated into sperm, increased with increasing CLC concentration, in a linear fashion, and each sperm incorporates a similar amount of cholesterol (coefficient of variation=12.9+/-0.7%). In addition, the cholesterol incorporates into all sperm membranes. Increasing membrane cholesterol levels, by adding CLCs to cells, prior to freezing, is a simple technology that increases the cryosurvival of bull sperm, and may benefit the cryosurvival of many cell types.     

Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios

This is the first study where the systematic application of theories and techniques used in mammalian sperm cryopreservation have been applied to honey bee (Apis mellifera L.) semen as a means to improve postthaw viability of cryopreserved sperm. Six newly designed diluents, three cryoprotectants (dimethyl sulfoxide, DMA, glycerol), and five diluent:semen ratios (1:1, 3:1, 6:1, 9:1, and 12:1) were tested. In addition, the sperm freezing tolerance of three honey bee strains was evaluated. Specific protocols were designed to control semen freezing and thawing rates. Sperm motility was assessed visually, whereas sperm viability was assessed using SYBR-14 and propidium iodide fluorescent stains. Diluent treatments did not affect fresh (nonfrozen) sperm viability yet affected fresh sperm motility (P < 0.05). Based on these assessments, two diluents were chosen and used in all successive cryopreservation experiments. Using the selected diluents, semen was collected at various diluent:semen ratios, along with one of the three cryoprotectants. Semen collected at high dilution ratios, using a hypotonic antioxidant diluent containing catalase, in combination with dimethyl sulfoxide, provided higher postthaw sperm viability than that of all other combinations tested (68.3 ± 5.4%; P < 0.05). Using this combination of dilution ratio, diluent, and cryoprotectant, there were no differences among honey bee strains for postthaw sperm viability (P = 0.805). Nevertheless, these new semen dilution and freezing methods improved postthaw viability of sperm to levels that could theoretically sustain worker populations in colonies, thus providing potential for further optimization of cryopreservation techniques for the genetic preservation and improvement of honey bee genotypes.     

Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm

Different cholesterol conjugates-loaded-cyclodextrin was added to bull sperm to improve sperm quality after freezing. Ejaculates from four bulls were diluted to 120 million cells/ml in Tris (T) diluent and then sub-divided into 10 treatments as follow: no additive (control); 1.5 mg CLC (positive control); 0.75 mg or 1.5 mg of cyclodextrin pre-loaded with cholesterol conjugated to heptanoate, palmitate, pelargonate or stearate, and incubated for 15 min at 22 °C. The samples were then diluted 1:1 with 20% egg yolk (EY) in T diluent and cooled to 5 °C over a 2 h. Upon reaching 5 °C, the samples were diluted 1:1 with T diluent containing 10% EY + 16% glycerol, and allowed to equilibrate for 15 min, and packaged into 0.5 ml straws and frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen for storage. Straws were thawed and the sperm motility, viability and number sperm binding to perivitelline membrane were determined. The ability of bull sperm to bind to the oocyte membranes was conducted using the chicken egg perivitelline membrane (CEPM) as described by Barbato et al. [G.F. Barbato, P.G. Cramer, R.H. Hammerstedt, A practical in vitro sperm-egg binding assay that detects subfertile males. Biol. Reprod. 58 (1998) 686-699] and modified by Amorim et al. [E. Amorim, J.K. Graham, B. Spizziri, M. Meyers, L. Amorim, C. Torres, The effect of adding cholesteryl-heptanoate, -palmitate, -pelargonate, or -stearate loaded cyclodextrin on bull sperm cryosurvival, in: Proceeding 40th Annual Meeting of the Society for the Study of Reproduction (SSR), July, San Antonio, TX, EUA, 2007], where these authors did not observe difference between bovine zona pellucide and CEPM. Higher percentages of motile and viable sperm were maintained after thawing from bull sperm treated with CLC and pelargonate compared to all other treatments (P < 0.05). Control samples and sperm treated with heptanoate, palmitate, or stearate loaded cyclodextrin exhibited similar motility percentages and viable sperm after freezing (P > 0.05). The percentage of motile sperm and number sperm binding to chicken egg perivitelline membrane was higher for CLC and pelargonate compared to control (P < 0.05). Therefore, adding either cholesterol or pelargonate to bull sperm membranes improved cell cryosurvival, whereas treatments with cyclodextrins pre-loaded with other cholesterol-like molecules did not.     

Effects of cholesterol-loaded cyclodextrin during freezing step of cryopreservation with TCGY extender containing bovine serum albumin on quality of goat spermatozoa

The susceptibility of mammalian spermatozoa to cold shock and freezing damage is due to changes in membrane lipid composition, particularly cholesterol depletion in plasma membrane during cryopreservation. The aim of this study was to investigate the effects of different concentrations of cholesterol-loaded cyclodextrin (CLC) and bovine serum albumin (BSA) on the cryopreservation of goat spermatozoa in tris-citrate egg yolk extender. Semen was collected from four mature goats and divided into seven aliquots prior to cryopreservation. The first aliquot remained untreated and was mixed with TCG, the second aliquot was mixed with TCG and egg yolk (TCGY), third aliquot was mixed with TCGY and 2.5% BSA (TCGYB) and other aliquots were mixed with TCGYB containing 0.75, 1.5, 2.5 and 3 mg/ml CLC. All samples were cryopreserved in straws over liquid nitrogen vapor and sperm motion Kinetics were measured by computer-assisted semen analysis (CASA) (percent motility (MOT), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and amplitude of lateral head displacement (ALH)). Acrosome status and vitality was observed by the triple-stain technique. CLC addition to extender resulted in significant (p < 0.05) enhancement of MOT, STR, and VCL of post-thawing sperm. Post-thawed motility, progressive motility and recovery rate were significantly (p < 0.05) higher in 1.5 mg/ml CLC with 2.5% BSA in TCGY extender compared to other groups. The 1.5 CLC sperm yielded a significant increase in percentage of spermatozoa with intact acrosome (P > 0.05). These results indicate that treating goat sperm with CLC and BSA in TCGY extender improved motility and vitality after freezing and thawing.     

In vitro sperm characterization and development of a sperm cryopreservation method using directional solidification in the killer whale (Orcinus orca)

Research was conducted to characterize seminal traits and to develop a sperm cryopreservation method using directional freezing (DF) for the killer whale (Orcinus orca). Experiments evaluated effects of: (i) freezing rate (SLOW, MED, FAST) by diluent (BF5F, Biladyl®, EYC) in 0.5 mL straws; and (ii) freezing method (straw or DF) by glycerol (3, 6, or 9% final concentration, v:v) on in vitro sperm quality. Fresh ejaculates (n = 161) were (mean ± SD) 7.8 ± 7.4 mL at 740 × 10⁶ sperm/mL with 92.2 ± 6.3% total motility (TM), 85.4 ± 6.9% progressive motility (PM), 89.6 ± 9.0% viability and 89.8 ± 9.2% acrosome integrity. Samples frozen using straws by the MED or SLOW method were improved (P < 0.05) over FAST across all diluents. At 3 h post thaw (PT), TM, PM, Rapid motility (RM), VAP, VCL, ALH and viability for 3% and 6% glycerol were improved (P < 0.05) over 9% glycerol. Directional freezing samples at 0 h and 3 h PT, at all glycerol concentrations, displayed higher (P < 0.001) TM, PM, RM, VAP, VSL, VCL and viability /intact acrosomes (PI/FITC-PNA) than straw. These data provided the first information on ejaculate characteristics and the development of a semen cryopreservation method using DF in the killer whale.     

Combined effects of glycerol concentration, cooling velocity, and osmolality of skim milk diluents on cryopreservation of ram spermatozoa

The interaction of glycerol concentration from 0 to 16% and cooling velocity from 1 to 100 degrees C/min on freeze-thaw survival of ram spermatozoa was studied using a diluent based on 15% skim milk (450 mOs/kg water). Optimal spermatozoa survival (percentage motility and rating) was obtained with 4 to 6% glycerol and freezing rates of 10 to 100 degrees C/min. Similar results were obtained with 8% glycerol at freezing rates of 5 to 30 degrees C/min. Although the ram spermatozoa tolerated several cooling velocities at each glycerol concentration, increasing the concentration of glycerol resulted in a downshift in the range of optimal cooling velocities. Glycerol concentrations above 8% were toxic and contributed greatly to the progressive decrease in spermatozoa survival. Comparison of the 15% skim milk diluent (450 mOs/kg water) with a 19% skim milk diluent (600 mOs/kg water) showed that optimal cryosurvival was obtained with 4 to 6% glycerol and freezing rates of 10 to 100 degrees C/min with both diluents.     

Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival

Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sperm were maintained after thawing when 1.5 mg CLC was added to sperm from stallions whose sperm do not survive freezing well, compared to control sperm from those same stallions (67% vs. 50%; P<0.05). Addition of CLCs increased the percentages of membrane intact sperm surviving cryopreservation compared to untreated sperm for all stallions (P<0.05). The amount of cholesterol that incorporated into the membranes of the sperm cells increased in a polynomial fashion (R2=0.9978) and incorporated into all sperm membranes. In addition, there was a significant loss of cholesterol from sperm membranes after cryopreservation; however, addition of CLCs to sperm prior to cryopreservation maintained higher cholesterol levels in the sperm after freezing and thawing than untreated sperm (P<0.05). Addition of CLCs also resulted in more sperm binding to the zona pellucida of bovine oocytes after cryopreservation than control sperm (48 vs. 15; P<0.05). In conclusion, CLCs improved the percentage of post-thaw viability in equine sperm as well as increased the number of sperm that bind to zona pellucida. Addition of CLCs to stallion sperm prior to cryopreservation is a simple procedure that increases the cryosurvival of cells.     

Cryopreservation of turkey semen: Effect of breeding line and freezing method on post-thaw sperm quality,fertilization, and hatching

Cryopreservation methods for poultry semen are not reliable for germplasm preservation, especially for turkeys, where fertility rates from frozen/thawed semen are particularly low. The objective was to evaluate cryopreservation methods for effectiveness in promoting cryosurvival and post-thaw function of sperm from five turkey lines: one commercial line and four research (RBC1; E; RBC2; F) lines from Ohio State University (OSU). The model for cryopreservation was set up as a 2 × 2 × 2 × 5 design for cryoprotectant (glycerol or dimethylacetamide (DMA)), cryopreservation medium (Lake or ASG), method of dilution (fixed dilution volume versus fixed sperm concentration) and turkey line, respectively. The final cryoprotectant concentrations were 11% glycerol or 6% DMA. Thawed sperm were evaluated for plasma membrane integrity and quality, motility, acrosome integrity and, after artificial insemination, for egg fertility and hatchability. Commercial turkey hens were used for all fertility trials, regardless of semen source. Turkey sperm frozen with glycerol exhibited higher membrane integrity and membrane quality upon thawing than turkey sperm frozen with DMA although no differences in total motility, and only minimal differences in progressive motility, were detected among the eight cryopreservation treatments. Within line, fertility was affected by cryoprotectant, medium and dilution method, where the overall highest percentages of fertile, viable embryos (Day 7) occurred for the DMA/ASG/fixed sperm concentration method, while high percentages (15.8–31.5%) of fertile, non-viable embryos (Day 1–6) were observed for multiple cryopreservation methods, including two glycerol treatments. From a single insemination, the duration of true and viable fertility in all lines was 10–13 weeks and 9–10 weeks, respectively. The duration of hatchability was 4–6 weeks after insemination for four of the turkey lines. The highest percentage of viable embryos was observed for the commercial line (9.5 ± 2.4%), followed by the E line (5.3 ± 1.3%), F line (3.7 ± 2.0%) and RBC2 line (2.6 ± 0.8%). For the RBC1 line, there was 100% embryonic death by Day 6 of incubation. Overall, better fertility results were obtained with the cryoprotectant DMA, the ASG diluent and fixed sperm concentration. However, the applicability of this method for preserving semen from research populations may be line dependent.     

Examination of some processing methods for freezing boar semen.

Five experiments were conducted to examine the effect of processing methods and diluents on survival and morphology of boar spermatozoa after freezing. Post-thawing survival of spermatozoa was better for Beltsville-F3 (BF3) than for tris-fructose-EDTA freezing diluent when the seminal plasma and glycerol were removed prior to freezing (method A). Both freezing diluents yielded similar viability results when the spermatozoa were frozen in the presence of siminal plasma and glycerol (method B). Viability of spermatozoa after thawing was better when glycerol concentration in the prefreezing diluent (method A) or in the freezing medium (method B) was 2-5 and 5-0 rather than 7-5%. Cooling of diluted semen to 5 degrees C beyond 4 h decreased the post-thawing survival of spermatozoa. The proportion of spermatozoa with undamaged acrosomes after processing and thawing by different methods was indistinguishable and relatively low. When the semen was frozen at cell concentrations ranging from 0-25 to 2-0 X 10(9)/ml, the viability of spermatozoa declined with increasing concentration following freezing in BF3, and S-1 diluents. Viability results were very similar for all cell concentrations examined when tris-fructose-EDTA diluent was used, indicating the possibility of freezing boar semen in a concentrated state.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

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.     

Effects of Cholesterol-Loaded Cyclodextrins on the Rate and the Quality of Motility in Frozen and Thawed Rabbit Sperm

The motility of sperm after freezing and thawing is critical for effective cryopreservation. It is known that supplementation with cholesterol-loaded cyclodextrin (CLC) improves cryosurvival of sperm in various animals. To clarify the effects of supplementation with CLC on rabbit sperm motility after freezing and thawing, rabbit sperm motility was analyzed using a computer-assisted sperm analysis system. Sperm motility with CLC supplementation was 29.4 ± 9.6% (mean ± SD), which was significantly higher than that of controls (20.8 ± 7.1%, P<0.05). The curvilinear velocity of sperm with CLC exceeded that of controls, whereas the values for linearity and wobble were significantly lower in sperm with CLC compared with controls. After artificial insemination, 44.3% of recovered ova were fertilized in the CLC-supplemented group, which was higher than the percentage in the control group (36.4%). The results indicate that supplementation with CLC improves the rate and quality of motility in rabbit sperm after freezing and thawing, and would be advantageous for successful cryopreservation.     

Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski’s horse (Equus ferus przewalskii)

Two studies were conducted to understand sperm cryosensitivity in an endangered equid, the Przewalski’s horse (Equus ferus przewalski), while testing the cryoprotectant ability of formamides. The first assessed the toxicity of permeating cryoprotectants (glycerol, methylformamide [MF] and dimethylformamide [DMF]) to Przewalski’s horse spermatozoa during liquid storage at 4 °C. The second examined the comparative influence of three diluents (with or without formamides) on cryosurvival of sperm from the Przewalski’s versus domestic horse. When Przewalski’s horse spermatozoa were incubated at 4 °C in INRA 96 with differing concentrations of glycerol, MF or DMF or a combination of these amides, cells tolerated all but the highest concentration (10% v/v) of MF alone or in combination with DMF, both of which decreased (P < 0.05) motility traits. There was no effect of cryoprotectants on sperm acrosomal integrity. In the cryosurvival study, average sperm motility and proportion of cells with intact acrosomes in fresh ejaculates were similar (P > 0.05) between the Przewalski’s (67%, 84%, respectively) and domestic (66%, 76%) horse donors. Sperm from both species were diluted in lactose–EDTA–glycerol (EQ), Botu-Crio (BOTU; a proprietary product containing glycerol and MF) or SM (INRA 96 plus 2% [v/v] egg yolk and 2.5% [v/v] MF and DMF) and then frozen over liquid nitrogen vapor. After thawing, the highest values recovered for total and progressive sperm motility, acrosomal integrity and mitochondrial membrane potential were 42.4%, 21.8%, 88.7% and 25.4 CN (CN = mean JC-1 fluorescence intensity/cell on a channel number scale), respectively, in the Przewalski’s and 49.3%, 24.6%, 88.9% and 25.8 CN, respectively, in the domestic horse. Although sperm progressive motility and acrosome integrity did not differ (P > 0.05) among treatments across species, mitochondrial membrane potential was higher (P < 0.05) in both species using EQ compared to BOTU or SM media. Additionally, Przewalski’s stallion sperm expressed higher (P < 0.05) post-thaw total motility in BOTU and SM compared to EQ, whereas there were no differences among freezing diluents in the domestic horse. In summary, Przewalski’s stallion sperm benefit from exposure to either MF or DMF as an alternative cryoprotectant to glycerol. Overt sperm quality appears similar between the Przewalski’s and domestic horse, although the total motility of cells from the former appears more sensitive to certain freezing diluents. Nonetheless, post-thaw motility and acrosomal integrity values for Przewalski’s horse spermatozoa mimic findings in the domestic horse in the presence of INRA 96 supplemented with 2% (v/v) egg yolk and a combined 2.5% concentration of MF and DMF.     

Cryopreservation of Atlantic cod Gadus morhua L. spermatozoa: Effects of extender composition and freezing rate on sperm motility, velocity and morphology

Broodstock selection programs are currently underway for Atlantic cod (Gadus morhua). To complement and further these selection programs we need to develop sperm cryopreservation procedures. This will allow genomic DNA from males from selected individuals or stocks to be frozen and conserved in perpetuity. In our study we used a full factorial ANOVA design to examine the effects of diluent (Mounib’s sucrose-based diluent, Hanks’ Balanced Salt Solution, Mounib’s sucrose-based diluent + hen’s egg yolk, and Hanks’ Balanced Salt Solution + hen’s egg yolk), cryoprotectant (propylene glycol, dimethyl sulphoxide, and glycerol), and freezing rate (−2.5, −5.0, −7.5, and −10.0 °C/min) on motility of cod frozen-thawed sperm. Sperm velocity and morphometric analyses of sperm heads and flagella were also assessed. We found that sperm motility-recovery index was strongly influenced by the presence of higher-order interactions of the factors we tested. The best cryoprotection used diluents that contained hen’s egg yolk. Generally, extenders containing propylene glycol yielded higher post-thaw sperm motilities than those with dimethyl sulphoxide or glycerol. In comparison to sperm from other frozen-thawed extenders, sperm from extenders supplemented with propylene glycol had significantly higher curvilinear velocity. Cryopreservation showed no impact on sperm head morphology parameters, however, considerable damage to frozen-thawed sperm flagella was observed. We believe that our experimental/statistical approach and our results add significantly new information to the study of semen biology/cryobiology in fishes. Our findings are also highly relevant to the development of cod mariculture and for aiding in conservation efforts of this very important marine species.     

The development of haddock and Atlantic cod sperm cryopreservation techniques and the effect of sperm age on cryopreservation success

Three cryoprotectants [dimethyl sulphoxide (DMSO), propylene glycol (PG) and glycerol], two diluents (sucrose‐ and saline‐based), two sperm collection times, two freezing rates and three times between thaw and activation (0, 30 and 60 min) were tested in order to develop a protocol for the cryopreservation of sperm of haddock Melanogrammus aeglefinus and Atlantic cod Gadus morhua . The faster freezing rate resulted in extremely low post‐thaw motility in comparison to the slower freezing rate, which was successful for sperm from both gadids. In both cases, the use of PG resulted in significantly higher post‐thaw sperm motility‐recovery indices than with DMSO or glycerol, which did not differ significantly from one another. Diluent had no effect on post‐thaw sperm motility for Atlantic cod or haddock. Sperm collected at the end of the spawning season tended to have reduced post‐thaw motility compared to that collected 2 weeks after the start of spawning. A 30 min delay between thaw and activation of haddock and Atlantic cod sperm resulted in a significant decrease in sperm motility. When PG was used as cryoprotectant, sperm motility continued to decrease between 30 and 60 min post‐thaw. With DMSO or glycerol as cryoprotectant, motilities were already very low after 30 min post‐thaw and did not decrease any further after 60 min. Cryoprotectant, diluent and time between thaw and activation had no effect on mean or maximum sperm swimming speeds for either Atlantic cod or haddock sperm. Fertilization success for haddock eggs, like sperm motility, was higher with PG‐frozen sperm than DMSO‐ or glycerol‐frozen sperm. These results constitute the first reported successful cryopreservation of haddock sperm and improve on previous methods used to cryopreserve sperm from Atlantic cod.     

Improved cryopreservation protocol for Blanca-Celtibérica buck semen collected by electroejaculation

The collection of sperm samples by electroejaculation (EE) leads to an increase of the production of seminal plasma which could modify the tolerance of spermatozoa to the cryopreservation procedure. This study aims to compare a standard sperm cryopreservation protocol for samples collected by artificial vagina (AV) with the same protocol and modifications to this for samples obtained by EE. Semen from six males of Blanca-Celtibérica goat breed was collected by AV (control) and EE, and three experiments were conducted. In Experiment 1, it was examined the effects of egg yolk concentration contained in freezing extender (0%, 1.5%, 10% and 20% of egg yolk); in Experiment 2, it was evaluated the cooling rate from 30 to 5 °C (fast: 10 min and slow: 90 min) and the temperature of glycerol addition (30 and 5 °C); and in Experiment 3, it was examined the time of equilibration at 5 °C (0, 1, 2 or 3 h). A heterologous in vitro fertilization test was carried out in order to compare the fertility of control samples with that resulting from the EE protocol which showed the highest sperm quality. Results showed greater sperm motility parameters after thawing for control samples cryopreserved in standard conditions in the three experiments. For samples collected by EE, extender with 20% egg yolk, a slow cooling rate and a longer equilibration time (3 h) provided higher sperm quality, and no differences were observed between temperatures of glycerol addition. Samples collected by EE and cryopreserved with the protocol which yielded the best sperm quality after thawing showed higher fertility compared to AV.     

Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria

Motility and cryopreservation of testicular sperm of European common frog, Rana temporaria were investigated. Collected testicular spermatozoa were immotile in solutions of high osmolalities: 300 mmol/l sucrose and motility inhibiting saline solution-MIS. Full sperm motility could be activated in distilled water or in a solution of 50 mmol/l NaCl, = 90 mosmol/kg, with 75-90% motility and 14-16 μm s⁻¹ swimming velocity. Spermatozoa activated in distilled water and kept at room temperature ceased the motility within a period of 1 h. But when they were kept at 4 °C, no significant decrease in sperm motility and velocity occurred over a period of 1 h. Incubation of testicular sperm diluted 1:2 with MIS containing 10% DMSO, 5% glycerol, 10% methanol, or 10% propandiol for a period of 40 min at 4 °C showed that propandiol was the most toxic cryoprotectant for spermatozoa of European common frog R. temporaria. However, methanol was not toxic to spermatozoa during the 40 min incubation period, it failed to protect spermatozoa during the freezing and thawing process. DMSO and glycerol were useful penetrating cryoprotectants that interacted with sperm diluents in cryodiluent efficacy. In combination with the sucrose diluent, DMSO was a better cryoprotectant than glycerol, while in combination with MIS, DMSO and glycerol were similarly useful. Sperm was frozen at two freezing levels above the surface of liquid nitrogen. Sperm frozen 5 cm above the surface of liquid nitrogen resulted in immotile and non-viable spermatozoa. However, sperm frozen at 10 cm above the surface of liquid nitrogen showed 40-45% viability and 30-35% motility, compared to the untreated freshly collected testicular sperm. Addition of hen egg yolk had no positive effect on the post-thaw sperm motility, viability and hatching rate when added to sucrose cryodiluents. However, addition of 5% egg yolk to the MIS containing 5% glycerol and 2.5% sucrose significantly improved the hatching rate than all other treatments. Therefore, we conclude that, MIS and 300 mmol/l sucrose are suitable diluents for immotile storage of testicular semen. For cryopreservation, dilution to a final concentration of 5-6 × 10⁶/ml in MIS with 5% glycerol, 2.5% sucrose and 5% egg yolk, frozen in liquid nitrogen vapour at 10 cm above its surface, and thawed at 22 °C for 40 s is a useful cryopreservation protocol for R. temporaria sperm. Further research is needed to determine the motility parameters and cryopreservation of spermatic urine of R. temporaria.     

Fertility test of frozen boar semen.

The fertility results of two experiments are presented. In experiment 1, the semen was frozen in tris-fructose-EDTA or BF3 diluents at 0-25 X 10(9)/ml sperm concentration and extended after thawing with either seminal plasma (SP) or the freezing medium (FM) containing no cryoprotective agent. In the second experiment the semen was glycerolated by two methods, frozen at 1-0 X 10(9)/ml sperm concentration, and extended wtih FM before insemination. Fertility after double insemination within one oestrus with semen frozen in tris-fructose-EDTA or BF3 diluents varied depending on the medium used for extension of thawed semen. The farrowing rates for semen frozen in the former diluent with FM and SP post-thawing media were 4/8 and 1/8 respectively, and for semen frozen BF3 diluent with FM and SP post-thawing extenders 1/8 and 5/8. The mean farrowing for the 32 animals inseminasted was 34-4%. Pregnancies for semen frozen in tris-fructose-EDTA and glycerolated at 30 or 5 degrees C were 5/12 and 4/12 respectively, and for single and double inseminations 6/12 and 3/12 respectively. Of 24 animals inseminated 37-5% farrowed.     

Effect of glycerol and dimethyl sulfoxide on cryopreservation of rhesus monkey (Macaca mulatta) sperm

Glycerol and dimethyl sulfoxide (DMSO) are widely used as penetrating cryoprotectants in the freezing of sperm, and various concentrations are applied in different species and laboratories. The present study aimed to examine the effect of these two cryoprotectants at different concentrations (2%, 5%, 10%, and 15% glycerol or DMSO) on rhesus monkey sperm cryopreservation. The results showed that the highest recovery of post-thaw sperm motility, and plasma membrane and acrosome integrity was achieved when the sperm was frozen with 5% glycerol. Spermatozoa cryopreserved with 15% DMSO showed the lowest post-thaw sperm motility, and spermatozoa cryopreserved with 15% glycerol and 15% DMSO showed the lowest plasma membrane integrity among the eight groups. The results achieved with 5% glycerol were significantly better for all parameters than those obtained with 5% DMSO. The functional cryosurvival of sperm frozen with 5% glycerol was further assessed by in vitro fertilization (IVF). Overall, 85.7% of the oocytes were successfully fertilized, and 51.4% and 5.7% of the resulting zygotes developed into morulae and blastocysts, respectively. The results indicate that the type and concentration of the penetrating cryoprotectant used can greatly affect the survival of rhesus monkey sperm after it is frozen and thawed. The suitable glycerol level for rhesus monkey sperm freezing is 5%, and DMSO is not suitable for rhesus monkey sperm cryopreservation.     

Effect of N-acetyl-D-glucosamine, and glycerol concentration and equilibration time on acrosome morphology and motility of frozen-thawed boar sperm.

A series of experiments were conducted to determine the effect of N-acetyl-D-glucosamine, glycerol concentration and equilibration time for the freezing of boar spermatozoa in 5 ml maxi-straws. The optimum final glycerol concentration in the diluent with 0.05% N-acetyl-D-glucosamine in the first diluent was 2-3% and the optimum glycerol equilibration time was 2-3h. In conclusion, we recommend the first diluent containing 11% lactose hydrate, 20% egg yolk and 0.05% N-acetyl-D-glucosamine in 100ml distilled water, and the second diluent containing 11% lactose hydrate, 20% egg yolk, 4% glycerol and 1% orvus es paste for the diluents of boar sperm freezing. Also, we found out that 0.05% soluble N-acetyl-D-glucosamine was the optimum concentration in the first diluent and a concentration of 0.05% soluble N-acetyl-D-glucosamine significantly enhanced the cryopreservation of boar spermatozoa.