Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.     

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.     

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

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

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.     

Cryopreservation of sperm from the endangered formosan landlocked salmon (Oncorhynchus masou formosanus)

The Formosan landlocked salmon (Oncorhynchus masou formosanus) are at a high risk of extinction, and the sustained maintenance of the population will soon depend on aquaculture systems, which use cryopreservation of spermatozoa to increase genetic diversity. We investigated the effectiveness of dimethyl sulfoxide (DMSO), dimethyl-acetamide (DMA), and methanol as cryoprotectants in combination with 300 mM glucose as extender on the freezing of Formosan landlocked salmon spermatozoa. We also evaluated the morphological changes of Formosan landlocked salmon spermatozoa after their immediate dilution in the 300 mM glucose-DMSO extender and after freeze-thawing. The spermatozoa frozen with DMSO as a cryoprotectant showed significantly higher post-thaw motility and fertility than spermatozoa frozen with DMA or methanol. The fertilization capacity of frozen-thawed Formosan landlocked salmon was comparable to that of fresh spermatozoa. Intersubspecies fertilization trials between cryopreserved Formosan landlocked salmon spermatozoa and Amago salmon eggs showed high fertilization rates. Based on the findings, the potential value of using sperm bank to safeguard this endangered species is discussed.     

The effect of cryoprotectant on kangaroo sperm ultrastructure and mitochondrial function

This study examined the effect of cryoprotectants (20% DMSO, a 10% DMSO/10% glycerol mixture, 20% glycerol and 1 M sucrose solution) on kangaroo sperm structure and function, along with the effect of varying concentrations of glycerol on sperm mitochondrial function. Eastern grey kangaroo cauda epididymidal spermatozoa were incubated for 10 min at 35 °C in each cryoprotectant and the plasma membrane integrity (PMI) and motility assessed using light microscopy. The same samples were fixed for TEM and the ultrastructural integrity of the spermatozoa examined. To investigate the effect of glycerol on the kangaroo sperm mitochondrial function, epididymidal spermatozoa were incubated with JC-1 in Tris–citrate media at 35 °C for 20 min in a range of glycerol concentrations (0%, 5%, 10%, 15% and 20%) and the mitochondrial membrane potential (MMP) and plasma membrane integrity determined. As expected, incubation of spermatozoa in 20% glycerol for 10 min resulted in a significant reduction in motility, PMI and ultrastructural integrity. Interestingly, incubation in 20% DMSO resulted in no significant reduction in motility or PMI but a significant loss of structural integrity when compared to the control spermatozoa (0% cryoprotectant). However, 20% DMSO was overall less damaging to sperm ultrastructure than glycerol, a combination of 10% glycerol and 10% DMSO, and sucrose. While all glycerol concentrations had an adverse effect on mitochondrial function, the statistical models presented for the relationship between MMP and glycerol predicted that spermatozoa, when added to 20% glycerol, would lose half of their initial MMP immediately at 35 °C and MMP would halve after 19.4 min at 4 °C. Models for the relationship between PMI and glycerol predicted that spermatozoa would lose half of their initial PMI after 1.8 min at 35 °C and PMI would halve after 21.1 min at 4 °C. These results suggest that if glycerol is to be used as a cryoprotectant for kangaroo spermatozoa then it is best administered at 4 °C and that mitochondrial function is more sensitive to glycerol than PMI. Future research should be directed at investigating strategies that reduce exposure of spermatozoa to glycerol during processing and that test the cryoprotective properties of 20% DMSO for kangaroo spermatozoa.     

Individual variability in post-thaw sperm survival in a captive koala population

This study documented the extent of individual animal variation with respect to two proven methods of sperm cryopreservation in a captive population of 22 koalas. Semen samples were collected by electroejaculation, diluted in Tris–citrate glucose and equilibrated to 4 °C before being further diluted and frozen in media containing a final concentration of either 14% (v/v) glycerol or 12.5% (v/v) dimethylacetamide (DMA). There were significant differences in post-thaw survival of spermatozoa from different animals that were independent of pre-freeze semen quality. Glycerol proved to be a better cryoprotectant than DMA in terms of maintaining motility, plasma membrane integrity and high mitochondrial membrane potential; however, there was no difference between cryoprotectants with regards to their ability to prevent chromatin relaxation. While a positive correlation was observed between motility and mitochondrial membrane potential, both before and after cryopreservation, the slopes of the pre-freeze regression lines of these relationships were significantly altered following cryopreservation, suggesting that the efficiency of energy generation by the mitochondria was lowered by the freeze–thaw process. Based on a cluster analysis of the post-thaw semen viability parameters, the koalas in this study could be divided into two distinct groups; Cluster 1 had significantly higher sperm viability compared to the other cluster, regardless of the cryoprotectant used. The unpredictability of assessing post-thaw survival from pre-freeze koala semen parameters requires further investigation but is likely to be associated with variation in ejaculate composition or inherent genetic differences between animals.     

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.     

Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa

Four experiments were conducted to investigate the effects of egg yolk during the freezing step of cryopreservation (namely, the process except for the cooling step), on the viability of goat spermatozoa. The effects of egg yolk on sperm motility and acrosome integrity during the freezing step were investigated in Experiment 1. Spermatozoa diluted with Tris-citric acid-glucose (TCG) solution containing 20% (v/v) egg yolk were cooled to 5 degrees C, washed, and then frozen in TCG with egg yolk (TCG-Y), TCG without egg yolk (TGG-NY), 0.370 M trehalose with egg yolk (TH-Y), or trehalose without egg yolk (TH-NY). All extenders contained glycerol. In frozen-thawed spermatozoa, the inclusion of egg yolk in the freezing extenders increased (P<0.05) percentages of motile sperm, progressively motile sperm, and the recovery rate (ratio of post-thaw to pre-freeze values), but decreased (P<0.05) acrosomal integrity. Moreover, extenders with trehalose had better (P<0.05) post-thaw sperm viability. In Experiment 2, the effects of egg yolk on acrosome status before and after freezing were studied. Egg yolk significantly decreased the proportion of intact acrosomes before freezing, leading to fewer (P<0.05) intact acrosomes post-thaw and lower (P<0.05) recovery rates for intact acrosomes. In Experiment 3, including sodium dodecyl sulfate (SDS) in a diluent containing egg yolk tended to preserve the acrosome compared with the egg yolk containing diluent free of SDS, however, spermatozoa had a lower (P<0.05) proportion of intact acrosomes than those in a yolk-free diluent. However, after cooling, spermatozoa were diluted with a glycerolated extender containing egg yolk. Therefore, the objective of Experiment 4 was to explore whether the egg yolk or glycerol was responsible for the reduced intact acrosome percentage. In this experiment, after cooling and washing the spermatozoa were diluted in TCG with glycerol and/or egg yolk. The combination of glycerol and egg yolk in the extender reduced (P<0.05) the proportion of intact acrosomes compared with egg yolk or glycerol alone. In conclusion, the inclusion of egg yolk significantly improved sperm motility, indicating its beneficial effects during the freezing step of cryopreservation; trehalose appeared to synergistically increase its cryoprotective effects. Furthermore, although neither glycerol nor egg yolk per se affected the proportion of intact acrosomes, the combination of the two significantly reduced the proportion of acrosome-intact spermatozoa.     

An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa

The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to investigate reasons for their respective post-thaw survival by examining the sperm's response to a range of osmotic media and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting a degree of structural inflexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5 microM), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of cell volume changes that typically occur during cryopreservation. In experiment 1, koala (n = 4) and wombat (n = 4) spermatozoa packaged in 0.25 mL straws were cryopreserved using two freezing rates (fast-3 cm above liquid N2 interface; slow-6 degrees C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris-citrate glucose buffer with 15% (v/v) egg yolk. Wombat spermatozoa showed better (P < 0.01) post-thaw survival (% motile, % intact plasma membranes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala spermatozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410 mOsmol/kg) than wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneficial effect on sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw survival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely to be associated with F-actin induced plasma membrane inflexibility.     

Strategies for the cryopreservation of microencapsulated cells

The major challenge in developing cryopreservation protocols for microencapsulated cells is that the relatively large size (300-400 microm) and the fragile semipermeable membrane of microcapsules makes them particularly prone to cryodamage. Rapid-cooling cryopreservation protocols with high DMSO concentrations (3.5M, 25% v/v) resulted in low post-thaw cell viability (<10%), which did not improve with higher concentrations (4.5M, 32% v/v) and longer exposure to DMSO, even though the majority of microcapsules (60-80%) remained intact. Subsequent investigations of slow cooling with a range of DMSO and EG concentrations resulted in a much higher post-thaw cell viability (80-85%), with the majority of the microcapsules remaining intact ( approximately 60%) when DMSO was used at a concentration of 2.8M (20% v/v) and EG at a concentration of 2.7M (15% v/v). The presence of 0.25M sucrose significantly improved post-thaw cell viability upon slow cooling with 2.8M (20% v/v) DMSO, although it had no effect on microcapsule integrity. Multistep exposure and removal of sucrose did not significantly improve either post-thaw cell viability or microcapsule integrity, compared to a single-step protocol. Ficoll 20% (w/v) also did not significantly improve post-thaw cell viability and microcapsule integrity. Hence, the optimal condition for microcapsule cryopreservation developed in this study is slow cooling with 2.8M (20% v/v) DMSO and 0.25M sucrose.     

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.     

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.     

French field results (1985-2005) on factors affecting fertility of frozen stallion semen

Results on procedures for freezing stallion semen and the subsequent fertility during 20 years are presented. The present system applied in French National Stud includes: (1) a freezing protocol (dilution in milk, centrifugation and addition of freezing extender (INRA82+egg yolk (2%, v/v)+glycerol (2.5%, v/v) at 22 degrees C, a moderate cooling rate to 4 degrees C and freezing at -60 degrees C/min in 0.5-ml straws); (2) selection of ejaculates showing post-thaw rapid motility >35%; and (3) an insemination protocol (mares examined once daily, two AI of 400 x 10(6) spermatozoa 24 h apart before ovulation, sufficient number of straws to have the possibility to perform six AI of 400 x 10(6) total spermatozoa, i.e. 2.4 x 10(9) total spermatozoa available per mare per season). This system was applied to >110 stallions per year, the average post-thaw motility of ejaculates was 50% (>1800 ejaculates) before selection. The semen freezability was defined as the number of selected ejaculates divided by the total number of ejaculates frozen. Of the stallions, 5, 4, 5, 21 and 64% had semen freezability of 0-10, 10-33, 33-60, 60-90 and over 90%, respectively. Per-cycle pregnancy rate was 45-48% (>1500 mares per year, 1.8 cycles per mare) and foaling rate 64%. In comparison, per-cycle pregnancy rate and foaling rate of mares hand-mated to stallions were 57-59% and 64%, respectively. The average number of straws used was 32-35 (1.75 x 10(9) total spermatozoa) per mare per season. According to our results and the literature, the most important factors for improving fertility of frozen equine semen include: (1) a low concentration of glycerol (2-3.5% final concentration); (2) a suitable base extender for freezing like Lactose-Glucose EDTA or INRA82; (3) a post-thaw motility >30-35%; and (4) a sufficient number of spermatozoa per mare per season (1.5-2 x 10(9) total spermatozoa for two to three cycles) divided into small units. Numbers of spermatozoa, lower than 750.10(6) total spermatozoa per cycle, could result in lower per-cycle pregnancy rate with higher additional costs for management of mares. Because there are no particular regulations on quality and quantity of equine semen in the European Community, there is a need for the uniformity of information about frozen semen. A codification is suggested, based on the number of spermatozoa available per mare per season, the post-thaw motility and the final glycerol concentration.     

The cryoprotectant used, its concentration, and the equilibration time are critical for the successful cryopreservation of rabbit sperm: Dimethylacetamide versus dimethylsulfoxide

This study was designed to identify a suitable freezing protocol for rabbit semen by comparing the effects of different concentrations and equilibration times of dimethylacetamide (DMA) and dimethylsulfoxide (DMSO) on the postthaw quality of the semen. After establishing the best protocols for each cryoprotectant, their efficacy was compared by examining the in vivo fertilizing capacity of the semen samples. Pooled semen samples diluted in freezing medium containing 4%, 6%, or 8% DMA or DMSO (all combined with 1% sucrose as a nonpermeating cryoprotectant) were loaded in straws and equilibrated for 5, 15, or 45 min before freezing in liquid nitrogen vapor. The variables assessed after thawing were sperm motility, viability, osmotic resistance, and acrosome and DNA integrity. Marked effects on these variables were shown by the cryoprotectant concentration and equilibration time, with best results obtained using DMA 6% or DMSO 8% and equilibration times of 45 min. These freezing protocols were selected to compare the two cryoprotectants in an insemination trial. Three groups of 114 rabbit does (28 nulliparous and 86 multiparous in each group) were inseminated with fresh semen or with semen frozen using the optimized DMA or DMSO protocols. Fertility rates and numbers of kids born were similar, respectively for the DMSO-frozen (79.8% and 7.7 ± 0.3 young per kindling) and fresh semen (81.6% and 8.6 ± 0.3) yet higher (P ≤ 0.05) than the rates returned using the DMA-frozen semen (47.4% and 6.7 ± 0.4). Moreover, the numbers of rabbits born alive when DMSO was used in the freezing protocol, despite being lower than those recorded using fresh semen, were higher than when DMA was used as the cryoprotectant (P < 0.05). The physiological status of the does (nulliparous or multiparous) had no influence on the fertility and prolificacy results. Our findings indicate that the cryosurvival of rabbit sperm frozen using DMSO or DMA as the cryoprotectant is highly influenced by the concentration of cryoprotectant used and the time the semen is exposed to the agent before freezing. According to our in vivo fertility and prolificacy data, DMSO emerged as more effective than DMA for the cryopreservation of rabbit sperm.     

Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing

Influence of different sugars supplemented to the extender on the motility, viability and intact acrosome rates of dog spermatozoa during dilution, equilibration and freezing was studied. The ejaculate was divided into 10 aliquots, which were diluted 1:3 with TRIS-citric acid extender containing 240 mMTRIS, 63 mM citric acid, 8% (v/v) glycerol, 20% (v/v) egg yolk and 70 mM sugar, which was either fructose, galactose, glucose, xylose (monosaccharide), lactose, trehalose, maltose, sucrose (disaccharide) or raffinose (trisaccharide). No sugar was added to the extender in the control group. Extended semen samples were cooled to 5 degrees C over 45 min, packaged in 0.25-mL straws, equilibrated for 2 h at 5 degrees C and frozen in liquid nitrogen vapor. Samples were thawed by placing straws into 37 degrees C water for 30 sec. Motility, viable sperm and intact acrosome rates decreased gradually in all groups after equilibration and consecutively freezing (P<0.001). The type of sugar significantly effected motility, viability and acrosomal integrity during equilibration and freezing (P<0.05). Galactose, lactose, trehalose, maltose and sucrose reduced damaged acrosome percentages in equilibrated samples (P<0.05). Sugar supplementation did not enhance motility and viability during equilibration. The disaccharides, except lactose, reduced post-thaw dead sperm and/or damaged acrosome percentages without promoting post-thaw motility (P<0.01), whereas monosaccharides, especially fructose and xylose, improved motility (P<0.05) along with viability and intact acrosome rates (P<0.05). Trehalose, xylose and fructose significantly increased total active sperm rates (motility x live sperm rate x normal acrosome rate) compared to other sugars (P<0.01) and control (P<0.0001) in frozen thawed samples. Therefore, sugar supplementation of the extender influenced post-equilibration and post-thaw sperm quality, and the type or locality of protective impact of the sugar on dog spermatozoa vary according to type of the sugar.     

Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena)

This study was conducted to identify optimal medium composition for freezing Oryzias dancena embryonic cell lines. Different freezing media consisting of various concentration of dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and trehalose were prepared and long-term cultured embryonic cell line was frozen in each freezing medium by conventional slow freezing program for 7 days. Through measurement of viability and growth of post-thaw cells frozen in each freezing medium, it was determined that optimal composition of three components was 10 % DMSO, 20 % FBS, and 0.1 M trehalose. The post-thaw cells frozen in optimal freezing medium showed similar morphology and growth rate with non-frozen cells. Next, this condition was applied to two different sets of experiment; (1) freezing of the same cells during expanded period (57 days) and (2) freezing of short-term cultured cells from other batches for 7 days. The viability of post-thaw cells was significantly low and comparable in set 1 and 2, respectively, when compared with the result of long term-cultured cells frozen in optimal freezing medium for 7 days and similar morphology and growth rate with non-frozen counterparts were detected in the post-thaw cells from both sets. In conclusion, this study first reports the optimal medium composition for freezing O. dancena embryonic cells, which can contribute to fish species preservation as well as improvement of cell-based biotechnology by providing stable cell storage.     

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

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

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 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.