Improvement of post-thaw sperm survivals using liquid nitrogen vapor in a spermcasting oyster Ostrea angasi

Low survival of cryopreserved sperm impedes the application of cryopreservation technique in spermcasting oyster species. This study developed a simple method of liquid nitrogen vapor freezing to improve post-thaw sperm survival in the spermcasting oyster Ostrea angasi. The results indicate that the permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were non-toxic to sperm up to 20% concentration and 90 min exposure whereas methanol at 10% or higher was toxic to sperm for any exposure over 30 min. Among the treatments with permeable cryoprotectants, 15% EG produced the highest post-thaw sperm motility. Sperm motility was further improved by the addition of non-permeable cryoprotectants (trehalose and glucose), with 15% EG + 0.2 M trehalose resulting in the highest post-thaw sperm motility among all the combinations evaluated. The durations of 20, 30 and 60 min equilibrations produced a higher post-thaw sperm motility and plasma membrane integrity (PMI) than 10 min. Higher post-thaw motility and PMI were achieved by freezing sperm at the 8 cm height from the liquid nitrogen surface than at the 2, 4, 6, 10 or 12 cm height. Holding sperm for 10 min in liquid nitrogen vapor produced higher post-thaw motility and PMI than for 2, 5 or 20 min. The cryopreservation protocol developed in this study improved both post-thaw motility and PMI of O. angasi sperm at least 15% higher than those cryopreserved using programmable freezing method. Liquid nitrogen vapor freezing might have greater applicability in improving post-thaw sperm quality of spermcasting oyster species.     

Effect of some permeating cryoprotectants on CASA motility results in cryopreserved bull spermatozoa

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility because they provide objective results for thousands of mammalian spermatozoa. Mammalian spermatozoa experience osmotic stress when the glycerol is added to the cells prior to freezing and removal from the cells after thawing. In order to minimize osmotic damage, cryoprotectants having lower molecular weights and greater membrane permeability than glycerol, were evaluated to determine their effectiveness for cryopreserving bull spermatozoa. The aim of this study was to compare the cryopreservation effects of low molecular weight cryoprotectants (ethylene glycol and methanol) to glycerol, on post-thaw CASA sperm parameters. Bull semen was diluted with tris-egg yolk extender containing 3% glycerol, 3, 2 and 1% ethylene glycol or 3, 2 and 1% methanol. Bull semen was frozen in 0.5 straws. Bull spermatozoa exhibited higher percentages (p<0.01) for total (Mot, 72.4%) and progressively (Prog, 29.5%) motilities when frozen in extender containing 3% glycerol compared to 3, 2 and 1% ethylene glycol or 3, 2 and 1% methanol. In conclusion, no advantages were found in using ethylene glycol or methanol to replace glycerol in bull semen freezing. Glycerol provided the best sperm characteristics for bull spermatozoa after freezing and thawing. The possibility of using ethylene glycol or methanol as permeating cryoprotectants for bull semen deserves further investigation, and these cryoprotectants should also be evaluated in extenders that contain disaccharides or cholesterol.     

Cryopreservation of a marine microalga, Nannochloropsis oculata (Eustigmatophyceae)

The cryopreservation of algae could prevent genetic drift and minimize labor costs compared to the current method of maintenance and subculturing. Clear, simple protocols for cryopreservation of marine microalga, Nannochloropsis oculata were developed and cryoprotectant choice and concentration optimized. The viability of the microalga was assessed directly after thawing, and algal concentration was measured after 2-30 days of growth. Five cryoprotectants (dimethyl sulphoxide, Me2SO; ethylene glycol, EG; glycerol, Gly; methanol, MeOH; and propylene glycol, PG) at five concentrations (10, 20, 30, 40, and 50%; v/v) were evaluated to determine the toxicity of various cryoprotectants to N. oculata. The toxicity of cryoprotectant (Me2SO, EG, MeOH, and PG) was observed only at higher concentrations of CPAs: > 20% for EG, > 30% for Me2SO and methanol, and > 40% for PG. Direct freezing of algae in liquid nitrogen resulted in a severe loss of viability and a modified cryopreservation protocol proved to be more appropriate for the preservation of N. oculata. Cryopreservation protocols developed and tested in the present study might be applied to cryopreserving other strains, or species, in this genus.     

Cryopreservation of some useful microalgae species for biotechnological exploitation

Culture collections of microalgae represent a biological resource for scientific research and biotechnological applications. When compared to the current methods of maintenance and sub-culturing, cryopreservation minimizes labor costs and is an effective method for maintaining a large range of species over long periods with high stability. In order to determine the best cryopreservation method for microalgae species with great biotechnological potential, three freezing protocols were employed using different cryoprotectants (dimethyl sulfoxide—Me₂SO; methanol—MeOH). Three marine microalgae species (Thalassiosira weissflogii; Nannochloropsis oculata, and Skeletonema sp.) were cooled by directly plunging into liquid nitrogen (?196°C) and with two-step controlled cooling protocols (?18°C and ?80°C pre-treatments). After storage periods ranging from 10 to 120 days, viability was determined by the ability of cells to actively grow again. Results obtained for T. weissflogii showed that this species could be preserved at ultra-low temperature (?196°C) for 10 and 30 days with 10?% Me₂SO and 5?% MeOH when employed a controlled cooling protocol (?80°C). N. oculata was successfully cryopreserved either by direct freezing or with controlled cooling protocols. N. oculata samples presented good responses when treated with 5?% Me₂SO, 10?% Me₂SO, 5?% MeOH and even without any cryoprotectant. Skeletonema sp. did not survive cryopreservation in any of the tested conditions. The results indicate the difficulty in establishing common protocols for different microalgae species, being necessary further studies for a better understanding of cell damages during freezing and thawing conditions for each species.     

New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos

This study was designed to test the efficiency of recently developed vitrification technology followed by microscope-free thawing and transfer of sheep embryos. In a first set of experiments, in vivo derived embryos at the morula to blastocyst stage were frozen in an automated freezer in ethylene glycol, and after thawing and removal of cryoprotectants, were transferred to recipient ewes according to a standard protocol (control group). A second group of embryos were loaded into open-pulled straws (OPS) and plunged into liquid nitrogen after exposure at room temperature to the media: 10% glycerol (G) for 5 min, 10% G+20% ethylene glycol (EG) for 5 min, 25% G+25% EG for 30s; or 10% EG+10% DMSO for 3 min, 20% EG+20% DMSO+0.3M trehalose for 30s. The OPS were thawed by plunging into tubes containing 0.5M trehalose. After this rapid thawing, the embryos were directly transferred using OPS as the catheter for the transplantation process. In a second set of experiments, in vivo derived and in vitro produced expanded blastocysts were vitrified in OPS and then transferred as described above. The lambing rates recorded (59% for the conventionally cryopreserved in vivo derived embryos, 56% for the vitrified in vivo derived embryos, and 20% for the vitrified in vitro produced embryos), suggest the suitability of the vitrification technique for the transfer of embryos obtained both in vivo and in vitro. This simple technology gives rise to a high embryo survival rate and will no doubt have applications in rearing sheep or other small ruminants.     

Ultrastructural features of agouti (Dasyprocta aguti) preantral follicles cryopreserved using dimethyl sulfoxide, ethylene glycol and propanediol

The objective was to develop an efficient protocol for cryopreservation of agouti (Dasyprocta aguti) ovarian tissue. Agouti ovarian fragments were placed, for 10 min, in a solution containing MEM and fetal bovine serum plus 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or propanediol (PROH); some of those fragments were subsequently cryopreserved in a programmable freezer. After exposure and/or thawing, all samples were fixed in Carnoy prior to histological analysis. To evaluate ultrastructure, follicles from the control and all cryopreserved treatments were fixed in Karnovsky and processed for transmission electron microscopy. After exposure and freezing, there was a significant decrease in the percentage of morphologically normal preantral follicles in all treatments when compared to the control (92.67 ± 2.79, mean ± SD). However, there were no significant difference when the exposure and freezing procedures were compared using the same cryoprotectant. Moreover, there was no significant difference among cryoprotectants at the time of exposure (DMSO: 64.7 ± 3.8; EG: 70.7 ± 11.2, PROH: 63.3 ± 8.5) or after freezing (DMSO: 60.6 ± 3.6, EG: 64.0 ± 11.9; PROH: 62.0 ± 6.9). However, only follicles frozen with PROH had normal ultrastructure. In conclusion, preantral follicles enclosed in agouti ovarian tissue were successfully cryopreserved using 1.5 M PROH, with satisfactory maintenance of follicle morphology and ultrastructure.     

TOLERANCE OF SIX MARINE MICROALGAE TO THE CRYOPROTECTANTS DIMETHYL SULFOXIDE AND METHANOL1

Knowledge of tolerance to cryoprotectants is important in determining viability after biological freezing of algae. Six taxonomically diverse marine microalgae were evaluated for their tolerance to the widely used cryoprotectants dimethyl sulfoxide (DMSO) and methanol. Tetraselmis chuii Butcher survived exposure to 30% (v/v) DMSO and 25% methanol for periods of up to 4 h. All other species were more sensitive to high concentrations of these cryoprotectants. DMSO was lethal at 25% after a 15-min exposure of Rhodomonas baltica Karsten, Isochrysis off. galbana (strain T-ISO) Parke, and Nannochloropsis gaditana Lubian. Nannochloris atomus Butcher could tolerate only a 1-min exposure at this concentration; Chaetoceros gracilis Schutt completely lost viability when exposed to 20% for 60 min. Safe concentrations for DMSO incubations were similar (about 5% lower) to lethal thresholds. Methanol incubations did not significantly decrease cell viability at concentrations of 5% (1 min) for R. baltica, 25% (up to 60 min) for T. chuii, 15% (up to 120 min) for I. galbana, 5% (up to 60 min) for N. gaditana, 15% (up to 240 min) for Ch. gracilis, and 15% (up to 120 min) for N. atomus. Nannochloris atomus has the potential to be cryopreserved without the need for any cryoprotectant. The other five species were clearly dependent on a 15% DMSO preincubation to achieve a growth response after thawing from ?196° C. Only N. atomus and N. gaditana could be grown after being cryopreserved in the presence of 5% methanol.     

Presence of free D-amino acids in microalgae

Several species of microalgae (phytoplankton), 4 species of freshwater algae and 4 species of marine diatoms, were cultured germ-free in the laboratory. The presence of free D-amino acids was verified in these species by a reversed-phase HPLC analysis. D-Aspartate was detected in all the microalgae examined, but D-alanine was only present in the marine diatoms. The D-amino acid content in Asterionella sp. of the marine diatoms increased from the exponential phase to the stationary phase and then decreased to the phase of decline.     

The fatty acid profile changes in marine invertebrate larval cells during cryopreservation

The development of cryopreservation methods for embryonic cells and larvae of sea animals offers a great potential for marine biotechnology. Larval cells of bivalves and sea urchins were frozen to −196 °C using traditional cryoprotectants (Me₂SO and trehalose) and the cryoprotective mixture developed by us. In addition to Me₂SO and trehalose, this mixture contained an exogenous lipid extract from mussel tissues and antioxidants. A positive effect of antioxidants (α-tocopherol acetate, ascorbic acid or echinochrome, the quinoid pigment of sea urchins) on cell viability became significant only in the presence of exogenous lipids. Antioxidants added to cryoprotective mixtures did not reveal visible cryoprotective activity when used separately. To better understand the mechanism of the protective effect of exogenous lipids on cell membranes of sea animals, a comparative analysis of the fatty acid (FA) composition of total lipids in larval cells before and after freezing was carried out using a gas–liquid chromatography. The results indicate that freezing–thawing has direct effects on the FA composition of major lipid classes in marine invertebrate cells, and these effects can vary depending on the provenance of the cells. We have found that (I) both cell viability and the FA profile of cell lipids after cryopreservation depend on the cryoprotectants used; (II) an amount of saturated, monoenic and polyenic FAs changes significantly after cryopreservation. We assume that the addition of the exogenous lipid extract in form of liposomes could promote a renewal of disturbance areas and prevent from membrane damages during freezing–thawing.     

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.     

Survival of IVF-derived bovine embryos frozen in glycerol or ethylene glycol

Survival of IVF-derived bovine embryos of different ages and stages of development, produced in 2 different co-culture systems and frozen in 2 different cryoprotectants, was investigated. In vitro-derived bovine embryos (n = 5,525) were utilized to study survival following exposure to cryoprotectants and after freezing. Survival of the frozen embryos was based on blastocyst re-expansion 24 h and hatching 72 h after thawing. There was no difference in survival when embryos were exposed to either glycerol (Gly) or ethylene glycol (EG) for 10 or 40 min with the cryoprotectant diluted with or without freezing. In 2 of 3 experiments in which a comparison was possible, more blastocysts frozen in 1.4 M glycerol than in 1.5 M ethylene glycol survived. Addition of 0.25 M sucrose to 1.5 M ethylene glycol in the freezing solution did not improve embryo survival. More blastocysts frozen on Day 7 of in vitro culture survived than those frozen on Day 6 or Day 8. On Days 6, 7 and 8, embryos in the most advanced stage of development survived better than those at less advanced stages. Post-thaw survival did not differ for embryos produced in co-culture with Buffalo Rat Liver (BRL) cells with either Menezo B2 Medium or Tissue Culture Medium 199 and frozen in 1.4 M glycerol.     

Cryopreservation of red snapper (Lutjanus argentimaculatus) sperm: Effect of cryoprotectants and cooling rates on sperm motility, sperm viability, and fertilization capacity

Sperm cryopreservation of red snapper (Lutjanus argentimaculatus) is essentially unexplored, although many species of the Lutjanidae family are considered to be high-value commercial species. The objective of this study was to develop a species-specific cryopreservation protocol for red snapper (L. argentimaculatus) sperm by optimizing cryoprotectants and cooling rates in the cryopreservation procedure. Ten cryoprotectants at four concentrations and two freezing protocols were examined in two separate experiments. In the first experiment, toxicity studies of dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG), formamide, methanol, ethanol, sucrose, trehalose, and dimethylacetamide (DMA) on sperm motility were performed. Semen diluted 1:1 in Ringer solution were exposed to cryoprotectants at four final concentrations of 5%, 10%, 15%, or 20% for periods of 10, 20, 30, 40, 50, 60, 90, and 120 min at room temperature (25 °C). The cryoprotectants and concentrations that showed the least toxic effect on sperm motility were selected for cryopreservation trials. In the second experiment, selected cryoprotectants were then assessed for freezing capacity of sperm as follows: DMSO 5% and 10%, PG 5% and 10%, EG 5% and 10%, ethanol 5%, and methanol 5%. Semen was diluted 1:1 in Ringer solution and equilibrated with selected cryoprotectants for 10 min at room temperature. Sperm were frozen in a controlled-rate programmable freezer at four cooling rates of 3, 5, 10, and 12 °C/min from an initial temperature of 25 °C to final temperatures of −40 or −80 °C before plunging into liquid nitrogen. Sperm equilibrated in 10% DMSO and cooled at a rate of 10 °C/min to a final temperature of −80 °C had the highest motility (91.1 ± 2.2%) and viability (92.7 ± 2.3%) after thawing. The fertilization rate of frozen-thawed sperm (72.4 ± 2.4%) was not different (P > 0.05) from that of fresh sperm (75.5 ± 2.4%). This study apparently represents the first reported attempt for cryopreservation of L. argentimaculatus sperm.     

Cryopreservation of primate embryonic stem cells with chemically-defined solution without Me2SO

Human embryonic stem (hES) cells are expected to be useful in the fields of regenerative medicine and tissue engineering due to their pluripotency. Therefore, it is necessary to establish highly efficient and reliable methods for the cryopreservation of hES cells. We have cryopreserved cynomolgus and human ES cells by the vitrification method, using a chemically-defined dimethyl sulfoxide (Me₂SO)-free and serum-free medium composed of Euro-Collins solution as a base medium and 40% (v/v) ethylene glycol (EG) and 10% (w/v) polyethylene glycol (PEG) as cryoprotectants. When the vitrification and the cryoprotectants were combined, the recovery ratio of hES cells was 22.9 ± 7.7%, compared to 0.4 ± 0.2% when the conventional slow-freezing method was used. After the cryopreservation and thawing cycle, hES cells were easily cultured and expressed undifferentiated cell markers such as Nanog, Oct-4, SSEA-4, and alkaline phosphatase activity after several subculturing steps. We also found that the pluripotency of hES cells was maintained, as demonstrated by teratoma formation of ES cells transplanted into severe combined immunodeficient (SCID) mice. Thus, we conclude that we have successfully cryopreserved primate ES cells with high efficiency using a Me₂SO-free, chemically-defined medium.     

Trehalose as cryoprotectant for the freeze preservation of carrot and tobacco cells

Suspension cultures of carrot (Daucus carota, line C1), tobacco (Nicotiana tabacum, line TX1), and Nicotiana plumbaginifolia (line NP) were frozen under controlled conditions with trehalose as the sole cryoprotectant. Maximal post-thaw viability (71-74%), measured by phenosafranin dye exclusion, was obtained with the C1 cells following a 24hour pretreatment with 5 or 10% trehalose and with 40% trehalose as the cryoprotectant during freezing. TX1 cells pretreated for 24 hours with 10% trehalose and cryoprotected with 40% trehalose during freezing showed 47% viability following thawing as determined by phenosafranin dye exclusion. The NP cells required a 3 to 6 day pretreatment with 10% trehalose and 40% trehalose as a cryoprotectant at the time of freezing for the recovery of viable cells. Growing cells were recovered when the C1 and NP cells treated as described were plated on agar-solidified medium following thawing.     

Infective Juveniles of the Entomopathogenic Nematode,Steinernema feltiae Produce Cryoprotectants in Response to Freezing and Cold Acclimation

Steinernema feltiae is a moderately freeze-tolerant entomopathogenic nematode which survives intracellular freezing. We have detected by gas chromatography that infective juveniles of S. feltiae produce cryoprotectants in response to cold acclimation and to freezing. Since the survival of this nematode varies with temperature, we analyzed their cryoprotectant profiles under different acclimation and freezing regimes. The principal cryoprotectants detected were trehalose and glycerol with glucose being the minor component. The amount of cryoprotectants varied with the temperature and duration of exposure. Trehalose was accumulated in higher concentrations when nematodes were acclimated at 5°C for two weeks whereas glycerol level decreased from that of the non-acclimated controls. Nematodes were seeded with a small ice crystal and held at -1°C, a regime that does not produce freezing of the nematodes but their bodies lose water to the surrounding ice (cryoprotective dehydration). This increased the levels of both trehalose and glycerol, with glycerol reaching a higher concentration than trehalose. Nematodes frozen at -3°C, a regime that produces freezing of the nematodes and results in intracellular ice formation, had elevated glycerol levels while trehalose levels did not change. Steinernema feltiae thus has two strategies of cryoprotectant accumulation: one is an acclimation response to low temperature when the body fluids are in a cooled or supercooled state and the infective juveniles produce trehalose before freezing. During this process a portion of the glycerol is converted to trehalose. The second strategy is a rapid response to freezing which induces the production of glycerol but trehalose levels do not change. These low molecular weight compounds are surmised to act as cryoprotectants for this species and to play an important role in its freezing tolerance.     

Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure

The study was designed to evaluate the efficiency of a modified (sealed) open pulled straw (mOPS) method for cryopreserving rabbit embryos by vitrification or rapid freezing. An additional objective was to determine whether the mOPS method could cause the vitrification of a cryoprotectant solution generally used in rapid freezing procedures. Two consecutive experiments of in vitro and in vivo viability were performed. In Experiment 1, the in vitro viability of rabbit embryos at the morula, compacted morula, early blastocyst and blastocyst stages was assessed after exposure to a mixture of 25% glycerol and 25% ethylene glycol (25GLY:25EG: vitrification solution) or 4.5 M (approximately 25% EG) ethylene glycol and 0.25 M sucrose (25EG:SUC: rapid freezing solution). Embryos were loaded into standard straws or mOPS and plunged directly into liquid nitrogen. The mOPS consisted of standard straws that were heat-pulled, leaving a wide opening for the cotton plug and a narrow one for loading embryos by capillarity. The embryos were aspirated into the mOPS in a column positioned between two columns of cryoprotectant solution separated by air bubbles. The mOPS were then sealed with polyvinyl-alcohol (PVA) sealing powder. The vitrification 25GLY:25EG solution became vitrified both in standard straws and mOPS, whereas the rapid freezing 25EG:SUC solution crystallized in standard straws, but vitrified in mOPS. The total number of embryos cryopreserved was 1695. Embryos cryopreserved after exposure to each solution in mOPS showed higher rates (88.2%) of survival immediately after thawing and removal of the cryoprotectant than those cryopreserved in 0.25 ml standard straws (78.8%; P < 0.0001). After culture, the developmental stage of the cryopreserved embryos significantly affected the rates of development to the expanded blastocyst stage. Regardless of the cryoprotectant used, lower rates of in vitro development were obtained when the embryos were cryopreserved at the morula stage, and higher rates achieved using embryos at blastocyst stages. Based on the results of Experiment 1, the second experiment was performed on blastocysts using the mOPS method. Experiment 2 was designed to evaluate the in vivo viability of cryopreserved rabbit blastocysts loaded into mOPS after exposure to 25GLY:25EG or 25EG:SUC. Embryos cryopreserved in mOPS and 25GLY:25EG solution gave rise to rates of live offspring (51.7%) not significantly different to those achieved using fresh embryos (58.5%). In conclusion, the modified (sealed) OPS method allows vitrification of the cryoprotectant solution at a lower concentration of cryoprotectants than that generally used in vitrification procedures. Rabbit blastocysts cryopreserved using a 25GLY:25EG solution in mOPS showed a similar rate of in vivo development after thawing to that shown by fresh embryos.     

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5–ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate.     

Effects of extenders, cryoprotectants and freezing methods on sperm quality of the threatened Brazilian freshwater fish pirapitinga-do-sul Brycon opalinus (Characiformes)

The objective was to develop a suitable freezing method to cryopreserve Brycon opalinus (Characiformes) sperm. Extenders (NaCl and glucose at 325 and 365 mOsm/kg), cryoprotectants (dimethyl sulfoxide = dimethyl sulfoxide (DMSO) and methyl glycol = methyl glycol (MG)), equilibration times (15 and 30 min), thawing temperatures (30 and 60 °C), and straw sizes (0.5 and 4.0 mL) were tested. Sperm were frozen in a liquid nitrogen vapor vessel at −170 °C and subsequently stored in liquid nitrogen. Post-thaw sperm quality was always evaluated in terms of motility (expressed as percentage of motile sperm), duration of motility and vitality (eosin-nigrosin staining, expressed as percentage of intact sperm). The best freezing method was also tested for fertility and hatching (expressed as the percentage of fertilized eggs). Post-thaw sperm quality was highest when sperm were cryopreserved in Glucose 365 mOsm/kg and MG, after a 30-min equilibration and thawed at 60 °C for 8 s, of regardless straw size: 74 ± 7% motile sperm, 47 ± 4 s of motility duration, 69 ± 3% intact sperm, 64 ± 4% fertilization and 63 ± 3% hatching. The freezing method developed in the present study was efficient and can be used to maximize larvae production for both aquaculture purposes and for conservational programs, since B. opalinus is a threatened species.     

Direct transfer of equine blastocysts frozen-thawed in the presence of ethylene glycol and sucrose

The present study was designed to examine the suitability of ethylene glycol as a cryoprotectant for equine embryos. Blastocysts recovered nonsurgically from Day 6 donor mares were cryopreserved by conventional 2-step freezing in the presence of 10% ethylene glycol (EG), 10% glycerol (Gly), or 10% ethylene glycol + 0.1M sucrose (EG + Suc). After thawing, the EG and Gly were removed by a 6-step manner, and the EG + Suc was diluted to one fourth in the freezing straw. The postthaw blastocysts were transferred nonsurgically into the uteri of recipient mares on Days 4 to 7 after ovulation. Pregnancy rates, based on Day 15 ultrasonography, were 25.0% (2/8) and 37.5% (3/8) for the blastocysts frozen in EG and Gly, respectively. Direct transfer following thawing and in-straw dilution of blastocysts frozen in EG + Suc resulted in a pregnancy rate of 63.6% (7/11). In fresh Day 6 blastocysts (control group), the pregnancy rate was 70.0% (7/10). These results indicate that the combined use of ethylene glycol and sucrose in a 2-step freezing regimen allows for the direct transfer of frozen-thawed blastocysts into recipient mares, with an acceptable pregnancy rate.     

A sperm cryopreservation protocol for the loach Misgurnus anguillicaudatus and its applicability for other related species

The aim of the present study was to establish a protocol of sperm cryopreservation in Misgurnus anguillicaudatus and verify the applicability of the obtained protocol in other loach species. We evaluated the following parameters: inseminating dose, thawing temperatures (20, 25 and 30 °C for 10 s), extenders (loach or cyprinid extenders), internal cryoprotectants (dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), glycerol (Gly), ethylene glycol (EG), and methanol (MeOH) at 0, 5, 10 and 15%), external cryoprotectants (bovine serum albumin 1 and 2%; sucrose 0.5 and 1%; glucose 0.5 and 1%; glycine 0.5 and 1%), activating solutions (distilled water, dechlorinated tap water, 25 mM NaCl and 50 mM NaCl), and hatchability of the eggs when fertilized with fresh or cryopreserved sperm. After the evaluation of these parameters, we optimized the cryopreservation using the following procedure: thawing temperature at 25 °C for 10 s; loach or cyprinid extenders; methanol at 10 or 15% as internal cryoprotectants; glycine 0.5% or bovine serum albumin 1% as external cryoprotectants and 50 mM NaCl for sperm activation. Using this procedure, the fertilizability of the post-thawed sperm was 47% in comparison to the fresh sperm, at the minimum inseminating dose (687.65 spermatozoa egg⁻¹ mL⁻¹). Based on this protocol, sperm from other loach species Lefua nikkonis, Misgurnus mizolepis and Barbatula toni were cryopreserved successfully.