Extender osmolality and sugar supplementation exert a complex effect on the cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa

We have carried out two experiments to study the cryobiology of red deer epididymal spermatozoa and to improve freezing extenders: (1) effect of extender (Tris-citrate-fructose) osmolality (300-600 mOsm/kg), and (2) effect of sugar (0.4M) supplementation to the extender (no sugar, glucose, fructose, mannose, sucrose, maltose, threalose and raffinose). Sperm quality was assessed pre-freezing, post-thawing, and after 2h at 37 degrees C post-thawing: sperm motility index (SMI), acrosome integrity and membrane integrity (HOS test) were assessed subjectively; mitochondrial activity (JC-1) and membrane stability (merocyanine 540) were assessed by flow cytometry. In experiment 2, DNA status was assessed using acridine orange and flow cytometry. To find an optimal osmolality, we fitted the data to a quadratic curve. Four hundred Osmolal per kilogram rendered better results pre-freezing and post-thawing. However, post-thawing viability and most parameters after 2h incubation fitted a linear model. Osmolalities above 425 mOsm/kg were deleterious (P<0.05). In experiment 2, fructose had a positive effect respect to control after 2h of incubation at 37 degrees C post-thawing. Di and trisaccharides were deleterious. Trehalose showed impaired DNA status after 2h incubation. In conclusion, the osmolality of the extenders should be around 400 mOsm/kg, although the change from quadratic to lineal may indicate a complex effect that must be further studied. Monosaccharides may enhance red deer epididymal sperm cryopreservation, especially fructose, whereas di and trisaccharides may not be adequate.     

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.     

Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation

Mouse sperm has proven to be more difficult to cryopreserve than sperm of other mammalian species. Published reports show that only three cryoprotectant agents (CPAs), alone or combined, have been studied: glycerol and dimethyl sulfoxide (DMSO), as permeating agents, and raffinose, as a nonpermeating agent. To date, the most consistent results for mouse sperm cryopreservation have been achieved by use of raffinose/skim milk as cryoprotectant with rapid cooling at 20 degrees C per minute. In this study, we compared the cryoprotection provided by permeating (glycerol, formamide, propanediol, DMSO, adonitol) or nonpermeating (lactose, raffinose, sucrose, trehalose, d-mannitol) compounds for freezing mouse sperm. Different solutions were made using 3% skim milk solution as the buffer or extender in which all different cryoprotectant agents were dissolved at a concentration of 0.3 M, with a final osmolality of approx. 400 mOsm. Sperm samples from CB6F1 (hybrid) and C57BL/6J (inbred) mice collected directly into each CPA were frozen/thawed under identical conditions. After thawing and CPA elimination (centrifugation) raffinose (59%), trehalose (61%), and sucrose (61%) sustained the best motility (P = < 0.1) of the nonpermeating agents, whereas the best of the permeating agents was DMSO (42%). Membrane integrity was analyzed and showed that the simple exposure (prefreeze) to sugars was less harmful than the exposure to glycols. Coincidentally, sperm frozen in trehalose (41%), raffinose (40.5%), and sucrose (37.5%) were the samples less injured among all different postthawed CPA tested. The in vitro fertilization results demonstrated that hybrid mouse spermatozoa frozen with sugars (lactose 80%, raffinose 80%, trehalose 79% of two-cell embryos production) were more fertile than those frozen with glycols (glycerol 11%).     

Maintenance of fertility in cryopreserved Indian gerbil (Tatera indica) spermatozoa

This study is the first attempt at sperm cryopreservation, as well as a further examination of frozen sperm fertility by the hamster test, applied to the maintenance of an Indian gerbil (Tatera indica) colony, which is a newly developing experimental animal.The osmotic tolerance of the spermatozoa was initially investigated by subjection to hypertonicity, up to 620 mOsm/kg, for 5 min at room temperature prior to freezing. Although the percentage of total motile sperm was not affected, that of progressive motile spermatozoa began to drop at 400 mOsm/kg, and a significant decrease was observed at 620 mOsm/kg (p < 0.01). According to these results, the osmolality of the solutions for the freezing experiment, in which 6–22% raffinose was present, was fixed at approximately 400 mOsm/kg. Sperm, suspended in a plastic straw, were frozen in liquid nitrogen vapor for 5 min, followed by immersion in liquid nitrogen. Motile sperm were recovered from all freezing conditions, and high survival was obtained when sperm were frozen in the presence of 14% and 18% raffinose, with a normalized motility higher than 40%. Fertility of cryopreserved Indian gerbil sperm was examined by the zona-free hamster test. Thawed sperm adhered to 88% of the zona-free hamster oocyte surface, and some oocytes were penetrated and exhibited swollen sperm heads or male pronuclei, which we used to define fertilization. Although the fertilization rate of cryopreserved sperm to zona-free hamster eggs was significantly lower than that of fresh sperm (6% vs. 30%, p < 0.01), we demonstrated that thawed Indian gerbil spermatozoa have the ability to maintain their fertility.     

Cryopreservation of epididymal sperm collected postmortem in the Tasmanian devil (Sarcophilus harrisii)

Effective sperm cryopreservation protocols are limited to a small number of marsupial species. In this study, postmortem gamete rescue (PMGR) epididymal sperm samples from Tasmanian devils (N = 34) euthanized due to the fatal Devil Facial Tumor Disease were used to develop long-term sperm storage techniques for the species. Cryoprotectant toxicity associated with equilibration of sperm samples in a TEST yolk diluent (TEST; 189 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 85 mM Trizma base [Tris], 11 mM glucose, 20% vol/vol egg yolk; pH 7.1, and 315.0 ± 5.0 mOsm/kg) with a final concentration of 0.06 M trehalose, or 4%, 10%, and 18% vol/vol of either glycerol or dimethyl sulfoxide (DMSO), was examined over 12 h at 15 °C. Trehalose supplementation resulted in an immediate decline (P < 0.05) of total motility. After 12 h, total motility was reduced (P < 0.05) in treatments containing 18% glycerol, and 10% and 18% dimethyl sulfoxide. The effects of final glycerol concentration (4% and 10%), glycerol equilibration duration (10 min 1 h, or 3 h) prefreeze, freezing rate and the addition of 0.10 M lactose or a combination of 0.10 M lactose and 0.11 M raffinose were assessed during three experiments on the cryopreservation of postmortem gamete rescue samples in TEST. In all experiments, motility and viability were reduced (P < 0.01 postthaw). Samples cryopreserved in TEST supplemented with lactose or lactose with raffinose using a fast freezing rate (−8 °C/min from 4 to −40 °C, then −65 °C/min until −165 °C) produced the highest (P < 0.05) postthaw motility (18.6 ± 5.5% and 16.9 ± 8.5%, respectively), which represented 35% to 48% retention of prefreeze motility. These results apparently were the best postthaw results of dasyurid sperm reported to date and will help lay the foundations for developing assisted reproductive technologies for marsupial species.     

The effect of supplementation of cryopreservation diluents with sugars on the post-thawing fertility of ram semen

This study was conducted to elucidate the effect of increasing the osmolality of a basic Tris, extender supplemented with sucrose, trehalose or raffinose on post-thawing ram semen quality (sperm motility, viability, acrosome integrity, total sperm abnormalities and membrane integrity). After primary evaluation of the collected ejaculates, only semen samples with more than 70% motile sperm, and a sperm concentration of higher than 3 × 10⁹ sperm/ml were used for cryopreservation. The semen samples were pooled and diluted (1:4) with a Tris-citric acid-fructose-yolk extender, supplemented with different concentrations (50, 70 or 100 mM) of sucrose, trehalose or raffinose. As control, semen was diluted and frozen in the base diluent, without additional sugars. Pooled semen samples were aspirated into 0.25 ml straws, cooled to 5 °C within 90 min and frozen by exposure to liquid nitrogen vapor (4-5 cm above the liquid nitrogen surface) for 10 min - before plunging into liquid nitrogen, for storage. After 24 h, straws were thawed in a water bath (37 °C) for 30 s. The frozen-thawed sperm characteristics were improved significantly (P < 0.05) by increasing the level of the sugars. Optimal results being obtained with 70 and 100 mM trehalose or raffinose. All extenders containing supplemental sugars were superior in terms of sperm quality to the control (P < 0.01) group. The highest sperm motility (60.6 ± 1.9%), viability (60.6 ± 2.5%) and membrane integrity (58.2 ± 2.1%) were recorded using 100 mM trehalose and the lowest with 50 mM sucrose (48.6 ± 1.9%, 51.4 ± 2.5% and 47.9 ± 2.1%, respectively). All sugar concentrations decreased the percentage of acrosomal and total sperm abnormalities (P < 0.05). The extenders containing 100 mM trehalose or raffinose significantly (P < 0.05) decreased the occurrence of sperm abnormalities, compared to the other treatments. The fertility rates obtained after cervical insemination of the frozen-thawed sperm were 46.8%, 44.1% and 16.7% for 100 mM trehalose, 100 mM raffinose and the control with supplementation of the diluents, respectively. The study showed that ram sperm can tolerate hyperosmotic diluents, and that a range of sugar concentrations (50-100 mM) may successfully be incorporated in the ram semen cryopreservation diluents, although further research is warranted.     

Development of a buffer system for dialysis of bovine spermatozoa before freezing. II. Effect of sugars and sugar alcohols on posthaw motility

Two experiments were conducted to evaluate the effect of different levels of sugars (glucose, lactose and raffinose) and the effect of those sugars (C(3) to C(6)) or their correspondent sugar alcohols on the dialysis of bovine semen. First, the effect of isosmotic solutions of glucose, lactose or raffinose at five different levels (0, 25, 50, 75, 95% V/V) on sperm motility of semen dialyzed prior to freezing were studied. These levels were used in extenders and dialysates, and the final volume was complemented with Piperazine-N-N-BIS (2-ethane sulfonic acid (PIPES) titrated to pH 7.0 with TRIS (hydroxymethyl) amino-methane (TRIS) to form PIPEST or a 1:1 (V/V) combination between PIPEST and sodium citrate solutions. In the second experiment, 30% of the buffer volume contained solutions of sugars (C(3) or C(6)) or their correspondent sugar alcohol, and the final volume was completed with PIPEST-citrate buffer. Semen aliquots were extended (1:10) and dialyzed (1:50) for 2 h while cooling from 37 to 5 degrees C in semipermeable dialysis bags of 12,000 to 14,000 molecular weight cut off. The samples were frozen in pellets 1 h after dialysis was terminated. Sperm survival was significantly higher in PIPEST-citrate than in PIPEST buffer alone (P<0.05). No significant difference (P>0.05) was obtained between the use of glucose or lactose or between lactose and raffinose. High levels of sugar appeared to be detrimental to sperm motility of fresh and thawed semen samples. Motility of cells extended in buffers containing 30% (V/V) isosmotic solutions of glucose, galactose, ribose, xylose, arabinose or their correspondent sugar alcohols was significantly higher (P<0.05) than their motility in extenders without sugar.     

Sugar compartmentation in frost-hardy and partially dehardened cabbage leaf cells

In frost-hardy and partially dehardened leaves of Brassica oleracea L. var. sabellica L. the distribution of cryoprotective sugars and of chloride between chloroplasts and the nonchloroplast part of leaf cells was investigated using the nonaqueous isolation technique as a means of cell fractionation. In chloroplasts of frost-hardy leaves high concentrations of sucrose and raffinose and comparatively low concentrations of chloride have been found. The ratios between sugars and chloride were so as to ascertain complete protection of the frost-sensitive thylakoid membranes during freezing. During dehardening, sugars decreased especially in the chloroplasts. There was a conversion of sucrose and raffinose into monosaccharides. This led to a large increase in the concentration of glucose and fructose in the nonchloroplast parts of the cells. There is evidence that the sugar concentration in the vacuole increased at the expense of sugars located in chloroplasts and cytoplasm. The quantity of sugars that remained in the chloroplasts did not appear to be sufficient for complete membrane protection at very low freezing temperatures.     

Enrichment in Specific Soluble Sugars of Two Eucalyptus Cell-Suspension Cultures by Various Treatments Enhances Their Frost Tolerance via a Noncolligative Mechanism

A cell-suspension culture obtained from the hybrid Eucalyptus gunnii/Eucalyptus globulus was hardened by exposure to lower temperatures, whereas in the same conditions cells from a hybrid with a more frost-sensitive genotype, Eucalyptus cypellocarpa/Eucalyptus globulus, were not able to acclimate. During the cold exposure the resistant cells accumulated soluble sugars, in particular fructose and sucrose, with a limited increase in cell osmolality. In contrast, the cell suspension that was unable to acclimate did not accumulate soluble sugars in response to the same cold treatment. To an extent similar to that induced after a cold acclimation, frost-hardiness of the cells increased after a 14-h incubation with specific soluble sugars such as sucrose, raffinose, fructose, and mannitol. Such hardening was also observed for long-term cultures in mannitol-enriched medium. This cryoprotective effect of sugars without exposure to lower temperatures was observed in both the resistant and the sensitive genotypes. Mannitol was one of the most efficient carbohydrates for the cryoprotection of eucalyptus. The best hardiness (a 2.7-fold increase in relative freezing tolerance) was obtained for the resistant cells by the cumulative effect of cold-induced acclimation and mannitol treatment. This positive effect of certain sugars on eucalyptus freezing tolerance was not colligative, since it was independent of osmolality and total sugar content.     

Effect of osmolality and oxygen tension on the survival of mouse sperm frozen to various temperatures in various concentrations of glycerol and raffinose

Cryopreserved mouse sperm are beginning to be used to meet the demand of a reliable cost-effective method for maintaining the rapidly expanding numbers of lines of mutant mice. However, successful and reproducible cryopreservation has proven to be a difficult problem. Furthermore, the underlying factors responsible for success or failure are mostly obscure. Several contributors to these difficulties have been identified. Our laboratory has found that mouse sperm are extremely susceptible to the mechanical stresses associated with pipetting, mixing, and centrifugation, and others have found that they are severely limited in their tolerance to osmotic volume changes. We have hypothesized two other contributors to the difficulties. One is that the concentrations of glycerol used in published protocols are substantially lower than those found to be optimal for most mammalian cells. The other hypothesis relates to the fact that mouse sperm membranes are especially susceptible to damage from oxygen-derived free radicals. That damage may reduce their ability to survive freezing. If so, survival ought to increase if the concentration of oxygen is kept low throughout the procedure. To achieve low levels, we have incorporated an Escherichia coli membrane fraction, Oxyrase, into all media. A previous report showed a protective effect. That is confirmed here under a broader range of conditions. The conditions studied have been the individual and interactive effects of the concentrations of glycerol, raffinose, and phosphate-buffered saline (PBS) on motility after freezing at 21 degrees C/min to -70 degrees C. Cryoprotection increased with increasing raffinose concentration, provided that the concentration of PBS was appropriately reduced to hold the total osmolality of nonpermeating solutes to within tolerated limits. Surprisingly, the best results were achieved in the total absence of glycerol. The highest motilities to date (68 +/- 8%) after freezing to -70 degrees C have been achieved using media containing Oxyrase, 0 M glycerol, and 18% raffinose in 14x strength modified PBS. We also determined the motility loss after freezing to intermediate temperatures, i.e., -10 and -30 degrees C. The major motility loss occurred by -10 degrees C, especially in the absence of Oxyrase. These results suggest that a major problem in the freezing of mouse sperm is the physical stress resulting from extracellular ice crystal formation. Oxyrase appears to lessen that damage substantially.     

Intracellular Delivery of Carbohydrates into Mammalian Cells through Swelling-activated Pathways

Volume changes of human T-lymphocytes (Jurkat line) exposed to hypotonic carbohydrate-substituted solutions of different composition and osmolality were studied by videomicroscopy. In 200 mOsm media the cells first swelled within 1–2 min and then underwent regulatory volume decrease (RVD) to their original isotonic volume within 10–15 min. RVD also occurred in strongly hypotonic 100 mOsm solutions of di- and trisaccharides (trehalose, sucrose, raffinose). In contrast to oligosaccharide media, 100 mOsm solutions of monomeric carbohydrates (glucose, galactose, inositol and sorbitol) inhibited RVD. The complex volumetric data were analyzed with a membrane transport model that allowed the estimation of the hydraulic conductivity and volume-dependent solute permeabilities. We found that under slightly hypotonic stress (200 mOsm) the cell membrane was impermeable to all carbohydrates studied here. Upon osmolality decrease to 100 mOsm, the membrane permeability to monomeric carbohydrates increased dramatically (apparently due to channel activation caused by extensive cell swelling), whereas oligosaccharide permeability remained very poor. The size-selectivity of the swelling-activated sugar permeation was confirmed by direct chromatographic measurements of intracellular sugars. The results of this study are of interest for biotechnology, where sugars and related compounds are increasingly being used as potential cryo- and lyoprotective agents for preservation of rare and valuable mammalian cells and tissues.This revised version was published online in June 2005 with a corrected cover date.     

Comparison of different osmolalities and egg-yolk composition in processing media for the cryopreservation of red wolf (Canis rufus) sperm

Successful cryopreservation of sperm and the maintenance of a sperm-based genome resource bank have been identified as priorities for the recovery of the endangered red wolf (Canis rufus). The objectives were to improve sperm processing and to determine the relative timing of damage to red wolf sperm during freezing and thawing. Fresh ejaculates (n=37) from adult red wolves (n=15, aged 2-13 y) were collected via electroejaculation and subjected to cooling, freezing and thawing in four TRIS-egg-yolk extender treatments varying in osmolality ( approximately 305 mOsm versus approximately 350 mOsm) and egg-yolk composition (0.8 microm-filtered versus unfiltered). Ejaculates were evaluated for sperm percentage motility, forward progressive motion, and morphological characteristics immediately upon collection and following extension, cooling (prior to freezing) and thawing. Although no single treatment consistently produced superior results, sperm suspended in approximately 305 mOsm extenders exhibited slight losses in motility post-thawing (13 and 7%). Also, sperm suspended in approximately 350 mOsm extenders tended to have slower rates of decline in motility in vitro post-thawing than those stored in approximately 305 mOsm extenders (P=0.55). Finally, extenders incorporating unfiltered egg yolk exhibited a slightly larger ratio of absent to partial acrosomes than did sperm frozen in extenders prepared with clarified egg yolk. For approximately 350 mOsm extenders, most motility loss occurred during the cooling rather than freezing and thawing. In conclusion, these data contribute to knowledge regarding cryopreservation of red wolf sperm.     

Factors affecting the survival of frozen-thawed mouse spermatozoa

Mouse epididymal spermatozoa were frozen in solutions containing various compounds with different molecular weights, and the factors affecting the postthawing survival were examined. Monosaccharides (glucose, galactose) had almost no protective effect regardless of the concentration and the temperature of exposure. On the other hand, disaccharides (sucrose, trehalose) and trisaccharides (raffinose, melezitose) resulted in higher survival rates, especially at a concentration of around 0.35 mol/kg H(2)O (0.381-0.412 Osm/kg). Macromolecules, such as PVP10, Ficoll 70, bovine serum albumin, and skim milk had almost no effect, but compounds with a molecular weight of about 800, such as metrizamide and Nycodenz, had some protective effect. When a raffinose solution was supplemented with 10% metrizamide, resulting in an osmolality of approximately 0.400 Osm/kg, a high survival rate was obtained. Solutions at about 0.400 Osm/kg containing trehalose alone, trehalose + metrizamide, raffinose alone, and raffinose + metrizamide, were all effective for sperm freezing; frozen-thawed sperm could fertilize oocytes, and the resultant embryos could develop to live young after transfer. For freezing mouse spermatozoa, aqueous solutions at approximately 0.400 Osm/kg containing a disaccharide or a trisaccharide seem to be effective.     

High-Throughput Cryopreservation of In Vivo-Derived Swine Embryos

Cryopreservation of swine embryos is inefficient. Our goal was to develop a non-invasive method for “relatively” high-throughput cryopreservation of in vivo-produced swine embryos. Since removal of the lipid droplets within early swine embryos improves cryosurvival we wanted to apply a technique of high osmolality treatment followed by centrifugation that was first developed for in vitro-produced swine embryos to in vivo-produced swine embryos. The first aim was to determine how sensitive the in vivo-produced zygote and 2-cell stage embryo was to various high osmolality conditions for a short duration. Culture for 6, 12 or 18 min at 300, 400 or 500 milliosmoles (mOsm) had no detectable affect on the resulting blastocyst stage embryos (number of inner cell mass nuclei, trophectoderm nuclei, total number of nuclei, ratio of the trophectoderm to inner cell mass nuclei or percent blastocyst). However there was an effect of gilt on each of these parameters. For the second aim we focused on 300 mOsm for 6 min, 400 mOsm for 12 min, 500 mOsm for 12 min, and 500 mOsm for 18 min. The embryos were centrifuged for the duration of high osmolality treatment, then cultured to the blastocyst stage and vitrified. After vitrification and thawing the 500 mOsm for 18 min had the highest percent re-expansion with no difference in the total number of nuclei. While requiring a different base culture medium than in vitro-produced embryos, in vivo-derived embryos also survive cryopreservation without damage to their zona pellucida.     

Effect of glutamine and sugars after bull spermatozoa cryopreservation

The objective of this study was to evaluate the effects of the addition of different sugars (raffinose, sucrose, and trehalose) on bull spermatozoa cryopreserved in a commercial extender (Optidyl) supplemented with glutamine on semen parameters, fertilizing ability and superoxide dismutase (SOD) activity. Nine ejaculates for each bull were used in the study. Semen was frozen in five different extenders: raffinose 25 mM plus glutamine 3 mM (RGO), sucrose 25 mM plus glutamine 3 mM (SGO), trehalose 25 mM plus glutamine 3 mM (TGO), glutamine 3 mM (GO) and control (O). Insemination doses were processed so that each 0.25 mL straw contained 15 x 10⁶sperm. Groups of GO and RGO resulted in the higher rates of subjective (54.0 ± 1.7% and 64.0 ± 1.1%; P < 0.01) and CASA motilities (53.0 ± 2.7% and 61.0 ± 4.4%; P < 0.001), respectively compared to the other groups. The supplementation of additives did not provide an effect on the level of post-thaw sperm CASA progressive motilities, the sperm motion characteristics and pregnancy rates. GO and RGO provided the better protective effect for sperm acrosome (4.0 ± 0.5% and 12.0 ± 0.6%) and total abnormalities (5.0 ± 0.3% and 13.0 ± 0.7%; P < 0.001), respectively. At the HOST values, the additives did not give to result the protective effect in comparison to Optydil extender without additives (P > 0.05). For pregnancy rates, there were no significant differences among the groups. The supplementation of additives did not provide any significant difference on the level of SOD activity (P > 0.05). It can be also thought that these sugars might have worked with glutamine in a synergy. Thereby, sugars such as raffinose and sucrose with glutamine in freezing extender may be recommended to facilitate bull semen freezability.     

Effect of sugar type on the survival of frozen-thawed rhesus monkey (Macaca mulatta) sperm

Sperm-freezing extenders supplemented with sugar or a combination of different sugars are widely used for the cryopreservation of nonhuman primate spermatozoa. Understanding which sugar or combination of sugars offers the highest level of cryoprotection would be beneficial for the development of sperm-freezing extenders. In the present study we aimed to investigate the effect of glucose, lactose, and raffinose separately or in combination on the cryosurvival of rhesus monkey spermatozoa. Toward that end, we prepared eight extenders by adding various types of sugars to a basic medium (BM): G-BM (0.3 M glucose), L-BM (0.3 M lactose), R-BM (0.3 M raffinose), LG-BM (0.15 M lactose+0.15 M glucose), RG-BM (0.15 M raffinose+0.15 M glucose), LR-BM (0.15 M lactose+0.15 M raffinose), and LRG-BM (0.1 M lactose+0.1 M raffinsoe+0.1 M glucose). A saline control (0.157 M sodium chloride) was also used. The results showed no significant difference in post-thaw motility when spermatozoa were frozen with G-BM, L-BM, LG-BM, RG-BM, and LRG-BM, but the post-thaw motility was significantly lower when it was frozen with R-BM, LR-BM, and the saline control. The highest plasma membrane integrity was achieved when spermatozoa were frozen with G-BM, L-BM, LG-BM, RG-BM, and LRG-BM, and the highest acrosome integrity was achieved with G-BM, L-BM, LG-BM, RG-BM, LRG-BM, and the saline control. The results indicate that the various sugars offered different protective effects. For the cryopreservation of rhesus monkey spermatozoa, glucose (monosaccharide) and lactose (disaccharide) were shown to be more suitable than raffinose (trisaccharide) for preserving spermatozoal motility, plasma membrane, and acrosome. Specifically, raffinose was detrimental to sperm acrosome integrity.     

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

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

不同渗透压的稀释液对猕猴精子低温冷冻保存的影响

以稀释液TTE(382mOsm/kg)为对照,研究了5种渗透压(688、389、329、166、43mOsm/kg)的TEST稀释液(TEST、mTEST1、mTEST2、mTEST3、mTEST4)在冷冻过程中对猕猴精子功能的影响。精液一步稀释于含甘油的防冻液中,甘油的终浓度为5%(v/v)。在冷冻前后分别检测精子的运动度和质膜完整性,后者用Hoechst33342和碘化丙锭双色标记流式细胞术分析。结果表明:冷冻之前,与鲜精相比,用TEST和mTEST4稀释的精子运动度和质膜完整性显著降低(P<0·001),其余组中除mTEST2稀释的精子质膜完整性显著降低(P<0·05)外,精子运动度无差异;冷冻复苏后,TTE、mTEST3和mTEST1冻存精子的运动度和质膜完整性最高,其次是mTEST2,TEST和mTEST4冷冻效果最差(P<0·05)。提示等渗、适当高渗或低渗的稀释液适合猕猴精子的冷冻保存;对精子产生高渗毒害作用是导致猕猴精子用TEST冷冻存活率低的主要原因。     

Regulation of sugar transport via the multiple sugar metabolism operon of Streptococcus mutans by the phosphoenolpyruvate phosphotransferase system.

In this report, we provide evidence that the transport of sugars in Streptococcus mutans via the multiple sugar metabolism system is regulated by the phosphoenolpyruvate phosphotransferase system. A ptsI-defective mutant (DC10), when grown on the multiple sugar metabolism system substrate raffinose, exhibited reduced growth, transport, and glycolytic activity with raffinose relative to the parent strain BM71. Inhibition of [3H]raffinose uptake was also observed in both BM71 and DC10 with increasing concentrations of glucose and the glucose analogs alpha-methyl glucoside and 2-deoxyglucose.     

Effect of storage method and extender osmolality in the quality of cryopreserved epididymal ram spermatozoa

Post-mortem sperm recovery and cryopreservation could be a complement to germplasm banking in sheep, especially for endangered breeds. This study is an attempt to identify factors for improving the success of cryopreserving ram epididymal spermatozoa, considering the decrease of sperm quality with post-mortem time. Epididymal spermatozoa from 9 rams were kept at 5°C using three storage methods: within the epididymes, undiluted sperm mass, and diluted in extenders of different osmolality (TES-Tris-fructose at 320, 370 or 420 mOsm/kg, 20% egg yolk, 8% glycerol). At 0, 24, 48 and 72h, spermatozoa were cryopreserved using each extender. Samples were analyzed before and after cryopreservation by CASA (motility) and flow cytometry (viability and acrosomal status). Post-mortem time decreased pre-freezing and post-thawing sperm quality. Some storage x extender combinations improved the effect of post-mortem time on sperm quality. Both epididymis storage combined with the 420 extender, and storing the spermatozoa diluted in the 320 extender improved post-thawing quality, especially at long post-mortem times. Storing the spermatozoa diluted in the 370 extender was detrimental for the acrosomal status. These findings have practical applications. The simplest storage method (within the epididymes) seems to be adequate if hyperosmotic extenders were used for freezing. An alternative method could be storing the spermatozoa diluted in a hypoosmotic extender. These recommendations are limited to the osmolalities tested in this study (420 mOsm/kg and 320 mOsm/kg); other osmolalities should be tested.