In vivo circulation of mouse red blood cells frozen in the presence of dextran and glucose

Loading with monosaccharide can improve the quality of human red blood cells (hRBCs) frozen with polymer. But in vivo life span of hRBCs frozen with polymer and sugar is not determined. In this study, following incubation with glucose, mouse red blood cells (mRBCs) were frozen in liquid nitrogen for 24 h using dextran as the extracellular protectant. After thawing, hemolysis, exposure of PS, and osmotic fragility of frozen mRBCs were determined in vitro. After transfusion of fluorescein isothiocyanate (FITC)-labeled mRBCs, the 24 h recovery and half life span of frozen mRBCs were determined. The data indicated the postthaw hemolysis of mRBCs frozen with dextran and glucose were significantly less than that of cells frozen with dextran (17.23% ± 5.21% vs 25.96% ± 10.07%, P = 0.034). But freezing can also result in exposure of phosphatidylserine and increase of osmotic fragility of mRBCs. After transfusion, the 24 h recovery of mRBCs frozen in the absence or presence of glucose was similar to that of the control cells (P = 0.748 and 0.971). However, the half life span of mRBCs frozen in the absence or presence of glucose was significantly less than that of the control cells (P = 0.000). In addition, incubation with glucose can not increase the life span of frozen red blood cells (7.16 ± 0.93 d vs 7.15 ± 0.34 d, P = 0.982). In conclusion, incubation with monosaccharide could significantly increase the recovery of mRBCs frozen with polymer. Although freezing can significantly shorten the half life span of frozen cells, it can not influence the 24 h recovery of frozen mRBCs. In addition, incubation with monosaccharide before freezing can not increase the life span of frozen mRBCs. So according to the above data, to increase the life span of hRBCs frozen with polymer and monosaccharide, the osmotic fragility of the frozen RBCs must be decreased in the future.     

Developing an in vitro method for Eimeria tenella attachment to its preferred and non-preferred intestinal sites

A frozen section method utilising chicken intestinal tissue was developed to study the Eimeria tenella attachment ex vivo. In order to examine Eimeria-epithelial cell attachment, 10⁵E. tenella sporozoites were incubated with each caecal frozen section (6, 10 and 14 μm) for 1 h in 5% CO₂ incubator at 41 °C. E. tenella sporozoites attached successfully to enterocytes in 14 μm thick of caecal sections. Sporozoite attachment to caecal sections was shown to be dependent on the number of parasites added. To evaluate the method, E. tenella sporozoites were incubated to its preferred (caecum) and non-preferred (duodenum and jejunum) intestinal sites. The number of sporozoites attached to the caecal enterocytes was significantly greater (P < 0.0001) in comparison with the limited number of sporozoites attached to enterocytes of non-preferred intestinal sites. This method was shown to be able to reveal differences in binding capability and allows for comparison of intestinal site attachment.     

Fixed-time AI pregnancy rate following insemination with frozen-thawed or fresh-extended semen in progesterone supplemented CO-Synch protocol in beef cows

The objective of this study was to compare fixed-time AI pregnancy rate in Angus crossbred beef cows inseminated with frozen-thawed or fresh-extended semen. Two ejaculates from each of two Angus bulls were collected by artificial vagina and pooled for each bull. The pooled semen from each bull was divided into two aliquots; Aliquot 1 was extended using Caprogen^® (LIC, Hamilton, New Zealand) to a concentration of 3 × 10⁶ sperm/straw and Aliquot 2 was extended using egg-yolk-glycerol extender to a concentration of 20 × 10⁶ sperm/straw. Semen extended with Caprogen^® was maintained at ambient temperature and semen extended with egg-yolk-glycerol extender was frozen and maintained at −196 °C until insemination. In each of two breeding seasons (Fall 2007 and Spring 2008), Angus-crossbeef cows (N = 1455) at 12 locations were randomly assigned within location to semen type [Fresh (N = 736) vs. Frozen (N = 719)] and sire [1 (N = 731) vs. 2 (N = 724)]. All cows were synchronized with 100 μg of GnRH im and a progesterone Controlled Internal Drug Release insert (CIDR) on Day 0, and on Day 7, 25 mg of PGF2_α im and CIDR removal. All cows received 100 μg of GnRH im and were inseminated at a fixed-time on Day 10, 66 h after CIDR removal. Timed-AI pregnancy rates were influenced by season (P < 0.05), cows detected in estrus prior to and at AI (P < 0.001), and dam age (P < 0.01). Pregnancy rates were not affected by semen type (Fresh = 51.5% vs. Frozen = 50.4%; P = 0.66) and there were no significant interactions of semen type by estrus expression, semen type by sire, or semen type by season (P > 0.1). In conclusion, commercial beef cows inseminated with fresh-extended semen (3 × 10⁶ sperm/straw) yielded comparable pregnancy rates to conventional frozen-thawed semen in a progesterone supplemented, CO-Synch fixed-time AI synchronization protocol and may provide an alternate to frozen semen for more efficient utilization of superior genetics.     

The validation of housekeeping genes as a reference in quantitative Real Time PCR analysis : Application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus

The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expression in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N = 13) and infected with CAEV (N = 13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240 days post-partum). The expression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of β-actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large subunit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were considered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of disease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and – according to the geNorm results – the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80 °C was also proved.     

Successful artificial insemination using semen frozen and stored by an ultrafreezer in the Majorera goat breed

The semen of five Majorera breed bucks was collected and processed to reach a final concentration of 200 × 10⁶ spermatozoa/straw in the extender containing 4% of glycerol and 12% of egg yolk. Two freezing techniques were assessed: (LN) straws were frozen and stored in liquid nitrogen, and (ULF) straws were frozen and stored in the ultra-low freezer at −152 °C. Semen quality (sperm motility, acrosome integrity and abnormal sperm cells percentages) was determined for different storage times (1, 30, 90 and 365 days of cryopreservation). Thereafter, 150 Majorera goats were assigned to four experimental groups: for groups LN-1 (n = 40) and LN-6 (n = 35), the goats were transcervically inseminated with frozen-thawed semen stored for 1 and 6 months in liquid nitrogen, respectively, while for groups ULF-1 (n = 40) and ULF-6 (n = 35), the goats were transcervically inseminated with frozen-thawed semen stored for 1 and 6 months in an ultra-low freezer at −152 °C, respectively. The pregnancy rate was determined by transabdominal ultrasound scanning; in addition, the kidding rate and prolificacy were recorded at parturition. In vitro results showed that the freezing protocol did not affect sperm quality with similar values for up to 1 year of cryopreservation. The kidding rates were not significantly different between experimental groups (43.6%, 38.5%, 42.8% and 40.0% for groups LN-1, ULF-1, LN-6 and ULF-6, respectively). In all experimental groups, the kidding rate and prolificacy were significantly higher (p < 0.01) in multiparous than in nulliparous goats. Therefore, the in vitro results and fertility trials confirmed the efficiency of the ULF technique for freezing and storage of goat semen.     

Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation

For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me₂SO). Me₂SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24 h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me₂SO. Although reduced (by 22 ± 2%, p = 0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me₂SO, where the doubling time was significantly reduced (by 41 ± 4%, p = 0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.     

Pregnancy loss in heifers after artificial insemination with frozen-thawed,sex-sorted,re-frozen-thawed dairy bull sperm

The use of sex-sorted sperm by the dairy industry is often limited by the geographical distance between potential sires and the sex-sorting facility. One method that may be used to overcome this limitation is sex-sorting sperm that have been previously frozen, or transported to the sorting facility as cooled liquid semen. In this study the in vivo fertility of frozen-thawed, sex-sorted, re-frozen-thawed (FSF) and cooled, sex-sorted, frozen-thawed (CSF) bull sperm was determined after artificial insemination (AI) of Holstein heifers. Semen from two bulls was frozen in straws, or transported to the sorting facility in an egg yolk diluent at 5 °C over 24 h. Thawed or re-warmed semen was processed through a PureSperm^® density gradient, and sperm were sorted for sex and frozen (2 or 4 × 10⁶ sperm/straw). Synchronised heifers (n = 183) were inseminated with either non-sorted control sperm (Control; 20 × 10⁶ dose) or with FSF or CSF ‘X’ sperm (2 or 4 × 10⁶/dose). Pregnancy rates (detected at 7–9 weeks) after AI with control sperm were higher than with FSF or CSF sperm (57.4 vs. 4.1 and 7.3% respectively; p < 0.001). There was a significant difference between bulls (Bull 1: Control 63.0%, FSF 8.6%, CSF 10.0%; Bull 2: Control 45.5%, FSF 0%, CSF 4.8%; p = 0.001). Five out of six (83.3%) pregnancies produced with sexed sperm were lost after pregnancy diagnosis. The exception was one heifer inseminated with CSF sperm (2 million sperm dose), which produced a heifer calf. In the non-sorted control group, three pregnancies were lost (8.3%) and three stillbirths occurred (8.3%). The low fertility and high rate of pregnancy loss in the sexed groups, in addition to environmental influences, may be attributed to impaired sperm function caused by sex-sorting and re-freezing, leading to poor embryo quality or altered gene expression. More precise timing of insemination and higher sperm doses might improve the fertility of FSF sperm. Moreover, the in vitro function of double-frozen sexed compared with non-sorted sperm requires further investigation.     

Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality

We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10 mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3 M trehalose or liposome solution, then cooled with slow (0.95 ± 0.02 °C/min), medium (73 ± 3 °C/min) and fast (265 ± 12 °C/min) cooling rates and storage in liquid nitrogen, followed by a 37 °C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66% ± 5% vs 29% ± 4%) compared to control RBCs (c-RBC, p = 0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p = 0.039 and p = 0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p = 0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p = 0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.     

Aminocarboxylate complexes of vanadium(III): Electronic structure investigation by high-frequency and -field electron paramagnetic resonance spectroscopy

Aminocarboxylate complexes of vanadium(III) are of interest as models for biologically and medicinally relevant forms of this interesting and somewhat neglected ion. The V(III) ion is paramagnetic, but not readily suited to conventional EPR, due to its integer-spin ground state (S = 1) and associated large zero-field splitting (zfs). High-frequency and -field EPR (HFEPR), however, has the ability to study such systems effectively. Three complexes, all previously structurally characterized: Na[V(trdta)] · 3H₂O, Na[V(edta)(H₂O)] · 3H₂O, and [V(nta)(H₂O)₃] · 4H₂O (where trdta stands for trimethylenediamine-N,N,N′,N′-tetraacetate and nta stands for nitrilotriacetate) were studied by HFEPR. All the investigated complexes produced HFEPR responses both in the solid state, and in aqueous solution, but those of [V(nta)(H₂O)₃] · 4H₂O were poorly interpretable. Analysis of multi-frequency HFEPR spectra yielded a set of spin Hamiltonian parameters (including axial and rhombic zfs parameters: D and E, respectively) for these first two complexes as solids: Na[V(trdta)] · 3H₂O: D = 5.60 cm⁻¹, E = 0.85 cm⁻¹, g = 1.95; Na[V(edta)(H₂O)] · 3H₂O: D = 1.4 cm⁻¹, E = 0.14 cm⁻¹, g = 1.97. Spectra in frozen solution yielded similar parameters and showed multiple species in the case of the trdta complex, which are the consequence of the flexibility of this ligand. The EPR spectra obtained in frozen aqueous solution are the first, to our knowledge, of V(III) in solution in general and show the applicability of HFEPR to these systems. In combination with very insightful previous studies of the electronic absorption of these complexes which provided ligand-field parameters, it has been possible to describe the electronic structure of V(III) in [V(trdta)]⁻ and [V(edta)(H₂O)]⁻; the quality of data for [V(nta)(H₂O)₃] does not permit analysis. Qualitatively, six-coordinate V(III) complexes with O,N donor atoms show no electronic absorption band in the NIR region, and exhibit relatively large magnitude zfs (D ? 5 cm⁻¹), while analogous seven-coordinate complexes do have a NIR absorption band and show relatively small magnitude zfs (D < 2 cm⁻¹).     

Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification

In this study, apical meristems from adventitious buds of three lily (Lilium L.) cultivars were successfully cryopreserved by droplet-vitrification. The most effective techniques were as follows. Excised meristems from in vitro plantlets which had been sub-cultured for about 2 months were cold-hardened at 4 °C for 1 week, precultured on MS medium supplemented with 0.3 mol L⁻¹ sucrose for 2 days, osmoprotected in loading solution for 20-40 min at room temperature and then soaked in PVS2 solution for 90-120 min at 0 °C, frozen in microdroplets of vitrification solution placed on aluminium foils, which were immersed rapidly in liquid nitrogen. The meristems were then rapidly rewarmed by dilution solution, transferred to regeneration medium and stored in the dark for two weeks at 20 °C, and then cultured under white fluorescent light at an intensity of 2000 lux, with a 16 h photoperiod at 20 °C. The highest post-thaw survival percentages of three cultivars ‘Siberia’ (Lilium × siberia), Lilium lancifolium Thunb. and ‘Snow Queen’ Lilium × longiflorum were 65.0%, 83.8% and 43.3%, and regeneration percentages were 62.0%, 67.6% and 35.0%, respectively. The study demonstrated that cryopreservation by droplet-vitrification increased survival and regeneration percentages of certain lily cultivars compared with vitrification. Thus to cryopreserve lily meristems, droplet-vitrification method is preferable to the vitrification method.     

4,4′-Bis(tert-butyl)-2,2′-bipyridinedichlorometal(II) - Synthesis, structure and EPR spectroscopy

Due to the better solubility of the 4,4′-substituted bipyridine ligand a series of 4,4′-bis(tert-butyl)-2,2′-bipyridinedichlorometal(II) complexes, [M(tbbpy)Cl₂], with M = Cu, Ni, Zn, Pd, Pt was synthesised and characterised. The blue copper complex 4,4′-bis(tert-butyl)-2,2′-bipyridinedichlorocopper(II) was isolated in two different polymorphic forms, as prisms 1 with a solvent inclusion and solvent-free as needles 2. Both structures were determined by X-ray structure analysis. They crystallise in the monoclinic space group P2₁/c with four molecules in the unit cell, but with different unit cells and packing motifs. Whereas in the prisms 1, with the unit cell parameters a = 12.1613(12), b = 10.6363(7), c = 16.3074(15) Å, β = 94.446(8)°, the packing is dominated by intra- and intermolecular hydrogen bonds, in the needles 2, with a = 7.738(1), b = 18. 333(2), c = 13.291(3) Å, β = 97.512(15)°, only intramolecular hydrogen bonds appear and the complex molecules are arranged in columns which are stabilised by π-π-stacking interactions. In both complexes the copper has a tetrahedrally distorted coordination sphere. These copper complexes were also studied by EPR spectroscopy in solution, as frozen glass and diamagnetically diluted powder with the analogue [Pd(tbbpy)Cl₂] as host lattice.     

Sperm characteristics and heterologous in vitro fertilisation capacity of Iberian ibex (Capra pyrenaica) epididymal sperm,frozen in the presence of the enzymatic antioxidant catalase

The aim of this work was to evaluate the protective effect of catalase (CAT) on frozen/thawed ibex epididymal sperm recovered post mortem, and to detect any harmful effect this might have on sperm fertilisation capacity. Epididymal spermatozoa were diluted using a Tris–citric acid–glucose medium (TCG) composed of 3.8% Tris (w/v), 2.2% citric acid (w/v), 0.6% glucose (w/v), 5% glycerol (v/v), and 6% egg yolk (v/v). Sperm masses from the right epididymis were diluted with TCG medium, while those from the left were diluted with TCG medium supplemented with 200 IU/mL CAT. Heterologous in vitro fertilisation (IVF) was used to assess the fertilisation capacity of this sperm. The addition of CAT to the extender did not improve frozen/thawed sperm variables. Moreover, a reduced fertilisation capacity was detected: sperm diluted with TCG provided 25.5% 2PN zygotes, while just 13.2% was recorded for that diluted with TCG-CAT (P < 0.01). The percentage of cleaved embryos at 48 hpi was higher (P < 0.01) with the TCG sperm than with the TCG-CAT sperm (16.7% vs. 7.6%). The use of 200 IU/mL CAT as an additive cannot, therefore, be recommended for the preservation of ibex epididymal sperm. Other antioxidants should, however, be tested in both this and related wild mountain ungulates.     

Cryopreservation of Greenshell™ mussel (Perna canaliculus) trochophore larvae

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16–20 h old). Toxicity tests showed that levels of 10–15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40–60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0 °C for 5 min, then cooling at 1 °C min⁻¹ to −10 °C, holding for a further 5 min, then cooling at 0.5 °C min⁻¹ to −35 °C followed by a 5 min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18 days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.     

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.     

Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality

The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against cryopreservation injuries on Iberian boar sperm. The sperm-rich fraction was collected and pooled from six sexually mature Iberian boars, and was frozen in different extenders containing glucose, lactose or fructose as sugar source and including Orvus ES Paste only in the freezing extender-2 (Glucose; Lactose and Fructose) or in both freezing extenders (Glucose2; Lactose2 and Fructose2). During the cryopreservation process, the supernatant was removed after the centrifugation step, then was extended with freezing extender-1 for the equilibration period and with freezing extender-2 immediately before freezing. Post-thaw sperm characteristics, such as plasma membrane integrity (SYBR-14/PI), mitochondrial function (Rhodamine 123) and acrosome integrity (NAR), were monitored. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in the different experimental treatments. Measurements were taken at 30 and 150 min post-thaw. The state of the acrosome after thawing did not show significant differences between the freezing extenders studied. Freezing–thawing caused a significant decrease (P < 0.001) in plasma membrane integrity and in mitochondrial activity in the spermatozoa frozen with Orvus ES Paste in both freezing extenders. Furthermore, spermatozoa frozen with Orvus ES Paste in both freezing extenders exhibited lower (P < 0.05) motility and kinematic parameters than those frozen in the absence of Orvus ES Paste in the first freezing extender. The spermatozoa frozen with the Lactose extender and with Orvus ES Paste only in the second freezing extender showed a better evolution of the motility and kinematic characteristics (P < 0.05) over time. The deterioration in post-thaw sperm motility and kinematic parameters were concurrent with reduced sperm characteristics. It can be suggested that in the Iberian pig, the beneficial effects of Orvus ES Paste during the freezing process of spermatozoa is time dependent. The analysis of different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, determined that the extenders studied in the present experiment affected the quality of frozen-thawed semen in Iberian boar.     

Cold tolerance and freeze-induced glucose accumulation in three terrestrial slugs

Cold tolerance and metabolic responses to freezing of three slug species common in Scandinavia (Arion ater, Arion rufus and Arion lusitanicus) are reported. Autumn collected slugs were cold acclimated in the laboratory and subjected to freezing conditions simulating likely winter temperatures in their habitat. Slugs spontaneously froze at about − 4 °C when cooled under dry conditions, but freezing of body fluids was readily induced at − 1 °C when in contact with external ice crystals. All three species survived freezing for 2 days at − 1 °C, and some A. rufus and A. lusitanicus also survived freezing at − 2 °C. ¹H NMR spectroscopy revealed that freezing of body fluids resulted in accumulation of lactate, succinate and glucose. Accumulation of lactate and succinate indicates that ATP production occurred via fermentative pathways, which is likely a result of oxygen depletion in frozen tissues. Glucose increased from about 6 to 22 μg/mg dry tissue upon freezing in A. rufus, but less so in A. ater and A. lusitanicus. Glucose may thus act as a cryoprotectant in these slugs, although the concentrations are not as high as reported for other freeze tolerant invertebrates.     

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

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

Sperm quality and the morphology of cryopreserved testicular tissues recovered post-mortem from diverse wild species

This study compared the effects of slow and fast freezing of testicular tissue of wild animals collected at post-mortem on testicular structure and testicular sperm. The testes of seven animals that had died in captivity; three felids (jungle cat, lion and leopard), two cervids (rusa deer and fea’s muntjac) and two bovids (Sumatran serows) were cryopreserved using slow- and fast-freezing protocols. There were greater reductions in the integrity of the sperm membrane and DNA in tissues cryopreserved using slow freezing compared to fast freezing (membrane integrity reduced by 21.5 ± 12.4% vs. 13.0 ± 6.9%, P = 0.11 and DNA integrity reduced by 22.7 ± 16.3% vs. 6.6 ± 6.3%, P = 0.13). Histologically, there were similar degrees of detachment and shrinkage of the seminiferous tubules whereas, TUNEL assay revealed a tendency towards more apoptotic changes in the intra-tubular cells of tissues frozen using fast freezing compared to slow freezing (P = 0.09). In conclusion, fast freezing tended to cause less damage to testicular sperm but its protective effect on intra-tubular cells was likely compromised. This is the first report of gamete recovery in the wild and of the comparison in various wildlife species, between testicular tissues cryopreserved using different protocols.     

Viability of respiratory pathogens cultured from nasopharyngeal swabs stored for up to 12 years at -70°C in skim milk tryptone glucose glycerol broth

Nasopharyngeal carriage studies are needed to monitor changes in important bacterial pathogens in response to vaccination and antibiotics. The ability to store original specimens frozen in skim milk tryptone glucose glycerol broth (STGGB) allows additional studies to be conducted without the need for further expensive field collection. Although sub-cultured isolates remain viable in this medium for many years, limited data are available to indicate viability of relatively low numbers of organisms present in nasopharyngeal specimens stored frozen over long periods of time. We conducted several studies whereby swabs stored in STGGB at − 70 °C for up to 12 years were thawed and aliquots cultured. Recovery of Streptococcus pneumoniae (72% positive from 269 swabs), Haemophilus influenzae (62% from 214) and Moraxella catarrhalis (81% from 162) was not significantly different from the original cultures: 69% (Risk Difference [RD] 3.0, 95% Confidence Interval [CI] − 4.7, 10.7), 66% (RD − 4.7, 95% CI − 13.8, 4.4) and 78% (RD 3.1, 95% CI − 5.7, 11.9) positive respectively. There was no trend in recovery from swabs stored for increasing lengths of time. We conclude that studies which rely on the viability of these respiratory pathogens can be conducted using original swabs stored at − 70 °C for at least 12 years.     

Synthesis of soluble coordination polymer nanoparticles using room-temperature ionic liquid

We report a new family of soluble cyano-bridged coordination polymer nanoparticles M₃[Cr(CN)₆]₂/[BMIM][BF₄] (where M²⁺ = Ni, Mn, V^IVO; BMIM = 1-butyl-3-methylimidazolium). These nanoparticles of ca. 6 nm were synthesised in the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate, which acts both as a stabilizing agent and a solvent. The magnetic properties of frozen colloids containing the nanoparticles show that the relaxation of magnetisation is strongly influenced by interparticle interactions leading to the appearance of spin-glass-like dynamics in these systems.