Slow cooling prevents cold-induced damage to sperm motility and acrosomal integrity in the black-footed ferret (Mustela nigripes)

The endangered black-footed ferret (Mustela nigripes) has benefited from artificial insemination; however, improved sperm cryopreservation protocols are still needed. The present study focused on identifying factors influencing gamete survival during processing before cryopreservation, including: (1) the presence or absence of seminal plasma; (2) temperature (25 degrees C v. 37 degrees C); (3) type of medium (Ham's F10 medium v. TEST yolk buffer [TYB]); (4) cooling rate (slow, rapid and ultra-rapid); and (5) the presence or absence of glycerol. Seminal plasma did not compromise (P > 0.05) sperm motility or acrosomal integrity. Sperm motility traits were maintained longer (P < 0.05) at 25 degrees C than at 37 degrees C in Ham's or TYB, but temperature did not affect (P > 0.05) acrosomal integrity. Overall, TYB maintained optimal (P < 0.05) sperm motility compared with Ham's medium, but Ham's medium maintained more (P < 0.05) intact acrosomes than TYB. Slow cooling (0.2 degrees C min(-1)) was optimal (P < 0.05) compared to rapid cooling (1 degrees C min(-1)), and ultra-rapid cooling (9 degrees C min(-1)) was found to be highly detrimental (P < 0.05). Results obtained in TYB with 0% or 4% glycerol were comparable (P > 0.05), indicating that 4% glycerol was non-toxic to ferret sperm; however, glycerol failed to ameliorate the detrimental effects of either rapid or ultra-rapid cooling. The results of the present study demonstrate that the damage observed to black-footed ferret spermatozoa is derived largely from the rate of cooling.     

Boar semen can tolerate rapid cooling rates prior to freezing

Two experiments were performed in the present study that demonstrated that boar spermatozoa are capable of surviving rapid cooling rates within a range of 15-5 °C before freezing. Boar ejaculates diluted in Beltsville thawing solution (BTS) (1:1, v/v) were held at 17-20 °C and shipped over a 24-h time period from two AI centres to a cryobiology laboratory, where they were pooled (Experiment 1) or cryopreserved individually (Experiment 2) using a standard 0.5-mL straw freezing protocol. The effects of cooling before freezing were assessed after thawing through the objective evaluation of sperm motility and flow cytometric analysis of membrane integrity, acrosomal status, changes in membrane lipid architecture monitored by merocyanine and annexin V binding and intracellular production of reactive oxygen species. In Experiment 1 (six replicates), two semen pools (five ejaculates per pool) were cooled from 15 to 5 °C at rates of 0.08, 0.13, 0.40 and 1.50 °C min(-1). These cooling rates did not result in any significant differences (P>0.05) in any of the post-thaw sperm assessments, even in thawed samples incubated under capacitation conditions. In Experiment 2, three individual ejaculates from 16 boars were slowly (0.08 °C min(-1)) or rapidly (1.5 °C min(-1)) cooled before freezing. A consistent interboar variability (P<0.01) was detected, which was independent of the cooling rate used. Cooling rate only significantly influenced (P<0.05) sperm assessments in four of 16 boars, which exhibited slightly higher percentages of motile cells and intact plasma and acrosomal membranes in the samples that had been cooled slowly. These findings demonstrate that boar spermatozoa undergoing cryopreservation can withstand rapid cooling rates before freezing.     

Dimethylacetamide can be used as an alternative to glycerol for the successful cryopreservation of koala (Phascolarctos cinereus) spermatozoa

Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of intermolecular disulfide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulfide bonds within their chromatin, but have been successfully cryopreserved. The present study examined the hypothesis that the cryoprotectants used for fish sperm cryopreservation would confer a similar degree of protection on koala spermatozoa. Three concentrations each of five cryoprotectants (dimethyl sulfoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide (DMA)) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Post-thaw assessment of progressive motility, plasma membrane integrity and mitochondrial membrane potential (MMP) revealed that protocols using 15% DMA achieved 62.2 +/- 3.6% (P < 0.05) sperm survival, of which 79% (P < 0.05) had high MMP, an improvement of 32% and 40%, respectively, over sperm frozen in 14% glycerol. The percentage of spermatozoa with swollen nuclei was also lowest when frozen in 15% DMA, both immediately after thawing (18.0 +/- 3.5%; P < 0.05) and after 2 h incubation at 35 degrees C (35.8 +/- 4.4%; P < 0.05). A second study was conducted to determine the optimal concentration of DMA for use in the cryopreservation of koala spermatozoa. High DMA concentrations (17.5% and 20%) resulted in significantly lower proportions of live spermatozoa showing high MMP immediately after thawing compared with spermatozoa frozen in the lower concentrations. The percentage of koala spermatozoa with swollen chromatin following cryopreservation was not affected by DMA concentration.     

Comparison of different sperm cryopreservation procedures on post-thaw quality and heterologous in vitro fertilisation success in the ocelot (Leopardus pardalis)

Cryopreservation of spermatozoa from free-living ocelots (Leopardus pardalis) could benefit their conservation by facilitating gene flow between in situ and ex situ populations without requiring removal of additional cats from the wild. The objective of this study was to investigate three different methods of ocelot sperm cryopreservation to identify the most appropriate technique for use in a field environment. Male ocelots (n = 10), housed in North American zoos, were anaesthetised with tiletamine-zolazepam (7 mg kg(-1) bodyweight; i.m.) and subjected to a regimented electroejaculation procedure. Recovered semen was evaluated for sperm concentration, motility and morphology and processed for cryopreservation by three methods: (1) pelleting on dry ice, (2) freezing in straws over liquid nitrogen vapour; and (3) freezing in straws in a dry shipper. Frozen samples were thawed and assessed for post-thaw acrosome status, viability, motility over time and ability to fertilize viable domestic cat oocytes. Although several post-thaw sperm parameters varied (P < 0.05) among freezing methods, frozen-thawed ocelot spermatozoa from all treatments showed a similar (P > 0.05) capacity to bind, penetrate and fertilize viable domestic cat oocytes. These findings suggest that spermatozoa collected from male ocelots under field conditions may be frozen in straws either using liquid nitrogen alone or in a charged dry shipper to retain adequate functional competence after thawing for use with assisted reproductive procedures.     

Ultrastructure, osmotic tolerance, glycerol toxicity and cryopreservation of caput and cauda epididymidal kangaroo spermatozoa

The aim of the present study was to compare cryopreservation, osmotic tolerance and glycerol toxicity between mature and immature epididymal kangaroo spermatozoa to investigate whether the lack of cryopreservation success of cauda epididymidal spermatozoa may be related to the increased complexity of the sperm ultrastructure acquired during epididymal transit. Caput and cauda epididymidal spermatozoa were recovered from red-necked wallabies (RNW; Macropus rufogriseus) and eastern grey kangaroos (EGK; M. giganteus). In Experiment 1, caput and cauda epididymidal spermatozoa were frozen and thawed using a standard cryopreservation procedure in Tris-citrate buffer with or without 20% glycerol. Although cryopreservation of caput epididymidal spermatozoa resulted in a significant increase in sperm plasma membrane damage, they were more tolerant of the procedure than spermatozoa recovered from the cauda epididymidis (P < 0.05). In Experiment 2, caput and cauda epididymidal EGK spermatozoa were diluted into phosphate-buffered saline media of varying osmolarity and their osmotic tolerance determined. Plasma membranes of caput epididymidal spermatozoa were more tolerant of hypo-osmotic media than were cauda epididymidal spermatozoa (P < 0.05). In Experiment 3, caput and cauda epididymidal RNW spermatozoa were incubated in Tris-citrate buffer with and without 20% glycerol at 35 and 4 degrees C to examine the cytotoxic effects of glycerol. At both temperatures, caput epididymidal spermatozoa showed less plasma membrane damage compared with cauda epididymidal spermatozoa when exposed to 20% glycerol (P < 0.05). These experiments clearly indicate that epididymal maturation of kangaroo spermatozoa results in a decreased ability to withstand the physiological stresses associated with cryopreservation.     

Oocyte quality determines bovine embryo development after fertilisation with hydrogen peroxide-stressed spermatozoa

Exposure of gametes to specific stressors at sublethal levels can enhance the gametes' subsequent performance in processes such as cryopreservation. In the present study, bull spermatozoa were subjected to H?O? for 4 h at 100-, 200- and 500-μM levels; computer-assisted sperm analysis (CASA) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay were used for evaluation of subsequent sperm motility and DNA integrity, respectively. Exposure of spermatozoa to H?O? did not affect sperm motility but DNA integrity was negatively affected by 500 μM H?O? compared with mock-exposed spermatozoa, whereas both motility and DNA integrity were affected compared with untreated spermatozoa. Nevertheless, insemination of oocytes with spermatozoa exposed to 200 μM H?O? increased fertilisation, cleavage and blastocyst rates (P < 0.05). Furthermore, the higher blastocyst yield after fertilisation of oocytes with spermatozoa exposed to 200 μM H?O? was related to oocyte diameter, with large-medium oocytes yielding higher blastocyst rates, while small-diameter oocytes consistently failed to develop into blastocysts. In conclusion, the results indicate that exposure of spermatozoa to 200 μM H?O? before sperm-oocyte interaction may enhance in vitro embryo production in cattle. However, this increased embryo production is largely dependent on the intrinsic quality of the oocytes.     

Cryopreservation of epididymal alpaca (Vicugna pacos) sperm: a comparison of citrate-, Tris- and lactose-based diluents and pellets and straws

Epididymal spermatozoa were harvested from male alpacas and frozen after extension and cooling to 4 degrees C in citrate-, Tris- and lactose-based diluents (Experiment 1) and as pellets in 0.25- and 0.5-mL straws on either dry ice or over liquid nitrogen vapour (Experiment 2) to determine the effects diluents and packaging on their motility and acrosome integrity. In Experiment 1, sperm motility was higher after cooling to 4 degrees C and after freeze-thawing (0 but not 3 h post-thaw) for spermatozoa extended in the lactose- than the citrate- or Tris-based diluent (P < 0.05). Post-thaw acrosome integrity after cooling to 4 degrees C and post-thaw (0 h) was reduced for spermatozoa frozen in citrate- compared with lactose- or Tris-based diluents, but was similar for all groups 3 h after thawing. In Experiment 2, sperm motility immediately after thawing was higher for pellet freezing than for 0.25- or 0.5-mL straws on dry ice or liquid nitrogen vapour (P < 0.05), although by 3 h post-thaw motility was similar for pellets and straws (P > 0.05). Acrosome integrity was similar for all groups immediately after thawing and 3 h post-thaw. Cryopreservation of epididymal alpaca spermatozoa is feasible, with retained motility and acrosome integrity post-thaw. Freezing as pellets in a lactose-based diluent is recommended.     

Superoxide dismutase affects the viability of thawed European mouflon (Ovis g. musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection

This study evaluated the effects of superoxide dismutase (SOD) on viability and acrosome integrity of European mouflon spermatozoa after cryopreservation and on the fertilization rates of sheep oocytes after i.v.f. or intracytoplasmatic sperm injection (i.c.s.i.). Frozen semen was thawed and washed with synthetic oviduct fluid supplemented with 0.6% bovine serum albumin. After centrifugation, the spermatozoa pellet was split into two culture systems: (i) without SOD; and (ii) in the presence of 1500 IU mL(-1) SOD. Sperm viability and acrosome integrity were evaluated simultaneously, immediately after thawing and after 3, 6 and 9 h of culture (5% CO2, 39 degrees C, 90% humidity), by incubating sperm with propidium iodide and fluorescein isothiocyanate-labelled Pisum sativum agglutinin. At the same time, sperm were assessed for motility using a standard scoring system (independent operators' observation of sperm) that graded degree of motility (i.e. 1 = immotile to 10 = maximum motility, as observed at the moment of thawing). For i.v.f., frozen-thawed semen derived from the two culture systems was placed in culture together with in vitro-matured sheep oocytes. For i.c.s.i., semen derived from the same culture systems as that for i.v.f. was used, and incubated for 1 h under standard conditions. The results showed a marked difference (P < 0.01) between the percentages of live spermatozoa in medium with SOD and those obtained in medium alone, after 3, 6 and 9 h of culture. The percentages of intact acrosome spermatozoa were higher in medium with SOD after 6 h (P = 0.05) of culture. Spermatozoa motility decreased significantly in SOD containing medium at 3 and 6 h of culture compared with motility in control medium. Fertilization rates were significantly lower in medium with SOD than in medium alone, whereas in the i.c.s.i. system fertilization rates were significantly higher in the presence of SOD. The results indicate that the addition of SOD to the culture media enhances the viability rates and the acrosome integrity of cryopreserved mouflon spermatozoa.     

Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca²⁺ concentration in boar spermatozoa during cryopreservation

Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 °C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation.     

Giant panda (Ailuropoda melanoleuca) spermatozoon decondensation in vitro is not compromised by cryopreservation

Natural breeding of giant pandas in captivity is compromised, making artificial insemination and spermatozoa cryopreservation essential for genetic management. This study examined the influence of freeze-thawing on traditional parameters such as motility and spermatozoon functionality, specifically decondensation in vitro. Giant panda spermatozoa were assessed before and after rapid cryopreservation (4 degrees C to -130 degrees C over 2 min) in liquid nitrogen vapour. Spermatozoa pre-incubated in medium for 6 h were co-incubated with cat zonae (2 zonae microL(-1)) for 30 min to effect capacitation and an acrosome reaction. Spermatozoa were then mixed with mature cat oocyte cytoplasm (2 cytoplasm microL(-1)) for 4 h and evaluated for decondensation. Frozen spermatozoa were less motile (P < 0.05) than fresh counterparts immediately post-thawing, but not after 6 h incubation. There were more (P < 0.05) spermatozoa with completely diffused chromatin post-thaw (10.4 +/- 1.3%; mean +/- s.e.m.) compared to fresh counterparts (5.1 +/- 1.0%). However, there was no overall difference (P > 0.05) in the incidence of decondensation between fresh (4 h, 69.8 +/- 5.9%) and thawed (4 h, 71.5 +/- 4.9%) spermatozoa after exposure to cat oocyte cytoplasm. It is concluded that the 'rapid' method now used to cryopreserve giant panda spermatozoa has little impact on spermatozoon decondensation.     

A simple glycerol-based freezing protocol for the semen of a marsupial Trichosurus vulpecula, the common brushtail possum

Frozen storage of semen and embryos is now a well established part of the breeding of many eutherian mammals but it has not been applied to marsupials. This paper reports the first successful technique for the frozen preservation of marsupial spermatozoa. Semen was collected by electroejaculation under anaesthesia from a pool of five brushtail possums. The ejaculated semen was diluted 1:1 with Krebs Henseleit Ringer, centrifuged at 800 g for 5 min, resuspended in the test cryoprotectant media at 1, 2 and 5 x 10(6) spermatozoa mL-1 and 7, 10.5, 14 and 17.5% glycerol and then drawn up into 0.25 mL plastic straws. The spermatozoa were rapidly frozen in the vapour phase, 6 cm above liquid nitrogen, for 30 min before the straws were plunged into the liquid. Sperm motility was assessed blind for coded straws by phase-contrast microscopy on a warmed stage (35 degrees C), before freezing and after rapid thawing in a water bath at 37 degrees C (10 s). The highest recovery of both percentage motility (around 50-60%) and progressive motility (around 0.5-1 unit lower than prefreeze) occurred when spermatozoa were frozen and thawed in the presence of 17.5% glycerol. Recovery of motility was greater at the higher sperm concentrations (2 and 5 x 10(6) mL-1). There was no evidence of acrosomal damage or loss after freezing and thawing in high concentrations of glycerol. The only defect detected in spermatozoa subjected to the protocol was a variable tendency to bending of the neck region. This ranged from heads inclined at a slight angle to the tail through to complete flexure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testosterone secretion, testicular histology and the cryopreservation of cauda epididymidal spermatozoa in the common ringtail possum (Pseudocheirus peregrinus)

The present study reports novel aspects of the reproductive biology of the male common ringtail possum (Pseudocheirus peregrinus). Plasma testosterone was measured through a stimulation test using the gonadotrophin-releasing hormone agonist, buserelin. Following intra-muscular administration of buserelin, there was an increase (P<0.05) in testosterone concentration in the peripheral circulation 4 h later. Quantitative testicular histology of this species was described for the first time. Eight stages of the seminiferous epithelium cycle were identified in 10 possums and their relative frequency determined. Spermatozoa were recovered from the cauda epididymides of hemi-castrated possums and cryopreservation conducted in straws (6 degrees C min(-1)) using final glycerol concentrations ranging between 2 and 20% in Tris-citrate egg yolk extender (v/v). Frozen straws were thawed and post-thaw motility, rate of motility, the percentage of live-dead spermatozoa and the percentage of sperm with swollen decondensed nuclei recorded. Similar to other marsupial sperm, common ringtail possum cauda epididymidal spermatozoa required high levels of glycerol (10-16%) in order to maintain post-thaw viability.     

Dry storage of sperm: applications in primates and domestic animals

Cryopreservation of spermatozoa, oocytes and embryos, as well as somatic cells or cell lines for cloning from cells, are all options for the long-term storage of unique genotypes and endangered species. Spermatozoal cryopreservation and storage currently require liquid nitrogen or ultralow refrigeration-based methods for long- or short-term storage, which requires routine maintenance and extensive space requirements. The preservation of stem cells also has strict requirements for long-term storage to maintain genetic integrity. Dessicated (lyopreserved) sperm and stem cells will provide an unprecedented type of long-term storage without the need for expensive and burdensome cryogenic conditions. Experiments were conducted to determine an effective intracellular concentration of the lyoprotectant trehalose. High-pressure liquid chromatography studies revealed that trehalose can be incorporated into mature sperm cells as well as spermatogonial stem cells from rhesus monkeys. In addition, using fourier transform infrared spectroscopy, we determined that thermotropic phase transitions for fresh ejaculates from rhesus monkey and stallion sperm occurred at 10-15, 33-37 and 55-59 degrees C. Preliminary studies in our laboratory have indicated that spermatogonial stem cells can be dried to <3 g g(-1) water and maintain viability following rehydration. Studies in our laboratory have provided preliminary results suggesting that the desiccated storage of sperm and spermatogonial stem cells may be a viable alternative to conventional cryopreservation.     

Activity of antioxidative enzymes in fresh and frozen thawed buffalo (Bubalus bubalis) spermatozoa in relation to lipid peroxidation and semen quality

ObjectiveTo investigate the activity of antioxidative enzymes in fresh and frozen thawed spermatozoa in relation to lipid peroxidation and semen quality in buffalo (Bubalus bubalis) bulls.MethodsForty two semen ejaculates from seven buffalo bulls were collected by artificial vagina method and were used for the study. Sperm motility, livability, plasma membrane and acrosomal integrity, buffalo cervical mucous penetration test were assessed in fresh and frozen thawed semen. Intracellular antioxidative enzymatic activity such as super oxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx) and reduced glutathione (GSH), reactive oxygen species (ROS) and lipid peroxidation (LPO) were estimated in fresh and frozen thawed semen.ResultsA significant (P<0.01) reduction in activity of antioxidative enzymes (SOD by 47.7%, GSHPx by 62.7% and GSH by 58.6%) in frozen thawed spermatozoa as compared to fresh spermatozoa was found. Although the catalase activity was varied from 0 to 3.8 IU/10⁹ sperm in fresh semen, but after freezing and thawing this activity was not detectable. These enzyme activities had a strong positive association with sperm motility, membrane integrity and distance traveled by vanguard spermatozoa in buffalo cervical mucus and negative correlation with LPO and ROS. However, no significant correlation with acrosomal integrity was found.ConclusionIt was concluded that loss of activity of intracellular antioxidative enzymes was evident after freezing and thawing and there was a strong association between the antioxidative enzyme activities, ROS, lipid peroxidation and sperm function in buffalo semen.     

Studies on the cryopreservation of common wombat (Vombatus ursinus) spermatozoa

In an attempt to develop a gamete-recovery protocol for the northern hairy nosed wombat ( Lasiorhinus krefftii ), spermatozoa were removed from the cauda epididymides of four common wombats ( Vombatus ursinus ) and cryopreserved following a variety of prefreeze storage conditions. Spermatozoa stored for 72 h at 4 degrees C within the testicle before cryopreservation tolerated the freeze-thaw procedure remarkably well, resulting in a higher post-thaw viability (% motile P < 0.01; rate of movement P < 0.01; % live P < 0.01) than sperm recovered on the day of post-mortem, stored in a test tube for 72 h at 4 degrees C and then frozen. The effect of post-thaw dilution with Tris citrate fructose (TCF) diluent on the survival of epididymal common wombat spermatozoa was also investigated. Motility (P < 0.05), rate of sperm movement (P < 0.01) and the percentage of live spermatozoa (P < 0.05) were all significantly greater when spermatozoa were thawed and diluted immediately in TCF than when thawed without dilution. The present study also reports, for the first time, a successful pellet method of freezing wombat spermatozoa on dry ice; volumes of 0.25 and 0.5 mL resulted in higher post-thaw survival compared with 0.1-mL pellets.     

In vitro characteristics of fresh and frozen-thawed ram spermatozoa after sex sorting and re-freezing

The in vitro function of sex-sorted, frozen-thawed ram spermatozoa derived from fresh or frozen semen was investigated. Sorted, frozen-thawed spermatozoa had higher (P < 0.05) motility, viability, acrosome integrity and mitochondrial activity than non-sorted, frozen-thawed controls immediately following thawing and after incubation at 37 degrees C for 3 and 6 h. Similarly, frozen-thawed, sorted, re-frozen-thawed spermatozoa outperformed (P < 0.05) non-sorted controls upon thawing (mitochondrial activity) and following a 3-h incubation (motility, viability/acrosome integrity and mitochondrial activity), but there were no differences after incubation for 6 h (P > 0.05). Velocity characteristics (computer assisted sperm assessment 0-6 h post-thaw) of sorted spermatozoa derived from either fresh or frozen semen remained inferior (P < 0.05) to non-sorted spermatozoa, as did their ability to penetrate artificial cervical mucus after thawing. Direct comparison of cryopreserved spermatozoa derived from either fresh or frozen semen revealed that frozen-thawed, sorted, re-frozen-thawed spermatozoa had comparable (P > 0.05) motility, viability/acrosome integrity, mitochondrial activity, average path velocity and oviducal binding capacity immediately post-thaw, but reduced (P < 0.05) quality after 3 and 6 h of incubation. These findings indicate that, under the tested in vitro conditions, sex-sorted spermatozoa derived from fresh semen are superior in some respects to those derived from frozen semen. Further, that the use of either technique, while reducing velocity characteristics and cervical mucus penetration, results in comparable, if not enhanced motility, membrane and mitochondrial function in the post-thaw population of spermatozoa when compared with non-sorted, frozen-thawed controls.     

Cryopreservation of macropodid spermatozoa: new insights from the cryomicroscope

This study examined the effects of cooling and cryopreservation upon macropod spermatozoa (eastern grey kangaroo, Macropus giganteus and red-necked wallaby, Macropus rufogriseus). Sperm survival during and after freezing to -30 degrees C or 70 degrees C in minimum essential medium (MEM) + 5, 10, 20 or 30% (v/v) glycerol, MEM + 10 or 20% (v/v) ethylene glycol and MEM containing a mixture of 7.5% (v/v) glycerol + 10% (v/v) dimethylsulphoxide was examined by cryomicroscopy. The MEM/glycerol mixtures permitted better post-thaw sperm recovery than the other cryoprotectants. After freezing to -30 degrees C at 10 degrees C min(-1) in 20% glycerol, then rewarming at 20 degrees C min(-1), flagellar activity resumed in more than 50% of spermatozoa when the temperature increased into the range 5-10 degrees C. However, as the temperature increased, into the range 20-25 degrees C, motility declined rapidly so that less than 5% motile cells were seen at 35 degrees C. Spermatozoa in MEM without cryoprotectant were also examined by cryomicroscopy to evaluate changes in flagellar configuration, swimming behaviour and viability during cooling from 35 degrees C to approximately -7 degrees C, and rewarming to 35 degrees C. Cooling from 35 to 28 degrees C induced kangaroo spermatozoa to exhibit rigid principal-piece bending and non-linear motility, which was reversed by further cooling and the spermatozoa resumed their normal linear movement. Rewarming induced principal-piece bending in the range of 20-30 degrees C, but this effect was reversed by further warming. Although red-necked wallaby spermatozoa showed these effects, they also exhibited a tendency to form rosette-like clusters during rewarming, especially when the temperature reached approximately 14 degrees C. The clusters were induced when the flagellar end-pieces became anteriorly reflected, producing hook-like flagellar conformations, which then became interlinked.     

VIABILITY AND FECUNDITY OF HUMAN SEMEN SPECIMENS CRYOSTORED AND TRANSPORTED AT 5°C USING THE BIO–TRANZTM SHIPPING

This study was designed to assess the viability and fecundity of semen stored at 5°C for 24 hours using the Bio-Tranz^TM shipping system. Semen specimens were assessed for motility and sperm membrane integrity at the time of collection and 24 hours after storage in the Bio-Tranz^TM. In group 1 (n = 61), specimens were diluted in TYB, processed and used for intrauterine insemination (IUI), leaving an aliquot for storage for 24 hours in the Bio-Tranz^TM. In group 2 (n = 67), specimens were diluted in TYB, stored for 24 hours in the Bio-Tranz^TM and then processed and used for IUI. In both groups, the total motile sperm used for IUI was similar and the women that underwent IUI were standardized for ovulation prediction and time of insemination. The overall sperm characteristics between the two groups were within normal range. Significant decreases were noted in sperm motility and membrane integrity in both groups after storage. Similar pregnancy rates were obtained between the two patient populations. The use of the Bio-Tranz^TM shipper is extremely convenient for patients requiring semen evaluation, cryostorage or IUI and other assisted reproductive technologies.     

Storage of cane toad (Bufo marinus) sperm for 6 days at 0 degrees C with subsequent cryopreservation

We investigated the recovery of motility of cane toad (Bufo marinus) sperm after storage for 6 days at 0 degree C (on ice) and after subsequent cryopreservation. Sperm suspensions were prepared from testes macerated in either simplified amphibian Ringer (SAR) or 10% (w/v) sucrose diluents, with 15% or 20% (v/v) glycerol or Me2SO as cryoprotectants, and were stored for 6 days. Alternatively, suspensions were prepared in either SAR or 10% (w/v) sucrose diluent and stored for 6 days, after which some of these suspensions were kept in diluents alone or, alternatively, had 15% or 20% (v/v) glycerol or Me2SO added. All treatments (suspensions) were then cryopreserved. Sperm motility was measured at Day 1 and Day 6 (before and after cryopreservation). A substantial and variable proportion (range 0%-40%) of sperm was immotile in suspensions immediately after preparation from testes macerates. Sperm stored in either SAR or 10% (w/v) sucrose diluent maintained approximately 75% motility for 6 days, but few sperm survived cryopreservation. After storage and cryopreservation, recovery of motility was substantially higher with Me2SO than in glycerol. However, both cryoprotectants exhibited toxicity at high concentrations. Glycerol was more toxic than Me2SO in 10% (w/v) sucrose than in SAR diluent, both before and after cryopreservation. The addition of some cryoprotectants to suspensions before storage gave greater recovery both before and after cryopreservation. After cryopreservation, the highest rate of sperm recovery was in suspensions with 10% (w/v) sucrose and 15% (v/v) Me2SO added prior to storage (mean (+/-SEM) 46 +/- 7% relative to initial; 39 +/- 6% absolute). Sperm were also stored for 6 days at 0 degrees C in suspensions or testes (with suspensions then prepared from testes) and cryopreserved. Sperm maintained higher recovery and membrane integrity both before and after cryopreservation when stored in suspensions rather than in testes.     

Improved sperm cryopreservation using cold cryoprotectant

It has generally been assumed that very rapid cooling above freezing point would be deleterious to human sperm because it would result in cold shock. Consequently, most routine cryopreservation protocols involve the use of warm (20-30 degrees C) cryoprotectant and slow cooling above the freezing point in order to minimise the risk of cold shock. In order to test this assumption, we added an equal volume of cold (4 degrees C) cryoprotectant in a single aliquot to warm (20, 30 or 37 degrees C) semen to induce rapid cooling. The results of this procedure were compared with those obtained using warm cryoprotectant or with the routine cryopreservation protocol used in this laboratory. The use of cold cryoprotectant resulted in a significant (P = 0.016) improvement (mean 63%, range 42%-79%) in post-thaw motility recovery compared with a standard procedure(mean 47%, range 35%-67%) and a significant (P = 0.016) improvement in post-thaw sperm velocity. A cold glycerol/egg yolk/citrate (GEYC) mixture also gave significantly higher motility recovery than GEYC equilibrated to either room temperature (20 degrees C) or body temperature (37 degrees C). Sperm frozen using the cold cryoprotectant protocol were as efficient at binding to and penetrating the human zona pellucida as sperm frozen with a standard protocol. The modified cryopreservation procedure may lead to improved pregnancy rates in donor insemination and in vitro fertilisation. Further investigation is required to determine how the cold cryoprotectant improves the cryopreservation outcome.