Relationships between reproductive characteristics in male Vimba vimba L. and the effects of osmolality on sperm motility

In Vimba vimba, GSI, sperm volume, and spermatozoa concentration range from 3.4-7.4 %, 0.1-1.1 ml, and 13.3-34.8 × 10⁹ spz ml⁻¹, respectively. Gonad mass (r = 0.90) and sperm volume (r = 0.35) significantly correlated with weight of males. Significant correlation was also found between gonad mass and length of males (r = 0.85). Sperm motility (r = 0.99) and velocity (r = 098) significantly decreased after activation in Tris-HCl 20 mM, pH 8.5. Osmolality of the seminal plasma was 273.2 mOsmol kg⁻¹. Sperm motility and velocity were significantly affected by the osmolality of the activation medium (P < 0.01). Hyper-osmolality compared to seminal plasma osmolality totally suppressed the sperm activation. At 15 s post-activation, the sperm motility significantly decreased at 240 mOsmol kg⁻¹ in KCl or NaCl media. The highest sperm motility and velocity (at 60 s post-activation) were observed at 200 mOsmol kg⁻¹ in NaCl, KCl, or sucrose media. In all treatments, the tip of the flagellum of spermatozoa became curled into a loop shape after activation of sperm in distilled water containing 20 mM Tris-HCl, pH 8.5 that shortened the flagellum.     

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

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

Effects of cooling and freezing on the motility of Ostrea edulis (L., 1758) spermatozoa after thawing

The aim of this work was to evaluate the effects of temperature, cryoprotectant agents and freezing curves on sperm motility of Ostrea edulis. All phases of cryopreservation were studied (evaluation of semen motility pattern, choice of cryoprotectants and freezing rates) to restore after thawing the motility characteristics distinctive of fresh semen.To assess the temperature effects on sperm motility, semen was activated using four different temperatures (25, 18, 10 and 3 °C). Sperm aliquots were maintained inactive at these temperatures for 1 and 3 h, then activated with FSW at same temperature of conservation. Sperm was activated and incubated to 3 °C with dimethylsulfoxide (Me₂SO), ethylene glycol (EG), 1–2 propylene glycol (PG) (5%, 7%, 10% and 15% final concentrations), glycerol (GlOH; 5%, 10% and 15% final concentrations) and methanol (MetOH; 4% and 10% final concentrations) for 10, 20 and 30 min. A first evaluation of freezing rates was made by testing four freezing curves: −1, −3, −6 and −10 °C/min. Then, an optimization was made by testing four freezing curves: −2.5, −3.0, −3.5 and −4 °C/min.The selected temperature for short term conservation has been 3 °C, because only this temperature has allowed good sperm motility conservation after 3 h of dry-storage; this is a time sufficient to conduct cryopreservation procedures. The sperm showed a particular sensitivity to GlOH and PG to all tested concentrations and to 15% Me₂SO. EG and MetOH to all concentrations and Me₂SO to concentrations lower than 15% have not shown significant toxic effects. The freezing rate −3 °C/min using 15% EG has shown an highest percentage of RVF (rapid, vigorous and forward) spermatozoa (class 3, about 75% of fresh semen) and an highest sperm motility duration.     

Cryopreservation of sperm of an indigenous endangered fish species Nandus nandus (Hamilton, 1822) for ex-situ conservation

This study dealt with the development of cryopreservation protocol for Nandus nandus, which entailed a number of experiments. Sperm was collected by sacrificing males. The collected sperm was suspended in extenders. Activation of sperm motility was evaluated in different osmolalities of NaCl. Motility of sperm decreased as the osmolality of the extender increased and was completely inhibited at almost 319 mOsmol/kg. To evaluate the toxicity of cryoprotectant, sperm was incubated with DMSO, methanol and ethanol at 5%, 10% and 15% concentrations, respectively, for 5–35 min. Five and ten percent of cryoprotectants produced better motility during 5 and 10 min incubation. Sperm incubated with 15% cryoprotectant seemed to be toxic and this concentration was excluded in the subsequent trials. Three extenders, namely, Alsever’s solution, egg-yolk citrate and urea egg-yolk and three cryoprotectants, DMSO, methanol and ethanol were employed to preserve the sperm. Alsever’s solution with 10% DMSO showed best performance producing 90.0 ± 1.8% and 75.0 ± 2.5% equilibration and post-thaw motility followed by that of 82.5 ± 4.2% and 62.5 ± 5.5% with Alsever’s solution plus methanol, respectively. Between two diluents, sperm preserved with Alsever’s solution plus DMSO produced highest fertilization (76.7 ± 3.3%) and hatching (43.8 ± 7.9%) while fresh sperm yielded 83.3 ± 6.7% and 64.0 ± 10.4% fertilization and hatching, respectively. The protocol developed through the study can be applied for long-term conservation of genetic materials of the endangered fish N. nandus and the cryopreserved sperm can be used in artificial breeding for generating new individuals.     

Sperm cryopreservation in the spermcasting Australian flat oyster Ostrea angasi by a programmable freezing method

Cryopreservation offers long-term storage of gametes without constraint from seasonal gamete maturation, provides opportunities to improve the efficiency of breeding and genetic programs, and protects endangered species from extinction due to epidemic diseases and natural disasters. In this study, a protocol for cryopreserving sperm of the spermcasting Australian flat oyster Ostrea angasi was developed by optimizing key factors influencing the quality of cryopreserved sperm. Dimethyl sulfoxide (DMSO) was non-toxic to sperm within the concentration and duration assessed in the toxicity experiment whereas 10% methanol or a higher concentration was toxic to sperm from the exposure duration of 30 min onwards. DMSO produced higher post-thaw sperm motility among the treatments with a single cryoprotectant. The inclusion of trehalose or glucose with DMSO further increased the post-thaw sperm motility (%) and plasma membrane integrity (PMI). Sperm equilibrated for 30 min showed higher post-thaw motility and PMI than those for 10 or 50 min. Higher post-thaw sperm motility and PMI were achieved at the freezing rate of −3 °C/min than at −7 °C/min. Sperm packaged in 0.5 ml straws had a higher post-thaw motility and PMI than those packaged in 0.25 ml straws. In this study, 44.4% post-thaw sperm motility and 49.2% PMI were achieved when sperm were equilibrated in 10% DMSO +0.45 M trehalose for 30 min, packaged in 0.5 ml straws, frozen at −3 °C/min from 4 °C to −80 °C, and thawed at 40 °C for 8 s. The availability of viable cryopreserved sperm would open an option for future breeding and genetic improvement programs for the spermcasting Australian flat oyster.     

Cryopreservation of sperm of an indigenous endangered fish species Nandus nandus (Hamilton, 1822) for ex-situ conservation

This study dealt with the development of cryopreservation protocol for Nandus nandus, which entailed a number of experiments. Sperm was collected by sacrificing males. The collected sperm was suspended in extenders. Activation of sperm motility was evaluated in different osmolalities of NaCl. Motility of sperm decreased as the osmolality of the extender increased and was completely inhibited at almost 319 mOsmol/kg. To evaluate the toxicity of cryoprotectant, sperm was incubated with DMSO, methanol and ethanol at 5%, 10% and 15% concentrations, respectively, for 5–35 min. Five and ten percent of cryoprotectants produced better motility during 5 and 10 min incubation. Sperm incubated with 15% cryoprotectant seemed to be toxic and this concentration was excluded in the subsequent trials. Three extenders, namely, Alsever’s solution, egg-yolk citrate and urea egg-yolk and three cryoprotectants, DMSO, methanol and ethanol were employed to preserve the sperm. Alsever’s solution with 10% DMSO showed best performance producing 90.0 ± 1.8% and 75.0 ± 2.5% equilibration and post-thaw motility followed by that of 82.5 ± 4.2% and 62.5 ± 5.5% with Alsever’s solution plus methanol, respectively. Between two diluents, sperm preserved with Alsever’s solution plus DMSO produced highest fertilization (76.7 ± 3.3%) and hatching (43.8 ± 7.9%) while fresh sperm yielded 83.3 ± 6.7% and 64.0 ± 10.4% fertilization and hatching, respectively. The protocol developed through the study can be applied for long-term conservation of genetic materials of the endangered fish N. nandus and the cryopreserved sperm can be used in artificial breeding for generating new individuals.     

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

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

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 cryopreservation of green swordtail Xiphophorus helleri, a fish with internal fertilization

Sperm cryopreservation for fishes with internal fertilization is essentially unexplored although many species of these fishes are valuable biomedical research models. To explore methods for sperm cryopreservation within the live-bearing genus Xiphophorus, this study used X. helleri to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio. Sperm motility and survival duration after thawing showed significant differences among different cryoprotectants with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as the cryoprotectant, the highest motility after thawing was observed with Hanks' balanced salt solution (HBSS) at 300 mOsmol/kg. Samples cooled from 5 to -80 degrees C at 20 degrees C/min yielded the highest post-thaw motility although no significant difference was found in the first 4h after thawing for cooling rates across the range of 20-35 degrees C/min. Evaluation of equilibration time revealed no significant difference between 20 min and 2h, but the highest motility at 10 min after thawing was found with a 20-min equilibration. Dilution ratios of sperm-to-extender at 1:20, 1:60, and 1:120 showed no significant differences in motility and survival duration after thawing, but the dilution of sperm solutions with HBSS (320 mOsmol/kg) immediately after thawing reduced the decline of sperm motility, and significantly prolonged the survival duration. Based on these findings, the highest average sperm motility (77%) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsmol/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS-glycerol of 1:20, equilibrated for 10 min, cooled at 20 degrees C/min from 5 to -80 degrees C before being plunged in liquid nitrogen, and thawed in a 40 degrees C water bath for 7s. If diluted immediately after thawing, sperm frozen by the protocol above retained continuous motility after thawing for more than 8 days when stored at 4 degrees C.     

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.     

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

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

Effect of low density lipoproteins in extender on freezability and fertility of buffalo (Bubalus bubalis) bull semen

This study was designed to determine whether low-density lipoporoteins (LDLs) extracted from egg yolk in extender improve the freezability and fertility of buffalo bull semen. Semen from three Nili-Ravi buffalo bulls was diluted at 37 °C with tris-citric acid extender (50 × 10⁶ motile spermatozoa mL⁻¹) containing LDLs 2.5%, 5%, 10%, and 15% extracted from egg yolk and extender containing 20% egg yolk was kept as control. Diluted semen was cooled to 4 °C in 2 h, equilibrated at 4 °C for 4 h, filled in 0.5 mL French straws, and kept on liquid nitrogen vapors for 10 min. Straws were then plunged and stored in liquid nitrogen (-196 °C). Sperm motility (visually; %), plasma membrane integrity (%; with supravital hypo-osmotic swelling test), and viability (%; with dual staining test using Trypan-blue Giemsa) were assessed at post-dilution, pre-freezing and post-thawing. At post-dilution and pre-freezing, sperm progressive motility, plasma membrane integrity and viability was similar (P > 0.05) in extender containing 10% LDLs or the control. However, at post-thaw the aforementioned parameters were higher (P < 0.05) in extender containing 10% LDLs compared with the control and other experimental extenders. The fertility rate of inseminations performed were higher (P < 0.05) with extender containing 10% LDLs than the control. It was concluded that LDLs (10%) in extender improved the freezability and fertility of buffalo bull spermatozoa.     

Development of a simplified and standardized protocol with potential for high-throughput for sperm cryopreservation in zebrafish Danio rerio

Sperm cryopreservation offers potential for long-term storage of genetic resources. However, the current protocols for zebrafish Danio rerio are cumbersome and poorly reproducible. Our objective was to facilitate adoption of cryopreservation by streamlining methods from sperm collection through thawing and use. First, sperm activation was evaluated, and motility was completely inhibited when osmolality of the extender was >/=295-300mOsmol/kg. To evaluate cryoprotectant toxicity, sperm were incubated with dimethyl sulfoxide (DMSO), N,N-dimethyl acetamide (DMA), methanol, or glycerol at 5, 10, and 15% concentrations. Based on motility, DMSO, DMA, and methanol (相似文献   

Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria

Motility and cryopreservation of testicular sperm of European common frog, Rana temporaria were investigated. Collected testicular spermatozoa were immotile in solutions of high osmolalities: 300 mmol/l sucrose and motility inhibiting saline solution-MIS. Full sperm motility could be activated in distilled water or in a solution of 50 mmol/l NaCl, = 90 mosmol/kg, with 75-90% motility and 14-16 μm s⁻¹ swimming velocity. Spermatozoa activated in distilled water and kept at room temperature ceased the motility within a period of 1 h. But when they were kept at 4 °C, no significant decrease in sperm motility and velocity occurred over a period of 1 h. Incubation of testicular sperm diluted 1:2 with MIS containing 10% DMSO, 5% glycerol, 10% methanol, or 10% propandiol for a period of 40 min at 4 °C showed that propandiol was the most toxic cryoprotectant for spermatozoa of European common frog R. temporaria. However, methanol was not toxic to spermatozoa during the 40 min incubation period, it failed to protect spermatozoa during the freezing and thawing process. DMSO and glycerol were useful penetrating cryoprotectants that interacted with sperm diluents in cryodiluent efficacy. In combination with the sucrose diluent, DMSO was a better cryoprotectant than glycerol, while in combination with MIS, DMSO and glycerol were similarly useful. Sperm was frozen at two freezing levels above the surface of liquid nitrogen. Sperm frozen 5 cm above the surface of liquid nitrogen resulted in immotile and non-viable spermatozoa. However, sperm frozen at 10 cm above the surface of liquid nitrogen showed 40-45% viability and 30-35% motility, compared to the untreated freshly collected testicular sperm. Addition of hen egg yolk had no positive effect on the post-thaw sperm motility, viability and hatching rate when added to sucrose cryodiluents. However, addition of 5% egg yolk to the MIS containing 5% glycerol and 2.5% sucrose significantly improved the hatching rate than all other treatments. Therefore, we conclude that, MIS and 300 mmol/l sucrose are suitable diluents for immotile storage of testicular semen. For cryopreservation, dilution to a final concentration of 5-6 × 10⁶/ml in MIS with 5% glycerol, 2.5% sucrose and 5% egg yolk, frozen in liquid nitrogen vapour at 10 cm above its surface, and thawed at 22 °C for 40 s is a useful cryopreservation protocol for R. temporaria sperm. Further research is needed to determine the motility parameters and cryopreservation of spermatic urine of R. temporaria.     

Effect of osmotic immobilization on refrigerated storage and cryopreservation of sperm from a viviparous fish, the green swordtail Xiphophorus helleri

In this study, refrigerated storage and cryopreservation of sperm from the green swordtail Xiphophorus helleri were investigated. Previous cryopreservation research in this species utilized motile sperm because unlike in most fish species, Xiphophorus sperm can remain continuously motile after collection for a week with refrigerated storage. However, this species reproduces by internal fertilization, and given the significant requirements for motility within the female reproductive tract and potential limitations on sperm energetic capacities, immobilization of sperm prior to insemination could be used to improve fertilization success. Thus, the goal in this study was to use osmotic pressure to inhibit the motility of sperm after collection from X. helleri, and to test the effect of immobilization on refrigerated storage and cryopreservation. The objectives were to: (1) estimate the motility of sperm at different osmotic pressures, and determine an osmotic pressure suitable for immobilization; (2) cryopreserve the immobilized sperm, and estimate the motility after thawing with or without dilution, and (3) compare motility of non-immobilized and immobilized sperm after thawing, centrifugation, and washing to remove cryoprotectant. Motility was determined when sperm were suspended in 11 different osmotic pressures (24-500 mOsmol/kg) of Hanks' balanced salt solution (HBSS). Motility was observed between 116 and 425 mOsmol/kg. Sperm were not motile when the osmolality was lower than 116 or higher than 425 mOsmol/kg. Motility of the immobilized (non-motile) sperm could be activated by changing the osmotic pressure to 291-316 mOsmol/kg, and motility of immobilized sperm from hypertonic HBSS (425 mOsmol/kg) was significantly higher than that from hypotonic HBSS (145 mOsmol/kg) after 48 h of storage. At an osmolality of 500 mOsmol/kg, HBSS was used as extender to maintain immobilized sperm during cryopreservation with glycerol as the cryoprotectant. High motility (approximately 55%) was obtained in sperm after thawing when cryopreserved with 10-15% glycerol, and dilution of thawed sperm in fresh HBSS (1:4; V:V) was found to decrease the motility significantly. No difference was found in the motility of thawed sperm cryopreserved with 14% glycerol and extended in 310 and 500 mOsmol/kg HBSS. Washing by centrifugation prolonged the motility of thawed sperm from 24 to 72 h in HBSS at 310 and 500 mOsmol/kg. This study showed that sperm from X. helleri could be immobilized by use of specific osmotic pressures, and that the immobilization did not affect sperm motility after thawing. The immobilization of sperm by osmotic pressure could minimize reduction of the energetic capacities necessary for insemination, traversal, and residence within the female reproductive tract, and fertilization.     

Cryopreservation of epididymal sperm in tree shrews (Tupaia belangeri)

Cryopreservation of sperm from tree shrews, which are considered primitive primates, would enhance genetic management and breeding programs. Epididymal sperm were surgically harvested from male tree shrews, cryopreserved in two Tes-Tris-based cryodiluents, and used in four experiments. In Experiment 1, there were no significant differences in motility and acrosome integrity among five concentrations of egg yolk in TTE after cooling to 4 °C. However, sperm frozen in TTE containing 20% egg yolk at −172 °C/min had better (P < 0.05) post-thaw motility and acrosome integrity. In Experiment 2, sperm held for 10 min prior to storage in liquid nitrogen had greater motility than those held for 5 or 15 min (P < 0.05), but acrosome integrity was not different (P > 0.05) among treatments. In Experiment 3, sperm frozen in TTE diluent had higher (P < 0.05) motility and acrosome integrity than those in TEST diluent. In Experiment 4, there were no differences (P > 0.05) in the fertilization rate of oocytes and the proportion of tree shrews yielding fertilized oocytes, following AI with fresh versus frozen sperm. In conclusion, tree shrew epididymal sperm were successfully cryopreserved, as assessed by post-thaw motility, acrosome integrity, and fertilizing ability.     

Semen cryopreservation in the Indian rhinoceros (Rhinoceros unicornis)

The objective was to identify an extender and cryoprotectant combination for Indian rhinoceros (Rhinoceros unicornis) sperm that yielded high post-thaw sperm quality. Male Indian rhinoceroses (n = 6; 7.5-34 yr old) were anesthetized and subjected to a regimented electroejaculation procedure (75-100 mAmps; 4-10 volts; 7-150 stimuli; total of 10 electroejaculation procedures). High quality semen fractions from each ejaculate were divided into four aliquots and a 2 x 2 factorial design used to compare the effect of two sperm extenders (standard equine [EQ] and skim milk-egg-yolk-sugar [SMEY]), and two cryoprotectants (glycerol and dimethylsulfoxide [DMSO]). Cyropreserved samples were thawed and assessed for motility, viability and acrosome integrity over time. Electroejaculate fractions processed for cryopreservation had high sperm concentration (516 × 10⁶/mL) and motility (79%). Post-thaw sperm characteristics were higher (P < 0.05) when semen was cryopreserved in EQ versus SMEY. Post-thaw motility of sperm cyropreserved in EQ averaged 50-55% compared to 22-37% in SMEY, with no significant differences in sperm characteristics of samples cyropreserved in glycerol and DMSO. In conclusion, sperm collected from Indian rhinoceroses via electroejaculation were cryopreserved using EQ extender with either glycerol or DMSO; post-thaw quality was adequate for use in assisted reproductive procedures.     

Physiological significance of behavioral hypothermia in hypoglycemic frogs (Rana catesbeiana)

Hypoxia elicits a number of compensatory responses in animals, including behavioral hypothermia. The hypothesis that hypoglycemia induces hypothermia in the bullfrog Rana catesbeiana was tested and that this behavioral response would be beneficial. Frogs equipped with a temperature probe were tested in a thermal gradient (10–40°C). Insulin (15 IU kg⁻¹) caused significant reduction of body temperature, from 25.0 to 17.8°C. A non-metabolizable glucose analogue, 2-deoxy-d-glucose (2-DG, 50 mg kg⁻¹),which blocks intracellular glucose utilization, was also injected and caused a similar drop in body temperature, despite an increase in plasma glucose levels. To assess the possible benefits of hypoglycemia-induced hypothermia, the effects of insulin and 2-DG injections were measured on plasma glucose concentration and on oxygen consumption of frogs equilibrated at 10, 20 and 30°C. The plasma glucose was elevated at higher temperatures and so was oxygen consumption. The insulin caused a significant reduction of plasma glucose concentration (about 1.22 μMol ml⁻¹) whereas 2-DG caused a significant increase (about 0.70 μMol ml⁻¹) at 30°C. Both drugs caused a reduction of oxygen consumption (approximately 0.388 and 0.382 ml min⁻¹ kg at 30°C after insulin and 2-DG injection, respectively). No effect of either insulin or 2-DG was observed when the animals were equilibrated at 10°C. In conclusion, hypothermia may be a beneficial response to hypoglycemia in frogs.     

Effect of cryopreservant combinations on the motility and morphology of curimba (Prochilodus lineatus) sperm

This study investigated the application of intra- and extra-cellular cryoprotectant combinations on the quality of curimba Prochilodus lineatus semen subjected to cryopreservation. Semen treatments were tested with 8% DMSO or methanol as intracellular cryoprotectant, 5% egg yolk or lactose as extracellular cryoprotectant and 5% BTS. These cryoprotectant combinations are suitable for curimba but have not been tested at the lesser concentrations proposed or in combination with BTS. Semen samples collected from 19 curimbas were diluted into one of four cryoprotectant combinations: DMSO+yolk; DMSO+lactose; methanol+yolk; and methanol+lactose. After dilution, semen samples were cryopreserved in 0.5 mL straws for 10 days in a liquid nitrogen tank. Semen was thawed in a water bath at 60°C for 8s. We evaluated the quality of fresh, diluted (pre-freezing) and post-freezing semen according to sperm motility rate (%) and duration (s). Sperm morphology was also analyzed in thawed semen. Sperm motility rate decreased progressively after dilution and thawing. The motility rate in post-freezing semen was higher in the treatments using DMSO+lactose and methanol+yolk. Sperm motility duration in post-freezing sperm was greater in the treatments using methanol rather than DMSO as intracellular cryoprotectant, irrespective of the extracellular cryoprotectant used. Abnormality frequency in thawed sperm was less in semen treated with egg yolk than with lactose. Thus the use of methanol intracellular cryoprotectant is recommended along with yolk extracellular cryoprotectant in the cryopreservation process for curimba semen.     

Fertilization environment of the non-copulating marine sculpin, Hemilepidotus gilberti

To clarify the extracellular environment for external fertilization in the non-copulating marine sculpin Hemilepidotus gilberti, sperm motility was measured in NaCl, KCl, mannitol solutions, seawater, and ovarian fluid. Spermatozoa of H. gilberti actively moved in seminal plasma the moment they were removed from the genital papilla. Spermatozoa showed higher motility in NaCl solution at osmolalities between 300–400 mOsmol kg^-1. In KCl and in mannitol solutions, spermatozoa actively moved at osmolalities between 500 and 800 mOsmol kg^-1, and at osmolality 300 mOsmol kg ^-1, respectively. The ovarian fluid was a transparent and viscous gelatinous material, rich in sodium with an osmolality of 340 mOsmol kg^-1. Sperm motility in the ovarian fluid lasted more than 90 min, which was six times longer than in seawater. This sperm motility under conditions isotonic to body fluid is similar to that of copulating marine sculpins rather than to other non-copulating marine fishes. In addition, eggs of H. gilberti could be fertilized in the ovarian fluid. This suggests that external fertilization takes place under physiological conditions similar to the internal conditions of the ovary provided by the ovarian fluid, which isolates the eggs from sea water for several hours after spawning. This manner of fertilization is thought to be one of the evolutionary pre-adaptations allowing copulation among marine sculpins.