Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47 ± 0.46 nmol/10⁸ cells. Following semen cryopreservation, GSH decreased to 1.62 ± 0.13 nmol/10⁸ cells, a 64% reduction (p < 0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p < 0.01). Addition of 1 mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5 mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze–thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.     

Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-frozen ram semen.

The influence oftemperature, addition of glycerol, initial freezing temperature, method of dilution, level of glycerol in the diluted semen, equilibration time and type of diluent on the survival and fertilizing capacity of deep-frozen according to the best conditions was compared with that of "fresh" semen. The addition of glycerol at plus30 degrees C resulted in a highly significant decrease in the mean proportion of motile spermatozoa immediately after thawing compared with the effect of addition at plus 4 degrees C. The immersion of the straws at minus55 degrees C significantly reduced the revival of the spermatozoa compared with initial freezing at lower temperatures. The exposure time to glycerol had no significant effect on the survival of spermatozoa after thawing and incubation, but fertility was significantly higher with 4% than with 2% glycerol. The I. N. R. A. diluent provided better sperm survival and a significantly higher conception rate than did lactose-egg yolk extender. The semen frozen according to the best conditions (about 50% of the samples) had a fertilizing ability similar to that of "fresh" semen when the proportion of motile spermatozoa before, and after 1 or 3 hr of incubation was equal to or above 45, 40 and 30% respectively.     

Cryopreservation of Plagiognathops microlepis sperm

Sperm was collected from cultured male fish and cryopreserved in 0.25 ml straws for the study of sperm cryopreservation. Different parameters were evaluated, including extender, dilution ratio, cryoprotectant type and concentration, equilibrium time, cooling height (in a two-step cooling protocol), and thawing temperature. The optimum result was obtained when the sperm was diluted at a 1:7 ratio in D-16 with 5% DMSO as a cryoprotectant, equilibrated for 20 min, held at 3 cm above liquid nitrogen for 10 min, and then stored in liquid nitrogen. After thawing in a water bath at 40 °C, the percentage of motile cells and fertilization rates of frozen-thawed sperm were 35.33 ± 2.52% and 39.00 ± 4.58%, respectively, while the corresponding rates for fresh sperm were 87.67 ± 3.06% and 88.67 ± 4.62%. We also used a programmed cooling protocol in which temperature was decreased from 4 °C to −80 °C by a rate of 30 °C/min, and then straws (0.25 ml) were placed above the surface of liquid nitrogen for 2 min before being stored in liquid nitrogen. This protocol provided a post-thaw activation rate of 36.67 ± 4.77%. Further parametric optimization is required to improve the quality of frozen-thawed sperm.     

Effects of dilution and centrifugation on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey semen

The aim of this work was to study the effects of dilution and centrifugation (i.e., two methods of reducing the influence of the seminal plasma) on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey (Equus asinus) semen. Fifty ejaculates from nine Catalonian jackasses were collected. Gel-free semen was diluted 1:1, 1:5 or 1:10 with Kenney extender. Another sample of semen was diluted 1:5, centrifuged, and then resuspended with Kenney extender until a final dilution of 25 × 10⁶ sperm/ml was achieved (C). After 24 h, 48 h or 72 h of refrigerated storage at 5 °C, aliquots of these semen samples were incubated at 37 °C for 5 min. The percentage of viable sperm was determined by staining with eosin-nigrosin. The motility characteristics of the spermatozoa were examined using the CASA system (Microptic, Barcelona, Spain). At 24 h, more surviving spermatozoa were seen in the more diluted and in the centrifuged semen samples (1:1 48.71%; 1:5 56.58%, 1:10 62.65%; C 72.40%). These differences were maintained at 48 h (1:1 34.31%, 1:5 40.56%, 1:10 48.52%, C 66.30%). After 72 h, only the C samples showed a survival rate of above 25%. The four known donkey motile sperm subpopulations were maintained by refrigeration. However, the percentage of motile sperms in each subpopulation changed with dilution. Only the centrifuged samples, and only at 24 h, showed exactly the same motile sperm subpopulation proportions as recorded for fresh sperm. However, the 1:10 dilutions at 24 and 48 h, and the centrifuged semen at 48 h, showed few variations compared to fresh sperm. These results show that the elimination of seminal plasma increases the survival of spermatozoa and the maintenance of motility patterns.The initial sperm concentration had a significant (P < 0.05) influence on centrifugation efficacy, but did not influence the number of spermatozoa damaged by centrifugation. In contrast, the percentage of live spermatozoa in the fresh semen significantly influenced the number of spermatozoa damaged by centrifugation, but not centrifugation efficacy.     

Gene banking of the neotropical fish Leporinus obtusidens (Valenciennes, 1836): a protocol to freeze its sperm in the field

A practical sperm cryopreservation protocol using a dry-shipper and a diluent of simple composition is described for the neotropical fish Leporinus obtusidens (Valenciennes, 1836). The cooling rate of the dry-shipper and its period of useful time, established under laboratory conditions, were respectively 25.7-30.8 degrees C/min (between 0 and -60 degrees C) and 9 days after charging. Sperm donors were selected on the basis of their hyperemic genital papilla and the ability to ooze milt under gentle manual pressure, during the reproductive months of November to January. Milt volume (1.3+/-0.3 mL; n=9 fish), fresh sperm motility rate (93.3+/-2.5%; n=6 fish), and sperm concentration (10.9+/-3.0 x 10(9)spermatozoa/mL of milt) were obtained. The sperm cryopreservation experiments were conducted with the following cryoprotectants (all at 10%, before mixing with milt): dimethyl sulphoxide (DMSO; n=10 fish), methanol (n=6 fish), propanediol (n=6 fish) and ethylene glycol (n=5 fish). Glucose (5%) and hen's egg yolk (10%) made up the diluents containing DMSO, ethylene glycol or propanediol. Milk powder (10%) replaced hen's egg yolk in the diluent containing methanol. Distilled water (up to 100%) completed the diluent solutions. Milt freezing (in 0.5-mL straws) was performed in the dry-shipper after 1:5 (milt:diluent) dilution. Thawed sperm cryopreserved in DMSO-containing diluent and activated by 119 mM NaHCO(3) gave the highest motility rate (62+/-14%). The fertilizing capacity of L. obtusidens sperm was tested using the combination of DMSO-containing diluent as the cryoprotectant and 119 mM NaHCO(3) as the activating solution. Oocytes were obtained from artificial spawning and fertilized with different proportions of spermatozoa. The greatest rate of fertilization (74%) occurred when the ratio of about 112,000 motile spermatozoa:oocyte was used. Thus, a protocol to freeze L. obtusidens sperm can be elaborated as follows. Milt (<1.5 mL fish(-1)) was readily available only in November to January; a simple solution, composed of 10% DMSO (concentration before adding milt), 5% glucose, and 10% hen yolk egg, in distilled water, was used as sperm diluent; cooling rate of 25-30 degrees C/min, yielded in a portable dry-shipper, was adequate to freeze diluted milt (1:5; milt:diluent), in 5-mL straws; about 112,000 thawed motile spermatozoa:oocyte activated by 119 mM NaHCO(3) assured a fertilization rate of 74%.     

Rooster spermatozoan motility,forward progression,and fertility after storage at 25, 5, or −196 °C in various extenders

Rooster spermatozoa were stored at 25, 5, or ?196 °C in either TC199, a pyruvate-lactate mouse ova culture medium, or as undiluted semen. There was a linear decrease in percent of motile sperm during storage at 25 or 5 °C in all cases, and a curvilinear decrease with increasing storage times at ?196 °C. Percent of motile sperm present after increasing storage time suggested pyruvate-lactate is a better extender than TC199 at the three storage temperatures studied. Pullets inseminated with 1 × 10⁸ motile sperm using fresh sperm diluted in TC199 or pyruvate-lactate, or stored 24 hr at 5 or ?196 °C produced 68.7, 74.1, 20.6, and 10.8% fertile eggs, respectively. The differences in fertility between controls or between samples stored at 5 and ?196 °C were not significant. However, fertility from sperm stored at 5 and ?196 °C was significantly lower (p < .05) than both control groups. Thus, it can be concluded that TC199 or pyruvate-lactate may be used to dilute fresh rooster semen collections prior to insemination. In contrast, fertility of rooster sperm is not satisfactorily maintained after 5 or ?196 °C storage for 24 hr in a pyruvate-lactate extender.     

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.     

In vitro capacitation of boar ejaculated spermatozoa: Effect of conditioned media prepared from preincubated sperm suspension

The fertilizing ability of boar ejaculated spermatozoa was examined in vitro after prcincubation at a concentration of 2.5 × 10⁸/ml for 4 hr in several conditioned media (CM). For preparation of CM, boar spermatozoa were incubated in a modified Krebs-Ringer bicarbonate solution (TYH) at concentrations of 20 to 40 × 10⁸/ml for several hours up to 4 hr; then their supernatant fluids were collected by centrifugation. When boar ejaculated spermatozoa were preincubated in TYH alone, 14.1% of oocytes were penetrated by them as we reported previously. On the other hand, preincubating them with CM, their fertilizing ability was elevated according as the incubation time of CM preparation was lengthened. The fertilization rate reached 75.0%, using 4 hr-incubated CM for the preincubation medium. The effect of CM was not deteriorated by heat treatments (56°C, 30 min, or 100°C, 5 min). The components of CM were separated at a molecular weight of 25,000 by ultrafiltration, and high fertilization rate (69.8%) was obtained when low molecular weight fraction was used for the preincubation medium. Sperm extracts prepared from directly frozen-thawed sperm suspension and 0.1–10 mM of taurine or hypotaurine had no effect on the fertilizing ability of boar spermatozoa. These results suggest that substances stimulating boar sperm capacitation were accumulated from viable spermatozoa into the medium during incubation and that the effective substances were heat-stable and of low molecular weight and were not taurine and hypotaurine.     

Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques

The factors that affect the survival of mouse lymphocytes throughout a procedure for storage at ?196 °C have been studied both for the improvement of recovery and the possible extension to the mouse system of cell selection by freezing. After thawing, the survival of cells cooled at different rates in dimethyl sulphoxide (DMSO, 5 or 10%, $\text{v}\text{v}$) was assessed from the [³H]thymidine incorporation in response to phytohaemagglutinin and concanavalin A. Before freezing the protection against freezing damage increased with time (up to 20 min) in DMSO (5%, $\text{v}\text{v}$) at 0 °C. Superimposed upon this effect was toxicity due to the DMSO. During freezing and thawing the cooling rate giving optimal survival was 8 to 15 °C/min for cells in DMSO (5%) and 1 to 3 °C/min for DMSO (10%). Omission of foetal calf serum was detrimental. Rapid thawing (>2.5 °C/min) was superior to slow thawing. After thawing dilution at 25 or 37 °C greatly improved cell survival compared with 0 °C; at 25 °C survival was optimal (75%) at a moderate dilution rate of 2.5 min for a 10-fold dilution in FCS (10%, $\text{v}\text{v}$) followed by gentle centrifugation (50g).Dilution damage during both thawing and post-thaw dilution may be due to osmotic swelling as DMSO and normally excluded solutes leave the cell. The susceptibility of the cell membrane to dilution damage may also be increased during freezing. The need to thaw rapidly and dilute at 25 °C after thawing is probably due to a decrease in dilution stress at higher temperatures. Optimisation of dilution procedures both maximised recovery and also widened the range of cooling rates over which the cells were recovered. These conditions increase the possibility of obtaining good recovery of a mixed cell population using a single cooling procedure. Alternatively, if cell types have different optimal cooling rates, stressful dilution may allow their selection from mixed cell populations.     

The effects of extenders and sperm‐egg ratios on fertilizing ability of cryopreserved testicular sperm of northern pike (Esox lucius L.)

The effects of four different extenders and two sperm‐to‐egg ratios on fertilizing ability of cryopreserved testicular sperm of northern pike (Esox lucius L.) were tested in the present study. Testicular sperm was diluted with each of the four extenders (0.6 m sucrose + 15% DMSO, 0.3 m glucose + 15% DMSO, 0.6 m sucrose + 10% methanol and 0.3 m glucose + 10% methanol, all supplemented with 10% egg yolk and 1.7 g KCl L^?1) and frozen in 0.5‐ml straws at 2 cm above the surface of liquid nitrogen and thawed at 25°C for 30 s. Then 125 μl or 50 μl of frozen‐thawed testicular sperm was poured onto about 1250 eggs for fertilization. The results showed that both sperm‐to‐egg ratio and diluent had no significant influence on cryopreservation efficiency of testicular sperm, whereas cryoprotectant had a significant influence. The highest fertilization rate (92.2%) was obtained from testicular sperm cryopreserved in glucose‐based extender containing 10% methanol at a sperm‐to‐egg ratio of 1 × 10⁶ spermatozoa per egg. The results indicated that glucose‐based extender containing 10% methanol, 10% egg yolk and 1.7 g KCl L^?1 was a useful combination.     

Semen characteristics, cryopreservation, and successful artificial insemination in the Blue rock pigeon (Columba livia)

The present study was undertaken in the Blue rock pigeon (Columba livia) to evaluate the annual semen characteristics, to identify a suitable extender for semen short-term storage, to determine a protocol for cryopreservation of semen and finally to check whether intracloacal insemination would lead to the birth of a chick. Semen characteristics such as semen volume, sperm concentration, sperm motility, and percentage of normal spermatozoa were maximum during the monsoon season. TALP was observed to be the most suitable semen extender and the sperm survived best at 37 degrees C at a dilution of 1:100 in TALP. Further, cryopreservation studies on pigeon semen indicated that 8% DMSO with or without egg yolk (20%) proved to be a better cryoprotectant compared to glycerol and polyethylene glycol. In addition, the slow freezing protocol was better than the fast-freezing protocol and about 40% of the cryopreserved spermatozoa were motile following thawing. Computer-aided semen analysis indicated that pigeon spermatozoa were extremely active immediately after dilution in TALP and exhibited linear trajectories persisting up to 9h. But, with time there was a time-dependent decrease in the velocity parameters (VAP, VSL, and VCL). Cryopreserved spermatozoa following thawing also exhibited linear trajectories but had reduced velocity as evident from the significant decrease in VAP, VSL, and VCL. Further, artificial inseminations using fresh semen resulted in 45% fertilization and birth of a live chick.     

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.     

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

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

Short-term storage sperm of coho salmon (Oncorhynchus kisutch) at 4 °C: Effect of sperm: Extender dilution ratios and antioxidant butyl-hydroxytoluene (BHT) on sperm function

Short-term storage of semen is a necessary key procedure in fish; it allows maximizing the use of gametes. Nevertheless, sperm quality decreases during storage has been associated with oxidative stress damage due to an increase in reactive oxygen species (ROS) during storage. This study was designed to optimize a short-term storage protocol for Coho salmon (Oncorhynchus kisutch) spermatozoa, evaluating the effect of extender dilution and the addition of butyl-hydroxytoluene (BHT) antioxidant on sperm function parameters. In the first experiment, fresh semen was diluted in Storfish®: extender dilution (1:2 and 1:3) and a control sample undiluted and stored at 4 °C for 7-days. In both experiments motility (MO), viability and integrity of plasma membrane, mitochondrial membrane potential (MMP) and superoxide anion level (O₂⁻) were evaluated at 0, 3 and 7 days. Result shows that, 1:3 dilution maintained a higher sperm function for a longer period time. In the second experiment, spermatozoa were suspended in Storfish® (1:3) supplemented with two different concentrations of BHT (1.0 mM and 2.0 mM) and a control sample without antioxidant and stored at 4 °C for 7 days. The results demonstrated that, antioxidant-supplemented samples greater MO than control samples (P < 0.05). The viability remained >75% during storage in all groups. MMP was higher in 2.0 mM BHT compared to 1.0 mM and control (P < 0.05), in addition, this concentration reduced O₂⁻ level (P < 0.05). In conclusion, sperm: extender dilution 1:3 and adding of 2.0 mM BHT in sperm storage extender may enhance protection sperm function in Oncorhynchus kisutch against effects harmful of the oxidative stress during the in vitro storage.     

Les spermatozoïdes immobiles frais ou décongelés en fécondation assistée

Contrasting with sperm count or morphology, complete lack of mobile sperm may seriously impair ICSI fertilization and pergnancy rate. In three cases with flagellar skeleton abnormalities [dynein arm absence] only immobile sperm were found in the ejaculate. Following repeated ejaculations, higher rates of viable spermatozoa and even some motile spermatozoa could be found. Some times, in nonobstructive azoospermia, extensive sperm search didn't allow us to find but immobile sperm mostly, with very few motile sperm cells, not enough for the microinjection of all oocytes. The third group of immobile sperm is iatrogenic, following freezing and thawing surgically retrieved, testicular or epididymal spermatozoa in order to avoid repeated surgical retrieval. Following thawing, one find frequently very few motile spermatozoa that may be not enough for all retrieved oocytes and it might be necessary to inject some eggs with immobile spermatozoa. The outcome of ICSI using mobile and immobile sperm was compared in the three above mentioned groups: 1-immobile ejaculated sperm with flagellar defects, 2-immobile sperm discovered in the ejaculate after extensive sperm search and 3- immobile frozen-thawed testicular or epididymal spermatozoa. The results of ICSI in these groups show that fertilizing ability of fresh or frozenthawed immobile spermatozoa is not significantly different from ICSI with mobile sperm from the same origin. More over, in the first group with flagellar abnormalities, repeated ejaculations allowed us significantly increase sperm viability and fertilization ability. Finding only immobile fresh or frozen-thawed sperm the day of egg retrieval should not lead us to ICSI cancellation. Pregnancies may occur with such immobile sperm.     

Motility and fertilizing capacity of fresh and frozen-thawed spermatozoa in sturgeons Acipenser Baeri and A. ruthenus

The Siberian sturgeon ( Acipenser baeri ) and the sterlet ( A. ruthenus ) were injected with dried sturgeon pituitary (2 mg kg⁻¹), yielding 24 h later respectively 1.71 ± 0.5 and 1.65 ± 0.5 10¹¹ spermatozoa kg⁻¹ body weight. Spermatozoa were best activated with a solution of Tris HC1 50 mM, pH 8.0. The percentage of activated cells was 88 ± 4.4 in A. baeri (n = 5) and 68 ± 19 in A. ruthenus. (n = 5). In A. baeri , immediately after activation, the beat frequency of the flagellum and the mean velocity were in the range of 48–52 Hz and 100–300 μm⁻¹s, respectively. The beat frequency declined to 15 Hz at 30–40 and velocity to 100 μm s⁻¹ at 60 s post-activation. Only a small percentage of the spermatozoa remained motile after 3–4 min. In all cases spermatozoa showed mostly quasi-linear trajectories. Sperm was frozen in liquid nitrogen vapor immediately after dilution 1 v: 1 v in a cryopreservation medium (23.4 mM saccharose, 118 mM Tris-HCl pH 8.0, 20% egg yolk to which 15% DMSO were added). After fast-thawing procedure (25 s at 40°C), the percentage of motile spermatozoa (once activated in 118 mM Tris-HCl, pH 8.0) decreased to 23 ± 8.8 in A. baeri and to 15 ± 11 in A. ruthenus. The fertilizing capacity also decreased: 53 ± 8.3% vs. 89 ± 7.6% in control (P < 0.05) in A. baeri and 23 ± 11% vs 53 ± 8.3% in control (P < 0.05) in A. ruthenus. The motility pattern of the surviving frozen/thawed spermatozoa was the same as in fresh spermatozoa.     

Characteristics and in vitro fertilizing ability of giant panda (Ailuropoda melanoleuca) frozen‐thawed epididymal spermatozoa obtained 4 hours postmortem: A case report

When Chu‐Lin, a male giant panda (studbook #249), died at Madrid Zoo, his reproductive tract was removed 4 hr postmortem and the epididymal spermatozoa were collected. Extended sperm were kept at 5°C for 4 hr, loaded into straws, and frozen for 7 min in liquid nitrogen vapor before the straws were plunged into liquid nitrogen. Two straws were thawed and evaluated. Sperm motility was assessed in fresh, refrigerated, and thawed spermatozoa (75%, 60%, 35%, respectively). Sperm viability and acrosome status were estimated using a triple‐stain technique (TST). The results showed 33% live sperm with intact acrosomes after thawing. A hypoosmotic swelling (HOS) test demonstrated the retention of membrane integrity in 72% of thawed sperm. To evaluate the in vitro fertilizing ability of thawed sperm, a sperm penetration assay (SPA) was performed. The values obtained for the percentage of penetration and the penetration index were 62% and 1.78 sperm/oocyte, respectively. The results obtained demonstrate that epididymal sperm recovered from a giant panda postmortem can be successfully cryopreserved. The sperm fertilizing ability demonstrated in vitro after thawing may provide a final opportunity for this male to contribute to the currently small germplasm reserves of this endangered species, and to reproduce in the future through assisted reproductive technology. Zoo Biol 23:279–285, 2004. © 2004 Wiley‐Liss, Inc.     

Sperm cryopreservation of the critically endangered olive barb (Sarpunti) Puntiussarana (Hamilton, 1822)

The present study focused on development of a sperm cryopreservation protocol for the critically endangered olive barb Puntiussarana (Hamilton, 1822) collected from two stocks within Bangladesh and reared in the Fisheries Field Laboratory, Bangladesh Agricultural University (BAU). The sperm were collected in Alsever’s solution prepared at 296 mOsmol kg⁻¹. Sperm were activated with distilled water (24 mOsmol kg⁻¹) to characterize motility. Maximum motility (90%) was observed within 15 s after activation, and sperm remained motile for 35 s. Sperm activation was evaluated in different osmolalities and motility was completely inhibited when osmolality of the extender was ?287 mOsmol kg⁻¹. To evaluate cryoprotectant toxicity, sperm were equilibrated with 5%, 10% and 15% each of dimethyl sulfoxide (DMSO) and methanol. Sperm motility was noticeably reduced within 10 min, when sperm were equilibrated with 15% DMSO, indicating acute toxicity to spermatozoa and therefore this concentration was excluded in further trials. Sperm were cryopreserved using DMSO at concentrations of 5% and 10% and methanol at 5%, 10% and 15%. The one-step freezing protocol (from 5 °C to −80 °C at 10 °C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL® CL-3300; Australia) and 0.25-ml straws containing spermatozoa were stored in liquid nitrogen for 7–15 days at −196 °C. The highest motility in thawed sperm 61 ± 8% (mean ± SD) was obtained with 10% DMSO. The fertilization and hatching rates were 70% and 37% for cryopreserved sperm, and 72% and 62% for fresh sperm. The protocol reported here can be useful for hatchery-scale production of olive barb. The use of cryopreserved sperm can facilitate hatchery operations, and can provide for long-term conservation of genetic resources to contribute in the recovery of critically endangered fish such as the olive barb.     

Intracellular signal transduction pathways in the regulation of fowl sperm motility: Evidence for the involvement of phosphatidylinositol 3‐kinase (PI3‐K) cascade

The possible role of PI3‐K in the reversible temperature‐dependent immobilization of fowl sperm motility was investigated by using PI3‐K inhibitor (LY294002) and its inactive analogue (LY303511). The existence of the PI3‐K in fowl spermatozoa was also confirmed by Western blotting analysis. Fowl sperm motility in TES/NaCl buffer remained negligible at the avian body temperature of 40°C but was maintained vigorously when the temperature was decreased to 30°C. At 30°C, no stimulation or inhibition of motility was observed after the addition of 2 mM CaCl₂ and 10 µM LY294002 or LY303511: around 70–80% of spermatozoa remained motile. In contrast, at 40°C, the motility of spermatozoa was activated immediately after the addition of Ca²⁺, but the subsequent addition of LY294002 inhibited the motility again. The addition of LY303511 did not appreciably affect the Ca²⁺‐supplemented sperm motility, which was maintained for at least 15 min. The ATP concentrations of spermatozoa after the addition of LY294002 + Ca²⁺ or LY303511 + Ca²⁺ were almost the same values compared with those of Ca²⁺ alone at 40°C, suggesting that the addition of LY294002 was not simply affecting membrane damage or inhibiting energy production in the spermatozoa, but may be acting on some part of the motility‐regulating cascade. Immunoblotting of sperm extract using an antibody to PI3‐K revealed a major cross‐reacting protein of 85 kDa, which corresponds to the molecular weight of the subunit of PI3‐K. These results suggest that PI3‐K may be positively involved in the calcium‐regulated maintenance of flagellar movement of fowl spermatozoa at 40°C. Mol. Reprod. Dev. 76: 603–610, 2009. © 2008 Wiley‐Liss, Inc.     

Effect of initial freezing temperature on the semen characteristics of frozen-thawed ram spermatozoa in a semi-arid tropical environment

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including initial freezing temperature. The present study was conducted to observe the effect of initial freezing temperature on post-thawing motility of ram spermatozoa of native and crossbred rams maintained in a semi-arid tropical environment. Good quality semen obtained from native Malpura and crossbred Bharat Merino rams were pooled within breed and diluted at a rate of 1000 million spermatozoa per milliliter in TEST—yolk–glycerol extender. Diluted semen samples were loaded in 0.25 ml straws and cooled to −25, −75 or −125 °C freezing temperature at the rate of −25 °C/min under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50 °C in a water bath for 10 s and motility characteristics of the frozen-thawed spermatozoa were assessed by a computer-assisted spermatozoa analysis technique. Initial freezing temperature significantly affected the post-thawing motility of sperm in both the breeds. The post-thawing % motility and rapid motile spermatozoa were significantly higher at initial freezing temperature of −125 °C and lower at −25 or −75 °C. The percentage medium motile sperm were similar at all three initial freezing temperatures. The percentage of slow motile and linearity of sperm varied (P<0.01) between the different freezing temperatures. The curvilinear velocity, average path velocity and straight line velocity of spermatozoa were higher (P<0.01) at −125 °C than −25 or −75 °C. Although the lateral head displacement of spermatozoa did not vary significantly between the different initial freezing temperatures, the stroke frequency was significantly lower at −25 °C than −75 or −125 °C. Except for % linearity, the average path velocity and straight line velocity, other spermatozoa characteristics were not significantly different between breeds. The interaction between freezing temperature and breed was significant only for the % motility and linearity of the spermatozoa. The study indicates that initial freezing temperature has a significant effect on spermatozoa motility and velocity following post-thawing. The best motile spermatozoa following thawing were achieved at −125 °C freezing temperature.