Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos

The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.     

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.     

In vitro development of domestic cat embryos following intra-cytoplasmic sperm injection with testicular spermatozoa

The objective was to assess the ability of testicular spermatozoa to fertilize in vitro matured domestic cat oocytes and support blastocyst formation in vitro following intra-cytoplasmic sperm injection (ICSI). After IVM, oocytes were randomly and equally allocated among treatment groups (ICSI with testicular spermatozoa, ICSI with ejaculated spermatozoa, sham ICSI, and control IVF). At 18 h after either injection or insemination, the percentage of fertilized oocytes (per total metaphase II oocytes) was approximately 65% after ICSI with testicular or ejaculated spermatozoa (P > 0.05), which was less (P < 0.05) than control IVF (approximately 90%). On Day 7, the percentage of cleaved embryos (per total metaphase II oocytes) was approximately 60% after ICSI with testicular or ejaculated spermatozoa (P > 0.05), which also was less (P < 0.05) than control IVF (approximately 85%). After ICSI with testicular spermatozoa, the percentage of blastocysts (per total cleaved embryos) was approximately 11.0%, which was less (P < 0.05) than ICSI with ejaculated spermatozoa (approximately 21.0%); the latter was less (P < 0.05) than control IVF (approximately 43.0%). No blastocyst formation was observed after sham ICSI. For the first time in the domestic cat, this study demonstrated the fertilizing ability and developmental potential of intra-testicular spermatozoa delivered directly into intra-ovarian oocytes matured in vitro.     

Sperm cryopreservation of African catfish, Clarias gariepinus: cryoprotectants, freezing rates and sperm:egg dilution ratio

Methods for cryopreserving spermatozoa and optimizing sperm:egg dilution ratio in African catfish Clarias gariepinus were developed. Five percent to 25% DMSO and methanol were tested as cryoprotectants, by diluting semen in Ginzburg fish ringer and freezing in 1-milliliter cryovials in a programmable freezer. To avoid an excess of spermatozoa per egg, post-thaw semen was diluted 1:20, 1:200 or 1:2,000 before fertilization. Highest hatching rates were obtained by spermatozoa frozen in 10% methanol and post-thaw diluted to 1:200. Then, slow freezing rates (-2, -5 or -10 degrees C/min) to various endpoint temperatures (range -25 to -70 degrees C) before fast freezing in liquid nitrogen (LN2) were evaluated. Hatching rates equal to control (P > 0.05) were obtained by spermatozoa frozen at -5 degrees C/min to -45 to -50 degrees C and at -10 degrees C/min to -55 degrees C. In 3-step freezing programs, at -5 degrees C/min, the effect of holding spermatozoa for 0, 2 or 5 min at -30, -35 or -40 degrees C before fast freezing in LN2 was analyzed. Hatching rates equal to control (P > 0.05) were produced by spermatozoa frozen to, and held at, -35 degrees C for 5 min and at -40 degrees C for 2 or 5 min. Finally, frozen spermatozoa (10% methanol, -5 degrees C/min, 5-min hold at -40 degrees C, LN2, post-thaw diluted to 1:200) were tested in on-farm fertilization conditions. Again, no difference (P > 0.05) in hatching rate was observed between frozen and fresh spermatozoa. Cryopreservation offers utility as a routine method of sperm storage and management for catfish.     

Principes de préparation et de congélation des spermatozoïdes testiculaires humains

The advantages and feasibility of human testicular spermatozoa cryoconservation for intracytoplasmic sperm injection (ICSI) have now been clearly demonstrated. However, the freezing protocol is based on empirical knowledge obtained from freezing of ejaculated spermatozoa. Testicular spermatozoa may not be fully mature gametes and may also be retrieved in only limited quantities. Little research has been conducted to determine whether they have the same cryobiological requirements as ejaculated spermatozoa. A better understanding of their cryobiological features and assessment of possible subcellular changes after thawing would help to optimize testicular preparations for cryopreservation (whole biopsies, seminiferous tubules, shredded suspension, single spermatozoa, etc.), freezing-thawing procedure, freezing media, and storage. Finally, there is a growing need for welldefined criteria (nuclear quality, etc.) to evaluate the tolerance of testicular spermatozoa to freezing-thawing procedure for ICSI     

Improvement of Sperm Motility of Sex-Reversed Male Rainbow Trout, Oncorhynchus mykiss, by Incubation in High-pH Artificial Seminal Plasma

Changes in the motility time of spermatozoa collected from the testes and the sperm duct of normal and sex-reversed male (XX) rainbow trout in physiological balanced salt solution were examined after incubation in artificial seminal plasmas of various pHs. Although untreated spermatozoa from the sperm duct retained motility for 60–90 s in the balanced salt solution, the spermatozoa collected from the testes were immotile. During the incubation in artificial seminal plasma of pH 7.0, the spermatozoa from the sperm duct hardly moved, similar to the testicular spermatozoa in the balanced salt solution. By suspending and incubating the testicular spermatozoa in artificial seminal plasma of pH 9.9 for 2 h at 4°C, the percentage of motile spermatozoa increased from 0–5% to 80%. The spermatozoa remained motile for at least 2 min after long-term incubation (12 h). When the full-sib eggs were inseminated with untreated testicular spermatozoa or testicular sperm treated for 2 h at high pH, the percentage survival increased from 5.5% to 53.8% at the eyed stage due to the high-pH treatment. The incubation of the spermatozoa in high-pH artificial seminal plasma improved the motility of the spermatozoa from the testes of the sex-reversed male that had lost its sperm duct. By this treatment, it is possible to markedly increase the mass production efficiency of all-female or all-female triploid sterile progenies.     

Thiourea-induced thyroid hormone depletion impairs testicular recrudescence in the air-breathing catfish, Clarias gariepinus

We used thiourea-induced thyroid hormone depletion as a strategy to understand the influence of thyroid hormones on testicular recrudescence of the air-breathing catfish, Clarias gariepinus. Treatment with 0.03% thiourea via immersion for 21 days induced hypothyroidism (thyroid hormone depletion) as evidenced by significantly reduced serum T(3) levels. Thiourea-treated males had narrowed seminiferous lobules with fewer spermatozoa in testis, very little or no secretory fluid, reduced protein and sialic acid levels in seminal vesicles when compared to controls. The histological changes were accompanied by reduction in serum and tissue levels of testosterone (T) and 11-ketotestosterone (11-KT), a potent male specific androgen in fish. Qualitative changes in the localization of catfish gonadotropin-releasing hormone (cfGnRH) and luteinizing hormone (LH, heterologous system) revealed a reduction in the distribution of immunoreactive neuronal cells and fibers in thyroid depleted fish. Interestingly, thiourea-withdrawal group showed physiological and histological signs of recovery after 21 days such as reappearance of spermatozoa and partial restoration of 11-KT and T levels. These data demonstrate that thyroid hormones play a significant role in testicular function of catfish. The mechanism of action includes modulating sex steroids either directly or through the hypothalamo (GnRH)-hypophyseal (LH) axis.     

Azoospermie et microinjection

In cases of azoospermia, testicular biopsy combined with cryopreservation of spermatozoa allows ICSI to be performed under good conditions. In this study, the authors present their results by emphasizing three major aspects:

- Retrieval of testicular spermatozoa by open biopsy or percutaneous needle aspiration: 40 patients with obstructive azoospermia underwent epididymal or testicular retrieval by open biopsy and 37 by percutaneous needle aspiration. All biopsies were positive. 133 patients with nonobstructive azoospermia underwent percutaneous needle aspiration and spermatozoa were successfully retrieved from 50 patients (38%).

- The freezing process was performed with a cryoprotective medium devoid of egg yolk after dilaceration of the testicular tissue using two sterile glass slides. No significant difference in the outcome of the ICSI procedure was observed between fresh and frozen-thawed spermatozoa. In cases of obstructive azoospermia, 13 pregnancies out of 41 ICSI cycles (31%) were obtained with the use of fresh testicular or epididymal spermatozoa and 24 pregnancies out of 115 ICSI cycles (20%) were obtained with the use of cryopreserved spermatozoa. In cases of non-obstructive azoospermia, 6 pregnancies out of 31 ICSI cycles (19%) were obtained with the use of fresh testicular spermatozoa and 12 pregnancies out of 33 ICSI cycles (36%) were obtained with the use of frozen-thawed spermatozoa.

- After the freezing-thawing process, the percentage motile testicular spermatozoa is very low (about 4%), with a weakly shaking motility making selection of live spermatozoa very long and difficult. The addition of pentoxifylline (3 mM) significantly increases this motility within 15 minutes, as 30% of spermatozoa have a progressive motility. Selection of viable motile spermatozoa is therefore easier and more rapid. Fertilization and pregnancy rates are comparable to those generally reported. No malformation was observed on 51 live births.

     

Cryopreservation of very low numbers of spermatozoa from male patients undergoing infertility treatment using agarose capsules

This study tried to cryopreserve low numbers of spermatozoa from men undergoing infertility treatments by inserting into agarose capsules. The capsules were transferred into a drop of cryoprotectant solution and injected 3–4 motile spermatozoa that were selected by the swim-up method by conventional intracytoplasmic sperm injection. These capsules were put on a Cryotop^® and frozen in liquid nitrogen vapor, and then submerged into liquid nitrogen and subsequently thawed and recovered. The motile spermatozoa in the capsules were counted. Eventually, we cryopreserved 2142 motile spermatozoa in 702 agarose capsules from 26 male patients and 1356 (63%) spermatozoa maintained their motility after thawing. The spermatozoa motility rates after thawing (MRAT) ranged from 20.0% (5/25) to 95.1% (58/61) among patients. The median MRAT was 68.3% (interquartile range 46.1–75.7). The total number of motile spermatozoa collected by swim-up method strongly correlated with MRAT (r = 0.746). It was possible to cryopreserve spermatozoa from male patients undergoing infertility treatment using agarose capsules. However, there were wide differences in MRAT among patients. It seems the spermatozoa from semen where there were many motile spermatozoa may have higher freezing resistance. Further studies using this method in cryptozoospermic semen, testicular and epididymal spermatozoa are required.     

Post-thaw plasma membrane integrity of bull spermatozoa separated with a Sephadex ion-exchange column

Previous experiments have established that filtration of bovine semen through a Sephadex ion-exchange column improves its quality before and after freezing. The present study was conducted to determine the post-thaw membrane integrity of bull spermatozoa separated with a Sephadex ion-exchange column and to determine the kind of protection to spermatozoa is provided by glycerol during freezing and thawing. Semen from Holstein bulls diluted in TEST-yolk extender (with and without glycerol) was filtered through a Sephadex ion-exchange column and frozen in liquid nitrogen (-196 degrees C). After thawing, there were more normal acrosomes in filtered spermatozoa than nonfiltered (P < 0.01). Post-thaw plasma membrane integrity and swelling ability in a hypoosmotic solution revealed that the filtered spermatozoa had a stronger (P < 0.005) plasma membranes than the nonfiltered. Filtered spermatozoa demonstrated higher zona-free hamster oocyte penetration than the nonfiltered (30.5 vs 11.5%; P < 0.0005). Spermatozoa extended in TEST-yolk without glycerol had the lowest (P < 0.001) normal acrosomes, intact plasma membranes and swelling ability. Plasma membrane over the post-acrosomal region of the head and post-midpiece region of the tail was more sensitive to damages caused by freezing and thawing than acrosomal and midpiece regions of spermatozoa. Glycerol in the extender provided significant (P < 0.05) protection to the sensitive regions of filtered and nonfiltered spermatozoa during freezing and thawing. Filtered plus glycerolated spermatozoa had the highest (P < 0.01) normal acrosomes, intact plasma membranes and swelling ability. In conclusion, the pre-freezing filtration of bovine semen harvested the spermatozoa possessing stronger plasma membranes which enabled them to endure freezing and thawing stresses. The addition of glycerol to the extender protected the post-acrosomal region of the head and post-midpiece region of the tail of spermatozoa from freezing and thawing shocks.     

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.     

Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa

The decline in boar semen quality after cryopreservation may be attributed to changes in intracellular proteins. Thus, the aim of the present study was to evaluate the change of protein profiles in boar spermatozoa during the process of cooling and after cryopreservation. A total of 9 sexually mature boars (mean age = 25.5+/-12.3 mo) was used. Samples for protein analysis were collected before chilling, after cooling to 15 degrees C, after cooling to 5 degrees C, following thawing after freezing to -100 degrees C, and following thawing after 1 wk of cryopreservation at -196 degrees C. Semen characteristics evaluated included progressive motility and the percentage of morphologically normal spermatozoa. Total proteins from 5x10(6) spermatozoa were separated and analyzed by SDS-PAGE. The results revealed that there was a substantial decrease of a 90 kDa protein in the frozen-thawed spermatozoa. Western blot analysis demonstrated that this protein was 90 kDa heat-shock protein (HSP90). Time course study showed that the decrease of HSP90 in spermatozoa initially occurred in the first hour during cooling to 5 degrees C. When compared with the fresh spermatozoa before chilling, there was a 64% decrease of HSP90 in spermatozoa after cooling to 5 degrees C. However, the motility and percentage of normal spermatozoa did not significantly decrease during this period of treatment. Both declined substantially as the semen was thawed after freezing from -100 degrees C. The results indicated that the decrease of HSP90 precedes the decline of semen characteristics. The length of time between a decrease of HSP90 and the decline in sperm motility was estimated to be 2 to 3 h. Taken together, the above results suggested that a substantial decrease of HSP90 might be associated with a decline in sperm motility during cooling of boar spermatozoa.     

Effects of egg yolk, glycerol and the freezing rate on the viability and acrosomal structures of frozen ram spermatozoa.

The influence of egg yolk, glycerol and the freezing rate on the survival of ram spermatozoa and on the structure of their acrosomes after freezing was investigated. Egg yolk was shown to be beneficial not only during chilling but also during freezing; of the levels examined, 1-5% gave the greatest protection. Although the presence of glycerol in the diluent improved the survival of spermatozoa, increasing concentrations produced significant deterioration of the acrosomes. With closely controlled linear cooling rates, no overall difference was detected in the survival of spermatozoa frozen at rates between 6 and 24 degrees C per min. However, a significant interaction between freezing rate and the inclusion of glycerol in the diluent showed that glycerol was less important at the highest freezing rate. A sudden cooling phase near to the freezing point following the release of the latent heat of fusion was not detrimental to spermatozoa.     

Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births

Data of cryoprotectant-free vitrification of human testicular and epididymal spermatozoa are limited. The aim of this investigation was to compare two aseptic technologies of TESE (testicular) and MESA (epididymal) spermatozoa cryopreservation: standard conventional freezing with the use of cryoprotectants and cryoprotectant-free vitrification. Sperm motility, capacitation-like changes, acrosome reaction and the mitochondrial membrane potential of frozen (5% glycerol, −10 °C/min) and vitrified (Human Tubal Fluid + 1% Human Serum Albumin+0.25 M sucrose, plunging into liquid nitrogen of capillaries with spermatozoa isolated from liquid nitrogen (aseptic method) were compared. The quality of the cryoprotectant-free vitrified MESA- and TESE-spermatozoa was higher than that of spermatozoa conventionally frozen with permeable cryoprotectants. Intracellular sperm injection (ICSI) was performed with vitrified spermatozoa. We report the birth of three healthy babies from two women following ICSI with motile MESA- and TESE-spermatozoa vitrified without cryoprotectants. This is the first report of full-term pregnancies and babies born after ICSI with epididymal and testicular spermatozoa vitrified without cryoprotectants. In conclusion, cryoprotectant-free vitrification can be successfully applied for the cryopreservation of motile TESE- and MESA-spermatozoa.     

Utilisation des spermatozoïdes testiculaires en ICSI. Intérêt de la culture in vitro. Revue de la littérature

The number of ICSI cycles performed with testicular spermatozoa has increased dramatically over recent years. However, one of the technical limitations of this approach concerns the extremely reduced motility of testicular spermatozoa. However, increased sperm motility was observed after incubating testicular samples for several hours. Therefore, in order to improve ICSI success rates, several authors have tested the effect of previous in vitro culture. We present a review of the literature on this subject. In vitro culture does not appear to be very useful in cases of obstructive azoospermia, as, apart from possible sperm “maturation” during this culture phase, a high proportion of motile spermatozoa is usually already observed prior to in vitro culture. The benefits of in vitro culture appear to be greater in the case of non-obstructive azoospermia, as when spermatozoa are present on the biopsy, they are usually immobile. However, discordant results have been published: after in vitro culture, spermatozoa have been reported to be either motile or mostly dead. Regardless of the type of azoospermia, the best results are obtained after 3–4 days of in vitro culture. Addition of recombinant FSH to the culture medium also appears to be effective. Cryopreservation of testicular biopsies may also be associated with in vitro culture and the in vitro culture/freezing sequence appears to give better results than the freezing/in vitro culture sequence. Very few studies have reported the results of ICSI using frozen in vitro cultured spermatozoa, as most published studies concern fresh spermatozoa, used after 1–2 days of in vitro culture with satisfactory fertilization and pregnancy rates. In vitro culture of testicular spermatozoa may therefore constitute an interesting research approach to improve the results of ICSI when the number of spermatozoa and/or motility are very low. In addition, in vitro culture of testicular spermatozoa appears to be a good tool to study the mechanisms of acquisition of motility, which are still poorly understood.     

Survival and fertility of ram spermatozoa frozen in pellets, straws and minitubes

The post-thaw survival and fertility of ram spermatozoa frozen in pellets, 0.25- and 0.5-ml PVC straws, and 0.25-ml minitubes were examined. In 5 experiments, a freezing height of 6 cm above the level of liquid nitrogen was optimal for 0.25- and 0.5-ml straws, whereas 4 cm was best for the 0.25-ml minitubes. Post-thaw motility of spermatozoa was lower for semen frozen in straws and minitubes than in pellets (Experiment 1: 43.7 vs 53.4%, P < 0.001), but after freezing was better in 0.5-ml straws and 0.25-ml minitubes than in 0.25-ml straws (Experiment 1: 44.9 vs 41.3%, P < 0.05; Experiment 2: 49.6 vs 46.8%, P < 0.01). Sperm motility was also better for 1:8 (semen:diluent) pre-freezing dilution rate (50.5%) than for 1:4 (45.6%, P < 0.01) and 1:2 (39.8%, P < 0.001) but not the 1:16 (49.5%) dilution rate. Dry ice was a better freezing medium than liquid nitrogen vapor (49.2 vs 46.9% motile spermatozoa, P < 0.001). The post-thaw motility of spermatozoa was similar for the three freezing packages if the semen was loaded at 5 degrees C, but motility was poorer for semen loaded into 0.25-ml straws than 0.25-ml minitubes at 30 degrees C (P < 0.05). In a fertility test, pregnancy rates were influenced by rams (3 rams, P < 0.05) and freezing package (pellets vs 0.25-ml minitube vs 0.25-ml straw vs 0.5-ml straw, P < 0.05) but not freezing medium (liquid nitrogen vapor vs dry ice). More ewes were pregnant after insemination with pellet-frozen semen (106/150, 71%) than with semen frozen in 0.25-ml straws (85/150, 57%; P < 0.05) and in 0.5-ml straws (83/150, 55%; P < 0.01) but not minitubes (98/150, 65%). It was concluded that minitubes provide a useful alternative to pellets as a storage package for ram spermatozoa, allowing for individual dose identification and easier storage while maintaining a fertility rate indistinguishable from that obtained with pellet-frozen semen.     

Peut-on optimiser la congélation des spermatozoïdes testiculaires? L’expérience du Centre Hospitalier de Poissy Saint-Germain

50% or more of non-obstructive azoospermic men have no spermatozoa in their testicular tissue, and no non-invasive predictor of spermatogenesis is yet available. For this reason, we therefore performed all TESE (74 TESE for non-obstructive azoospermia and 37 TESE for obstructive azoospermia) prior to initiating ovarian stimulation. 34% (25/74) of TESE performed for non-obstructive azoospermia were successful. Spermatozoa were retrieved in 100% of cases of obstructive azoospermia. When TESE were positive, spermatozoa were frozen in 25–50 μl micro-droplets (several straws). 60 ICSI cycles (25 couples) were treated for non obstructive azoospermia. The clinical pregnancy rate per ICSI cycle was 18%, and the implantation rate per embryo transferred was 9.2%. 81 ICSI cycles (37 couples) were treated for obstructive azoospermia. The fertilization rate was 54%, and embryo transfer was performed in 89% (72/81) of cycles. The clinical pregnancy rate per embryo ICSI cycle was 26%, and the implantation rate per embryo transferred was 16%. This management of azoospermic patients, including TESE and multiple testicular tissue freezing in micro-droplets prior to ovarian stimulation, avoids ova pick-up cancellation and multiple TESE, as several ICSI can be performed after a single TESE. Our results show that this micro-technique for freezing testicular tissue is effective not only for obstructive azoospermia, but also for non-obstructive azoospermia when only very few spermatozoa can be extracted from the testis.     

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.     

French field results (1985-2005) on factors affecting fertility of frozen stallion semen

Results on procedures for freezing stallion semen and the subsequent fertility during 20 years are presented. The present system applied in French National Stud includes: (1) a freezing protocol (dilution in milk, centrifugation and addition of freezing extender (INRA82+egg yolk (2%, v/v)+glycerol (2.5%, v/v) at 22 degrees C, a moderate cooling rate to 4 degrees C and freezing at -60 degrees C/min in 0.5-ml straws); (2) selection of ejaculates showing post-thaw rapid motility >35%; and (3) an insemination protocol (mares examined once daily, two AI of 400 x 10(6) spermatozoa 24 h apart before ovulation, sufficient number of straws to have the possibility to perform six AI of 400 x 10(6) total spermatozoa, i.e. 2.4 x 10(9) total spermatozoa available per mare per season). This system was applied to >110 stallions per year, the average post-thaw motility of ejaculates was 50% (>1800 ejaculates) before selection. The semen freezability was defined as the number of selected ejaculates divided by the total number of ejaculates frozen. Of the stallions, 5, 4, 5, 21 and 64% had semen freezability of 0-10, 10-33, 33-60, 60-90 and over 90%, respectively. Per-cycle pregnancy rate was 45-48% (>1500 mares per year, 1.8 cycles per mare) and foaling rate 64%. In comparison, per-cycle pregnancy rate and foaling rate of mares hand-mated to stallions were 57-59% and 64%, respectively. The average number of straws used was 32-35 (1.75 x 10(9) total spermatozoa) per mare per season. According to our results and the literature, the most important factors for improving fertility of frozen equine semen include: (1) a low concentration of glycerol (2-3.5% final concentration); (2) a suitable base extender for freezing like Lactose-Glucose EDTA or INRA82; (3) a post-thaw motility >30-35%; and (4) a sufficient number of spermatozoa per mare per season (1.5-2 x 10(9) total spermatozoa for two to three cycles) divided into small units. Numbers of spermatozoa, lower than 750.10(6) total spermatozoa per cycle, could result in lower per-cycle pregnancy rate with higher additional costs for management of mares. Because there are no particular regulations on quality and quantity of equine semen in the European Community, there is a need for the uniformity of information about frozen semen. A codification is suggested, based on the number of spermatozoa available per mare per season, the post-thaw motility and the final glycerol concentration.     

Species variation in osmotic, cryoprotectant, and cooling rate tolerance in poultry, eagle, and peregrine falcon spermatozoa

Potential factors influencing spermatozoa survival to cryopreservation and thawing were analyzed across a range of the following avian species: domestic chicken (Gallus domesticus), domestic turkey (Meleagris gallopavo), golden eagle (Aquila chrysaetos), Bonelli's eagle (Hieraaetus fasciatus), imperial eagle (Aquila adalberti), and peregrine falcon (Falco peregrinus). Studies focused on spermatozoa tolerance to the following: 1) osmotic stress, 2) different extracellular concentrations of the cryoprotectant dimethylacetamide (DMA), 3) equilibration times of 1 versus 4 h, 4) equilibration temperature of 4 versus 21 degrees C, and 5) rapid versus slow cooling before cryopreservation and standard thawing. Sperm viability was assessed with the live/dead stain (SYBR-14/propidium iodine). Sperm viability at osmolalities >/=800 mOsm was higher (P: < 0.05) in raptor than poultry semen. Return to isotonicity after exposure to hypertonicity (3000 mOsm) decreased (P: < 0.05) number of viable spermatozoa in chicken, turkey, and golden and Bonelli's eagle spermatozoa but not in imperial eagle or peregrine falcon spermatozoa. Differences were found in spermatozoa resistance to hypotonic conditions, with eagle species demonstrating the most tolerance. Semen, equilibrated for 1 h (4 degrees C) in diluent containing DMA (> or =2.06 M), experienced decreased (P: < 0. 05) spermatozoa survival in all species, except the golden eagle and peregrine falcon. Number of surviving spermatozoa diminished progressively with increasing DMA concentrations in all species. Increased equilibration temperature (from 4 to 21 degrees C) markedly reduced (P: < 0.05) spermatozoa survival in all species except the Bonelli's eagle and turkey. Rapid cooling was detrimental (P: < 0.05) to spermatozoa from all species except the imperial eagle and the chicken. These results demonstrate that avian spermatozoa differ remarkably in response to osmotic changes, DMA concentrations, equilibration time, temperature, and survival after fast or slow freezing. These differences emphasize the need for species-specific studies in the development and enhancement of assisted breeding for poultry and endangered species.