Vitrification of dog spermatozoa: Effects of two cryoprotectants (sucrose or trehalose) and two warming procedures

Sperm vitrification is a low cost and simple technique that does not require special equipment and may represent an attractive alternative to the costly and time consuming conventional dog spermatozoa cryopreservation techniques. The objective of this study was to evaluate different cryoprotectants and warming temperatures on the vitrification of dog spermatozoa. Pooled semen samples from 10 beagle dogs were vitrified with four extenders, based on Tris, citric acid and glucose, 20% egg yolk (TCG-20% EY) and different combinations of sucrose and/or trehalose: 250 mM sucrose; 250 mM trehalose; 125 mM sucrose + 125 mM trehalose; 250 mM sucrose + 250 mM trehalose. Samples were vitrified by dropping 50 μL of sperm suspension directly into liquid nitrogen. After vitrification, warming was done either fast (at 65 °C for 2–5 s) or slow (at 37 °C for one minute). Motility was assayed using a computer-aided sperm analysis (CASA) system; membrane integrity and acrosomal status were analyzed by fluorescence microscopy. For comparison, samples were also conventionally frozen in liquid nitrogen vapor using a TCG-20% egg yolk extender plus 5% glycerol. Frozen straws were thawed in a water bath at 37 °C for 30 s. Poorer motility results (P < 0.05) but similar viability were obtained when vitrification was performed, compared to conventional freezing (P > 0.05). When vitrification was used, cryoprotectants containing either 250 mM sucrose or 250 mM trehalose and warmed at 37 °C returned the best sperm quality variables.     

Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: stability of mitochondrion as criterion of effectiveness

The aim of the present investigations was to test a novel technology comprising cryoprotectant-free vitrification of the spermatozoa of rainbow trout and to study the ability of sucrose and components of seminal plasma to protect these cells from cryoinjuries. Spermatozoa were isolated and vitrified using five different mediums: Group 1: standard buffer for fish spermatozoa, Cortland(?)-medium (CM, control); Group 2: CM+1% bovine serum albumin (BSA); Group 3: CM+1% BSA+0.125 M sucrose; Group 4: CM+1% BSA+40% seminal plasma; and Group 5: CM+1% BSA+40% seminal plasma+0.125 M sucrose. For cooling, 20 μL suspensions of cells from each group were dropped directly into liquid nitrogen. For warming, the spheres containing the cells were quickly submerged in CM+1% BSA at 37 °C with gentle agitation. The quality of spermatozoa before and after vitrification was analysed by the evaluation of motility, cytoplasmic membrane integrity (SYBR-14/propidium iodide staining technique), and mitochondrial membrane integrity (JC-1 staining). Motility (86%, 71%, 80%, 81%, and 82%, for Groups 1, 2, 3, 4, and 5, respectively) and cytoplasmic membrane integrity (90%, 82%, 83%, 84%, and 87%, respectively) of spermatozoa in all the 5 groups were not decreased significantly. All tested solutions can be used for vitrification of fish spermatozoa with good post-warming motility and cytoplasmic membrane integrity. However, mitochondrial membrane potentials of the spermatozoa in Groups 1, 2, 3, 4, and 5 were changed significantly (6%, 50%, 37%, 55%, and 34%, respectively) (P(1,2,3,4,5)<0.001; P(2,3,4,5) <0.01)(P(3-5)>0.1). This rate was maximal in Group 4 (CM+1% BSA+40% seminal plasma). In conclusion, this is the first report about successful cryoprotectant-free cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen (vitrification). Vitrification of fish spermatozoa without permeable cryoprotectants is a prospective direction for investigations: these cells can be successfully vitrified with 1% BSA+40% seminal plasma without significant loss of important physiological parameters.     

First birth of a healthy infant following intra-cytoplasmic sperm injection using a new permeable cryoprotectant-free sperm vitrification protocol

PurposeThe purpose of this study is to present the first birth of healthy infant born following ICSI using the new permeable cryoprotectant-free sperm vitrification protocol Easy-Sperm^®.Principal resultsA 39 years old woman and his 40 years old partner underwent egg donation treatment at IVF-Spain Alicante (Spain). Half of the mature oocytes obtained from a young and healthy donor were fertilized by ICSI, using slow-frozen spermatozoa and the other half with vitrified spermatozoa. A total of 5 blastocysts were obtained on day 5 (3 resulting from vitrified spermatozoa and 2 from frozen sperm). The best embryo, with AA quality (derived from one of the oocytes fertilized with vitrified sperm) was transferred. The woman conceived and, following a normal pregnancy, delivered a healthy boy.ConclusionsTo the best of our knowledge, this is the first case report of a successful pregnancy and delivery of a healthy infant from ICSI with permeable vitrified spermatozoa in an oocyte donation program with transfer on blastocyst stage.     

Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability

Human spermatozoa can be successfully cryopreserved avoiding the use of cryoprotectants through vitrification at very high cooling rates (up to 7.2 x 10(5) degrees C/min). This is achieved by directly plunging a copper cryoloop loaded with a sperm suspension into liquid nitrogen. After storage, vitrified spermatozoa are instantly thawed by melting in an agitated, warm medium. The goal of the present study was to compare the quality of spermatozoa cryopreserved using this rapid vitrification method with that of spermatozoa cooled relatively slowly by preexposure of the loaded cryoloop to liquid nitrogen vapor (-160 degrees C) with speed in the range 150-250 degrees C/min) before immersion into liquid nitrogen. Both cooling modes led to comparable results in terms of the motility, fertilization ability, and DNA integrity of the warmed spermatozoa. In both cases, instant thawing by melting in a warm medium was essential for successful cryopreservation. Our findings suggest that optimal regimes for the cryoprotectant-free cryopreservation of spermatozoa need not be restricted to very fast cooling before storage in liquid nitrogen, a wide range of cooling rates being acceptable. Herein, we discuss the implications of this finding in the light of the physics of extra- and intracellular vitrification.     

Comparison of cryosurvival and spermatogenesis efficiency of cryopreserved neonatal mouse testicular tissue between three vitrification protocols and controlled-rate freezing

Grafting of cryopreserved testicular tissue is a promising tool for fertility and testicular function preservation in endangered species, mutant animals, or cancer patients for future use. In this study, we aimed to improve the whole neonatal mouse testicular tissue cryopreservation protocols by comparing cryosurvival, spermatogenesis, and androgen production of grafted testicular tissue after cryopreservation with three different vitrification protocols and an automated computed controlled-rate freezing. Whole neonatal mouse testes were vitrified with various vitrification solutions (V1) 40% EG + 18% Ficoll + 0.35 M Sucrose, (V2) DAP 213 (2 M DMSO + 1 M Acetamid + 3 M PG), or (V3) 15% EG + 15% PG + 0.5 M Sucrose (total solute concentration V1:74.34%, V2:44.0%, and V3:49.22% wt/vol). Alternatively, neonatal testicular tissue was also frozen in 0.7 M DMSO +5% fetal bovine serum using controlled-rate freezing and compared to fresh grafted testicular tissue, sham grafted controls, and the vitrification protocol groups. Fresh (n = 4) and frozen-thawed (n = 4) testes tissues were grafted onto the flank of castrated male NCr Nude recipient mouse. The grafts were harvested after three months. Fresh or frozen-thawed grafts with controlled-rate freezing had the highest rate of tissue survival compared to other vitrified protocols after harvesting (p < 0.05). Both controlled-rate freezing and V1 protocol groups displayed the most advanced stages of spermatogenesis with elongated spermatids and spermatozoa in 17.6 ± 1.3% and 16.3 ± 1.9% of seminiferous tubules based on histopathological evaluation, respectively. Hosts of the testicular graft from controlled-rate freezing had higher levels of serum testosterone compared to all other vitrified-thawed graft groups (p < 0.05). This study shows that completed spermatogenesis from whole neonatal mouse testes were obtained when frozen with controlled-rate freezing and V1 vitrification solution and that testicular cryopreservation efficacy vary with the protocol and vitrification technique.     

Pentoxifylline increase sperm motility in devitrified spermatozoa from asthenozoospermic patient without damage chromatin and DNA integrity

The freeze–thaw process results in reduced motility, viability and fertilization potential of human spermatozoa. So, a variety of substances were evaluated in order to enhance human sperm resistance to the stress of cryopreservation, such as Pentoxifylline (PTX) for improving the Intracytoplasmic sperm injection (ICSI) outcomes. The aim was to investigate the effect of PTX on sperm parameters and chromatin/DNA integrity of asthenozoospermic semen post vitrification. A total of 30 semen specimens were obtained from infertile men with asthenozoospermia. The cryoprotectant-free vitrification was performed for the samples after assessment of sperm parameters. After warming, each sample was exposed for 30 min to 3.6 mmol/l PTX in experimental group and the control group without any treatment apposing at 37 °C for 30 min in regard, to repeat all in vitro analysis (sperm parameters and DNA integrity assay). Regardless of the vitrification devastating impacts on sperm parameters, incubation of post vitrified samples with PTX increased the rate of progressive motility (P < 0.01). Moreover, PTX addition did not significantly damage DNA integrity of asthenozoospermic sperm samples. The data showed that PTX was able to improve sperm movement without any adverse effects on sperm chromatin/DNA integrity in vitrification program.     

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.

     

Generation of Live Piglets for the First Time Using Sperm Retrieved from Immature Testicular Tissue Cryopreserved and Grafted into Nude Mice

Cryopreservation of immature testicular tissues is essential for increasing the possibilities of offspring generation by testicular xenografting for agricultural or medical purposes. However, successful production of offspring from the sperm involved has never been reported previously. In the present study, therefore, using intracytoplasmic sperm injection (ICSI), we examined whether xenogeneic sperm obtained from immature pig testicular tissue after cryopreservation would have the capacity to produce live piglets. Testicular fragments from 9- to 11-day-old piglets were vitrified after 10- or 20-min immersion in vitrification solution containing ethylene glycol (EG), polyvinyl pyrrolidone (PVP) and trehalose as cryoprotectants, and then stored in liquid nitrogen for more than 140 days. Thirty nude mice were assigned to each immersion-time group. Testicular fragments were transplanted under the back skin of castrated mice immediately after warming and removal of the cryoprotectants. Blood and testicular grafts were then recovered from the recipient mice on days 60, 120, 180 and 230−350 (day 0 =  grafting). Histological assessment of the testicular grafts and analyses of inhibin and testosterone production revealed no significant differences between the two immersion-time groups, indicating equal growth activity of the cryopreserved tissues. A single sperm obtained from a mouse in each group on day 230−350 was injected into an in vitro-matured porcine oocyte, and then the ICSI oocytes were transferred to the oviducts of estrus-synchronized recipient gilts. One out of 4 gilts that had received oocytes fertilized using sperm from the 10-min immersion group delivered 2 live piglets, and one of another 4 gilts from the 20-min group delivered 4 live piglets. Thus, we have successfully generated porcine offspring utilizing sperm from immature testicular tissues after cryopreservation and transplantation into nude mice. The present model using pigs will be applicable to many large animals, since pigs are phylogenetically distant from the murine recipients.     

Comment identifier le spermatozoïde vivant?

Testicular sperm extraction (TESE) has been used to retrieve spermatozoa in patients with secretory azoospermia for intracytoplasmic sperm injection (ICSI). However, testicular spermatozoa have poor motility that significantly decreases after cryopreservation and thawing. The major difficulty with testicular spermatozoa is to distinguish between living and dead spermatozoa, as most spermatozoa are immotile. The aim of this study was firstly to report the various methods used to explore spermatozoa vitality. Most tests assess the functional and structural integrity of the sperm membrane, such as staining methods and hypo-osmotic swelling test (HOS-test). We then evaluates the potential of pentoxifylline (PTX), a phosphodiesterase inhibitor of the methylxanthine group, to improve the distinction between living and dead immotile testicular spermatozoa by increasing the number of post-thawed motile spermatozoa. We also analysed the results of 100 ICSI cycles performed with frozen-thawed testicular (n=72) and epididymal (n=28) spermatozoa treated with 3.5 mM PTX. To test the effect of PTX on motility, 14 samples of frozen-thawed testicular spermatozoa from eight patients with secretory azoospermia and six patients with excretory azoospermia were divided into three equal samples: one sample treated with 3.5 mM PTX, one sample initially migrated on two-layer Percoll gradient and then divided into two aliquots (one treated with 3.5 mM PTX, one without treatment), and the last sample without migration and without PTX treatment. The number of motile spermatozoa was evaluated in 10 μL of each sample with an inverted microscope at 15, 30, 60, 120 minutes and 24 hours. We also compared the outcome of ICSI in 100 cycles using frozen-thawed epididymal or testicular spermatozoa between secretory and excretory patients. PTX significantly increased the number of motile frozen-thawed testicular spermatozoa in secretory and excretory azoospermia. In excretory azoospermia, the number of motile spermatozoa was further increased when PTX was associated with migration on Percoll gradient, while PTX alone gave the best results in secretory azoospermia. Fertilization and pregnancy rates as well as embryo quality and division stages were comparable in the two groups. By increasing the number of motile frozen-thawed testicular spermatozoa, PTX improves the selection of living spermatozoa.     

Cryopreservation of Bufotes viridis embryos by vitrification

Loss of biodiversity among amphibians is a current concern. Our hypothesis is that the embryos of amphibian species at risk of extinction could be cryopreserved by vitrification, using methods which have proved successful with fish oocyte. To test this hypothesis, samples of four cryoprotectants - methanol (MeOH), dimethyl sulphoxide (Me2SO), propylene glycol (PG) and polyethylene glycol (PEG), some singly, some in combination, were plunged in liquid nitrogen for 5 min to find the best solution for vitrification. To find the least toxic of these solutions, blastulae and stage G17 embryos of Bufotes Viridis, a typical amphibian, were exposed to solutions at different concentrations (0.5–10 M) for different lengths of time (15–30 min), with and without their normal protective jelly coats. In each case the number of survivors, which reached stage G25 was counted. Finally a series of embryos was vitrified in liquid nitrogen using the most efficient and least toxic cryoprotectants.Propylene glycol had the best vitrification characteristics, but MeOH vitrified at higher concentrations. The optimum regime, with the least toxic ctyoprotectants, consisted of 1M Me2SO for 15 min and a combination of 15% PEG_(w/v) + 3M PG + 2M Me2SO for 3 min, with the jelly coat intact, followed by vitrification. This gave a survival percentage of 87.6% immediately after vitrification. Methods designed for cryopreservation of fish embryos make a good starting point for cryopreservation of the embryos of amphibian.     

Long-term (24h) cooling of ovarian fragments in the presence of permeable cryoprotectants prior to freezing: Two unsuccesful IVF-cycles and spontaneous pregnancy with baby born after re-transplantation

Cancer is the second major cause of death in the world. The problem of post-cancer infertility plays a significant role, because chemotherapy can be gonadotoxic. Cryopreservation of ovarian tissue before cancer therapy with re-implantation after convalescence is the potential key solution to this problem. The aim of this study was to test the viability of cryopreserved human ovarian cortex after long-term cooling in culture medium composed of permeable cryoprotectants. Ovarian fragments from sixteen patients were randomly divided into two groups. After the operation, tissue pieces assigned to both groups were cooled to 5 °C for 22–24 h, frozen and thawed. Group 1 pieces (n = 32) were cooled before cryopreservation in the standard culture medium, and Group 2 pieces (n = 32) were cooled in the freezing medium (culture medium+6% ethylene glycol+6% dimethyl sulfoxide+0.15 M sucrose). Freezing was performed in standard 5 ml cryo-vials with ice formation at −9 °C, cooling from −9 to −34 °C at a rate of −0.3 °C/min and plunging at −34 °C into liquid nitrogen. After thawing in a 100 °C (boiling) water bath, the removal of cryoprotectants was performed in 0.5 M sucrose with 20 min exposure in sucrose and 30 min stepping rehydration. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development of follicles (histology). Six months after the autotransplantation, oocytes from the twenty-seven-year old, hormonally stimulated patient were retrieved and fertilized with her partner sperm through the intracytoplasmic spermatozoa injection (ICSI). For groups 1 and 2, 93.5 ± 1.9% and 96.4 ± 2.0% of the preantral follicles, respectively, were morphologically normal (P > 0.1) (with a tendency toward increasing in quality in Group 2). Six months after the auto-transplantation, two ICSI cycles resulted in the gathering and transplantation of high quality embryos, but no pregnancy had been established. Thirteen months after the auto-transplantation, the patient became spontaneously pregnant and delivered a healthy baby girl at term. Long-term (24 h) cooling of ovarian tissue to 5 °C before cryopreservation in the presence of permeable cryoprotectants simplifies the protocol of cryopreservation and has a tendency of increasing of the cells viability after thawing.     

Comparison among different cryoprotectants for cryopreservation of epididymal sperm from agouti (Dasyprocta leporina)

We verify the effects of different cryoprotectants on the cryopreservation of agouti (Dasyprocta leporina) epididymal sperm. We used 16 pairs of testes–epididymis complexes of sexually mature animals. We immediately evaluated epididymal sperm obtained by retrograde flushing for concentration, motility, vigor, viability, osmotic response, and morphology. Samples were extended in a coconut water extender plus 20% egg yolk, containing glycerol, ethylene glycol, dimethylsulfoxide – DMSO, or dimethylformamide. Finally, samples were stored in 0.25 mL straws, frozen in liquid nitrogen, and thawed after one week, being reevaluated and assessed for membrane integrity using fluorescent probes. The higher values for postthawing sperm motility, vigor, and membrane integrity were achieved by the usage of glycerol, when compared to ethylene glycol and dimethylformamide (P < 0.05); however, no differences were found between glycerol and DMSO (P > 0.05). All cryoprotectants provided a similar effect on the preservation of sperm morphology, osmotic response, and viability (P > 0.05). Therefore, here onwards, there was testing of glycerol and DMSO at 3 and 6% concentrations using the same freezing–thawing protocol reported previously. As the main result, DMSO at 6% concentration provided a decrease in sperm parameters, as well as in the chromatin integrity and in the binding capability of sperm. In conclusion, glycerol 3 or 6% and DMSO 3% can be used as alternative cryoprotectants for agouti epididymal sperm cryopreservation.     

Post-coital sperm recovery and cryopreservation in the Sumatran rhinoceros (Dicerorhinus sumatrensis) and application to gamete rescue in the African black rhinoceros (Diceros bicornis)

Sumatran rhinoceros (Dicerorhinus sumatrensis) sperm samples were collected from a post-copulatory female and characterized to determine their potential for sperm preservation and future use in artificial insemination. Five samples of acceptable quality from one male were used to compare the effect of two cryoprotectants (glycerol and dimethyl sulfoxide (DMSO)) and two post-thaw protocols (untreated and glass wool column) on sperm quality. The percentage of motile spermatozoa, sperm motility index (0-100) and sperm morphology were evaluated subjectively, and viability and acrosomal status were assessed using fluorescent markers. Evaluations of frozen-thawed spermatozoa were performed over a 6 h incubation interval. Post-coital semen samples (n = 5; 104.0 +/- 9.1 ml; 2.5 +/- 0.8 x 10(9) total spermatozoa; mean +/- SEM) exhibited a sperm motility index of 56.7 +/- 3.3, and contained 40.2 +/- 6.3%, 72.0 +/- 3.2% and 79.8 +/- 6.5% normal, viable and acrosome-intact spermatozoa, respectively. Glycerol and DMSO were equally effective as cryoprotectants and, regardless of post-thaw protocol, samples retained greater than 80% of all pre-freeze characteristic values. Processing semen samples through glass wool yielded higher quality samples, but only half the total number of motile spermatozoa compared with untreated samples. High values for pre-freeze sperm characteristics were also maintained after cryopreservation of epididymal spermatozoa from one black rhinoceros (Diceros bicornis) using the same protocol. In summary, Sumatran rhinoceros spermatozoa of moderate quality can be collected from post-copulatory females. Rhinoceros sperm samples show only slight reductions in quality after cryopreservation and thawing and have potential for use in artificial insemination.     

Maintenance of fertility in cryopreserved Indian gerbil (Tatera indica) spermatozoa

This study is the first attempt at sperm cryopreservation, as well as a further examination of frozen sperm fertility by the hamster test, applied to the maintenance of an Indian gerbil (Tatera indica) colony, which is a newly developing experimental animal.The osmotic tolerance of the spermatozoa was initially investigated by subjection to hypertonicity, up to 620 mOsm/kg, for 5 min at room temperature prior to freezing. Although the percentage of total motile sperm was not affected, that of progressive motile spermatozoa began to drop at 400 mOsm/kg, and a significant decrease was observed at 620 mOsm/kg (p < 0.01). According to these results, the osmolality of the solutions for the freezing experiment, in which 6–22% raffinose was present, was fixed at approximately 400 mOsm/kg. Sperm, suspended in a plastic straw, were frozen in liquid nitrogen vapor for 5 min, followed by immersion in liquid nitrogen. Motile sperm were recovered from all freezing conditions, and high survival was obtained when sperm were frozen in the presence of 14% and 18% raffinose, with a normalized motility higher than 40%. Fertility of cryopreserved Indian gerbil sperm was examined by the zona-free hamster test. Thawed sperm adhered to 88% of the zona-free hamster oocyte surface, and some oocytes were penetrated and exhibited swollen sperm heads or male pronuclei, which we used to define fertilization. Although the fertilization rate of cryopreserved sperm to zona-free hamster eggs was significantly lower than that of fresh sperm (6% vs. 30%, p < 0.01), we demonstrated that thawed Indian gerbil spermatozoa have the ability to maintain their fertility.     

Rhesus monkey sperm cryopreservation with TEST-yolk extender in the absence of permeable cryoprotectant

Recently, there has been increased interest in ultra-rapid freezing with mammalian spermatozoa, especially for vitrification in the absence of cryoprotectants. Sperm cryopreservation in non-human primates has been successful, but the use of frozen-thawed sperm in standard artificial insemination (AI) remains difficult, and removal of permeable cryoprotectant may offer opportunities for increased AI success. The present study intended to explore the possibility of freezing rhesus monkey sperm in the absence of permeable cryoprotectants. Specifically, we evaluated various factors such as presence or absence of egg yolk, the percentage of egg yolk in the extenders, and the effect of cooling and thawing rate on the success of freezing without permeable cryoprotectants. Findings revealed that freezing with TEST in the absence of egg yolk offers little protection (<15% post-thaw motility). Egg yolk of 40% or more in TEST resulted in decreased motility, while egg yolk in the range of 20-30% yielded the most motile sperm. Cooling at a slow rate (29 °C/min) reduced post-thaw motility significantly for samples frozen with TEST-yolk alone, but had no effect for controls in the presence of glycerol. Similarly, slow thawing in room temperature air is detrimental for freezing without permeable cryoprotectant (<2% motility). In addition to motility, the ability of sperm to capacitate based on an increase in intracellular calcium levels upon activation with cAMP and caffeine suggested no difference between fresh and frozen-thawed motile sperm, regardless of treatment. In summary, the present study demonstrates that ejaculated and epididymal sperm from rhesus monkeys can be cryopreserved with TEST-yolk (20%) in the absence of permeable cryoprotectant when samples were loaded in a standard 0.25-mL straw, cooled rapidly in liquid nitrogen vapor at 220 °C/min, and thawed rapidly in a 37 °C water bath. This study also represents the first success of freezing without permeable cryoprotectant in non-human primates.     

Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling

This report describes the feasibility of using vitrification for fish sperm. Vitrification can be used to preserve samples in the field and offers an alternative to conventional cryopreservation, although it has not been systematically studied for sperm of aquatic species. The overall goal of the project was to develop streamlined protocols that could be integrated into a standardized approach for vitrification of aquatic species germplasm. The objectives of the present study in channel catfish (Ictalurus punctatus) were to: (1) evaluate the acute toxicity of 5%, 10%, 20% and 30% methanol, N,N-dimethyl acetamide, dimethyl sulfoxide, 1,2-propanediol, and methyl glycol; (2) evaluate a range of devices commonly used for cryopreservation and vitrification of mammalian sperm; (3) compare vitrification with and without cryoprotectants; (4) evaluate the post-thaw membrane integrity of sperm vitrified in different cryoprotectant solutions, and (5) evaluate the ability of vitrified sperm to fertilize eggs. Cryoprotectant concentrations of higher than 20% were found to be toxic to sperm. Methanol and methyl glycol were the least toxic at a concentration of 20% with an exposure time of less than 5 min. We evaluated a method reported for human sperm, using small volumes in loops (15 μl) or cut standard straws (20 μl) with and without cryoprotectants plunged into liquid nitrogen. Cryoprotectant-free vitrification using loops did not yield fertilization (assessed by neurulation), and the fertilization rates observed in two trials using the cut standard straws were low (∼2%). In general, fertilization values for vitrification experiments were low and the use of low concentrations of cryoprotectants yielded lower fertilization (<10%) than the use of vitrification solutions containing high cryoprotectant concentrations (as high as 25%). The highest neurulation obtained was from a mixture of three cryoprotectants (20% methanol + 10% methyl glycol + 10% propanediol) with a single-step addition. This was reflected in the flow cytometry data from which the highest membrane integrity using loops was for 20% methanol + 10% methyl glycol + 10% propanediol (∼50%). We report the first successful sperm vitrification in fish and production of offspring from vitrified sperm in channel catfish. Although the fertilization values were low, at present this technique could nevertheless be used to reconstitute lines (especially in small aquarium fishes), but it would require improvement and scaling up before being useful as a production method for large-bodied fishes such as catfish.     

Japanese flounder (Paralichthys olivaceus) embryos are difficult to cryopreserve by vitrification

The first successful cryopreservation of fish embryos was reported in the Japanese flounder by vitrification [Chen and Tian, Theriogenology, 63, 1207-1219, 2005]. Since very high concentrations of cryoprotectants are needed for vitrification and fish embryos have a large volume, Japanese flounder embryos must have low sensitivity to cryoprotectant toxicity and high permeability to water and cryoprotectants. So, we investigated the sensitivity and the permeability of Japanese flounder embryos. In addition, we assessed the survival of flounder embryos after vitrification with solutions containing methanol and propylene glycol, following Chen and Tian's report. The embryos were relatively insensitive to the toxicity of individual cryoprotectants at lower concentrations, especially methanol and propylene glycol as their report. Although their permeability to water and cryoprotectants could not be measured from volume changes in cryoprotectant solutions, the embryos appeared to be permeable to methanol but less permeable to DMSO, ethylene glycol, and propylene glycol. Although vitrification solutions containing methanol and propylene glycol, which were used in Chen and Tian's report, were toxic to embryos, a small proportion of embryos did survived. However, when vitrified with the vitrification solutions, no embryos survived after warming. The embryos became opaque during cooling with liquid nitrogen, indicating the formation of intracellular ice during cooling. When embryos had been kept in vitrification solutions for 60 min after being treated with the vitrification solution, some remained transparent during cooling, but became opaque during warming. This suggests that dehydration and/or permeation by cryoprotectants were insufficient for vitrification of the embryos even after they had been over-treated with the vitrification solutions. Thus, Chen and Tian's cryopreservation method lacks general application to Japanese flounder embryos.     

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.     

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.     

Immature cat oocyte vitrification in open pulled straws (OPSs) using a cryoprotectant mixture

Cryopreservation of gametes is an important tool in assisted reproduction programs to optimise captive breeding programmes of selected felid species. In this study the vitrification was evaluated in order to cryopreserve the immature domestic cat oocytes by assessing the survival of cumulus-oocyte complexes (COC), and the development competence after IVM and IVF by fresh cat epididymal sperms. From a total of 892 COC obtained from queens after ovariectomy were divided into two groups: Experiment 1 for viability evaluation (150 vitrified and 100 control COC) and Experiment 2 for assessing the developmental competence (414 vitrified and 228 control COC). The viability was evaluated by double staining with carboxyfluorescein and Trypan blue, while the developmental competence was evaluated by in vitro maturation (IVM), in vitro fertilisation (IVF) by fresh epididymal spermatozoa and in vitro culture (IVC). The vitrification was performed in OPS into sucrose medium (1 M sucrose in HSOF + 6% BSA) containing dimethyl sulfoxide (DMSO) (16.5% final concentration) and ethylene glycol (EG) (16.5% final concentration) as cryoprotectants. Percentage of non-viable COC was significantly higher in Experimental 1 vs Control 1 (11% vs 54.5%; P < 0.01), while cleavage rate were significantly lower for vitrified oocytes (Experimental 2) than control 2 (18.6% vs 48.2%; P < 0.01). Blastocyst rate on day 8 was higher for control oocytes than vitrified counterparts (4.3% vs 20.6% P < 0.01). This vitrification protocol ensured a development to blastocyst stage and it is the first report of development of vitrified GV COC.