DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development.

Sperm freeze-drying is a revolutionary technique, which has been gaining prominence in recent years. The first related significant result was Wakayama and Yanagimachi's demonstration in 1998 of the birth of healthy mouse offspring by Intracytoplasmic Sperm Injection (ICSI), using epididymal freeze-dried spermatozoa. Mouse, rat, and hamster models were the first small mammals born from lyophilized epididymal spermatozoa, whereas most other studies in this field used ejaculated spermatozoa. In this work, we applied this technique to ram epididymal spermatozoa, checking the correlation between DNA integrity and embryo development following ICSI. To do this, epididymal sperm from four rams was lyophilized in a trehalose, glucose, KCl, HEPES, and Trolox media. To evaluate DNA damage and fragmentation after rehydration, samples were processed for Sperm Chromatin Dispersion test (SCD), Two-Tailed Comet Assay, and were used for ICSI. Ram #2 had a higher rate of spermatozoa with intact DNA compared with rams #1, #3, and #4 (28% vs. 3.8%, 2.8%, and 5%, respectively) and the lowest rate of Single-Strand Breaks (SSBs) (70% vs. 95.9%, 92.6%, and 93% respectively). Ram #3 had a higher level of Double-Strand Breaks (DSBs) compared to Ram #1 (4.6% vs. 0.33%, respectively). Embryo development to the blastocyst stage following ICSI was only reached from rams whose sperm had higher level of intact DNA - Rams #2 and #4 (6%, 5/147 and 6.3%, 4/64, respectively). Definitively, the impact of sperm DNA damage on embryonic development depends on the balance between sperm DNA fragmentation extent, fragmentation type (SSBs or DSBs), and the oocyte's repair capacity.

Rewinding the process of mammalian extinction.

With only three living individuals left on this planet, the northern white rhinoceros (Ceratotherium simum cottoni) could be considered doomed for extinction. It might still be possible, however, to rescue the (sub)species by combining novel stem cell and assisted reproductive technologies. To discuss the various practical options available to us, we convened a multidisciplinary meeting under the name "Conservation by Cellular Technologies." The outcome of this meeting and the proposed road map that, if successfully implemented, would ultimately lead to a self-sustaining population of an extremely endangered species are outlined here. The ideas discussed here, while centered on the northern white rhinoceros, are equally applicable, after proper adjustments, to other mammals on the brink of extinction. Through implementation of these ideas we hope to establish the foundation for reversal of some of the effects of what has been termed the sixth mass extinction event in the history of Earth, and the first anthropogenic one. Zoo Biol. 35:280-292, 2016. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc.

Sperm rescue in wild African elephants.

This study investigated ways of improving the usefulness of ~1700mL of poor-quality frozen semen collected from wild African elephant (Loxodonta africana) bulls. Ten semen samples from six bulls, frozen with 5% glycerol in Berliner cryomedium, with or without prior removal of the seminal plasma by centrifugation, were tested. All samples were subjected to the following density-gradient centrifugation treatments: no centrifugation (control), sham centrifugation, Percoll, OptiPrep, Isolate and PureSperm. Sample evaluation included motility, concentration, viability, acrosome integrity and normal morphology after thawing and after gradient centrifugation. Motility was also evaluated 3h after thawing. While all treatments were similar to the Control in acrosome integrity and normal morphology, significant differences were noted in concentration, viability and motility. Samples treated by Percoll showed the best motility, which was maintained unchanged over 3h of incubation (37°C). Correlations between manual and automated evaluations of concentration were high (cytometer; rho=0.92), but were lower for viability (cytometer; rho=0.57) and motility (computer-aided sperm analysis; rho=0.66). By performing density centrifugation, the quality of these sperm samples may be improved to a level suitable for artificial insemination in elephants. Although a sizeable proportion of cells are lost in the process, combining samples may still allow for multiple inseminations.

CRYOPRESERVATION OF ONAGER (EQUUS HEMIONUS ONAGER) EPIDIDYMAL SPERMATOZOA.

Genetic diversity is a primary component of adaptive evolution, and its loss or reduction can decrease the long-term survival probability of populations. Utilization of cryopreserved semen may be considered a perfect tool to improve genetic diversity, reduce inbreeding, and avoid animal translocation for breeding. The present study aimed at finding a reliable epididymal sperm freezing protocol for the critically endangered onager (Equus hemionus onager). Six testicles from three animals were processed postmortem. The effects of two transportation temperatures (22°C and 4°C; testicles submerged in saline), two cryopreservation techniques (conventional liquid nitrogen vapor freezing in straws and directional freezing in 8-ml HollowTubes(TM)), and two postthaw incubation temperatures (22°C and 37°C; evaluated after 0.5, 1, 2, and 3 hr) were tested in a 2×2×2 experimental design. Sperm samples were evaluated for motility, viability, acrosome integrity, and sperm morphology. The resulting optimal freezing protocol includes transportation of testicles at 4°C, cryopreservation by directional freezing, and, if needed, postthaw incubation at 22°C. With this combination of transportation temperature and cryopreservation technique, the authors obtained the following postthaw values normalized to prefreezing values: 60.3±8.8% motility, 60.7±13.3% viability, 75.3±9.5% acrosome integrity, and 94.7±2.9% normal morphology (excluding defects due to the epididymal origin of the sperm). After incubation at 22°C, motility values for the above combination were 40±5.7%, 30.3±5.2%, 28.3±4.4%, and 16.7±4.4% for 0.5, 1, 2, and 3 hr, respectively. In conclusion, with this protocol, good quality semen can be stored for future use in artificial inseminations when and where needed.

Cloning the mammoth: a complicated task or just a dream?

Recently there has been growing interest in applying the most advanced embryological tools, particularly cloning, to bring extinct species back to life, with a particular focus on the woolly mammoth (Mammuthus primigenius). Mammoth's bodies found in the permafrost are relatively well preserved, with identifiable nuclei in their tissues. The purpose of this chapter is to review the literature published on the topic, and to present the strategies potentially suitable for a mammoth cloning project, with a frank assessment of their feasibility and the ethical issues involved.

Directional freezing of spermatozoa and embryos.

Directional freezing is based on a simple thermodynamic principle whereby the sample is moved through a predetermined temperature gradient at a velocity that determines the cooling rate. Directional freezing permits a precise and uniform cooling rate in small- and large-volume samples. It avoids supercooling and reduces mechanical damage caused by crystallisation. Directional solidification was used to date for slow and rapid freezing, as well as for vitrification of oocytes and embryos by means of the minimum drop size technique: small drops are placed on a microscope slide that is moved at high velocity from the hot base to the cold base. Sperm samples from a wide range of domestic and wild animals were successfully cryopreserved using the directional freezing method. The bovine sexed semen industry may benefit from the increased survival of spermatozoa after directional freezing.

Reviving vacuum-dried encapsulated ram spermatozoa via ICSI after 2 years of storage.

Introduction: Freeze-drying techniques give alternative preservation mammalian spermatozoa without liquid nitrogen. However, most of the work has been conducted in the laboratory mouse, while little information has been gathered on large animals that could also benefit from this kind of storage. Methods: This work adapted a technique known as vacuum-drying encapsulation (VDE), originally developed for nucleic acid conservation in anhydrous state, to ram spermatozoa, and compared it to canonical lyophilization (FD), testing long-term storage at room temperature (RT) and 4°C. Results and discussion: The results demonstrated better structural stability, namely lipid composition and DNA integrity, in VDE spermatozoa than FD ones, with outcomes at RT storage comparable to 4°C. Likewise, in VDE the embryonic development was higher than in FD samples (12.8% vs. 8.7%, p < 0.001, respectively). Our findings indicated that in large mammals, it is important to consider dehydration-related changes in sperm polyunsaturated fatty acids coupled with DNA alterations, given their crucial role in embryonic development.

Medetomidine-ketamine-isoflurane anaesthesia in pygmy hippopotami (Choeropsis liberiensis)--a case series.

HISTORY: Medical knowledge of pygmy hippopotami is limited. Anaesthesia has been considered a challenge because of the anatomy, semi-aquatic life style and aggressive behaviour. Polycystic kidney disease (PKD) has been described and can contribute to active kidney disease potentially affecting anaesthesia. PHYSICAL EXAMINATION AND MANAGEMENT: Fourteen pygmy hippopotami were anaesthetized for general health assessment and reproductive procedures. Animals (estimated bodyweight 250 kg) were darted intramuscularly with 0.08 mg kg(-1) medetomidine and 1.2 mg kg(-1) ketamine. After endotracheal intubation, anaesthesia was maintained with isoflurane delivered either by circle system (100% oxygen) or by Triservice apparatus (air or air/oxygen admixture). Heart rate (HR) respiratory rate (f(R) ), oxygen saturation (SpO(2)) and end tidal CO(2) were recorded at 5-minute intervals. Atipamezole was administered intramuscularly (0.4 mg kg(-1)) at the end of the procedure. Statistical analysis was performed using anova (p < 0.05). Most animals rapidly became recumbent although five hippopotami needed additional drugs to assure acceptable immobilization. There were no statistical differences in mean HR between animals with or without PKD (PKD: 34 ± 8 beats minutes(-1); no PKD: 33 ± 6 beats minutes(-1)), f(R) (PKD: 15 ± 7 breaths minutes(-1); no PKD; 12 ± 5 breaths minutes(-1)) and end tidal CO(2) (PKD: 7.1 ± 1.3 kPa; no PKD: 7.8 ± 1.4 kPa). SpO(2) was higher in animals receiving 100% oxygen or air with oxygen (92 ± 8% and 91 ± 9% respectively) compared with animals receiving air only (77 ± 5%) (p = 0.003). Recovery was uneventful after atipamezole administration. FOLLOW-UP: There were no apparent adverse effects after anaesthesia during a 24-hour follow-up period. DISCUSSION AND CONCLUSIONS: Medetomidine-ketamine-isoflurane induced satisfactory anaesthesia in this species. Incremental induction doses were related to remote injection and the animals' thick skin. There were no differences in anaesthetic parameters in animals with or without PKD. Supplemental oxygen should be mandatory during anaesthesia in this species.

Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification.

Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans, cows, and mice. Two basic cryopreservation techniques rule the field--controlled-rate freezing, the first to be developed, and vitrification, which, in recent years, has gained a foothold. While much progress has been achieved in human medicine, the cattle industry, and in laboratory animals, this is far from being the case for most other mammals and even less so for other vertebrates. The major strides and obstacles in human and other vertebrate oocyte and embryo cryopreservation will be reviewed here.

Reproductive biotechnology and critically endangered species: Merging in vitro gametogenesis with inner cell mass transfer.

A fifth of mammalian species face the risk of extinction. A variety of stresses, and lack of sufficient resources and political endorsement, mean thousands of further extinctions in the coming years. Once a species has declined to a mere few individuals, in situ efforts seem insufficient to prevent its extinction. Here we propose a roadmap to overcome some of the current roadblocks and facilitate rejuvenation of such critically endangered species. We suggest combining two advanced assisted reproductive technologies to accomplish this task. The first is the generation of gametes from induced pluripotent stem cells, already demonstrated in mice. The second is to 'trick' the immunological system of abundant species' surrogate mothers into believing it carries conceptus of its own species. This can be achieved by transferring the inner cell mass (ICM) of the endangered species into a trophoblastic vesicle derived from the foster mother's species. Such synthesis of reproductive biotechnologies, in association with in situ habitat conservation and societal changes, holds the potential to restore diversity and accelerate the production of animals in the most endangered species on Earth.

Dry biobanking as a conservation tool in the Anthropocene.

Species are going extinct at an alarming rate, termed by some as the sixth mass extinction event in the history of Earth. Many are the causes for this but in the end, all converge to one entity - humans. Since we are the cause, we also hold the key to making the change. Any change, however, will take time, and for some species this could be too long. While working on possible solutions, we also have the responsibility to buy time for those species on the verge of extinction. Genome resource banks, in the form of cryobanks, where samples are maintained under liquid nitrogen, are already in existence but they come with a host of drawbacks. Biomimicry - innovation inspired by Nature, has been a huge source for ideas. Searching methods that Nature utilizes to preserve biological systems for extended periods of time, we realize that drying rather than freezing is the method of choice. We thus argue here in favor of preserving at least part of the samples from critically endangered species in dry biobanks, a much safer, cost-effective, biobanking approach.

Do physical forces contribute to cryodamage?

To achieve the ultimate goal of both cryosurgery and cryopreservation, a thorough understanding of the processes responsible for cell and tissue damage is desired. The general belief is that cells are damaged primarily due to osmotic effects at slow cooling rates and intracellular ice formation at high cooling rates, together termed the "two factor theory." The present study deals with a third, largely ignored component--mechanical damage. Using pooled bull sperm cells as a model and directional freezing in large volumes, samples were frozen in the presence or absence of glass balls of three different diameters: 70-110, 250-500, and 1,000-1,250 microm, as a means of altering the surface area with which the cells come in contact. Post-thaw evaluation included motility at 0 h and after 3 h at 37 degrees C, viability, acrosome integrity, and hypoosmotic swelling test. Interactions among glass balls, sperm cells, and ice crystals were observed by directional freezing cryomicroscopy. Intra-container pressure in relation to volume was also evaluated. The series of studies presented here indicate that the higher the surface area with which the cells come in contact, the greater the damage, possibly because the cells are squeezed between the ice crystals and the surface. We further demonstrate that with a decrease in volume, and thus increase in surface area-to-volume ratio, the intra-container pressure during freezing increases. It is suggested that large volume freezing, given that heat dissipation is solved, will inflict less cryodamage to the cells than the current practice of small volume freezing.

Exploring dry storage as an alternative biobanking strategy inspired by Nature.

Biobanking is a rapidly growing industry, covering diverse fields such as human medicine, farm animal production, laboratory animals record keeping, and wildlife conservation. Presently, biobanking is done almost exclusively by cryopreservation, followed by maintenance of the samples under liquid nitrogen. Cryopreservation has satisfactory efficiency but it comes with a host of problems, and the process is highly species-specific. Like in many other walks of life, we turn to Nature in search for better alternatives. Nature opted for controlled drying rather than water preservation via freezing when long-term preservation is desired, a strategy known as 'anhydrobiosis'. To achieve reversible drying, anhydrobiotic organisms utilise an assortment of protective materials, including disaccharides, late embryogenesis abundant proteins, anhydrin, heat shock protein, and more. Once dry, desiccation-tolerant organisms can survive extended periods of time and be resistant to extreme environmental stressors. Over the past 70 years researchers attempted applying this idea to preserve desiccation-sensitive mammalian cells in the dry form. At present dried cells mostly do not resume biological activity upon rehydration. The DNA, however, is often well preserved to allow utilisation in advanced reproductive techniques. Spermatozoa are by far the most commonly dried cell type, primarily from mice and bulls. A number of drying approaches have been applied, with freeze-drying taking the lead. To date offspring have been produced from dried spermatozoa in mouse, rat, hamster, rabbit, and horse. No offspring were produced from dried somatic cells. Desiccation experiences a sharp increase in interest and research output in recent years. Presented here is an overview of dry preservation, its possible applications, the open questions the field is still facing, and some suggested directions for the future.

Differential detection of tuberculous and non-tuberculous mycobacteria by qPCR in lavage fluids of tuberculosis-suspicious white rhinoceros.

Tuberculosis (TB) occurs in a wide range of mammalian species and thus poses a health risk to humans living or working in close proximity with TB infected animals. Despite a high incidence of M. bovis infections in domestic or wildlife species tuberculosis infections in rhinoceros have so far been very limited. Over the past 53 years, tuberculosis of the respiratory tract has been confirmed in just 22 rhinoceros, most of those infected not by M. bovis but M. tuberculosis. However, because of the zoonotic risk TB testing is recommended or becomes even mandatory in endangered species. The dilemma in rhinoceros and many other wildlife species; non-validated tests are highly inconsistent in their ability to identify TB infection. Current lack of TB diagnostics may result in TB positive rhinoceros living with the infection, transmitting it to those around them or in euthanasia of animals found unconfirmed at necropsy. This is an unacceptable diagnostic status considering that some species are critically endangered and therefore should not be euthanized in order to confirm suspicion of disease. To overcome this shortcoming we used bronchoscopy to detect mycobacteria in respiratory fluids of TB suspicious rhinoceros. Fluids from seven, TB suspicious white rhinoceros were harvested during 21 bronchoscopies. Our new approach: In addition to bacterial culture a dual quantitative PCR system tested for the general presence of DNA from NTM and more specifically for DNA from MTC. Both, bacterial culture and qPCR were negative for MTC in respiratory fluids of all rhinoceros (7/7). At the same time, respiratory fluids from six rhinoceros tested positive for the presence of NTM or other closely related bacteria (6/7). M. tuberculosis was found only once in an oesophageal aspirate. The high incidence of mycobacterial DNA in the respiratory tract suggests that white rhinoceros, as strict grazers, are immensely exposed to environmental bacteria of this genus. Presence of NTM in the respiratory or intestinal system could possibly cause false positive results in intradermal tests. A wider use of bronchoalveolar lavage is warranted to further elucidate immunologic response to NTM and exposure to, incidence and prevalence of MTC infections in rhinoceros.

Evaluation of lidocaine treatment and risk factors for death associated with gastric dilatation and volvulus in dogs: 112 cases (1997-2005).

OBJECTIVE: To determine clinical features, outcome, risk factors for death, and efficacy of IV administration of lidocaine as a prophylactic treatment for ischemic reperfusion injury in gastric dilatation and volvulus (GDV) in dogs. DESIGN: Retrospective case series. ANIMALS: 112 dogs with GDV. PROCEDURES: Data pertaining to breed; time lag to admission; clinical, clinicopathologic, and surgical findings; lidocaine treatment; and postoperative complications were assessed for association with outcome. RESULTS: German Shepherd Dogs (28.6%) and Great Danes (17%) were significantly over-represented. Risk factors for death included time lag (> or = 5 hours vs < 5 hours) from onset of clinical signs to admission (46.0% vs 11.3%), rectal temperature (< or = 38 degrees C vs > 38 degrees C [< 100.4 degrees F vs > 100.4 degrees F]) at admission (40.0% vs 14.9%), presence or absence of ARF (67.0% vs 23.3%), presence or absence of suspected gastric wall necrosis (59.3% vs 16.0%), and untreated gastric wall necrosis, compared with treated gastric wall necrosis (100% vs 47.6%). Overall mortality rate was 26.8%; no significant differences were detected in mortality rate or postoperative complications between dogs that received lidocaine IV prior to surgical intervention (52.0%) and dogs that did not (48.0%). Mean +/- SD hospitalization time was longer in the lidocaine treatment group (3.5 +/- 1.9 days vs 2.5 +/- 1.4 days). CONCLUSIONS AND CLINICAL RELEVANCE: Presence of the identified risk factors should warrant aggressive treatment. Lidocaine treatment was not associated with mortality rate or postoperative complications, but was associated with prolonged hospitalization time.

Follicular size predicts success in artificial insemination with frozen-thawed sperm in donkeys.

In asses, semen collection, cryopreservation, and artificial insemination (AI) with frozen-thawed semen have been scarcely described and success rate, particularly following AI, is reportedly low. In the absence of reliable protocols, assisted reproductive technologies cannot support the conservation efforts aimed at endangered wild ass species and domestic donkey breeds. Two experiments were conducted in this study. In experiment 1 we evaluated freezing Abyssinian donkey (N = 5, 4 ejaculates each) spermatozoa using three freezing extenders (Berliner Cryomedium + glycerol, BC+G; BotuCrio, BOTU; INRAFreeze, INRA) and two cryopreservation techniques (liquid nitrogen vapour, LNV; directional freezing, DF). Post-thaw evaluation indicated that BOTU and INRA were similar and both superior to BC+G (P ≤ 0.004 for all motility tests), and that DF was superior to LNV (P < 0.002 for all evaluation parameters). In experiment 2, relying on these results, we used Abyssinian donkey sperm frozen in BOTU and INRA by DF for AI (N = 20). Prior to AI, thawed samples were diluted in corresponding centrifugation media or autologous seminal fluids at 1:1 ratio. No difference was found between BOTU and INRA or between the addition of seminal fluids or media, all resulting in ~50% pregnancy, and no differences were noted between males (N = 4). The size of pre-ovulatory follicle was a significant (P = 0.001) predictor for AI success with 9/10 pregnancies occurring when follicular size ranged between 33.1-37.4 mm, no pregnancy when it was smaller, and only one when larger. A number of ass species face the risk of extinction. Knowledge gained in this study on the Abyssinian donkey can be customised and transferred to its closely related endangered species and breeds.

Post-mortem semen cryopreservation and characterization in two different endangered gazelle species (Gazella gazella and Gazella dorcas) and one subspecies (Gazella gazelle acaiae).

Both Gazella gazella and Gazella dorcas are endangered species with continually dwindling population size, yet basic knowledge on their spermatozoa is missing. Semen collected post-mortem (PM) from the cauda epididymis of five adult gazelles (three Gazella gazella gazella, one Gazella gazella acaiae and one G. dorcas) was cryopreserved using directional freezing of large volumes (8 mL) with egg-yolk-free extender. Sperm size measurements and SYBR-14/propodium iodide (PI) viability stain validation for use in gazelles were conducted. Post-thaw characterization included motility, viability, acrosome damage evaluation, computerized motility characterization and morphology and sperm motility index (SMI) was calculated. Extracted sperm motility was 71.67+/-11.67% (mean+/-S.E.M.). Post-thaw motility ranged between 15% and 63%, viability was 57.49+/-3.24%, intact acrosome was detected in 63.74+/-2.6% (median 64.8%, upper/lower quartiles 71.79%, 61.82%), and normal morphology ranged between 41% and 63%. Motility characterization showed two sub-groups-highly active and progressively motile spermatozoa with SMI of 62.75+/-0.38 and low activity and poorly progressive with SMI of 46.16+/-1.53. Our results indicate that PM preservation of gazelle spermatozoa with satisfactory post-thaw viability is possible and cryobanking is achievable.

Heat stroke in dogs: A retrospective study of 54 cases (1999-2004) and analysis of risk factors for death.

The medical records of 54 dogs presented to the Hebrew University Veterinary Teaching Hospital and diagnosed with heat stroke were retrospectively reviewed. Data abstracted included history, clinical and clinicopathological signs at admission, treatment, disease progression, and outcome. Exertional and environmental heat stroke were present in 63% (34 of 54) and 37% (20 of 54) of the dogs, respectively, and 78% (42 of 54) were examined between June and August. The mean temperature and heat discomfort index in the particular days of heat stroke were significantly increased (P < .001, P < .001, respectively) compared with their corresponding average daily values. In 27 dogs the body temperature was > or = 41 degrees C (105.8 degrees F). Belgian Malinois (15%, odds ratio [OR] = 24, 95% confidence interval [CI95%] 8.2-64.5), Golden and Labrador Retrievers (21%, OR = 2.08, CI95% 0.95-4.2), and brachycephalic breeds (25%, OR = 1.7, CI95%], 0.81-3.21) were overrepresented, whereas small breeds (<8 kg) were underrepresented (2%, OR = 0.08, CI95%, 0.002-0.48). Thrombocytopenia (45 of 54 dogs) and prolongation of the prothrombin (PT) and activated thromboplastin (aPTT) times (27 of 47 dogs) were recorded during hospitalization. Disseminated intravascular coagulation (P = .013) and acute renal failure (P = .008), diagnosed in 28 of 54 and 18 of 54 of the cases, respectively, were risk factors for death. The overall mortality rate was 50%. Hypoglycemia (<47 mg/dL, P = .003), prolonged PT (>18 seconds, P = .05), and aPTT (>30 sec, P < .001) at admission were associated with death. Serum creatinine >1.5 mg/dL (P = .003) after 24 hours, delayed admission (>90 minutes, P = .032), seizures (P = .02), and obesity (P = .04) were also risk factors for death. Heat stroke in dogs results in serious complications and high fatality rate despite appropriate treatment.

Directional freezing of sperm and associated derived technologies.

Directional freezing has now completed 30 years of development since it was first introduced to cryobiology. In the field of sperm cryopreservation, directional freezing has been shown to be advantageous over slow freezing for numerous domestic and wildlife species. In particular, it was shown that freezing of large volume is possible. Furthermore, double freezing of sperm and freezing of sex-sorted sperm are possible and became the routine in the sex sorted sperm industry. In wild animals, our labs and others showed that sperm from a wide range of terrestrial and aquatic species can be successfully cryopreserved using directional freezing. Finally, we will describe for the first time the successful freeze-drying of human sperm in an aseptic method. Using a device that produces clean liquid air, we froze human sperm in small droplets and then dried them in a bench top lyophilizer that was sterilized prior to use. More than 80% of DNA integrity was found after rehydration.