RESUMO
Vitrification of mammalian oocytes and embryos is typically a two-step procedure involving two solutions of increasing concentrations of cryoprotectants. In the present study, we report a simple vitrification protocol that uses low cryoprotectant concentration and a single medium (LCSM). This medium, along with the traditional high concentration two media (HCTM) protocol, was used to vitrify mouse oocytes, zygotes, and blastocysts using silica capillary, cryotop, cryolock, and 0.25 ml straws. Survival rates, two-cell rates, and blastocyst formation rates were compared for oocytes and zygotes vitrified using both protocols. Results show that the LCSM protocol was as good as or better than the traditional HCTM protocol for vitrifying mouse MII oocytes and zygotes using silica capillary, cryotop, and cryolock. On the other hand, for blastocysts, only silica capillary using LCSM had comparable results with the traditional HCTM protocol while cryolock and cryotop had significantly lower percentages of re-expanded and hatched blastocysts. Collapsing blastocysts prior to vitrification or longer duration for better cryoprotectant distribution in multicellular embryos may improve the outcome. In conclusion, the LCSM protocol, with one medium of much lower cryoprotectant concentrations and shorter equilibration time, reduces exposure to cryoprotectant toxicity while improves efficiency, consistency and reliability for mammalian oocyte and embryo preservation.
Assuntos
Criopreservação , Vitrificação , Animais , Blastocisto , Criopreservação/métodos , Crioprotetores/farmacologia , Camundongos , Oócitos , Reprodutibilidade dos TestesRESUMO
Cryopreservation of human spermatozoa is a commonly used technique in assisted reproduction, however freezing low concentrations of sperm while maintaining adequate post-thaw motility remains a challenge. In an effort to optimize post-thaw motility yields, low volumes of human sperm were frozen in polyimide-coated fused silica micro-capillaries using 0.065 M, 0.125 M, 0.25 M, or 0.5 M trehalose as the only cryoprotectant. Micro-capillaries were either initially incubated in liquid nitrogen vapor before plunging into liquid nitrogen, or directly plunged into liquid nitrogen. Post thaw sperm counts and motility were estimated. Spermatozoa that were initially incubated in liquid nitrogen vapor had greater post thaw motility than those plunged immediately into liquid nitrogen independent of trehalose concentration. The protective effect of 0.125 M d-glucose, 3-O-methyl-d-glucopyranose, trehalose, sucrose, raffinose, or stachyose were evaluated individually. Trehalose and sucrose were the most effective cryoprotectants, recovering 69.0% and 68.9% of initial sperm motility, respectively.
Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/fisiologia , 3-O-Metilglucose/farmacologia , Animais , Congelamento , Glucose/farmacologia , Humanos , Masculino , Oligossacarídeos/farmacologia , Rafinose/farmacologia , Sacarose/farmacologia , Trealose/farmacologiaRESUMO
The purpose of this study is to develop a mouse sperm preservation method based on evaporative drying. Mouse sperm were evaporatively dried and stored at 4°C and ambient temperature for 3 months to 2 years. Upon rehydration, a single sperm was injected into a mature oocyte to develop into a blastocyst after culture or a live birth after embryo transfer to a recipient female. For the samples stored at 4°C for 3, 6, 12, 18, and 24 months, the blastocyst formation rate was 61.5%, 49.1%, 31.5%, 32.2%, and 41.4%, respectively. The blastocyst rate for those stored at ambient temperature (â¼22°C) for 3, 6, 12, and 18 months was 57.8%, 36.2%, 33.6%, and 34.4%, respectively. Fifteen, eight and three live pups were produced from sperm stored at room temperature for 12, 18, and 24 months, respectively. This is the first report of live offspring produced from dried mouse sperm stored at ambient temperature for up to 2 years. Based on these results, we suggest that evaporative drying is a potentially useful method for the routine preservation of mouse sperm.
Assuntos
Dessecação , Nascido Vivo , Preservação do Sêmen/métodos , Temperatura , Animais , Animais Recém-Nascidos , Células Cultivadas , Transferência Embrionária/veterinária , Feminino , Nascido Vivo/veterinária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Gravidez , Preservação do Sêmen/veterinária , Injeções de Esperma Intracitoplásmicas/veterinária , Fatores de TempoRESUMO
PURPOSE: To determine the pattern of expression of parathyroid hormone-related protein (PTHrP) and its receptor, parathyroid hormone receptor 1 (PTHR1), in mouse embryos in different stages of preimplantation development. METHODS: Embryos were cultured from the pronuclear zygote stage and harvested as 2-cell, 4-cell and 8-cell embryos, morulae and blastocysts. RT-PCR was carried out on mRNAs of these and of trophoblast outgrowths for detection of PTHrP and PTHR1. Whole mounted embryos intact or stripped of zonae pellucidae were immunofluorescently stained for PTHrP and PTH receptor and observed with confocal microscopy. RESULTS: PTHrP mRNA was present in the pronuclear zygote, not present in 2-cell, 4-cell and uncompacted 8-cell embryos, present in the 8-cell compacting embryo, and not detected in 16-cell morulae or blastocysts. The mRNA was present in trophoblasts growing on fibronectin beds. mRNA for PTHR1 was detected in the pronuclear zygote, then undetected until the compacted 8-cell stage and thereafter. PTH receptor protein was observed in 2-cell embryos, morulae and in the inner cell mass and trophectoderm of blastocysts. PTHrP was observed dispersed in the cytoplasm of 2-cell, 4-cell and uncompacted 8-cell embryos, and in distinct foci near the nuclei of morulae. In blastocysts, PTHrP appeared on the apical surface of only trophoblast cells which had extruded from the zona pellucida. Fully hatched blastocysts expressed the protein on the apical side of all trophoblasts. When morulae were prematurely stripped of their zonae, PTHrP was observed on the embryos' outer surface. CONCLUSIONS: PTHrP protein is expressed throughout early embryo development, and its receptor PTHR1 is expressed from the morula stage. Embryo hatching is associated with translocation of PTHrP to the apical plasma membrane of trophoblasts. PTHrP may thus have autocrine effects on the developing blastocyst.
Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário , Proteína Relacionada ao Hormônio Paratireóideo/análise , Animais , Camundongos , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Transporte Proteico , Zona Pelúcida/metabolismo , Zona Pelúcida/ultraestruturaRESUMO
A comparison was made of the development of human zygotes in either a one-step (Global® medium) or two-step culture system (Quinn's Advantage®). A total of 257 normally fertilized 2PN zygotes from 28 patients were used in the study. The study was broken down into two parts: the first concerned the development of embryos from Days 1 to 3 in Global® medium and Quinn's Advantage® cleavage medium; the second consisted of a comparison of the development of embryos from Day 3 to 5/6 in Global® medium and Quinn's Advantage® blastocyst medium. There were no significant differences between the two culture media with respect to embryo quality throughout the preimplantation phase of human embryo development as determined by the extent and variability of the cell counts, fragmentation, and nucleation. A difference was noted in the blastomere symmetry of Day 2 embryos in the two media, but was no longer apparent on examination of Day 3 embryos. No differences were noted in the rates of blastocyst development, inner cell mass (ICM), and trophectoderm (TE) scores in the two culture media. Finally, no significant differences were noted with either the proportion of blastocysts chosen for transfer or cryopreservation (vitrification). The findings support the view that two-step sequential media protocols are sufficient but not necessary to support the complete in vitro development of human preimplantation embryos.
Assuntos
Blastocisto/fisiologia , Meios de Cultura , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário , Zigoto/crescimento & desenvolvimento , Blastocisto/ultraestrutura , Blastômeros/ultraestrutura , Transferência Embrionária , Feminino , Fertilização in vitro/métodos , Humanos , Gravidez , Irmãos , Resultado do TratamentoRESUMO
The prevalence of genetically engineered mice in medical research has led to ever increasing storage costs. Trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing. Using multi-isotope imaging mass spectrometry, we are able to image and measure trehalose in individual spermatozoa. We provide the first evidence that trehalose penetrates the nucleus of a mammalian cell, permitting tolerance to desiccation. These results have broad implications for long-term storage of mammalian cells.
Assuntos
Núcleo Celular/diagnóstico por imagem , Espectrometria de Massas/métodos , Isótopos de Oxigênio/farmacologia , Preservação do Sêmen/métodos , Espermatozoides/diagnóstico por imagem , Trealose/química , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , Dessecação/métodos , Engenharia Genética/métodos , Imageamento Tridimensional/métodos , Masculino , Camundongos , Cintilografia , Espermatozoides/metabolismo , TemperaturaRESUMO
IVF and embryo transfer for the treatment of human infertility has now resulted in the birth of over 4 million babies. The technique did not arise as a quantum event but was built on the efforts of many earlier workers in the fields of reproductive endocrinology and development. One should remember the famous saying of Isaac Newton: 'If I have seen further than most, it is because I have stood on the shoulder's of giants'. Ethical and moral issues have always arisen when investigators study early mammalian development, particularly human development. This paper documents these earlier studies and also draws attention to the ethical and moral arguments that inevitably arose.
Assuntos
Transferência Embrionária/história , Transferência Embrionária/métodos , Fertilização in vitro/história , Fertilização in vitro/métodos , Técnicas de Reprodução Assistida/ética , Técnicas de Reprodução Assistida/história , História do Século XX , História do Século XXI , HumanosRESUMO
Apolipoprotein E (Apoe)-deficient knockout mice were used to test the hypothesis that mutant mice preserved as evaporatively dried (ED) spermatozoa, stored at -80â°C for 6 months, and then recovered by ICSI will exhibit the same phenotype as before preservation. The birth rate of mice recovered by ICSI of evaporatively dried spermatozoa was lower than that of fresh spermatozoa (17.5 vs 38.0%). Progeny of mice preserved using evaporatively dried spermatozoa were reproductively sound. From these, the second generation of mice produced by natural mating showed lesions typical of APOE deficiency, including severe hypercholesterolemia, hypertriglyceridemia, markedly increased plasma low-density lipoprotein level, and extensive and severe atherosclerotic lesions in the aorta. We conclude that the expected phenotype caused by an induced genetic mutation can be faithfully recapitulated and sustained in subsequent generations of mice preserved and stored as ED spermatozoa and recovered using ICSI. Because it is simpler, faster, and cheaper than conventional (cryopreservation) and nonconventional (freeze-drying) preservation procedures, evaporative drying is a viable, cost-effective, and efficient method for preserving and storing valuable mutant mouse strains.
Assuntos
Injeções de Esperma Intracitoplásmicas , Espermatozoides , Preservação de Tecido , Animais , Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/patologia , Feminino , Lipídeos/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , FenótipoRESUMO
With the fast advancement in the genetics and bio-medical fields, the vast number of valuable transgenic and rare genetic mouse models need to be preserved. Preservation of mouse sperm by convective drying and subsequent storing at above freezing temperatures could dramatically reduce the cost and facilitate shipping. Mouse sperm were convectively dried under nitrogen gas in the Na-EGTA solution containing 100 mmol/L 3-O-methyl-D-glucose and stored in LiCl sorption jars (Relative Humidity, RH, 12%) at 4°C and 22°C for up to one year. The functionality of these sperm samples after storage was tested by intracytoplasmic injection into mouse oocytes. The percentages of blastocysts produced from sperm stored at 4°C for 1, 2, 3, 6, and 12 months were 62.6%, 53.4%, 39.6%, 33.3%, and 30.4%, respectively, while those stored at 22°C for 1, 2, and 3 months were 28.8%, 26.6%, and 12.2%, respectively. Transfer of 38 two- to four-cell embryos from sperm stored at 4°C for 1 year produced two live pups while 59 two- to four-cell embryos from sperm stored at 22°C for 3 months also produced two live pups. Although all the pups looked healthy at 3 weeks of age, normality of offspring produced using convectively dried sperm needs further investigation. The percentages of blastocyst from sperm stored in the higher relative humidity conditions of NaBr and MgCl(2) jars and driest condition of P(2)O(5) jars at 4°C and 22°C were all lower. A simple method of mouse sperm preservation is demonstrated. Three-O-methyl-D-glucose, a metabolically inactive derivative of glucose, offers significant protection for dried mouse sperm at above freezing temperatures without the need for poration of cell membrane.
Assuntos
3-O-Metilglucose/farmacologia , Dessecação/métodos , Preservação do Sêmen/métodos , Espermatozoides/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Blastocisto , Transferência Embrionária/métodos , Transferência Embrionária/estatística & dados numéricos , Feminino , Modelos Lineares , Modelos Logísticos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Endogâmicos , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Injeções de Esperma Intracitoplásmicas/métodos , Injeções de Esperma Intracitoplásmicas/estatística & dados numéricos , Espermatozoides/citologia , Temperatura , Fatores de TempoRESUMO
OBJECTIVE: To evaluate the validity of collecting day 3 embryo morphology variables into the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System (SART CORS). DESIGN: Retrospective. SETTING: National database-SART CORS. PATIENT(S): Fresh autologous assisted reproductive technology (ART) cycles from 2006-2007 in which embryos were transferred singly (n=1,020) or in pairs (n=6,508) and embryo morphology was collected. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Relationship between live birth, maternal age, and morphology of transferred day 3 embryos as defined by cell number, fragmentation, and blastomere symmetry. Logistic multiple regressions and receiver operating characteristic curve analyses were applied to determine specificity and sensitivity for correctly classifying embryos as either failures or successes. RESULT(S): Live birth rate was positively associated with increasing cell number up to eight cells (<6 cells: 2.9%; 6 cells: 9.6%; 7 cells: 15.5%; 8 cells: 24.3%; and >8 cells: 16.2%), but was negatively associated with maternal age, increasing fragmentation, and asymmetry scores. An area under the receiver operating curve of 0.753 (95% confidence interval 0.740-0.766) was derived, with a sensitivity of 45.0%, a specificity of 83.2%, and 76.4% of embryos being correctly classified with a cutoff probability of 0.3. CONCLUSION(S): This analysis provides support for the validity of collecting morphology fields for day 3 embryos into SART CORS. Standardization of morphology collections will assist in controlling for embryo quality in future database analyses.
Assuntos
Blastômeros/citologia , Fase de Clivagem do Zigoto/citologia , Bases de Dados Factuais , Embrião de Mamíferos/citologia , Taxa de Gravidez , Técnicas de Reprodução Assistida , Sociedades Médicas/organização & administração , Adulto , Contagem de Células , Forma Celular , Coleta de Dados/métodos , Feminino , Humanos , Recém-Nascido , Masculino , Prole de Múltiplos Nascimentos/estatística & dados numéricos , Gravidez , Estudos Retrospectivos , Resultado do Tratamento , Estados UnidosAssuntos
Pesquisas com Embriões/história , Transferência Embrionária/história , Fertilização in vitro/história , Tomada de Decisões Gerenciais , Pesquisas com Embriões/economia , Transferência Embrionária/efeitos adversos , Transferência Embrionária/ética , Fertilização in vitro/efeitos adversos , Fertilização in vitro/ética , História do Século XX , Humanos , Revisão da Pesquisa por Pares , Reino UnidoRESUMO
Mouse spermatozoa can be evaporatively dried under a stream of nitrogen gas at ambient temperature and stored for 5 months at -80 degrees C. The method as it stands at present is available for practical use, although its value would be greatly increased if the spermatozoa could be stored at ambient temperature. The need for further research is stressed.
Assuntos
Preservação do Sêmen/veterinária , Animais , Criopreservação/veterinária , Dessecação , Masculino , Camundongos , Nitrogênio , Preservação do Sêmen/métodos , TemperaturaRESUMO
BACKGROUND: This study was undertaken to determine what characteristics should be recorded on which days to build a predictive model for selection of Day 3 embryos. METHODS: Embryos failing to form a clinical sac or that formed a viable fetus (to > or =12 weeks), and transferred singly (n = 269) or in pairs (n = 1326) were scored for early cleavage and pronuclear status on Day 1, and cell number, fragmentation, and symmetry on Days 2 and 3, with number of nuclei per blastomere also recorded on Day 2. Seven candidate models were identified using a priori clinical knowledge and univariate analyses. Each model was fit on a training-set and evaluated on a test-set with resampling, with discrimination assessed using the area under the ROC curve (AUC) and calibration assessed using the Hosmer-Lemeshow statistics. RESULTS: Models built using Day 1, 2 or 3 scores independently on the 30 resampled data sets showed that Day 1 evaluations provided the poorest predictive value (median AUC = 0.683 versus 0.729 and 0.725, for Day 2 and 3). Combining information from Day 1, 2 and 3 marginally improved discrimination (median AUC = 0.737). Using the final Day 3 model fitted on the whole dataset, the median AUC was 0.732 (95% CI, 0.700-0.764), and 68.6% of embryos would be correctly classified with a cutoff probability equal to 0.3. CONCLUSIONS: Day 2 or Day 3 evaluations alone are sufficient for morphological selection of cleavage stage embryos. The derived regression coefficients can be used prospectively in an algorithm to rank embryos for selection.
Assuntos
Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos , Desenvolvimento Embrionário , Fertilização in vitro/métodos , Adulto , Algoritmos , Transferência Embrionária/métodos , Feminino , Humanos , Idade Materna , Valor Preditivo dos Testes , Gravidez , Análise de Regressão , Medição de Risco , Injeções de Esperma Intracitoplásmicas , Fatores de Tempo , Resultado do TratamentoRESUMO
It has been shown in the past that mouse spermatozoa could be dried under a stream of nitrogen gas at ambient temperature and stored at 4 degrees C or 22 degrees C for up to 3 months and was capable of generating live-born offspring. In previous desiccation work, dried sperm were stored in a vacuum-sealed plastic bag placed in a vacuum-packed Mylar bag. However, dried specimens stored in this way often lost moisture, particularly in samples stored at higher temperatures (22 degrees C) compared to lower temperatures (4 degrees C). The present report describes a method which minimizes this water loss from the dried sperm samples. Its use is described in a preliminary study on the effect of supplementing the trehalose with glycerol. The results have demonstrated that mouse sperm can be stored at 4 degrees C over saturated NaBr without the uptake of water which occurs when they are stored in Mylar packages. In addition, we were able to get some survival of sperm (9-15%) at room temperature storage after 3 months. The addition of glycerol to trehalose had little effect on the survival of dried mouse sperm stored over NaBr for 1 and 3 months.
Assuntos
Criopreservação/métodos , Espermatozoides/fisiologia , Animais , Citoplasma/metabolismo , Dessecação , Feminino , Gases , Glicerol/química , Masculino , Camundongos , Nitrogênio/química , Preservação do Sêmen/métodos , Espermatozoides/metabolismo , Temperatura , Fatores de Tempo , Trealose/químicaRESUMO
OBJECTIVE: To analyze critically the reasons justifying the choice of two-step protocols requiring two media for the culture of human preimplantation embryos from the zygote to the blastocyst. DESIGN: Literature review. RESULT(S): Two types of protocol are used for the culture of human preimplantation embryos from the zygote to the blastocyst, using either one medium (one-step protocol) or two media of different composition (two-step protocol). Two-step protocols are the most widely used, largely because all but one of the commercially available protocols are of this type. The reasons for the adoption of two-step protocols are described and critically analyzed. They are based on considerations of the functions of glucose, ethylenediaminetetraacetic acid (EDTA), glutamine, and amino acids that are included in the media. A reappraisal of the reasons for selecting two-step protocols is important because recent animal experiments and clinical observations have raised doubts as to whether the more complex, two-step protocols have any advantage over one-step protocols. The analyses show that all of conclusions reached should be considered equivocal. CONCLUSION(S): Clinical embryologists should evaluate the justification for selecting two-step protocols for the culture of human preimplantation embryos from the zygote to the blastocyst.
Assuntos
Blastocisto/fisiologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Técnicas de Cultura Embrionária/métodos , Técnicas de Cultura Embrionária/tendências , Meios de Cultura/classificação , HumanosRESUMO
Intracytoplasmic sperm injection (ICSI) is an important assisted reproductive technology (ART). Due to deployment difficulties and low efficiency of the earlier (conventional) version of ICSI, especially in the mouse, a piezo-assisted ICSI technique had evolved as a popular ART methodology in recent years. An important and remaining problem with this technique, however, is that it requires small amounts of mercury to stabilize the pipette tip when piezoelectric force pulses are applied. To eliminate this problem we developed and tested a completely different and mercury-free technology, called the "Ros-Drill" (rotationally oscillating drill). The technique uses microprocessor-controlled rotational oscillations on a spiked micropipette without mercury or piezo. Preliminary experimental results show that this new microinjection technology gives high survival rate (>70% of the injected oocytes) and fertilization rate (>80% of the survived oocytes), and blastocyst formation rates in early trials (approximately 50% of the survived oocytes). Blastocysts created by Ros-Drill ICSI were transferred into the uteruses of pseudopregnant surrogate mothers and healthy pups were born and weaned. The Ros-Drill ICSI technique is automated and therefore; it requires a very short preliminary training for the specialists, as evidenced in many successful biological trials. These advantages of Ros-Drill ICSI over conventional and piezo-assisted ICSI are clearly demonstrated and it appears to have resolved an important problem in reproductive biology.
Assuntos
Blastocisto/citologia , Microinjeções/instrumentação , Microinjeções/métodos , Oócitos/citologia , Injeções de Esperma Intracitoplásmicas/instrumentação , Injeções de Esperma Intracitoplásmicas/métodos , Espermatozoides/citologia , Animais , Transferência Embrionária/instrumentação , Transferência Embrionária/métodos , Feminino , Masculino , Mercúrio , CamundongosRESUMO
Combination of evaporative drying and frozen storage at -80 degrees C has been used successfully to preserve hybrid B6D2F1 mouse spermatozoa. To determine whether this method can be applied equally well to inbred mice, spermatozoa of C57BL/6J and FVB/ NJ mice were evaporatively dried and stored for 1 mo at -80 degrees C before being used for intracytoplasmic sperm injection (ICSI) to produce live offspring. After weaning, 1 male and 1 female mouse from each litter were randomly selected at 8 wk of age for natural mating to produce live offspring. Results showed that spermatozoa from both inbred strains that had been evaporatively dried and subsequently stored at -80 degrees C could be used successfully to derive live, healthy, and reproductively sound offspring by ICSI. No significant differences were found in embryo transfer rate (number of pups born/number of embryos transferred), litter size, weaning rate, body weight, number of pathologic lesions, and amount of contamination by pathogens of mice produced by ICSI using evaporatively dried spermatozoa compared with mice produced by natural mating or by ICSI using fresh (that is, nonpreserved) spermatozoa. Progeny produced by mating mice generated from ICSI using evaporatively dried spermatozoa were normal. Therefore, spermatozoa from inbred mouse strains C57BL/6J and FVB/NJ can be preserved successfully after evaporative drying and frozen storage at -80 degrees C.
Assuntos
Criopreservação , Camundongos Endogâmicos/fisiologia , Preservação do Sêmen , Espermatozoides/fisiologia , Animais , Peso Corporal , Cruzamento , Desenvolvimento Embrionário , Feminino , Tamanho da Ninhada de Vivíparos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos/anatomia & histologia , Camundongos Endogâmicos/embriologia , Fenótipo , Preservação do Sêmen/instrumentação , Razão de Masculinidade , Injeções de Esperma IntracitoplásmicasRESUMO
To determine if mouse spermatozoa could be preserved long-term without using liquid nitrogen, mouse spermatozoa in trehalose-EGTA solution were partially evaporatively dried under nitrogen gas (5 min at flow rate10 l/min) and stored for 1 week and 5 months at 4, -20, and -80 degrees C before intracytoplasmic sperm injection. Fertilization rates were neither different with spermatozoa stored at 4, -20, or -80 degrees C for 1 week or 1, 3, and 5 months respectively, nor blastocyst formation rates with spermatozoa stored for 1 week and 1 month. However, spermatozoa stored at 4 and -20 degrees C for 3 months resulted in fewer blastocysts (35.1 and 54.3% respectively) when compared with spermatozoa stored at -80 degrees C (74.4%). Blastocyst formation rates using spermatozoa stored for 5 months at -20 degrees C (57.4%) or -80 degrees C (74.5%) were not significantly different from those stored for 3 months at the same temperatures respectively, but were significantly better than those stored for 5 months at 4 degrees C (10.2%). Blastocysts derived from spermatozoa stored for 3 and 5 months at -20 and -80 degrees C respectively, were then transferred to pseudopregnant mothers to develop into healthy liveborn offspring. No significant differences were found in embryo transfer rates (number of pups born/number of embryos transferred), weaning rates, or sex ratios of resultant pups, which were healthy and reproductively sound. These results demonstrate for the first time that partially evaporatively dried mouse spermatozoa in trehalose-EGTA solution can be preserved for long term at -20 and -80 degrees C. The possibility that the storage temperature must be less than the glass transition temperature is discussed.