This study reports the development of a reliable method for cryopreservation of rat epididymal spermatozoa and the production of live young by artificial insemination using these cryopreserved spermatozoa. The motility and membrane integrity of rat spermatozoa were investigated after spermatozoa had been subjected to physical stress and frozen with various concentrations of glycerol (0, 3 and 6%) either in the presence or absence of Equex Stem as cryoprotective agents. The ability of cryopreserved spermatozoa to generate normal offspring by intrauterine insemination was also evaluated. Rat spermatozoa that had been centrifuged at 700 g for 5 min showed a significant decrease in motility compared with non-centrifuged spermatozoa. In addition, after centrifugation three times the percentage of membrane-intact spermatozoa decreased to approximately 0%. The percentage of membrane-intact spermatozoa was significantly higher (P < 0.01) in semen samples that had been frozen in medium without glycerol than in samples frozen in medium with 3% glycerol. Although the addition of 0.7% Equex Stem to medium without glycerol or with 3% glycerol did not influence rates of sperm motility after freezing and thawing, the percentage of membrane-intact spermatozoa was improved by the presence of 0.7% Equex (P < 0.05). Therefore, rat spermatozoa were handled gently to avoid physical stress and were frozen in medium containing 23% egg yolk, 8% lactose monohydrate and 0.7% Equex Stem, at pH 7.4 adjusted with 10% Tris(hydroxymethyl)aminomethane solution. Thirteen female rats were inseminated into the oviductal end of both uterine horns with frozen-thawed spermatozoa. Forty-one normal live offspring were obtained from nine of the inseminated females. These results indicate that frozen-thawed rat spermatozoa can generate normal offspring. To our knowledge, this procedure is the first successful production of offspring using spermatozoa cryopreserved in liquid nitrogen.
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E Nakatsukasa, T Inomata, T Ikeda, M Shino, and N Kashiwazaki
Ming-Wen Li, Brian Baridon, Amanda Trainor, Esi Djan, Amanda Koehne, Stephen M Griffey, John D Biggers, Mehmet Toner, and K C Kent Lloyd
Apolipoprotein E (Apoe)-deficient knockout mice were used to test the hypothesis that mutant mice preserved as evaporatively dried (ED) spermatozoa, stored at −80 °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.
M Arias-Álvarez, R M García-García, L Torres-Rovira, A González-Bulnes, P G Rebollar, and P L Lorenzo
Extreme body mass indexes may impair reproductive outcome in assisted reproductive technologies. Leptin reflects the amount of body fat and could act as a modulator of oocyte quality through activation of specific transcription factors. The aim of this work was to establish whether: 1) leptin influences meiotic and cytoplasmic oocyte maturation; 2) STAT3 and MAPK mediate the effects of leptin and 3) leptin modulates steroid secretion by cumulus–oocyte complexes (COC) during in vitro maturation (IVM). We confirmed immunolocalisation of leptin receptor in oocytes, cumulus/granulosa cells during the peri-ovulatory period. The confocal study showed that COC supplemented with 1, 10 and 100 ng/ml leptin had a significantly higher metaphase II (MII) percentage than those IVM without leptin (P<0.05) and a similar MII index compared to the group supplemented with 10% FCS. Leptin did not increase the percentage of cytoplasmically matured oocytes in terms of cortical granule migration rate, whereas a significantly higher index was found in the FCS group (P<0.001). Oestradiol concentrations in spent media were higher in the FCS group compared to other treatments (P<0.001). Leptin-stimulated nuclear oocyte maturation was significantly impaired when leptin-induced JAK2/STAT3 and MEK 1/2 activation was suppressed by the inhibitors (P<0.001). Steroid secretion of COC was not affected by leptin activation of JAK2/STAT3 or MEK 1/2 pathways. In conclusion, JAK2/STAT3 and MEK 1/2 pathways mediate the enhancement of nuclear oocyte maturation by leptin; however, neither cytoplasmic oocyte maturation nor steroidogenic response of COC were improved in the present rabbit model.
N. Tada, M. Sato, J. Yamanoi, T. Mizorogi, K. Kasai, and S. Ogawa
Summary. When mouse epididymal spermatozoa were rapidly frozen in two steps (37 to −70°C for solid CO2 and −70 to −196°C for liquid nitrogen) as pellets, 18% raffinose provided the greatest protection to ICR mouse spermatozoa against cold-shock; sperm motility and fertilizing ability were 43% and 22·4%, respectively. A small proportion of spermatozoa frozen with 10% sucrose was motile but incapable of fertilizing ovulated oocytes. Glycerol and dimethylsulphoxide were less effective at any concentration examined. However, the fertilizing ability of frozen–thawed ICR spermatozoa was significantly improved (35·5%) by addition of glycerol (1·75% final concentration) to medium containing 18% raffinose. Spermatozoa from one outbred (ddY) and 5 inbred (C57BL/6N, C3H/HeN, DBA/2N, BALB/c and kk) strains of mice were successfully frozen in the presence of 18% raffinose and 1·75% glycerol, although the fertilization rates of frozen–thawed spermatozoa varied among strains (13% for C57BL/6N to 64% for DBA/2N). A small fraction of mouse eggs resulting from fertilization by frozen–thawed spermatozoa developed normally in vitro (37% in C57BL/6N to 71% in ICR) to the blastocyst stage and in vivo (19% for C57BL/6N spermatozoa and ddY oocytes) to Day 18 of gestation.
Keywords: mouse; spermatozoa; cryopreservation; pellet method; cryoprotectant; raffinose
A. M. Donoghue, L. A. Johnston, U. S. Seal, D. L. Armstrong, L. G. Simmons, T. Gross, R. L. Tilson, and D. E. Wildt
Summary. Electroejaculates from tigers were collected and half was used fresh to inseminate tiger eggs in vitro and domestic cat eggs stored in a hypertonic salt solution. The remainder was pelleted, frozen in a solution of 20% egg yolk, 11% lactose and 4% glycerol, thawed and cultured with tiger and domestic cat eggs. The motility index ((sperm % motility) + (status rating × 20))/2 for thawed spermatozoa was about 86% of that in fresh aliquots. Of the 49 tiger oocytes inseminated in vitro with fresh spermatozoa, 34 (69·4%) cleaved, compared with 33 of 47 oocytes (70·2%) cultured with thawed spermatozoa (P > 0·05). Embryos generated by either sperm treatment could develop in vitro to the 16-cell or morula stage. Fresh and thawed tiger spermatozoa were equally capable (P > 0·05) of binding and penetrating the outer and inner zona pellucida of domestic cat eggs. These results demonstrate the ability of frozen–thawed tiger spermatozoa to (i) penetrate homologous and heterologous eggs and (ii) result in conspecific, advanced development of preimplantation embryos in vitro.
Keywords: tiger; spermatozoa; cryopreservation; in vitro fertilization; embryo
S. J. Fuller and D. G. Whittingham
Attempts to freeze mouse spermatozoa in liquid nitrogen (−196°C) have met with limited success. In an attempt to identify the factor(s) that damage mouse spermatozoa during cryopreservation, the effect of slow cooling to 4°C was examined. Epididymal spermatozoa were collected into a variety of media at 37°C, cooled slowly to 4°C over 4 h and warmed in a water bath at 37°C for 5 min. Survival of spermatozoa was assessed by motility, membrane integrity and acrosome status. Labelling with chlortetracycline showed that > 80% of spermatozoa were capacitated and had intact acrosomes immediately after warming compared with < 20% of freshly collected (control) spermatozoa. The rate of fertilization in vitro was similar using spermatozoa cooled in Dulbecco's phosphate-buffered saline and then mixed with oocytes immediately after warming and with control spermatozoa incubated for 2 h before mixing with oocytes (85%). Fewer oocytes were fertilized with spermatozoa cooled in either a modified HEPES-buffered Tyrode's medium or a simple HEPES-buffered medium with a high osmolarity (D3), 63% and 58%, respectively. Two-cell embryos were transferred to the oviducts of pseudopregnant recipients. Implantation was similar in all groups 81–88% and 54–74% of embryos formed normal late stage fetuses.
A. P. Byers, A. G. Hunter, U. S. Seal, G. A. Binczik, E. F. Graham, N. J. Reindl, and R. L. Tilson
Summary. Electroejaculates from 5 tigers were split and half of each was assayed fresh while the remainder was frozen and thawed before being assayed. Preincubation time, temperature and removal of seminal plasma were evaluated for their effect on in-vitro capacitation. Ability of spermatozoa to penetrate oocytes, as measured by the zona-free hamster egg–sperm penetration assay (SPA), was used as verification of capacitation. Results of the experiments with fresh semen indicate that: (1) preincubation time affects the fertilizability of tiger spermatozoa with 2 h appearing optimal, (2) a preincubation temperature of 37°C results in significantly higher penetration rates than does a 22°C treatment, and (3) tiger seminal plasma does not appear to contain decapacitation factors, as has been reported for several other species. Frozen semen experiments indicate that (1) frozen–thawed tiger spermatozoa must be removed from the environment of the semen extender before capacitation can take place, and (2) the freeze–thaw procedure results in a shortening of the required capacitation time.
Keywords: capacitation; Siberian tiger; spermatozoa; cryopreservation
Anna M Petrunkina, Barbara Gröpper, Anne-Rose Günzel-Apel, and Edda Töpfer-Petersen
Due to the similarity of plasma membrane changes induced by capacitation and cryopreservation, the parameters describing sperm response to capacitating conditions can be used for evaluating the cryopreservation response in many animal systems. In dog sperm, the response of the total sperm population to ionophore treatment has been shown to be an indication of the freezability of semen samples. Another sperm functional characteristic decisive for cryopreservability is cell volume regulation, due to the generation of essential osmotic gradients across the plasma membrane during the freeze-thaw cycles. In the present study, cryopreservation-induced changes in the membrane functional integrity were examined by monitoring the osmotically induced response of cell volume and the response to an ionophore in live cell populations. Cell volume measurements were performed on Percoll-washed suspensions of freshly diluted and frozen-thawed dog spermatozoa. The proportion of live acrosome-reacted cells was evaluated by flow cytometry after incubation under capacitating conditions in the presence of the calcium ionophore, A23187. During freezing-thawing, significant membrane changes occurred related to the disturbance of volume control ability and the loss of a proportion of live acrosome-reacted cells (P < 0.05). There were significant differences between individuals with respect to the degree of functional and structural membrane changes after thawing. Significant correlations were found between acrosomal integrity and functional membrane integrity. When assessed in freshly diluted semen, these parameters correlated with those of frozen-thawed semen samples, pointing to the similarities between mechanisms of cryopreservation-related changes and those mechanisms that mediate changes in membrane permeabilities and in cell volume regulation. The detection of changes in the sperm plasma membrane by monitoring the sperm cell volume represents a simple, rapid and sensitive method to estimate sperm quality after the cryopreservation procedure. The individual variability in response to osmotic stress or to calcium ionophore treatment appears to reflect the subtle differences in the sperm membrane functionality which are crucial for the prediction of cryopreservability.
E Blesbois, I Grasseau, and F Seigneurin
The ability to survive cryopreservation varies in spermatozoa from different bird species. Among the biological factors potentially responsible for such differences, species variations in membrane fluidity have a role in the restoration of the physiological state after freezing. Membrane fluidity may be assessed by measuring fluorescence polarization anisotropy with a fluorescent dye. Anistropy values are proportional to membrane rigidity and consequently inversely proportional to membrane fluidity. In the present study, polarization anisotropy of spermatozoa originating from species differing in the freezability of their semen (chicken, turkey and guinea fowl) was measured in addition to lipid composition (cholesterol/phospholipid ratio), sperm viability (membrane permeability to eosine) and morphological integrity before and after cryopreservation.
The percentages of viable and normal spermatozoa in fresh sperm were highest in the chicken (87%), lowest in guinea fowl (64%), and intermediate in turkeys (69%). Anisotropy values were highest in guinea fowl (0.205), lowest in chickens (0.155), and intermediate in turkeys (0.180). As a consequence, membrane fluidity was highest in chickens and lowest in guinea fowl. Cryopreservation significantly decreased sperm viability and morphological integrity and increased anisotropy in all species but did not change the inter species hierarchy. Initial cholesterol/phospholipid ratios were lower in chickens than in guinea fowl, and intermediate in turkeys (0.25, 0.26 and 0.29, respectively). Cryopreservation induced a severe decrease in cholesterol/phospholipid ratios in turkeys and guinea fowl.
Sperm membrane fluidity in chickens, turkeys and guinea fowl behaves as an indicator of sperm freezability in these species. Inter species differences for this parameter may be partly explained by differences in initial cholesterol/phospholipids content of spermatozoa. On the other hand, the rigidifying process induced by cryopreservation is not related to lipid damage by the same mechanisms.
Hsiao Yun Yang, Shae-Lee Cox, Graham Jenkin, Jock Findlay, Alan Trounson, and Jillian Shaw
Ovarian tissue cryopreservation and subsequent transplantation can restore fertility in cancer patients. This study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity, the kidney capsule or subcutaneous) influences the number, fertilization rate and developmental potential of oocytes recovered from grafts and whether using a standard gonadotrophin stimulation protocol would increase oocyte yield from the grafts. Mouse ovarian tissue was grafted into four week old mice and collected three weeks later. Graft recipients were treated either with or without exogenous gonadotrophin stimulation prior to graft collection. Grafted ovaries yielded oocytes that were either at the germinal vesicle (GV) stage or mature metaphase II (MII) stage at collection. These GV oocytes were matured before in vitro fertilization (IVF), while the MII oocytes underwent IVF immediately. Oocytes collected from the oviducts of non-grafted superovulated mice of the same age served as controls. Two-cell embryos were transferred to pseudopregnant recipients and recovered at day 15 of gestation or left to go to term. Graft retrieval and the number of oocytes from each graft were lowest from the subcutaneous graft site. The number of two-cell embryos produced was significantly higher for oocytes from the grafts to the bursa as compared with the other sites. All graft sites gave rise to embryos with comparable implantation rates and developmental potential to fetuses and offspring following transfer. However, the oocytes from grafted ovaries had a significantly lower developmental potential when compared with the control group. Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation and development. In conclusion, graft site affects the number and quality of oocytes produced from ovarian grafts.