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1.
The development of in vitro fertilization (IVF) techniques in pigs as well as in other species is of great importance because of the possible applications of this technology in different research fields. Methods of IVF vary in different incubation periods and temperatures, in the hormone concentrations used, and in the treatment of the sperm samples. It has been particularly difficult to succeed in the achievement of fertilization in the pig. In the present study we used FSH and LH concentrations of 2 IU/ml for oocyte maturation, an incubation temperature of 37°C, and dilution of spermatozoa for capacitation, and we achieved a high fertilization rate (50 to 75%) with no cases of polyspermy. 相似文献
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In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 x 10(6) sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophore-treated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures. 相似文献
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C. Kirdmanee Y. Kitaya T. Kozai 《In vitro cellular & developmental biology. Plant》1995,31(3):144-149
Summary
Eucalyptus camaldulensis shoots were cultured photoautotrophicallyin vitro for 6 wk with four different types of supporting materials (agar matrix, Gelrite matrix, plastic net, or vermiculite) under
CO2-nonenriched or CO2-enriched conditions. Plantlets from each treatmentin vitro were then grownex vitro in a greenhouse for 4 wk. The growth and net photosynthetic rate of plantletsin vitro, as well as subsequent growth, survival percentage, transpiration rate, and net photosynthetic rate of plantletsex vitro were evaluated. CO2 enrichment significantly increased growth (total dry weight and number of primary roots) and net photosynthetic rate of plantletsin vitro, as well as the growth and survival percentage of plantletsex vitro regardless of the type of supporting materials. The growthin vitro was greatest in the vermiculite, followed by the plastic net, Gelrite matrix, and agar matrix (in descending order) under
either the CO2-nonenriched or CO2-enriched conditions. The growth and survival percentage of plantletsex vitro were highest in the vermicultie under the CO2-enriched condition. The extensive root system producedin vitro was necessary for growth and survival of plantletsex vitro. 相似文献
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Sheep oocytes that matured and fertilized in vitro were cultured to evaluate their cleavage to the 8- to 16- cell stage and further development in five different media as follows: 1) CPMW (TCM199 + 20% ewe serum + 0.4% BSA), 2) Ham's F-10 + 10% ewe serum, 3) Brinster's pyruvate medium + 0.1% glucose (BPM-G), 4) co-culture with sheep oviduct epithelial cells in TCM199 + 10% fetal calf serum, and 5) co-culture with sheep granulosa cells in the same medium as 4. The culture duration was 4 or 7 d for 8- to 16-cell or further development. The proportions of 8- to 16-cell eggs were 1) 16% (8 49 ), 2) 25% (12 49 ), 3) 52% (58 112 ), 4) 63% (105 167 ) and 5) 45% (27 60 ). The co-culture with sheep oviduct cells resulted in a significantly (P < 0.05) higher rate of cleavage than the other media, except BPM-G. The proportion of noncompacted morula (35%, 24 68 ) was also significantly (P < 0.05) higher in the co-culture of sheep oviduct cells than the other media. The 8- to 16-cell eggs produced by BPM-G (n=38) and the co-culture with sheep oviduct cells (n=42) were transferred into the uterus of recipient ewes, but no elongated blastocysts were obtained 13 d later. On the other hand, 8 out of 55 one-cell eggs (15 to 18 h after in vitro insemination) transferred to the oviduct of recipient ewes were elongated blastocysts (24% of 34 recovered eggs). The data show that the co-culture of in vitro fertilized eggs with sheep oviduct epithelial cells could support development of 8- to 16-cell embryos or early morula, but their viability is still questionable. 相似文献
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J. Bobek 《Biologia Plantarum》1971,13(5-6):405-407
Excised anthers ofNicotiana tabacum L. were culturedin vitro at the stage of pollen tetrads, which proved in our experiments to be the most suitable initial stage for cultivation, up to the stage of mature pollen grains, using Ito and Stern's nutrient medium, or this medium supplemented with uracil. Germinating capacity of the pollen grains formed and the lengths of pollen tubes were quantitatively evaluated. 相似文献
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M A B JORGE A I ROBERTSON A B MASHINGAIDZE E KEOGH 《The Annals of applied biology》2000,137(3):311-319
Cassava is one of the most important food crops in Africa. Meristem culture is an effective method of eliminating viruses and other systemic diseases spread through the vegetative propagation of stems. However, in semi‐arid conditions, survival of ex vitro plants in the field is often disappointing. When an increasing range of light regimes in vitro was provided, the fresh and dry masses more than doubled their values between 29 and 369 mmol s?1 m?2 PPFDs. Increases in numbers of senescent leaves and stem thickness were also recorded with increasing PPFD. However, PPFD above 101 mmol s?1 m?2 resulted in 30–70% reduction in plant survival, with the thin plants with the smallest fresh and dry masses being the ones with highest survival rates. High light and temperature levels in the greenhouse were also found to be critical for plant survival. It was also shown that transpirational loss from detached leaves and epicuticular wax deposits were not good indicators for predicting survival of ex vitro cassava plantlets during acclimatisation. 相似文献
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Shoot proliferation has been achieved in Garcinia mangostana L. using seed explants. Maximum mean number of shoots per explant (16.8) was obtained from cultures on Murashige and Skoog medium supplemented with 40 mM 6- benzyladenine, and 2.5 mM -naphthaleneacetic acid and kept at 30 °C under an 8 hour photoperiod. Cultures on the same medium but supplemented with 2 g l-1 activated charcoal produced fewer shoots. However, growth of these shoots was more organized and 75% rooting was obtained. Woody Plant Medium was not a suitable medium for shoot proliferation. Ex vitro establishment was best obtained on planting medium consisting of sand, soil and organic material (3:2:1).Abbreviations BA
6-benzyladenine
- IBA
indole-3-butyric acid
- NAA
-naphthaleneacetic acid
- MS
Murashige & Skoog (1962) basal medium
- WPM
Woody Plant basal medium (Lloyd & Mc Cown 1980) 相似文献
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An in vitro DNA packaging system was used to encapsulate T7 DNA that had been synthesized by extracts prepared from gently lysed Escherchia coli infected with bacteriophage T7 carrying amber mutations in gene 3 or in both genes 3 and 6. Isopycnic centrifugation of density-labeled wild-type DNA was employed in an effort to separate product from template; suppressor-free indicator bacteria were used to eliminate contributions from endogenous DNA or contaminating phage. Additional controls indicated that fragmented DNA is packaged in vitro only with very low efficiency and that the frequency of recombination during packaging is too low to affect interpretation of these experiments. T7 DNA replicated by extracts prepared using T7 mutants deficient in both genes 3 and 6 could be packaged in vitro with an efficiency comparable to that found when highly purified virion T7 DNA was used. When T7 deficient in the gene 3 endonuclease but with normal levels of the gene 6 exonuclease was used, fast-sedimentingconcatemer-like DNA structures were formed during in vitro DNA synthesis. Electron microscopy revealed many branched and highly complex DNA structures formed during this reaction. This concatemer-like DNA was encapsulated in vitro with an efficiency significantly greater than that found for DNA the length of a single T7 genome. 相似文献
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Rodrigues Bde A dos Santos LC Rodrigues JL 《Molecular reproduction and development》2004,67(2):215-223
In several studies, early cleavage stage canine embryos have been derived from in vitro fertilized oocytes cultured under various conditions. Despite these results, IVF protocols for canine oocytes have yielded low fertilization rates. In this study, Experiment I compared the effects of tissue culture medium (TCM)-199 supplemented with either (A) 1 microg/ml estradiol or (B) 20 microg/ml estradiol + 1 microg/ml human somatotropin (hST) on the in vitro nuclear maturation rate of canine oocytes. Meiotic progression to the metaphase I and II (MI/MII) stages at 72 hr of in vitro culture (IVC) was 10.2% (11/108) in medium A versus 14.1% (30/142) in medium B (P = 0.802). In Experiment II, cleavage rate was determined among oocytes recovered from ovaries of bitches at different reproductive stages. Oocytes (n = 888) were retrieved from bitches at the follicular, anestrous, and luteal stages and selected for high morphological quality. Oocytes were matured for 48 hr in TCM-199 supplemented with 1 microg/ml hST + 20 microg/ml estradiol. Oocytes were in vitro fertilized with fresh canine spermatozoa that had been isolated on a Percoll gradient, and were cultured in synthetic oviduct fluid (SOF) medium with bovine serum albumin (BSA; 4 mg/ml) up to 5 days in 5% CO(2) in air at 37 degrees C. A proportion of oocytes (30.6%) with identifiable nuclear material had cytoplasm penetrated or fertilized by sperm. The percentage of oocytes developing into early stage embryos was 10.1% (27/267). Although pronuclear development was observed to be higher for oocytes recovered at the follicular phase, the cleavage rate was similar among oocytes recovered from bitches at the follicular, anestrus, and luteal stages. There was no correlation between the proportion of capacitated or acrosome reacted spermatozoa and pronuclei formation and/or percent cleavage. It was concluded that TCM-199 supplemented with 1 microg/ml hST and estradiol (20 microg/ml) supports nuclear and cytoplasmic maturation of canine oocytes. In this study, meiotic competence was verified by the in vitro production (IVP) and development of embryos up to the 8 cell-stage. Furthermore, the results indicate that, under the described conditions and despite the influence of reproductive status of the bitch on the developmental competence of in vitro fertilized oocytes to the pronuclei stage, cleavage was independent of donor's reproductive estrous cycle stage. 相似文献
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目的 在SOF +PVA(合成输卵管液 +聚乙烯醇 )这一化学成分明确培养系统条件下 ,观察了葡萄糖、丙酮酸和乳酸三种碳水化合物对牛体外受精胚胎体外发育的影响 ,以便为今后进一步探讨影响牛早期胚胎体外发育的因素提供实验依据。方法 牛卵母细胞体外成熟和体外受精后 ,在化学成分明确培养系统内进行体外发育培养。结果 实验 1将牛体外受精卵培养于不含有葡萄糖的SOF +PVA培养系统中 ,培养 12 0h后分别移入含有 0、1 5 0、3 30、5 0 0mmol L的SOF +PVA培养系统中 ,对照组胚胎一直在含有 1 5 0mmol L葡萄糖的SOF +PVA中培养 ,结果囊胚的发育率分别为 9 2 % a、12 1%、19 2 % b、18 9%和 11 7% (a