Factors affecting the efficiency of nuclear transplantation in the rabbit embryo

Procedures to improve nuclear transplantation efficiency in the rabbit were evaluated. We report the influence of recipient oocyte age on the different steps of nuclear transplantation. The effect of multiple pulses and the influence of manipulation medium and cytochalasin B in the post-fusion/activation medium on activation and development were studied. Recently ovulated oocytes were enucleated at a higher rate (60%) than aged oocytes (3%, p less than 0.005); they also fused at a higher rate (85% vs. 26%, p less than 0.001). Activation was low with freshly ovulated oocytes compared to aged oocytes (3% vs. 37%, respectively; p less than 0.005), but was increased by using multiple pulses (85% vs. 68%, p less than 0.05). Multiple pulses also improved development to blastocysts (48% vs. 5%, p less than 0.001). Incubation of oocytes in a bicarbonate-buffered medium with 10% fetal calf serum for manipulation also enhanced rates of activation (100% vs. 89%, p less than 0.05) and development of oocytes to blastocysts (77% vs. 26%, p less than 0.001). Furthermore, 7.5 micrograms/ml cytochalasin B in the post-fusion/activation medium increased activation rates (78% vs. 50%, p less than 0.05) and development to blastocysts of manipulated embryos (46% vs. 11%, p less than 0.001). When the above modifications were applied, 10% (23/230) of the total nuclear transplant embryos (8-16-cell-stage donor nuclei) or 21% (23/110) of those transferred to recipients developed to offspring, rates similar to the development of nonmanipulated control embryos (10%, 4/41, p greater than 0.1).     

Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%).     

Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods

We examined possible genotype effects on the survival of 8- to 16-cell mouse embryos isolated from four inbred strains (C57BL/6N, BALB/cAnN, DBA/2N, and C3H/HeN), a outbred stock (ICR), and various crosses after cryopreservation by vitrification or conventional slow freezing using glycerol solutions. The rates of in vitro development of C57BL/6N, BALB/cAnN, C3H/HeN, and ICR embryos to expanded blastocysts ranged from 86% to 94% after slow freezing and 85% to 97% after vitrification. The cryopreservation method did not significantly influence in vitro embryo survival after thawing (P >0.05). Although genotype significantly influenced the in vitro survival of embryos (P = 0.008), this presumably resulted from an increased difficulty in assessing the quality grade of C3H/HeN embryos prior to cryopreservation. The rates in vivo development of C57BL/6N, BALB/cAnN, C3H/HeN, DBA/2N, and ICR embryos to normal day 18–19 fetuses ranged from 19% to 64% after slow freezing and from 18% to 63% after vitrification. The in vivo development of cryopreserved embryos was significantly influenced by cryopreservation method and genotype (P = 0.01 and P = 0.001, respectively). Vitrification yielded significantly higher rates of in vivo development than that after slow freezing (P > 0.05). In vivo development rates of DBA/2N and ICR♀ X B6D2F1 ♂ embryos after cryopreservation were significantly higher than that of embryos from BALB/cAnN and C3H/HeN mice (P < 0.05). These results indicate that parental genotype exerts little or no effect on the ability of embryos to develop in vitro after vitrification or slow freezing. Differences in the ability of cryopreserved embryos to develop normally in vivo may reflect inherent genotype related differences in their post-implantation developmental potential and not their sensitivity to cryoinjury. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Emerging applications of sperm, embryo and somatic cell cryopreservation in maintenance, relocation and rederivation of swine genetics

Advances in porcine assisted reproductive technology (ART) make it possible to use cryopreserved sperm, embryos and somatic cells in the maintenance, relocation and regeneration of swine genetics. In this review, development of key application-limiting technology is discussed in each cell type, focusing on the efficiencies, ease of storage and transportation, and minimization of pathogen transmission. Methods to regenerate swine genetics and/or models using frozen sperm, embryos and somatic cells in combination with other porcine ARTs, such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transplantation (SCNT), are also discussed. The applications of these ARTs utilizing cryopreserved cells will greatly increase the efficiency as well as biosecurity for maintenance, relocation and rederivation of swine genetics/models.     

Factors affecting the efficiency of embryo transfer in the domestic ferret (Mustela putorius furo)

Embryo transfer (ET) to recipient females is a foundational strategy for a number of assisted reproductive technologies, including cloning by somatic cell nuclear transfer. In an attempt to develop efficient ET in domestic ferrets, factors affecting development of transferred embryo were investigated. Unilateral and bilateral transfer of zygotes or blastocysts in the oviduct or uterus was evaluated in recipient nulliparous or primiparous females. Developing fetuses were collected from recipient animals 21 days post-copulation and examined. The percentage of fetal formation was different (P<0.05) for unilateral and bilateral transfer of zygotes (71%) in nulliparous females with bilateral transfer (56%) in primiparous recipients. The percentage (90%) of fetal formation in nulliparous recipients following unilateral transfer of blastocysts was higher (P<0.05) than that observed in primiparous recipients with bilateral ET (73%). Notably, the percentage of fetal formation was higher (P<0.05) when blastocyts were transferred as compared to zygotes (90% versus 71%). Transuterine migration of embryos occurred following all unilateral transfers and also in approximately 50% of bilateral transfers with different number of embryos in each uterine horn. These data will help to facilitate the development of assisted reproductive strategies in the ferret and could lead to the use of this species for modeling human disease and for conservation of the endangered Mustelidae species such as black-footed ferret and European mink.     

Factors affecting protoplast electrofusion efficiency

The electrofusion efficiency of protoplasts isolated from a carrot (Daucus carota) suspension culture was increased by treatment with 0.1 mg/ml lysolecithin, 2.5% dimethylsulfoxide (DMSO), or 0.5 mM Ca²⁺. The lysolecithin and DMSO treatments substantially increased protoplast lysis, whereas calcium treatment did not. The enzymes used for protoplast isolation were also found to have a dramatic effect on the efficiency of fusion. A mixture of Cellulysin and Driselase led to a two-fold enhancement of fusion as compared with Driselase alone. The stimulation by Cellulysin appears to be due to enzymatic modification of the cell surface. However, comparison of the time course for wall digestion with the development of susceptibility to electrofusion suggests that the effect of Cellulysin is not simply due to removal of the cell wall. Brief treatment of the cells with pronase or proteinase K also doubled the efficiency of fusion. Taken together, these results indicate that electrofusion efficiency can be enhanced by the method used for protoplast isolation; they also suggest that modification of membrane/cell-surface proteins during protoplast isolation may be particularly important in determining electrofusion efficiencies.Abbreviations a.c. alternating current - d.c. direct current - DMSO dimethylsulfoxide - NAA naphthaleneacetic acid - PEG polyethylene glycol     

Factors affecting the electrofusion efficiency ofPorphyra protoplasts

The effects of various factors on the electrofusion efficiencies ofPorphyra protoplasts were investigated. These factors were protoplast stabilizing reagents, divalent cations, membrane digestive enzymes and cold storage of the protoplasts. Fusion efficiencies were dependent on the concentrations of reagents used to adjust the osmotic pressure of the medium. With mannitol or sorbitol the maximum fusion efficiency (approximately 16%) was observed at concentrations of 0.6 to 0.7 M; glucose was less effective. Brief treatment of the protoplasts with pronase stimulated electrofusion, whereas treatment with proteinase K, trypsin, phospholipase C or lipase repressed fusion. The addition of Ca²⁺ at 10^-5 to 10^-4 M in the protoplast medium enhanced the fusion efficiency to approximately four times that of the non-treated control. Sr²⁺ and Co²⁺ also stimulated electrofusion, but less effectively than Ca²⁺. The fusion capacity of the protoplasts remained stable for about 3 h when kept on ice, but decreased gradually when left at room temperate.     

Factors affecting the success of surgical and nonsurgical equine embryo transfer

Nonsurgical embryo recovery was attempted from light-horse and draft mares. Embryo recovery rates were not affected (P>.05) by technician or stallion but were lower (P<.05) from draft mares (44%) than light-horse mares (67%). Sham transfer of embryos on day 8 post-ovulation did not (P>.05) increase the number of mares returning to estrus by 22 days post-ovulation. Method of embryo transfer greatly affected pregnancy rates. Embryos transferred surgically during March–June resulted in 0 of 12 pregnancies versus 13 of 25 pregnancies obtained during July–September, This strongly suggests a seasonal influence on pregnancy rates. Technician influenced (P<.05) the success of nonsurgical transfer (46.2% vs. 7.7%). In addition, protection of the insemination rod with a sheath (guarded method) appeared to provide some advantage over an unguarded method of nonsurgical transfer (54% vs. 23%). Lastly, a preliminary experiment was conducted to evaluate transfer of embryos via flank incision. Four of 5 embryos transferred by this method resulted in a pregnancy at 50 days post estrus.     

Factors affecting the cryosurvival of mouse two-cell embryos

A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors affecting the survival of frozen-thawed mouse spermatozoa

Mouse epididymal spermatozoa were frozen in solutions containing various compounds with different molecular weights, and the factors affecting the postthawing survival were examined. Monosaccharides (glucose, galactose) had almost no protective effect regardless of the concentration and the temperature of exposure. On the other hand, disaccharides (sucrose, trehalose) and trisaccharides (raffinose, melezitose) resulted in higher survival rates, especially at a concentration of around 0.35 mol/kg H(2)O (0.381-0.412 Osm/kg). Macromolecules, such as PVP10, Ficoll 70, bovine serum albumin, and skim milk had almost no effect, but compounds with a molecular weight of about 800, such as metrizamide and Nycodenz, had some protective effect. When a raffinose solution was supplemented with 10% metrizamide, resulting in an osmolality of approximately 0.400 Osm/kg, a high survival rate was obtained. Solutions at about 0.400 Osm/kg containing trehalose alone, trehalose + metrizamide, raffinose alone, and raffinose + metrizamide, were all effective for sperm freezing; frozen-thawed sperm could fertilize oocytes, and the resultant embryos could develop to live young after transfer. For freezing mouse spermatozoa, aqueous solutions at approximately 0.400 Osm/kg containing a disaccharide or a trisaccharide seem to be effective.     

Animal oocyte and embryo cryopreservation

Cryopreservation of oocytes and embryos is a crucial step for the widespread and conservation of animal genetic resources. However, oocytes and early embryos are very sensitive to chilling and cryopreservation and although new advances have been achieved in the past few years the perfect protocol has not yet been established. All oocytes and embryos suffer considerable morphological and functional damage during cryopreservation but the extent of the injury as well as differences in survival and developmental rates may be highly variable depending on the species, developmental stage and origin (for example, in vitro produced or in vivo derived, micromanipulated or not). Currently, there are two methods for gamete and embryos cryopreservation: slow freezing and vitrification. We have experienced both techniques but vitrification has become a viable and promising alternative to traditional approaches especially when dealing with in vitro produced or micromanipulated embryos and oocytes. Recently new strategies based on emerging studies in the field of lipid research have been used to reduce intracellular lipid content in bovine in vitro produced embryos and therefore increase their tolerance to micromanipulation and cryopreservation. The addition of a conjugated isomer of linoleic acid, the trans-10, cis-12 octadecadienoic acid to embryo culture medium more than twice improved embryo post-thawing viability after micromanipulation and vitrification. Vitrification was also used for the cryopreservation of embryos belonging to the Portuguese Animal Germplasm Bank project presently running at our facilities. Presented at the International Consensus Meeting “New Horizons in Cell and Tissue Banking” on May 2007 at Vale de Santarém, Portugal.     

Factors affecting electrofusion of 2-cell mouse embryos

Parameters of electrofusion of 2-cell mouse embryos were optimized for application as a model for nuclear transplantation. There was considerable lysis of embryos with M(2) as the medium for fusion; however, 100% fusion (n = 58) was obtained with a single 0.31-kv / cm, 1280-musec pulse. With mannitol and sucrose solutions as the medium, a wide range of field strengths (0.31-1.41 kv / cm for 0.26 M sucrose solution and 0.31 to 2.04 kv / cm for 0.3 M mannitol solution) and durations of the electrical pulse (10-1280 musec) resulted in high rates of fusion (often 100%). Likewise, osmolarity of sucrose and mannitol solutions did not affect the rate of fusion using a 0.47-kv / cm pulse. With a field strength of 2.04 kv / cm, the proportion of embryos that fused in mannitol solution increased (P<0.05) and the proportion that were lysed decreased (P<0.05) as osmolarity increased. Both fused (162 642 , 25%) and control embryos (32 72 , 44%) continued to develop in culture for 48 h, after which they began to compact. Fused embryos were only at the 4-cell stage by this time, while control embryos were at the 8-cell stage. Optimal pulse durations are plotted for field strengths between 0.31 and 1.41 kv / cm with 0.26 M sucrose as fusion medium.     

Factors affecting the survival of mouse embryos cryopreserved by vitrification

Preimplantation stage mouse embryos have been used to examine the response of a simple multicellular system to cryopreservation by the complete vitrification of the suspension. Successful vitrification requires the use of a solution of cryoprotectants that is sufficiently concentrated to supercool and solidify into a glass at practicable cooling rates. Factors that influence the survival of embryos include the concentration and composition of the vitrification solution, the procedure used to equilibrate embryos in this solution, the cooling and warming conditions, and the procedure used to dilute embryos from the vitrification solution. High rates of survival are obtained when embryos are dehydrated prior to vitrification in solutions composed of saline plus multimolar concentrations of either mixtures of permeating cryoprotectants (e.g. dimethyl sulphoxide-acetamide-propylene glycol) or single permeating cryoprotectants (propylene glycol or glycerol). Full permeation of cryoprotectants into the cells is not necessary and may lead to chemical toxicity and osmotic injury. Partial permeation and osmotic shrinkage concentrates the endogenous cytoplasmic macromolecules and greatly increases the likelihood of intracellular vitrification. Vitrification is a practical approach for embryo cryopreservation and offers new opportunities to examine fundamental aspects of cryoprotection and cryoinjury in the absence of freezing.