Survival of rabbit embryos after culture or culture/freezing

Five-hundred-and-ninety-five rabbit embryos at the 2- to 4-cell stage were cultured for 48 h to the morula stage. One-hundred-and-sixty-three embryos were transferred directly after culture while the rest (432) were frozen to −196°C. The development of these embryos was tested by transfer into synchronized pseudopregnant recipients or into pseudopregnant recipients 24 h before synchrony. The results were determined at day 17 of pregnancy. The transfer of cultured embryos into synchronized recipients gave a higher survival rate than transfer into asynchronized recipients (51 vs. 15%; P<0.05). The freezing of cultured embryos affected in vitro and in vivo development. Only 56% of the frozen-thawed morulae developed to the blastocyst stage compared with 89% in the control group (P<0.005). The survival rate after synchronous transfer was only 14%. Our results indicate that rabbit embryos need asynchronous conditions when they are frozen and cultured. Embryo survival rate was enhanced by 38% (P<0.07) when these cultured frozen-thawed embryos were transferred into pseudopregnant recipients in an earlier physiological stage (−24 h).     

An attempt at embryo transfer as a means of controlling Bordetella bronchiseptica infection in the rabbit

Embryo transfer was attempted in order to control disease in rabbits. Embryos were collected by flushing of the oviducts of donor rabbits on Day 2 of gestation, into small tubes containing the medium, transported within the body warmth of the person carrying the tubes and transferred into the oviducts of SPF pseudopregnant recipients. The time between embryo collection and transfer was 7-8 hours. Ten of 56 embryos derived from Bordetella bronchiseptica infected animals developed into newborns. As a result of bacteriological examination of intranasal exudate in six weanlings, no pathogens were detected. We suggest that embryo transfer is an effective and simple alternative to caesarean operation in Bordetella bronchiseptica infected rabbits.     

Effect of various procedures on the viability of mouse embryos containing half the normal number of blastomeres

Half embryos produced from 8-cell or compacted stages were cultured in vitro for 1-2 days and transferred to oviducts or uteri of recipients at different stages of pseudopregnancy. The proportion of live fetuses was low (8-12%), except for one group (27%) in which half embryos were cultured in vitro for 1 day and transferred into oviducts on the 1st day of pregnancy. Monozygotic twin production rate, however, was low (1 out of 10) even in this group. Fetal weight on the 18th day of gestation was significantly lower after transfer of half embryos than after transfer of similarly treated but undivided embryos. Half embryos produced from the 2-cell stage were inserted into empty zonae, embedded in agar, cultured in ligated mouse oviducts for 2-4 days and transferred to oviducts of recipient females on the 1st day of pregnancy or pseudopregnancy. When twin embryos cultured for 2-3 days were transferred to pseudopregnant recipients together with control embryos, 4 sets of monozygotic twins and 5 singletons out of 10 sets of twin embryos were obtained on Days 18-19 of gestation, giving a survival rate of 65%.     

Transfer of cultured rabbit embryos

Embryo transfer experiments were carried out to study the developmental capacity of cultured rabbit embryos when transferred to recipients of variable postovulatory maturity. Rabbit embryos were flushed from the oviduct at 26 hours postcoitum (pc) and cultured in a modified Ham's F-10 medium supplemented with bovine serum albumin (BSA) for a period of 70 hours. At 96 hours pc the cultured embryos, which ranged from the early morula to the expanding blastocyst stage, were transferred to pseudopregnant recipients mated to vasectomized males 36 to 96 hours prior to the transfer procedure. Greatest embryo survival occurred when transfers were made to either the oviducts or uterine horns of recipients at 48 hours pc. Intermediate results for both implantation rates and number of young born were obtained with recipients at 36, 60, 72, and 84 hours pc. Transferred embryos consistently failed to survive the uterine environment of recipients 96 hours pc at transfer although this group was synchronous with embryonic chronological age. Oviductal transfers were generally more successful than uterine transfers. Markedly higher rates of embryo survival resulted from embryos that were collected 60 and 72 hours pc and transferred directly to synchronous recipients without an interim period of culture. Dissimilarity of development for in vivo grown rabbit embryos and those cultured in synthetic medium is demonstrated.     

Effect of RU486 on development and implantation of rat embryos

This study evaluated the effects of postcoital treatment with the antiprogestin RU486 on transport, development and implantation of rat embryos. Doses of 0.1, 0.5, 1.0, 2.0, or 3.0 mg/rat of RU486 were injected subcutaneously on days 1, 1 + 2, or 4 of pregnancy. Autopsies were carried out on days 5 or 12 of pregnancy. RU486 provoked a significant dose-related reduction in the number of recovered embryos and inhibited their development (day 5) and decreased the number and size of implanted embryos (day 12). Treatment on day 4 was the least effective. Blastocysts recovered from RU486-treated rats exhibited comparable rate of trophoblastic outgrowth in vitro as the controls. Blastocysts transferred from RU486-treated rats to synchronous untreated pseudopregnant recipients yielded implanted embryos 12 days later in all recipients, although at a significantly lower rate than the controls. Blastocysts transferred from control pregnant rats to RU486-treated pseudopregnant recipients failed to implant completely when the dose was greater than or equal to 1.0 mg. The interceptive mechanism of postcoital treatment with RU486 in the rat involves loss of embryos from the reproductive tract and altered development prior to implantation. Endometrial receptivity or the ability of the uterus to retain the embryos until the time of implantation are also impaired by RU486. The embryos that survive these effects may experience delayed implantation in their mothers.     

Cleavage rates of diploid and tetraploid mouse embryos during the preimplantation period

Despite the fact that spontaneous tetraploidy is a rare phenomenon in mice, such embryos may be produced experimentally by a variety of means, though only a very limited degree of postimplantation development has been achieved. Despite this apparent limitation, much data on the rate of development of preimplantation tetraploid embryos has been published. However, the findings from these studies has often been conflicting. In the light of the recent successful achievement of advanced postimplantation tetraploid development in our laboratory, we decided it was an opportune time to re-evaluate the preimplantation development of these embryos in as near to optimal conditions as we could achieve. Three groups were studied, namely 1) control (diploid) embryos developing in vivo, 2) control (diploid) embryos that had been isolated at the 2-cell stage, briefly retained in culture, then transferred to the oviducts of pseudopregnant recipients, and 3) tetraploid embryos produced by electrofusion of blastomeres at the 2-cell stage, then transferred to the oviducts of pseudopregnant recipients. Embryos were isolated from females from each group at specific times after the HCG injection to induce ovulation. The total cell number of each embryo was established and the log mean values were plotted against time. From the gradients of the lines it was possible to establish that there was a significant difference between the cell doubling time of the transferred controls (group 2) compared to the in vivo controls (group 1) with cell doubling times of 15.86 +/- 1.45 h and 10.27 +/- 0.24 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

昆明小鼠精子冷冻的研究（简报）

胚胎工程技术是动物品种、品系培育,种质资源保存及转基因动物制备、保种的重要手段。配子的冷冻保存技术目前广泛应用于胚胎工程。和胚胎冷冻相比小鼠精子冷冻技术方便、高效尤其适用于转基因及突变系小鼠的保种。成功的精子冷冻要求复苏后通过体外受精(IVF)获得胚胎,再移植入受     

Correlation of increased concentration of ovine endometrial cyclooxygenase 2 with the increase in PGE2 and PGD2 in the late luteal phase

The effect of intraoviductal embryos on endometrial receptivity was studied by intraendometrial and intrauterine embryo transfer. Five-week-old female ICR mice were mated after superovulation; a vaginal plug confirmed day 1 of pregnancy. On day 4 (90 h after hCG injection), blastocysts were collected and transferred to pseudopregnant female mice and to recipient mice in which the uterotubal junction had been ligated bilaterally on day 1 of pregnancy. Three embryos per uterine horn, a total of six embryos per recipient mouse at days 1-6, were transferred to the endometrium or uterine cavity and implantation and pregnancy rates were calculated. The implantation rate for intraendometrial embryo transfer to recipients of days 3, 5 and 6 was significantly higher for uterotubal junction-ligated mice (72.2, 20.8 and 9.7%, respectively) than for pseudopregnant mice (55.0, 8.3 and 0.0%, respectively). The implantation rate for intrauterine embryo transfer to recipients at days 2, 5 and 6 was significantly higher for uterotubal junction-ligated mice (11.1, 25.0 and 8.3%, respectively) than for pseudopregnant mice (0.0, 3.3 and 0.0%, respectively). Uterotubal junction-ligated mice achieved implantation and bore neonates by intrauterine embryo transfer on days 2 and 6, whereas no implantation was achieved in pseudopregnant mice. The difference in implantation rate could not be explained by a difference in progesterone concentration between the groups. The distribution of proliferating cells in the endometrium was also studied immunohistochemically by use of anti-proliferating cell nuclear antigen (PCNA) antibody in the recipient mice. PCNA-positive cells were more abundant in uterotubal junction-ligated mice and demonstrated a marked extension from the epithelium to the stroma over time, in contrast to those in pseudopregnant mice. These findings indicate that an intraoviductal embryo exerts a biological effect by sending a signal to the endometrial epithelium and stroma, thus facilitating endometrial receptivity to the embryo and improving the rate of implantation.     

Factors influencing efficient production of transgenic rabbits

Factors that influence the efficient production of transgenic rabbits are described. The effects of the number of embryos transferred to the recipient, of recipient age, of a variety of gene constructs and of a dual use of donors as recipients (donor-recipient (DR) method) were statistically evaluated from the data collected in three experiments with three different genes. Higher survival rates of microinjected embryos were obtained in younger recipients (6-17 months), while the rates were-markedly decreased in recipients over 18 months old. Integration efficiencies (transgenic rabbits per newborn) were significantly different from the gene constructs used, but not related to either the number of embryos transferred or the number of newborns obtained. No significant differences in the survival rate of embryos of injected embryos and the integration efficiency were observed in both the DR embryo transfer method and the traditional method using pseudopregnant recipients (PR). Our results suggest that the gene construct and the survival rate of injected embryos were important factors affecting the efficiency of producing transgenic rabbits, and the age of recipients was one of the important factors affecting the survival rate of the injected embryos. The DR method was useful for reducing the number of animals required for production of transgenic rabbits.     

Survival rate to term of chimeric morulae produced by aggregation of five to nine embryos in the mouse, Mus musculus

Chimeric morulae from five to nine embryos were produced by aggregation after removal of the zonae pellucidae and brief incubation in phytohemagglutinin-P. After 28 hr of in vitro culture, these aggregates were transferred to the left uterine horn of pseudopregnant recipient female mice. Each recipient also received control embryos in the right uterine horn. Genetic markers were included so that aggregate-derived offspring could be distinguished from control offspring. Aggregate-derived embryos survived to term, but at a much reduced rate. In the 25 recipients that produced litters, 6.2% (N=211) of the aggregated embryos developed into liveborn young. Survival-to-term for control embryos was 38.8% (N=387). Survival-to-term was independent of the number of embryos aggregated. The aggregate-derived embryos were apparently undergoing size regulation following implantation.     

Birth of rats following nuclear exchange at the 2-cell stage

We report full-term development of nuclear transfer embryos following nuclear exchange at the 2-cell stage. Nuclei from 2-cell rat embryos were transferred into enucleated 2-cell embryos and developed to term after transfer to recipients (NT2). Pronuclear exchange in zygotes was used for comparison (NT1). Zygotes and 2-cell embryos were harvested from 4-week-old female Sprague-Dawley rats. Nuclear transfer was performed by transferring the pronuclei or karyoplasts into the perivitelline space of recipient embryos followed by electrofusion to reconstruct embryos. Fused couplets were cultured for 4 or 24 h before being transferred into day 1 pseudopregnant recipients (Hooded Wistar) at the 1- or 2-cell stage. In vitro culture was also carried out to check the developmental competence of the embryos. In vitro development to the blastocyst stage was not significantly different between the two groups (NT1, 34.3%; NT2, 45.0%). Two of three recipients from NT1 and two of five recipients from NT2 became pregnant. Six pups (3 from NT1, 3 from NT2) were delivered from the four foster mothers. Three female pups survived; 2 from NT1 and 1 from NT2. At 2 months of age these pups appeared healthy, and were mated with Sprague-Dawley males. One rat derived from NT1 delivered 15 pups (5 males, 10 females) as did the rat from NT2 (7 males, 8 females). Our results show that by using karyoplasts from 2-cell stage embryos as nuclear donors and reconstructing them with enucleated 2-cell embryos, healthy rats can be produced.     

Effect of mitochondrial DNA (mtDNA) type on pregnancy rate after embryo transfer in rats

Because the mtDNA is maternally inherited, embryos resulting from matings or artificial inseminations have the same type of mtDNA as that of the mother. However, when embryos are transferred the mtDNA of the embryos may be different to that of the recipient and this may interfere with maternal recognition and the establishment of pregnancy. This study was done to determine whether differences in the mtDNA between embryos and recipients would influence the survival to term of transferred embryos.A total of 1,220 rat embryos were recovered from non-superovulated donors of known mtDNA type. The number and distribution of developmental stages of embryos collected from 51 rats of mtDNA type A (n = 595) were not different (P>0.05) from those collected from 50 rats of mtDNA type B (n = 625). The overall pregnancy rate after transfer of embryos to pseudopregnant rats was 54% (26 48 ). The pregnancy rate was not affected (P>0.05) by the type of mtDNA of the recipient or of the embryo, and the interaction between mtDNA type of embryos and recipients was also not significant (P>0.05). Embryonic survival to birth was low (78 622 , 12.5%) but was not affected (P>0.05) by the type of mtDNA of the recipient (A = 28 250 ; B = 50 372 ) or of the embryo (A = 41 306 ; B = 37 316 ). Survival of pups to weaning was affected by the type of mtDNA of the embryo (P < 0.01) but not by the type of mtDNA of the recipient (P>0.05) and the interaction between mtDNA type of embryos and recipients was also not significant (P>0.05). More pups (P < 0.005) derived from donor rats of mtDNA type A (34 41 ) survived to weaning age than pups from donor rats of mtDNA type B (18 37 ). These results indicate that differences in the type of mtDNA between embryos and recipients do not interfere with establishment of pregnancy in pseudopregnant recipients.     

Embryo survival in pseudopregnant and in pregnant but genetically semi-sterile recipients after nonsurgical embryo transfer in the mouse

A new nonsurgical embryo transfer technique was used in the mouse that yielded survival rates of between 40 and 70% depending on embryo stage and, possibly, on the degree of synchrony between the embryo and recipient. Three variables were tested using this embryo transfer technique: a) pseudopregnant recipients vs pregnant but genetically semi-sterile recipients, b) embryos resulting from superovulation vs embryos from natural ovulation, and c) 12-hour vs 24-hour asynchrony between donors and recipients. None of these variables significantly affected the pregnancy rate or the percentage of transferred embryos developing to term. The pregnancy rates were between 77 and 90% in 6 experimental groups of 8 to 13 females. Survival rates were between 41 and 63% when all recipients were considered and between 53 and 68% when only the pregnant recipients were included. The embryo transfer procedure influenced litter size composition of the endogenous conceptuses of the semi-sterile recipients. Too many females were devoid of these. Recipients of expanded blastocysts had significantly better transfer results than recipients that also received morulae and early blastocysts. It was concluded that the transfer success rates were influenced by the recipients and possibly by their preparation for transfer.     

Mouse fetuses by nuclear transfer from embryonic stem cells

At present, two methods for cloning mammals by nuclear transfer are employed. The first is based on cell fusion and has been applied to domestic animals, such as sheep, cows, and goats. While, nuclear microinjection has been used in mice only. Cloning by nuclear transfer has been reported mainly with cells from primary culture and freshly isolated cells. Here, using ES cell line TT2, we tried to produce clone mouse embryos by the two methods. With ES cell line TT2 (10-13 passaged), 16% of reconstructed oocytes microinjected with the nuclei developed in vitro to the morula/blastocycst stage, and 50% of these embryos developed to fetuses until 14 dpc when transferred to pseudopregnant females. At 20 dpc implanted sites were degenerated and absorbed. Also, in vitro development of embryos reconstructed by electrofusion shown similar results. But, when transferred to recipients, subsequent development of embryos showed lower rates, as compared with embryos microinjected and from recipients live-born pups could not be obtained.     

Cleavage rate of diandric triploid mouse embryos during the preimplantation period.

The postimplantation development of human and animal triploid embryos is well documented, but there is little informative data on their preimplantation development. An analysis of cell number at appropriate times during this period and thus their cleavage rate would give an indication of the potential triploids have for further development and may explain some problems associated with their postimplantation development. To rule out any effects of technical procedures on cleavage rate, appropriate controls were used. Diandric triploid embryos were produced using standard micromanipulatory techniques, which involved the injection of a male pronucleus into a recipient one-cell-stage embryo. The karyoplast was fused to the cytoplasm by electrofusion, and the resulting tripronucleate diandric triploid embryos were transferred to appropriate pseudopregnant recipients. At specific times after the transfer, the embryos were recovered and cell numbers established. The results were plotted and regression lines drawn. Three controls were used 1) micromanipulated diploid embryos from which the male pronucleus had been removed and immediately reinserted and fused to restore diploidy, 2) diploid embryos that had been briefly incubated in cytochalasin D and colcemid to find out the effects these agents had on development, and 3) diploid embryos that had been isolated and briefly incubated in tissue culture medium. All embryos were subsequently transferred to recipients. After isolation at specific times during the preimplantation period, cell numbers were also established and the results plotted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible expansion of "Window of Implantation" in pseudopregnant mice: time of implantation of embryos at different stages of development transferred into the same recipient

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and maternal environment. It is believed that the growth of transferred embryos of different ages is synchronized during preimplantation development and that such embryos are implanted in the uterus at the same time. To define the time of synchronization for developing embryos of different ages, embryos at two different stages of development were transferred separately into the oviducts of the same recipient. We then examined the subsequent development of the embryos at various time intervals after transfer. Pronucleus (PN) stage eggs were transferred separately to the right or left oviduct of recipients on Day 0, while eight-cell embryos (8C) were transferred to the other oviduct. For 8C, 5%, 63%, and 74% of transferred embryos were implanted in the uterus at 42, 66, and 90 h posttransfer, respectively. In contrast, none of the transferred PN was implanted until 90 h posttransfer. At 90 h posttransfer, 59% of the PN had successfully implanted. Histological examination revealed that developmental stage of the embryos in both groups synchronized around 162 h posttransfer, even though the implantation was accelerated in 8C compared with PN. Our results indicate that embryos of advanced stage transferred to the oviduct implant in the uterus in advance of younger embryos and that the uterine development is synchronized at the neural plate, presomite stage. Our results strongly suggest that uterine receptivity for implantation is expandable in pseudopregnant mice.     

Cleaning of Sendai virus-infected mice by embryo transfer technique

Mouse embryos (8-16 cells) were collected from Sendai virus (SV)-infected mice at 5 or 7 weeks after inoculation. All donors having embryo(s) were positive when tested by CF test or ELISA for SV antibody at the time of embryo collection. Most of the morphologically normal embryos developed (90.6%, 259/286) to morulae or blastocysts after culture for 26-28 hr. A total of 76 embryos cultured were transferred to the uteri of SV-free pseudopregnant recipients. Forty-seven young were obtained from these recipients (61.8% of development rate) and 46 young were successfully reared up to 10th week of age. All the recipients and the young were negative by testing SV antibody. These results indicate that the embryo transfer technique is useful for cleaning of SV-infected mice.     

Survival after transfer of “sexed” mouse embryos exposed to H-Y antisera

Immunological means were used to determine the sex of mouse embryos prior to transfer to pseudopregnant recipients. Antisera to histocompatibility-Y (H-Y) antigen were prepared in adult C57BL/6 female mice by repeated intraperitoneal injections of spleen cells from males of the same strain. Eight-to 16-cell embryos were cultured in BMOC-3 alone or BMOC-3 without bovine serum albumin to which one of the following had been added: H-Y antiserum and normal guinea pig serum (NGPS), NGPS alone, normal mouse serum alone or normal mouse serum and NGPS. After 24 hr of culture, embryos were classified as either affected or unaffected. An embryo was classified as affected if degeneration of the embryo or breakdown of one or more cells was observed. A total of 1000 embryos were cultured in BMOC-3 with H-Y antiserum and NGPS (treated embryos). Two hundred and fifty embryos were cultured in each of the other four media (control embryos). Eighty-seven (9%) of the control embryos and 479 (48%) of the treated embryos were classified as affected after culture. Unaffected embryos, approximately 12 each, were transferred to pseudopregnant recipients. One-hundred forty control embryos (17%) survived to term with 67 females (48%) and 73 males (52%) born. Fifty-eight treated embryos (14%) survived to term, producing 50 females (86%) and 8 males (14%). Percentage of females from embryos cultured in antiserum was greater than for embryos cultured in any other media (P<0.001). These results demonstrate that detection of H-Y antigen on preimplantation embryos may be a useful and effective method of determining sex of an embryo prior to transfer.     

Cryopreservation of spermatozoa of a transgenic mouse]

Spermatozoa of a homozygous transgenic mouse, in which the firefly luciferase gene was expressed under the control of beta-actin promoter, were frozen at -196 degrees C. One fourth of the frozen sperm was later thawed and used for in vitro fertilization. Thirty-six of 65 oocytes (55.4%) developed to the 2-cell stage. All the 2-cell embryos were transferred to the oviducts of pseudopregnant recipients and 23 young (63.9%, 23/36) were born. All of young analyzed carried the transgene and showed the luciferase gene expression.     

Viability of mouse half-embryos in vitro and in vivo

Mouse embryos at the 2-, 4-, 8-cell, and morula stage were divided in half by using microsurgical procedures and were either grown in vitro up to the blastocyst stage or transferred at the late morula stage into the uteri of pseudopregnant recipients. A relatively high percentage of the half embryos from 2-cell (70%), 4-cell (75%), 8-cell (93%), or morula stage embryos (75%) developed into blastocysts in vitro. However, the overall development in vivo of half embryos was low, as 3%, 13%, 8%, and 1% of half embryos from the 2-cell, 4-cell, 8-cell, and morula stages, respectively, developed into live fetuses. Embryos which were divided in half at different stages developed at different rates in vitro. This determined the stage of embryonic development at the time of transfer, which might have interacted with the stage of pseudopregnancy of the recipients to influence embryo survival in vivo.