Autotransfer of Day 4 embryos from oviduct to oviduct versus oviduct to uterus in the mare

Embryo autotransfer is defined as the collection of an embryo from and the transfer of this embryo into the same animal. The objectives of this study were to: 1) test the hypothesis that oviduct transport of the equine embryo from the oviduct into the uterus is not dependent on a unilateral embryo-corpus luteum interaction, 2) develop an embryo autotransfer technique for the mare and 3) compare the success rates of Day 4 embryos surgically autotransferred from the oviduct ipsilateral to ovulation to either the oviduct (n=10 mares) or the uterine horn (n=10 mares) contralateral to ovulation. Seventy percent (7 10 ) of the Day 4 embryos which were autotransferred to the oviduct contralateral to ovulation were transported through the oviduct and subsequently developed into embryonic vesicles detectable by ultrasonography between 10 and 21 days postovulation. This finding supported the hypothesis that oviductal embryo transport is not dependent upon the ipsilateral corpus luteum. Overall, sixty percent (12 20 ) of the autotransfers were successful. The success rate of uterine-transferred embryos was not significantly less (P>0.3) than that of oviductal-transferred embryos (5 10 vs 7 10 , respectively). Therefore, the Day 4 equine embryos were apparently mature enough to survive in the mare's uterus.     

Intrauterine transfer of early canine embryos

The success rate of in vitro fertilization (IVF) in dogs is low and only early embryos have been obtained. In the present study, we investigated the use of intrauterine transfer of early-stage canine embryos to obtain pups. Twenty-three female dogs, in which the date of ovulation (based on plasma progesterone concentrations) differed by +/-1 day, were used (10 donors and 13 recipients). The uterine tube was extirpated under general anesthesia 1-4 days after mating (5-7 days after ovulation), and descending perfusion was done to collect embryos. Embryos were examined and transferred into the uterine horn of a recipient, ipsilateral to the ovary with the most corpora lutea. Pregnancy was established in one of eight bitches that received early embryos (zygote to 4-cell embryos); she received two zygotes and one 2-cell embryo and delivered two puppies. Although intrauterine transfer of early embryos (zygote to 4-cell embryos) was difficult, pregnancy was achieved, suggesting that uterine tube transfer is appropriate for these early-stage embryos.     

Production of identical twins by separating two-cell rat embryos

Rat identical twins were produced from two-cell embryos. In the presence of cytochalasin B, rat two-cell embryos could be separated efficiently into two blastomeres by micromanipulation. Isolated blastomeres, embedded in agar cylinders and cultivated in ligated rat oviducts for 3 days, developed to the morula or blastocyst stage. After removing the agar, pairs of developed one-half embryos were transferred into Day 1 oviducts or Day 4 uteri of pseudopregnant rats. The percentage of embryos, separated either in the presence or absence of cytochalasin B, that developed into live fetuses was higher in cases of uterine transfer than in cases of oviduct transfer (38% vs. 18%, 31% vs. 15%, respectively). Throughout the present experiment, nine pairs of identical twins were successfully produced. This is the first report of the production of identical rat twins by separating two-cell embryos.     

三种胚胎移植技术方法比较

以近交系FVB、DBA／2小鼠作为供体胚小鼠,封闭群CD-1小鼠作为受体小鼠,对实验小鼠胚胎移植的三种方法进行比较,即：对小鼠受精卵1细胞期胚(或2细胞期胚)经由受体小鼠输卵管口移植、经由受体小鼠输卵管壁移植及对小鼠进行子宫移植(8细胞期胚)的方法进行了实验研究,并对各方法适用于使用的动物对象、实验要求、产仔率、优势与不足等做了进一步探讨,为今后的实验动物提供参考依据。结果表明,输卵管壁移植优于输卵管伞口移植和子宫移植。     

Successful transfer of biopsied equine embryos

Embryo biopsy has been used to detect inherited disorders and to improve the phenotype by analyzing of linkages between marker loci and the desired characteristics. Unfortunately, early procedures required the removal of a large portion (one-half) of the embryo for analysis, and the transfer of bisected equine embryos has not been particularly successful. Recent discovery of the polymerase chain reaction (PCR) has made possible the detection of specific DNA sequences from only a few cells. We investigated whether the removal of a small biopsy would allow for successful PCR and normal embryonic development. In the study reported here, 14 microbladebiopsied Day 6 to 7 equine embryos were transferred nonsurgically into recipient mares. The sex of each embryo was determined from the biopsy by means of restriction fragment length polymorphism analysis of the ZFY/ZFX loci after PCR amplification. The embryos were sexed as 8 females and 6 males on the basis of PCR assay results. Two embryos were biopsied using a needle aspiration technique, but no PCR amplification products resulted from these attempts. Eight intact control embryos were transferred to recipient mares using the same method. Pregnancy rates were 3 14 and 6 8 for the microblade biopsy and control groups, respectively. All of the microblade biopsy group pregnancies were females. One was aborted for cytogenetic analysis. Two were born after normal gestation. With improved pregnancy rates, this technique could be used for preimplantation diagnostics of equine embryos. As gene mapping advances and associations between particular DNA sequences and inherited traits become established, a rapid PCR technique could be used to select embryos before transfer.     

A simple technique for nonsurgical embryo transfer in mice

A simple method was developed for nonsurgical transfer of mouse embryos which enabled transfer to both uterine horns. Embryos were picked up in a modified capillary tube and transferred through the cervix into each uterine horn of an unanesthetized mouse. A glass speculum was used to facilitate location of the cervix. The technique was found to be as successful (up to 60% of embryos transferred developed to term) as surgical methods yet was simpler and eliminated surgical trauma to the recipient mouse.     

Early developing embryos affect the gene expression patterns in the mouse oviduct

Fertilization and development of mouse embryos occur in the ampullae of oviduct. We hypothesize that fetal-maternal communication exists in the preimplantation period, allowing optimal development of embryos. It is known that embryotrophic factors from oviduct affect the development of embryos. Although embryos affect their own transport in the oviduct, the mechanism of action is unknown. As a step toward understanding the action of embryos on oviductal physiology, we adopted suppression subtractive hybridization (SSH) to compare the gene expression in the mouse oviduct containing early embryos with that of oviduct containing oocytes. Ten to twelve 1-cell mouse embryos were transferred to one oviduct of a foster mother and similar number of oocytes were transferred to the contralateral oviduct. The animals were sacrificed after 48 h and their oviducts were excised for mRNA study. Using SSH, we screened out 250 putative positive clones from the subtracted embryo-containing oviduct library and 97 of them were screened positive by reverse dot-blot analysis. DNA sequence analysis identified genes that shared high homology with sequences in GenBank/EMBL database with unknown functions. Overall, 13 of the 90 high-quality sequences (14%) were homologous to 6 different genes previously described. Reverse Northern analysis confirmed that the expression of these genes were higher in the embryo-containing oviduct than in the oocyte-containing oviduct. About 12% of these clones (11/90) were novel. This article is the first to report identification of genes in the oviduct that are upregulated in the presence of embryos during the preimplantation period.     

Survival of mouse embryos after being frozen in glycerol-sucrose mixture

Random bred female albino mice (6-8 weeks old) were used as a source of embryos. 8- to 16 cell embryos were dehydrated in glycerol-sucrose mixture in 0.25 ml straws at room temperature. Straws were cooled at the rate of 5 degrees C/min to -7 degrees C. Seeding was induced by touching the out side of the straw at -7 degrees C. Straws were further cooled at 0.5 degree C/min down to -35 degrees C and then plunged into liquid N2. Thawing of straws was done by direct transfer into water at 35 degrees C. Frozen-thawed embryos were cultured in a CO2 incubator maintained at 39 degrees C. Out 190 embryos (8-16 cell) initially frozen, 169 (88.94%) were recovered on thawing. 158 (93.5%) out of 169 were apparently normal and used for culture. 75 (47.46%) developed to morulae/early blastocysts and 72 (45.56%) to expanded blastocysts on 24 and 48 hr culture respectively. In conclusion, the incorporation of sucrose in the freezing medium at a concentration of 0.25 M has led us to propose a freezing, thawing and transfer method without dilution of glycerol. The technique being quite simple is worth trying in farm animals where importance of this technique in non-surgical transfer of frozen-thawed embryos will be a boon.     

In vivo culture of embryos in the immature mouse oviduct

In-vitro culture of mammalian preimplantation embryos is associated with subsequent decreased viability. This phenomenon is more pronounced with the domestic species embryos as culture conditions are at present unable to sustain cleavage of early preimplantation embryos for more than one or two cell divisions. In this study, the immature mouse oviduct is shown to be capable of supporting cleavage and morphological development of rabbit and porcine embryos. The immature mouse oviduct was shown to be comparable to in vitro culture as 76% and 60% of the transferred zygotes developed to the morula stage after 2 and 3 d respectively. The porcine zygotes, however, failed to develop beyond the 4-cell stage in either the immature mouse oviduct or in vitro. Porcine morula showed better tolerance of the oviduct environment and when recovered after 2 d contained an average of 64 cells, which was significantly more than in in vitro cultured morulae (40 cells). Early porcine blastocysts transferred to the mouse oviduct had over a two-fold increase in cell division (104 cells) over comparable blastocysts grown in vitro (57 cells). The immature mouse oviduct is, therefore, a potential surrogate environment for short-term storage of embryos of other species.     

Culture of one- and two-cell bovine embryos to the blastocyst stage in the ovine oviduct

The ovine oviduct was evaluated as a culture system for early bovine embryos. One- to two-cell embryos were collected from superovulated heifers killed 36 or 48 h after the onset of estrus, embedded in agar cylinders, and transferred to oviducts ligated at the uterotubal junction. After 5 d (6.5 to 7.0 d after donor estrus), embryos were recovered and evaluated for development to the late morula or blastocyst stage. In Experiment 1, 86 embryos were cultured in 10 ewes in which the onset of estrus was synchronized with that of the donors. Fifty-eight embryos (68%) were recovered; of these, 31 (53%) had continued normal development. In Experiment 2, development in ovariectomized versus intact cyclic ewes was compared. Recovery from ovariectomized ewes (26/39, 67%) did not differ from intact cyclic ewes (26/35, 74%) and the proportion developing normally also did not differ (ovariectomized: 7/26, 27%; intact cyclic: 11/26, 42%). In Experiment 3, embryo development was compared in anestrous versus ovariectomized ewes. Recovery rate (anestrous: 22/43, 51%; ovariectomized: 20/51, 39%) and the proportion developing normally (anestrous: 8/22, 37%; ovariectomized: 9/20, 45%) did not differ between treatments. Developmental competence of oviduct-cultured embryos was tested by transfer to 16 synchronous heifers, of which eight (50%) became pregnant; five delivered calves. Results indicate that the ovine oviduct provides an adequate site for the culture of early bovine embryos.     

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.     

Phospholipid transfer protein (PLTP) mRNA expression is stimulated by developing embryos in the oviduct

In mammal, fertilization and early preimplantation embryo development occurs in the oviduct. Evidence is accumulating that the oviductal epithelia secrete various biomolecules to the lumen during the secretory phase of the estrus cycle to enhance embryo development. This secretory activity of the oviduct is under the regulation of steroid hormones. Observations also suggested that the gametes and embryos modulate the physiology and gene-expressing pattern of the oviduct. However, the underlying molecular changes remain elusive. We hypothesize that the developing embryos interact with the surrounding environment and affect the gene expression patterns of the oviduct, thereby modulating the oviductal secretory activity conducive to the preimplantation embryo development. To test this hypothesis, suppression subtractive hybridization (SSH) was used to compare the gene expressions in mouse oviduct containing transferred in vitro cultured preimplantation embryos with that of oviduct containing oocytes during the preimplantation period. We reported here the identification and characterization of phospholipids transfer protein (PLTP), which is highly expressed in the embryo-containing oviduct and localized at the oviductal epithelium by in situ hybridization. PLTP contains signal peptide putative for secretory function. More importantly, PLTP mRNA increases in the oviductal epithelia of pregnant, but not pseudo-pregnant mice when assayed by real-time PCR. Taken together, our data suggested that PLTP may play important role(s) during in vivo preimplantation embryo development. This molecule would be a target to delineate the mechanisms and the roles of oviductal secretory proteins on early embryonic development.     

In vivo development of vitrified rabbit embryos: Effects on prenatal survival and placental development

The aim of this work is to study the effect of the vitrification procedure on prenatal survival and on placental development at the end of gestation in rabbits (Oryctolagus cuniculus). One hundred eighty-one females were slaughtered at 72 h of gestation. Morphologically normal embryos recovered at 72 h of gestation were kept at room temperature until transfer or vitrification. Vitrified embryos (320 embryos) were transferred into a total of 24 does and fresh embryos (712 embryos) were transferred into a total of 43 does. Females were induced to ovulate 72 h before transfer when fresh embryos were transferred and 60 to 63 h before transfer when vitrified embryos were transferred. Each recipient doe received eight embryos into the left oviduct and eight embryos into the right oviduct. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at Day 14 of gestation. Recipient females were slaughtered by stunning and exsanguination 25 d after the transfer, and fetuses were classified according to their status. Live fetuses and fetal and maternal placenta were weighed Pregnancy rate was defined as the total number of females having at least one live fetus at Day 28 of gestation divided by the total number of females. Prenatal survival was estimated as live fetuses at Day 28 of gestation divided by the number of transferred embryos. The pregnancy rate after transfer of vitrified embryos (92%) was similar to that achieved with fresh embryos (86%), but prenatal survival was lower for vitrified than for fresh embryos (53% vs. 34%). We did not find differences in embryo survival from 72 h to implantation. Transfer of vitrified embryos reduced fetal survival from implantation to Day 28 (57% vs. 82%). Differences in the number of live fetuses at Day 28 of gestation were mainly due to the higher fetal mortality observed soon after implantation in pregnancies resulting from the transfer of vitrified embryos. A higher percentage of decidual reactions and atrophic maternal placentas (27.5% vs. 8.3%) and also of atrophic fetal and maternal placentas (12.1% vs. 5.3%) were observed after transfer of vitrified embryos. Both treatments showed similar percentage of dead fetuses (3.3% vs. 4%). Maternal placenta of the fetuses from fresh embryos was 15% heavier than maternal placenta of fetuses from vitrified embryos.     

The freezability of biopsied bovine embryos

A biopsy of 1-5 blastomeres was taken from 86 bovine compacted morulae, using a technique, that did minimal damage to the zona pellucida. As soon as possible after the micromanipulation the embryos were frozen, without sealing the penetrated zona pellucida. In order to evaluate the viability of the biopsied frozen-thawed embryos, half of them were transferred to syncronized recipients while the remaining half were co-cultured to evaluate the developmental capacity. A control group of 43 intact embryos was subjected to the same procedures. This study revealed a slight, but not significant, decrease in the pregnancy rate of the biopsied embryos after freezing and thawing, although the embryos by co-culture with bovine oviduct epithelial cells revealed normal morphology and developmental rate. It is concluded that the described biopsy technique did not compromise the freezability of 7-day-old bovine embryos.     

Intrafallopian transfer of gametes and early stage embryos for in vivo culture in cattle

It may be possible to avoid inadequate in vitro culture conditions by incubating gametes or embryos in the oviducts for a short time. Ideally, an optimized procedure should be devised, combining in vitro and in vivo systems, in order to achieve synchronization in cattle. We transferred gametes as well as embryos in various stages of development and placed them into the oviducts. Embryos were recovered on Day 7 by flushing of oviducts and uterine horns. Blastocyst rates were determined on Day 7 and on Day 8. Experimental designs included transfer of in vitro matured cumulus oocyte complexes into previously inseminated heifers (COCs group), transfer of in vitro matured COCs simultaneously with capacitated spermatozoa (GIFTs group), transfer of four to eight cell stage embryos developed in vitro after IVM/IVF (Cleaved Stages group) and a group of solely in vitro produced embryos (IVP control group). Our results indicate that in vivo culture of IVM/IVF embryos in the homologous bovine oviduct has a positive influence on subsequent pre-implantation development. In addition, we have evidence that in vitro maturation and in vivo fertilization cannot be synchronized.     

Full-term development of rat after transfer of nuclei from two-cell stage embryos

Cloning technology would allow targeted genetic alterations in the rat, a species which is yet unaccessible for such studies due to the lack of germline-competent embryonic stem cells. The present study was performed to examine the developmental ability of reconstructed rat embryos after transfer of nuclei from early preimplantation stages. We observed that single blastomeres from two-cell embryos and zygotes reconstructed by pronuclei exchange can develop in vitro until morula/blastocyst stage. When karyoplasts from blastomeres were used for the reconstruction of embryos, highest in vitro cleavage rates were obtained with nuclei in an early phase of the cell cycle transferred into enucleated preactivated oocytes or zygotes. However, further in vitro development of reconstructed embryos produced from blastomere nuclei was arrested at early cleavage stages under all conditions tested in this study. In contrast, immediate transfer to foster mothers of reconstructed embryos with nuclei from two-cell embryos at an early stage of the cell cycle in preactivated enucleated oocytes resulted in live newborn rats, with a general efficiency of 0.4%-2.2%. The genetic origin of the cloned offspring was verified by using donor nuclei from embryos of Black Hooded Wistar rats and transgenic rats carrying an ubiquitously expressed green fluorescent protein transgene. Thus, we report for the first time the production of live cloned rats using nuclei from two-cell embryos.     

Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova.

Almost 30 years after the first successful in vitro fertilization (IVF) in golden hamsters (Mesocricetus auratus), we report that IVF hamster embryos can develop in a chemically defined, protein-free culture medium into morulae and blastocysts, and produce normal offspring after transfer to recipients. When examined 96 h post-insemination, 82% (160/200) of IVF ova had cleaved to at least 2 cells, 55% (97/200) had developed beyond the 4-cell stage, and 22% (38/200) had developed into morulae/blastocysts. In vitro development of IVF embryos to greater than or equal to 8 cells was absolutely dependent on hypotaurine. Twenty living offspring were produced from transfer of IVF embryos to recipients, with an overall success rate of 5% and 17% for oviductal (2-cell) and uterine (8-cell/morulae) transfers, respectively. In vivo-fertilized pronucleate embryos collected 3 h after egg activation were less able to develop in vitro than embryos collected only 6 h later, revealing a critical influence of the oviduct within the first hours of embryo development. Hypotaurine partly compensated for the decreased oviductal exposure of early 1-cell embryos. Establishment of a key role for hypotaurine in hamster embryo development, support of IVF embryos to morula/blastocyst stages in vitro, and production of living offspring after IVF embryo transfer are significant steps towards the goal of obtaining comparative data on preimplantation embryogenesis.     

Successful direct transfer of vitrified sheep embryos

The use of a simple cryopreservation method, adapted to direct transfer of thawed embryos may help to reduce the costs of embryo transfer in sheep and increase the use of this technique genetic improvement of this species. Two experiments were made to test a vitrification method that is easy to apply in field conditions. All embryos were collected at Day 7 of the estrous cycle of FSH-stimulated donor ewes and were assessed morphologically, washed in modified PBS and incubated for 5 min in 10% glycerol, for 5 min in 10% glycerol and 20% ethylene glycol and were transferred into the vitrification solution (25% glycerol and 25% ethylene glycol). All solutions were based on mPBS. Embryos were loaded in straws (1 cm central part, the remaining parts being filled with 0.8 M galactose in mPBS) and plunged into liquid N2 within 30 sec of contact with the vitrification solution. The straws were thawed (10 sec at 20 degrees C) and the embryos were either transferred directly or after 5 min of incubation in the content of the straw (followed by washing in PBS) into the uterus of a recipient ewe. In Trial 1, the pregnancy rates at term (72 vs. 72%) as well as the embryo survival rates (60 vs 50% respectively) were not different between fresh (n = 48 embryos) and vitrified (n = 50) embryos. In a second trial no difference was observed between vitrified embryos transferred after in vitro removal of the cryoprotectant (n = 86 embryos) or directly after thawing (n = 72) both in terms of lambing rate (67 vs. 75%, respectively) and embryo survival rate (lambs born/embryos transferred; 49 vs. 53%). This method of sheep embryo cryopreservation provided high pregnancy and embryo survival, even after direct transfer of the embryos.     

Aberrant cannabinoid signaling impairs oviductal transport of embryos

Ectopic pregnancy is a major reproductive health issue. Although other underlying causes remain largely unknown, one cause of ectopic pregnancy is embryo retention in the fallopian tube. Here we show that genetic or pharmacologic silencing of cannabinoid receptor CB1 causes retention of a large number of embryos in the mouse oviduct, eventually leading to pregnancy failure. This is reversed by isoproterenol, a beta-adrenergic receptor agonist. Impaired oviductal embryo transport is also observed in wild-type mice treated with methanandamide. Collectively, the results suggest that aberrant cannabinoid signaling impedes coordinated oviductal smooth muscle contraction and relaxation crucial to normal oviductal embryo transport. Colocalization of CB1 and beta2-adrenergic receptors in the oviduct muscularis implies that a basal endocannabinoid tone in collaboration with adrenergic receptors coordinates oviductal motility for normal journey of embryos into the uterus. Besides uncovering a new regulatory mechanism, this study could be clinically relevant to ectopic pregnancy.