Numerical simulation of embryo transfer: how the viscosity of transferred medium affects the transport of embryos

Background

Embryo transfer (ET) is a key step of assisted reproductive procedures, where the transferred medium containing the embryos is injected into the uterine cavity through a transcervical catheter and blended with intrauterine fluid in the uterine cavity. This procedure determines the delivery sites of embryos in the uterine cavity and has crucial impact on the implantation. Due to practical restrictions and ethical issues, it is often difficult to perform an in vivo study in humans to examine factors that affect the motions and delivery of embryos during ET. Alternatively, mathematical modeling is a powerful tool to that end.

Results

A computational model is developed to simulate the intrauterine mixing flow and track the embryo motions. Two important factors affecting the intrauterine flow are studied via this model: the viscosity of the transferred medium and the injection speed. Numerical results show that the dispersion pattern and the final delivery sites of the embryos are significantly influenced by the viscosity of the transferred medium. Specially, increasing the transferred medium viscosity close to that of the uterine fluid can enhance the probability that the embryos are delivered close to the fundus and keep them from being dragged backward to the cervix during catheter withdrawal. In addition, a slow injection speed can lower the driving force on the embryo during ET, which can prevent the embryo from being injured.

Conclusions

Based on our study, the practice of using a transferred medium with similar viscosity to that of the uterine fluid and a slow injection speed is recommended for real embryo transfer procedures in clinic.

     

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.     

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.     

Successful implantation of in vitro cultured rabbit embryos after uterine transfer: A role for mucin

The functional role of the mucin layer for development of rabbit embryos was examined by uterine transfer of embryos with different thicknesses of mucin. Embryos collected at various intervals after human chorionic gonadotropin (hCG) injection were cultured until 90 hr post-coitum (p.c.) and transferred to the uterus of synchronized recipients. When embryos collected at 20 or 25 hr p.c. were used for transfer, no implantation occurred. By contrast, embryos collected at 35 or 40 hr p.c. developed to term at high rates (53 and 80%, respectively). The thickness of the mucin layer on the embryos was different between these two groups. Embryos collected before 25 hr p.c. have less than 11.2 ± 0.2 μm of thickness of mucin and embryos collected after 35 hr p.c. have more than 34.3 ± 5.5 μm. To examine whether mucin deposition is required for in vitro cultured rabbit blastocysts to continue development after uterine transfer, embryos were collected at 20 hr p.c., cultured for 60 or 70 hr in vitro, and then temporarily transferred to the oviducts of recipient does to add mucin. These embryos were recovered from the oviducts at 24 hr after transfer, classified according to the thickness of mucin deposition, and transferred again to the uterus of synchronized recipients. Twenty live young were obtained from 67 embryos with a 20–40 μm thick mucin layer. No live young were obtained from 57 embryos with less than a 20 μm thick mucin. The thickness of the mucin layer appears to be an important factor for successful implantation of rabbit embryos. © 1996 Wiley-Liss, Inc.     

Production of piglets from in vitro-produced embryos following non-surgical transfer

The objective of this study was to enhance procedures for producing piglets derived from in vitro-produced (IVP) pig embryos by non-surgical embryo transfer (ET). The effects of insertion length for the catheter, asynchrony between the age of donor IVP blastocysts and the recipient estrous cycle, and volume of transfer medium were investigated. The IVP blastocysts at 5 days after in vitro fertilization were placed into porcine zygote medium (PZM)-5 supplemented with 10% (v/v) fetal bovine serum (PZM+FBS) in a 0.25 mL plastic straw (21-40 blastocysts per straw) and then transferred into one uterine horn of recipients using the Takumi(?) catheter for deep intrauterine insertion. Successful production of piglets derived from IVP embryos was achieved following non-surgical ET when the catheter was inserted at more than 30 cm anterior to the spiral guide spirette. The efficiency of piglet production (percentage number of piglet(s) born based on the number of embryos transferred) was greater (P<0.05) in recipients whose estrous cycle was asynchronous to that of donors with a 1-day delay (8.3%) than in those with a 2-day (1.5%) or 3-day (0.9%) delay, while pregnancy and farrowing rates (10-40%) did not differ among treatments. When blastocysts were transferred into recipients with 1.0 or 2.5 mL PZM+FBS, there were no significant differences in farrowing rate (30-40%) or average litter size (4.5-6.7) between treatments. The results of the present study indicate that the insertion length of the deep intrauterine catheter and the degree of asynchrony between donor embryos and recipient estrous cycle influenced on pregnancy and birth outcome following non-surgical transfer of IVP blastocysts.     

A hierarchical Bayesian approach to modeling embryo implantation following in vitro fertilization

In vitro fertilization and embryo transfer (IVF-ET) is considered a method of last resort for treating infertility. Oocytes taken from a woman are fertilized in vitro, and one or more resulting embryos are transferred into the uterus, with the hope that at least one will implant and result in pregnancy. Successful implantation depends on both embryo viability and uterine receptivity. This has led to the development of the EU model for embryo implantation, wherein uterine receptivity is characterized by a latent binary variable U and embryo viability is characterized by a latent binomial variable E representing the number of viable embryos among those selected for transfer. The observed number of implantations is the product of E and U. Zhou and Weinberg (1998) developed a regression formulation of the EU model in which embryo viabilities are independent within patients. We extend their methodology to a Bayesian hierarchical framework that allows for correlation between the embryo viabilities and gives explicit characterization of patient-level heterogeneity. When some subjects have zero implantations, the likelihood for the hierarchical EU model is relatively flat and therefore using prior information for key parametersis needed. This provides a key motivation for adopting a Bayesian approach. The model is used to assess the effect of hydrosalpinx on embryo implantation in a cohort of 288 women undergoing IVF-ET because of tubal disease. Hydrosalpinx is a build-up of fluid in the Fallopian tubes, which sometimes leaks to the uterus and may reduce the likelihood of implantation. The EU model is well suited to this question because hydrosalpinx is thought to affect implantation by reducing uterine receptivity only. Our analysis indicates substantial subject-level heterogeneity with respect to embryo viability, suggesting the utility of a multi-level model.     

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.     

Aberrant expression of retinol-binding protein, osteopontin and fibroblast growth factor 7 in the porcine uterine endometrium of pregnant recipients carrying embryos produced by somatic cell nuclear transfer

The technique of somatic cell nuclear transfer (NT) is a useful tool to produce cloned animals for various purposes, but the efficiency to generate cloned animals using this technique is still very low. To improve the low efficiency in production of cloned pigs it is critical to understand the reprogramming process during development of cloned embryos, but it is also essential to understand the uterine function interacting with the transferred cloned embryos during implantation and placentation. Thus, to understand the uterine responsiveness to NT cloned embryos during pregnancy, we investigated expression of retinol-binding protein (RBP), osteopontin (OPN) and fibroblast growth factor 7 (FGF7), which play important roles in implantation and/or maintenance of pregnancy as a transport protein, an extracellular matrix protein and a growth factor, respectively, in the uterine endometrium in pigs. The uterine tissue samples were obtained by C-section from pigs with NT cloned normal (NT-normal) embryos and NT cloned abnormal (NT-abnormal) embryos and pigs with non-NT (Non-NT) embryos at term. Immunoblot analysis showed that expression of RBP and FGF7 decreased in the uterine endometrium of recipient gilts carrying NT embryos than in the endometrium of gilts carrying Non-NT embryos. Levels of OPN protein of 70 and 45kDa were not different in between the uterine endometrium of gilts carrying Non-NT and NT-normal embryos, but in the uterine endometrium of gilts carrying NT-abnormal embryos 70 and 45kDa OPN proteins increased compared to those in the endometrium of gilts carrying Non-NT embryos. Immunohistochemistry results showed that RBP expression was lower in the endometrial glandular epithelial cells, while OPN expression was higher in the endometrial luminal epithelial cells of the uterus of gilts carrying NT embryos than in the uterus of gilts carrying Non-NT embryos. Results of this study showed that maternal uterine genes were aberrantly expressed in the uterine endometrium of gilts carrying NT cloned embryos in varying degrees depending on the normality of the developing embryos. These results indicate that abnormal maternal-fetal interactions of the uterus carrying the developing NT cloned embryos may cause problems in development of cloned embryos.     

Ultrastructural studies on maternal-embryonic cell interaction during experimentally induced implantation of rat blastocysts to the endometrium of the mouse.

Rat blastocysts collected around noon on Day 5 of pregnancy were transferred to the uterus of the mouse on Day 3 of pseudopregnancy. Out of 430 rat blastocysts transferred, a total of 37 were recovered as xenogeneic implants from the recipient mice killed 36, 48, 52, 58, 72, 96, and 120 hr after transfer. None of the transferred blastocysts was found surviving in the host uterus beyond 96 hr after transfer. Electron microscopic examination of the recovered implants revealed that rat blastocysts can successfully undergo the stages of ovum implantation in the mouse uterus from the early attachment to the initial phase of the trophoblastic invasion of the endometrium. During the late attachment stage, desmosomes (maculae adhaerentes), intermediate junctions (zonulae adhaerentes), and gap junctions (nexuses) were formed xenogeneically between the foreign trophoblast and the uterine epithelial cells of the host. Trophoblast cells of xenogeneic implants were destroyed shortly after the penetration of the basement membrane of the luminal epithelium of the host endometrium, leading to the degeneration and sloughing off of the transferred 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.     

Successful nonsurgical deep uterine embryo transfer in pigs

At present, it is possible to transfer pig embryos directly into the uterine body of sows by nonsurgical procedures. The aim of this study was to develop a procedure for nonsurgical embryo transfer (ET) into the upper part of one uterine horn in gilts and sows. In experiment 1, 29 gilts and 43 sows were used. Intrauterine insertions took place for each female at days 4-6 of the estrous cycle (D0 = onset of estrus). An artificial insemination (AI) spirette was inserted into the cervix to assist with the guidance of a modified flexible catheter originally developed for deep intrauterine insemination in pigs. The flexible catheter length inserted anterior to the inserted AI spirette was 43.0 +/- 1.7 cm. The time required to complete the procedure was affected by the type of female (P < 0.001) and by the difficulties encountered for inserting the catheter (P < 0.001). However, when no or minor difficulties were encountered during the insertion of the catheter (in approximately 70 and 80% of gilts and sows, respectively), the time required to complete the procedure did not differ between gilts (2.5 +/- 0.1 min) and sows (2.3 +/- 0.1 min). In experiment 2, 24 to 31 fresh morulae and/or blastocysts were transferred to each of 24 recipients. Seventeen animals (70.8%) farrowed an average of 6.9 +/- 0.7 piglets, of which 0.6 +/- 0.3 piglets were born dead. In conclusion, the procedure described in this study offers new possibilities to transfer embryos nonsurgically to the uterine horn of pigs.     

Pregnancy rates and gravid uterine parameters in single, twin and triplet pregnancies in naturally bred ewes and ewes after transfer of in vitro produced embryos

The objectives of this study were to: (1) evaluate the pregnancy rates after transfer of embryos produced in the presence or absence of epidermal growth factor (EGF) during in vitro maturation, and (2) compare several variables of the gravid uterus on day 140 after fertilization in single, twin and triplet pregnancies in ewes (n = 12) bred naturally and in ewes (n = 18) after transfer of embryos produced in vitro. Oocytes collected from FSH-treated ewes (n = 18) were collected from all visible follicles and cultured in maturation medium with or without EGF. Oocytes were then fertilized in vitro by frozen-thawed semen. On day 5 after fertilization, embryos with > or = 16 cells were transferred to recipient ewes (n = 39). In addition 12 ewes were bred naturally. Pregnancy was verified by real-time ultrasonography on day 45 or later after embryo transfer (ET) or breeding. On day 140 of pregnancy, the reproductive tract was collected from all ewes and the following parameters were determined: the number, sex, weight and crown to rump length (CRL) of fetuses, weights of gravid uterus and fetal membranes, and weight and number of placentomes. Presence of EGF in maturation medium increased (P < 0.04) cleavage rates (78% versus 59%) and percentage of > or = 16 cell embryos on day 5 after fertilization (62% versus 40%). Pregnancy rates tended to be greater (P < 0.1) after transfer of embryos matured in the presence of EGF (52%) than in the absence of EGF (39%). EGF presence in maturation medium did not affect any variables of gravid uterus or fetal weight. For single pregnancies in naturally bred ewes and ewes after ET all uterine variables were similar. For twin pregnancies, weight of gravid uterus, weight of uterus plus fetal membranes, total weight of placentomes/ewe, mean weight of individual placentome, mean weight of fetus, total fetal weight/ewe and CRL were greater (P < 0.0001-0.04) for ewes after ET than for ewes bred naturally. The weights of gravid uterus, fluid, uterus plus fetal membranes, fetal membranes, total placentomes/ewe, mean weight of individual placentome and total fetal weight/ewe were greater (P < 0.0001-0.08) for triplet pregnancies in ewes after ET than single and twin pregnancies in ewes naturally bred or after ET. The number of placentomes/fetus was greatest (P < 0.0001-0.06) in single pregnancies in ewes bred naturally and after ET fewer in twin pregnancies in ewes bred naturally and after ET and fewest in triplet pregnancies in ewes after ET. The total number of placentomes/ewe was greatest (P < 0.0001-0.06) for twin pregnancies in ewes naturally bred, fewer in single pregnancies in ewes naturally bred and twin and triplet pregnancies after ET, and fewest in single pregnancies in ewes after ET. The mean weight of fetus was greater (P < 0.0001-0.07) in single pregnancies in ewes naturally bred or after ET than in twin or triplet pregnancies in ewes naturally bred or after ET. The CRL was the lowest (P < 0.01) in twin pregnancies in ewes bred naturally. For pregnancies after natural breeding and after ET, the number of fetuses/ewe was negatively correlated (P < 0.03-0.0001) with the weight of placentomes/fetus, the number of placentomes/fetus, the mean weight of the fetus and CRL, and was positively correlated (P < 0.0001-0.05) with weight of gravid uterus, the total number of placentomes/ewe and total fetal weight/ewe. These data demonstrate that the presence of EGF in maturation medium increases the rates of cleavage and early embryonic development, and has a tendency to enhance rates of pregnancy but does not affect variables of the gravid uteri in ewes after transfer of in vitro produced embryos. Transfer of embryos produced in vitro affected some uterine variables in twin but not single pregnancies to compare with pregnancies after natural breeding. In addition, culture conditions in the present experiment did not create large offspring syndrome. The low number of placentomes/fetus seen in triple pregnancies appears to be compensated for by the increase in the weight of each individual placentome.     

The Impact of Maternal Uterine Genotype on Postnatal Growth and Adult Body Size in Mice

Embryo transfers were used to demonstrate that the genotype of the mother providing the uterine developmental environment significantly influences postnatal growth and adult body size of her progeny. Irrespective of their own genotype, mouse embryos transferred into the uterus of an inbred strain with large body size (C3H) had greater body weights, longer tails and higher growth rates than those transferred into the uterus of a strain with small body size (SWR). Uterine heterosis on body size was smaller than progeny heterosis, and both progeny and uterine heterosis persisted in adult mice. Uterine litter size was significantly negatively associated with body weight, tail length, growth rate and the timing of developmental events. The inbred SWR strain was more sensitive to the embryo transfer procedure than the C3H strain, but effects due to embryo transfer were moderate. Prenatal uterine effects have ramifications for biotechnologies utilizing embryo transfer as well as predictions about evolutionary change by selection.     

Effects of asynchrony on rabbit blastocyst development

The development of synchronously and asynchronously transferred rabbit morulae (recovered at Day 3 p.c.) and blastocysts (recovered at Day 4 p.c.) was investigated before the anticipated time of implantation. The results obtained with various techniques (evaluation of gross morphology, measurement of diameter, thymidine incorporation, light and electron microscopy) led basically to the same conclusions. Embryos being asynchronously transferred to the uterus of recipient rabbits survived, at least in terms of certain cellular functions like cell proliferation, for more than 2 days. Development, however, was clearly retarded and ultrastructural examination revealed substantial cell damage. Some blastocysts showed, even after 3 days, normal growth and cell proliferation indicating considerable differences between individuals in the ability to compensate for suboptimal developmental conditions before implantation. In general, this ability was greater in the transferred Day-3 morulae than in the Day-4 blastocysts. Embryonic growth and the ability to dissolve the zona pellucida, to synthesize crystalloid bodies and to differentiate extraembryonic endoderm indicated the maintenance of some developmental functions under asynchronous conditions. Blastocyst development was influenced by the progestational stage of the recipient. At 1 day after transfer into asynchronous older uteri, blastocyst diameters were larger and cell proliferation was increased compared with all other groups, suggesting an attempt of the blastocyst to adjust to the more advanced maternal milieu. Development in asynchronous younger uteri was delayed. No comparable differences in development were found in cultured embryos for which the media had been supplemented with flushings from the same progestational uterine stages as used for transfer. Thymidine incorporation in cultured embryos did not differ between the various supplements (P greater than 0.05) and was generally lower than in chronologically aged asynchronously transferred embryos (P less than 0.05 for Day-3 and P less than 0.001 or P greater than 0.05 for Day-4 embryos).     

Effects of leukaemia inhibitory factor on endometrial receptivity and its hormonal regulation in rabbits

The effects of hormones on production of leukaemia inhibitory factor (LIF) and the uterine receptivity in rabbits were studied. In ovariectomised rabbits, LIF protein was not detected in control but upregulated by progesterone alone. Oestrogen had a slightly negative effect when the rabbits were treated with both oestrogen and progesterone. Mifepristone (Mi) inhibited the progesterone-stimulated production of LIF in rabbit uterus. The transfer of embryos to LIF-treated recipients significantly increased pregnancy rate (70%) and implantation rate (27%) as compared with control (pregnancy rate=40% and implantation rate=17%). The transfer of embryos to LIF and mifepristone-treated recipients significantly decreased pregnancy rate (30%) and implantation rate (9%). The results indicated that LIF protein had a beneficial effect on uterine receptivity and mifepristone prevented this effect.     

The effect of partial weaning on the rebreeding performance of primiparous Hereford heifers

One or two 6–7 or 9–10 day old blastocysts were transferred to 39 pure-bred or cross-bred Charolais heifer recipients.The embryos were collected after slaughter of the donors. An insemination catheter (Cassou gun) was used to transfer the blastocysts through the cervix into the uterus. Placement was approximately 5cm beyond the body of the uterus.Pregnancy was determined by slaughter of the recipients on the 45th day of gestation. Nineteen of the 39 recipients were pregnant. The survival rate of embryos transferred was as follows: 6–7 day old embryos: 7 out of 23 (30.4%); 9–10 day old embryos: 18 out of 36 (50%). All embryos: 25 out of 59 (42.3%). The difference between the 6–7 day old embryos and the 9–10 day old embryos was not significant. Twelve of the 20 recipients receiving a bilateral transfer were pregnant, and 6 were carrying twins. This is a satisfactory technique for use in field conditions for the transfer of bovine blastocysts.     

Invitro uptake of estradiol and progesterone in the hamster uterus relative to the time of embryo transfer and stage of development

The normal time for cleavage to the 2- and 8-cell stages of development of naturally ovulated hamster embryos is 25–27.5 and 59–61 h respectively, after ovulation and mating. The corresponding values for PMS-hCG treated hamsters were 33–35.5 and 62–64 h respectively. When 2-cell embryos, obtained by this timing method, were transferred to the oviductal bursa on Day 2 of the cycle, and when 8-cell embryos were transferred to the uterus on Day 3 of the cycle, implantation rates of 61.5 and 64.0% respectively, were obtained. The current study was conducted to determine the effects of transfer on estradiol and progesterone uptake by the uterine tissue on Day 14 after mating. A 2-fold increase in estradiol uptake was observed in superovulated, nonpregnant uteri when compared with nonsuperovulated animals. This level was also significantly higher than with nonsuperovulated pregnant animals and of animals receiving 2-cell embryo transfers. Estradiol uptake increases of 3.2 and 1.2-fold were noted for animals receiving 8-cell embryo transfers in naturally and superovulated groups respectively. Superovulation resulted in increased progesterone uptake. Transfer of 2- and 8-cell embryos resulted in a 15.8 and 109.9% increase in progesterone uptake respectively in naturally ovulated hamsters. Similar values for superovulated hamsters were 16.2 and 87.6% respectively. The 8-cell embryos, however, were transferred about four hrs prior to the time for normal 8-cell cleavage and this, coupled with increased estradiol uptake by the embryos themselves, resulted in an elevated estradiol and progesterone uptake in the uterine tissue even when measured on Day 14 of pregnancy. The degree of increase was less with superovulated animals receiving 8-cell embryos, reflecting higher levels of estradiol and progesterone uptake in control tissues. This could account for the slight delay in development of superovulated embryos.     

Exposure to warmer postoperative temperatures reduces hypothermia caused by anaesthesia and significantly increases the implantation rate of transferred embryos in the mouse

Embryo transfer (ET) is among the key factors determining the overall efficiency of transgenic technology in the mouse. A successful ET depends among other factors on the quality of the transferred embryos, foster mothers and anaesthetic reagents and on the transfer techniques. Anaesthesia-caused deaths and suboptimal ET procedures are factors which reduce the success of transgenic experiments and mouse colony maintenance. Here we compared the effects of two anaesthetic reagents-a ketamine/xylazine combination, and tribromoethanol (Avertin)-on the rates of implantation and development to term of mouse zygotes transferred into the oviducts of CD-1 foster mothers, and evaluated whether hypothermia caused by anaesthetics after the ET operation could be overcome by postoperative incubation of the foster mothers. We established two experimental groups of fosters, one of which was kept at room temperature (RT, 21 degrees C) with the other in an incubator (33 degrees C) overnight after ET. Rates of implantation, resorption and development to normal fetuses were evaluated by sacrificing the foster mothers on the 15th day of their pregnancy. Our results showed that regardless of the anaesthetic reagents used, the rates of implantation and of development to normal fetuses can be significantly improved by exposing the foster mothers to warmer temperatures (33 degrees C) immediately after the ET operation. These results may have important implications in increasing the success rate of ET with micromanipulated embryos.     

Embryonic and calving losses in bovine mixed-breed twins induced by transfer of in vitro-produced embryos to bred recipients

One or two in vitro-produced (IVP) Japanese Black (JB) cattle embryos at 8 days after in vitro fertilization were transferred to the contralateral uterine horn of previously bred Japanese Shorthorn (JSH) or JSH-JB cross recipients, and then the occurrence of early embryonic death, abortion during mid- and late gestation, and calving loss were recorded. The survival rate of embryos, including indigenous ones, was not affected by the number of embryos transferred, and a significantly higher twinning rate (68% of pregnant recipients at 80 days after transfer) was achieved when two IVP embryos were transferred, as compared with the rate when one IVP embryo was transferred (24%). In late ET (recipients at 8.5-9.0 days after the onset of oestrus), the embryo survival rate (22%) and the pregnancy rate (42%) at 80 days after ET were significantly lower than those rates in the synchronous ET (recipients at 8.0 days after the onset of oestrus; 47 and 79%, respectively). In the early ET (recipients at 6.0-7.5 days after the onset of oestrus), no significant differences from the synchronous ET were detected in these rates. Twenty-six percent of twin pregnant recipients were aborted during mid- or late-pregnancy, and 39% of twin calves were stillborn. The mean gestation length of the twin-bearing JSH dams (276 days) was 1 week shorter than that of the single-bearing JSH dams, and it was 2 weeks shorter than that of the JB dams bearing a single JB calf derived from the IVP embryos. The longer gestation length of single JB calves derived from IVP embryos resulted in a significantly higher mean birth weight than that of in vivo control calves with the standard length of gestation. In conclusion, the number of embryos to be transferred did not affect the embryo survival rate, and the transfer of two IVP embryos to previously inseminated recipients induced a significantly higher twinning rate during early pregnancy than that of one IVP embryo transfer. The incidence of embryonic losses during early pregnancy increased when Day 8 embryos were transferred to the recipients later in the oestrous cycle (>8.0 days). The results suggested that one cause of the high rate of abortions and stillbirths in twin-bearing dams is the difference in the mean gestation length between the native JSH and JB foetuses derived from transferred IVP embryos.     

Survival of equine embryos transferred to normal and subfertile mares

To test the hypothesis that an abnormal uterine environment was a cause of early embryonic loss in subfertile mares, morphologically normal embryos were transferred to normal mares (n = 20) and subfertile mares (n = 20), and embryo survival rates were compared. Embryos were recovered nonsurgically at Days 7 to 8 postovulation and transferred surgically to normal and subfertile mares that had ovulated on the same day or within 2 d after a donor. Survival of transferred embryos was monitored by ultrasonography of the recipient mare's uterus from Day 9 through Day 28 postovulation. There were no significant differences (P > 0.5) in the embryo survival rates at Day 12 (11 20 vs 9 20 ) or Day 28 (10 20 vs 8 20 ) for normal or subfertile mares, respectively. The uterine environment of subfertile mares was apparently adequate to support the development of transferred embryos from Days 7 or 8 through Day 28 postovulation.