Antagonists of embryo-derived platelet-activating factor act by inhibiting the ability of the mouse embryo to implant.

This study utilized the transfer of preimplantation embryos to pseudo-pregnant mice to determine whether PAF-antagonists act primarily on the maternal or embryonic components of implantation. The first experiment used reciprocal embryo transfers, in which blastocysts from mice treated with PAF antagonist (SRI 63-441) or saline (controls), from Days 1 to 4 of pregnancy, were transferred to Day-3 pseudo-pregnant recipients which were also treated with SRI 63-441 or saline on Days 1-4 of pregnancy. The antagonist (40 micrograms) was administered at 16:00 h on Day 1 and at 09:00 h on Days 2-4 of pregnancy. The percentage of the transferred embryos which implanted was determined on Day 8 of pregnancy. Treatment of the recipient or the donor female with SRI 63-441 resulted in a reduction in implantation rate, from a control level of 45% to 33.8% or 34.7% (P less than 0.0002, P less than 0.007) respectively. These results suggest that the PAF antagonist affected implantation at the embryonic and maternal levels. However, when the blastocysts were transferred to Day-4 pseudopregnant recipients, treatment of the donor female had a dramatic effect on the implantation rate, resulting in a reduction of 64% (from 40% to 14.3%, P less than 0.04), while treatment of the recipient female had no significant effect. In this later experiment the transferred embryos were exposed to the recipient uterine environment for a shorter period before implantation. These results suggest that PAF antagonists affected implantation at the embryonic level and did not adversely affect maternal physiology.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of superovulation induction on embryonic development on day 5 and subsequent development and survival after nonsurgical embryo transfer in pigs

To evaluate the effects of eCG dosage on recovery and quality of Day 5 embryos and on subsequent development and survival after embryo transfer, batches of 5 to 10 donor sows were treated with 1000 or 1500 IU eCG. Recipients from the same batch were synchronously treated with 800 IU eCG. Ovulation was induced with 750 IU hCG (72 h after eCG) in donors and recipients. Donors were inseminated and embryos were collected at 162 h after hCG (120 h after ovulation). Ovulation rate was lower using 1000 IU eCG (28.5+/-11.7; n=48) than 1500 IU eCG (45.7+/-20.3; n=32; P<0.0001). Embryo recovery rate (82.9+/-16.9%) and percentage expanded blastocysts (56.2+/-31.4%) were similar (P>0.05). Expanded blastocysts from each group of sows were pooled into 2 groups within eCG treatment, containing embryos from normally ovulating sows (< or = 25 corpora lutea [CL]) or from superovulated sows (> 25 CL). Average diameter and number of cells of a random sample of the expanded blastocysts per pool were recorded. The average diameter of blastocysts (160.5+/-11.5 microm) was not affected by eCG dosage or ovulation rate (P>0.10). The average number of cells per embryo was higher in the 1000 IU eCG group (84.3+/-15.3) than in the 1500 IU eCG group (70.2+/-1.9; P<0.05) but was similar for normal and superovulated donors within each eCG group (P>0.10). Of the 4 groups, litters of 28 to 30 blastocysts were nonsurgically transferred to 27 synchronous recipients. Pregnant recipients were slaughtered on Day 37 after hCG treatment to evaluate embryonic development and survival. Pregnancy rate for the 1000 and 1500 IU eCG donor groups was 71% (10/14) and 46% (6/13; P>0.10), respectively. The number of implantations and fetuses for the 1000 IU eCG groups was 12.9+/-3.0 and 11.1+/-2.7, and 14.2+/-7.0 and 10.5+/-4.6, respectively, for the 1500 IU eCG groups (P>0.10). After post-priory categorizing the litters of blastocysts to below or above the average diameter (158 microm) of the transferred embryos, irrespective of eCG dosage or ovulation rate, the pregnancy rate was 43% (6/14) and 77% (10/13; P<0.10), respectively. Post-priory categorizing the transferred litters to below or above the average number of cells per embryo litter, irrespective of eCG dosage or ovulation rate, showed no differences in pregnancy rates or number of implantations and fetuses (P>0.10). It was concluded that eCG dosage affects embryonic development at Day 7 after hCG, and this effect was not due to ovulation rate. Embryonic survival after nonsurgical transfer was not related to eCG dosage but tended to be related to the diameter of the blastocysts.     

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.     

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.     

Egg transfer in the Mongolian gerbil (Meriones unguiculatus) during lactational delay of implantation

Lactating pregnant gerbils in which one oviduct was ligated during early pregnancy were used on Day 6-14 p.c. as recipients for eggs (morulae and blastocysts) recovered on Day 6-12 p.c. from lactating donors. Eggs were transferred to the ligated (experimental) horn and the litter was removed. About half of the recipients had implantations in the non-ligated (control) horn 15 days after litter removal. Of these 40 females, 37 had implantations in the experimental horn and 32 had some normal fetuses present. Of 179 eggs transferred into the ligated uteri, 61% implanted and 41% developed into normal fetuses. The time after copulation of recipient and donor did not appear to influence the number of implantations or post-implantation development of the transferred eggs.     

Effects of transferring in vitro-cultured rabbit embryos to recipient oviducts on mucin coat deposition, implantation and development

A mucin coat is deposited on rabbit embryos during passage through the oviduct; rabbit blastocysts cultured from the 1-cell stage in vitro have no mucin coat. When cultured blastocysts are transferred to recipients, the lack of mucin coat might account in part for subsequent failure of pregnancy. We have investigated the possibility that mucin coat deposition is induced following transfer of in vitro 72 h-cultured blastocysts to oviducts of asynchronous or synchronous recipients. One-cell embryos were collected by flushing oviducts 19-20 h post-coitus and were cultured in vitro for 72 h until they reached the blastocyst stage. The blastocysts were transferred to the oviducts of recipients that were synchronized either with the donors (synchronous) or 1 day later than the donors (asynchronous). They were recovered after 24-48 h and the mucin coat thickness and embryo degeneration rate were measured. The degeneration rate of blastocysts recovered from uteri of synchronous recipients was higher than that from asynchronous recipients (72.2% vs 40.0%). The mucin coats around embryos recovered from oviducts of asynchronous recipients after 48 h were thicker than those from synchronous recipients. More asynchronous recipients were pregnant and gave birth to more pups than synchronous recipients. These results indicate that the oviducts of asynchronous recipients secreted more mucin around the transferred embryos, causing higher rates of implantation of the in vitro-cultured blastocysts.     

Capillary permeability changes in the uteri of recipient rabbits after transfer of blastocysts from indomethacin-treated donors

Blastocysts recovered from control or indomethacin-treated (10 mg/kg s.c. twice daily starting on Day 4.5 of pregnancy) donor rabbits were transferred to the uteri of Day-6 or 6.5 pseudopregnant recipients. The minimal time required to cause an increase in capillary permeability in the endometrium underlying control blastocysts was approximately 9 h. Blastocysts derived from the indomethacin-treated donors were depleted of PGE and PGF (determined by RIA) and were unable to produce any increase in capillary permeability during the same time period, although after 46 h in vivo the diameters of the implantation swellings related to control or indomethacin-treated blastocysts were not different. This suggests that, in the untreated recipients, blastocysts depleted of PGs can become replenished and then release these PGs in a site-directed manner. Indomethacin thus causes a delay rather than a complete inhibition of implantation. Incubation of the indomethacin-treated blastocysts in vitro led to replenishment with PGs, but such replenished blastocysts failed to induce an increase in capillary permeability within the same time-frame as control blastocysts. Evidence is presented that indomethacin is probably not the cyclooxygenase inhibitor of choice, since it interferes with PG uptake and efflux. Such an action could explain the failure of the replenished blastocysts to induce a normal increase in capillary permeability.     

Reduced survival in utero from transferred mouse blastocysts compared with morulae

Studies on the survival of mouse embryos revealed that fewer offspring were produced when blastocysts, rather than morulae, were transferred to foster mothers. Approximately 8–10 h after fertilization F1 hybrid eggs (C57BL/6J × LT/Sv) were collected and cultured to morulae (day 4) or blastocysts (day 5 ) before transfer into uteri of day 3 foster mothers. A few recipients were killed on day 8 of gestation and deciduae were examined histologically. Embryos developing from transferred morulae were found to lie deep within the deciduae and were surrounded by numerous, large blood islands. Conversely, embryos developing from transferred blastocysts implanted more distally to the maternal blood vessels with only a few blood islands surrounding the embryos. These observations, suggesting abnormal implantation with insufficient embyro nourishment, were confirmed when uteri of foster mothers were examined on day 19 of gestation. Although the proportion of implantations from transferred morulae or blastocysts was similar (42 % and 47%, respectively), significantly more of the implantations were resorbed after transfer of blastocysts (78%) as compared with morulae (15%). These results demonstrate that transfer of day 5 cultured blastocysts into uteri of foster mothers increases embryonic mortality as a consequence of improper implantation.     

Developmental potential of mouse follicular epithelial cells and cumulus cells after nuclear transfer.

The developmental potential after nuclear transfer of mouse follicular epithelial cells cultured in vitro was examined. Follicular epithelial cells surrounding growing oocytes (type 5, diameter of oocytes, 62.6 +/- 5.9 microm; n = 14) were obtained from ovaries of adult mice. Before nuclear transfer, cells were cultured for several passages and subjected to serum starvation for several days. When the nuclear-transfer oocytes were at the 2-cell stage, serial nuclear transfer was performed. Additionally, cumulus cells surrounding ovulated oocytes were used as nuclear donors, with or without thermal stimulation (from -25 degrees C to 60 degrees C for 10 min) before nuclear transfer. Nuclear-transfer oocytes with follicular epithelial cells developed into blastocysts (34%) after serial nuclear transfer, and 4 living fetuses on Day 10.5 (25%, 16 transferred) and 1 dead fetus on Day 19.5 of pregnancy (3%, 30 transferred) were obtained after transfer to recipients. Although blastocysts (20%) were obtained after serial nuclear transfer of cumulus cells, only one implantation site without a fetus was observed on Day 10.5 of pregnancy. Thermal stimulation of cumulus cells before nuclear transfer did not enhance the ability to develop into fetuses or blastocysts.     

Transfer of vitrified blastocysts from one or two superovulated Large White Hyperprolific donors to Meishan recipients: reproductive parameters at Day 30 of pregnancy

The present study was designed to determine the effect of pooling embryos from two donors on the reproductive success of transfer of vitrified/warmed porcine blastocysts. Intact blastocysts were collected from superovulated Large White Hyperprolific gilts (n = 24) on Days 5-5.5 after artificial insemination. Embryos were recovered by flushing the uterine horns, and unhatched blastocysts were selected. Vitrification and warming were performed as described by Berthelot et al. [Cryobiology 41(2000) 116]. To evaluate in vitro development, 37 vitrified/warmed blastocysts were cultured, non-vitrified embryos (n = 48) were used as controls. Embryo transfers were conducted in asynchronous (-24 h) Meishan gilts (n = 20). Twenty vitrified/warmed blastocysts were surgically transferred into one uterine horn. Ten recipients received embryos from one donor (Group 1) and the other 10 transfers were performed with mixed embryos from two donors (Group 2). Pregnancy was assessed ultrasonographically at Day 25 after estrus and recipients were slaughtered at Day 30 after transfer. In vitro survival rate of the vitrified/warmed blastocysts was lower (P < 0.01) than that from control embryos (73.0% versus 93.7%). The pregnancy rate for Group 1 (70%) was not different (P > 0.05) than that from Group 2 (90%). No significant differences were detected between Groups 1 and 2 for in vivo embryo development (number fetuses/transferred embryos in pregnant recipients) or in vivo embryo survival (number viable fetuses/transferred embryos in pregnant recipients). However, the in vivo efficiency (number viable fetuses/total transferred embryos) was higher (P < 0.05) when transfers were performed with embryos from two donors (19.5% versus 30.5%). These results indicate that pooling embryos from two donors increases the in vivo efficiency after transfer of vitrified/warmed porcine blastocysts.     

In vivo effect of leukemia inhibitory factor (LIF) and an anti-LIF polyclonal antibody on murine embryo and fetal development following exposure at the time of transcervical blastocyst transfer

Leukemia inhibitory factor (LIF) enhances in vitro murine preimplantation development in a time- and dose-dependent fashion. Knockout experiments have demonstrated that endometrial LIF is essential for in vivo murine implantation. We assessed the impact of LIF and an anti-LIF polyclonal antibody (pab) on in vivo development and developed a novel and successful nonsurgical method of embryo transfer for this species, a transcervical blastocyst transfer technique. The objectives of this study were to evaluate the effects of LIF and the anti-LIF pab on 1) implantation, resorption, pregnancy, and viability rates and 2) the overall structural and skeletal development. Two-cell embryos were recovered from superovulated mated donors, cultured to the expanded blastocyst stage, and transferred transcervically into pseudopregnant recipients. Exposure to 5000 U/ml LIF resulted in significant increases in implantation, pregnancy, and viability rates compared with controls. A similar dose of pab produced overall inhibitory effects with a significant decrease in implantation rate. Paradoxically, lower pab doses resulted in significantly increased viability rates. Exposure to LIF had no effect on fetoplacental development. However, pab treatments had variable but significant negative effects on placental length, ossification of the exoccipital bone, and vertebral space width compared with controls. Exposure of murine blastocysts to LIF at the time of transcervical transfer resulted in pronounced positive effects on implantation and pregnancy rates without affecting fetal development. A similar pab dose dramatically reduced implantation and pregnancy rates; at high and low doses, pab produced deleterious effects on placental and skeletal development.     

Factors affecting success of embryo collection and transfer in a transgenic goat program

During a goat transgenic program that took place in Israel from July 1995 to February 1996, Saanen (n = 343) and Nubian x Damascus (n = 378) crossbred goats of mixed ages were used as donors (n = 433) and recipients (n = 288). The effects of season, age, number of surgical procedures, previous hormonal treatments and ovulation rate on the number of microinjectable embryos collected were studied. Likewise, the effects of these parameters on the pregnancy rate as well as the number of embryos transplanted, endogenous progesterone concentrations and exogenous progesterone supplementation were studied in recipient does. Following superovulation with ovine follicle stimulating hormone, 85% of the does responded with 13.6 +/- 5.7 (mean +/- S D) ovulations/doe. Age, month and number of previous hormonal treatments significantly affected the ovulation rate. The average recovery rate was 70%, and it was affected only by the ovulation rate. Pronuclei were visualized in about 30% of the flushed embryos (including unfertilized ova), and those were microinjected with human serum albumin gene construct. About 68% of the injected embryos underwent at least one division during an overnight incubation, and those embryos were transferred, giving about 2.0 transferred embryos per ovulated donor. Of the recipients, 86% responded following synchronization with 3.1 +/- 1.6 (mean +/- S D) ovulations per doe. Breed and month had a significant effect on the ovulation rate. Two or three microinjected embryos were transferred to each recipient, resulting in more than a 40% pregnancy rate during September to November. Lower pregnancy rates were obtained before and after that period. By monitoring plasma progesterone concentrations in the recipients it was found that progesterone concentration was correlated with the ovulation rate. However, the pregnancy rate was not affected by progesterone concentration. During January and February, 30 to 50% of the recipients failed to develop functional corpora lutea (CL) following embryo transfer, which explained the lower pregnancy rate in those months. Of the 86 kids born 4 were transgenic.     

Requirement for progesterone priming and its long-term effects on implantation in the mouse

At least 48 hr of progesterone (P4) priming has been documented to be essential for P4 and estrogen to initiate implantation in the rat. However, the length of this P4 priming requirement for implantation in the mouse has not been experimentally defined. Therefore, our first objective was to determine the length of P4-priming requirement for implantation in the mouse. Day 4 blastocysts were transferred into the uteri of Day 5 or Day 6 pseudopregnant mice that were ovariectomized on Day 1 (= vaginal plug) and treated with a single injection of P4 and 17 beta-estradiol (E2) only on Day 5, or a single injection of P4 on Day 5 followed by a second injection of P4 plus E2 on Day 6, respectively. Although none of the transferred blastocysts implanted in the uteri of P4-unprimed recipients, 46% of the transferred blastocysts implanted into the uteri of all recipients that were first primed with P4 24 hour prior to a second injection of P4 and E2. These results suggest that in contrast to the rat, the mouse uterus requires at most 24 hr of P4 priming before P4 and estrogen can initiate implantation. Our second objective was to determine whether P4 priming has a long-term effect on implantation in the mouse. Our present results and those of others suggest that the mouse uterus is exposed to rising P4 levels for 24 hr prior to implantation on Day 4 of pregnancy. Therefore, in the present investigation, induction of implantation by an injection of P4 and E2 following 5 days of ovariectomy performed on Day 4 of pregnancy clearly suggests that once exposed to P4 for 24 hr, the mouse uterus retains a long-term effect, i.e., following P4 withdrawal for several days, 24 hr of initial P4 priming is no longer required for P4 and estrogen to initiate implantation. Our next objective was to explore whether this long-term effect of P4 priming on implantation can be prolonged and potentiated by increasing the length of initial P4 priming. Thus, when the mice were ovariectomized on Day 4 of pregnancy and treated with P4 beginning on Day 5 for 4 days, the long-term effect on implantation was prolonged (8 days vs 5 days following P4 withdrawal) and potentiated (94% vs 0% mice with implantation following 8 days of P4 withdrawal) as compared with those with no P4 priming after ovariectomy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Viability and development of 'tube-locked' mouse embryos

'Tube-locked' morulae and blastocysts were recovered from the ampulla of the oviduct of centchroman-treated mice between Days 4 and 12 post coitum and transferred to the uteri of pseudopregnant female mice. Pregnancy and implantation rates were lower and the post-implantation resorption rate was higher in the treated than in the control group. There was little difference in the pregnancy or implantation rates between embryos recovered on Days 4 or 12 post coitum, but the resorption rate increased with increasing duration of embryos in the oviducts and was 100% for the Day-12 embryos. The resorption rate was similar even when these embryos were transferred to the sterile uterine horn of unilaterally pregnant mice. Centchroman did not produce any deleterious effect on embryos which survived until Day 19 of pregnancy in foster mothers. The average fetal weight was also comparable to those of control fetuses.     

Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae

Embryos were collected nonsurgically from n?turally-cycling or superovulated donors 7 d after estrus. Forty-four morulae, early blastocysts and blastocysts classified as good to excellent were bisected using a fine glass needle to produce forty-four identical demi-embryos. The bisected demi-embryos, without zonae pellucidae, were nonsurgically transferred, either by twin or single transfer. An additional forty-eight embryos were collected from the same donors and transferred as a control. Among the twin transfers, 8 of the 13 recipients became pregnant (61.5%). Seven of them conceived twin fetuses (87%) and one a single fetus. However, only two sets of normal identical twin calves were obtained. Among the single transfers, 72.6% (45/62) of bisected embryos without zonae pellucidae resulted in pregnancy, of which 48.4% (30/62) were identical twins, and 24.2% (15/62) were singletons. Another 27.4% (17/62) of the recipients did not became pregnant. The pregnancy rate for whole embryos with zonae pellucidae was 72.9% (35/48). These data show that there was no significant difference between the pregnancy rates of bisected embryos without zonae pellucidae and whole embryos with zonae pellucidae transferred 7 d after estrus. Bisection of bovine embryos was simplified and even morula stage embryos were transferred without zonae pellucidae.     

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.     

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).     

Transfer of European mink (Mustela lutreola) embryos into hybrid recipients

The European mink is considered as a highly endangered Mustelidae species. The objective of this study was to explore the intriguing possibility of embryo transfer from European mink to closely related Mustelidae recipient females. To overcome interspecies pregnancy failure, embryos of European mink (Mustela lutreola) were transferred into hybrid females obtained after mating of European polecat (Mustela putorius) males and European mink (M. lutreola) females and vice versa. A total of 32 blastocysts were surgically flushed from the uteri of nine European mink donors and surgically transferred into six pseudopregnant hybrid recipients. One of the recipients received a single embryo and did not whelp. The remaining five recipients each received five to eight embryos and delivered kits. The overall success rate was 50% (16 kits/32 transferred embryos). For both male and female offspring, the average birth weight was lower in ET group when compared with naturally bred control population of European mink. The postnatal mortality rate was significantly higher in ET group as compared to controls: only 9 of 16 kits survived past the first week. At 10 days of age, the average weight for male offspring from the ET and control groups did not differ, although differences still persisted at this age for female offspring. At 3 months of age, the weight of male and female offspring in the ET group did not differ from European minks born after natural mating. We propose that transfer of European mink embryos to hybrid recipients be considered as a new experimental tool within the framework of ex situ approach conservation of this aboriginal European mustelid.     

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.     

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.