Decreased embryonic survival of in-vitro fertilized oocytes in rats is due to retardation of preimplantation development

Immature female rats (60-65 g) were injected with 4 i.u. PMSG on Day -2 and allocated to 3 groups. On the evening of Day 0, rats in Groups I and II were allowed to mate. Embryos were collected on Day 4 (Group I, control morulae) or Day 5 (Group II, control blastocysts) and were transferred into the oviduct or uterine horn of Day-4 pregnant recipient rats. On the transfer side of the recipients, the bursa had been peeled from around the ovary to prevent endogenous oocytes from entering the oviduct. For Group III, unmated donors were killed 65-67 h after PMSG injection. Ovulated oocytes recovered from the oviducts were fertilized in vitro and transferred 16-18 h later. Embryos developing from in-vitro fertilized (IVF) oocytes were recovered on Day 5, separated into morulae (Group IIIm) and blastocysts (Group IIIb) and transferred into Day-4 pregnant recipients similar to control embryos. Some embryos from each group were used to determine the mean number of cells/embryo. Embryo recipients were killed on Day 20. After transfer, the development of IVF oocytes was retarded compared to control embryos. IVF morulae contained significantly fewer cells/embryo than did control morulae but were able to implant and grow to fetuses, in proportions similar to controls, if transferred into the oviduct of the recipients. These results suggest that the developmental potential of rat oocytes fertilized in vitro is limited due to asynchrony between the embryo and the uterine environment at the time of implantation, rather than possible defects incurred by the oocyte during the fertilization procedure.     

Oviductal and uterine influence on the development of Day-2 equine embryos in vivo and in vitro

The objective of this experiment was to contrast the influence of the oviductal and uterine environments on development of Day-2 embryos. Embryos were transferred to oviducts or uteri of synchronous recipient mares, or were incubated in oviductal co-culture, in uterine co-culture or in defined culture medium. Significantly more (P < 0.02) embryos transferred to the oviduct versus the uterus survived until Day 11 after ovulation (5 7 vs 0 7 , respectively). Significantly more (P < 0.001) embryos developed to expanded and hatched blastocysts in uterine co-culture than in culture medium (6 7 vs 0 7 , respectively). The rate of embryo development to expanded blastocysts was not significantly different (P > 0.1) in oviductal co-culture versus uterine co-culture (3 7 vs 6 7 , respectively), or in oviductal co-culture versus culture in medium (3 7 vs 0 7 , respectively). Three of 7 and 6 of 7 embryos developed to hatched blastocysts greater than 2000 mum in diameter during oviductal and uterine co-culture, respectively, while 0 of 7 embryos cultured in medium expanded to greater than 500 mum in diameter. Proportions of embryos that developed for at least 9 days.     

Survival of equine embryos co-cultured with equine oviductal epithelium from the four- to eight-cell to the blastocyst stage after transfer to synchronous recipient mares

In this study we examined the ability of equine oviductal epithelial cells (OEC) to support the development of four- to eight-cell equine embryos in vitro and investigated the ability of co-cultured embryos to continue normal development after transfer to synchronous recipient mares. Equine embryos obtained at Day 2 after ovulation were cultured with or without OEC for 5 days. Those OEC co-cultured embryos that reached the blastocyst stage and embryos recovered from the uterus at Day 7 were surgically transferred to synchronous recipient mares. Co-culture with OEC improved (P < 0.01) development of four- to eight-cell embryos to blastocysts compared to medium alone (11/15 vs 0/6) during 5 days in vitro. Embryos co-cultured with OEC were smaller (P < 0.05) and more delayed in development than Day-7 uterine blastocysts. There was no difference in the Day-30 survival rate of co-cultured blastocysts (3/8) or Day-7 uterine blastocysts (5/8) after transfer to recipient mares. These results indicate that co-culture with OEC can support development of four- to eight-cell equine embryos in vitro and that co-cultured embryos can continue normal development after transfer to recipient mares.     

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.     

Effect of asynchronous superinduction on embryo survival and range of blastocyst development in swine

The importance of uniform development of blastocysts was examined by comparing the effects of asynchronous superinduction (Day 6 embryos into Day 7 pregnant recipients and Day 7 embryos into Day 6 pregnant recipients) on the range of embryo development at Days 12 and 13 to subsequent survival to Day 30. Twenty gilts were used to produce five Day 7 recipients that received Day 6 embryos and five Day 6 recipients that received Day 7 embryos. Embryos from the Day 7 and Day 6 recipients were examined 6 days later. Recovered embryos ranged morphologically from spherical to filamentous blastocysts. This range of embryos was within the limits of that previously observed for naturally mated sows. However, recovered blastocysts from the Day 6 embryos transferred into Day 7 recipients were morphologically more variable and proportionately less developed than the blastocysts from the Day 7 embryos transferred into Day 6 recipients. Forty additional gilts were subsequently utilized to generate 20 recipients (10 recipients per transfer group) that were examined on Day 30. More Day 7 embryos transferred into Day 6 recipients survived (p less than 0.05) than Day 6 embryos transferred into Day 7 recipients. These experiments suggested that greater variation in early development of embryos, within litters, subsequently resulted in greater mortality of embryos.     

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)     

Survival of day-4 embryos from young, normal mares and aged, subfertile mares after transfer to normal recipient mares

The estimated embryonic loss rate between Days 4 and 14 after ovulation for young, normal mares (9%) was significantly lower (P less than 0.01) than the estimated embryonic loss rate for aged subfertile mares (62%). Fertilization rates, which were based on the recovery of embryos at Day 4 after ovulation, were 96% and 81% (P less than 0.1) for normal and subfertile mares, respectively. Day-4 embryos were collected from the oviducts of normal and subfertile donors mares. These embryos were transferred to the uteri of synchronized, normal recipient mares to test the hypothesis that the high incidence of embryonic loss in subfertile mares was related to embryonic defects. The hypothesis was supported because embryo survival rates were significantly higher (P less than 0.05) for Day-4 embryos from normal compared to subfertile mares. These defects may have been intrinsic to the embryo or might have arisen due to the influence of the oviducal environment before Day 4 after ovulation.     

Production of identical twins by bisection of blastocysts in the cow

Day-8 embryos were recovered by a non-surgical method from superovulated crossbred heifers. Normal expanded blastocysts with a distinct inner cell mass and a trophoblast were released from the zona pellucida and bisected along a sagittal plane into two 'half' blastocysts. Each 'half' blastocyst was replaced in an empty zona pellucida and cultured for 2 h in B2 medium. After culture the 'half' blastocysts were directly transferred to recipient heifers via the cervix. From 11 blastocysts, 11 monozygotic 'half' blastocyst pairs were transferred to 11 recipients: 8 recipients became pregnant, 4 carried twins and one delivered a normal calf and an acardiacus amorphus monster consisting of disorganized embryonic tissues. A further 11 'half' blastocysts were transferred as singletons to 11 recipients. Five recipients were apparently pregnant at Day 42. One returned to oestrus at Day 45, 3 were carrying normal fetuses and 1 a pair of normal twin fetuses when slaughtered at Day 128. It is concluded that even after the first irreversible cellular differentiation which occurs at the blastocyst stage it is still possible to produce identical cattle twins by bisection of the Day-8 blastocyst.     

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.     

Abnormal expression of matrix metalloproteinase-2 and -9 in interspecific pregnancy of rat embryos in mouse recipients

The high failure rate of interspecific pregnancy is a major obstacle to the successful interspecific cloning of mammals. Embryo transfer between rats and mice provides a unique model for studying the causes of such failures. Previous research has shown that the upper time limit for the survival of rat embryos in mouse uteri was the seventh day of pregnancy (Day 7). To study the reasons for the failure of interspecific pregnancy between rats and mice, we transferred rat blastocysts into mouse uteri on the third day of pseudopregnancy. Unexpectedly, intact rat embryos could still be observed in mouse uteri on Day 9 and the implantation rate was as high as 30.6%. However, compared with mouse embryos, the further development of transferred rat embryos in mouse uteri was retarded. On Day 10, transferred rat embryos shrank with much blood. From Day 11 on, they lost their intact structure and the recipient uteri developed dropsy. On Day 12, the embryos shrank further and completely separated from the mouse uteri. By Day 13, they had been absorbed without any remains. In an in vitro co-culture (CT) system, the attachment rate of rat embryos on a monolayer of mouse uterine epithelial cells was similar to that of mouse embryos, but the outgrowth rate of rat embryos was significantly lower. Further investigation by gelatin zymography showed that matrix metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) activities in transferred rat embryos was significantly less than in mouse embryos. The same result was obtained in the in vitro CT assay. These results suggest that rat embryos can complete adhesion but not the invasion when transferred into mouse uteri. The reduced invasive ability, and especially, the associated reduction of MMP-2 and -9 activity, is one of the reasons for the failure of interspecific pregnancy.     

Effect of variation in embryo stage on the establishment of pregnancy, and embryo survival and growth in ewes with two embryos

Embryos at different stages of development were transferred to recipient ewes on Day 6 to investigate the effect of variation in stage of development on embryo survival and growth. Three groups of ewes received 2 embryos that were at the same stage of development, Day 4, Day 6 or Day 8. A fourth group received 1 Day-4 and 1 Day-8 embryo. At autopsy on recipient Day 34 there were no significant differences in embryo survival (Day 4, 34%; Day 6, 50%; Day 8, 46%; and Day 4 and 8, 48%). Fetuses developing from Day-8 embryos were heavier than others (Day 4, 1.10 +/- 0.06 g; Day 6, 1.15 +/- 0.06 g; Day 8, 1.41 +/- 0.08 g; P less than 0.05). In Group 4 neither survival nor growth of embryos was significantly affected by the presence of an embryo at a different stage of development. The ability of the uterus to stimulate development of a relatively retarded embryo is confirmed. Apparently the uterus has less effect in slowing the development of advanced embryos.     

The effect of recipient age on the success of embryo transfer in mice

Eight-hundred-sixteen morulae and blastocysts were transferred to the uteri of 102 recipients of various ages on Day 3 or 4 of pseudopregnancy (Day 1 is the day of the vaginal plug). Day 3 recipients had significantly higher pregnancy rates and embryo survival rates than Day 4 recipients. Recipient age had little effect on pregnancy rates, but had a significant effect on embryo survival in Day 3 recipients. Day 3 recipients of 11-13 weeks of age had the highest pregnancy rate (100%) and embryo survival (75%). The results suggest that recipient age should be considered an important factor in embryo transfer experiments.     

The effects of cryopreservation and transfer on embryonic development in the common marmoset monkey, Callithrix jacchus

Embryos were collected at the 4-10-cell stage from the oviducts (Day 4; Day 1 = ovulation) or as morulae (Day 7) from the uterus of marmosets and frozen in 1.5 M-DMSO (Days 4 and 7) or 1.0 M-glycerol (Day 4 only), using a slow freezing and thawing technique. Of 22 Day-4 embryos frozen in DMSO, 18 were recovered and 16 of these were transferred to 10 synchronized recipients; 7 recipients became pregnant compared with all 7 control recipients receiving 10 unfrozen embryos. Fifteen frozen-thawed morulae were transferred to 9 Day-6 recipients; the pregnancy rate (55.6%) was lower than for control embryos (85.7%). Embryos frozen in glycerol suffered severe osmotic stress during glycerol addition and removal. Of 8 recipients, 3 (37.5%) became pregnant but only one fetus was carried to term. These results on embryo collection, freezing and transfer in the marmoset have important implications for developing improved methods for freezing human embryos and the breeding of endangered primates.     

Survival of biopsied and sexed bovine demi-embryos

The viability of sex-diagnosed bovine demi-embryos was investigated after transfer. Day-7 morulae and blastocysts were subjected to splitting and biopsy in PBS + 4mg/ml polyvinylpyrrolidone + 200mM sucrose using a microblade. The biopsy (approximately 2 to 8 blastomeres) was transferred to a tube, and its presence in the tube was verified by examination under a stereomicroscope. After proteinase K treatment, repetetive male-specific DNA was amplified by the polymerase chain reaction (PCR). No autosomal control primers were used in the PCR. Instead, the absence of a characteristic Y-specific product together with the amplification of non-specific products was considered an indication of a female sample. The biopsied demi-embryos were transferred either singly or in pairs to synchronous heifer or cow recipients 6 to 10 h after flushing. Sex diagnosis was carried out within 6 to 7 h. Of 19 original embryos, 7 were diagnosed as males and 5 as females. The DNA of the biopsies of the remaining 7 embryos did not result in any amplification products. Since 5 of these samples were seen in the tubes prior to PCR, the corresponding embryos were considered "potential females." The sex of the last 2 samples could not be determined. Nine of 10 embryos were correctly sexed as revealed by calving data. Of the 38 transferred demi-embryos, 16 had developed to live fetuses as detected by ultrasonography on Day 65 of pregnancy. Eleven live calves and three stillborn calves were delivered. After bisection, biopsy and single transfer, 6 live calves were born from 7 original embryos (86%). After transfer of both halves into the same recipient, only 5 live calves from 12 original embryos were produced (42%). None of the 4 manipulated Grade-2 embryos survived to term, nor did any of the 4 manipulated blastocysts. Of the 14 original Grade-1 morulae manipulated and transferred, 15 were live fetuses at Day 65, and 11 live calves were born.     

Cellular composition and viability of demi- and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro

The cellular composition and viability of intact, IVP embryos were compared with those of demi- and quarter-embryos produced by bisection of IVP morulae and blastocysts. Embryos were produced by established techniques from oocytes harvested from slaughterhouse ovaries. In Experiment 1, morulae at Day 6 or blastocysts at Day 7 were bisected on an inverted microscope using a microsurgical steel blade. Demi-embryos were then cultured without a zona pellucida until Day 8, when they were morphologically assessed for quality (viability). A higher proportion of demi-embryos made from blastocysts than from morulae were classified as viable (381/420, 91% vs 164/267, 61%; P < 0.001). In Experiment 2, only Day 7 blastocysts were bisected, and some of the resulting demi-embryos were bisected a second time 24 h later to produce quarter-embryos. The remaining demi-embryos, the quarter-embryos, and control intact embryos were cultured until Day 9, at which time they were assessed for quality and subjected to immunosurgery and differential staining to count inner cell mass (ICM) and trophectoderm cells. A higher proportion of demi-embryos than quarter-embryos was classified as viable (408/459, 89% vs 223/319, 70%, respectively; P < 0.001). Total cell numbers decreased with successive bisections, but the proportion of surviving cells found in the ICM was significantly (P < 0.05) higher in the best quality demi- and quarter-embryos (35 and 32%, respectively) than in the controls (22%). Transfer of all 12 quarter-embryos derived from 3 blastocysts, in pairs, into 6 recipient heifers resulted in 2 pregnancies, each with a single viable fetus at 90 d of gestation. The fetuses originated from 2 different blastocysts. The results suggest that bisection of intact IVP embryos into demi-embryos and bisection of those into quarter-embryos can increase the number of transferable embryos by as much as 178 and 235%, respectively.     

Viability of frozen-thawed bovine embryos bisected in sucrose: A preliminary report

A method for producing identical twin calves is described in which Day 7 frozen-thawed bovine embryos in 12.5% sucrose solution were bisected using a fine microsurgical blade. The resulting bisected embryos were transferred nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (+/-1 d), two bisected embryos per recipient. The pregnancy rate when both halves remained in the same zona pellucida was 50% (5 10 ); the pregnancy rate was 1 5 for morulae and 4 5 for blastocysts. The pregnancy rate for unfrozen morulae bisected in PBS and transferred without zona pellucida was 27% (4 15 ). The in vitro survival rate of embryos bisected in 12.5% sucrose when both halves remained in the original zona pellucida was 82% (18 22 ), which was higher than when embryos were bisected in PBS (53%, 9 17 ).     

Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in-vitro matured follicular oocytes

Bovine follicular oocytes surrounded by cumulus cells for more than one-third of their surface were matured, fertilized and developed in vitro utilizing a co-culture system with bovine cumulus cells. Embryos developed into blastocysts were non-surgically transferred to the uteri of cows at Day 6, 7 or 8 (Day 0 = oestrus). Out of 6 recipient cows (19 blastocysts transferred), 3 became pregnant. One of the 3 pregnant cows carried twins. The results of this study demonstrated the viability of embryos obtained from in-vitro maturation of bovine oocytes followed by in-vitro fertilization and culture to the blastocyst stage in vitro.     

Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles

Early bovine embryos (1- to 8-cell stages) were recovered from superovulated heifers at slaughter on Days 2 or 3. Embryos were cultured for 3-4 days in Medium B2 supplemented with 15% (v/v) fetal calf serum in the absence (B2SS, 106 embryos) or presence of trophoblastic vesicles (B2SS + TV, 190 embryos). At the end of culture, there were more (P less than 0.001) morulae (greater than or equal to 16 cells) in B2SS X TV (46%) than in B2SS alone (18%) irrespective of the initial cell stage. More 8-cell embryos reached the 16-cell stage than did embryos with less than 8 cells (30% vs 15% in B2SS, P greater than 0.05; 70% vs 41% in B2SS + TV, P less than 0.005). After culture, 102 morulae were transferred non-surgically to temporary recipient heifers (84 embryos cultured in B2SS + TV and 18 in B2SS). After 2 or 3 days, 14 out of 58 embryos from the B2SS + TV group and 3 out of 10 embryos from the B2SS group were recovered as blastocysts. Most blastocysts were deep-frozen and stored for several weeks. After thawing, 10 apparently normal embryos from the B2SS + TV group were transferred non-surgically into 10 recipient heifers. Four pregnancies were induced, but only one embryo survived to term (birth of a normal male calf). It is concluded that trophoblastic vesicles release one or several unknown compound(s) normally present in vivo, promoting the cleavage of early bovine embryos.     

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.     

Embryo survival and conceptus growth after reciprocal embryo transfer between Chinese Meishan and Landrace x Large White gilts

Embryos were transferred between Meishan and Landrace x Large White (control) gilts on Day 4 or 5 to establish approximately equal numbers of all four possible combinations of donor breed and recipient breed. The breed of the donor gilt significantly (P less than 0.01) affected embryo survival with 44.5% of transferred Meishan embryos and 69.6% of transferred control embryos surviving to Day 30 +/- 1. There was no influence of the breed of the recipient gilt on the proportion of embryos which survived. These differences in embryo survival between the two breeds could not be explained by differences in (1) the number of embryos transferred, (2) the stage of development of the embryos transferred, (3) the interval between ovulation and transfer or (4) the degree of asynchrony between donor and recipient gilt. On Day 30 +/- 1 embryos from control donors developed into longer fetuses (P less than 0.01) with larger allantoic sacs (P less than 0.05) than did embryos from Meishan donors. Fetuses in control recipients were longer (P less than 0.01), heavier (P less than 0.001) and had larger allantoic sacs (P less than 0.05) than fetuses occupying Meishan uteri. The interaction between breed of donor gilt and breed of recipient gilt did not significantly affect conceptus growth. These results suggest that Meishan pig embryos may be less tolerant to routine embryo transfer procedures than those of control gilts, that the genotype of the dam does not affect the proportion of embryos surviving to Day 30 +/- 1, and that both fetal and maternal factors affect conceptus growth.