Post-implantation development of demi-embryos and induction of decidual cell reaction in mice

Mouse demi-embryos that developed from bisected morulae were transferred to recipients. The eu-blastocysts (distinct inner cell mass and well-developed trophectoderm) contained cells equal to 51% of the controls that developed from zona-free morulae. The rate of decidual cell reaction induced by the eu-blastocysts was not significantly different from that of the controls, but the size of the deciduum containing the egg cylinder was significantly smaller on Day 5.5 of pregnancy (P < 0.001). A significant increase in embryonic loss was observed from Day 7.5 to Day 9.5 in the eu-blastocysts (P < 0.05), while the controls exhibited no significant difference. Although the embryos from the eu-blastocysts showed retardation of developmental stages and decreased size, they attained normal stages and size regulation up to 90% of that of the control on Day 10.5. The pseudo-blastocysts (poorly developed inner cell mass enclosed by trophectoderm) contained cells equal to 25% of those of the controls and showed less than a 10% developmental rate to the egg cylinder stage. The trophectodermal vesicles (no enclosed cells) and nonintegrated forms (disorganized clusters of cells) contained cells less than 18% of those of the controls. They showed lower rates of decidual cell reaction than those in the controls (P < 0.05), and no egg cylinder was found in the deciduum. The results indicate that a severe decrease in the number of embryonic cells affects the regulation of embryonic development and decidual cell reaction in the uterus.     

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 ).     

Lamb production using superovulation, embryo bisection, and transfer

In this study, 39 embryos from donor ewes superovulated with follicle stimulating hormone-pituitary (FSH-P) were bisected to produce pairs of monozygotic twin lambs for experimentation. Each pair obtained by bisecting 8-, 9- or 10-day-old embryos was immediately transferred surgically into a recipient ewe at the same physiological stage. Of the 39 recipients which received a pair of half-embryos by transfer into the uterine horn ipsilateral to the corpus luteum, 28 (72%) lambed. Eighteen of 28 recipients lambing (64%) produced pairs, i.e., 7 male and 11 female pairs. Ten of 28 lambings produced a single lamb, i.e., six males and four females. Overall yield (the number of lambs produced in relation to the number of embryos used) was 118%. This percentage tended to increase, depending on the day of collection (Day 8, 100%; Day 9, 118%; and Day 10, 131%).     

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.     

Sexing of half-embryos produced by microsurgical bisection of mouse morulae and production of chimeric mouse of defined sex composition by aggregating two sexed half-embryos

Using the halved morulae of mice obtained with microsurgical technique, the following two experiments were performed. 1) Sexing of half-embryos by chromosomal analysis and transfer of the half-embryos after determining the sex of the other monozygotic half. One half of the bisected embryo was cultured in Colcemid solution (0.04 micrograms/ml) to be ensured for chromosomal preparation. More than 50% (152/270) of the blastulated embryos from the halves could be sexed by direct sex chromosome analysis. Thirty-nine of the half-embryos of which the co-twin halves were sexed, were transplanted in to the uterine horns of 18 pseudopregnant mice, and twelve became pregnant. The autopsies of them on Day 18 to 20 of pregnancy, revealed the presence of 16 fetuses. The morphological sex of these fetuses thus obtained coincided completely with the previous judgement based on the chromosomal sexing. 2) Production of chimeras of defined sex composition by aggregating two half-morulae of defined sex. Out of 147 pairs of half-morulae of two different strains (ICR and C3H/He), which were replaced in pairs into empty zona pellucidae, 107 (72.8%) were aggregated successfully and developed in vitro into full expanding blastocysts of typical form. Among the 107 aggregate blastocysts, 31 were sexed for both component embryos by chromosomal analysis on the co-twin half-embryos. When these 31 blastocysts were transferred, 11 living offspring including 4 chimeras were obtained. Transfer of 12 male-male and 5 female-female aggregate blastocysts resulted in 8 males and 1 female, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pregnancy rates with bisected bovine embryos

A method for producing identical twin calves is described in which 5.5 to 7.5 day bovine embryos were bisected using a fine microsurgical blade. The resulting demi-embryos were transferred surgically or nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (+/- 2 days), one demi-embryo per recipient. The mean pregnancy rate per demi-embryo was 48% (157/330), and per whole embryo split, it was 95% (157/165). Pregnancy rate was dependent on the stage of embryonic development. The pregnancy rate from bisected early morulae, 16% (7/44), was lower than that from bisected early blastocysts, 60% (58/96) P<0.01. The rate of identical twin production per embryo split reflected these pregnancy rates. Bisected blastocysts resulted in more identical twin sets, 39% (24/62), than early morulae, 9% (2/22) P<0.01. When used with normal embryo transfer procedures, this technique provides a method for routine production of identical twin calves. Furthermore, more calves are produced per two demi-embryos than per whole embryo.     

Production of normal piglets from microsurgically split morulae and blastocysts

The embryo splitting technique was applied to pig embryos, and the developmental ability of the split embryos was examined by means of in vitro culture and transfer. Morulae, early blastocysts and blastocysts were collected from Landrace x Large White F(1) gilts which had been mated to Duroc boars. The embryos were bisected with a fine glass or alloy (PtIr) needle after the softening of zonae pellucidae. The halved-embryos, which had either been placed in zonae pellucidae or not, were transferred to recipient gilts immediately after the micromanipulation (Experiment 1) or after cultivation for 15 to 20 h (Experiment 2). In Experiment 1, two fetuses were obtained from one of three recipients which had received 12 half-embryos. In Experiment 2, three of five recipients became pregnant, and in one recipient, seven piglets of a litter were obtained from 12 zona-free half-embryos produced from the original seven blastocysts. The results obtained indicate that a simple method not requiring the encasing of split embryos into zonae pellucidae is satisfactory to produce viable half-embryos.     

Microsurgery on bovine embryos at the morula stage to produce monozygotic twin calves

Mature Brangus donor cows were superovulated with follicle stim-ulating hormone administered twice daily in intramuscular injections. On day 6.5 to 7 post-estrus, embryos were collected non-surgically using a phosphate-buffered saline medium. A total of 37 ova was collected, of which 28 were advanced morulae and early blastocysts. Twenty of these embryos were selected for micromanipulation with a radial-type Leitz micromanipulator. While the embryos were in a holding medium containing 10% fetal calf serum, three glass microinstruments were used to open the zona pellucida, remove the mass of blastomeres and bisect the embryo on a vertical plane. Halved embryos were inserted into bovine zonae and placed either as single half-embryos or twin half-embryos in 0.25 ml French straws with fresh holding medium. The micromanipulated embryos (demi-embryos) were then non-surgically transplanted, either as a single demi-embryo or as a twin demi-embryo pair, into the uterine horn of day 6.5 to 8 recipient beef females ipsilateral to the existing corpus luteum. Of the 14 micromanipulated embryos that were transplanted to recipients, pregnancy rates were 16.6% for the single demi-embryos and 62.5% for the twin demi-embryos. No pregnancies resulted from bisected blastocysts.     

Quick-splitting of bovine embryos

Described is a simplified method of bovine embryo bisection amenable to on-farm embryo transfer. Using a microblade operated by a hand-held micromanipulator, Day 7 bovine embryos were bisected while in the zona pellucida. With a vertical motion, the embryo was pinned between the blade and the bottom of a plastic petri dish and bisected. Demi-embryos were transferred nonsurgically (without zonae pellucidae) into synchronized recipients. Pregnancy rates were normal with 5 13 (38%) and 9 20 (45%) of recipients confirmed pregnant 70 to 80 d after receiving either twin or single half embryos, respectively. This compared to 12 28 (43%) of recipients becoming pregnant from transfer of whole embryos. These data confirm that bovine demi-embryos do not need zonae pellucidae on Day 7 and that simplified field methods of bisection give normal pregnancy results.     

Sex-dependent loss of bisected bovine morulae after culture and freezing.

The objective of this study was to determine the post-transfer survival rate of bovine embryos cultured between the time of bisection and freezing. In this experiment 158 morulae were bisected and both portions were cultured for 24-44 h either in vivo after transfer to sheep oviducts (n = 80 morulae) or in vitro (n = 78 morulae) in Ham's F10 medium with 20% fetal calf serum, with bovine oviduct cells or in medium collected from oviduct cultures (conditioned medium). After culture, half of each morula was fixed for cytogenetic sex determination (n = 125) and the other half was frozen. The frozen halves were later thawed and transferred (n = 115) to recipients, who were, if pregnant, slaughtered to determine the sex of the fetus. The culture resulted in better pregnancy rates than those previously reported for embryos frozen immediately after bisection. The sex of 49 (33 males, 16 females) of the fixed demi-morulae was determined, and 38 of the transferred demi-morulae established pregnancies (23 males, 10 females and 5 fetuses that were not recovered). The male:female ratio in in vivo and in vitro culture groups was significantly different from the expected ratio of 1:1 and suggests that manipulation and culture of embryos results in a preferential loss of female embryos.     

Bisection of Bovine Morulae and Blastocysts from Superovulated Danish Dairy Cows

Sixtyfour compacted morulae and blastocysts were bisected with a microscalpel. The majority of the demi-embryos (n= 122) were reinserted into separate zona pellucidae (ZP) before non-surgical transfer to 113 syncronized recipients, as singles (n = 98) (DE-S) or in pairs (n = 30) (DE-P). Thirty non-manipulated embryos (E) were transferred during the same period and served as controls. Pregnancies were diagnosed by rectal palpation 4–7 weeks after transfer. The pregnancy rates for DE-S, DE-P and E were 32%, 53% and 40%, respectively (P > 0.05). A substantial number of abortions were recorded between 50 and 250 days of pregnancy among the recipients with DE-S. The fetal survival rate for DE-S was reduced to 21% and significantly lower (p < 0.05) than the survival rates of DE-P (43%) and E (40%). The quality of DE and the presence of ZP did not significantly influence the results. No conclusive reasons for the fetal loss could be found but different possibilities are discussed.     

Successful production of piglets derived from vitrified morulae and early blastocysts using a microdroplet method

This study was conducted to determine the efficiency of vitrification using the microdroplet (MD) method for early stage porcine embryos. Embryos at compacted morulae to early blastocyst stage were vitrified in a vitrification solution containing 40% (v/v) ethylene glycol, 0.6M sucrose and 2% (w/v) polyethylene glycol in M2 (ESP) without any pretreatment. The equilibration and dilution were carried out in third and fourth steps, respectively, at 38 degrees C. The survivability of the cryopreserved embryos was assessed for both in vitro culture (Experiment 1) and by embryo transfer (Experiment 2). In Experiment 1, the embryos were vitrified within a microdroplet or 0.25 ml straw (ST) and fresh embryos were used as a control group. The survival rates after 24h culture in the MD, ST and control groups were 21/23, 14/20 and 20/20, respectively. The hatching rates of the embryos after 48 h incubation were 14/23, 4/20 and 16/20, respectively. In Experiment 2, 171 vitrified embryos were transferred to 5 recipient gilts, and 17 healthy piglets were produced from 2 recipients (3 recipients aborted) in Group 1. In Group 2, 81 vitrified embryos and 16 fresh embryos in total were transferred to 4 recipient gilts, and 10 healthy piglets from the vitrified embryos were produced from 3 recipients. These results indicated that porcine embryos of compacted morulae to early blastocyst stage can survive cryopreservation using the microdroplet method without any special intracellular manipulation or treatment.     

Survival of bisected rabbit morulae transferred to synchronous and asynchronous recipients

The rabbit was used as a model to test the concept that temporal asynchrony is required to establish physiological synchrony when embryos are bisected to produce demiembryos. In preliminary studies with intact embryos it was confirmed that embryos harvested on days 2, 3, 4, or 5 (day 0 = day of breeding) can be transferred with +/- 1 day of asynchrony to the uteri of recipient rabbits. Three experiments were conducted with bisected embryos. In experiment 1, 192 bisected and 194 control day 3 embryos were transferred to uteri of day 2, 2.5, and 3 recipients (ovulated 0, 12, and 24 h after the donors), with 14% of the bisected and 39% of the intact embryos (P less than .05) resulting in young. Only 4% (2/48) of the day 3 bisected embryos vs. 39% (P less than .05) of the intact day 3 embryos survived in the uteri of day 2 recipients. In experiment 2, day 3 bisected and intact embryos were transferred to the oviducts of day 3, 3.5, or 4 recipients, the speculation being that the oviduct might provide a more neutral environment than the uterus. However, embryo survival was very low, except for the intact embryos transferred to synchronized recipients (42% young born). In experiment 3, 150 intact and 162 (81 pairs) bisected day 3 embryos collected from uteri were transferred to uteri of day 2.5, 3.0, and 3.5 recipient does. Significantly more pregnancies (100% vs. 47%, P less than .01) and young born (56% vs. 19%, P less than .01) resulted from intact embryos than from bisected embryos, irrespective of the uterine age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and survival after transfer of cow embryos cultured from 1-2-cells to morulae or blastocysts in rabbit oviducts or in a simple medium with bovine oviduct epithelial cells

This study compares development of bovine 1-2-cell embryos in bovine oviduct epithelial cell co-culture (Group EC) with a glucose- and serum-free simple medium (CZB), or after surgical transfer to ligated oviducts of rabbits (Group RO). Embryos were surgically collected from superovulated donor cows 40-48 h after the beginning of oestrus and randomly distributed between the two groups. Embryos were cultured or incubated for 5 days. In Exp. 1, embryo quality scores and total numbers of cells in the two groups were compared. In Exp. 2, pairs of similarly treated morulae were transferred to each of 10 or 12 recipients in the Groups RO and EC, respectively. Total cell counts per embryo in both groups averaged 52 (P greater than 0.05), and the in-vitro culture system was equivalent to the rabbit oviducts in promoting embryo development for all characteristics measured. Embryo survival, as determined by ultrasound between Days 39 and 43 after oestrus, in 13 ideal recipients was 57% for embryos in Group EC and 58% for embryos Group RO. None of the 9 less desirable recipients was pregnant for either group. These results establish that cattle zygotes can develop to morulae in culture with bovine oviduct epithelial cells in a simple medium and can produce normal pregnancy rates.     

In vitro culture of goat morulae to blastocysts before freezing

The results of transfer of frozen-thawed caprine embryos that were collected either as blastocysts or morulae and cultured to the blastocyst stage prior to freezing were compared. After thawing, the embryos collected as blastocysts appeared to be of marginally better quality than those that had been cultured from morulae (89 vs 72% rated as good; P > 0.05). The transfer of 24 frozen-thawed embryos collected as blastocysts to 12 recipients resulted in a pregnancy rate of 83% (10/12) and an embryo survival rate of 67%. Corresponding results for frozen-thawed blastocysts that had been cultured from morulae and were transferred to 11 recipients were 54% (6/11) and 41%, respectively. Since an earlier investigation had shown that the transfer of frozen caprine morulae yields very poor results, in our laboratory all morulae are now cultured to the blastocyst stage before being cryopreserved.     

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.     

Survival of sheep demi-embryos in vivo and in vitro

Sheep embryos (morulae and blastocysts) were bisected either by microscalpel or by microneedle after dissolving the zona pellucida with acidified Tyrode's solution. Fourteen and 11 cryopreserved demi-embryos failed to develop when transferred to recipients or placed in culture, respectively. When fresh demi-embryos were cultured in Dulbecco's phosphate buffered saline (DPBS) plus fetal calf serum (FCS) or Whitten's medium, the survival rate was 26% compared to 68% for whole embryos (P<0.01), and there was a suggestion that the presence of a zona pellucida was beneficial to survival. When two demi-embryos each within a zona pellucida were transferred into each of 10 ewes, six of them lambed to produce a total of eight lambs, including two sets of identical twins. Of 10 ewes receiving two demi-embryos without zonae pellucidae, three lambed to produce a total of four lambs, including one set of identical twins. Of 10 ewes that each received two whole embryos, 10 lambed to produce a total of 16 lambs. There was a suggestion that the zona pellucida might enhance the survival of demi-morulae but not demi-blastocysts.     

Viability of bovine demi- and quarter-embryos after transfer

The viability of bovine demi- and quarter-embryos was investigated. Early compacting morulae were nonsurgically flushed from superovulated donor cows and were bisected by two microneedles. One of the halves was then split further into two quarters. Each demi- and quarter-embryo was placed in an evacuated zona pellucida. One demi- or two quarter-embryos were transferred non-surgically into cow or heifer recipients. Viability was measured by ultrasound scanning of the fetuses on Days 35, 48 and 60 of pregnancy. The pregnancy rates at Day 60 were 46.2% (6 13 ) for heifers and 33.3% (4 12 ) for cows after the transfer of a single demi-embryo. The transfer of two quarter-embryos resulted in a pregnancy rate of 61.5% (8 13 ) for heifers and 8.3% (1 12 ) for cows. Seven (53.8%) and four (33.3%) live fetuses were found on Day 60 following the transfer of demi-embryos into heifers and cows, respectively. The transfer of quarter-embryos resulted in 10 fetuses (38.5%) in the heifer recipients and only one fetus (4.2%) in the cow recipients. The results of this study suggest that heifers are more suitable than cows as recipients for quarter-embryos.     

Factors affecting the survival of bisected sheep embryos in vivo

Two experiments were carried out to examine the effects of different factors on the survival of split sheep embryos. In Experiment 1, embryos collected on Day-6, Day-7 or Day-8 were bisected and transferred into recipient ewes in pairs. The proportions of Day-6, Day-7 and Day-8 demi-embryos developing to lambs were 26% (14 54 ), 30% (31 102 ) and 32% (24 74 ), respectively. Replacement of bisected late morula to expanded late blastocyst stage embryos into zonae did not affect their survival rate (P>0.5). The proportion of demi-embryos developing to lambs in recipients with two or more ovulations was higher (35%, 53 152 ) than in recipients with a single ovulation (21%, 16 78 ; P<0.05). In Experiment 2, Day-6 embryos were split with or without exposure to 0.25 M of sucrose and were transferred into recipients in pairs or singly. Exposure to 0.25 M of sucrose decreased the proportion of split embryos developing to lambs compared with that of the controls (31%, 22 70 vs 49%, 34 70 ; P<0.05). The effects of the number of demi-embryos transferred or the stage of development on the survival rate were not significant (P>0.05). The number of lambs born per original embryo was the highest when the embryos were split without exposure to sucrose and transferred into recipients singly (106%, 17 16 ).     

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

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