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.     

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.     

山羊品种间体细胞核移植研究

萨能奶山羊是著名的奶用山羊品种,波尔山羊则是世界著名的肉用山羊品种。为了研究波尔山羊体细胞在奶山羊卵母细胞中的去分化,我们针成年波尔山羊的颗粒细胞或耳皮肤成纤维细胞作为供核细胞（试验组）,移入奶山羊中Ⅱ期的去核卵母细胞透明带下,经电融合和离子霉素与6－二甲基氨基嘌呤－DMAP）激活,直接移入同期发情奶山羊输卵管或经体内培养,将发育的重构胚移入同期发情羊子宫内。妊娠早期作B超诊断,确立妊娠的观察至足月。同时将奶山羊的35日龄胎儿成纤维细胞作供核细胞（对照组）,按试验组同样方法处理,将重构胚直接移入同期发情的奶山羊输卵管内。结果：试验组,波尔羊颗粒粒细胞与耳皮肤成纤维2细胞的融合率分别为78．2％（115／147）,57．4％（116／202）,重构胚卵裂率为85．8％（115／134）,桑椹胚,囊胚的发育率38．8％（52／134）,早期妊娠三头,分别于妊娠40,60,60日龄终止妊娠。对照组,融合率为89．5％（136／152）,早期妊娠率为42．9％（6／14）,四头受体足月分娩,产四头公羊羔,其中三头存活,一头分娩时死于肺不扩张,并体重过大,显示胎儿过大综合症。经基因型鉴定证实,这四头克隆羔羊均源于同一胎儿成纤维细胞系。以上结果表明,波尔羊体细胞核在奶山羊卵母细胞中能够去分化,并维持一定程度的发育。     

Birth of normal calves resulting from bovine oocytes matured, fertilized, and cultured with cumulus cells in vitro up to the blastocyst stage

Bovine oocytes matured in vitro were fertilized in high proportions (92% of matured oocytes) by sperm capacitated with Ca ionophore A23187. Eight percent of inseminated oocytes that were denuded 96 h after insemination developed to the morula stage when cultured for 6-120 h after insemination with cumulus cells from the original oocytes. Inseminated oocytes denuded 96 h after insemination developed to the blastocyst stage when cultured with or without cumulus cells or in the conditioned medium from 96 h to 168-216 h after insemination (9.0%, 8.1%, and 6.8% of inseminated oocytes respectively). Six frozen-thawed blastocysts were transferred nonsurgically to 3 recipients (2 embryos/recipient). Two of the 3 recipients became pregnant, with one delivering live twins at term. Seven fresh blastocysts were transferred nonsurgically to 6 recipients (1-2 embryos/recipient). Three of the 6 recipients became pregnant, with 2 delivering live calves.     

山羊品种间体细胞核移植研究

萨能奶山羊是著名的奶用山羊品种,波尔山羊则是世界著名的肉用山羊品种.为了研究波尔山羊体细胞在奶山羊卵母细胞中的去分化,我们将成年波尔山羊的颗粒细胞或耳皮肤成纤维细胞作为供核细胞(试验组),移入奶山羊中Ⅱ期的去核卵母细胞透明带下,经电融合和离子霉素与6-二甲基氨基嘌呤(6-DMAP)激活,直接移入同期发情奶山羊输卵管或经体内培养,将发育的重构胚移人同期发情羊子宫内.妊娠早期作B超诊断,确立妊娠的观察至足月.同时将奶山羊的35日龄胎儿成纤维细胞作供核细胞(对照组),按试验组同样方法处理,将重构胚直接移入同期发情的奶山羊输卵管内.结果试验组,波尔羊颗粒粒细胞与耳皮肤成纤维细胞的融合率分别为78.2%(115/147)、57.4%(116/202),重构胚卵裂率为85.8%(115/134),桑椹胚、囊胚的发育率38.8%(52/134),早期妊娠三头,分别于妊娠40、60、60日龄终止妊娠.对照组,融合率为89.5%(136/152),早期妊娠率为42.9%(6/14),四头受体足月分娩,产四头公羊羔,其中三头存活,一头分娩时死于肺不扩张,并体重过大,显示胎儿过大综合症.经基因型鉴定证实,这四头克隆羔羊均源于同一胎儿成纤维细胞系.以上结果表明,波尔羊体细胞核在奶山羊卵母细胞中能够去分化,并维持一定程度的发育.     

Micromanipulation of sheep morulae to produce monozygotic twins

Donor ewes were treated with an intravaginal sponge containing 30 mg fluorogestone acetate (FGA) which was removed 12 days later. On the mornings of days 10, 11, and 12, each animal received 30 mg horse anterior pituitary (HAP) extract. Donors were mated twice daily during oestrus. Egg recovery was attemped seven days after progestagen with-drawal, when late morula/early blastocysts could be expected. The zona pellucida was opened with the aid of a micromanipulator, and the embryo was removed by gentle positive pressure from an evacuating pipette. After removal from the zona pellucida, the embryo was bisected using a fine glass needle; each demi-embryo was immediately placed either into the original zona pellucida or into one from an evacuated oocyte. The original opening was closed and both halves were transferred bilaterally into previously synchronized recipients. From 17 embryos split and transferred to 17 ewes, nine became pregnant and produced seven sets of monozygotic twins and two singletons.     

Intergeneric somatic cell nucleus transfer in marbled cat and flat-headed cat

Intergeneric nucleus transfer (ig-NT) is a promising technique to produce offspring of endangered species. The objectives of this study were to (1) investigate the in vitro development of marbled cat (MC; Pardofelis marmorata) and flat-headed cat (FC; Prionailurus planiceps) ig-NT embryos reconstructed from domestic cat (DC; Felis catus) oocytes (Experiment 1), (2) evaluate the effect of individual FC donor cell lines on NT success (Experiment 2), and (3) assess the developmental ability of FC-cloned and DC-IVF embryos in vitro and in vivo after oviductal transfer (Experiment 3). In Experiment 1, the morula rate of FC-reconstructed embryos was significantly higher than those of MC and DC embryos but lower than that of parthenogenic DC embryos. However, blastocyst rate was not different. In Experiment 2, FC-ig-NT embryos reconstructed from female muscular tissue had significantly higher morula rate in comparison with those derived from other donor cell lines. However, there was no difference in blastocyst rate among cell lines. In Experiment 3, in vitro development of FC-ig-NT embryos was lower than that of DC-IVF embryos. The competency of in vivo development of FC-ig-NT and/or DC-IVF embryos was investigated by assessing pregnancy rate after their transfer into DC recipients. Domestic cat recipients receiving only FC-ig-NT embryos, FC-ig-NT embryos in one side of the oviduct and DC-IVF embryos contralaterally (co-transfer), and only DC-IVF embryos were observed. No pregnancy was detected in all recipients receiving FC-ig-NT embryos. One recipient receiving co-transferred embryos became pregnant, then delivered DC-IVF dead fetuses (n = 2) and live kittens (n = 6). All recipients receiving DC-IVF embryos became pregnant, and three of six recipients delivered five DC-IVF kittens. These results illustrate the developmental capacity of MC- and FC-ig-NT embryos up to the blastocyst stage. Individual donor cell line affects the developmental success up to the morula stage of FC-ig-NT embryos. Improving the developmental competence and quality of FC-ig-NT embryos may be required for implantation and development to term of FC-ig-NT offspring.     

In vitro development to blastocysts of early porcine embryos produced in vivo or in vitro

The objective of this study was to compare the development of porcine embryos from the 2- and 4-cell stages to the blastocyst stage after in vivo or in vitro fertilization and in vivo or in vitro culture. Early-stage embryos were collected either from superovulated gilts 36 h after the second mating or after in vitro fertilization (IVF) of in vivo-matured oocytes, both followed by in vitro culture to the blastocyst stage. Blastocysts collected from superovulated donors served as controls. In the first experiment, a total of 821 2- and 4-cell embryos derived from in vivo-fertilized oocytes was cultured either in medium NCSU 23, modified Whittens' medium or modified KRB for 5 d. Significantly (P < 0.05 and P < 0.001) more embryos overcame the 4-cell block and developed to the blastocyst stage in medium NCSU 23 than in the 2 other culture media. Hatching was only observed in medium NCSU 23. In the second experiment, embryos derived from in vivo-matured oocytes fertilized in vitro were cultured in medium NCSU 23. Of 1869 mature oocytes 781 (41.8%) cleaved within 48 h after in vitro fertilization. A total of 715 embryos was cultured to the morula and blastocyst stages, and 410 (57.3%) overcame the developmental block stage, with 358 embryos (50.1%) developing to the morula and blastocyst stages. None of the embryos hatched, and the number of nuclei was significantly (P < 0.05) lower compared with that of in vivo-fertilized embryos (18.9 +/- 9.8 vs 31.2 +/- 5.8). In the third experiment, 156 blastocysts derived from in vitro fertilization and 276 blastocysts derived from in vivo fertilization and in vitro culture were transferred into synchronized recipients, while 164 blastocysts were transferred immediately after collection into 6 recipients, resulting in a pregnancy rate of 83.3%, with 35 piglets (on average 7.0) born. From the in vitro-cultured embryos, 58.3% (7/12) of the recipients remained pregnant at Day 35 after transfer, but only 33.3% maintained pregnancy to term, and 14 piglets (on average 3.5) were born. In contrast, the transfer of embryos derived from in vitro-fertilized oocytes did not result in pregnancies. It is concluded that 1) NCSU 23 is superior to modified Whittens' medium and modified KRB and 2) blastocysts derived from in vitro fertilization have reduced viability as indicated by the lower number of nuclei and failure to induce pregnancy upon transfer into recipients.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

Effect of donor stimulation, frozen semen and heparin treatment on the efficiency of in vitro embryo production in goats

Investigations on in vitro embryo production in goats in comparison with other domestic species, especially cattle, have been the subject of few reports despite their usefulness for both basic research and commercial application. The objectives of this study were to compare the efficiency of IVP in goats using immature follicular oocytes recovered from FSH-primed and control goats. After IVM, oocytes were fertilized with fresh or frozen-thawed semen capacitated with or without heparin. Mature oocytes were fertilized in vitro with fresh and frozen-thawed sperm of a single buck. Sperm preparation included swim-up separation and heparin treatment (50 micrograms/ml of sperm suspension for 45 min) before spermatozoa were added to oocytes in TALP-IVF. After IVF, the zygotes were cultured for 24h and cleaved embryos were further cultured with goat oviduct epithelial cells or transferred to synchronized recipients. Mean number of oocytes recovered from FSH-primed goats (24.5 +/- 8.6) was significantly higher (P < 0.01; t test) in comparison to control does (14.7 +/- 4.7). Irrespective of fresh or frozen semen, no differences were observed in blastocyst yield when sperm was treated with heparin. However, the highest cleavage rate (99/126; 79.4%) as well as blastocyst yield (47/126; 37.3%) was obtained after IVF with fresh sperm capacitated without heparin. Contrary to fresh sperm, heparin treatment of frozen-thawed sperm significantly improved (P < 0.01) embryo cleavage. No differences between in vivo developmental competence of embryos related to sperm origin were found after transferring into recipients. Overall, more than 60% of the recipients became pregnant and 20% of all transferred embryos survived delivering 13 healthy kids. Our caprine IVP system allows obtaining embryos with developmental competence comparable to bovine IVP.     

Invitro fertilization of cow ova

Embryos were recovered from superovulated heifers and transferred non-surgically through the cervix of heifers via an ureteral catheter. The ureteral catheter was passed into the anterior portion of the uterine horn ipsilateral to the corpus luteum through a series of concentric stainless steel tubes. One morula or blastocyst was transferred to each of 95 heifers.Eighteen heifers (19%) were pregnant 55 to 60 days later. The pregnancy rate was not significantly influenced by treatment of the vagina with antibiotics before transfer; whether a morula or blastocyst was transferred; or whether ovulation occurred from the left or right ovary. There were no significant differences among the three technicians. Fifty of the 77 recipients that were not pregnant at 60 days of gestation were observed in estrus after 18–23.5 or 37–44.5 days, representing normal or double the normal estrous cycle lengths. However, 11 showed a shortened return to estrus (9–26 days), and 16 had a delayed return to estrus (25–33 or 48–55 days). We conclude that this technique results in premature luteolysis in some recipients and probably in more early embryonic death than normal.     

Successful in vitro and in vivo development of in vitro fertilized two- to four-cell cat embryos following cryopreservation, culture and transfer

In vitro and in vivo survival of in vitro-derived 2- to 4-cell cat embryos following cryopreservation was examined. Prefreeze 1- vs 2-step cryoprotectant exposure (Experiment 1) and warming method (Experiment 2) on zona pellucida damage and development in vitro were compared. To determine viability in vivo, frozen/thawed embryos were cultured in vitro to the morula/early blastocyst stage and transferred to synchronous recipients (Experiment 3). At 24 to 26 h after IVF, embryos were cryopreserved in 1.4 M propanediol (Pr) + 0.125 M sucrose (Su) by cooling at 0.3 degrees C/min from -6 degrees C to -30 degrees C and storing in liquid nitrogen. Autologous embryos were cultured in vitro for 7 d. After warming for 5 sec in air and 10 sec at 37 degrees C in water (Experiments 1 to 3), or at room temperature air (22 degrees C; Experiment 2), the cryoprotectant was removed and embryos were cultured in vitro for 6 d (Experiments 1 and 2). Development was assessed after staining by counting cell numbers/embryo and determining the percentages at the 2- to 4-cell (nonsurvivor), pre (5 to 15), early (16 to 32), mid (33 to 50), late (>50) morula or blastocyst stages. Post-thaw development to late morula/blastocyst after 1-step exposure (68%, 15 min Pr + Su) was higher (P< 0.05) than that after 2-step exposure (36%, 15 min Pr and 15 min Pr + Su). Both warming methods produced similar percentages of embryos with damaged zonae (13 to 15%) and equivalent development to morula/blastocyst (64 to 69%). Development in vitro to early morula/blastocyst of frozen embryos with intact zonae was similar to that of nonfrozen embryos. Following cryopreservation, most 2- to 4-cell cat embryos retained their capability for in vitro development to morula/blastocyst, and in vivo viability was demonstrated by the birth of 3 live kittens to 2 of 4 recipients following the transfer of 58 embryos.     

Assessment of nuclear totipotency of fetal bovine diploid germ cells by nuclear transfer

Nuclear transfer was used to study nuclear reprogramming of fetal diploid bovine germ cells collected at two stages of the fetal development. In the first case, germ cells of both sexes were collected during their period of intragonadal mitotic multiplication at 48 days post co?tum (d.p.c.). In the second case, only male germ cells were collected after this period, between 105 and 185 d.p.c. Isolated germ cells were fused with enucleated oocytes. Reconstituted embryos were cultured in vitro and those reaching the compacted morula or blastocyst stage were transferred into synchronous recipient heifers. Of 511 reconstituted embryos with 48 d.p.c. germ cells (309 males and 202 females), 48% (247/511 ) cleaved; 2.7% (14/511 ) reached the compacted morula stage and 8 of them the blastocyst stage (1.6%). No difference was observed between sexes. All 14 compacted morulae/blastocysts were transferred into 6 recipients and one pregnancy was initiated. This recipient was slaughtered at Day 35 and an abnormal conceptus (extended trophectoderm and degenerated embryo) was collected. Its male sex, genetically determined, corresponded to that of donor fetus. Of 380 reconstituted embryos with male 105 to 185 d.p.c. germ cells, 72.1% (274/380 ) cleaved, 2.1% (8 380 ) reached the compact morula stage and 7 of these the blastocyst stage (1.8%). Three blastocysts and one morula were transferred into 4 recipients. Two became pregnant at Day 21 but only one at Day 35 which aborted around Day 40. Our results show that the nucleus of diploid bovine germ cells of both sexes can be reprogrammed. However, in the absence of further development of these reconstituted embryos, nuclear totipotency of bovine diploid germ cells remains to be evidenced.     

两种2-细胞鼠胚体外培养方法比较

目的应用鼠胚质控中的小鼠胚胎体外培养模型,探讨两种胚胎培养方式（四孔皿与微滴法）在单胚观察时间上的差异以及对2-细胞鼠胚体外发育潜能的影响。方法取6-8周龄的昆明白雌性小鼠。采用HMG10IU促排卵,48 h后注射HCG 10IU促卵泡成熟,取形态正常的2-细胞鼠胚。每5-10个胚胎培养在含500μL培养基的四孔皿中（A组）,或单个胚胎接种在含50μL的培养微滴中（B组）。培养后,每隔24 h在倒置显微镜下观察一次,计算单胚观察时间,并检测24 h时的≥4细胞胚形成率、48 h的融合胚形成率7、2 h的囊胚与扩张囊胚形成率、96 h囊胚孵化率。结果两种培养方式于同一试验条件下分别试验5次,A组培养83个胚胎,B组培养69个2-细胞鼠胚。在每一个观察点上,微滴培养的单胚观察时间远超过四孔皿培养（P〈0.001）。但两组各时间点的胚胎发育率相似,无显著差异（P〉0.05）。结论尽管微滴单胚培养方式的胚胎暴露培养箱外时间长,但与四孔皿多胚培养方式比较,两者间2-细胞鼠胚的体外发育潜能相似。     

Open pulled straw vitrification of goat embryos at various stages of development

This investigation addresses the question whether it is possible to apply the open pulled straw (OPS) vitrification method, found to be effective for cryopreserving caprine (Capra aegagrus hircus) blastocysts, to other embryonal stages. Morulae, blastocysts and hatched blastocysts were cryopreserved by way of OPS vitrification and blastocysts and hatched blastocysts by conventional freezing. Morulae were not included with conventional freezing because in our experience the survival rate is very low. To assess the viability of the cryopreserved embryos, they were transferred to synchronized does; in most cases, two embryos per doe. After OPS vitrification, of nine does receiving morulae, not a single one became pregnant; of 11 does receiving blastocysts, nine (82%) became pregnant (all of which kidded and gave birth to, on average, 1.8 kids); and of nine does receiving hatched blastocysts, three (33%) became pregnant (two of which [22%] kidded, giving birth to a single kid each). After conventional freezing, of 10 does receiving blastocysts, five became pregnant (four of which [40%] carried to term and gave birth to a pair of twins each); and of nine does receiving hatched blastocysts, three (33%) became pregnant (and gave birth to a single kid each). Embryo survival (kids born/embryos transferred) after vitrification for morulae, blastocysts, and hatched blastocysts was 0, 70% (16 of 23), and 13% (2 of 16), respectively, and after conventional freezing for blastocysts and hatched blastocysts was 42% (8 of 19) and 19% (3 of 16), respectively. The difference in pregnancy and kidding rate between vitrified and conventionally frozen blastocysts was significant, and so was the difference in pregnancy rate between hatched and nonhatched blastocysts, regardless whether OPS-vitrified or conventionally frozen. The results of the current study indicate that OPS vitrification is a very effective means of cryopreserving caprine blastocysts. Unfortunately, the superiority of OPS vitrification over conventional freezing does not apply to caprine morulae and hatched blastocysts.     

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.     

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 ).     

Impact of adding 5.5 mM glucose to SOF medium on the development,metabolism and quality of in vitro produced bovine embryos from the morula to the blastocyst stage

Although toxic for early stages of embryo development, glucose is a physiological metabolic substrate at the morula and blastocyst stages. We evaluated the effect of adding 5.5 mM glucose from the morula stage on bovine blastocyst development and quality. In vitro matured and fertilised bovine oocytes were cultured in modified Synthetic Oviduct Fluid medium containing 5% fetal calf serum, but without added glucose, up to day 5 post-insemination (pi). Morulae were selected and further cultured in the presence or absence of 5.5 mM glucose. Blastocyst and hatched blastocyst rates were recorded. Oxygen, glucose and pyruvate uptakes as well as lactate release were evaluated. The quality of the resulting blastocysts was evaluated by the cell allocation to the inner cell mass (ICM) and trophectoderm (TE) and by the apoptotic index. Adding glucose increased the blastocyst rate at day 8 pi (80% vs 65%) but had no impact on hatching rate (25% vs 28%). A 22% decrease in oxygen uptake was observed in the presence of glucose, concomitant with an increase in lactate release, although no change was observed in pyruvate uptake. A slight decrease in blastocyst cell number was observed at day 7 in the presence of glucose while neither the ICM/TE cell ratio nor the apoptotic index were affected. In conclusion, adding 5.5 mM glucose from the morula stage has a limited impact on blastocyst rate and quality although important modifications were observed in embryo metabolism. It remains to be determined whether those modifications could influence embryo viability after transfer.     

Colcemid‐treatment of heifer oocytes enhances nuclear transfer embryonic development,establishment of pregnancy and development to term

Four experiments were designed to examine the effects of colcemid, a microtubule assembly inhibitor, on the development of bovine nuclear transfer (NT) embryos in vitro and in vivo. Recipient oocytes matured at different times were exposed to colcemid. Approximately 80–93% of the exposed oocytes, with or without the first polar body (PB1), developed obvious membrane projections. In Experiment 1, oocytes matured for either 14–15 or 16–17 hr, treated with colcemid and used as recipient cytoplasm for NT resulted in over 40% blastocyst development. In Experiment 2, oocytes matured for 16–17 hr were treated with either 0.2 or 0.4 µg/ml colcemid for 2–3 or 5–6 hr, respectively. The percentages of blastocyst development (39–42%) were not statistically different among the different colcemid treatment groups, but were both higher (P < 0.05) than the control group (30%). Colcemid concentrations and length of colcemid treatment of oocytes did not affect their ability to support NT embryo development to the blastocyst and hatched blastocyst stages. Results from Experiment 3 indicate that semi‐defined medium increases morula and blastocyst development of NT embryos derived fromcolcemid‐treated oocytes under 5% CO₂ in air atmosphere. In addition, cell numbers of blastocysts in colcemid‐treated groups were numerically higher than the control groups. After embryo transfer, higher (P < 0.05) pregnant rates were obtained from the colcemid‐treated group than the nontreated group. Five of 40 recipients (12.5%) which received embryos from colcemid‐treated oocytes delivered healthy calves, significantly higher than those recipients (3.3%) that received embryos derived from nontreated oocytes. Mol. Reprod. Dev. 76: 620–628, 2009. © 2009 Wiley‐Liss, Inc.     

Early embryonic development in Thai swamp buffalo (Bubalus bubalis )

A total of 33 nonsurgical embryo collections was carried out to investigate early embryo development in Thai swamp buffalo. Collections were performed on Days 5.5, 6.0, 6.5, 7.0 and 7.5. The different stages of embryo development on these days were the 16-cell stage, compact morula, blastocyst, hatched blastocyst and hatched expanding blastocyst, respectively. In addition, some degenerating embryos and unfertilized ova were also recovered. A higher recovery rate was obtained with single embryo collection after natural estrus than after induced estrus or superovulation, 78% (7 9 ) vs 46% (6 13 ) vs 54.5% (6 11 ), respectively. A higher percentage of normal embryos was also obtained with single embryo collection after either natural or induced estrus than after superovulation, 71% (5 7 ), 83% (5 6 ) and 38% (6 16 ), respectively.