Viability of blastocysts produced by aggregation of two half-embryos in the mouse

Although methods for production of chimeras from early cleavage stages have been well established, little research has been directed toward production of genetically identical chimeric offspring. This study was designed to examine survival of blastocysts produced by aggregation of two halved eight-cell stage embryos from two different mouse strains. Four blastomeres of an eight-cell embryo from a pigmented strain were aggregated with four blastomeres of an eight-cell embryo from a nonpigmented strain. Aggregates were cultured for 48 h and transferred as blastocysts to synchronized recipients of three treatment groups. Viability was determined by examining the number of offspring produced relative to the number of blastocysts transferred. Thirty-nine pups were born from 375 transferred blastocysts (10%), with 16 pups displaying coat-color chimerism. Both nonmanipulated eight-cell embryos cultured for 48 h (P < 0.05) and chimeric blastocysts (P < 0.001) displayed lower embryo survival after transfer to recipients than noncultured, nonmanipulated blastocysts used as controls. Viability of chimeric blastocysts was also lower than that of nonmanipulated embryos cultured for the same period and transferred to the same recipients (P < 0.001). Although posttransfer survival of chimeric blastocysts was low, the birth of morphologically normal offspring demonstrated that production of chimeras from half embryos was compatible with survival. Improvements in this procedure may be useful for production of tenetically identical chimeras from outbred populations, such as those commonly found in domestic livestock species.     

Maternal serum reactivity to species-specific antigens in sheep-goat interspecific pregnancy.

Three models were used to test the hypothesis that interspecific pregnancy failure between the sheep and goat is due to a species-specific, maternal antibody response. Interspecific pregnancies were established in ewes and does, sheep in equilibrium goat chimeric conceptuses produced by injection of ovine blastocysts were transferred to ovine recipients, and ovine and caprine pregnancies were established in interspecific chimeras. Complement-mediated lymphocytotoxic and hemolytic assays were used to monitor onset and titer of antibodies. Sera from 3 of 8 injection-chimera recipients reacted with all caprine peripheral blood lymphocytes (PBL) and red blood cells (RBC) tested (n = 18). Sera from 3 of 6 ewes and 7 of 7 does also were pancytotoxic to PBL of the other species (n greater than or equal to 20). Absorptions with xenogeneic RBC generally removed the reactivity. The data were consistent with responses to species-specific, monomorphic antigens expressed on PBL and RBC, and probably trophoblast. The response preceded or coincided with interspecific pregnancy failure in does, but not in ewes. Accordingly, no xenoreactivity was observed in chimera sera but caprine pregnancies were resorbed (n = 16) and ovine pregnancies developed to term (n = 11). The data did not support the hypothesis that failure of caprine pregnancy in ewes or chimeras is due to a species-specific, maternal antibody response. In contrast, a maternal, cytotoxic antibody response to species-specific antigen(s) may contribute to failure of hybrid or ovine pregnancy in does.     

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.     

Survival of porcine inner cell masses in culture and after injection into blastocysts

Isolation of embryonic stem cells has been documented only in the mouse and perhaps the hamster and cow. We report results of experiments designed to determine the effect of age of porcine embryos (6 through 10 d after the first day of estrus) on isolation of cell lines with embryonic stem cell-like morphology. The capacity of fresh and short-term cultured inner cell mass (ICM) cells to differentiate into normal tissues after injection into blastocysts was also measured. Few Day-6 ICM survived in culture to the first passage onto fresh feeder cells, but cell lines with embryonic stem cell-like morphology developed from Day-7 through Day-10 ICM. Isolation of embryonic stem cell-like colonies was achieved at a higher frequency from ICM isolated from older embryos, but embryonic stem cell-like colonies from older embryos also tended to differentiate spontaneously in culture. Viable porcine chimeras were born after injection of fresh ICM into blastocysts that were transferred to recipients for development to term; no chimeras were born from blastocysts injected with ICM subjected to short-term (1 to 6 d) culture. Germ-cell chimerism was confirmed in one of the chimeras. These results document that undifferentiated cells can be removed from porcine blastocysts, transplanted to other embryos, and contribute to development of normal differentiated tissues, including germ cells. Cells with embryonic stem-like morphology can be isolated in culture from ICM at various embryonic ages, but ICM from young blastocysts (e.g., Day-7 embryos) yield embryonic stem cell-like colonies at lower frequency than do ICM from older blastocysts (e.g., Day-10 embryos).     

Interspecies nuclear transfer of Tibetan antelope using caprine oocyte as recipient

Interspecies nuclear transfer is an invalulable tool for studying nucleus-cytoplasm interactions; and at the same time, it provides a possible alternative to clone endangered animals whose oocytes are difficult to obtain. In the present study, we investigated the possibility of cloning Tibetan antelope embryos using abattoir-derived caprine oocytes as recipients. Effects of culture conditions, enucleation timing, and donor cell passages on the in vitro development of Tibetan antelope-goat cloned embryos were studied. Maternal to zygotic transition timing of interspecies Tibetan antelope embryos was also investigated using two types of cloned embryos, Tibetan antelope-rabbit and Tibetan antelope-goat embryos. Our results indicate that: (1) goat oocyte is able to reprogram somatic cells of different genus and supports development to blastocyst in vitro. (2) Coculture system supported the development of Tibetan antelope-goat embryos to blastocyst rate stage (4.0%), while CR1aa alone did not. (3) When MII phase enucleated caprine cytoplast and TII phase enucleated caprine cytoplast were used as recipients, the fusion rate and blastocyst rate of hybrid embryos were not statistically different (73.9% vs. 67.4%; 4.0% vs. 1.1%). (4) When donor cells at 3-8 passages were used, 2.9% hybrid embryos developed to blastocysts, while none developed to blastocysts when cells at 10-17 passages were used. (5) There may be a morula-to-blastocyst block for Tibetan antelope-goat, while there may be an 8- to 16-cell block for Tibetan antelope-rabbit embryos.     

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。     

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.     

Differences in the incidence of apoptosis between in vivo and in vitro produced blastocysts of farm animal species: a comparative study

The occurrence of pregnancies and births after embryo transfer (ET) of in vivo produced embryos is generally more successful compared to that of embryos produced in vitro. This difference in ET success has been observed when embryos of morphological equal (high) quality were used. The incidence of apoptosis has been suggested as an additional criterion to morphological embryo evaluation in order to assess embryo quality and effectively predict embryo viability. In this study, equine, porcine, ovine, caprine and bovine in vivo and in vitro produced morphologically selected high quality (grade-I) blastocysts were compared for the occurrence of apoptosis in blastomeres. The total number of cells per embryo and the number of cells with damaged plasma membranes, fragmented DNA and fragmented nuclei per embryo were assessed in selected blastocysts by combining Ethidium homodimer (EthD-1), terminal dUTP nick end labeling (TUNEL) and Hoechst 33342 staining. In general, the level of blastomere apoptosis was low. A higher level of apoptosis was observed in in vitro produced equine, porcine and bovine blastocysts compared to their in vivo counterparts. Interestingly, 4 of the initially selected 29 bovine in vitro produced blastocysts exhibited extensive signs of apoptosis affecting the inner cell mass (ICM), which is not compatible with a viable conceptus. Repeated occurrence of this observation may explain the lower ET outcome of in vitro produced bovine embryos compared to in vivo produced embryos. It is concluded that, although in morphologically high quality blastocysts of several farm animal species a significant difference exists in the percentages of apoptotic cells between in vivo and in vitro produced embryos, the incidence of apoptosis at the blastocyst stage is at such a low level that it cannot reflect the substantial differences in embryo viability that have been described between in vivo and in vitro produced blastocysts following ET.     

Interactions between diploid embryonal carcinoma cells and early embryonic cells

Two diploid embryonal carcinoma (EC) cell lines, P10 and P19, differ in their response to the embryonic environment. P10 produces mostly normal chimeras following injection into blastocysts, whereas P19 produces mostly abnormal chimeras. In this study, P10 cells were aggregated with morulae, and all resulting fetuses were chimeric with very large contributions from the EC cells. However, all embryos were abnormal. Following aggregation of P19 cells with morulae, very few embryos were recovered and they were all non-chimeric. Both P10 and P19 were capable of forming functional gap junctions with morula cells and with the ICM of the blastocyst but not with trophoblast, showing that differences in the ability to make junctional contact with the embryo cannot explain the differences between the two cell lines.     

Identical triplets and twins developed from isolated blastomeres of 8- and 16-cell mouse embryos supported with tetraploid blastomeres

We studied the developmental potential of single blastomeres from early cleavage mouse embryos. Eight- and sixteen-cell diploid mouse embryos were disaggregated and single blastomeres from eight-cell embryos or pairs of sister blastomeres from sixteen-cell embryos were aggregated with 4, 5 or 6 tetraploid blastomeres from 4-cell embryos. Each diploid donor embryo gave eight sister aggregates, which later were manipulated together as one group (set). The aggregates were cultured in vitro until the blastocyst stage, when they were transferred (in sets) to the oviducts of pseudopregnant recipients. Eighteen live foetuses or pups were obtained from the transfer (11.0% of transferred blastocysts) and out of those, eleven developed into fertile adults (one triplet, one pair of twins and four singletons). In all surviving adults, pups and living foetuses, only diploid cells were detected in their organs and tissues as shown by analysis of coat pigmentation and distribution of glucose phosphate isomerase isoforms. In order to explain the observed high rate of mortality of transferred blastocysts, in an accompanying experiment, the diploid and tetraploid blastomeres were labelled with different fluorochromes and then aggregated. These experiments showed the diploid cells to be present not only in the inner cell mass (ICM) but also in the trophectoderm. The low number of diploid cells and the predominance of tetraploid cells in the ICM of chimaeric blastocysts might have been responsible for high postimplantation mortality of our experimental embryos.     

A caprine chimera produced by injection of embryonic germ cells into a blastocyst

This report details a chimeric goat derived by injecting caprine embryonic germ (EG) cells into a host blastocyst. The EG cells, isolated from the primordial genital ridge of white Guanzhong goat fetuses (28-42 days of pregnancy), had alkaline phosphatase activity and several stem cell markers, including SSEA-1, c-kit, and Nanog. Ten to 20EG cells were microinjected into the blastocoelic cavity of a host blastocyst collected from a black goat following natural service. Twenty-nine injected blastocysts were transferred into nine white surrogate goats. One of the recipients maintained pregnancy to term and gave birth to three kids: one male, one female, and a dead, malformed fetus of undetermined gender; all three fetuses were black, but the female and the malformed fetus each had a large white spot on their head. Based on PCR and microsatellite DNA assay, the female and the malformed fetus were monozygotic twins and chimeras. Microsatellite assay on various tissues from the dead fetus (including skin, blood, liver, placenta, lung, heart, spleen, muscle, and brain), revealed that these tissues and organs were chimeric and contained cells derived from EG cells. In conclusion, caprine EG cells differentiated into all three germ layers in vivo.     

A simple vitrification technique for sheep and goat embryo cryopreservation

The aim of this study was to evaluate pregnancy and embryo survival rate of vitrified in vivo produced Merino sheep and Criolla goat (morulae and blastocysts) embryos, using the plastic tips of micropipettes, as containers (Cryo-tips). The embryos were exposed, at room temperature, to two successive equilibration solutions for a period of 5 min and then to a vitrification solution (VS) for 30 s. Then embryos were then loaded in 1 μl VS, into a plastic micropipette tip, and plunged into liquid nitrogen. On thawing, the embryos were warmed (37 °C) and placed into cryoprotectant dilutions (three-step-process). In the ovine, the morula and blastocyst pregnancy rates (47.1% vs 50%) and embryo survival rates (41.2% vs 50%) recorded were similar for both embryonic stages. Unlike the sheep, no pregnancies were recorded in goat vitrified/thawed morulae embryos, following transfer. However, in contrast, goats receiving blastocysts recorded high rates of pregnancy and embryo survival (64% and 64%, respectively). This technique allows for easy handling of cryopreserved embryos, is simple and efficient in both ovine embryo stages and also for goat vitrified blastocysts. The technique has definite potential application.     

Effect of coculture with oviduct epithelial cells on viability after transfer of vitrified in vitro produced goat embryos

This study evaluates the effect of coculture with goat oviduct epithelial cells (GOEC) on the pregnancy rate, embryo survival rate and offspring development after direct transfer of vitrified/thawed caprine in vitro produced (IVP) embryos. Oocytes were recovered from slaughterhouse goat ovaries, matured and inseminated with frozen/thawed capacitated semen, and presumptive zygotes were randomly cultured in synthetic oviduct fluid (SOF) (n=352) or GOEC (n=314). The percentage of cleaved embryos reaching the blastocyst stage was 28% and 20% in SOF and GOEC, respectively (P<0.05). Overall, 26 blastocysts of SOF were transferred freshly in pairs to recipient goats, whereas 58 of SOF and 36 of GOEC were vitrified and transferred directly in pairs to recipient goats after thawing without removal of cryoprotectants or morphological evaluation. The kidding rate was 92% for SOF fresh, 14% for SOF vitrified (P<0.001) and 56% for GOEC vitrified (P<0.05); the difference was also significant between vitrified groups (P<0.01). The embryo survival rate was 62% for SOF fresh, 9% for SOF vitrified (P<0.001) and 33% for GOEC vitrified (P<0.05) with a significant difference between vitrified groups (P<0.01). The results showed that the coculture of IVP goat embryos with GOEC significantly improves the pregnancy and embryo survival rates and leads to the birth of healthy offspring. However, further research using more defined GOEC coculture is required to confirm its capacity to increase the success rate of IVP embryo technology in goat.     

Chimeras between parthenogenetic or androgenetic blastomeres and normal embryos: allocation to the inner cell mass and trophectoderm

A series of chimeras was generated by injecting single normal, parthenogenetic, or androgenetic blastomeres carrying transgenic markers under the zona pellucida of nontransgenic eight-cell embryos. These chimeras were cultured to the blastocyst stage and sectioned, and the transgenic component was detected by in situ hybridization. No statistically significant difference was found among the normal, parthenogenetic, and androgenetic chimeras in the number of chimeric blastocysts with a transgenic contribution to the inner cell mass (ICM), the trophectoderm, or both the ICM and trophectoderm. Since androgenetic and parthenogenetic cells were present in chimeras at a high frequency in both the ICM and trophectoderm at the blastocyst stage, but not in similar chimeras at late gastrulation, these cells must not respond normally to developmental signals subsequent to blastocyst formation and prior to late gastrulation.     

The ovine uterus as a host for in vitro-produced bovine embryos

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.     

Evaluation of mouse blastocyst implantation rate by morphology grading

The aim of our study is to observe the relationship between the blastocyst morphology and the implantation rate for mice. Mouse embryos obtained from the superovulated-ICR mice were cultured in vitro from 1-cell zygotes to blastocysts. Mouse blastocysts were then classified into 3 grades: grade I, small blastocysts; grade II, large blastocysts; grade III, hatching blastocysts. They were independently transferred into the uterus of recipient females mated with vasectomized male mice on 96 hours after the zygotes were cultured in vitro. The successful implantation was checked by injection of Chicago Sky Blue 6B on the second day after embryo transfer. Although there was no significant difference in the implantation rates between the grade III and grade II, grade I was significantly decreased, as compared with grade III. Grade I and grade II was also significantly decreased in both the diameter of blastocysts and cell number of inner cell mass (ICM) and trophectoderm (TE), as compared with grade III. These findings indicate that the expanded and hatching blastocyst selections for embryo transfer in in vitro fertilization were evaluated with the high implantation rate.     

Localization and binding of transforming growth factor-beta isoforms in mouse preimplantation embryos and in delayed and activated blastocysts.

The stage and cell-specific accumulation of mammalian isoforms of transforming growth factor-beta (TGF-beta 1, TGF-beta 2, and TGF-beta 3) and TGF-beta binding were examined in the preimplantation embryo and in progesterone (P4)-treated delayed or P4 plus estradiol-17 beta (E2)-treated activated blastocysts in the mouse. Immunocytochemical studies revealed that while all three immunoreactive TGF-beta isoforms were present in one-cell embryos, very little or no immunostaining was observed in two-cell embryos. However, distinct immunostaining of these isoforms was again observed in four-cell embryos and persisted through the blastocyst stage. Among the isoforms studied, TGF-beta 2 immunostaining showed a unique pattern in late morulae. In many of these morulae, the staining was primarily observed in outside cells. However, in blastocysts, immunostaining for all three isoforms was present both in the inner cell mass (ICM) and trophectoderm (Tr). Immunostaining in sectioned blastocysts and immunosurgically isolated ICMs confirmed immunostaining in Tr and ICM cells. To ascertain whether preimplantation embryos can produce TGF-beta isoforms, immunostaining was performed in embryos grown in vitro from two-cell stage in simple balanced salt solution. Immunoreactive TGF-beta s 1-3 were present in embryos at all stages of development examined (four-cell embryos through blastocysts). The virtual absence of immunoactive TGF-beta s in two-cell embryos but their accumulation in embryos at later stages of development in vitro provides evidence that these growth factors were produced by embryos. In order to assess at what stages of development preimplantation embryos could be responsive to TGF-beta s, specific binding of [125I]TGF-beta 1 and [125I]TGF-beta 2 was performed in embryos and examined by autoradiography. Low levels of binding were first detected in eight-cell embryos. The binding increased in morulae followed by a further increase in blastocysts. Analysis of binding of [125I]TGF-beta 2 in immunosurgically isolated ICMs indicated that binding was primarily evident in Tr cells. Affinity labeling of TGF-beta 1 or TGF-beta 2 in Day 4 blastocysts revealed three classes of binding proteins with approximate molecular sizes of 65 kDa (type I), 90 kDa (type II), and greater than 250 kDa (type III), in addition to a doublet of 130 and 140 kDa proteins. This observation is similar to those reported for other cell types. The data suggest that embryos are likely to be responsive to TGF-beta s after the third cleavage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of bovine and porcine embryonic teratomas in athymic mice

Inner cell masses (ICM) and embryonic discs from bovine and porcine blastocysts of various ages were transplanted under the kidney capsule of athymic (nude) mice to evaluate growth of teratocarcinomas containing both differentiated tissues and undifferentiated stem cells. Inner cell masses were isolated immunosurgically from Day 8, Day 9 and Day 10 porcine blastocysts and from Day 8, Day 10 and Day 12 bovine blastocysts. Embryonic discs were mechanically dissected from Day 11 and Day 12 porcine embryos and from Day 14 bovine embryos. Day 6 egg cylinders were dissected from embryos and from hybrid embryos of a cross between BALB/C and an outbred strain of mouse. Two to four ICM, embryonic discs or egg cylinders were transplanted under the kidney capsule of each athymic host. After 8 weeks, graft hosts were killed and their tumors removed, fixed and prepared for histological and immunohistochemical examination. Embryonic teratomas developed at high frequency from murine egg cylinders and from Day 11 and Day 12 porcine and Day 14 bovine embryos. Tumors were observed only infrequently from younger bovine and porcine blastocysts. Murine embryonic tumors were composed of numerous differentiated cell types of ectodermal, mesodermal and endodermal origins, but representation of the three embryonic germ layers was somewhat more restricted in bovine and porcine embryonic tumors. No undifferentiated stem cells were detected in tumors of any of the three species. These results demonstrate that teratomas will develop from bovine and porcine embryos when grafted to an immunocompromised host, but the presence of undifferentiated teratocarcinoma stem cells from these species has yet to be achieved.     

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.     

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.