Developmental rate and allocation of transgenic cells in rabbit chimeric embryos

The objective of this study was to compare developmental capacity of rabbit chimeric embryos and the allocation of the EGFP gene expression to the embryoblast (ICM) or embryonic shield. We produced chimeric embryos (TR< >N) by synchronous transfer of two or three blastomeres at the 16-cell stage from transgenic (TR) into normal host embryos (N) at the same stage. In the control group, two to three non-transgenic blastomeres were used to produce chimeric embryos. The TR embryos were produced by microinjection of EGFP into both pronuclei of fertilized rabbit eggs. The developmental rate and allocation of EGFP-positive cells of the reconstructed chimeric embryos was controlled at blastocyst (96 h PC) and embryonic shield (day 6) stage. All chimeric embryos (120/120, 100%) developed up to blastocyst stage. Using fluorescent microscope, we detected green signal (EGFP expression). In 90 chimeric (TR< >N) embryos (75%). Average total number of cells in chimeric embryos at blastocyst stage was 175+/-13.10, of which 58+/-2.76 cells were found in the ICM area. The number of EGFP-positive cells in the ICM area was 24+/-5.02 (35%). After the transfer of 50 chimeric rabbit embryos at the 16-cell stage, 20 embryos (40%) were flushed from five recipients on day 6 of pregnancy, of which five embryos (25%) were EGFP positive at the embryonic shield stage. Our results demonstrate that transgenic blastomeres in synchronous chimeric embryos reconstructed from TR embryos have an ability to develop and colonize ICM and embryonic shield area.     

Development of mouse enucleated oocytes receiving a nucleus from different stages of the second cell cycle.

The influence of the stage of the cell cycle of donor nuclei on the development of mouse oocytes enucleated at telophase I was examined. After nuclear transplantation and activation, a high proportion of the oocytes remodelled a nucleus, emitted a polar body and formed a pronuclear-like nucleus. Most of the reconstituted embryos that received an interphase nucleus 30-32 h or 34-36 h after treatment with human chorionic gonadotrophin (hCG) arrested at the 2-cell stage. The reconstituted embryos were able to develop to blastocysts when nuclei from late 2-cell embryos (44-46 and 48-50 h after hCG) were transferred to the oocytes. The resulting blastocysts were transferred to recipients and ten live young were obtained from the embryos that formed a pronuclear-like nucleus after extrusion of a polar body. Thus, the developmental ability of the reconstituted embryos was critically influenced by the stage of the cell cycle of the donor nuclei.     

AGE-DEPENDENT CHANGE IN THE TRANSDIFFERENTIATION ABILITY OF CHICK NEURAL RETINA IN CELL CULTURE*

We examined how the transdifferentiation ability of neural retinal cells into lens and/or pigment cells in call culture is changed with the development of the donor. Cells dissociated from neural retinas of chick embryos ranging from 3-day-old to the stage immediately before hatching and of 3-day-old chicks were cultured for about 60 days. The results clearly indicated that the transdifferentiation ability decreased with age. The latest developmental stage at which the differentiation of lens cells took place was in 18-day-old embryos. A gradual decrease in this ability was shown by the comparison of crystallin content in cultures prepared from embryos at different stages. The differentiation of pigment cells was recognized in cultures of neural retinas earlier than in 15-day-old embryos. Such loss of the ability of neural retinal cells to transdifferentiate into pigment cells earlier than into lens cells can be partially attributed to inhibitory factors accumulated in medium conditioned with many neuronal cells present in cultures.     

A method to obtain avian germ-line chimaeras using isolated primordial germ cells.

Primordial germ cells (PGCs), collected from the blood of 2-day-old chick embryos, were concentrated by Ficoll density centrifugation. The blood contained 0.048% PGCs and the concentrated fraction contained 3.9% PGCs in blood cells. The PGCs were picked up with a fine glass pipette, and one hundred were then injected into the terminal sinuses of 2-day-old Japanese quail embryos (24 somites); bubbles were then inserted to prevent haemorrhage. The embryos were further incubated at 38 degrees C for 24 h, and then fixed. Serial sections were stained with the periodic acid-Schiff reagent (PAS) to demonstrate chicken PGCs and with Feulgen stain to identify quail cells. On the basis of the differences in staining properties, 63.6 +/- 5.3 chick PGCs were detected in the quail embryo in the area where the gonads develop. Furthermore, 39.3 +/- 4.5 chick PGCs were incorporated into the quail germinal epithelium within 24 h of the injection. A similar percentage of the host (quail) PGCs had also migrated to the germinal epithelium at the same stage of development. This technique for obtaining germ-line chimaeras will facilitate research on avian germ-line differentiation.     

Cloning of bovine embryos by multiple nuclear transfer

The in vitro development of multiple generation bovine nuclear transferred embryos to blastocysts and their survival ability after freezing and thawing were examined. Parent donor embryos which had 20 to 50 cells were recovered from superovulated cows. Follicular oocytes matured in vitro were used as recipient oocytes. The recipient oocytes enucleated at 22 to 24 h after the onset of maturation were preactivated at 33 h. Enucleated oocytes with a donor blastomere were fused 9 h after activation by an electric stimulus and the fused oocytes were cultured in vitro (first generation). Reconstituted oocytes that had developed to the 8- to 16-cell stage 3 to 4 d after fusion were used as donor embryos for the next generation. Recloning procedures were performed twice (second and third generations). The proportion of recipient oocytes successfully fused with a blastomere increased with the cycle of nuclear transfer. Eighty to 86% of fused oocytes developed to the 2-cell stage and there was no significant difference with the generation. The proportion of reconstituted embryos receiving blastomeres derived from first generation embryos had higher developmental ability in vitro, than those derived from other generations (43 vs 31% for 8 to 16-cell stage, 37 vs 20 and 21% for blastocyst stage). The number of cloned blastocysts increased with repeated nuclear transfer (once: 6.2 +/- 4.3, twice: 19.8 +/- 9.2 and three times: 30.0 +/- 14.7) but varied greatly with each parent donor embryo. The in vitro viability of cloned blastocysts after freezing and thawing (59%) was low but not significantly different from that obtained for in vitro fertilized blastocysts (72%). After transfer of either fresh or frozen-thawed cloned blastocysts to 21 recipients, 10 of them were pregnant on Day 60. Four and 3 offspring were produced from 20 fresh and 14 frozen-thawed blastocysts,respectively.     

Observations on in vitro development of bovine morulae in Ham's F-10 and Dulbecco's phosphate buffered saline supplemented with normal steer serum

This study was conducted to compare in vitro development of bovine morulae in Ham's F-10 and Dulbecco's phosphate buffered saline (D-PBS) media supplemented with 10% (v/v) normal steer serum. Fifty-three excellent and good embryos were obtained by superovulating 15 non-lactating Holstein cows. Embryos were placed randomly in culture with Ham's F-10 or D-PBS media and development was recorded at 12-h intervals for the duration of culture. All embryos reached early blastocyst, blastocyst and expanded blastocyst stage. Nineteen of 27 embryos (70.1%) cultured in Ham's F-10 developed to hatched blastocyst stage in contrast to three out of 26 in D-PBS (11.5%). The mean developmental scores at 24, 48, 72, 96 and 120 h of culture were significantly (P<0.001) higher for embryos cultured in Ham's F-10. Also, the mean times to reach early blastocyst (25.84 +/- 6.65 vs 46.67 +/- 9.99 h), blastocyst (44.57 +/- 11.45 vs 61.89 +/- 16.62 h) and expanded blastocyst stage (65.00 +/- 13.20 vs 73.41 +/- 15.80 h) were significantly (P<0.001) shorter for embryos cultured in Ham's F-10. No difference was observed in the mean time to reach hatching (90.00 +/- 10.85 vs 84.00 +/- 16.97 h) and hatched blastocyst stage (97.26 +/- 18.71 vs 96.00 +/- 0.00 h). The results obtained support the concept that Ham's F-10 and normal steer serum provide for optimal bovine embryo development and suggest that 10% normal steer serum could be used as a protein supplement with D-PBS for short term storage and culture of bovine embryos.     

Analysis of corneal development with monoclonal antibodies. I. Differentiation in isolated corneas

Monoclonal antibodies highly selective for developmentally regulated antigens present in the cornea (Zak and Linsenmayer, Dev. Biol. 99, 373-381, 1983) have been used to immunohistochemically evaluate differentiation in intact chick corneas cultured on the chorioallantoic membrane (CAM) of host embryos. One antibody is directed against the epithelial cell layer and the other is against the corneal stromal matrix. It has been established that both antigens recognized by the antibodies are expressed de novo in young explanted corneas and that the stromal matrix antigen is a product of the corneal fibroblasts. Thus expression of the antigens can be used as criteria for overt differentiation of the respective cell types. The antibodies have been employed to assess when the corneal epithelial and stromal cells become capable of autonomous differentiation within isolated corneas. To accomplish this, corneas of various ages were explanted with and without adjacent pericorneal tissues. The results indicate that, under the culture conditions employed, corneal stromal differentiation is dependent on the presence of the lens until stage 28 (51/2-6 days of development), which is the time when invasion of the stroma by pericorneal mesenchymal cells is initiated. After stage 28, the stromal matrix antigen was expressed by isolated corneas irrespective of the presence of the lens. Possibly the lens acts by maintaining the integrity of the corneal endothelial monolayer and thus promoting normal migration of pericorneal mesenchymal cells into the primary corneal stroma, where they undergo differentiation. Conversely, differentiation of the corneal epithelium was independent of any pericorneal structure from the earliest stage examined (41/2-5 days of development). It was even independent of overt stromal differentiation, thus suggesting an early and strong determination for this tissue.     

In vitro developmental competence of domestic cat embryos after somatic cloning: a preliminary report

This work was undertaken in order to study the developmental competence of nuclear transfer feline embryos with regard to the recipient-cytoplast's age and type of somatic cells used as donor nuclei. Oocytes were recovered by mincing the ovaries in HEPES-buffered TCM-199. Selected cumulus-oocyte complexes (COCs) with compact cumulus cell mass and a dark, homogenous ooplasm were cultured for maturation in the modified medium TC-199 for 24, 35, and 43 h, and after enucleation were used as a source of recipient cytoplasts for exogenous somatic nuclei. Two experiments were carried out. In Experiment 1, the source of recipient cytoplasts was oocytes matured in vitro for 24 h (Group 1), 35 h (Group 2), and 43 h (Group 3), while the source of donor nuclei was cycling fetal fibroblasts. Somatic cell-cytoplast complexes (SC-CCs) were fused electrically by double DC pulses of 2.0 kV/cm for 15 micros. The reconstructed embryos were cultured in B2 medium for 72 h after NT, then co-cultured with BRL cells in the same medium supplemented with 10% FBS at 38.5 degrees C under 5% CO2 in air. In Groups 1, 2, and 3, the fusion rates were 71.4 (25/35), 74.6 (47/63), and 57.5% (46/80), respectively. The cleavage rates in Groups 1, 2, and 3 were 80.0 (20/25), 55.3 (26/47), and 60.8% (28/46), respectively. The development to morula and blastocyst stages was higher in Groups 1 and 2 compared to Group 3 (morula stage 14/25 (56.0%), 16/47 (34.0%), and 13/46 (28.2%); blastocyst stage 2/20 (8.0%), 4/47, (8.5%), and 0/46, respectively). In Experiment 2, the oocytes matured for 24-35 h were used as a source of recipient cytoplasts and cycling fetal fibroblasts and cumulus cells derived from mature COCs were used as a source of donor nuclei. The fusion rates were 115/193 (59.6%) versus 65/143 (45.4%) for fetal fibroblasts and cumulus cells, respectively. The cleavage rate was 72/115 (62.6%) versus 48/65 (73.8%), and the development to blastocyst stage 6/115 (5.2%) versus 5/65 (7.7%), for fetal fibroblast and cumulus cells, respectively. In conclusion, a prolonged maturation period of cat oocytes decreases developmental competence of reconstructed embryos, especially the ability to reach the blastocyst stage. The in vitro development of reconstructed embryos with either nuclei of fetal fibroblasts or cumulus cells was at approximately the same level.     

The possibility of lens formation in explants of the retina and pigment epithelium of the eye in chick embryos

Undissociated tissue explants of the retina and retinal pigment epithelium (RPE) of 3,5-, 4-, 5- and 8-day-old chick embryos were cultured in vitro. After 7 days in culture, lentoids were observed in explants of either retina or RPE from 3,5-, 4- and 5-day-old embryos. As demonstrated by immunohistochemistry, these lentoids contained specific chick lens proteins (alpha-, beta- and delta-crystallins). No crystallin-containing cells were found in eye tissue explants from 8-day-old embryos. However, when 5-bromo-deoxyuridine (25 microM) was introduced into the medium at the beginning of culturing (for 12 h), large eosinophilic cells containing alpha-, beta- and delta-crystallins were detected in retinal explants of the 8-day old embryos. Thus, retina and RPE of 3,5-5-day-old chick embryos are capable of lens differentiation after explantation in vitro without dissociation into individual cells. This capacity is lost during development.     

Nucleolar changes in bovine nucleotransferred embryos.

This study focused on nucleolar changes in bovine embryos reconstructed from enucleated mature oocytes fused with blastomeres of morulae or with cultured, serum unstarved bovine fetal skin fibroblasts (embryonic vs. somatic cloning). The nucleotransferred (NT) embryos were collected and fixed at time intervals of 1-2 h (early 1-cell stage), 10-15 h (late 1-cell stage), 22-24 h (2-cell stage), 37-38 h (4-cell stage), 40-41 h (early 8-cell stage), 47-48 h (late 8-cell stage), and 55 h (16-cell stage) after fusion. Immunocytochemistry by light and electron microscopy was used for structure-function characterization of nucleolar components. Antibodies against RNA, protein B23, protein C23, and fibrillarin were applied. In addition, DNA was localized by the terminal deoxynucleotidyl transferase (TdT) technique, and the functional organization of chromatin was determined with the nick-translation immunogold approach. The results show that fully reticulated (active) nucleoli observed in donor cells immediately before fusion as well as in the early 1-cell stage after fusion were progressively transformed into nucleolar bodies displaying decreasing numbers of vacuoles from the 2- to 4-cell stage in both types of reconstructed embryos. At the late 8-cell stage, morphological signs of resuming nucleolar activity were detected. Numerous new small vacuoles appeared, and chromatin blocks reassociated with the nucleolar body. During this period, nick-translation technique revealed numerous active DNA sites in the periphery of chromatin blocks associated with the nucleolar body. Fully reticulated nucleoli were again observed as early as the 16-cell stage of embryonic cloned embryos. In comparison, the embryos obtained by fetal cloning displayed a lower tendency to develop, mainly during the first cell cycle and during the period of presumed reactivation. Correlatively, the changes in nucleolar morphology (desegregation and rebuilding) were at least delayed in many somatic NT embryos in comparison with the embryonic NT group. It is concluded that complete reprogramming of rRNA gene expression is part of the general nuclear reprogramming necessary for development after NT.     

Effect of ooplast activation on the development of oocytes following nucleus transfer in cattle

We assessed the effect of ooplast (enucleated oocytes) activation prior to receiving a donor nucleus on the development of nucleus transferred oocytes in cattle. The ooplasts were activated by electric stimulus at 30, 33, 36 and 39 h after being placed in culture medium for meiotic maturation. The activated ooplasts were further cultured in vitro, for a total 42 h from the beginning of maturation, 16- to 32-cell stage embryos produced by in vitro fertilization were used as donor embryos. The nucleus transferred oocytes were co-cultured with bovine oviductal epithelial cells in vitro. The fusion rate was not different between the activated (90%) and aged (94%) ooplasts 42 h after culture. Activated ooplasts receiving a donor nucleus showed a higher developmental rate than the aged ooplasts. Maximal development of the oocytes was obtained if the ooplast was activated at 9 h prior to receiving a donor nucleus. Thirty-nine percent developed to morulae and 24% to blastocysts. This compares (P<0.01) with 13% of the aged ooplasts developing to morulae and 8% to blastocysts. Of the activated ooplasts at 3, 6 and 12 h prior to fusion with a donor blastomere, 12, 16 and 13% developed to blastocysts, respectively. Of the 17 recipient cows receiving nucleus transferred embryos, 9 (53%) were diagnosed pregnant by palpation per rectum examination, and 3 normal offspring were obtained.     

A study of the inductive capacity of post-nodal primitive-streak pieces after treatment with follicle-stimulating hormone

Summary Chick embryos at the primitive-streak stage were treatedin vitro with 0.002 and 0.008% follicle-stimulating hormone (FSH)(NIH-FSH-S1) for 24 hours. Post-nodal primitive-streak pieces 0.8 mm behind the node level were grafted into host chick embryos at the primitive-streak stage to assess the capacity of the grafted pieces to produce inductions.Control grafts from donors maintained under identical conditions were unable to cause induction; all of them were resorbed into the host embryo. The post-nodal pieces treated with FSH acquired capacity to induce neural tissue. The grafts seemed to induce foregut formation also. FSH appeared to have supported differentiation of the grafts into somites and mesenchyme.     

Probing teleost eye development by lens transplantation

Experimental manipulation and other lines of evidence indicate that the lens plays a prominent role in the growth and differentiation of the vertebrate eye. Here we describe a lens transplantation method for studying the role of the lens in teleost eye development. The method involves three steps: (1) preparing embryos for the operations by embedding them in agar, (2) microsurgery with tungsten needles to remove the lens from a donor embryo and insert it into the optic cup of a host embryo lacking its own lens, and (3) a recovery period allowing surface ectoderm to close over the wound left by insertion of the lens into the host embryo. A movie illustrating the method can be found at http://www.life.umd.edu/labs/jeffery. A troubleshooting guide and summary of assays for evaluating the development of the transplanted lens and its effects on other eye parts, including the retina, are presented. Finally, some current applications of the lens transplantation method are briefly described: (1) determination of the autonomy of zebrafish lens mutants and (2) investigation of the role of the lens in eye degeneration in the cavefish Astyanax. The transplantation method will help characterize the mechanisms through which vertebrate eye development is regulated by the lens.     

Differences in the effects of treatment of uncompacted and compacted mouse embryos with phorbol esters on pre- and postimplantation development

Abstract. Differences are described in the effects of treatment of preimplantation mouse embryos with low levels (0.01–1 n M ) of phorbol myristate acetate (PMA), during three different periods of a 48-h culture from the 2-cell stage, on pre- and postimplantation development. Treatment of embryos with PMA for 48 h (first group) or 24 h (second group) from the 2-cell stage caused premature cavitation (prior to the 16-cell stage) and it also reduced the size and alkaline phosphatase (ALPase) activity of inner cell masses (ICMs), as well as the numbers of cells in blastocysts, in a dose-dependent manner. Treatment of early morulae with PMA for 24 h (third group) did not have the abovementioned effects on embryos but inhibited the formation and subsequent enlargement of the blastocoel. The blastocysts that were allowed to develop in the three treatment groups were examined for postimplantation development. Implantation was unaffected in all groups. The survival rate after implantation was low in the first and second groups but relatively high in the third group. The results indicate that an embryo exposed to PMA for 24 h from the 2-cell stage forms a premature blastocoel, and, in such an embryo, quantitative and qualitative differentiation into the ICM is blocked but qualitative differentiation into trophectoderm is uninhibited. Consequently, the embryo can implant but does not survive for a long time. When embryos were exposed to PMA for 24 h from the early morula stage, the formation and enlargement of the blastocoel were inhibited even though the treatment had a minimal effect on other developmental events. It is suggested that the effects of PMA on early mouse development are specific to each period at which the drug is applied.     

Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos

This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.     

Nuclear totipotency of cultured rabbit morulae to support full-term development following nuclear transfer.

The rabbit was used as a model for nuclear transfer. A critical step in nuclear transfer is oocyte activation, which was evaluated in this research. Optimal field strength of an electric stimulus for activation was examined. A significantly higher activation rate in all criteria tested was achieved when oocytes were activated electrically with a field strength of 2.4 kV/cm versus 1.2 or 1.8 kV/cm. Also, electrical stimulation with combined alternating current (AC) and direct current (DC) was superior to DC stimulation alone for activation. In another study involving 586 oocytes, exposure of oocytes to cytochalasin B for 1 h followed by activation with electrical stimulation significantly improved development of the oocytes to blastocyst stage compared to oocytes without cytochalasin B pre-exposure (38% vs 26%, p less than 0.05). Cytochalasin B exposure alone (control), however, had no effect on activation. Exposing oocytes to activation medium without electrical stimulation also activated some oocytes. In the nuclear transfer experiment, blastomeres from 8-cell embryos cultured for 20-24 h to the 32-64-cell stage were used as nuclear donor cells. Of 491 oocytes used, 459 (93%) survived the enucleation and fusion procedure, 370 (81%) fused, and 284 (77%) developed into 2-4-cell embryos. A total of 243 of these 2-4-cell embryos were transferred to 15 pseudopregnant recipients and produced 8 young (3%). Although the efficiency is low, this study demonstrated that rabbit morulae cultured for 20-24 h to the 32-64-cell stage as nuclear donors for transfer remain totipotent.     

Oviductal fluid and growth factors failed to enhance development of porcine embryos

Porcine embryos (1-, 2- and 4-cell) were cultured in a basal medium consisting of Krebs-Ringer bicarbonate buffer supplemented with oviductal fluid and several growth factors and observed for further development. Oviducts were flushed at either 48 h (Experiment 1) or 96 h (Experiment 2) after the onset of estrus. Observations were made every 48 h (Experiment 1) or 12 h (Experiment 2) until failure of the embryos to develop for 2 consecutive observations. Embryos were scored 0 = no development, 1 = cleavage, 2 = morula, 3 = blastocyst, or 4 = hatched blastocyst. In the first experiment, development of 1-, 2- and 4-cell embryos (n=282) in the basal medium supplemented with oviductal fluid (4:1) or 3 sets of growth factors, was less or equal to one cleavage stage. Those embryos cultured in the basal medium supplemented with bovine serum albumin (fatty acid free) (BSA) advanced to the blastocyst stage. In the second experiment, 96 h aged embryos (n=142) were cultured in the basal medium supplemented with IGF-1 and - 2 and EGF, or with BSA alone or with BSA and the three growth factors. In the treatments without BSA, the embryonic development was less than one cleavage, whereas in those treatments with BSA, embryos advanced beyond hatching and began to expand. We conclude that for culture of porcine embryos, supplementation with several growth factors or with oviductal fluid, in the concentration used in this study, was of little benefit at this stage of development. However, the type of BSA significantly affected development. More than 90% of the embryos reached the morula and blastocyst stages in medium than included BSA (fatty acid free).     

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.     

The influence of culture and transport on pretransfer development and posttransfer survival of sheep embryos

Sheep embryos were collected under sterile conditions in Dulbecco's Phosphate Buffered Saline (DPBS) + 10% Fetal Calf Serum (FCS) and cultured in DPBS + 20% FCS at 37 degrees C. They were classified according to their stage of development, from the lowest stage, 13 (less than a morula) to the highest stage, 20 (hatched blastocyst). Morphology was graded from 1 for excellent to 5 for degenerated. All embryos placed in the experiment had a development stage classification of 14 (morula) or higher and morphology classification of 1 to 3. Thirty-four embryos cultured in the laboratory for 24 h (Treatment B) and 32 embryos handled the same way except that they were transported for approximately 12 h (Treatment C) increased an average of 1.8 development classification scores and decreased -0.84 and -0.58 morphology classification scores for the two treatments, respectively. Twelve embryos in Treatment D (the same as Treatment C except that they were cultured in the laboratory for an additional 24 h) progressed 2.2 development classification scores during 48 h culture and did not change (-0.08) in the morphology classification score. The development estimates related to length of culture time but showed no affect of transport. Seventy-five percent of 20 embryos transferred in Treatment C and 42% of 12 embryos in Treatment D survived to parturition, demonstrating that the procedures used to transport and culture them for 24 and 48 h, respectively, maintained embryos capable of post transfer survival. Forty-one percent of the 22 embryos transferred in Treatment A (noncultured) and 20% of 20 embryos in Treatment B survived.     

Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization

The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.