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PubMed2006

Proteomic analyses of somatic and zygotic embryos of Cyclamen persicum Mill. reveal new insights into seed and germination physiology.

In the horticulturally important ornamental species Cyclamen persicum Mill., somatic embryogenesis is an efficient vegetative propagation method and the development of artificial seeds is an ultimate aim. This study aims at a systematic comparison of the proteomes of zygotic embryos, somatic embryos grown in liquid medium containing 30 or 60 g l(-1) sucrose, germinating embryos of both types and endosperm in order to obtain novel insights into seed and germination physiology. Using high resolution two-dimensional isoelectric focussing/sodium dodecylsulfate polyacrylamide gel electrophoresis (2D IEF/SDS PAGE), 74% of the proteins expressed in zygotic embryos were found in similar abundance in somatic embryos grown in 60 g l(-1) sucrose. Somatic embryos grown in 30 g l(-1) sucrose accumulated fewer protein species than those grown in 60 g l(-1). Selected proteins were identified following mass spectrometry (nano-LC-MS/MS). Four enzymes involved in glycolysis (UDP-glucose pyrophosphorylase, fructose bisphosphate aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase GAPDH) were specifically induced in somatic embryos. 11S globulin proteins identified by MS were present in high levels in somatic embryos, zygotic embryos and endosperm, whereas 7S globulins were detected mainly in endosperm and zygotic embryos. These are the first storage proteins identified in C. persicum. Xyloglucans are known to be another group of seed storage compounds in C. persicum. Interestingly, xyloglucan endotransglycosylases were found to be highly expressed in endosperm tissue. We discuss the physiological implications of these observations.

Culture Media↗
PubMed2004

Effects of growth hormone on the ultrastructure of bovine preimplantation embryos.

Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.

Animals↗
PubMed2002

High accumulation of legumin and Lea-like mRNAs during maturation is associated with increased conversion frequency of somatic embryos from pedunculate oak (Quercus robur L.).

The expression patterns of the storage protein legumin gene and Em- and dehydrin-like homologues were investigated in somatic embryos from Quercus robur L. The effect of different maturation treatments (1% agar, 6% sorbitol, or 5% polyethylene glycol) and partial desiccation on transcript accumulations as well as conversion capacity of somatic embryos was also investigated. Differential expression of putative Em- and two dehydrin-like homologues (designated as Dhn1a and Dhn1b) was detected in somatic oak embryos with heterologous probes. Low expression levels of legumin, Em- and dehydrin-like mRNAs were detected in somatic embryos prior to maturation treatment. A high accumulation of these transcripts was found in embryos that had been cultured on media supplemented with 6% sorbitol or 1% agar. These embryos also showed a high conversion frequency into plantlets. In contrast, no improvement in plant conversion as well as a low accumulation of legumin, Em-like and Dhn1b-like transcripts was observed in embryos that were matured on polyethylene glycol medium. Partial-desiccation treatment significantly enhanced the plant conversion. Nevertheless, a decline in expression of legumin, Em-like and Dhn1a-like homologues was detected upon dehydration. In contrast, Dhn1b and oak homologues to peach dehydrin were also strongly expressed in desiccated embryos. In addition, the treatment of embryos with abscisic acid promoted the accumulation of all investigated transcripts. These results suggest that the regulation of the legumin storage protein gene and Lea-like homologues in somatic oak embryos is under developmental control and that their regulation can be influenced by manipulating the culture conditions.

Abscisic Acid↗
PubMed2006

Human papilloma virus DNA exposure and embryo survival is stage-specific.

PURPOSE: Human papillomavirus (HPV) has been shown to disrupt late-stage implanting embryos. The objectives were (a) to assess the development of early embryos exposed to HPV DNA and (b) to analyze the blastocyst hatching process after HPV exposure. METHODS: The study involved exposing two-cell and 4-8-cell mouse embryos to DNA fragments from either HPV type 16, type 18 or DQA1 (control). The embryos were incubated for 120 h and assessed. RESULTS: HPV 16 and 18 inhibited two-cell embryo development. In contrast, delaying the exposure of HPV DNA until the 4-8-cell stage resulted in further embryonic development. There was 25.9% less blastocyst formed with HPV 16 exposure. Additionally, there were 25.9-31.8% more degenerated embryos with HPV 16 exposure. CONCLUSIONS: The study demonstrated embryo stage-specific effects of HPV on early development. The results suggested HPV exposure was linked to two-cell embryo demise and delaying the exposure of HPV until later embryo stages permitted embryo development. HPV 16 was shown to decrease blastocyst formation while HPV 18 inhibited the blastocyst hatching process.

Animals↗
PubMed1983

Expression of cytokeratin polypeptides in mouse oocytes and preimplantation embryos.

The presence and distribution of intermediate filament proteins in mouse oocytes and preimplantation embryos was studied. In immunoblotting analysis of electrophoretically separated polypeptides, a distinct doublet of polypeptides with Mr of 54K and 57K, reactive with cytokeratin antibodies, was detected in oocytes and in cleavage-stage embryos. A similar doublet of polypeptides, reactive with cytokeratin antibodies, was also detected in late morula-and blastocyst-stage embryos, and in a mouse embryo epithelial cell line (MMC-E). A third polypeptide with Mr of 50K, present in oocytes only as a minor component, was additionally detected in the blastocyst-stage embryos. No cytokeratin polypeptides could be detected in granulosa cells. Immunoblotting with vimentin antibodies gave negative results in both cleavage-stage and blastocyst-stage embryos. In electron microscopy, scattered filaments, 10-11 nm in diameter, were seen in detergent-extracted cleavage-stage embryos. Abundant 10-nm filaments were present in the blastocyst outgrowth cells. In indirect immunofluorescence microscopy (IIF) of oocytes and cleavage-stage embryos, diffuse cytoplasmic staining was seen with antibodies to cytokeratin polypeptides but not with antibodies to vimentin, glial fibrillary acidic protein, or neurofilament protein. Similarly, the inner cell mass (ICM) cells in blastocyst outgrowths showed diffuse cytokeratin-specific fluorescence. We could not detect any significant fibrillar staining in cleavage-stage cells or ICM cells by the IIF method. The first outgrowing trophectoderm cells already had a strong fibrillar cytokeratin organization. These immunoblotting and -fluorescence results suggest that cytokeratin-like polypeptides are present in mouse oocytes and preimplantation-stage embryos, and the electron microscopy observations show that these early stages also contain detergent-resistant 10- to 11-nm filaments. The relative scarcity of these filaments, as compared to the high intensity in the immunoblotting and immunofluorescence stainings, speaks in favor of a nonfilamentous pool of cytokeratin in oocytes and cleavage-stage embryos.

Animals↗
PubMed1986

The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein synthesis.

The role of the suspensor in the early development of the dicot embryo has been described as merely an anchor or, conversely, as the major route of nutrients into the embryo. In order to further elucidate the role of the suspensor we have examined protein synthesis in early 0.2-mm and late heart stage 0.5-mm Phaseolus vulgaris (var. Taylor's Horticultural) embryos in tissue culture. Protein synthesis was examined in embryos and suspensors. Our results showed that in 0.2-mm embryos virtually all protein synthesis was dependent on an attached suspensor. Maximum protein synthesis in 0.5-mm embryos was observed when embryos were cultured attached to the suspensor. The levels were moderately decreased when the embryo was cultured detached from or without the suspensor. Gibberellic acid at 10(-6) to 10(-7) M elicited the same protein diversity and greater [35S]methionine incorporation than did the attached suspensor in 0.2-mm embryos. Embryos of 0.5 mm did not appear to be differentially responsive to various gibberellin concentrations.

Culture Techniques↗
PubMed1985

A new procedure for the cryopreservation of equine embryos.

Early equine blastocysts and blastocysts were collected nonsurgically at six days post-ovulation. Thirty-two embryos were randomly assigned to a 2x2 factorial design. Factors were: 1) 0.5-ml straws or 1-ml glass ampules; and 2) plunging into liquid nitrogen (IN(2)) at -33 C or -38 C. Cryoprotectant, 10% glycerol in PBS plus 5% fetal calf serum (FCS) was added in two steps, 5% then 10%. Embryos were cooled at 4 C/min to -6 C and then seeded, 0.3 C/min to -30 or -35 C and 0.1 C/min to -33 or -38 C. Samples were thawed in 37 C water and glycerol removed in six steps, 10 min per step. Embryo quality and stage of development were evaluated prior to freezing, immediately post-thaw and after 24 h culture in Ham's F10 with 5% FCS. The mean post-thaw quality of embryos plunged at -33 C was superior (P<0.05) to that of embryos plunged at -38 C (2.0 vs 2.9). Embryos frozen in ampules and plunged at -38 C were of poorer quality (P<0.05) than those frozen in ampules and plunged at -33 C or frozen in straws and plunged at -33 C. After 24 h of culture, more embryos developed if frozen in straws compared to ampules, and plunging at -33 C resulted in higher quality embryos than plunging at -38 C. In Experiment 2, 23 embryos were packaged in straws and plunged at -33 C as described in Experiment 1. Six of the 23 surgically transferred frozen embryos were degenerate at thawing and the remaining 17 surgically transferred were via flank incision. Pregnancy rate at 50 days post-ovulation was 53% (nine of 17). Early blastocysts resulted in a higher (P<0.05) pregnancy rate (8 10 , 80%) than expanded blastocysts (1 7 , 14%).

Journal Article↗
PubMed1985

Use of a uterine-cell monolayer culture system for micromanipulated bovine embryos.

The objective of this study was to evaluate the growth of micromanipulated bovine embryos in two in vitro culture systems. Sixty ova (day 7 from estrus) were collected in Dulbecco's phosphate-buffered saline (PBS), with 2% fetal calf serum, and transferred to a PBS holding medium containing 10% fetal calf serum to prepare for micromanipulation. Forty embryos (morula to expanded blastocyst stages) were selected for embryo splitting using a modified microsurgery procedure. Thirty-nine of these embryos were successfully bisected into demi-embryos (DE) and the halves allotted by post-manipulation quality grades into one of two treatment groups (Trt). DE in Trt A were cultured in Ham's F-10 medium with 10% FCS (HF-10) while the remaining DE halves from each embryo were cocultured in HF-10 on a monolayer of endometrial fibroblasts (8 x 10(4) viable fibroblast cells plated three days prior to culture) in Trt B. Embryo development, recorded at 12-hour intervals, was evaluated by a split-plot analysis of variance. Results indicated that embryo viability decreased (P<0.001) over time in culture. Overall viability was greater (P<0.001) for DE in Trt B than in Trt A, with a significant (P<0.05) Trt x Time interaction, indicating that embryo viability decreased more rapidly across time in HF-10 than in the monolayer coculture system. The percentage of DE developing at 12, 24, 36, 48, 60 and 72 hours in culture was: 44%, 41%, 33%, 28%, 21% and 18% for Trt A and 69%, 69%, 69%, 67%, 62% and 62% for Trt B. Fourteen of the DE in Trt B attached to fibroblast monolayer and initiated trophoblastic outgrowth and four additional DE remained viable for up to 17.5 days in vitro as intact blastocysts. These findings are the first reported that demonstrate that the zona-free bovine DE will develop during in vitro culture. Also, the bovine endometrial fibroblast monolayer system proved to be excellent for both short term (</=12 hours) and long term (up to 72 hours) culture of halved bovine embryos.

Journal Article↗
PubMed1987

Factors affecting survivability of transferred whole and demi-embryos in a commercial dairy herd.

Sixty Holstein donor cows were superovulated and embryos were collected during a 6-d (27 cows) and a 4-d (33 cows) period approximately 60 d apart. Forty-three donor cows yielded embryos. Ninety-one embryos graded 1 or 2 were split and transferred to 181 recipient Holsteins. Demi-embryos were graded 2, 2-, 3 and 3- prior to transfer. Pregnancy and calving percentages were similar for all demi-embryo grades, averaging 59 and 53% from the two donor groups, respectively. Twin demi-embryo pregnancies averaged 36 and 19% for embryos split at the compacted morula and blastocyst stages, respectively. Twin demi-embryo calvings averaged 30 and 15% for these same groups. Progesterone levels of recipients (of either whole or demi-embryos) of second period donors were measured. Pregnancy rate increased generally with level of progesterone; however, calving percentage was slightly greater for recipients with intermediate levels of progesterone (2-6 ng/ml). Multiparous cow (20) recipients of demi-embryos had 45% pregnancy and 40% calving, while nulliparous heifer (161) recipients averaged 59 and 53% pregnancy and calving, respectively.

Journal Article↗
PubMed1988

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.

Journal Article↗
PubMed1989

Effect of gossypol and its metabolite on in vitro early development of mouse embryos.

The purpose of this study was to examine the effect of gossypol and its metabolite on early in vitro mouse embryo development. One hundred and thirty-eight excellent quality mouse blastocysts were randomly assigned to five different treatments. Culture media were supplemented with 10% (V/N) normal steer serum. The embryos were cultured at 37 degrees C with an atmosphere of 5% O(2), 5% CO(2) and 90% N(2), and embryo development was examined and recorded at 12-h intervals for 72 h. The percentage of embryos that developed to expanded blastocyst (92%), hatching blastocyst (84%), and hatched blastocyst (76%) stages in control Ham's F-10 media was not different from that of embryos cultured in media containing 0.1 and 5 mug of gossypol; however, none of the embryos treated with 265 ng of gossypol metabolite (GM) developed beyond the blastocyst stage. A substantial decrease in the percentage of embryos reaching hatching blastocyst (29%) and hatched blastocyst (29%) stages was observed in the embryos cultured with 5.3 ng of GM. At both light and electron microscopic levels, the embryos appeared to be affected even by a lower concentration of GM in vitro. Our results suggest that GM has a much greater potency than the parent gossypol in inhibiting the early development of mouse embryos in vitro.

Journal Article↗
PubMed1989

Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae.

Embryos were collected nonsurgically from nåturally-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.

Journal Article↗
PubMed1991

Fusion and development rates of single blastomere pairs of mouse two- and four-cell embryos using the electrofusion method.

The present study was undertaken to find suitable conditions for blastomere fusion of mouse two- and four-cell embryos using the electrofusion method to simplify the nuclear transfer procedure. Single blastomeres of ICR and F1 (C57BL/6J x CBA/N) two-cell embryos or ICR four-cell embryos and F1 two-cell embryos were paired and treated with electric stimulus under different fusion conditions. Two hours after electrofusion treatment, the fused blastomere pairs were encapsulated in alginate gel and cultured for 96 hours to observe their developmental potential. When the single blastomere pairs of two-cell embryos were exposed to electric pulses of 1.0, 1.5 and 2.0 kV/cm for 30, 60 and 90 mu sec, high fusion rates were obtained (84.6 to 100%). However, when two-cell blastomere were paired with four-cell blastomere and then treated under the same conditions, the fusion rates (27.5 to 87.5%) were lower than that of single blastomere pairs of two-cell embryos regardless of the duration and strength of the d.c. pulses. The blastocyst developmental rate after in vitro culture of the fused blastomere pairs of two-cell embryos using the above electrofusion conditions was high (81.8 to 100%). Lower blastocyst developmental rates were obtained on the fused blastomere pairs of two- and four-cell embryos (46.4 to 76.2%). Based on the results of this study, a pulse duration of 60 mu sec and a pulse strength of 1.0kV/cm were the most suitable conditions for single blastomere pair fusion of two-cell or two- and four-cell embryos. The study further showed that alginate gel is a good substitute for zonae pellucidae for encapsulating zona-free embryos.

Journal Article↗
PubMed1991

Failure of embryos from bluetongue infected cattle to transmit virus to susceptible recipients or their offspring.

Sixty heifers were infected with bluetongue virus (BTV) by the bites of the vector and by inoculation with insect origin virus. During the acute and convalescent stages of the infection, embryos were collected nonsurgically from these animals and washed according to the recommendations of the International Embryo Transfer Society (1). No BTV was isolated from 77 of these embryos when they were inoculated onto cell culture and into embryonating chicken eggs. There was no evidence of lateral BTV transmission when 231 of these embryos were transferred into susceptible recipients, nor was there evidence of vertical BTV transmission to the 88 calves resulting from these transfers. Another six donors that were assumed to have recovered from a natural infection of BTV, were added to the study to increase the probability of obtaining embryos from a persistently infected BTV carrier. However, it was determined later that these animals had not been infected with BTV but with the closely-related epizootic hemorrhagic disease virus (EHDV). Embryos were collected from these donors and washed as above. Neither BTV nor EHDV was isolated from 26 of these embryos by the inoculation of cell culture and embryonating chicken eggs. There was no evidence of lateral BTV or EHDV transmission to recipients of 15 of these embryos or of vertical BTV or EHDV transmission to the resulting 7 calves. However, two recipients of embryos from one of these donors developed antibodies to BTV 6 to 9 months after transfer. Passive antibodies to BTV were also detected in their calves. There is good evidence that these two recipients acquired BTV from natural exposure to infected insect vectors and not from the transferred embryos.

Journal Article↗
PubMed1991

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).

Journal Article↗
PubMed1992

Number and viability of embryos collected in vivo or from the excised uteri of slaughtered donor cows.

Embryos were collected from superovulated donor cows on Day 7 of the cycle either in vivo by a standard nonsurgical method (A) or in vitro from the excised uterus after slaughter of the donor cow (B). In Method B, the time between slaughter and flushing of the uterus ranged from 0.5 to 4 hours. Flush yield was 5.6 +/- 5.0 and 8.4 +/- 5.3 embryos (P<0.01); the recovery rate was 0.6 +/- 0.4 and 0.8 +/- 0.3 (P<0.05) for Methods A and B, respectively. Method B resulted in more Grade 3 (P<0.001) and 4 embryos, while Method A resulted in more Grade 1 and 2 embryos. The correlation between the percentage of Grade 1 and 2 embryos and the time interval between slaughter of the donor cow and flushing the excised uterus was -0.42 for in vitro flushes. Viability of fresh and frozen-thawed embryos, as determined by in vitro culture of Grade 1 and 2 morulae and early blastocysts, was considerably lower for Method B than Method A. The percentage of embryos developing into expanded blastocysts was 100% (10 10 ) and 40% (4 10 ) for fresh embryos (P<0.01) and 52.7% (29 55 ) and 0% (0 25 ) for the frozen-thawed embryos (P<0.001) for Methods A and B, respectively. This reduction in viability might be the result of a postmortem pH decrease in the uterine fluids within the first 30 minutes from 7.0 to 5.8 and 6.0. Flushing of the uterus directly after slaughter (within 5 to 10 minutes) may prevent the possible detrimental effect of a low pH on the embryos.

Journal Article↗
PubMed1993

Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide.

The influence of equilibration time before vitrification on the viability of vitrified morula- to blastocyst-stage bovine embryos and in vivo viability of vitrified embryos following transfer to recipients were investigated. In experiment 1, the embryos were exposed to an equilibration solution (50% VSED) containing 12.5% v/v ethylene glycol and 12.5% v/v dimethyl sulfoxide in modified Dulbecco's phosphate buffered saline with 4 mg/ml BSA (m-PBS) for 1, 2 and 5 minutes at room temperature (22 to 24 degrees C). The embryos were then placed in 15mul vitrification solution (VSED) consisting of 25% v/v ethylene glycol and 25% v/v dimethyl sulfoxide in m-PBS and were loaded into 0.25 ml plastic straws at room temperature. After 30 seconds, the straws were placed in liquid nitrogen (LN(2)) vapor for 2 minutes, plunged and stored in LN(2). To thaw, the straws were warmed in water at 20 degrees C for 15 seconds and the contents of the straws were expelled into a plastic dish. The embryos were diluted in 0.5 M sucrose + m-PBS for 5 minutes and were cultured in TCM-199 supplemented with bovine oviductal epithelial tissue. Viability of the embryos was assessed by the forming or reforming of the blastocoele after 24 hours of culture. High in vitro survival rates (73 approximately 90%) of vitrified embryos were obtained after 1 and 2 minute equilibrations, but was reduced (P<0.05) after 5 minute equilibration. In Experiment 2, morula- to blastocyst-stage embryos were vitrified after 1 minute equilibration in 50% VSED and 30 seconds of exposure to VSED. The vitrified-warmed embryos were transferred to recipient heifers at 7 days after estrus (1 embryo per recipient). Five (38%) of 13 (40%) of 10 recipients that had received blastocysts were diagnosed as pregnant using ultrasonography 60 days following transfer.

Journal Article↗
PubMed1994

Effects of hyaluronic acid on the development of 1- and 2-cell porcine embryos to the blastocyst stage in vitro.

The purpose of this study was to evaluate the ability of hyaluronic acid to improve the development of 1- and 2-cell porcine embryos to the blastocyst stage in a simple medium. In Experiment 1, we confirmed the ability of Whitten's medium supplemented with 15 mg/ml BSA to support the development of porcine embryos to the blastocyst stage under our experimental conditions. Embryos collected from oviducts were cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in humidified air up to 6 d. After 2 d of culture, 82 and 78% of embryos reached the 4-cell stage or beyond in TCM199 supplemented with 10% fetal calf serum (FCS) and in Whitten's medium with BSA, respectively. However, no embryo developed to the morula stage in TCM199 after 6 d of culture. On the other hand, 26 and 15% of embryos developed to the morula and the blastocyst stage in Whitten's medium, respectively. In Experiment 2, we determined whether supplementation of hyaluronic acid in Whitten's medium would improve the development of porcine embryos to the blastocyst stage. After 6 d of culture, development of the embryos to the blastocyst stage was best supported in Whitten's medium with 4 mg/ml BSA and 0.5 mg/ml hyaluronic acid (70%). The proportion of degenerated embryos was lower in the presence than in the absence of hyaluronic acid. These results indicate that the supplementation of Whitten's medium with hyaluronic acid improves the development of 1- and 2-cell porcine embryos to the blastocyst stage.

Journal Article↗