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PubMed2004

Enhanced inflammatory response in neural tubes of embryos derived from diabetic mice exposed to a teratogen.

Exposure of embryos to the teratogen cyclophosphamide (CP) and maternal diabetes is linked to pathogenesis of neural tube defects during development. Maternal diabetes aggravates the teratogen-induced inflammatory reaction leading to increased risk of neural tube defects in mouse embryos. The inflammatory reaction in the developing neural tube has been characterized by the presence of activated amoeboid microglia/brain macrophages and altered expression levels of cytokines. Although there were no obvious anomalies observed in the neural tubes of embryos from CP-treated non-diabetic mice, the frequency of neural tube defects was increased significantly in embryos of CP-treated diabetic mice. Moreover, there were more activated amoeboid microglia in the forebrain of CP-treated diabetic embryos compared to that in CP-treated non-diabetic mice. The expression of cytokines such as tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) in the fetal brain of normal and diabetic embryos was induced in the neural tubes after CP treatment. Furthermore, the mRNA expression levels of both genes were increased markedly in the neural tube of CP-treated diabetic embryos compared to that of CP-treated non-diabetic embryos as measured by quantitative real-time PCR. Immunohistochemically, more TNF-alpha- and TGF-beta1-positive cells, which included neurons and amoeboid microglia, were detected in CP-treated diabetic embryos than in CP-treated normal embryos. Maternal diabetes aggravates teratogen-induced inflammation, which is characterized in the developing neural tube by increased amoeboid microglia and enhanced expression of inflammatory cytokines. Although a definite link has yet to be elucidated, it is suggested that the increased rate of neural tube defects observed in CP-treated diabetic embryos may be due to upregulation of proinflammatory cytokines caused by maternal diabetes.

Animals↗
PubMed2002

Induced thermotolerance in bovine two-cell embryos and the role of heat shock protein 70 in embryonic development.

Induced thermotolerance is a phenomenon whereby exposure to a mild heat shock can induce heat shock proteins (HSP) and other cellular changes to make cells more resistant to a subsequent, more severe heat shock. Given that the 2-cell bovine embryo is very sensitive to heat shock, but can also produce HSP70 in response to elevated temperature, experiments were conducted to test whether 2-cell embryos could be made to undergo induced thermotolerance. Another objective was to test the role of the heat-inducible form of heat shock protein 70 (HSP70i) in development and sensitivity of bovine embryos to heat shock. To test for induced thermotolerance, 2-cell bovine embryos were first exposed to a mild heat shock (40 degrees C for 1 hr, or 41 degrees C or 42 degrees C for 80 min), allowed to recover at 38.5 degrees C and 5% (v/v) CO2 for 2 hr, and then exposed to a severe heat shock (41 degrees C for 4.5, 6, or 12 hr). Regardless of the conditions, previous exposure to mild heat shock did not reduce the deleterious effect of heat shock on development of embryos to the blastocyst stage. The role of HSP70i in embryonic development was tested in two experiments by culturing embryos with a monoclonal antibody to the inducible form of HSP70. At both 38.5 degrees C and 41 degrees C, the proportion of 2-cell embryos that developed to blastocyst was reduced (P < 0.05) by addition of anti-HSP70i to the culture medium. In contrast, sensitivity to heat shock was not generally increased by addition of antibody. In conclusion, bovine 2-cell embryos appear incapable of induced thermotolerance. Lack of capacity for induced thermotolerance could explain in part the increased sensitivity of 2-cell embryos to heat shock as compared to embryos at later stages of development. Results also implicate a role for HSP70i in normal development of bovine embryos.

Animals↗
PubMed2003

Nucleolar proteins and ultrastructure in bovine in vivo developed, in vitro produced, and parthenogenetic cleavage-stage embryos.

In the present study, ribosomal RNA (rRNA) gene activation, monitored through nucleolus development, was studied by autoradiography following (3)H-uridine incubation, transmission electron microscopy, and immunofluorescence confocal laser scanning microscopy of key nucleolar proteins involved in rRNA transcription (topoisomerase I, upstream binding factor, and RNA polymerase I) and processing (fibrillarin, nucleolin, and nucleophosmin) in in vivo developed, in vitro produced, and parthenogenetic bovine embryos. In general, in vivo developed embryos displayed formation of fibrillo-granular nucleoli during the 4th post-fertilization cell cycle. During the previous stages of development, nucleolus precursor bodies (NPBs) were observed. However, on some occasions the initial steps of nucleolus formation were observed already at the 2- and 4-cell stage in cases where such embryos were collected from superovulated animals together with later embryonic stages presenting nucleolar development and autoradiographic labeling. The in vitro produced embryos displayed very synchronous formation of fibrillo-granular nucleoli and autoradiographic labeling during the 4th cell cycle. In vivo developed and in vitro produced embryos displayed allocation of nucleolar proteins to fibrillar and granular compartments of the developing nucleoli during the 4th cell cycle. The parthenogenetic embryos typically displayed formation of fibrillo- granular nucleoli during the 5th cell cycle and autoradiographic labeling was not observed until the morula stage. Moreover, the 1-, 2-, and 4-cell parthenogenetic embryos practically lacked NPBs. On the other hand, parthenogenetic embryos displayed allocation of nucleoar proteins to nuclear entities during the 4th cell cycle. In conclusion, both in vivo developed and in vitro produced bovine embryos displayed activation of transcription and nucleolar development during the 4th cell cycle. However, in vivo developed embryos flushed together with later developmental stages displayed premature activation of these processes. Parthenogenetic bovine embryos, on the other hand, displayed a delayed activation.

Animals↗
PubMed1987

Identification of a male-specific histocompatibility protein on preimplantation porcine embryos.

An indirect immunofluorescence assay was used to detect the presence of male-specific protein(s) on various stages of preimplantation porcine embryos. Embryos were collected at slaughter from the reproductive tracts of day-2.5, -4, -5, -6, and -8 (day 0 = first day of estrus) sows and gilts. Embryos were placed in medium containing an anti-male primary antibody, washed, and transferred to culture drops containing a fluorescein isothiocyanate (FITC)-labeled secondary antibody. Embryos were classified as either fluorescent (H-Y positive) or nonfluorescent (H-Y negative), transferred to coded drops, and karyotyped to examine sex chromosomes. A total of 91 eight-cell to blastocyst stage embryos were evaluated; of these, 46% were classified as fluorescent and 54% as nonfluorescent. Of readable metaphase spreads (65%) from these embryos, 81% (48 of 59, P less than 0.005) were correctly sexed by immunological detection of the male-specific antigen. Although 13% (2/15) of four-cell embryos evaluated were classified as fluorescent, the accuracy with which embryos at this stage were sexed by detection of H-Y antigen was not different from 50%. Fifty percent of eight-cell embryos were classified as H-Y positive with 78% of embryos correctly sexed. It was concluded that the eight-cell embryo is the earliest stage of development for which there is evidence for expression of H-Y antigen. Detection of the male-specific protein was difficult at the expanded blastocyst stage.

Animals↗
PubMed1989

Sex difference in digit and palate formation of mouse embryos at midgestation.

We investigated whether there is a difference in timing between the sexes of mouse embryos with regard to digit and palatal formation at midgestation by using a simple method of sex chromatin analysis for rodent embryos. At day 14.4 of gestation, although the mean body weight of male embryos was greater than that of female embryos, digit and palatal formation of female embryos was found to be more advanced than in males when compared according to their body weight. This was contrary to our previous finding of digit formation at the earlier stage (day 12.0 of gestation): namely, that digit development was more advanced in male embryos than in female embryos even when two sexes were compared according to their body weight. Thus, the development of female embryos catches up with that of male embryos at the later stages of midgestation. The period for digit development must be longer in male embryos than in female embryos. If mothers are exposed to some teratogens, a sex difference in incidence of digital defects might be produced.

Animals↗
PubMed1995

Glutathione redox cycle-driven recovery of reduced glutathione after oxidation by tertiary-butyl hydroperoxide in preimplantation mouse embryos.

The purpose of this study was to determine whether mouse embryos at the two-cell to blastocyst stage have the capacity to reduce glutathione disulfide (GSSG) and to elucidate the mechanism that embryos utilize to recover from tertiary-butyl hydroperoxide (tBH)-induced oxidative stress. Experiments were conducted on embryos in vitro and tBH was used to induce oxidation of embryonic reduced glutathione (GSH). After derivatization of extracted embryo samples with dansyl chloride, GSH and GSSG were measured at picomole levels by fluorometric HPLC. Two-cell- and blastocyst-stage embryos were able to recover their GSH levels within 45 min after depletion of GSH by incubation in tBH for 15 min. Addition of 1,3-bis(2-chloroethyl)-1-nitrosourea to the culture medium blocked recovery of GSH and resulted in continued elevation of GSSG. Addition of buthionine sulfoximine (BSO) to the culture medium did not affect GSH levels in two-cell-stage embryos, but did reduce GSH levels in blastocysts by 1.5 h. Culture of two-cell embryos in the presence of BSO for 45 h decreased embryonic GSH content and percentage of embryos developing to the blastocyst stage. These results indicate that preimplantation mouse embryos have the capacity to reduce GSSG and suggest that under normal conditions, depletion and synthesis of GSH occur to a greater extent in the blastocyst than in the two-cell-stage embryo. A major protective role for glutathione reductase during specific stages of embryo development is indicated.

Animals↗
PubMed1996

Beneficial effects of coculture with cumulus cells on blastocyst formation in a prospective trial with supernumerary human embryos.

PURPOSE: We reported previously on the use of coculture with cumulus cells in insemination medium for the development of human embryos in vitro. Here we describe a prospective trial to determine if this procedure has a significant beneficial effect. METHODS: On the day after insemination, zygotes were randomized for culture in either a fresh drop of medium without (- cum) or were left in their insemination drop with (+ cum) cumulus cells. Embryos with the best morphological quality were replaced on the third day of development at the eight-cell stage. The remaining embryos were cultured for a further 3 days and cryopreserved if they reached the fully expanded blastocyst (FEB) stage. Three different culture media were used over the period of this study. RESULTS: In 11 patients, supernumerary embryos were available only for continued culture in +cum and three patients had embryos cultured in only -cum. Thirty-nine other patients had embryos assigned to both +cum and -cum treatments. In the +cum group, 98 blastocysts developed from 216 embryos cultured for 6 days (45%), and this was significantly greater (P < 0.01) than the 48 blastocysts from 156 embryos (31%) developing in the absence of cumulus cells. In basal HTF medium (HTF medium with EDTA and glutamine) and basal XI HTF medium (similar to basal HTF but devoid of glucose and phosphate), culture of embryos with cumulus cells produced significantly more FEBs than in the absence of cumulus cells. There was no significant difference between the two culture treatments when regular HTF medium was used. Preliminary results indicate that pronectin-coated dishes provide a good substratum for cumulus cell attachment and embryo development. CONCLUSIONS: The culture of human embryos with their cumulus cells in insemination drops of medium produces a significantly greater proportion of FEBs than when the zygotes are transferred to fresh culture drops devoid of cumulus cells. This is the first report of a significantly higher blastocyst rate with coculture in which a real comparison has been made between two culture treatments which differ only in the presence or absence of homologous cumulus cells in insemination drops.

Blastocyst↗
PubMed1996

Evaluation of synthetic serum substitute versus serum as protein supplementation for mouse and human embryo culture.

PURPOSE: Our purpose was to determine the effect of Synthetic Serum Substitute (SSS) versus serum supplementation on fertilization rates and subsequent development of embryos from patients undergoing IVF. PROCEDURE: Experiment I compared the effects of SSS to human serum on mouse embryo development. Two hundred one-cell B6D2F1 mouse embryos were cultured in 100-microliter droplets of human tubal fluid (HTF) containing either (1) no protein (control; n = 37), (2) 15% serum from women with tubal infertility (n = 44), (3) 15% serum from women with endometriosis (n = 49), (4) 15% fertile donor serum (n = 33), or (5) 15% SSS (n = 37). Experiment II compared the effects of SSS to human serum on the development of embryos from patients undergoing IVF. Thirty-three women were included in this study. A total of 371 oocytes was cultured in HTF containing either (1) maternal or donor serum (n = 140) or (2) 15% SSS (n = 231). Embryo development was evaluated 48 hr after fertilization. RESULTS: In Experiment I, the rate of blastocyst development was evaluated at 48, 72, and 96 hr of culture. Sixty-four and nine-tenths percent of embryos cultured in SSS were morulae at 48 hr of culture (versus 5.4, 0, 8.2, and 6.1 in Groups 1, 2, 3, and 4, respectively). By 72 hr, 29.7% of these embryos had developed into blastocysts (versus 0, 0, 8.2, and 3.0, for Groups 1, 2, 3, and 4, respectively). This percentage increased to a total of 83.7 after 96 hr (versus 27.0, 20.4, 38.8, and 39.4 for Groups 1, 2, 3, and 4, respectively). Forty-three and two-tenths percent of the blastocysts cultured in SSS had hatched from their zonae by 96 hr. With the exception of Group 5, which had a rate of 9.1%, embryo hatching was not observed in any of the groups at the termination of culture (96 hr). In Experiment II there were no differences in cell stage or quality of human embryos cultured in SSS or serum, but fertilization rates tended to be better (P = 0.07) for oocytes inseminated in media containing SSS (70.0%, vs 55.0% for serum). CONCLUSIONS: SSS appears to be a superior protein source for mouse embryo growth and is as good as serum from fertile donors in promoting in vitro human embryo development.

Animals↗
PubMed2005

Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp].

The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anti-clinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of pro-embryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anti-clinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of pro-embryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient pro-cambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the pro-cambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of pro-cambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo.

Cells, Cultured↗
PubMed1983

Chromatin structure in pre- and postblastula embryos of Drosophila.

Early in embryogenesis of Drosophila melanogaster, DNA synthesis is extremely rapid while RNA synthesis is virtually undetectable. We have examined the chromatin structure of nuclei from preblastula embryos to determine whether these unusual rates of replication and transcription correlate with any alteration in the chromatin. DNase I-hypersensitive sites at the 5' end of genes have been postulated to be necessary but not sufficient for activity of the associated gene and have been shown to be established prior to the onset of transcription. In order to ascertain whether the apparent transcriptional incompetence of the early embryos is the result of the absence of such chromatin structure, we have examined nuclei from cleavage-stage embryos to determine whether the DNase I-hypersensitive sites have been established. A variety of genes, including inducible heat-shock genes, a constitutively expressed ribosomal protein gene, and two developmentally regulated genes, have been examined. In every case the pattern of DNase I-hypersensitive sites in preblastula embryos duplicates that in the later (6-18 hr after oviposition) embryos. In addition, two extra sites are observed in the early embryos, one at the 5' end of the hsp 70 gene and one in a gene at chromosomal locus 67B1. These sites do not correlate with any known function; however, neither can functional significance be ruled out. In a further investigation of the chromatin structure of early embryos, a nucleosomal array was generated. The pattern produced from nuclei of early embryos is extremely similar to that from 6- to 18-hr embryos, but somewhat less distinct. Both nucleosomal arrays and DNase I-hypersensitive sites must therefore be established very rapidly following DNA replication. The chromatin structure of cleavage-stage embryos detected by these tests appears to be essentially the same as that of older embryos, both in general, and at specific loci.

Animals↗
PubMed1983

Control of gap junction formation in early mouse embryos.

Intercellular communication via gap junctions begins in the eight-cell stage in early mouse embryos. We have studied the timing of this event in relation to compaction, and have begun to explore some of the possible control mechanisms underlying it. Gap junction formation was inferred by measuring ionic coupling as well as by observing the intercellular transfer of fluorescent dye. Embryos were obtained early on Day 3 of pregnancy by flushing the oviducts of HA/ICR mice that had been mated with CB6F1/J males. Gap junctions were detected only in those embryos which had achieved the fully compacted state. Inhibition of protein synthesis by cycloheximide treatment beginning as early as the late four-cell stage failed to block compaction or the acquisition of gap junctions, demonstrating that the necessary proteinaceous components are present in advance of these events. In order to test the possibility that gap junctions could be induced to form prematurely, fully compacted, communication-competent eight-cell embryos were aggregated with two- or four-cell embryos. Even after 10 hr of aggregation, no interembryonic gap junctions could be detected. Fully compacted eight-cell embryos when aggregated with each other, however, became ionically coupled within 3-5 hr. The number of interembryonic junctional channels was judged to be effectively small, since the aggregated embryos exhibited obvious ionic coupling but very weak dye coupling. In contrast to gap junction formation within embryos, junction formation between embryos was blocked by cycloheximide. These results demonstrate that gap junction formation in early mouse embryos is under precise temporal control, involving the assembly or mobilisation of preexisting components. This stockpile of components is either unavailable or insufficient to allow the formation of additional gap junctions between aggregated communication-competent embryos without new protein synthesis.

Animals↗
PubMed1984

Regulation of cytoplasmic mRNA prevalence in sea urchin embryos. Rates of appearance and turnover for specific sequences.

Complementary DNA clones representing cytoplasmic poly(A) RNAs of sea urchin embryos were hybridized with metabolically labeled cytoplasmic RNA preparations and the rates of appearance and of decay for each transcript species were determined at the blastula-gastrula stage of development. The prevalence of the transcripts chosen for this study ranged, on average, from about one molecule per cell to a few hundred molecules per cell. The embryos were labeled continuously for 18 hours with [3H]guanosine, beginning at 24 hours post-fertilization. The amount of cytoplasmic [3H]poly(A) RNA that hybridized to each cloned sequence was determined and the specific activity of the [3H]GTP pool was measured in the same embryos. Rate constants for the entry of each transcript species into the cytoplasm, and for its decay were extracted from these data. The embryo transcript species identified by the cloned probes displayed a range of stabilities. Half-lives of only a few hours were measured both for a very rare sequence and for a moderately prevalent sequence. Other newly synthesized transcripts, including sequences that first appear during embryonic development, as well as sequences also represented in maternal RNA, are far more stable. We conclude that cytoplasmic RNA turnover rate is a major variable in the determination of the cytoplasmic level of expression of embryo genes. The entry rates of the transcripts into the cytoplasm also varied, from a few molecules per embryo per minute to several hundred, depending on the sequence. By comparing the mass of transcripts of a given sequence in the embryo to the mass of transcripts of that sequence accumulating as a result of new synthesis, the point at which embryo transcription accounts for the major fraction of the cytoplasmic molecules could be estimated. This calculation showed that for some sequences maternal transcripts persist well beyond gastrulation, while other embryo poly(A) RNA species are largely the product of transcription in the embryo nuclei from the blastula stage onwards. There is no single stage at which all maternal transcripts are suddenly replaced by newly synthesized embryo transcripts. Primary transcription rates were measured for two sequences by determining accumulation of label in these RNA species soon after addition of [3H]guanosine to the cultures. Comparing these rates to the cytoplasmic entry rates, we did not detect a significantly greater nuclear transcription of the sequence homologous to the cloned probe.

Animals↗
PubMed1983

Cytolytic and fluorescent detection of H-Y antigen on preimplantation mouse embryos.

Monoclonal antibodies to histocompatibility (H-Y) antigen, of IgM subclass, were used to immunologically determine the sex of mouse embryos prior to transfer to pseudopregnant recipients. Two experiments were performed, one using cytolysis of H-Y positive embryos and the other using binding of a Fluorescein Isothiocyanate-(FITC) labeled second antibody. Eight- to 16-cell embryos used in the cytolytic assay were cultured in Whitten's Medium without bovine serum albumin (WM), to which monoclonal antibody and normal guinea pig serum were added. Embryos were classified as affected or unaffected, based on morphology of the embryo and its blastomeres. A total of 550 embryos were cultured; 294 (53.5%) were scored as unaffected and 263 of these were transferred to recipients. Forty-three (81.1%) of 53 pups born were female. Morulae and early blastocysts were used in the FITC-labeled second antibody assay. Embryos were cultured in WM containing monoclonal antibody, washed and placed in drops of WM containing FITC-labeled anti-IgM. Following another wash embryos were individually evaluated at 200X for fluorescence. Fifty-five percent (169 of 305) of the embryos displayed cell-specific fluorescence. A total of twenty-three pups, 18 males (78.3%) and five females (21.7%), were born following transfer of 156 fluorescing embryos. Four male (17.4%) and nineteen female (82.6%) pups resulted from embryos classified as non-fluorescing.

Journal Article↗
PubMed1985

Development of one-cell ovine embryos in two culture media under two gas atmospheres.

The objective of this study was to develop a successful system for culturing one-cell ovine embryos through several cleavage divisions. One hundred and four one-cell embryos were collected from synchronized, FSH-treated ewes 48 hr after the onset of estrus and randomly placed in one of four culture treatments. The effect of glucose supplementation and reduced oxygen tension (20% vs. 5%) on embryo development was studied. Embryo development was quantitated by a cleavage index based on the number of completed cell divisions. The number of embryos completing at least two cell divisions when cultured in Brinster's Pyruvate Medium (BPM) was 7 26 and 9 26 , under 5% CO(2) in air and 90% N(2), 5% CO(2), 5% O(2), respectively, while 22 26 and 20 26 embryos divided when cultured in BPM supplemented with 0.1% glucose (BPM-G) under similar atmospheres. Mean cleavage indices for embryos cultured in BPM were 1.2 and 1.6 under 5% CO(2) in air and 90% N(2), 5% CO(2), 5% O(2), respectively, while embryos cultured in BPM-G had mean cleavage indices of 4.6 and 4.0, respectively. Results of this study indicate that one-cell ovine embryos can be successfully cultured through several cleavage divisions. Glucose supplementation was beneficial for one-cell ovine embryo development. Reducing the oxygen tension from 20 to 5% had no effect on embryo development, and there was no media x gaseous atmosphere interaction.

Journal Article↗
PubMed1988

The integration of the transfer of frozen embryos with artificial insemination in a commercial beef herd.

This trial was designed to examine the integration of the transfer of frozen embryos and artificial insemination (A.I.) in a commercial herd of beef cows. Embryos were collected nonsurgically from 15 superovulated cows and all embryos judged viable (n = 69) were frozen in 0.25 ml plastic A.I. straws. Glycerol was used as a cryoprotectant and the embryos were frozen with omission of the traditional "seeding" step. On three consecutive days immediately preceding the onset of a 60-day breeding season, 54 embryos were transferred to multiparous cows with calves at foot. The remaining 82 cows in the same herd were used as controls. Cows receiving embryos were maintained under observation, but not inseminated for 21 days, after which cows exhibiting signs of estrus were inseminated up to Day 60. Cows in the control group were inseminated at observed estrus throughout the 60-day period. The overall pregnancy rate for the experimental and control groups did not differ (77.8 % and 73.2 % respectively). Of the experimental cows, 24.1 % conceived to the embryo transfer, and 53.7 % to A.I. The mean calving to conception interval for the experimental cows was 96.3 days (median 101 days) and for the control cows it was 92.3 days (median 88 days). The calving pattern in the experimental cows was biphasic, calvings resulting from embryo transfer of A.I. accounting for the two peaks. Reconception rates in the following breeding season were not different between the embryo transfer or A.I. groups or between experimental and control herds. Weaning masses of calves born of embryo transfer were significantly higher than the other groups, but calves resulting from A.I. in the experimental herd were not different from calves in the control herd. It was concluded that the transfer of frozen embryos could be successfully integrated into an A.I. program with a limited breeding season without detrimentally affecting the overall reproductive performance of the recipient herd.

Journal Article↗
PubMed1991

Determination of pentose phosphate and Embden-Meyerhof pathway activities in bovine embryos.

Quantitative determination was made of the activity of pentose phosphate pathway (PPP) and Embden-Meyerhof pathway (EMP) in individual bovine embryos from the six-cell to the hatched blastocyst stage. Embryos were collected from superovulated cross-bred heifers and classified into good and poor categories. A single embryo in 1 microl of medium was mixed with 2 microl of medium containing 3 to 30 nCi radiolabeled glucose previously placed on a detached lid of the 1.5-ml polypropylene microcentrifugé vial. The lid was then fitted to its vial which had been loaded in advance with 1.5 ml of 0.1 N NaOH. Vials were then incubated at 37 degrees C for 3 h. At the end of the incubation period, a 1.5-ml NaOH trap was inverted and placed into a 20-ml scintillation vial containing 10 ml of aqueous counting solution and counted in a liquid scintillation spectrophotometer. The PPP activity in good-quality embryos was greatest at the six-cell stage and decreased with increasing embryo development. The EMP activity showed the reverse trend. Poor- quality embryos had a lower glucose metabolism and higher PPP activity. Similar measurements were made on embryos following 24 h of culture, and total glucose metabolism and percentage of PPP activity were increased. In conclusion, these data suggest that in good quality bovine embryos total glucose utilization is low until 16-cell stage, with PPP being the predominant pathway. Total glucose utilization increases significantly at the morula stage; EMP activity increases with increasing embryo development; and PPP activity increases significantly in poor quality embryos and in embryos 24 h in culture.

Journal Article↗
PubMed1994

Sheep embryo cryopreservation by vitrification and conventional freezing.

The survival of ovine embryos (morulae and blastocysts) either frozen by a conventional method or vitrified was investigated in culture. In Experiment I, embryos were vitrified using a solution containing 25% propylene glycol and 25% glycerol. A group of embryos (simulated control) was processed without freezing to evaluate the toxicity of the vitrification solution. In Experiment II, embryos were exposed to a solution of PBS containing 10% glycerol and 0.25 M sucrose placed horizontally in a programmable freezer. Automatic seeding was applied at -7 degrees C in 2 positions on straws and cooled at -0.3 degrees C/min to -25 degrees C and then stored in liquid nitrogen. In vitro development rates of vitrified embryos were 12% (morulae) and 19% (blastocysts). Simulated embryos showed a higher rate of survival than embryos cryopreserved by vitrification (67 and 63%, morulae and blastocysts respectively). In conventional cooling, the blastocysts showed the highest viability percentage (67%) of all the experimental groups but these values decreased significantly in morulae (31%). Differences in temperature between straws placed in distinct positions in the freezing chamber and thermic deviation were observed when automatic seeding was applied. Embryo viability differed from 51 to 75% according the relative position of the embryos within the chamber. Survival was higher when automatic seeding was applied on the meniscus of the embryo column versus the central point of this column (65 vs 21%). The damage of both cryopreservation methods on zona pellucida integrity (27 and 35% in vitrified and conventionally frozen embryos, respectively) had no effect on the in vitro survival.

Journal Article↗
PubMed1994

Splitting of porcine embryos at the four-cell or morula stage.

Porcine embryos obtained from estrus-induced prepuberal gilts were split at the 4-cell and morula stage. The in vitro development of the demi-embryos was compared with that of intact control embryos. The intact control embryos developed according to expectation. The in vitro survival of the demi-embryos was inferior to that of the respective controls. The splitting of 4-cell embryos was easier to accomplish and more efficient than the splitting of morulae. The in vitro development of the 4-cell embryos was also slightly better, although this difference was not significant. In vitro development of demi-embryos originating from morulae split at room temperature was slightly although nonsignificantly inferior to that of demi-embryos from morulae split on a warming stage at 37 degrees C. Between 14 and 18 demi-embryos were transferred to synchronous recipient gilts after 24 to 48 h of culture. Of 3 gilts receiving split 4-cell embryos, 1 gilt aborted 2 normal fetuses on Day 90 of pregnancy and 1 carried a single piglet to term. Of the 4 gilts receiving split morulae, 1 gilt had 4 normal and 3 degenerated fetuses upon slaughter on Day 35 of pregnancy.

Journal Article↗