Biomedical subjects

T Greve

Publications and source records attributed to T Greve.

At least 37 records · Page 2Linked to original sources

PubMed2001

Downregulation of p56(lck) tyrosine kinase activity in T cells of squirrel monkeys (Saimiri sciureus) correlates with the nontransforming and apathogenic properties of herpesvirus saimiri in its natural host.

Herpesvirus saimiri is capable of transforming T lymphocytes of various primate species to stable growth in culture. The interaction of the T-cellular tyrosine kinase p56(lck) with the transformation-associated viral protein Tip has been shown before to activate the kinase and provides one model for the T-cell-specific transformation by herpesvirus saimiri subgroup C strains. In contrast to other primate species, squirrel monkeys (Saimiri sciureus) are naturally infected with the virus without signs of lymphoma or other disease. Although the endogenous virus was regularly recovered from peripheral blood cells from squirrel monkeys, we observed that the T cells lost the virus genomes in culture. Superinfection with virus strain C488 did not induce growth transformation, in contrast to parallel experiments with T cells of other primate species. Surprisingly, p56(lck) was enzymatically inactive in primary T-cell lines derived from different squirrel monkeys, although the T cells reacted appropriately to stimulatory signals. The cDNA sequence revealed minor point mutations only, and transfections in COS-7 cells demonstrated that the S. sciureus lck gene codes for a functional enzyme. In S. sciureus, the tyrosine kinase p56(lck) was not activated after T-cell stimulation and enzymatic activity could not be induced by Tip of herpesvirus saimiri C488. However, the suppression of p56(lck) was partially released after administration of the phosphatase inhibitor pervanadate. This argues for unique species-specific conditions in T cells of S. sciureus which may interfere with the transforming activity and pathogenicity of herpesvirus saimiri subgroup C strains in their natural host.

Amino Acid Sequence↗

PubMed2001

Ribosomal RNA gene expression and chromosome aberrations in bovine oocytes and preimplantation embryos.

This review focuses on the key features of development of the bovine oocyte and embryo, with comparisons of the developmental characteristics of embryos produced in vivo and in vitro. The oocyte is transcriptionally quiescent in the primordial and primary follicle. In the secondary follicle transcription is initiated in the oocyte and a ribosome-synthesizing nucleolus is established in this cell. Transcription and nucleolar activity are enhanced in the tertiary follicle during oocyte growth. When the oocyte reaches approximately 110 microm in diameter, corresponding to a follicle of about 3 mm in diameter, transcription ceases and the nucleolus is inactivated, forming a dense spherical remnant. During the final phase of follicular dominance this remnant becomes vacuolated and, in conjunction with resumption of meiosis, it disperses. The rRNA genes are apparently re-activated during the four-cell stage, that is, the third cell cycle after fertilization, but a nucleolus is not formed. During the subsequent cell cycle, that is, during the eight-cell stage, ribosome-synthesizing nucleoli are again established. Bovine embryos produced in vitro apparently display the same pattern of nucleolus development as that in embryos developed in vivo. Examination of the ploidy of embryonic cells using fluorescence in situ hybridization has revealed that the production of bovine embryos in vitro is associated with increased chromosome aberrations in the embryos. Blastocysts produced in vitro display a significantly higher rate of mixoploidy, that is, when the embryo consists of both normal diploid and abnormal polyploid cells, than that in embryos developed in vivo. The rate of mixoploidy among embryos produced in vitro increases with increasing developmental stage. Moreover, after fertilization in vitro, initially there is a high rate of 'true' polyploidy, that is, when all cells of the embryos are polyploid. However, the polyploid embryos are eliminated before they cleave beyond the eight-cell stage, the stage at which major activation of the embryonic genome occurs in cattle.

Animals↗

PubMed2001

Embryonic developmental stages in relation to the LH peak in dogs.

Blood samples were collected from nine Beagle bitches every 6 h during pro-oestrus and oestrus to measure plasma concentrations of progesterone, oestradiol and LH. The number of ovarian follicles was estimated once a day using transcutaneous ultrasonography. According to the concentrations of plasma progesterone, the bitches were mated once and subsequently ovariohysterectomized 3-7 days after mating. The number of corpora lutea was counted, and oocytes and embryos were collected by flushing of the oviducts. In ovaries that had more than three follicles, the number of follicles observed using ultrasonography was underestimated, whereas the recovery rate (number of oocytes and embryos flushed/number of corpora lutea counted) was 99%. Embryos were processed for sectioning and the developmental stages were determined by counting the number of blastomeres under bright field microscopy. Potentially fertilized oocytes and a zygote were observed on day 7 after the LH peak and the developmental rate was about one cell cycle in 24 h until day 12 after the LH peak. The timing of embryonic development was significantly correlated with the time of the LH peak.

Animals↗

PubMed2000

A novel approach for in vitro production of bovine embryos: use of the Oxoid atmosphere generating system.

The importance of the incubator type is often overlooked when protocols for in vitro production of embryos are evaluated. In this study the ability of a standard CO2 Heraeus incubator and the Oxoid CO2Gen atmosphere-generating system to support bovine in vitro oocyte maturation, fertilization and embryo development is described for the first time. The Oxoid CO2Gen gas generating system, originally designed for the growth of bacteria, is based on the chemical reaction of ascorbic acid and air. When the sachet with ascorbic acid is placed in the confined volume of the airtight AnaeroJar, an atmosphere of 6% CO2 in 15% O2 is created, which is comparable to the 5% CO2 and 20% O2 used for standard in vitro production of bovine embryos. In the first set of experiments oocyte in vitro maturation (IVM), fertilization (IVF) and embryo culture (IVC) were allocated to one or the other of the culture systems. In the second set of experiments IVM and IVF took place in the Heraeus incubator, while IVC was allocated either to the Heraeus or to the AnaeroJar. During experiments the AnaeroJar was placed in the Heraeus incubator to ensure identical incubation temperatures of 38.8 degrees C. A standard protocol was used for production of embryos: 23 h of IVM in TCM-199, 20 h of IVF with frozen-thawed washed spermatozoa in TALP medium and 7 days of IVC (8 days after insemination) in B2 medium with bovine oviduct epithelial cells. In the first set of experiments, based on a total of 766 inseminated oocytes, the Day 8 blastocyst rates were the same in the Heraeus incubator and the AnaeroJar: 30% vs. 30% with oviduct cell coculture, and 21% vs. 18% without coculture. In the second set of experiments, based on 1963 inseminated oocytes, the average blastocyst rates were 27% vs. 32% from the Heraeus incubator and the AnaeroJar. In 2 of 6 replicates blastocyst rates were lower in the Heraeus incubator than in the jar; in the remaining replicates they were alike. No differences were noted in blastocyst kinetics or morphology. In conclusion, the Oxoid gas generating system seems to be a cheap, convenient and stable alternative to expensive CO2 incubators, not only for the growth of bacteria, but also for in vitro production of bovine embryos.

Animals↗

PubMed2000

Steroid concentrations in follicular fluid aspirated repeatedly from transitional and cyclic mares.

The objectives of the present study were to determine follicular progesterone (P4) and estradiol-17beta (E2) in transitional mares and to compare follicular steroid concentrations between transitional and cyclic mares. Follicles > 8 mm were aspirated under transvaginal ultrasound-guidance 4 times at 3 to 4 day intervals (T1-T4) in Norwegian pony mares during vernal transition. During the breeding season, follicular aspirations were conducted in each mare on Day 6, Day 14 and Day 18 after ovulation of 3 separate estrous cycles (Day of ovulation = Day 0). Plasma and follicular fluids were analyzed for P4 and E2 with ELISA and RIA, respectively. Plasma P4 concentrations remained below 1 ng/mL throughout T1-T4, while the follicular P4 concentrations increased significantly to cyclic levels after the first transitional aspiration. Plasma E2 concentrations similarly remained at low levels during the course of the transitional aspirations, while the follicular E2 concentrations increased gradually over the 4 aspirations to cyclic concentrations. The mares ovulated on average 9.8 +/- 1.6 (mean +/- SEM) days after the last transitional aspiration, and 16.6 +/- 0.2, 11.3 +/- 1.5 and 23.2 +/- 4.4 days after aspirations conducted on Day 6, 14 and 18, respectively. The present study demonstrates that in the transitional mare newly developing follicles exhibit biosynthesis of P4 and E2. Furthermore, an increase in follicular steroid concentrations is not necessarily reflected in the peripheral steroid concentrations.

Animals↗

PubMed2000

Activation of ribosomal RNA genes in preimplantation cattle and swine embryos.

Transcription of ribosomal RNA (rRNA) genes occurs in the nucleolus resulting in ribosome synthesis. In cattle and swine embryos, functional ribosome-synthesizing nucleoli become structurally recognizable towards the end of the fourth and third post-fertilization cell cycle, respectively. In cattle, a range of important nucleolar proteins become localized to the nucleolar anlage over several cell cycles and this localization is apparently completed towards the end of the fourth cell cycle. In swine, the localization of these proteins to the anlage is more synchronous and occurs towards the end of the third cell cycle and is apparently completed at the onset of the fourth. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus, serve to evaluate the developmental potential of embryos originating from different embryo technological procedures. By this approach, we have demonstrated that in vitro produced porcine embryos display a lack of localization of nucleolar proteins to the nucleolar anlage as compared with in vivo developed counterparts. Similarly, bovine embryos produced by nuclear transfer from morulae display such deviations as compared with in vitro produced counterparts. Collectively, this information may help to explain the appearance of abnormalities seen in a certain proportion of offspring derived from in vitro produced embryos and after cloning.

Animals↗

PubMed2000

Ease of calving, blood chemistry, insulin and bovine growth hormone of newborn calves derived from embryos produced in vitro in culture systems with serum and co-culture or with PVA.

Blood chemistry (pH, pCO2, pO2, glucose, lactate) as well as plasma insulin and growth hormone of calves derived from embryos produced under 2 different in vitro culture systems (modified SOFaa with 20% serum and co-culture with bovine oviduct epithelial cells [IVP serum, n=8] or with 3 mg/mL PVA [IVPdefined, n=6]) were compared with those of calves derived from AI (n=5). Calvings were classified according to the ease (unassisted, light traction, heavy traction). Blood samples were taken from the jugular vein of calves at 5, 15, 30 and 60 min, and at 2, 3, 6, 12, 18 and 24 h after delivery, then daily for 6 d. At the second day of life after 4 feedings and a 4-h fasting period, a glucose tolerance test was performed to evaluate glucose metabolism and insulin secretion. Calves in the IVP serum group had higher birth weights than AI calves (LS mean +/- SEM, IVP serum: 45.2 +/- 1.4 kg vs AI: 40.4 +/- 1.7 kg; P < 0.05), while the birth weights of calves in the IVP defined group were in between (IVPdefined: 41.9 +/- 1.6 kg). More IVP serum calves (75%) needed assistance than IVP defined (33%) or AI (40%) calves. The effect of ease of calving vs the effect of embryo culture was compared in relation to blood parameters at birth. There was an effect of ease of calving but not of embryo culture conditions on blood pH, lactate and PCO2. Calves requiring heavy traction had lower pH during the first 3 h after calving, a higher lactate during the first 60 min after calving and a higher pCO2 the first 2 h after calving than calves born unassisted. Calves requiring heavy traction also had lower pH the first 2 h and higher lactate the first 3 h after calving than calves born by light traction. IVP defined calves had lower lactate than IVP serum calves the first 60 min after calving. At 6 h after delivery, all blood parameters had stabilized. There was no effect of either embryo culture or ease of calving on basal insulin and growth hormone level, or the ability of the calves to handle glucose postnatally and during a glucose tolerance test.

Animals↗

PubMed2000

New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system.

Culture of mammalian zygotes individually and in small groups results in lower developmental rates than culture of large groups. Zona-free zygotes also have impaired developmental potential in current culture systems. This paper describes a new approach to resolve the problems, the Well of the Well (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 x 3 factorial experiment was performed. Bovine presumptive zygotes were cultured from day 1 to day 7 (day 0: day of insemination) using three modules (single embryos, embryo groups of five, or single zona-digested embryos) and three different culture systems (400 microl medium, 200 microl drops, or WOWs). An additional control group consisted of 40 to 50 embryos cultured in 400 microl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59%; group of five: 61%; single zona-digested: 53%) than the culture in drops or in wells (P < 0.05 for all). The developmental rate was independent of the number of WOWs per well. The cell number of blastocysts cultured in the WOW system did not differ from that of the controls. Apart from its theoretical value in revealing the role of different factors influencing embryo development in vitro, the WOW system may have immediate practical consequences in certain areas of mammalian embryo production.

Animals↗

PubMed2000

Effects of ethanol and dimethylsulphoxide on nuclear and cytoplasmic maturation of bovine cumulus-oocyte complexes.

The influence of small doses of ethanol or dimethylsulphoxide (DMSO) on in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COC) was examined, either after spontanous maturation or after inhibition of meiosis with 6-dimethylaminopurine (6-DMAP) or 3-isobutyl-1-methylxanthine (IBMX). Subsequent to IVM for 23 hr in semidefined serum-free Earle's TCM199 medium, nuclear maturation was assessed cytogenetically, while the combined cytoplasmic and nuclear maturation was measured indirectly by the oocytes' ability to undergo fertilization and further embryonic development. Embryo development was followed until the blastocyst stages at day 9 after insemination. Neither spontanous nuclear maturation nor cleavage was compromised by IVM in </=1% (v/v) ethanol or </=1% (v/v) DMSO, nor was the frequency of polyspermy altered. However, IVM in 0.3% or 1% (v/v) ethanol or in 0.4 or 1% (v/v) DMSO negatively affected blastocyst formation, compared to 0% in the control groups (22% and 23% vs. 34%, P < 0.0001, and 29% and 22% vs. 34%, P < 0.01, respectively), whereas the speed of blastocyst formation, assessed as the D7/D9 blastocyst proportion, was not compromised. In oocytes meiotically inhibited with 2 mM 6-DMAP, the presence of ethanol (0. 5%, 1%, and 2% [v/v]) induced germinal vesicle breakdown in a dose-dependent manner (32%, 45%, and 68%, vs. 22%, P < 0.0001), however, the oocytes exhibited no further meiotic progression. In oocytes inhibited with 1 and 2 mM IBMX, the presence of ethanol (0. 5%, 1%, and 2% [v/v]) significantly (P < 0.05) enhanced the inhibitory effect in a dose-dependent manner by reducing the proportion of the mature (AI-MII) stages (77%, 68%, and 56% vs. 79%, and 33%, 29%, and 18% vs. 39%, respectively). It is concluded that even small doses of ethanol or DMSO can cause profound negative effects on bovine in vitro maturation and subsequent embryo development.

1-Methyl-3-isobutylxanthine↗

PubMed2000

Body dimensions and birth and organ weights of calves derived from in vitro produced embryos cultured with or without serum and oviduct epithelium cells.

Body dimensions, birth and organ weights of calves derived from embryos produced in 2 in vitro culture systems (modified SOFaa with 20% cattle serum and co-cultured with oviduct-epithelium cells [IVPserum, n=8], and modified SOFaa with 3 mg/mL PVA [IVPdefined, n=6]) were compared with calves originating from artificial insemination (AI, n=85). Three additional IVP calves were included which had been vitrified as mature oocytes by the open pulled straw (OPS) method, warmed, fertilized and cultured to the blastocyst stage in modified SOFaa with 5% cattle serum, then again OPS-vitrified and warmed prior to transfer (IVPops, n=3). At birth, gestation length and birth weights were registered for all calves. At 1 wk of age all 17 IVP and 7 of the AI calves were killed, and their body dimensions and organ weights recorded. Birth weight was higher for the IVPserum and IVPops calves than for AI control calves (kg +/- SEM: IVPserum 46.9+/-1.8, IVPops 50.6+/-2.4, AI 41.8+/-0.8; P < 0.002). There was no difference between IVP and AI calves regarding gestation length and no effect of culture conditions on body dimensions or organ weights, except for longer hind legs in IVPdefined calves compared with AI calves (cm +/- SEM: IVPdefined 93+/-2, AI 87+/-2; P < 0.04). The IVPops calves had an increased liver weight compared with AI and the other IVP calves (g +/- SEM: IVPops 1.457+/-59; AI 1,117+/-37; IVPserum 1,159+/-34, IVPdefined 1,073+/-39; P < 0.0003). It is concluded that in vitro culture of bovine embryos in the presence of serum and oviduct epithelium cells increased birth weight but not organ weight and body dimension in 1-wk-old calves. However, vitrification of the ova as oocyte and again as blastocysts increased birth weight and liver size. This possible effect of cryopreservation of oocytes on subsequent fetal development awaits further investigation.

Animals↗

PubMed2000

Effect of parity, season and FSH treatment on the calving interval of Ayrshire cows in the tropics.

A total of 607 calving intervals on 99 Ayrshire cows with parities from 1 to 10 and belonging to one dairy herd at Iringa in the southern highlands of Tanzania were analysed to study the effect of parity and season or month of calving on the subsequent calving interval. In addition, 22 calving intervals incorporating a superovulatory cycle were compared with those of 52 controls that had calved around the same period. The average (mean +/- SEM) age at first calving was 985 +/- 16 days. The effect of parity on calving interval was highly significant (p < 0.001). Cows that calved in December, at the beginning of the rainy season, exhibited the shortest calving interval (404 +/- 13), while those that calved in September/October, in the dry season, exhibited the longest average calving interval (466 +/- 20 days), although this difference was not significant. Prior treatment with follicle-stimulating hormone to induce superovulation, significantly lengthened the average calving interval (579 +/- 28 vs 457 +/- 15 days; p < 0.001). It was concluded that both parity and FSH treatment affected the calving interval. However, season (rainy vs dry) or month of calving did not influence this interval.

Animals↗

PubMed2000

Ploidy of bovine nuclear transfer blastocysts reconstructed using in vitro produced blastomere donors.

The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro development. Because little is known about ploidy errors in nuclear transfer embryos, this was examined using embryos reconstructed from in vitro produced embryo donors. In vitro matured oocytes were enucleated and then activated using calcium ionophore A23187 followed by 6-dimethylaminopurine (6-DMAP). Subsequently, embryos were reconstructed using blastomeres from day 4-5 in vitro produced donors. The embryos were cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 +/- 20.2 (mean % +/- SD) nuclei/embryo were examined. Of these 16, 7 embryos (43.8%) were potentially abnormal because in these, 1.1%, 1.4%, 5.3%, 7.5%, 26.3%, 30.4%, and 66.2% % of the nuclei had a chromosome composition deviating from the diploid condition, indicating a wide degree of variation between embryos. These errors comprised mainly triploid (8.2 +/- 10.3 [0-26.3]: % +/- SD [range]) and tetraploid (10.6 +/- 19.9 [0-54.9]) nuclei with other ploidy combinations accounting for only 0.9 +/- 2.1 [0-2.1]% of deviant nuclei. The proportion of completely normal nuclear transfer embryos was no less than those produced by in vitro fertilization but the distribution of chromosome abnormalities was different (p = 0.0002). In conclusion, nuclear transfer embryos reconstructed using blastomere cells can produce over 50% blastocysts with a diploid chromosome complement. However, the contribution of chromosome abnormalities to embryonic loss in the remaining embryos deserves further investigation.

Adenine↗

PubMed2000

Pre-ovulatory graafian follicles are cooler than neighbouring stroma in pig ovaries.

Using an infra-red camera, domestic pig ovaries were thermo-imaged almost instantaneously at laparotomy or within a closed abdomen by endoscopy. Rectal and jugular vein temperatures were recorded using thermo-probes. Graafian follicles (7-10 mm diameter) were cooler than ovarian stroma in all experimental models examined, and both compartments were cooler than rectal and jugular temperatures. The mean difference between follicles and stroma in 73 observations was 1.3 +/- 0.1 degrees C. When thermo-imaged under the fimbriated extremity of the Fallopian tube, follicles and stroma could still be distinguished. Follicles cooled slightly more rapidly than adjoining stroma during the first 10 s of a 60 s recording interval, after which curves for the two tissues remained parallel. Arresting ovarian blood supply for 5 min had a negligible influence on the temperature differentials. Endoscopy in three models recorded mean differentials between follicles and stroma of 0.6 +/- 0.1 degrees C to 1.1 +/- 0.1 degrees C. It is concluded that temperature gradients do exist in the ovarian tissues of mature animals, and that these are generated at least in part as a consequence of endothermic reactions within Graafian follicles.

Animals↗

PubMed2000

Risks of in-vitro production of cattle and swine embryos: aberrations in chromosome numbers, ribosomal RNA gene activation and perinatal physiology.

In cattle, in-vitro production (IVP) of embryos has become a standardized technique; however, increased frequencies of calving problems and larger calves have been reported. In swine, IVP has resulted in only a limited number of piglets. In this paper we present information on cattle and swine embryos produced in vitro by oocyte maturation, fertilization and further embryo culture to the blastocyst stage in vitro. Control in-vivo developed embryos were collected after superovulation. The cattle embryos were processed for fluorescence in-situ hybridization (FISH) with two chromosome-specific probes to detect numerical chromosome aberrations. The swine embryos were processed for transmission electron microscopy and immunocytochemistry with an antibody against RNA polymerase I [essential for ribosomal RNA (rRNA) gene transcription] in order to highlight the post-fertilization development of the nucleolus as a marker for rRNA gene activation. The FISH analyses of the cattle embryos revealed that 72% of IVP blastocysts were mixoploid, i.e. contained both diploid and polyploid cells, versus 25% in vivo. Chromosome abnormalities were observed from the 2-cell stage onwards. The immunocytochemical analyses of the swine embryos revealed that during in-vivo development, RNA polymerase I became localized to multiple foci in the developing nucleoli late during the 4-cell stage. This focal localization of RNA polymerase I was not observed in IVP embryos. In conclusion, IVP embryos may display aberrations in chromosome numbers and rRNA gene activation. The significance of these deviations for fetal and perinatal viability, however, remains unknown. The survival of most calves derived from IVP indicates that a considerable number of these embryos are able to compensate for the adverse effects of the in-vitro procedures.

Animals↗

PubMed2000

Nucleolar proteins and nuclear ultrastructure in preimplantation bovine embryos produced in vitro.

The aim of the present investigation was to describe the basic cell biology of the postfertilization activation of rRNA genes using in vitro-produced bovine embryos as a model. We used immunofluorescence confocal laser scanning microscopy and transmission electron microscopy to study nucleolar development in the nuclei of embryos up to the fifth postfertilization cell cycle. During the first cell cycle (1-cell stage), fibrillarin, upstream binding factor (UBF), nucleolin (C23), and RNA polymerase I were localized to distinct foci in the pronuclei, and, ultrastructurally, compact spherical fibrillar masses were the most prominent pronuclear finding. During the second cell cycle (2-cell stage), the findings were similar except for a lack of nucleolin and RNA polymerase I labeling. During the third cell cycle (4-cell stage), fibrillarin, UBF, nucleophosmin, and nucleolin were localized to distinct foci. Ultrastructurally, spherical fibrillar masses that developed a central vacuole over the course of the cell cycle were observed. Early in the fourth cell cycle (8-cell stage), fibrillarin, nucleophosmin, and nucleolin were localized to small bodies that with time developed a central vacuole. UBF and topoisomerase I were localized to clusters of small foci. Ultrastructurally, spherical fibrillar masses with a large eccentric vacuole and later small peripheral vacuoles were seen. Late in the fourth cell cycle, nucleophosmin and nucleolin were localized to large shell-like bodies; and fibrillarin, UBF, topoisomerase I, and RNA polymerase I were localized to clusters of small foci. Ultrastructurally, a presumptive dense fibrillar component (DFC) and fibrillar centers (FCs) were observed peripherally in the vacuolated spherical fibrillar masses. Subsequently, the presumptive granular component (GC) gradually became embedded in the substance of this entity, resulting in the formation of a fibrillo-granular nucleolus. During the fifth cell cycle (16-cell stage), a spherical fibrillo-granular nucleolus developed from the start of the cell cycle. In conclusion, the nucleolar protein compartment in in vitro-produced preimplantation bovine embryos is assembled over several cell cycles. In particular, RNA polymerase I and topoisomerase I are detected for the first time late during the fourth embryonic cell cycle, which coincides with the first recognition of the DFC, FCs, and GC at the ultrastructural level.

Animals↗

PubMed2000

Blood chemistry, nutrient metabolism, and organ weights in fetal and newborn calves derived from in vitro-produced bovine embryos.

Calves born after in vitro production (IVP) of embryos often show reduced perinatal viability. The present experiment investigated a series of physiological variables in the immediate prenatal and postnatal period of IVP dairy calves. Fetal IVP and control calves (each n = 7) were prepared with vascular catheters at 248+/-1 day gestation (term = 280 days), and blood samples were taken for five days before premature delivery by cesarean section. IVP fetuses compared with controls had significantly elevated arterial hemoglobin and oxygen content (8.41 vs. 7.52% and 5.75 vs. 3.79%, respectively) whereas lactate level was lowered (1.89 vs. 2.26 mM). The umbilical venous-arterial concentration differences in oxygen, lactate, and glucose indicated that IVP fetuses relied more on lactate and less on glucose as oxidative substrates. The fetal glucose tolerance, and the basal and adrenocorticotropin-stimulated cortisol levels were similar between the groups. In the immediate postnatal period, IVP calves showed elevated venous blood pH (7.294 vs. 7.270), hemoglobin (9.06 vs. 8.25%), oxygen contents (6.33 vs. 4. 64%), K(+) levels (4.89 vs. 4.56 mM), and rectal temperature (38.9 vs. 37.4 degrees C), and lowered blood Na(+) (139.9 vs. 141.0 mM), Cl(-) (100.2 vs. 103.1 mM) and glucose levels (2.86 vs. 3.11 mM). There were no differences in body dimensions and organ weights, except that the fore legs and hind legs were slightly longer in the IVP group (76.1 vs. 72.4 cm and 93.4 vs. 88.8 cm, respectively). Although prenatal and neonatal IVP calves differed from control calves in a number of variables, the effects were relatively minor and provide no direct evidence for the hypothesis that IVP calves have an impaired capacity to adapt to life ex utero. In fact, several parameters indicated enhanced rather than retarded maturation of IVP calves when data from premature calves were compared with data from a group of control calves delivered at term.

Animal Nutritional Physiological Phenomena↗

PubMed2000

Chromosome aberrations in in vitro-produced bovine embryos at days 2-5 post-insemination.

Availability of embryos of high quality is required to obtain satisfactory embryonic developmental rates and normal calves following transfer of in vitro-produced (IVP) bovine embryos. One relevant quality parameter is the frequency of chromosome aberrations, which can be evaluated using multicolor fluorescent in situ hybridization (FISH) with chromosome 6- and chromosome 7-specific probes in cattle. In this study, interphase nuclei (n = 3805) were analyzed from 426 bovine IVP embryos. We found that 73%, 72%, 81%, and 58% of the embryos from Days 2, 3, 4, and 5 post-insemination (pi), respectively, displayed a normal diploid chromosome number in all cells. When looking at the types of chromosome aberrations, the percentages of mixoploidy at Days 2, 3, 4, and 5 pi were 22%, 15%, 16%, and 42%, respectively, whereas the percentages of polyploidy (i.e., all nuclei in an embryo were analyzed and were polyploid) were 5%, 13%, 3%, and 0%, respectively. In conclusion, numerical chromosome aberrations were detected as early as Day 2 pi. The development of polyploid embryos is slow and is apparently arrested during the third cell cycle, whereas the mixoploid embryos seem to continue development.

Animals↗

PubMed2000

Effect of administering a crude equine gonadotrophin preparation to mares on follicular development, oocyte recovery rate and oocyte maturation in vivo.

In mares, the shortage of oocytes and the variability in nuclear maturation at a certain time of the oestrous cycle hinders the optimization of methods for in vitro maturation and in vitro fertilization. Increasing the number of small-to-medium-sized follicles available for aspiration in vivo may increase the overall oocyte yield. The aims of the present study were to investigate whether administration of crude equine gonadotrophins affects follicular development, oocyte recovery rate, in vivo oocyte maturation and follicular concentrations of meiosis-activating sterols. During oestrus, all follicles >/= 4 mm were aspirated from 19 pony mares (first aspiration: A1). Over the next 8 days, the mares were treated daily with either 25 mg crude equine gonadotrophins (n = 10) or physiological saline (n = 9). Between day 1 and day 8, follicular growth was monitored by ultrasonography. On day 8, all follicles >/= 4 mm were evacuated (second aspiration: A2) and nuclear maturation of the recovered oocytes was assessed after orcein staining. Follicular growth between A1 and A2, as well as the number and size of follicles at A2 were similar for control mares and mares treated with crude equine gonadotrophins. The oocyte recovery rates at A1 and A2 were similar. At A2, the oocyte recovery rate and oocyte maturation in vivo were not affected by treatment with crude equine gonadotrophins. The number of expanded cumulus oophorus complexes recovered from follicles </= 29 mm was significantly higher at A1 than at A2. The number of oocytes at the germinal vesicle stage was significantly higher at A2 (41.5%) than at A1 (17.8%). Meiosis-activating sterols (FF-MAS and T-MAS) were identified in follicular fluid recovered at A2. Follicular concentrations of FF-MAS and T-MAS were unaffected by treatment with crude equine gonadotrophins. The present study demonstrates that follicular aspiration during oestrus allowed a new follicular population to develop and resulted in a higher degree of synchronization of oocyte development with respect to cumulus expansion and nuclear maturation. The availability of a more homogeneous population of oocytes might facilitate a better optimization of in vitro maturation and in vitro fertilization techniques in mares. Administration of crude equine gonadotrophins during early dioestrus did not affect the growth of small follicles, the oocyte yield after aspiration or oocyte maturation in vivo.

Animals↗