Effects of developmental stage, embryonic interferon-tau secretion and recipient synchrony on pregnancy rate after transfer of in vitro produced bovine blastocysts.

Three separate trials of bovine embryo transfers were performed consisting of 32, 41 and 33 transfers, respectively, to examine the effects of (a) the developmental stage of in vitro-derived blastocysts, (b) the amount of interferon-tau (IFN-tau) they secreted during culture and (c) the cyclic stage of the recipient at the time of transfer on the probability of establishment of pregnancy. One blastocyst was transferred into the ipsilateral uterine horn to the CL. At the time of transfer, blastocysts were classified into one of three developmental stages (early blastocyst, blastocyst and expanded blastocyst) and the cyclic stage of each cow was assessed (-12 h, on time, +12 h, +24 h, >24 h). Prior to the second and third trials, blastocysts were individually cultured for 24 h in 50 microl medium droplets and the IFN-tau concentration in the droplet was determined. Logistic regression analyses revealed that expanded blastocysts had a significantly higher likelihood of establishing pregnancy (p = 0.009), and that there was a significant interaction with the cyclic stage of the recipient in this group with lower rates of pregnancy resulting from decreasing synchrony with the recipient (p = 0.033). IFN-tau secretion during culture was significantly higher in expanded blastocysts than in the other two groups (p < 0.05). A significant effect of the pre-transfer level of IFN-tau secretion was found only in the 'Blastocyst' group where transfer of embryos with lower IFN-tau production prior to transfer resulted in higher pregnancy rates (p = 0.047). These results demonstrate that IFN-tau secretion may be a useful tool to predict pregnancy outcome, but only within certain developmental stages.

TUNEL analyses of bovine blastocysts after culture with EGF and IGF-I.

Experiments were carried out to investigate the beneficial effects of IGF-I or EGF on bovine embryo development in chemically defined embryo culture media and resultant incidences of nuclear DNA fragmentation as an indication of embryo quality. Presumptive IVF zygotes were randomly cultured in either control (with no added growth factor) or treatment groups, i.e., with 50 ng/ml IGF-I (experiment 1) or 5 ng/ml EGF (experiment 2). IGF-I supplemented to culture media significantly improved proportions of blastocysts from oocytes inseminated compared to untreated controls (38.0% vs. 28.5%). Only embryos reaching the blastocyst stage on day 8 showed significant effects of IGF-I treatment by resulting in higher blastocyst cell numbers (162 vs. 141) and lower percentages of TUNEL positive nuclei (2.1% vs. 3.3%) when compared to controls. Blastocyst development from oocytes was also improved by EGF supplementation compared to untreated controls (38.5% vs. 30.7%). Cell numbers of either day 7 or day 8 blastocysts were not affected by EGF treatment, nor were percentages of TUNEL positive nuclei when compared with controls. Similar proportions of parthenogenetically activated oocytes developed to blastocysts as for inseminated oocytes (28.8%). Parthenogenetic blastocysts contained fewer cells (93) and an increased percentage of TUNEL positive nuclei (5.7%) than were found for IVF embryos.

Influences of epidermal growth factor and insulin-like growth factor-I on bovine blastocyst development in vitro.

Experiments were carried out to investigate putative beneficial effects of adding epidermal growth factor (EGF) or insulin-like growth factor-I (IGF-I) for bovine embryo culture in chemically defined media. Presumptive zygotes (18 h post-insemination) were randomly assigned to culture treatments. In experiment 1, treatments involved additions of recombinant human EGF to provide concentrations of 0 ng (control), 1, 5, and 25 ng/ml. No differences were seen in numbers of 4-cell stage embryos between groups. A concentration of 5 ng/ml EGF but not 1 or 25 ng/ml during embryo culture improved percentages of 4-cell stage embryos reaching blastocysts compared to the control (P<0.05). Numbers of inner cell mass (ICM) cells and trophoblast cells of day 8 blastocysts were similar for the control and 5 ng/ml EGF-treated groups. In experiment 2, culture with recombinant human IGF-I in concentrations of 0 ng (control), 2, 10, and 50 ng/ml resulted in no differences in numbers of 4-cell stage embryos between groups. When compared to controls, IGF-I treatments at 10 and 50 ng/ml improved proportions of 4-cell stage embryos that reached blastocysts (P<0.05). In experiment 3, numbers of ICM cells of day 8 blastocysts were significantly higher after being cultured with 50 ng/ml of IGF-I compared to those of the controls (P<0.05). No additive effect of combining EGF (5 ng/ml) and IGF-I (50 ng/ml) was seen when results were compared to those following supplementation of the media with either EGF or IGF-I alone. In conclusion, both EGF and IGF-I could independently enhance bovine preimplantational development in chemically defined media and IGF-I but not EGF may play a mitogenic role during early bovine development.

Effect of leukemia inhibitory factor on bovine embryos produced in vitro under chemically defined conditions.

The objective of these experiments was to assess putative embryotrophic effects of leukemia inhibitory factor (LIF) on bovine preimplantation development in chemically defined media. Recombinant human LIF was added to embryo culture media at a concentration of 100 ng/ml. When added for culture of morulae LIF had no positive effect on the proportion of embryos reaching the blastocyst stage. However, LIF significantly reduced development to the blastocyst stage when added for culture of 4-cell stage embryos (P<0.05). In contrast, a positive effect was found for progression of blastocyst development. In vitro blastocyst hatching rates were significantly improved in the presence of LIF (P<0.02). Number of total cells and of inner cell mass (ICM) cells were increased in LIF-treated blastocysts. In vitro survival of frozen-thawed blastocysts was not improved by adding LIF to morula stage embryos before cryopreservation. The pregnancy rate after direct transfer of cryopreserved LIF-treated embryos was not different from that for untreated control embryos. Data indicate that addition of LIF has no major beneficial effect on bovine embryos produced in these chemically defined conditions.

Offspring resulting from direct transfer of cryopreserved bovine embryos produced in vitro in chemically defined media.

The present work was designed to study the in vitro and in vivo viability, as assessed by blastocyst formation, pregnancy rate and term delivery of bovine embryos produced under completely defined conditions with or without insulin-like growth factor I (IGF-I) following direct transfer after cryopreservation. Slaughterhouse-derived bovine oocytes were matured for 24h, fertilized with frozen-thawed spermatozoa and cultured in vitro under completely defined conditions with or without exposure to IGF-I (5 ng/ml). Only those embryos classified as excellent or good quality blastocysts were frozen. Each blastocyst was individually loaded into a straw, seeded and pre-cooled to -7 degrees C. After 10 min at -7 degrees C straws were frozen further to -30 degrees C at a rate of 0.3 degrees C/min and then plunged into liquid nitrogen. Synchronized recipient cows received one embryo in the horn ipsilateral to the corpus luteum (CL). Pregnancies were diagnosed by ultrasonography 35-45 days after embryo transfer (ET). IGF-I failed to improve cleavage rate, as well as blastocyst production, when added during in vitro culture (IVC). Pregnancy outcome was not significantly improved in cows that received an IGF-I-treated embryo compared with controls (4/10 versus 3/10, respectively). Five out of six calves delivered to date were born alive and healthy. We have shown that it is possible to obtain healthy live offspring from frozen-thawed embryos produced under chemically defined conditions after direct transfer.

Photosensitive chemical and laser light treatments decrease epizootic hemorrhagic disease virus associated with in vitro produced bovine embryos.

Photoinactivation was employed to eliminate EHDV-2 from in vitro produced bovine embryos experimentally exposed to this virus. Immature oocytes were matured, fertilized, and cultured in chemically defined conditions. All treatments were performed on zygotes. Developmental potential of zygotes and cell numbers of resulting hatched blastocysts were assessed after exposure to a 1 mW helium neon laser (633 nm, red) for 1, 5, 10, and 15 min; the photosensitive chemicals hematoporphyrin (15 microM) and hypericin (1 and 10 microM) for 15 min; a combination of 10 microM hypericin and laser light for 1, 3, or 5 min; and a combination of 15 microM hematoporphyrin and laser light for 1, 2, or 3 min. There were no significant differences among proportions of embryos developing or cell numbers after treatment with or without exposure to laser light alone for up to 10 min. No differences were observed after exposure of zygotes to photosensitive chemicals alone. Exposure to 10 microM hypericin and 5 min of laser light or 15 microM hematoporphyrin and 2 min of laser light compromised zygote developmental potential. After exposure to 10(6) TCID50/mL EHDV-2 for 90 min groups of 10 zygotes were exposed to 10 microM hypericin or 15 microM hematoporphyrin and laser light to inactivate the virus. Hematoporphyrin was effective with 3 min light exposure at reducing the percentage of EHDV-2 contaminated zygote pools (16.7%) as compared to EHDV-2 exposed pools without treatment (88.9%) but hematoporphyrin + 1 min light was ineffective. Hypericin + 3 min light provided an intermediate effect (55.6%).

Reduction of infectious epizootic hemorrhagic disease virus associated with in vitro produced bovine embryos by non-specific protease.

Infectious viruses bind more tenaciously to the zonae pellucidae of in vitro produced bovine embryos than to zonae of in vivo derived embryos. Currently, the International Embryo Transfer Society recommends that all in vivo derived embryos be subjected to a rigorous washing procedure in combination with exposure to trypsin to remove viruses adherent to the zonae. In contrast to in vivo derived embryos, this method is not effective for disinfecting in vitro produced embryos. Our hypothesis was that a more potent, non-specific protease from Streptomyces griseus (S. griseus) would provide a more effective treatment for virus removal from in vitro produced bovine embryos. Bovine oocytes were matured, fertilized, and cultured in completely defined in vitro conditions. Zygotes were washed according to the procedure outlined by the International Embryo Transfer Society, replacing trypsin with the experimental protease. Experimental incubations were with 0.1% (4 units/ml) protease for 0, 30, 45, 60 and 75s intervals. Embryos were able to withstand exposure to this enzymatic treatment for only 45s before their developmental potential was significantly reduced; 60s exposure was detrimental (P<0.05). Oocytes were exposed to epizootic hemorrhagic disease virus serotype 2 (EHDV-2, 10(6) TCID(50)/ml) during in vitro maturation. Resulting zygotes were washed according to the International Embryo Transfer Society procedure and either exposed to trypsin or protease. Exposure to EHDV-2 prevented cumulus expansion and markedly reduced embryonic development (P<0.05). There were no differences in development among virus exposed groups receiving no treatment or treatment with trypsin or protease. However, proportions of infected embryos were reduced after protease treatment versus positive controls and trypsin treated embryos.

Chlortetracycline staining patterns of frozen-thawed bull spermatozoa treated with beta-cyclodextrins, dibutyryl cAMP and progesterone.

Efforts to achieve complete chemical definition of media used for in vitro capacitation of bovine spermatozoa including removal of heparin purified from porcine intestinal mucosa are presented. Fluorescent staining with chlortetracycline (CTC), known to reflect changes coincident with sperm capacitation in certain species, was studied following treatments of frozen-thawed bull spermatozoa with beta-cyclodextrins, dibutyryl cAMP (dbcAMP) and progesterone in comparison with heparin. The CTC staining patterns (F, B and AR) were confirmed to correlate with known conditions that effectively prepare cryopreserved bull spermatozoa for fertilisation in vitro. In the absence of glucose, the routinely employed heparin-containing capacitating medium caused an increase in spermatozoa displaying the AR pattern. Both progesterone (100 microM) and dbcAMP (0.01-0.1 mM) were able to increase the proportion of B pattern stained sperm cells more than after exposure to control (mDM) conditions without a significant reduction in motility. Exposure to either dbcAMP or beta-cyclodextrins was accompanied by an increase in proportions of spermatozoa displaying the AR pattern over those seen in controls. Exposure to beta-cyclodextrins did not increase the proportion of B pattern stained spermatozoa. Comparison of spermatozoa from two bulls revealed differential responses of spermatozoa from different males to treatments with heparin and progesterone. In vitro fertilisation results demonstrated that previously cryopreserved bull spermatozoa could be capacitated in chemically defined conditions devoid of heparin or other biological components.

Acute acetaminophen toxicity in transgenic mice with elevated hepatic glutathione.

Previous studies demonstrated that elevation of hepatic glutathione (GSH) concentrations protect against acetaminophen (APAP) hepatotoxicity in mice. Employing transgenic mice overexpressing glutathione synthetase, this study was conducted to determine if sustained elevation of hepatic GSH concentrations could ameliorate or prevent APAP toxicity. International Cancer Research transgenic mouse males and matched (ie same strain, sex, and age) control nontransgenic mice were pretreated ip with GSH synthetase substrate gamma-glutamylcysteinyl ethyl ester (gamma-GCE) or with saline. After a 16-h fast, mice received a single dose of 500 mg APAP/kg bw in saline ip and were sacrificed 4 h later. Other mice similarly pretreated were killed without APAP challenge. The elevated GSH concentrations in transgenic mice livers did not lessen APAP hepatotoxicity. Instead higher degrees of hepatotoxicity and nephrotoxicity were observed in transgenic mice than in controls as indicated by higher serum alanine aminotransferase activity and more severe histopathological lesions in transgenic mice livers and kidneys. Pretreatment with gamma-GCE did not affect either initial or post-APAP treatment tissue GSH concentrations or observed degrees of toxicity. Detection of a higher level of serum APAP in transgenic mice and the histopathological lesions found in transgenic mice kidneys together with no observable nephrotoxicity in control mice indicated early kidney damage in transgenic mice. Our findings suggest that high levels of GSH-APAP conjugates resulting from increased GSH concentrations in the livers of transgenic mice caused rapid kidney damage. Compromised excretory ability may have caused retention of APAP, which, in effect, elicited higher hepatotoxicity than that observed in nontransgenic mice.