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PubMed2005

Comparative embryotoxicity and proteotoxicity of three carrier solvents to zebrafish (Danio rerio) embryos.

The present study examines the effects of ethanol (ETOH), dimethyl sulfoxide (DMSO), and acetone on zebrafish embryos and the implications of the observed results on the use of these solvents to zebrafish early life stage tests. The embryos were exposed to different concentrations (0.0, 0.0001, 0.001, 0.01, 0.1, 0.05, 1, 1.5, and 2.0% v/v) of the respective solvents by diluting reagent-grade solvent with reconstituted water [DIN 38415-6-Suborganismische Testverfahren (Gruppe T) Teil 6: Giftigkeit gegenüber Fischen. Deutsches Institute für Normung e.V]. The following endpoints were investigated (mortality, hatching rate, abnormalities, heart rate, and hsp 70 induction). No effect on survival was recorded for both acetone and DMSO even up to the highest concentration. On the other hand, embryos exposed to 1.5% and 2.0% ethanol showed a significant reduction in survival rate. No developmental defects occurred with any of the solvents at the 0.1% concentration. However, starting with 1.0%, weak to very pronounced abnormalities (weak pigmentation, edema, crooked bodies, eye defect, tail defect, reduced heartbeat, and abnormal hatching) were observed depending on the solvent type and the concentration used. Ethanol has been shown to be the most embryotoxic solvent while DMSO and acetone have comparably lesser effects. Heat shock protein 70 was induced by all solvents but at different concentration ranges. DMSO has been shown to be the most potent inducer of stress proteins. Based on the study, the chemicals tested here may be used as carrier solvents in the zebrafish embryo assay at levels below 1.5, 1.5, and 1% v/v for acetone, DMSO, and ethanol, respectively. For stress protein analysis of the exposed embryos, however, the solvent levels should be below 0.1%, 0.01%, and 1.5%, respectively. Additional and separate investigations utilizing other biomarkers should be carried out to further validate the suitability of using these solvents in a typical zebrafish embryo assay.

Abnormalities, Drug-Induced↗
PubMed2006

Potential role of toll-like receptor 3 in a murine model of polyinosinic-polycytidylic acid-induced embryo resorption.

OBJECTIVE: To investigate the potential role that toll-like receptor 3 (TLR3) may play in a murine model of polyinosinic-polycytidylic acid (polyIC)-induced embryo resorption. DESIGN: An animal model-based study on induced embryo resorption. SETTING: University animal laboratory. ANIMAL(S): Pathogen-free animals housed under barrier conditions and monitored for health status. Pregnant BALB/c mouse mated by C57BL/6 male was used as a model of allogeneic gestation. INTERVENTION(S): The administration of polyIC was performed to establish a murine model of induced embryo resorption, with or without TLR3 blocking by multiple injection of mAb against this receptor. MAIN OUTCOME MEASURE(S): Flow cytometric analysis was performed to detect the percentage of CD45(+)DX5+ and DX5(+)CD69+ cell subsets, with the DX5 antigen used as a common natural killer (NK) cell marker, and CD69 as a marker for activated NK cells, respectively. RESULT(S): In the allogeneic mating model, BALB/cxC57BL/6, both the CD45(+)DX5+ and DX5(+)CD69+ cell percentages were significantly elevated upon polyIC stimulation at the absence of anti-TLR3 administration but were kept unchanged if the female mice were pretreated with anti-TLR3 monoclonal antibody. Accordingly, the resorption rate of embryos was boosted by polyIC administration, but this effect could be abrogated by pretreatment of anti-TLR3 mAb. CONCLUSION(S): The engagement of polyIC with TLR3 may be critical to the activation of NK cells infiltrated at the feto-maternal interface, subsequently resulting in an increase in embryo resorption.

Animals↗
PubMed2006

In vivo and in vitro investigations of the effects of the antimalarial drug dihydroartemisinin (DHA) on rat embryos.

Artemisinin derivatives are clinically effective and safe antimalarials, but are not recommended during the first trimester of pregnancy because of the resorptions and abnormalities seen in animal reproduction studies. Understanding how, when and what toxicity occurs is crucial to any assessment of clinical relevance. Previously, DHA has been shown in the rat whole embryo culture (WEC) to primarily affect primitive red blood cells (RBCs) causing subsequent tissue damage and dysmorphogenesis. To verify the primary target of DHA in vivo and to detect consequences induced by early damage on embryo development, pregnant female rats were orally treated on gestation days (GD) 9.5 and 10.5 with 7.5 or 15 mg/kg/day DHA and caesarean sectioned on GD11.5, 12.5, 13.5, 15 and 20. A parallel in vitro WEC study evaluated the role of oxidative damage and examined blood islands and primitive RBCs. In accordance with the WEC results, primitive RBCs from yolk sac hematopoiesis were the target of DHA in vivo. The resulting anemia led to cell damage, which depending on its degree, was either diffuse or focal. Embryonic response to acute anemia varied from complete recovery to malformation and death, depending on the extent of cell death. Malformations occurred only in litters with embryonic deaths. DHA induced low glutathione levels in RBCs, indicating that oxidative stress may be involved in artemisinin toxicity; effects were extremely rapid, with altered RBCs seen as early as GD10. In establishing the relevance of these findings to humans, one should consider differences in the development of rodents and humans. While yolk sac hematopoiesis occurs similarly in the two species, early placentation and extent of exposure differ. In particular, early hematopoiesis takes only 7 days in rats (during which RBCs expand in a clonal fashion) compared with 6 weeks in humans; thus the susceptible period in relation to the duration of exposure to an artemisinin-based treatment may be substantially different.

Abnormalities, Multiple↗
PubMed2005

Post-insemination milk progesterone concentration and embryo survival in dairy cows.

Logistic regression analysis was used to evaluate the relationship between post-insemination milk progesterone concentration and embryo survival, and between milk yield and milk progesterone concentration. Milk samples were collected on Days 1, 4, 5, 6, and 7 (insemination=Day 0) following 871 inseminations in spring-calving dairy cows. Milk progesterone concentrations were measured by enzyme-immunoassay and pregnancy diagnosis was conducted with transrectal ultrasonography at approximately Day 30. There was a negative linear relationship (P<0.01) between milk progesterone concentration on Day 4 and embryo survival while, in contrast, there was a positive linear and quadratic relationship between milk progesterone concentration on Days 5, 6 and 7 (P<0.05) and also between the rate of change in progesterone concentrations between Days 4 and 7 inclusive and embryo survival (P<0.05). There was a weak negative linear relationship between average daily milk yield at the time of insemination and milk progesterone concentrations (P<0.001). There was no association between many production parameters, including liveweight and body condition score measured at various stages between calving and insemination, and milk progesterone concentration between Days 4 and 7 inclusive (P>0.05). In conclusion, low progesterone during Days 5-7 (after insemination) was associated with low fertility in dairy cows and there were indications of a range of progesterone concentrations within which embryo survival was maximal.

Animals↗
PubMed2005

Factors affecting the success of oocyte transfer in a clinical program for subfertile mares.

Oocyte transfer is a potential method to produce offspring from valuable mares that cannot carry a pregnancy or produce embryos. From 2000 through 2004, 86 mares, 19.2 +/- 0.4 yr of age (mean +/- S.E.M.), were used as oocyte donors in a clinical program at Colorado State University. Oocytes were collected from 77% (548/710) of preovulatory follicles and during 96% (548/570) of cycles. Oocytes were collected 21.0+/-0.1h after administration of hCG to estrous donors and cultured 16.4 +/- 0.2 h prior to transfer into recipients' oviducts. At 16 and 50 d after transfer, pregnancies were detected in 201 of 504 (40%) and 159 of 504 (32%) of recipients, respectively, with an embryo-loss rate of 21% (42/201). Pregnancy rates were similar (P > 0.05) for cyclic and noncyclic recipients and for recipients inseminated with cooled, fresh or frozen semen. One or more recipients were detected pregnant at 16 and 50 d, respectively, for 80% (69/86) and 71% (61/86) of donors. More donors <20 than > or = 20 yr (mean ages +/- S.E.M. of 15.5 +/- 0.4 and 23.0 +/- 0.3 yr, respectively) tended (P = 0.1) to have one or more pregnant recipients at 50 d (36/45, 80%; 28/45, 62%, respectively). Results of the program confirm that pregnancies can consistently be obtained from older, subfertile mares using oocyte transfer.

Aging↗
PubMed2005

Improving cryopreservation systems.

Cryopreservation of oocytes and embryos is a crucial step for widespread practical application of other techniques in domestic animal embryology. However, in contrast to the rapid development of procedures in the eighties and early nineties of the last century, new advancements with profound practical consequences have only been recently achieved. As a result of a long and controversial development, an alternative group of methods, vitrification, has proved its efficiency and practicality in the past few years. The aim of this short review is to characterize strategies to prevent cryoinjuries, summarize the development of vitrification, overview its recent achievements, and provide a perspective about possible application. Authors strongly believe that the future of mammalian oocyte and embryo cryopreservation will be mainly based on vitrification, and the rate of advancement will be determined by the rate by which embryologists learn and acknowledge this new approach.

Animals↗
PubMed2006

A study on reproductive performance and related factors of zebu cows in pastoral herds in a semi-arid area of Tanzania.

A prospective longitudinal study was carried out from September 2001 to June 2004 in three adjacent villages in a semi-arid area of Tanzania. The objectives of this study were to measure the intervals between calving and either resumption of cyclical activity or confirmation of pregnancy, to estimate calving intervals, and to investigate the effect of factors assumed to be related to postpartum reproductive performance. A total of 275 lactation periods from 177 Tanzanian Shorthorn Zebu cows managed in a traditional pastoral system in 46 households were initially included. Animals were initially screened for brucelosis and thereafter examined by palpation per rectum at 2-week intervals. Body condition score (scale 1 to 5) was assessed and girth measurement (cm) taken. Occurrence of other reproductive events such as calving, abortion, death of calf, culling and reason for culling were recorded. In a subset of 98 lactation periods from 91 cows milk samples for progesterone (P4) determination were collected twice per week from day 7 after calving to the time of confirmed pregnancy or until milk production ceased before pregnancy. The data were analysed both univariately and in multivariable Cox proportional hazard (frailty) models. The mean (+/-S.E.M.) calving interval was 500+/-13.6 days. Positive reactors in the brucellosis test were 15.6% of the tested animals. Milk P4 analysis showed the rate of abortion/late embryo loss to be 14.3%. Calf mortality rates varied between 14.6 and 17.4%. A positive relationship was found between the outcome variables likelihood of cyclical activity and likelihood of pregnancy in the Cox model, and the explanatory variables: parity and body condition score (BCS) at calving. A negative relationship was found between the outcome variables, and the explanatory variables: maximum BCS loss and calf survival/mortality. Calving in the rainy season was associated with an increased likelihood of pregnancy.

Animals↗
PubMed2000

Cytochrome c deficiency causes embryonic lethality and attenuates stress-induced apoptosis.

Cytochrome c released from mitochondria has been proposed to be an essential component of an apoptotic pathway responsive to DNA damage and other forms of cell stress. Murine embryos devoid of cytochrome c die in utero by midgestation, but cell lines established from early cytochrome c null embryos are viable under conditions that compensate for defective oxidative phosphorylation. As compared to cell lines established from wild-type embryos, cells lacking cytochrome c show reduced caspase-3 activation and are resistant to the proapoptotic effects of UV irradiation, serum withdrawal, or staurosporine. In contrast, cells lacking cytochrome c demonstrate increased sensitivity to cell death signals triggered by TNFalpha. These results define the role of cytochrome c in different apoptotic signaling cascades.

Animals↗
PubMed2000

Embryotoxic effects adjacent and opposite to the early regressing bovine corpus luteum.

Early luteal regression in cattle has an embryotoxic effect that is not overcome by replacement with progesterone, but is prevented by removal of the regressing CL. Two experiments were designed to test the null hypothesis that the luteal component of the embryotoxic effect is delivered by a systemic pathway. Beef heifers and cows (n = 39) received two good quality embryos, one placed into each uterine horn on Day 6 or 7 of the estrous cycle. Treated animals (n = 20) received 15 mg of PGF2alpha three times per day from Day 7 (n = 11; Experiment 1) or 5 (n = 9; Experiment 2) through 8; controls (n = 19) received saline. Progestogen replacement therapy (12 mg flurogestone acetate daily, s.c.) was provided from Day 6 (Experiment 1) or 4 (Experiment 2) until ultrasonographic diagnosis of embryo survival on Day 35 after estrus. The effects of treatment, location of the embryo and location by treatment interaction on embryo survival were tested by Chi square. In Experiment 1, there was no significant difference in embryo survival rate between PGF2alpha-treated and control recipients. In Experiment 2, only 6 of 18 embryos survived to Day 35 when transferred to animals treated with PGF2alpha compared to 12 of 18 in control animals (P< 0.05). The survival of embryos did not differ with location (adjacent or opposite to the regressing CL) or location by treatment interaction. Thus no evidence was obtained to support a local effect of the regressing CL. The embryo mortality associated with luteolytic doses of PGF2alpha in cows receiving replacement therapy with progestogen probably involves compounds that either act systemically or are transported via the uterine lumen to the uterine horn contralateral to the regressing CL.

Animals↗
PubMed2003

Associations between the manipulation of patterns of follicular development and fertility in cattle.

The wave-like patterns of ovarian follicular development in cattle can be manipulated by shortening the luteal phase with prostaglandin F2alpha (PGF), lengthening the period of follicle dominance with progesterone or curtailing follicle development with GnRH or oestradiol as 17beta, benzoate or cypionate. These hormones can also be used to synchronise ovulation allowing timed inseminations without detected oestrus. Progesterone, PGF, GnRH and oestradiol benzoate have each been used to increase conception rates in some situations, but their use has reduced them in others. For example, inseminations made within 96 h of a single injection of PGF administered during the luteal phase were associated with increased conception rates in dairy cows whereas double injection protocols reduced conception rates. The three forms of oestradiol and GnRH have greater effects on follicular development following divergence and dominance than following wave emergence. This can mean that follicles of differing maturity will be present about 7 days later and can result in varied intervals to the onset of oestrus following a PGF injection. The consequent variation in ovulation time can be reduced by injecting GnRH or an oestradiol during pro-oestrus. This means that some less mature follicles will ovulate, forming corpus luteum (CL) associated with a slower rise in plasma progesterone and lower mid-luteal concentrations. The lower conception rates recorded with single timed inseminations with synchronised ovulations have been associated with increased prevalences of short cycles in lactating dairy cows (with GnRH), with long luteal phases in cows and heifers (with oestradiol benzoate) and with embryo loss following positive pregnancy diagnosis (as with Ovsynch in lactating Holstein cows). Extensive Canadian studies have demonstrated that these same hormones can be successfully used without these limitations and reliably obtaining conception rates over 50% and up to 70% in beef cattle that have been supplemented with a progestin during the period of ovarian follicle synchronisation. The inherently lower fertility of Holstein cows during early lactation may be contributing to the reduced effectiveness of hormonal treatments for synchronised follicle development and ovulation. The role of reduced dose rates of GnRH in compromising this effectiveness needs to be determined if the potential of these treatments realised with beef cattle is to be achieved with lactating Holstein cows.

Animals↗
PubMedDate not supplied

In vivo and in vitro developmental toxicity in LPS-induced zinc-deficient rabbits.

Lipopolysaccharide (LPS) was used to induce maternal hypozincemia in order to test the hypothesis that altered zinc homeostasis is developmentally toxic in the rabbit. Treatment of dams on Gestation Day (GD) 8 with LPS (0.67 microgram/kg i.v.) caused total resorption of 78% (7 of 9) of the litters whereas GD 10 treatment increased the percentage of resorbed implantations (18-fold), but resulted in only 14% (1 of 7) totally resorbed litters. Cotreatment with zinc oxide (ZnO) on GD 10 decreased the resorption rate by 44%, indicating that hypozincemia was partially responsible for the resorptions. However, ZnO had no effect on resorption rate in GD 8 LPS-treated dams. No malformations were observed with LPS dosing on either gestation day. To determine whether LPS-induced Zn deficiency had any direct effects on rabbit embryos, normal GD 9 embryos were cultured for 48 h in serum from LPS-treated dams (0.53 +/- 0.01 microgram/mL Zn) or from controls (1.74 +/- 0.07 micrograms/mL Zn). Embryo growth and development were normal in both groups, indicating a lack of any direct embryo effects of Zn deficiency. Finally, maternal plasma progesterone and the Zn content of conceptus tissues were measured 24 h after LPS injection on GD 10. Despite a marked decrease in maternal serum Zn, no significant changes in embryo, visceral yolk sac, yolk sac cavity-exoceolomic fluid, or placental Zn were found. However, maternal progesterone levels were decreased 33 and 28% in the LPS and LPS + ZnO groups, respectively. Taken together, these results indicate that rabbits may differ from rodent species in their lesser susceptibility to the teratogenic potential of transient maternal Zn deficiency, as well as in their resistance to conceptus Zn changes. Nonetheless, Zn deficiency may be responsible for an increase in resorption rate in the rabbit.

Animals↗
PubMed2002

Zebrafish DeltaNp63 is a direct target of Bmp signaling and encodes a transcriptional repressor blocking neural specification in the ventral ectoderm.

Bone morphogenetic proteins (Bmps) promote ventral specification in both the mesoderm and the ectoderm of vertebrate embryos. Here we identify zebrafish DeltaNp63, encoding an isoform of the p53-related protein p63, as an ectoderm-specific direct transcriptional target of Bmp signaling. DeltaNp63 itself acts as a transcriptional repressor required for ventral specification in the ectoderm of gastrulating embryos. Loss of DeltaNp63 function leads to reduced nonneural ectoderm followed by defects in epidermal development during skin and fin bud formation. In contrast, forced DeltaNp63 expression blocks neural development and promotes nonneural development, even in the absence of Bmp signaling. Together, DeltaNp63 fulfills the criteria to be the neural repressor postulated by the "neural default model."

Animals↗
PubMed2003

Identification of possible mediators of embryonic mortality caused by mastitis: actions of lipopolysaccharide, prostaglandin F2alpha, and the nitric oxide generator, sodium nitroprusside dihydrate, on oocyte maturation and embryonic development in cattle.

PROBLEM: Mastitis and immunization against constituents of organisms causing mastitis can reduce fertility of cattle and sheep, respectively. For the current experiments, it was hypothesized that these effects are mediated via actions of lipopolysaccharide (LPS), prostaglandin F2alpha (PGF2), and nitric oxide on oocyte maturation and embryonic development. METHOD OF STUDY: To evaluate effects on oocyte maturation, oocytes were matured with various concentrations of LPS, PGF2alpha, or the nitric oxide (NO) generator, sodium nitroprusside (SNP). Following maturation, oocytes were fertilized and cultured until day 8 after fertilization. To test effects on embryo growth, oocytes were matured and fertilized and cultured after fertilization with LPS, PGF2alpha, or SNP. RESULTS: Addition of 100 and 1000 ng/mL LPS and 50 and 100 ng/mL PGF2alpha to oocyte maturation medium reduced the proportion of oocytes that became blastocysts at day 8 after fertilization. When added after fertilization, in contrast, neither LPS nor PGF2alpha reduced development to the blastocyst stage. Unlike for LPS and PGF2alpha, addition of SNP during oocyte maturation was without effect on the proportion of oocytes that became blastocysts at day 8 after fertilization. However, addition of 10 microM SNP to culture medium after fertilization completely prevented development to the blastocyst stage while 0.1 and 1 microM SNP did not affect development. CONCLUSIONS: Results indicate that increased local concentrations of LPS, PGF2alpha, and NO can have deleterious consequences on oocyte function (LPS, PGF2alpha) and embryonic development (NO). Thus, these molecules are putative mediators of effects of infectious disease or inflammation, including mastitis, on fertility of cattle.

Animals↗
PubMed2003

Actions of tumor necrosis factor-alpha on oocyte maturation and embryonic development in cattle.

PROBLEM: Infertility can accompany mastitis in cattle. Involvement of tumor necrosis factor-alpha (TNF-alpha) in this phenomenon is suggested by observations that circulating concentrations of TNF-alpha are elevated after intramammary infection or infusion of endotoxin. It was hypothesized that (1) TNF-alpha acts on the oocyte during maturation to decrease the percent of oocytes that cleave and develop following fertilization; (2) exposure of embryos to TNF-alpha after fertilization reduces development to the blastocyst stage; and (3) TNF-alpha increases the proportion of blastomeres that undergo apoptosis in a stage-of-development dependent manner. METHOD OF STUDY: In one experiment, oocytes were matured with various concentrations of TNF-alpha and then fertilized and cultured without TNF-alpha. In another study, embryos were cultured with TNF-alpha for 8 days beginning after fertilization. Finally, embryos were collected at the two or four-cell stage (at 28-30 hr after insemination) or when > or = 9-cells (at day 4 after insemination) and cultured +/- TNF-alpha for 24 hr. The proportion of blastomeres undergoing apoptosis was then determined by the TUNEL procedure. RESULTS: Addition of TNF-alpha to maturation medium did not affect the proportion of oocytes that cleaved. However, the percent of oocytes that developed to the blastocyst stage at day 8 after insemination was reduced (P = 0.05) at all TNF-alpha concentrations tested (0.1-100 ng/mL). When added during embryo culture, there was no significant effect of TNF-alpha on the proportion of oocytes that became blastocysts. In addition, TNF-alpha did not induce apoptosis in two and four-cell embryos. For embryos > or = 9-cells, however, 10 and 100 ng/mL TNF-alpha increased (P < 0.05) the percent of blastomeres labeling as TUNEL-positive. CONCLUSION: TNF-alpha can have deleterious actions on oocyte maturation that compromise development of the resultant embryo. While exposure of fertilized embryos to TNF-alpha did not inhibit development to the blastocyst stage, TNF-alpha increased the percentage of blastomeres undergoing apoptosis when exposure occurred for embryos > or = 9-cells. Increased blastomere apoptosis could conceivably compromise subsequent embryo survival.

Animals↗
PubMed1999

Free radical-mediated oxidative DNA damage in the mechanism of thalidomide teratogenicity.

The sedative drug thalidomide ([+]-alpha-phthalimidoglutarimide), once abandoned for causing birth defects in humans, has found new therapeutic license in leprosy and other diseases, with renewed teratological consequences. Although the mechanism of teratogenesis and determinants of risk remain unclear, related teratogenic xenobiotics are bioactivated by embryonic prostaglandin H synthase (PHS) to a free-radical intermediates that produce reactive oxygen species (ROS), which cause oxidative damage to DNA and other cellular macromolecules. Similarly, thalidomide is bioactivated by horseradish peroxidase, and oxidizes DNA and glutathione, indicating free radical-mediated oxidative stress. Furthermore, thalidomide teratogenicity in rabbits is reduced by the PHS inhibitor acetylsalicylic acid, indicating PHS-catalyzed bioactivation. Here, we show in rabbits that thalidomide initiates embryonic DNA oxidation and teratogenicity, both of which are abolished by pre-treatment with the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (PBN). In contrast, in mice, a species resistant to thalidomide teratogenicity, thalidomide does not enhance DNA oxidation, even at a dose 300% higher than that used in rabbits, providing insight into an embryonic determinant of species-dependent susceptibility. In addition to their therapeutic implications, these results constitute direct evidence that the teratogenicity of thalidomide may involve free radical-mediated oxidative damage to embryonic cellular macromolecules.

Animals↗