Metabolomics has been found to be applicable to a wide range of fields, including the study of gene function, toxicology, plant sciences, environmental analysis, clinical diagnostics, nutrition, and the discrimination of organism genotypes. This approach combines high-throughput sample analysis with computer-assisted multivariate pattern-recognition techniques. It is increasingly being deployed in toxico- and pharmacokinetic studies in the pharmaceutical industry, especially during the safety assessment of candidate drugs in human medicine. However, despite the potential of this technique to reduce both costs and the numbers of animals used for research, examples of the application of metabolomics in veterinary research are, thus far, rare. Here we give an introduction to metabolomics and discuss its potential in the field of veterinary science.

Repeated daily dosing of mice with 4-vinylcyclohexene diepoxide (VCD) causes a gradual onset of ovarian failure, providing a model for perimenopause. Because increasing numbers of women are delaying starting a family, infertility in aging women is of concern. This study was designed to determine the effects of impending ovarian failure on fertility in VCD-treated mice. Female C57BL/6J mice were dosed daily (17 d) with vehicle control or VCD (160 mg/kg, intraperitoneally) to deplete primordial follicles and then were divided into 2 groups. Group 1 was mated soon after dosing; group 2 was mated on day 20 after dosing, during impending ovarian failure. Fertility was evaluated on gestational day 16. In group 1, cycle length, pregnancy rate, and number of live fetuses did not differ between VCD-treated animals and controls, but VCD-treated mice required more matings to become pregnant and had more resorptions. In group 2, VCD-treated mice demonstrated proestrus and copulatory plugs, but only 1 animal became pregnant, and she had no viable fetuses. Ovaries from pregnant and nonpregnant controls contained similar numbers of follicles and corpora lutea. Ovaries from VCD-treated animals contained no follicles, and corpora lutea were seen only in pregnant animals. In VCD-treated mice mated soon after dosing, conception was more difficult and more resorbed fetuses were seen, whereas in those mated closer to impending ovarian failure, no successful pregnancies were achieved. These results demonstrate that VCD-treated mice can be used to model infertility in perimenopausal women.

To establish a small animal model of severe acute respiratory syndrome (SARS), we developed a mouse model of human severe acute respiratory syndrome coronavirus (SARS-CoV) infection by introducing the human gene for angiotensin-converting enzyme 2 (hACE2) (the cellular receptor of SARS-CoV), driven by the mouse ACE2 promoter, into the mouse genome. The hACE2 gene was expressed in lung, heart, kidney, and intestine. We also evaluated the responses of wild-type and transgenic mice to SARS-CoV inoculation. At days 3 and 7 postinoculation, SARS-CoV replicated more efficiently in the lungs of transgenic mice than in those of wild-type mice. In addition, transgenic mice had more severe pulmonary lesions, including interstitial hyperemia and hemorrhage, monocytic and lymphocytic infiltration, protein exudation, and alveolar epithelial cell proliferation and desquamation. Other pathologic changes, including vasculitis, degeneration, and necrosis, were found in the extrapulmonary organs of transgenic mice, and viral antigen was found in brain. Therefore, transgenic mice were more susceptible to SARS-CoV than were wild-type mice, and susceptibility was associated with severe pathologic changes that resembled human SARS infection. These mice will be valuable for testing potential vaccine and antiviral drug therapies and for furthering our understanding of SARS pathogenesis.

Myogenesis is one of the critical developmental processes in mammals. Several transcription factors from the dermomyotome affect embryonic myogenesis. Among these, Dmrt2 and Pax3 were tested for genetic and functional interactions during embryonic myogenesis by evaluating myogenin and desmin expression patterns in Dmrt2–Pax3 mutant mouse embryos. In doubly homozygous mutant embryos, myogenin expression was reduced, and the expression pattern was altered dramatically. In Pax3-knockout mouse embryos, the pattern of Dmrt2 expression was altered, suggesting that Pax3 is important in maintaining the epaxial dermomyotome. Even though Pax3 and Dmrt2 are expressed in similar tissue- and developmental-stage-specific manners during dermomyotomal development, they appear to have independent roles in mammalian myogenesis. The processes characteristic of embryonic myogenesis are similar to those occurring during muscle regeneration in adults. Therefore, these results may provide insight into the pathogenesis of innate muscular dystrophy and may lead to the development of drugs to promote muscle repair after injury.

Combination of evaporative drying and frozen storage at –80 °C has been used successfully to preserve hybrid B6D2F1 mouse spermatozoa. To determine whether this method can be applied equally well to inbred mice, spermatozoa of C57BL/6J and FVB/ NJ mice were evaporatively dried and stored for 1 mo at –80 °C before being used for intracytoplasmic sperm injection (ICSI) to produce live offspring. After weaning, 1 male and 1 female mouse from each litter were randomly selected at 8 wk of age for natural mating to produce live offspring. Results showed that spermatozoa from both inbred strains that had been evaporatively dried and subsequently stored at –80 °C could be used successfully to derive live, healthy, and reproductively sound offspring by ICSI. No significant differences were found in embryo transfer rate (number of pups born/number of embryos transferred), litter size, weaning rate, body weight, number of pathologic lesions, and amount of contamination by pathogens of mice produced by ICSI using evaporatively dried spermatozoa compared with mice produced by natural mating or by ICSI using fresh (that is, nonpreserved) spermatozoa. Progeny produced by mating mice generated from ICSI using evaporatively dried spermatozoa were normal. Therefore, spermatozoa from inbred mouse strains C57BL/6J and FVB/NJ can be preserved successfully after evaporative drying and frozen storage at –80 °C.

Diabetes is chronic disease that is accompanied by a rapid thymus involution. To investigate the factors responsible for thymic involution in a model of STZ-induced diabetes, mice were injected with STZ alone or in combination with the cyclooxygenase 2 inhibitor indomethacin (INDO). Thymus weight, glycemia and serum corticosterone were measured, and apoptosis in thymus and thymocyte cultures was analyzed by flow cytometry. Although earlier studies report that streptozotocin (STZ) is toxic to lymphoid tissues, in our experiments even massive doses of STZ did not negatively affect thymocyte cultures. Cultured thymocytes also seemed unaffected by high glucose concentrations, even after 24 h of exposure. Administration of INDO concomitantly with STZ reduced thymic involution but did not prevent the onset of hyperglycemia or reduce established hyperglycemia. When INDO was given before STZ, the same degree of thymic involution occurred; however, hyperglycemia was reduced, although normoglycemia was not restored. INDO also reduced serum corticosterone. Because thymocytes are known to be sensitive to glucocorticoids, this finding suggests that cyclooxygenase 2 inhibition may retard thymic involution by reducing serum glucocorticoids. In conclusion, our results show that STZ and hyperglycemia are not toxic to thymocytes and that cyclooxygenase 2-mediated mechanisms are involved in thymic involution during diabetes.

Insulin promotes early embryonic development, but whether this action affects postimplantation fetal development and alters the expression of imprinted genes remain to be determined. This study analyzed the expression and methylation levels of the growthrelated imprinted genes H19 and insulin-like growth factor 2 (Igf2) in fetuses exposed to insulin before implantation. We cultured 2-cell embryos in either 0 or 0.25 μg/ml insulin until the blastocyst stage and then transferred them into pseudopregnant recipient mice. The number of embryos developing to blastocysts after insulin exposure was 16.4% higher than that of the control, and the birth body weight of the insulin-exposed group was 17.8% higher than that of the control group. Real-time reverse transcription– polymerase chain reaction analysis revealed that exposure of preimplantation embryos to insulin increased the mRNA expression of both Igf2 and H19 in embryonic day (E) 14 fetuses. Bisulfite genomic sequencing demonstrated that the methylation level of the H19–Igf2 imprint control region was 19.3% lower in insulin-exposed E14 fetuses than in controls. The present study indicates that insulin exposure during the preimplantation stage alters the expression of imprinted genes and affects fetal development.

In evaluating discrepant results between experiments in our laboratory, we collected data that challenge the notion that anthelminthic drugs like FBZ do not alter inflammatory responses. We found that FBZ significantly modulates inflammation in F344 rats intrastriatally injected with LPS. FBZ treatment of LPS-injected rats significantly increased weight loss, microglial activation, and dopamine loss; in addition, FBZ attenuated the LPS-induced loss of astrocytes. Therefore, FBZ treatment altered the effects of LPS injection. Caution should be used in interpreting data collected from rats treated with LPS and FBZ.

Gender-associated differences in pathophysiology and treatment of disease are an evolving area in human medicine that should be addressed in animal models. The aim of this study was to characterize gender differences in metabolic parameters of Göttingen minipigs and to determine which gender has the metabolic profile that is most appropriate as a model for human metabolic syndrome. Blood samples were collected from fasted, lean male and female Göttingen minipigs at 8 wk and 8 mo of age. Samples were analyzed for glucose, fructosamine, insulin, C-peptide, glucagon, triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-c), free fatty acids, leptin, testosterone, and 17β-estradiol. Insulin sensitivity and beta cell function were estimated by homeostasis model assessment and degree of obesity by measuring the abdominal circumference. Male minipigs had higher concentrations of both testosterone and estradiol. Female minipigs had a larger abdominal circumference and higher concentrations of C-peptide, insulin, triglyceride, total cholesterol, HDL-c and leptin but a lower concentration of free fatty acids and lower HDL-c:total cholesterol ratio. Compared with male minipigs, female minipigs were more insulin-resistant and had a higher beta-cell function. No gender-associated differences were found in any of the other investigated parameters. In conclusion, female minipigs were more obese and insulin-resistant and had a more atherogenic plasma profile than did their male counterparts and therefore may be better models for metabolic syndrome. Their high concentrations of both testosterone and estradiol may protect male minipigs from obesity and metabolic disturbances.

Excessive preformed vitamin A (VA) intake is contraindicated during pregnancy because of teratogenic concerns. Recent studies have provided biochemical and histologic evidence of chronic hypervitaminosis A in captive Old World monkeys consuming laboratory diets containing high concentrations of retinyl acetate. To investigate the effects of maternal chronic overconsumption of preformed VA on VA storage in early fetal liver, we analyzed monkey fetal livers ranging from 35 to 93 d gestational age (comparable with mid-first to late second trimester in humans) for VA (n = 19) and retinoic acid (n = 9). Retinyl esters were identified in all fetal livers, and retinol, on a percentage basis, was more abundant in younger fetuses. Liver VA concentration increased with gestational age, ranging from 0.0011 to 0.26 μmol/g in the youngest (35 d) and oldest fetuses (93 d), respectively. Liver VA concentrations (mean ± 1 standard deviation) were 0.023 ± 0.008 μmol/g in early gestation and 0.19 ± 0.06 μmol/g in midgestation fetuses. All-trans retinoic acid concentrations were higher in early gestation (99.2 ± 57.0 pmol/g, n = 6) than in midgestation (18.2 ± 6.1 pmol/g, n = 3) but were variable. Liver VA concentrations from midgestation fetuses were higher than those in fetal human and monkey livers from later stages of development, when growth and VA accumulation rates are assumed to be highest. Therefore, excessive intake of preformed VA by the mothers results in amplified early fetal liver retinyl ester storage.