Establishment and maintenance of pregnancy in equids is only partially understood. To provide new insights into early events of this process, we performed a systematic analysis of transcriptome changes in the endometrium at Days 8 and 12 of pregnancy. Endometrial biopsy samples from pregnant and nonpregnant stages were taken from the same mares. Composition of the collected biopsy samples was analyzed using quantitative stereological techniques to determine proportions of surface and glandular epithelium and blood vessels. Microarray analysis did not reveal detectable changes in gene expression at Day 8, whereas at Day 12 of pregnancy 374 differentially expressed genes were identified, 332 with higher and 42 with lower transcript levels in pregnant endometrium. Expression of selected genes was validated by quantitative real-time RT-PCR. Gene set enrichment analysis, functional annotation clustering, and cocitation analysis were performed to characterize the genes differentially expressed in Day 12 pregnant endometrium. Many known estrogen-induced genes and genes involved in regulation of estrogen signaling were found, but also genes known to be regulated by progesterone and prostaglandin E2. Additionally, differential expression of a number of genes related to angiogenesis and vascular remodeling suggests an important role of this process. Furthermore, genes that probably have conserved functions across species, such as CRYAB, ERRFI1, FGF9, IGFBP2, NR2F2, STC1, and TNFSF10, were identified. This study revealed the potential target genes and pathways of conceptus-derived estrogens, progesterone, and prostaglandin E2 in the equine endometrium probably involved in the early events of establishment and maintenance of pregnancy in the mare.

The findings argue against a persistent level of pronounced distress and suggest a classification of the kindling paradigm as a model with moderate severity based on a longer-lasting mild impact on animal behavioral patterns. This suggestion provides a basis for a prospective and retrospective case-by-case severity assessment.

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Progesterone (P4) measurement in the peripheral blood is an objective parameter for determination of reproductive functions in the bitch. This study evaluates an enzyme-linked fluorescence assay (ELFA) (Biomerieux, France) for the determination of progesterone validated for use in human. The ELFA is to be performed on the MiniVidas automated analyser which provides quantitative results within 45 min. Blood samples from a total of 27 female dogs of different breeds were used. To test the correctness of the ELFA 15 blood samples with a range of 0.3-40.0 ng/ml were compared to a radioimmunoassay (RIA) validated in the dog. The values obtained with the MiniVidas showed a high agreement (mean deviation 15%), deviations were in both directions and the correlation coefficient was 0.989. The coefficient of correlation according to Passing-Bablok test was 0.995. The intra-assay reproducibility in the MiniVidas system was tested on five samples (mean values 61.8, 6.8, 51.4, 43.7 and 1.1 ng/ml). The coefficients of variation (CV; 10-12 replicates) were 3.4%, 6.7%, 2.6%, 3.1% and 25.4%, respectively. Four serum samples (mean value 47.0, 15.1, 49.1 and 4.0 ng/ml) from different bitches were assayed singly in 10 separate series to test the inter-assay variability. The corresponding CV was 2.1%, 2.2%, 3.1% and 4.3% respectively. Samples from three dogs were used to test the accuracy of the assay. These samples were diluted (1/2, 1/4, 1/8 and 1/16) with charcoal-stripped human serum (Biomerieux, France) and tested in three runs. The expected values were met in a range of 60-75%. Measurement of progesterone for the detection of ovulation as well as prediction of parturition provided meaningful results. As a conclusion the use of the MiniVidas system for determination of P4 in peripheral blood of the bitch provides rapid and reliable results.

ObjectiveThe prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INSC94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates.MethodsFemale MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics.ResultsMIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples.ConclusionsThe broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension.

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A limited number of reports is available on cryopreservation of in vitro fertilization (IVF)-derived cat blastocysts. In the present study, IVF-derived domestic cat embryos which reached the blastocyst stage either on day 6 or day 7 were cryopreserved by vitrification using Cryotop as a cryodevice. Fresh control and post-warm surviving blastocysts were examined by differential cell staining with Hoechst 33342 and propidium iodide to determine total cell number and inner cell mass (ICM) ratio, and the post-warm survival rate was determined by re-expansion of the blastocoel during 24 h of in vitro culture. In fresh control, the mean number of total cells of day 7 blastocysts (61.4 cells) tended to be smaller than that of day 6 blastocysts (81.9 cells, p = 0.096). The post-warm survival rates of day 6 and day 7 blastocysts were not statistically different (73.8%; 31 of 42 vs 66.7%; 18 of 27). There were no significant differences in the total cell number and ICM ratio between fresh control and vitrified blastocysts, although the ICM ratio of surviving day 7 blastocysts was significantly smaller than that of fresh controls (stained at day 8, 18.9% vs 28.9%, p < 0.05). These results indicate that IVF-derived domestic cat embryos that reached the blastocyst stage earlier can survive the Cryotop vitrification without a reduction in the parameters studied.

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