Unveiling how vitrification affects the porcine blastocyst: clues from a transcriptomic study.

BACKGROUND: Currently, there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes. To date, vitrification (VIT) is the most efficient method for pig embryo cryopreservation. Despite a high number of embryos survives in vitro after vitrification/warming procedures, the in vivo embryo survival rates after embryo transfer are variable among laboratories. So far, most studies have focused on cryoprotective agents and devices, while the VIT effects on porcine embryonic gene expression remained unclear. The few studies performed were based on vitrified/warmed embryos that were cultured in vitro (IVC) to allow them to re-expand. Thus, the specific alterations of VIT, IVC, and the cumulative effect of both remained unknown. To unveil the VIT-specific embryonic alterations, gene expression in VIT versus (vs.) IVC embryos was analyzed. Additionally, changes derived from both VIT and IVC vs. control embryos (CO) were analyzed to confirm the VIT embryonic alterations. Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA-sequencing: (1) VIT embryos (vitrified/warmed and cultured in vitro), (2) IVC embryos and (3) CO embryos. RESULTS: RNA-sequencing revealed three clearly different mRNA profiles for VIT, IVC and CO embryos. Comparative analysis of mRNA profiles between VIT and IVC identified 321, differentially expressed genes (DEG) (FDR < 0.006). In VIT vs. CO and IVC vs. CO, 1901 and 1519 DEG were found, respectively, with an overlap of 1045 genes. VIT-specific functional alterations were associated to response to osmotic stress, response to hormones, and developmental growth. While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects. CONCLUSIONS: Our findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs. IVC embryos and second by an integrative transcriptome analysis including in vivo control embryos. The identified VIT alterations might reflect the transcriptional signature of the embryo cryodamage but also the embryo healing process overcoming the VIT impacts. Selected validated genes were pointed as potential biomarkers that may help to improve vitrification.

Uterine fluid proteome changes during diapause and resumption of embryo development in roe deer (Capreolus capreolus).

The uterine microenvironment during pre-implantation presents a pro-survival milieu and is essential for embryo elongation in ruminants. The European roe deer (Careolus capreolus) pre-implantation embryo development is characterised by a 4-month period of reduced development, embryonic diapause, after which the embryo rapidly elongates and implants. We investigated the uterine fluid proteome by label-free liquid chromatography tandem mass spectrometry at four defined stages covering the phase of reduced developmental pace (early diapause, mid-diapause and late diapause) and embryo elongation. We hypothesised that embryo development during diapause is halted by the lack of signals that support progression past the blastocyst stage. Three clusters of differentially abundant proteins were identified by a self-organising tree algorithm: (1) gradual reduction over development; (2) stable abundance during diapause, followed by a sharp rise at elongation; and (3) gradual increase over development. Proteins in the different clusters were subjected to gene ontology analysis. 'Cellular detoxification' in cluster 1 was represented by alcohol dehydrogenase, glutathione S-transferase and peroxiredoxin-2. ATP-citrate synthase, nucleolin, lamin A/C, and purine phosphorylase as cell proliferation regulators were found in cluster 2 and 'cortical cytoskeleton', 'regulation of substrate adhesion-dependent cell spreading' and 'melanosome' were present in cluster 3. Cell cycle promoters were higher abundant at elongation than during diapause, and polyamines presence indicates their role in diapause regulation. This study provides a comprehensive overview of proteins in the roe deer uterine fluid during diapause and forms a basis for studies aiming at understanding the impact of the lack of cell cycle promoters during diapause.

Deciphering the oviductal extracellular vesicles content across the estrous cycle: implications for the gametes-oviduct interactions and the environment of the potential embryo.

BACKGROUND: The success of early reproductive events depends on an appropriate communication between gametes/embryos and the oviduct. Extracellular vesicles (EVs) contained in oviductal secretions have been suggested as new players in mediating this crucial cross-talk by transferring their cargo (proteins, mRNA and small ncRNA) from cell to cell. However, little is known about the oviductal EVs (oEVS) composition and their implications in the reproductive success. The aim of the study was to determine the oEVs content at protein, mRNA and small RNA level and to examine whether the oEVs content is under the hormonal influence of the estrous cycle. RESULTS: We identified the presence of oEVs, exosomes and microvesicles, in the bovine oviductal fluid at different stages of the estrous cycle (postovulatory-stage, early luteal phase, late luteal phase and pre-ovulatory stage) and demonstrated that their composition is under hormonal regulation. RNA-sequencing identified 903 differentially expressed transcripts (FDR < 0.001) in oEVs across the estrous cycle. Moreover, small RNA-Seq identified the presence of different types of ncRNAs (miRNAs, rRNA fragments, tRNA fragments, snRNA, snoRNA, and other ncRNAs), which were partially also under hormonal influence. Major differences were found between post-ovulatory and the rest of the stages analyzed for mRNAs. Interesting miRNAs identified in oEVs and showing differential abundance among stages, miR-34c and miR-449a, have been associated with defective cilia in the oviduct and infertility. Furthermore, functional annotation of the differentially abundant mRNAs identified functions related to exosome/vesicles, cilia expression, embryo development and many transcripts encoding ribosomal proteins. Moreover, the analysis of oEVs protein content also revealed changes across the estrous cycle. Mass spectrometry identified 336 clusters of proteins in oEVs, of which 170 were differentially abundant across the estrous cycle (p-value< 0.05, ratio < 0.5 or ratio > 2). Our data revealed proteins related to early embryo development and gamete-oviduct interactions as well as numerous ribosomal proteins. CONCLUSIONS: Our study provides with the first molecular signature of oEVs across the bovine estrous cycle, revealing marked differences between post- and pre-ovulatory stages. Our findings contribute to a better understanding of the potential role of oEVs as modulators of gamete/embryo-maternal interactions and their implications for the reproductive success.

Serum microRNA profiles in cats with hypertrophic cardiomyopathy.

The role of microRNAs (miRNAs) in the pathogenesis of heart diseases of humans and rodents, as well as their diagnostic potential, has recently received much attention, but comparable studies for spontaneous disease models in the domestic cat are missing. Hypertrophic cardiomyopathy (HCM) is the most common heart disease in cats. The pathology is largely unknown, but is suspected to be influenced by genetic background. In this study, we examined the miRNA profiles in the serum of cats with stable congestive heart failure caused by HCM (n = 11) and healthy control cats (n = 12) using miRNA arrays. 965 out of 2026 miRNAs could be detected in at least six samples of either of the groups. Eleven mammalian miRNAs were differentially expressed between the groups with a fold change ≥ 1.6. Hierarchical cluster analysis resulted in distinct separation of the two groups. After correction for multiple testing (adjusted p < 0.05), a higher expression of miR-381-3p, miR-486-3p, miR-4751, miR-476c-3p, miR-5700, miR-513a-3p, and miR-320e in the HCM group was confirmed. Additionally, miR-1246 was found to be upregulated 3-fold in the HCM group using quantitative RT-PCR. Software analysis of the significantly regulated miRNAs revealed 49 mRNA targets involved in cardiac hypertrophy. Cats with primary HCM show a distinct miRNA profile that includes miRNAs that have already been shown to be differentially regulated in human patients and rodent models for cardiac disease. Studying HCM as a spontaneous cardiac disease of the cat may help to reveal additional pathophysiologic pathways.

Exploration of relationships between production and fertility traits in dairy cattle via association studies of SNPs within candidate genes derived by expression profiling.

The objective of this work was to integrate findings from functional genomics studies with genome-wide association studies for fertility and production traits in dairy cattle. Association analyses of production and fertility traits with SNPs located within or close to 170 candidate genes derived from two gene expression studies and from the literature were performed. Data from 2294 Holstein bulls genotyped for 39557 SNPs were used. A total of 111 SNPs were located on chromosomal segments covered by a candidate gene. Allele substitution effects for each SNP were estimated using a mixed model with a fixed effect of marker and a random polygenic effect. Assumed covariance was derived either from marker or from pedigree information. Results from the analysis with the kinship matrix built from marker genotypes were more conservative than from the analysis with the pedigree-derived relationship matrix. From sixteen SNPs with significant effects on both classes of traits, ten provided evidence of an antagonistic relationship between productivity and fertility. However, we found four SNPs with favourable effects on fertility and on yield traits, one SNP with favourable effects on fertility and percentage traits, and one SNP with antagonistic effects on two fertility traits. While most quantitative genetic studies have proven genetic antagonisms between yield and functional traits, improvements in both production and functionality may be possible when focusing on a few relevant SNPs. Investigations combining input from quantitative genetics and functional genomics with association analysis may be applied for the identification of such SNPs.

Microarray analysis of equine endometrium at days 8 and 12 of pregnancy.

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.

Creating new knowledge for ruminant reproduction from rapidly expanding and evolving scientific databases.

Declining fertility is a major problem for the dairy industry. Recent developments of Omics-technologies facilitate a comprehensive analysis of molecular patters in gametes, embryos and tissues of the reproductive tract which may help to identify the reasons for impaired fertility. Large Omics-datasets require appropriate bioinformatics analysis in the context of rapidly expanding and evolving scientific databases. This overview summarizes the current status of ruminant genome projects, describes currently existing resources for ruminant genomics, transcriptomics and proteomics as well as databases and tools for the interpretation and exploitation of transcriptomics and proteomics datasets. Gene set enrichment analysis (GSEA) and transcription factor binding site (TFBS) analyses are strategies for the identification of regulatory genes. In general, the comprehensive analysis of molecular traits by Omics-technologies can enhance the interpretation of genome-wide association studies, providing insights into the biological pathways linking genotype and phenotype, and their modulation by endogenous and environmental factors.

Transcriptome studies of bovine endometrium reveal molecular profiles characteristic for specific stages of estrous cycle and early pregnancy.

The endometrium undergoes marked functional changes during estrous cycle and pregnancy. As the adjacent environment of the conceptus, it represents the maternal interface for embryo-maternal communication, which is essential to maintain pregnancy. Transcriptome studies provide the unique opportunity to assess molecular profiles changing in response to endocrine or metabolic stimuli or to embryonic pregnancy recognition signals. Here we review the current state of transcriptome profiling techniques and the results of a series of transciptome studies comparing bovine endometrium samples during the estrous cycle or endometrium samples from pregnant vs. non-pregnant animals. These studies revealed specific mRNA profiles which are characteristic for the functional status of the endometrium. Transcriptome studies of endometrial samples recovered during the pre-attachment period identified many interferon-stimulated genes, genes that are possibly involved in embryo-maternal immune modulation ( C1S, C1R, C4, SERPING1, UTMP, CD81, IFITM1, BST2), as well as genes affecting cell adhesion ( AGRN, CD81, LGALS3BP, LGALS9, GPLD1, MFGE8, and TGM2) and remodeling of the endometrium ( CLDN4, MEP1B, LGMN, MMP19, TIMP2, TGM2, MET, and EPSTI1). The results of these transcriptome studies were compared to those of similar microarray analyses in human, mouse and Rhesus monkey to identify similarities in endometrial biology between mammalian species and species-specific differences. Future studies will cover dynamic transcriptome changes between different stages of early pregnancy, the relationship between metabolic problems in dairy cows and the functionality of reproductive tissues as well as endometrium transcriptome profiles in recipients of somatic cell nuclear transfer embryos.

Technical note: Bovine oviduct and endometrium array version 1: a tailored tool for studying bovine endometrium biology and pathophysiology.

Fertility problems are the main reason for slaughter of high-performance milk cows, because elongated calving intervals result in financial losses for the farmer and retard genetic progress. Genetic improvement of fertility would be of great benefit, but functional traits for effective selection are missing. Recent advances in functional genomics tools like DNA microarrays could be the key to identify gene expression patterns in the endometrium that correlate with maternal fertility. Therefore, a first version of a bovine oviduct and endometrium cDNA array was established that contains a set of 1,440 cDNA clones and long oligonucleotides representing 950 different genes. The major part of these genes has been identified in a series of differential gene expression studies in endometrium (different stages of the estrous cycle, d 18 pregnant vs. nonpregnant) and in oviduct epithelial cells (different stages of the estrous cycle) using a combination of subtracted cDNA libraries and cDNA array hybridization. Furthermore, cDNA clones of genes, which showed no changes in their mRNA levels in the analyzed tissues, were added as controls. Reproducibility of the array hybridization, a comparison with the Affymetrix bovine genome array, and confirmation of differential gene expression with reverse transcription-quantitative PCR is shown. Potential future applications include systematic studies of interactions between metabolic status and functionality of the endometrium to identify genes that could be used for differential diagnosis of fertility problems. Further, endometrium transcriptome profiles may serve as novel traits to improve fertility by genetic selection.

Region-specific expression of nitric oxide synthases in the bovine oviduct during the oestrous cycle and in vitro.

Nitric oxide synthases (NOS) account for the endogenous production of nitric oxide (NO), a small and permeable bioreactive molecule. NO is known to act as a paracrine mediator during various processes associated with female reproduction. In the present study, the mRNA expression of the endothelial (eNOS) and inducible (iNOS) NO synthases were examined in bovine oviduct epithelial cells (BOEC) during the oestrous cycle. In addition, eNOS and iNOS mRNA and protein were localised by in situ hybridisation and immunocytochemistry respectively. Furthermore, the effects of exogenously applied oestradiol-17beta and progesterone on NOS mRNA regulation were studied in a suspension culture of BOEC. The eNOS mRNA abundance was low around ovulation (day 0) and increased significantly until pro-oestrus (day 18) in the ampulla. Immunoreactive protein of eNOS was detected predominantly in endothelial cells as well as in secretory oviduct epithelial cells at pro-oestrus. The iNOS mRNA concentration was significantly reduced in the isthmus at pro-oestrus (day 18) and oestrus (day 0) compared with persistently high levels in the ampulla. By in situ hybridisation, specific iNOS transcripts were additionally demonstrated in the oviduct epithelium. Immunoreactive iNOS protein was localised in secretory epithelial cells as well as in the lamina muscularis. The in vitro stimulation showed that both NOS were stimulated by progesterone, but not by oestradiol-17beta. The region-specific modulated expression of eNOS and iNOS provides evidence for an involvement of endogenously produced NO in the regulation of oviductal functions.

Functional genomics: tools for improving farm animal health and welfare.

The first genome sequence assemblies of farm animal species are now accessible through public domain databases, and further sequencing projects are in rapid progress. In addition, large collections of expressed sequences have been obtained, which will aid in constructing annotated transcript maps for many economically important species. Thus, the breeding of farm animals is entering the post-genome era. Functional genomics, defined as applying global experimental approaches to assess gene function, by using the information and reagents provided by structural genomics (i.e. mapping and sequencing), has become the focus of interest. Combining a holistic view of phenotypes at the molecular level with genetic marker data seems a particularly promising approach for improving health and welfare traits in farm animals. These traits are often difficult to define. They suffer from low heritabilities and a corresponding lack of genetic gain in conventional selection and breeding programmes. At the same time, genomic information from micro-organisms and parasites offers the potential for new vaccines and therapeutics. This review describes major functional genomics tools, lists genomic resources available for farm animals and discusses the prospects and challenges of functional genomics in improving the health and welfare of farm animals.

Gene expression profiling of bovine endometrium during the oestrous cycle: detection of molecular pathways involved in functional changes.

The endometrium plays a central role among the reproductive tissues in the context of early embryo-maternal communication and pregnancy. It undergoes typical changes during the sexual/oestrous cycle, which are regulated by the ovarian hormones progesterone and oestrogen. To identify the underlying molecular mechanisms we have performed the first holistic screen of transcriptome changes in bovine intercaruncular endometrium at two stages of the cycle--end of day 0 (late oestrus, low progesterone) and day 12 (dioestrus, high progesterone). A combination of subtracted cDNA libraries and cDNA array hybridisation revealed 133 genes showing at least a 2-fold change of their mRNA abundance, 65 with higher levels at oestrus and 68 at dioestrus. Interestingly, genes were identified which showed differential expression between different uterine sections as well. The most prominent example was the UTMP (uterine milk protein) mRNA, which was markedly upregulated in the cranial part of the ipsilateral uterine horn at oestrus. A Gene Ontology classification of the genes with known function characterised the oestrus time by elevated expression of genes, for example related to cell adhesion, cell motility and extracellular matrix and the dioestrus time by higher expression of mRNAs encoding for a variety of enzymes and transport proteins, in particular ion channels. Searching in pathway databases and literature data-mining revealed physiological processes and signalling cascades, e.g. the transforming growth factor-beta signalling pathway and retinoic acid signalling, which are potentially involved in the regulation of changes of the endometrium during the oestrous cycle.

Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle.

The oviduct epithelium undergoes marked morphological and functional changes during the oestrous cycle. To study these changes at the level of the transcriptome we did a systematic gene expression analysis of bovine oviduct epithelial cells at oestrus and dioestrus using a combination of subtracted cDNA libraries and cDNA array hybridisation. A total of 3072 cDNA clones of two subtracted libraries were analysed by array hybridisation with cDNA probes derived from six cyclic heifers, three of them slaughtered at oestrus and three at dioestrus. Sequencing of cDNAs showing significant differences in their expression levels revealed 77 different cDNAs. Thirty-seven were expressed at a higher level at oestrus, for the other 40 genes expression levels were higher at dioestrus. The identified genes represented a variety of functional classes. During oestrus especially genes involved in the regulation of protein secretion and protein modification, and mRNAs of secreted proteins, were up-regulated, whereas during dioestrus particularly transcripts of genes involved in transcription regulation showed a slight up-regulation. The concentrations of seven selected transcripts were quantified by real-time RT-PCR to validate the cDNA array hybridisation data. For all seven transcripts, RT-PCR results were in excellent correlation (r>0.92) with the results obtained by array hybridisation. Our study is the first to analyse changes in gene expression profiles of bovine oviduct epithelial cells during different stages of the oestrous cycle, providing a starting point for the clarification of the key transcriptome changes in these cells.

Embryo-maternal communication in bovine - strategies for deciphering a complex cross-talk.

Early embryonic development, implantation and maintenance of a pregnancy are critically dependent on an intact embryo-maternal communication. So far, only few signals involved in this dialogue have been identified. In bovine and other ruminants, interferon tau is the predominant embryonic pregnancy recognition signal, exhibiting antiluteolytic activity. However, this is just one aspect of the complex process of embryo-maternal signalling, and a number of other systems are more likely to be involved. To gain a more comprehensive understanding of these important mechanisms, integrated projects involving specialists in embryology, reproductive biotechnology and functional genome research are necessary to perform a systematic analysis of interactions between pre-implantation stage embryos and oviduct or uterine epithelial cells, respectively. State-of-the-art transcriptomic and proteomic technologies will identify reciprocal signals between embryos and their maternal environment and the respective downstream reaction cascades. For in vivo studies, the use of monozygotic twins as recipient animals provides elegant model systems, thus eliminating genetic variability as a cause of differential gene expression. In addition, suitable systems for the co-culture of oviduct epithelial or endometrium cells with the respective embryonic stages need to be established for functional validation of candidate genes potentially involved in the dialogue between embryos and their maternal environment. The knowledge of these mechanisms should help to increase the pregnancy rate following embryo transfer and to avoid embryonic losses. Candidate genes involved in embryo-maternal communication will also be used to define new quality criteria for the selection of embryos for transfer to recipients. Another application is the supplementation of embryotrophic factors or components of embryo-maternal signalling in optimized formulations, such as bioartificial matrices. As a long-term goal, signalling mechanisms identified in bovine will also be functionally evaluated in other species, including the human.

Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs.

With the complete human genomic sequence being unraveled, the focus will shift to gene identification and to the functional analysis of gene products. The generation of a set of cDNAs, both sequences and physical clones, which contains the complete and noninterrupted protein coding regions of all human genes will provide the indispensable tools for the systematic and comprehensive analysis of protein function to eventually understand the molecular basis of man. Here we report the sequencing and analysis of 500 novel human cDNAs containing the complete protein coding frame. Assignment to functional categories was possible for 52% (259) of the encoded proteins, the remaining fraction having no similarities with known proteins. By aligning the cDNA sequences with the sequences of the finished chromosomes 21 and 22 we identified a number of genes that either had been completely missed in the analysis of the genomic sequences or had been wrongly predicted. Three of these genes appear to be present in several copies. We conclude that full-length cDNA sequencing continues to be crucial also for the accurate identification of genes. The set of 500 novel cDNAs, and another 1000 full-coding cDNAs of known transcripts we have identified, adds up to cDNA representations covering 2%--5 % of all human genes. We thus substantially contribute to the generation of a gene catalog, consisting of both full-coding cDNA sequences and clones, which should be made freely available and will become an invaluable tool for detailed functional studies.

A hybrid outer membrane protein antigen for vaccination against Pseudomonas aeruginosa.

Recently a hybrid protein containing parts of the outer membrane proteins OprF (aa 190-342) and OprI (aa 21-83) from Pseudomonas aeruginosa fused to the glutathione-S-transferase was shown to protect mice against a 975-fold 50% lethal dose of P. aeruginosa. To omit the use of the GST-protein, the hybrid protein OprF-OprI was expressed in E. coli using distinct modifications which have not to be eliminated after its expression. Using different signal peptides, the yield of the hybrid protein OprF-OprI in E. coli could be increased to 30% of the total cell protein, however, only a very small amount of the hybrid preprotein was processed and could be isolated from the periplasm of the host. A construct containing an N-terminal extension of 11 amino acids from the original OprF gene gave rise to a significantly higher expression in the cytoplasm. Purification was facilitated by the addition of a five histidine tag at the C-terminus. An even higher expression was obtained by a construct in which a six histidine tag was attached to the N-terminus of the hybrid protein. The N-terminal extended OprF-OprI as well as the N-terminal his-tagged OprF-OprI hybrid antigens were purified by immobilized-metal affinity chromatography under native and denaturing conditions and can now be tested for protectivity against P. aeruginosa in animal model systems.

Effects of different levels of dietary selenium and vitamin E on the humoral immunity of rats.

The aim of the present study was to investigate whether the level of selenium and selenium/vitamin E supply influences the humoral immunity of rats. In order to detect the effect of Se supply and age, 36 weaned Sprague-Dawley rats divided into two equal groups were killed after 22 or 45 experimental days by decapitation (Exp. I). In Exp. II 9 groups of 10 rats each were exposed to each combination of deficient, normal or excessive selenium with a vitamin E supply and killed after 44 days. The basic (deficiency) diet which was the same in both experiments contained 0.04mg Se and 8mg vitamin E per kg dry matter. The supplementation per kg diet was 0 or 0.2mg Se and 30mg vitamin E in Exp. I and 0, 0.2 or 1mg Se and 0, 30 or 200mg vitamin E in Exp. II. The concentration of selenium in serum, liver and spleen samples and the activity of glutathione peroxidase, which were determined to define the selenium status of the animals, corresponded well to the required supply situation. The immunoglobulins of type IgA, IgM and IgG with the subtypes IgG1, IgG2a, IgG2b and IgG2c were measured by immunoelectrophoresis. In both experiments selenium deficiency decreased the values of the IgG groups only nominally, IgA was not changed. IgM was significantly reduced, especially with prolonged selenium deficiency and simultaneous vitamin E deficiency. An excessive selenium supply compensated to a great extent for the effects of vitamin E deficiency on IgG and IgA.

[Hematological parameters, selenium concentration and glutathione peroxidase activities in serum and the liver of rats at different selenium and vitamin E levels]. / Hämatologische Parameter sowie Selenkonzentration und GSH-Px-Aktivität in Serum und Leber der Ratte bei unterschiedlicher Selen- und Vitamin-E-Versorgung.

The aim of the both experiments was to determine whether selenium or selenium/vitamin E supply of rats significantly influences the most important hematological criteria. With experiment 1 the influence of Se deficiency should be determined at two different times of growing. So 36 weaned rats were divided into 2 groups of 18 animals each, the half of them being decapitated at day 22, the rest on day 45. In experiment 2 with the aim to investigate a combination of deficient, adequate and excessive Se and vitamin E supply 90 weaned rats in 9 groups were decapitated at day 44. The basic diet contained 0.04 mg Se and 8 mg vitamin E per kg dry matter and was supplemented in exp. 1 with 0 mg or 0.2 mg Se and 30 mg vitamin E and in exp. 2 with 0 mg, 0.2 mg or 1.0 mg Se and 0 mg, 30 mg or 200 mg vitamin E. With Se deficiency Se concentration and GSH-Px activity in serum and liver were significantly reduced. With excessive Se supply Se concentration in serum was higher; there was no effect on GSH-Px activity. Vitamin E supply had no influence neither on Se content nor on GSH-Px activity in serum or in liver. In exp. 1 Se deficiency caused no clear changes of the analysed hematological criteria although the increase of MCV (+3%) and hematocrit (+7%) on day 22 and the increase of leucocytes (+43%) and the decrease of MCH (-3%) and MCHC (-6%) on day 45 were statistically significant. In exp. 2 these results could not be repeated. The vitamin E supply was without significant effects on the examined hematological parameters.