Effect of periconceptional nutrition on the growth, behaviour and survival of the neonatal lamb.

Periconceptional nutrition (PCN) can influence foetal hypothalamo-pituitary adrenal (HPA) axis function and alter cortisol secretion with possible consequences for maturation and growth of major organs, gestation length and behaviour. We examined effects of PCN on phenotype and survival of the neonatal lamb in 466 Merino ewes allocated to treatments providing 70%, 100% and 150% respectively, of maintenance requirements for 17 days prior and 6 days after insemination. Gestation length and birth weight for lambs in PCN treatment groups was similar (P > 0.05) but low PCN decreased the size of the neonate (crown-rump-length and metacarpal length P < 0.05). A subset of lambs euthanased at 5 days of age further showed that low PCN decreased the amount of peri-renal fat (P < 0.05) and increased liver mass (P < 0.05) while high PCN increased neck thymus and ovary mass (P < 0.05). Neonatal lambs from low PCN ewes returned faster to their mothers after release (P < 0.05) and contacted the udder in the shortest time (P < 0.05). Significant interactions between PCN treatment and sex (P < 0.05) and between PCN treatment and ewe age (P < 0.05) were also observed for time lambs took to follow the ewe. Survival of lambs was similar but potential differences may have been masked by favourable weather conditions. In conclusion, this study provides evidence of significant changes in lamb growth and development dependent on PCN and, for the first time, links these changes with significant changes in behaviour of the neonate. The impact of these effects on lamb survival and potential reproductive capacity of female offspring remains to be determined.

Haplogroup relationships between domestic and wild sheep resolved using a mitogenome panel.

Five haplogroups have been identified in domestic sheep through global surveys of mitochondrial (mt) sequence variation, however these group classifications are often based on small fragments of the complete mtDNA sequence; partial control region or the cytochrome B gene. This study presents the complete mitogenome from representatives of each haplogroup identified in domestic sheep, plus a sample of their wild relatives. Comparison of the sequence successfully resolved the relationships between each haplogroup and provided insight into the relationship with wild sheep. The five haplogroups were characterised as branching independently, a radiation that shared a common ancestor 920,000 ± 190,000 years ago based on protein coding sequence. The utility of various mtDNA components to inform the true relationship between sheep was also examined with Bayesian, maximum likelihood and partitioned Bremmer support analyses. The control region was found to be the mtDNA component, which contributed the highest amount of support to the tree generated using the complete data set. This study provides the nucleus of a mtDNA mitogenome panel, which can be used to assess additional mitogenomes and serve as a reference set to evaluate small fragments of the mtDNA.

Distinct patterns of gene-specific methylation in mammalian placentas: implications for placental evolution and function.

The placenta has arisen relatively recently and is among the most rapidly evolving tissues in mammals. Several different placental barrier and structure types appear to have independently evolved common functional features. Specific patterns of gene expression that determine placental development in humans are predicted to be accompanied by specific profiles of epigenetic modification. However, the stratification of epigenetic modifications into those involved in conserved aspects of placental function, versus those involved in divergent placental features, has yet to begin. As a first step towards this goal, we have investigated the methylation status of a small number of gene-specific methylation events recently identified in human placenta, in a panel of placental tissue from baboon, marmoset, cow, cat, guinea pig and mouse. These represent disparate placental barrier types and structures. In this study we hypothesized that specific epigenetic markings may be associated with placental barrier type or function, independent of phylogeny. However, in contrast to our predictions, the majority of gene-specific methylation appears to track with phylogeny, independent of placental barrier type or other structural features. This suggests that despite the likelihood of epigenetic modification playing a role in the functioning and evolution of different placental subtypes, there is no evidence for an involvement of the gene-specific methylation profiles we have identified, in specifying these differences. Further studies, examining larger numbers of epigenetic modifications across phylogeny, are required to define the role of specific epigenetic modifications in the evolution of distinct placental structures.

IFPA Meeting 2009 workshops report.

Workshops are an important part of the annual meeting of the International Federation of Placenta Associations (IFPA). At IFPA Meeting 2009 diverse topics were discussed in twelve themed workshops. Topics covered included: immune response to pregnancy; signaling between fetus and placenta; bioactive lipids in placenta; placenta in agricultural species; epigenetics and placentation; trophoblast deportation; glucocorticoids and placental function; endothelium; placental transport; genes and placenta; uteroplacental blood flow and placental stem cells. This report is a full summary of the various topics covered.

In vitro systems for intercepting early embryo-maternal cross-talk in the bovine oviduct.

A comprehensive understanding of the complex embryo-maternal interactions during the preimplantation period requires the analysis of very early stages of pregnancy. These are difficult to assess in vivo due to the small size of the embryo exerting local paracrine effects. Specifically designed experiments and holistic transcriptome and proteome analyses to address the early embryo-maternal cross-talk in the oviduct require sufficient numbers of well-defined cells in a standardized experimental environment. The pronounced estrous cycle-dependent changes in gene expression and morphology of bovine oviduct epithelial cells (BOECs) clearly show that a precise definition of the stage of estrous cycle is essential for obtaining a well-defined homogenous population of functional cells. The number of intact cells isolated from individual ampullae by solely mechanical means was 10-fold higher than previously reported cell yields after enzymatic treatment, and the purity was comparable. Bovine oviduct epithelial cells have been cultured as monolayers or in suspension. Proliferating cells grown in monolayers dedifferentiated, with a concomitant loss of important morphologic characteristics. After several days in culture, BOECs in monolayers are less likely to mimic the oviduct environment in vivo than BOEC vesicles formed of epithelial sheets in short-term suspension culture. A 24-h culture system for BOECs isolated on Day 3.5 of the estrous cycle showed excellent preservation of morphologic criteria, marker gene expression, and hormone responsiveness. The short-term BOEC culture system provides well-defined and functional BOECs in sufficient quantities for studies of early embryo-maternal interactions in experiments that mimic the environment in the oviduct in vivo.

Complete mitochondrial genomes of Bos taurus and Bos indicus provide new insights into intra-species variation, taxonomy and domestication.

The taurine and zebuine cattle breeds comprise the majority of the world cattle population but their taxonomic status is still controversial. The two forms of cattle are currently classified as Bos taurus and Bos indicus species and are differentiated primarily by the presence or absence of a hump. However, these two species hybridize readily, producing fully fertile offspring. We have determined and analyzed complete B. taurus and B. indicus mitochondrial genome sequences to investigate the extent of sequence divergences and to study their taxonomic status by molecular dating. The sequences encompassed 16,338 and 16,339 nucleotides, respectively, and differed at 237 positions. Estimated divergence times indicated that the two cattle lineages separated 1.7-2.0 million years ago. Combined phylogenetic analyses of 18 new and 130 previously reported extant B. taurus and B. indicus control region sequences with data from 32 archaeological specimens of the extinct wild aurochs (Bos primigenius) identified four major maternal lineages. B. primigenius haplotypes were present in all but the B. indicus lineage, and one B. taurus sequence clustered with B. primigenius P haplotypes that were not previously linked with domestic cattle. The B. indicus cluster and a recently reported new B. primigenius haplotype that represents a new lineage were approximately equidistant from the B. taurus cluster. These data suggest domestications from several differentiated populations of B. primigenius and a subspecies status for taurine (B. primigenius taurus) and zebuine (B. primigenius indicus) cattle.

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.

In vitro embryo production efficiency in cattle and its association with oocyte adenosine triphosphate content, quantity of mitochondrial DNA, and mitochondrial DNA haplogroup.

Mitochondria have a broad range of functions that affect reproduction, and structural as well as quantitative variation in mtDNA has been associated with gamete quality and reproductive success. To investigate the mitochondria effect on in vitro embryo production, we collected oocytes by ultrasound-guided follicular aspiration from donor cows known to differ in the developmental capacity, measured by the blastocyst formation rate, of their oocytes. To evaluate the potential effects of mtDNA and mitochondrial function on oocyte quality, the donor cows' mtDNA control region was sequenced and, after pairwise comparisons of polymorphisms, animals were grouped into two major haplogroups. The number of mtDNA molecules per oocyte was quantified by real-time PCR, and the adenosine triphosphate (ATP) content was measured in each oocyte to identify variations between haplogroups. Overall, ATP stocks in oocytes of the two haplogroups differed significantly (P < 0.05; means +/- SEM) both at the germinal vesicle and metaphase II stages (2.8 +/- 0.06 pmol vs. 2.6 +/- 0.07 pmol and 2.9 +/- 0.1 pmol vs. 2.3 +/- 0.06 pmol, respectively). The proportion of development to blastocyst was significantly different between haplogroups (22.3 +/- 2.1 % vs. 36.7 +/- 2.9 %). The number of mtDNA molecules per oocyte was highly variable (377 327 +/- 14 104, ranging from 2.0 x 10(3) to 1.2 x 10(6)) but not significantly different between the two haplogroups; significant differences were observed between animals without any apparent relationship to blastocyst production. These data suggest that mitochondria and mtDNA haplogroup affect the developmental capacity of bovine oocytes in vitro.

Mapping of QTL for Body Conformation and Behavior in Cattle.

Genome scans for quantitative trait loci (QTL) in farm animals have concentrated on primary production and health traits, and information on QTL for other important traits is rare. We performed a whole genome scan in a granddaughter design to detect QTL affecting body conformation and behavior in dairy cattle. The analysis included 16 paternal half-sib families of the Holstein breed with 872 sons and 264 genetic markers. The markers were distributed across all 29 autosomes and the pseudoautosomal region of the sex chromosomes with average intervals of 13.9 cM and covering an estimated 3155.5 cM. All families were analyzed jointly for 22 traits using multimarker regression and significance thresholds determined empirically by permutation. QTL that exceeded the experiment-wise significance threshold (5% level) were detected on chromosome 6 for foot angle, teat placement, and udder depth, and on chromosome 29 for temperament. QTL approaching experiment-wise significance (10% level) were located on chromosome 6 for general quality of feet and legs and general quality of udder, on chromosome 13 for teat length, on chromosome 23 for general quality of feet and legs, and on chromosome 29 for milking speed. An additional 51 QTL significant at the 5% chromosome-wise level were distributed over 21 chromosomes. This study provides the first evidence for QTL involved in behavior of dairy cattle and identifies QTL for udder conformation on chromosome 6 that could form the basis of recently reported QTL for clinical mastitis.

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.

The mitochondrial genome in embryo technologies.

The mammalian mitochondrial genome encodes for 37 genes which are involved in a broad range of cellular functions. The mitochondrial DNA (mtDNA) molecule is commonly assumed to be inherited through oocyte cytoplasm in a clonal manner, and apparently species-specific mechanisms have evolved to eliminate the contribution of sperm mitochondria after natural fertilization. However, recent evidence for paternal mtDNA inheritance in embryos and offspring questions the general validity of this model, particularly in the context of assisted reproduction and embryo biotechnology. In addition to normal mt DNA haplotype variation, oocytes and spermatozoa show remarkable differences in mtDNA content and may be affected by inherited or acquired mtDNA aberrations. All these parameters have been correlated with gamete quality and reproductive success rates. Nuclear transfer (NT) technology provides experimental models for studying interactions between nuclear and mitochondrial genomes. Recent studies demonstrated (i) a significant effect of mtDNA haplotype or other maternal cytoplasmic factors on the efficiency of NT; (ii) phenotypic differences between transmitochondrial clones pointing to functionally relevant nuclear-cytoplasmic interactions; and (iii) neutral or non-neutral selection of mtDNA haplotypes in heteroplasmic conditions. Mitochondria form a dynamic reticulum, enabling complementation of mitochondrial components and possibly mixing of different mtDNA populations in heteroplasmic individuals. Future directions of research on mtDNA in the context of reproductive biotechnology range from the elimination of adverse effects of artificial heteroplasmy, e.g. created by ooplasm transfer, to engineering of optimized constellations of nuclear and cytoplasmic genes for the production of superior livestock.

Quantitative trait loci mapping of functional traits in the German Holstein cattle population.

A whole-genome scan to detect quantitative trait loci (QTL) for functional traits was performed in the German Holstein cattle population. For this purpose, 263 genetic markers across all autosomes and the pseudoautosomal region of the sex chromosomes were genotyped in 16 granddaughter-design families with 872 sons. The traits investigated were deregressed breedingvalues for maternal and direct effects on dystocia (DYSm, DYSd) and stillbirth (STIm, STId) as well as maternal and paternal effects on nonreturn rates of 90 d (NR90m, NR90p). Furthermore, deregressed breeding values for functional herd life (FHL) and daughter yield deviation for somatic cell count (SCC) were investigated. Weighted multimarker regression analyses across families and permutation tests were applied for the detection of QTL and the calculation of statistical significance. A ten percent genomewise significant QTL was localized for DYSm on chromosome 8 and for SCC on chromosome 18. A further 24 putative QTL exceeding the 5% chromosomewise threshold were detected. On chromosomes 7, 8, 10, 18, and X/Yps, coincidence of QTL for several traits was observed. Our results suggest that loci with influence on udder health may also contribute to genetic variance of longevity. Prior to implementation of these QTL in marker assisted selection programs for functional traits, information about direct and correlated effects of these QTL as well as fine mapping of their chromosomal positions is required.

Mapping of the bovine blood group systems J, N', R', and Z show evidence for oligo-genetic inheritance.

Genes determining the bovine erythrocyte antigens were mapped by linkage analysis. In total 9591 genotypes of 20 grandsire families with 1074 sires from a grand-daughter design were elucidated for the genes determining the erythrocyte antigens EAA, EAB, EAC, EAF, EAJ, EAL, EAM, EAN', EAR', EAS, EAT', and EAZ according to standard paternity testing procedures in the blood typing laboratories. Linkage analyses were performed with 248 microsatellite markers, eight SSCP markers and four polymorphic proteins and enzymes covering the 29 autosomes and the pseudoautosomal region of the sex chromosomes. The number of informative meioses for the blood group systems ranged from 76 to 947. Blood group systems EAM and EAT' were non-informative. Most of the erythrocyte antigen loci showed significant linkage to a single chromosome and were mapped unequivocally. The genes determining erythrocyte antigen EAA, EAB, EAC, EAL, and EAS were mapped to chromosomes 15, 12, 18, 3, and 21, respectively. Lod-score values ranged from 11.43 to 107.83. Moreover, the EAF system could be mapped to chromosome 17. However, the EAN' system previously known as part of the EAF system could be mapped to chromosome 5. In addition, the blood group systems EAJ, the new EAN', EAR', and EAZ, showed significant linkage to microsatellite markers on various chromosomes and also to other blood groups. The appearance of a single blood group system might be therefore either dependent on the existence of other blood group systems or because of an interaction between different loci on various chromosomes as is known in humans and in pigs.

A whole genome scan for differences in recombination rates among three Bos taurus breeds.

Twenty paternal half-sib families of a granddaughter design were genotyped for 265 genetic markers, most of them microsatellites. These were 16 Holstein families, 3 Simmental families, and 1 Brown Swiss family. The number of sires per breed was 872, 170, and 32, respectively. Two-point recombination rates were estimated both jointly for all breeds and each single breed separately. Of 1168 marker intervals, 865 provided estimates for at least two breeds. Differences between breeds were tested by likelihood ratio tests. Four marker intervals, representing three genomic regions on BTA19, BTA24, and BTA27, show a significant impact of the breed at a false discovery rate of 0.23 and indicate a genetic component of observed heterogeneity of recombination. The variability of recombination rates between cattle breeds might not be a common feature of the whole genome, but rather might be restricted to certain chromosomal segments. Thus, attention should be paid to heterogeneities when pooling data of such regions from different breeds.

An enhanced linkage map of the sheep genome comprising more than 1000 loci.

A medium-density linkage map of the ovine genome has been developed. Marker data for 550 new loci were generated and merged with the previous sheep linkage map. The new map comprises 1093 markers representing 1062 unique loci (941 anonymous loci, 121 genes) and spans 3500 cM (sex-averaged) for the autosomes and 132 cM (female) on the X chromosome. There is an average spacing of 3.4 cM between autosomal loci and 8.3 cM between highly polymorphic [polymorphic information content (PIC) > or = 0.7] autosomal loci. The largest gap between markers is 32.5 cM, and the number of gaps of > 20 cM between loci, or regions where loci are missing from chromosome ends, has been reduced from 40 in the previous map to 6. Five hundred and seventy-three of the loci can be ordered on a framework map with odds of > 1000 : 1. The sheep linkage map contains strong links to both the cattle and goat maps. Five hundred and seventy-two of the loci positioned on the sheep linkage map have also been mapped by linkage analysis in cattle, and 209 of the loci mapped on the sheep linkage map have also been placed on the goat linkage map. Inspection of ruminant linkage maps indicates that the genomic coverage by the current sheep linkage map is comparable to that of the available cattle maps. The sheep map provides a valuable resource to the international sheep, cattle, and goat gene mapping community.