Sex diagnosis of ovine and bovine embryos by enzymatic amplification and digestion of DNA from the ZFY/ZFX locus

A PCR-based sex determination assay for sheep and cattle embryos was developed using mouse embryos for optimizing the protocol. Samples were lysed either enzymatically or by alkaline treatment followed by enzymatic amplification of DNA from the ZFY/ZFX locus. Sex diagnosis could be done after the digestion of the amplified product by restriction endonucleases. Ovine and bovine embryos could be sexed from biopsies as small as 1-4 cells. Some embryos were split into 2-4 sections, which were amplified separately. Blind trials with such samples demonstrated that the method was highly accurate, even when embryo biopsy was done under farm conditions. The protocol involves an in-built control. This eliminates the need for autosomal control primers, which often inhibit the amplification of the Y-chromosome-specific DNA, especially when a small amount of template is used

Gene Expression Profiling of Corpus luteum Reveals Important Insights about Early Pregnancy in Domestic Sheep

The majority of pregnancy loss in ruminants occurs during the preimplantation stage, which is thus the most critical period determining reproductive success. Here, we performed a comparative transcriptome study by sequencing total mRNA from corpus luteum (CL) collected during the preimplantation stage of pregnancy in Finnsheep, Texel and F1 crosses. A total of 21,287 genes were expressed in our data. Highly expressed autosomal genes in the CL were associated with biological processes such as progesterone formation (STAR, CYP11A1, and HSD3B1) and embryo implantation (e.g., TIMP1, TIMP2 and TCTP). Among the list of differentially expressed genes, sialic acid-binding immunoglobulin (Ig)-like lectins (SIGLEC3, SIGLEC14, SIGLEC8), ribosomal proteins (RPL17, RPL34, RPS3A, MRPS33) and chemokines (CCL5, CCL24, CXCL13, CXCL9) were upregulated in Finnsheep, while four multidrug resistance-associated proteins (MRPs) were upregulated in Texel ewes. A total of 17 known genes and two uncharacterized noncoding RNAs (ncRNAs) were differentially expressed in breed-wise comparisons owing to the flushing diet effect. The significantly upregulated TXNL1 gene indicated potential for embryonic diapause in Finnsheep and F1. Moreover, we report, for the first time in any species, several genes that are active in the CL during early pregnancy (including TXNL1, SIGLEC14, SIGLEC8, MRP4, and CA5A).202

A simple culture system for time-lapse video recording of bovine embryos

Continuous observation of embryonic growth can improve understanding of the early developmental events and allow us to use parametric statistical analyses with time as a parameter. A cinematographic study such as that reported here utilizes time-lapse video recording. Previously published methods for time-lapse video recording have involved building an incubator around a microscope, a process that is both expensive and laborious. Here we present a simplified method for time-lapse video recording of early bovine embryo development. The embryos were cultured during a 24-hour period in a standard pregassed tissue culture bottle, which was darkened and placed on the heating stage of an inverted microscope for recording through a red filter. The control embryos were cultured in a conventional CO2 incubator. After 10 replicates we could not find a statistically significant difference between the cell numbers of these two treatments (P=0.95), suggesting that the culture setup is appropriate for continuous observation of early cleavage of the cattle embryo

Functional profiling of the endometrium transcriptome during preimplantation development in Finnsheep, Texel and their F1 crosses

Carefully coordinated interaction between the endometrium and embryo is critical for the establishment and maintenance of pregnancy in mammals. By exploring the gene expression dynamics of this tissue during preimplantation development, we may be able to get insight into the genetic mechanisms of reproduction during early pregnancy. Here, we have performed comparative transcriptome profiling of the endometrium in response to spherical (Day 7 to Day 12) and elongated (Day 13 to Day 17) embryos in Finnsheep, Texel and their F1 crosses using RNA sequencing (RNA-seq) approach. A total of 21125 genes were expressed in our dataset of which 554 were significantly (absolute log2 fold change > 2.5; adjusted p-value < 0.01) upregulated in the endometrium with elongated embryos. Highly abundant autosomal genes in the endometrium were associated with biological processes such as facilitation of maternal recognition of pregnancy, trophoblast elongation and implantation (LGALS15, CST3, CST6, and EEF1A1). Several endogenous retroviruses (ERVs) including a novel ERV gene located in a reduced FecL locus potentially associated with sheep prolificacy were expressed in our dataset. Comparative transcriptome profiling of the endometrium having spherical and elongated embryos revealed distinct gene expression patterns. Genes that were upregulated in response to elongated embryos indicated the importance of immune system at the maternal-embryo interface prior to implantation

Differences in Adipose Gene Expression Profiles between Male and Female Even Reindeer (Rangifer tarandus) in Sakha (Yakutia)

Reindeer are native to harsh northern Eurasian environments which are characterized by long and cold winters, short summers, and limited pasture vegetation. Adipose tissues play a significant role in these animals by modulating energy metabolism, immunity, and reproduction. Here, we have investigated the transcriptome profiles of metacarpal, perirenal, and prescapular adipose tissues in Even reindeer and searched for genes that were differentially expressed in male and female individuals. A total of 15,551 genes were expressed, where the transcriptome profile of metacarpal adipose tissue was found to be distinct from that of perirenal and prescapular adipose tissues. Interestingly, 10 genes, including PRDM9, which is known to have an important role in adaptation and speciation in reindeer, were always upregulated in all three tissues of male reindeer

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Transmission of Mycoplasma bovis infection in bovine in vitro embryo production

Mycoplasma bovis (M. bovis) causes several costly diseases in cattle and has a negative effect on cattle welfare. There is no effective commercial vaccine, and antimicrobial resistance is common. Maintaining a closed herd is the best method to minimize the risk of introduction of M. bovis. Assisted reproduction is crucial in a closed herd to make genetic improvements. M. bovis has been found in commercial semen, and contaminated semen has been the source of disease in naïve dairy herds. The objective of this study was to evaluate M. bovis transmission in bovine in vitro embryo production (IVP) using several possible exposure routes. We used a wild-type M. bovis strain isolated from semen at a final concentration of 106 CFU/mL to infect cumulus–oocyte complexes, spermatozoa, and 5-day-old embryos. We also used naturally contaminated semen in fertilization. Blastocysts were collected on day 7–8 and zona pellucida (ZP)-intact embryos were either washed 12 times, including trypsin washes as recommended by the International Embryo Technology Society (IETS), or left unwashed. Washed and unwashed embryos, follicular fluids, maturation medium, cumulus cells, fertilization medium, and G1 and G2 culture media, as well as all wash media were analyzed using enrichment culture followed by real-time PCR detection of M. bovis. Altogether, 76 pools containing 363 unwashed embryos and 52 pools containing 261 IETS washed embryos were analyzed after oocytes, spermatozoa, or 5-day-old embryos were infected with M. bovis or naturally contaminated semen was used in fertilization. We could not detect M. bovis in any of the embryo pools. M. bovis was not found in any of 12 wash media from different exposure experiments. M. bovis did not affect the blastocyst rate, except when using experimentally infected semen. Contrary to an earlier study, which used a cell co-culture system, we could not demonstrate M. bovis in embryo wash media or tight adherence of M. bovis to ZP-intact embryos. Naturally infected semen did not transmit M. bovis to embryos. We conclude that by using our IVP system, the risk of M. bovis transmission via IVP embryos to recipient cows is very low

Ovarian mRNA and miRNA Transcriptome Profiling of Domestic Sheep Breeds

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Integrated ovarian mRNA and miRNA transcriptome profiling characterizes the genetic basis of prolificacy traits in sheep (Ovis aries)

Background: The highly prolific breeds of domestic sheep (Ovis aries) are globally valuable genetic resources for sheep industry. Genetic, nutritional and other environmental factors affect prolificacy traits in sheep. To improve our knowledge of the sheep prolificacy traits, we conducted mRNA-miRNA integrated profiling of ovarian tissues from two pure breeds with large (Finnsheep) vs. small (Texel) litter sizes and their F1 crosses, half of which were fed a flushing diet. Results: Among the samples, 16,402 genes (60.6% known ovine genes) were expressed, 79 novel miRNAs were found, and a cluster of miRNAs on chromosome 18 was detected. The majority of the differentially expressed genes between breeds were upregulated in the Texel with low prolificacy, owing to the flushing diet effect, whereas a similar pattern was not detected in the Finnsheep. F1 ewes responded similarly to Finnsheep rather than displaying a performance intermediate between the two pure breeds. Conclusions: The identification and characterization of differentially expressed genes and miRNAs in the ovaries of sheep provided insights into genetic and environmental factors affecting prolificacy traits. The three genes (CST6, MEPE and HBB) that were differentially expressed between the group of Finnsheep and Texel ewes kept in normal diet appeared to be candidate genes of prolificacy traits and will require further validation.Peer reviewe

Integrated ovarian mRNA and miRNA transcriptome profiling characterizes the genetic basis of prolificacy traits in sheep (Ovis aries)

Abstract Background The highly prolific breeds of domestic sheep (Ovis aries) are globally valuable genetic resources for sheep industry. Genetic, nutritional and other environmental factors affect prolificacy traits in sheep. To improve our knowledge of the sheep prolificacy traits, we conducted mRNA-miRNA integrated profiling of ovarian tissues from two pure breeds with large (Finnsheep) vs. small (Texel) litter sizes and their F1 crosses, half of which were fed a flushing diet. Results Among the samples, 16,402 genes (60.6% known ovine genes) were expressed, 79 novel miRNAs were found, and a cluster of miRNAs on chromosome 18 was detected. The majority of the differentially expressed genes between breeds were upregulated in the Texel with low prolificacy, owing to the flushing diet effect, whereas a similar pattern was not detected in the Finnsheep. F1 ewes responded similarly to Finnsheep rather than displaying a performance intermediate between the two pure breeds. Conclusions The identification and characterization of differentially expressed genes and miRNAs in the ovaries of sheep provided insights into genetic and environmental factors affecting prolificacy traits. The three genes (CST6, MEPE and HBB) that were differentially expressed between the group of Finnsheep and Texel ewes kept in normal diet appeared to be candidate genes of prolificacy traits and will require further validation