Analysis of genetic variation contributing to measured speed in Thoroughbreds identifies genomic regions involved in the transcriptional response to exercise.

Despite strong selection for athletic traits in Thoroughbred horses, there is marked variation in speed and aptitude for racing performance within the breed. Using global positioning system monitoring during exercise training, we measured speed variables and temporal changes in speed with age to derive phenotypes for GWAS. The aim of the study was to test the hypothesis that genetic variation contributes to variation in end-point physiological traits, in this case galloping speed measured during field exercise tests. Standardisation of field-measured phenotypes was attempted by assessing horses exercised on the same gallop track and managed under similar conditions by a single trainer. PCA of six key speed indices captured 73.9% of the variation with principal component 1 (PC1). Verifying the utility of the phenotype, we observed that PC1 (median) in 2-year-old horses was significantly different among elite, non-elite and unraced horses (P < 0.001) and the temporal change with age in PC1 varied among horses with different myostatin (MSTN) g.66493737C>T SNP genotypes. A GWAS for PC1 in 2-year-old horses (n = 122) identified four SNPs reaching the suggestive threshold for association (P < 4.80 × 10-5 ), defining a 1.09 Mb candidate region on ECA8 containing the myosin XVIIIB (MYO18B) gene. In a GWAS for temporal change in PC1 with age (n = 168), five SNPs reached the suggestive threshold for association and defined candidate regions on ECA2 and ECA11. Both regions contained genes that are significantly differentially expressed in equine skeletal muscle in response to acute exercise and training stimuli, including MYO18A. As MYO18A plays a regulatory role in the skeletal muscle response to exercise, the identified genomic variation proximal to the myosin family genes may be important for the regulation of the response to exercise and training.

Genetic contributions to precocity traits in racing Thoroughbreds.

Adaptation to early training and racing (i.e. precocity), which is highly variable in racing Thoroughbreds, has implications for the selection and training of horses. We hypothesised that precocity in Thoroughbred racehorses is heritable. Age at first sprint training session (work day), age at first race and age at best race were used as phenotypes to quantify precocity. Using high-density SNP array data, additive SNP heritability (hSNP2) was estimated to be 0.17, 0.14 and 0.17 for the three traits respectively. In genome-wide association studies (GWAS) for age at first race and age at best race, a 1.98-Mb region on equine chromosome 18 (ECA18) was identified. The most significant association was with the myostatin (MSTN) g.66493737C>T SNP (P = 5.46 × 10-12 and P = 1.89 × 10-14 respectively). In addition, two SNPs on ECA1 (g.37770220G>A and g.37770305T>C) within the first intron of the serotonin receptor gene HTR7 were significantly associated with age at first race and age at best race. Although no significant associations were identified for age at first work day, the MSTN:g.66493737C>T SNP was among the top 20 SNPs in the GWAS (P = 3.98 × 10-5 ). Here we have identified variants with potential roles in early adaptation to training. Although there was an overlap in genes associated with precocity and distance aptitude (i.e. MSTN), the HTR7 variants were more strongly associated with precocity than with distance. Because HTR7 is closely related to the HTR1A gene, previously implicated in tractability in young Thoroughbreds, this suggests that behavioural traits may influence precocity.

Fertility and genomics: comparison of gene expression in contrasting reproductive tissues of female cattle.

To compare gene expression among bovine tissues, large bovine RNA-seq datasets were used, comprising 280 samples from 10 different bovine tissues (uterine endometrium, granulosa cells, theca cells, cervix, embryos, leucocytes, liver, hypothalamus, pituitary, muscle) and generating 260 Gbases of data. Twin approaches were used: an information-theoretic analysis of the existing annotated transcriptome to identify the most tissue-specific genes and a de-novo transcriptome annotation to evaluate general features of the transcription landscape. Expression was detected for 97% of the Ensembl transcriptome with at least one read in one sample and between 28% and 66% at a level of 10 tags per million (TPM) or greater in individual tissues. Over 95% of genes exhibited some level of tissue-specific gene expression. This was mostly due to different levels of expression in different tissues rather than exclusive expression in a single tissue. Less than 1% of annotated genes exhibited a highly restricted tissue-specific expression profile and approximately 2% exhibited classic housekeeping profiles. In conclusion, it is the combined effects of the variable expression of large numbers of genes (73%-93% of the genome) and the specific expression of a small number of genes (<1% of the transcriptome) that contribute to determining the outcome of the function of individual tissues.

N-glycan profiling of bovine follicular fluid at key dominant follicle developmental stages.

Follicular fluid (FF), an important microenvironment for the development of oocytes, contains many proteins that are glycosylated with N-linked glycans. This study aimed i) to present an initial analysis of the N-linked glycan profile of bovine FF using hydrophilic interaction liquid chromatography, anion exchange chromatography, high performance liquid chromatography (HPLC)-based separations and subsequent liquid chromatography-mass spectrometry/mass spectrometry analysis; ii) to determine differences in the N-glycan profile between FF from dominant and subordinate follicles from dairy heifers and lactating dairy cows and iii) to identify alterations in the N-glycan profile of FF during preovulatory follicle development using newly selected, differentiated (preovulatory) and luteinised dominant follicles from dairy heifers and lactating cows. We found that the majority of glycans on bovine FF are based on biantennary hypersialylated structures, where the glycans are sialylated on both the galactose and N-acetylglucosamine terminal sugars. A comparison of FF N-glycans from cows and heifers indicated higher levels of nonsialylated glycans with a lower proportion of sialylated glycans in cows than in heifers. Overall, as the follicle develops from Selection, Differentiation and Luteinisation in both cows and heifers, there is an overall decrease in sialylated structures on FF N-glycans.

Expression of microRNAs and their target genes and pathways associated with ovarian follicle development in cattle.

Development of ovarian follicles is controlled at the molecular level by several gene products whose precise expression leads to regression or ovulation of follicles. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through sequence-specific base pairing with target messenger RNAs (mRNAs) causing translation repression or mRNA degradation. The aim of this study was to identify miRNAs expressed in theca and/or granulosa layers and their putative target genes/pathways that are involved in bovine ovarian follicle development. By using miRCURY microarray (Exiqon) we identified 14 and 49 differentially expressed miRNAs (P < 0.01) between dominant and subordinate follicles in theca and granulosa cells, respectively. The expression levels of four selected miRNAs were confirmed by qRT-PCR. To identify target prediction and pathways of differentially expressed miRNAs we used Union of Genes option in DIANA miRPath v.2.0 software. The predicted targets for these miRNAs were enriched for pathways involving oocyte meiosis, Wnt, TGF-beta, ErbB, insulin, P13K-Akt, and MAPK signaling pathways. This study identified differentially expressed miRNAs in the theca and granulosa cells of dominant and subordinate follicles and implicates them in having important roles in regulating known molecular pathways that determine the fate of ovarian follicle development.

Physiological status alters immunological regulation of bovine follicle differentiation in dairy cattle.

Lactation in dairy cattle is associated with a multitude of endocrine, metabolic and immunological changes that not only influence animal health, but also affect fertility, and in particular ovarian function. We have previously generated a global transcriptomic profile of bovine follicular tissue using RNA sequencing. This study aimed to: identify key immune-related transcriptional changes that occur during follicle differentiation and luteinisation using ingenuity pathway analysis (IPA); and determine if a compromised model of stress (i.e. lactation) influences the temporal expression of these genes. Ovarian follicular tissue from Holstein-Friesian non-lactating heifers (n=17) and lactating cows (n=16) was compared at three stages of preovulatory follicle development: (A) the newly selected dominant follicle in the luteal phase (Selection); (B) the follicular phase before the LH surge (Differentiation), and (C) the preovulatory phase after the LH surge (Luteinisation). IPA revealed an over-representation of immune-related pathways in theca compared with granulosa cells during differentiation; these were related to leucocyte extravasation and chemotaxis. Conversely, luteinisation was characterised by over-representation of immune-related pathways in granulosa compared with theca cells; these were related to inflammation and innate immune response. Notably, comparison of follicles from heifers and lactating cows revealed a large number of differentially expressed genes associated with immune cell subpopulations and chemotaxis. In conclusion, identification of immune-related canonical pathways during follicle development supports the hypothesis that ovulation is an inflammatory event. This process is influenced by the physiological status of lactation and likely contributes to compromised peri-ovulatory follicle function by impairing the inflammatory process of ovulation.

Evidence for an early endometrial response to pregnancy in cattle: both dependent upon and independent of interferon tau.

The aims of this study were to 1) identify the earliest transcriptional response of the bovine endometrium to the presence of the conceptus (using RNAseq), 2) investigate if these genes are regulated by interferon tau (IFNT) in vivo, and 3) determine if they are predictive of the pregnancy status of postpartum dairy cows. RNAseq identified 459 differentially expressed genes (DEGs) between pregnant and cyclic endometria on day 16. Quantitative real-time PCR analysis of selected genes revealed PARP12, ZNFX1, HERC6, IFI16, RNF213, and DDX58 expression increased in pregnant compared with cyclic endometria on day 16 and were directly upregulated by intrauterine infusion of IFNT in vivo for 2 h (P < 0.05). On day 13 following estrous endometrial expression of nine genes increased [ARHGAP1, MGC127874, LIMS2, TBC1D1, FBXL7, C25H16orf71, LOC507810, ZSWIM4, and one novel gene (ENSBTAT00000050193)] and seven genes decreased (SERBP1, SRGAP2, AL7A1, TBK1, F2RL2, MGC128929, and WBSCR17; P < 0.05) in pregnant compared with cyclic heifers. Of these DEGs, significant differences in expression between pregnant and cyclic endometria were maintained on day 16 for F2RL2, LIMS2, LOC507810, MGC127874, TBC1D1, WBSCR17, and ZSWIM4 (P < 0.05) both their expression was not directly regulated by IFNT in vivo. Analysis of the expression of selected interferon-stimulated genes in blood samples from postpartum dairy cows revealed a significant increase (P < 0.05) in expression of ZXFX1, PARP12, SAMD9, and HERC6 on day 18 following artificial insemination in cows subsequently confirmed pregnant compared with cyclic controls. In conclusion, RNAseq identified a number of novel pregnancy-associated genes in the endometrium of cattle during early pregnancy that are not regulated by IFNT in vivo. In addition, a number of genes that are directly regulated by short term exposure to IFNT in vivo are differentially expressed on day 18 following estrus detection in the blood of postpartum dairy cows depending on their pregnancy status.

Effect of the metabolic environment at key stages of follicle development in cattle: focus on steroid biosynthesis.

Cellular mechanisms that contribute to low estradiol concentrations produced by the preovulatory ovarian follicle in cattle with a compromised metabolic status are largely unknown. To gain insight into the main metabolic mechanisms affecting preovulatory follicle function, two different animal models were used. Experiment 1 compared Holstein-Friesian nonlactating heifers (n = 17) and lactating cows (n = 16) at three stages of preovulatory follicle development: 1) newly selected dominant follicle in the luteal phase (Selection), 2) follicular phase before the LH surge (Differentiation), and 3) preovulatory phase after the LH surge (Luteinization). Experiment 2 compared newly selected dominant follicles in the luteal phase in beef heifers fed a diet of 1.2 times maintenance (M, n = 8) or 0.4 M (n = 11). Lactating cows and 0.4 M beef heifers had higher concentrations of ß-hydroxybutyrate, and lower concentrations of glucose, insulin, and IGF-I compared with dairy heifers and 1.2 M beef heifers, respectively. In lactating cows this altered metabolic environment was associated with reduced dominant follicle estradiol and progesterone synthesis during Differentiation and Luteinization, respectively, and in 0.4 M beef heifers with reduced dominant follicle estradiol synthesis. Using a combination of RNA sequencing, Ingenuity Pathway Analysis, and qRT-PCR validation, we identified several important molecular markers involved in steroid biosynthesis, such as the expression of steroidogenic acute regulatory protein (STAR) within developing dominant follicles, to be downregulated by the catabolic state. Based on this, we propose that the adverse metabolic environment caused by lactation or nutritional restriction decreases preovulatory follicle function mainly by affecting cholesterol transport into the mitochondria to initiate steroidogenesis.

Conceptus-induced changes in the endometrial transcriptome: how soon does the cow know she is pregnant?

This study sought to determine the earliest response of the bovine uterine endometrium to the presence of the conceptus at key developmental stages of early pregnancy. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7, and 13 postestrus, but the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). Of these 514 genes, MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD9, EIF4E, and IFIT2 increased to the greatest extent in pregnant endometria (>8-fold log2 fold change increase). The expression of OXTR, Bt.643 (unofficial symbol), and KCNMA1 was reduced the most, but short-term treatment with recombinant ovine interferon tau (IFNT) in vitro or in vivo did not alter their expression. In vivo intrauterine infusion of IFNT induced the expression of EIF4E, IFIT2, IFI44, ISG20, MX2, RSAD2, SAMD9, and USP18. These results revealed for the first time that changes that occur in the endometrial transcriptome are independent of the presence of a conceptus until pregnancy recognition. The differentially expressed genes (including MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD, and EIF4E) are a consequence of IFNT production by the conceptus. The identified genes represent known and novel early markers of conceptus development and/or return to cyclicity and may be useful to identify the earliest stage at which the endometrial response to the conceptus is detectable.

Clustal W and Clustal X version 2.0.

SUMMARY: The Clustal W and Clustal X multiple sequence alignment programs have been completely rewritten in C++. This will facilitate the further development of the alignment algorithms in the future and has allowed proper porting of the programs to the latest versions of Linux, Macintosh and Windows operating systems. AVAILABILITY: The programs can be run on-line from the EBI web server: http://www.ebi.ac.uk/tools/clustalw2. The source code and executables for Windows, Linux and Macintosh computers are available from the EBI ftp site ftp://ftp.ebi.ac.uk/pub/software/clustalw2/