Genomic imprinting is an epigenetic mechanism that leads to parental-allele-specific gene expression. Approximately 150 imprinted genes have been identified in humans and mice but less than 30 have been described as imprinted in cattle. For the purpose of de novo identification of imprinted genes in bovine, we determined global monoallelic gene expression in brain, skeletal muscle, liver, kidney and placenta of day ∼105 Bos taurus indicus × Bos taurus taurus F1 conceptuses using RNA sequencing. To accomplish this, we developed a bioinformatics pipeline to identify parent-specific single nucleotide polymorphism alleles after filtering adenosine to inosine (A-to-I) RNA editing sites. We identified 53 genes subject to monoallelic expression. Twenty three are genes known to be imprinted in the cow and an additional 7 have previously been characterized as imprinted in human and/or mouse that have not been reported as imprinted in cattle. Of the remaining 23 genes, we found that 10 are uncharacterized or unannotated transcripts located in known imprinted clusters, whereas the other 13 genes are distributed throughout the bovine genome and are not close to any known imprinted clusters. To exclude potential cis-eQTL effects on allele expression, we corroborated the parental specificity of monoallelic expression in day 86 Bos taurus taurus × Bos taurus taurus conceptuses and identified 8 novel bovine imprinted genes. Further, we identified 671 candidate A-to-I RNA editing sites and describe random X-inactivation in day 15 bovine extraembryonic membranes. Our results expand the imprinted gene list in bovine and demonstrate that monoallelic gene expression can be the result of cis-eQTL effects.

The objective was to determine the accuracy of a pregnancy test for predicting nonpregnant cattle based on the evaluation of corpus luteum (CL) blood flow at 20 d (CLBF-d20) after timed artificial insemination (TAI). Crossbred Holstein-Gir dairy heifers (n=209) and lactating cows (n=317) were synchronized for TAI using the following protocol: intravaginal implant (1.0 g of progesterone) and 2mg of estradiol benzoate i.m. on d -10, implant removal and 0.526 mg of sodium cloprostenol i.m. on d -2, 1mg of estradiol benzoate i.m. on d -1, and TAI on d 0. On d 20, animals underwent grayscale ultrasonography (US) to locate the CL and color flow Doppler to evaluate CLBF-d20 using a portable ultrasound equipped with a 7.5-MHz rectal transducer. Based only on a visual, subjective CLBF evaluation, the animals were classified as pregnant or not pregnant. On d 30 to 35, blinded from results of the previous diagnosis, the same operator performed a final pregnancy diagnosis using US to visualize the fetal heartbeat (gold standard; US-d30). A second evaluator also analyzed the CLBF-d20 in the same animals by watching 7-s recorded videos. Blood samples were collected from a subset of 171 females to determine, by RIA, plasma progesterone (P4) concentrations, which indicate CL function. The final pregnancy outcome (US-d30) was retrospectively compared with the CLBF-d20 diagnoses and then classified either as correct or incorrect. The number of true positive, true negative, false positive, and false negative decisions were inserted into a 2 × 2 decision matrix. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the CLBF-d20 test were calculated using specific equations. Binomial variables (pregnancy rate and proportions) were analyzed using Fisher's exact test for the effect of parity and to compare between evaluators and tests (CLBF-d20 vs. plasma P₄). The kappa values were calculated to quantify the agreement between CLBF-d20 and the gold standard (US-d30) and between evaluators. The performance parameters of CLBF-d20 test were as follows: sensitivity=99.0%, specificity=53.7%, positive predictive value=65.1%, negative predictive value=98.5%, and accuracy=74.8%. False negatives represented only 0.4% of the exams. No differences existed in these parameters between evaluators (no. 1 vs. no. 2) and tests (CLBF-d20 vs. plasma P4). Moreover, a high level of agreement was observed between evaluators (0.91). In conclusion, visual evaluation of CLBF-d20 represents a quick, reliable, and consistent diagnostic test that enables the early detection of nonpregnant cattle.

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Abnormal fetuses, neonates, and adult offspring derived by assisted reproductive technologies have been reported in humans and mice and have been associated with increased likelihood of certain adult diseases. To test the hypothesis that bovine females derived by assisted reproductive technologies have altered postnatal growth and adult function, a retrospective cohort study evaluated survival, growth, and production traits of offspring derived by in vitro embryo production (IVP) with conventional (IVP-conv) or reverse X-sorted semen (IVP-sexed), multiple ovulation and embryo transfer, and artificial insemination (AI) in a large dairy herd. Live calves produced by IVP were born slightly heavier compared with AI calves. In addition, IVP-sexed calves had a higher cumulative mortality from 90 to 180 d of age compared with AI offspring. Mortality of IVP-conv and multiple ovulation and embryo transfer offspring was intermediate and not different from AI or IVP-sexed offspring. The altered phenotype of offspring from IVP-sexed extended to adult milk production. Cows derived by IVP-sexed produced less milk, fat, and protein in their first lactation compared with dairy cows derived by AI. Additionally, females born to nulliparous dams had a distinct postnatal phenotype compared with offspring from parous dams even when data were restricted to offspring of surrogate females. In conclusion, procedures associated with in vitro production of embryos involving use of reverse-sorted spermatozoa for fertilization result in an alteration of embryonic programming that persists postnatally and causes an effect on milk production in adulthood. Thus, some benefits of reverse-sorted semen for genetic improvement may be offset by adverse programming events.

Knowledge of the molecules used by the maternal reproductive tract to regulate development of the preimplantation embryo is largely incomplete. The goal of the present experiment was to identify candidates for this function. The approach was to assess expression patterns in the endometrium and oviduct of 93 genes encoding for hormones, growth factors, chemokines, cytokines, and WNT-related molecules. Results show that all of the genes were expressed in the reproductive tract. Expression in oviduct was affected by day of the estrous cycle for 21 genes with 11 genes having highest expression at estrus (CCL21, CTGF, CXCL10, CXCL16, DKK3, FGF10, IL18, IL33, IL34, PGF, and SFRP2), 1 gene at d 3 (WNT4), 8 at d 5 (BMP7, HGF, IL6, SFRP1, TGFB1, WIF1, WNT2, and WNT5A), and 1 at d 7 (IK). For endometrium, expression of 34 genes was affected by day of the estrous cycle with 11 having highest expression at d 0 (BMP7, CCL14, CCL21, CCL26, CTGF, CXCL12, IGF2, IL16, IL33, SFRP2, and WIF1), 2 at d 3 (HDGF, IL15), 14 at d 5 (CSF2, CX3CL1, CXCL3, FGF1, FGF2, GRO1, HGF, IGF1, IL1B, IL8, SFRP1, SFRP4, WNT5A, and WNT16), and 7 at d 7 (CXCL16, FGF13, HDGFRP2, TDGF1, VEGFB, WNT7A, and WNT11). Results are consistent with a set of genes regulated by estradiol early in the estrous cycle and another set regulated by progesterone later in the cycle. The cell-signaling genes identified here as being expressed in the oviduct and endometrium could serve to regulate early embryonic development in a stage-of-pregnancy-specific manner.

We tested whether gene expression of the bovine morula is modified by CSF2 in a sex-dependent manner and if sex determines the effect of CSF2 on competence of embryos to become blastocysts. Embryos were produced in vitro using X- or Y-sorted semen and treated at Day 5 of culture with 10 ng/mL bovine CSF2 or control. In experiment 1, morulae were collected at Day 6 and biological replicates (n = 8) were evaluated for transcript abundance of 90 genes by RT-qPCR using the Fluidigm Delta Gene assay. Expression of more than one-third (33 of 90) of genes examined was affected by sex. The effect of CSF2 on gene expression was modified by sex (P < 0.05) for five genes (DDX3Y/DDX3X-like, NANOG, MYF6, POU5F1 and RIPK3) and tended (P < 0.10) to be modified by sex for five other genes (DAPK1, HOXA5, PPP2R3A, PTEN and TNFSF8). In experiment 2, embryos were treated at Day 5 with control or CSF2 and blastocysts were collected at Day 7 for immunolabeling to determine the number of inner cell mass (ICM) and trophectoderm (TE) cells. CSF2 increased the percent of putative zygotes that became blastocysts for females, but did not affect the development of males. There was no effect of CSF2 or interaction of CSF2 with sex on the total number of blastomeres in blastocysts or in the number of inner cell mass or trophectoderm cells. In conclusion, CSF2 exerted divergent responses on gene expression and development of female and male embryos. These results are evidence of sexually dimorphic responses of the preimplantation embryo to this embryokine.

The developmental program of the embryo displays a plasticity that can result in long-acting effects that extend into post-natal life. In mammals, adult phenotype can be altered by changes in the maternal environment during the preimplantation period. One characteristic of developmental programming during this time is that the change in adult phenotype is often different for female offspring than for male offspring. In this paper, we propose the hypothesis that sexual dimorphism in preimplantation programming is mediated, at least in part, by sex-specific responses of embryos to maternal regulatory molecules whose secretion is dependent on maternal environment. The strongest evidence for this idea comes from the study of colony-stimulating factor 2 (CSF2). Expression of CSF2 from the oviduct and endometrium is modified by environmental factors of the mother, in particular seminal plasma and obesity. Additionally, CSF2 alters several properties of the preimplantation embryo and has been shown to alleviate negative consequences of culture of mouse embryos on postnatal phenotype in a sex-dependent manner. In cattle, exposure of preimplantation bovine embryos to CSF2 causes sex-specific changes in gene expression, interferon-τ secretion, and DNA methylation later in pregnancy (day 15 of gestation). It is likely that several embryokines can alter postnatal phenotype through actions directed towards the preimplantation embryo. Identification of these molecules and elucidation of the mechanisms by which sexually-disparate programming is established will lead to new insights into the control and manipulation of embryonic development.

A single missense mutation at position 159 of coenzyme Q9 (COQ9) (G→A; rs109301586) has been associated with genetic variation in fertility in Holstein cattle, with the A allele associated with higher fertility. COQ9 is involved in the synthesis of coenzyme COQ10, a component of the electron transport system of the mitochondria. Here we tested whether reproductive phenotype is associated with the mutation and evaluated functional consequences for cellular oxygen metabolism, body weight changes, and ovarian function. The mutation in COQ9 modifies predicted tertiary protein structure and affected mitochondrial respiration of peripheral blood mononuclear cells. The A allele was associated with low resting oxygen consumption and high electron transport system capacity. Phenotypic measurements for fertility were evaluated for up to five lactations in a population of 2273 Holstein cows. There were additive effects of the mutation (P < 0.05) in favor of the A allele for pregnancy rate, interval from calving to conception, and services per conception. There was no association of genotype with milk production or body weight changes postpartum. The mutation in COQ9 affected ovarian function; the A allele was associated with increased mitochondrial DNA copy number in oocytes, and there were overdominance effects for COQ9 expression in oocytes, follicle number, and antimullerian hormone concentrations. Overall, results show how a gene involved in mitochondrial function is associated with overall fertility, possibly in part by affecting oocyte quality.