RhoA/ROCK signaling antagonizes bovine trophoblast stem cell self-renewal and regulates preimplantation embryo size and differentiation.

Exponential proliferation of trophoblast stem cells (TSC) is crucial in Ruminantia to maximize numerical access to caruncles, the restricted uterine sites that permit implantation. When translating systems biology of the undifferentiated bovine trophectoderm, we uncovered that inhibition of RhoA/Rock promoted self-renewing proliferation and substantially increased blastocyst size. Analysis of transcripts suppressed by Rock inhibition revealed transforming growth factor ß1 (TGFß1) as a primary upstream effector. TGFß1 treatment induced changes consistent with differentiation in bTSCs, a response that could be replicated by induced expression of the bovine ROCK2 transgene. Rocki could partially antagonize TGFß1 effects, and TGFß receptor inhibition promoted proliferation identical to Rocki, indicating an all-encompassing upstream regulation. Morphological differentiation included formation of binucleate cells and infrequent multinucleate syncytia, features we also localize in the in vivo bovine placenta. Collectively, we demonstrate a central role for TGFß1, RhoA and Rock in inducing bTSC differentiation, attenuation of which is sufficient to sustain self-renewal and proliferation linked to blastocyst size and preimplantation development. Unraveling these mechanisms augments evolutionary/comparative physiology of the trophoblast cell lineage and placental development in eutherians.

Machine learning to identify endometrial biomarkers predictive of pregnancy success following artificial insemination in dairy cows†.

The objective was to identify a set of genes whose transcript abundance is predictive of a cow's ability to become pregnant following artificial insemination. Endometrial epithelial cells from the uterine body were collected for RNA sequencing using the cytobrush method from 193 first-service Holstein cows at estrus prior to artificial insemination (day 0). A group of 253 first-service cows not used for cytobrush collection were controls. There was no effect of cytobrush collection on pregnancy outcomes at day 30 or 70 or on pregnancy loss between days 30 and 70. There were 2 upregulated and 214 downregulated genes (false discovery rate < 0.05, absolute fold change >2-fold) for cows pregnant at day 30 versus those that were not pregnant. Functional terms overrepresented in the downregulated genes included those related to immune and inflammatory responses. Machine learning for fertility biomarkers with the R package BORUTA resulted in identification of 57 biomarkers that predicted pregnancy outcome at day 30 with an average accuracy of 77%. Thus, machine learning can identify predictive biomarkers of pregnancy in endometrium with high accuracy. Moreover, sampling of endometrial epithelium using the cytobrush can help understand functional characteristics of the endometrium at artificial insemination without compromising cow fertility. Functional characteristics of the genes comprising the set of biomarkers is indicative that a major determinant of cow fertility, at least for first insemination after calving, is immune status of the uterus, which, in turn, is likely to reflect the previous history of uterine disease.

Provision of choline chloride to the bovine preimplantation embryo alters postnatal body size and DNA methylation†.

Choline is a vital micronutrient. In this study, we aimed to confirm, and expand on previous findings, how choline impacts embryos from the first 7 days of development to affect postnatal phenotype. Bos indicus embryos were cultured in a choline-free medium (termed vehicle) or medium supplemented with 1.8 mM choline. Blastocyst-stage embryos were transferred into crossbred recipients. Once born, calves were evaluated at birth, 94 days, 178 days, and at weaning (average age = 239 days). Following weaning, all calves were enrolled into a feed efficiency trial before being separated by sex, with males being slaughtered at ~580 days of age. Results confirm that exposure of 1.8 mM choline chloride during the first 7 days of development alters postnatal characteristics of the resultant calves. Calves of both sexes from choline-treated embryos were consistently heavier through weaning and males had heavier testes at 3 months of age. There were sex-dependent alterations in DNA methylation in whole blood caused by choline treatment. After weaning, feed efficiency was affected by an interaction with sex, with choline calves being more efficient for females and less efficient for males. Calves from choline-treated embryos were heavier, or tended to be heavier, than calves from vehicle embryos at all observations after weaning. Carcass weight was heavier for choline calves and the cross-sectional area of the longissimus thoracis muscle was increased by choline.

Machine-learning methods applied to integrated transcriptomic data from bovine blastocysts and elongating conceptuses to identify genes predictive of embryonic competence.

Early pregnancy loss markedly impacts reproductive efficiency in cattle. The objectives were to model a biologically relevant gene signature predicting embryonic competence for survival after integrating transcriptomic data from blastocysts and elongating conceptuses with different developmental capacities and to validate the potential biomarkers with independent embryonic data sets through the application of machine-learning algorithms. First, two data sets from in vivo-produced blastocysts competent or not to sustain a pregnancy were integrated with a data set from long and short day-15 conceptuses. A statistical contrast determined differentially expressed genes (DEG) increasing in expression from a competent blastocyst to a long conceptus and vice versa; these were enriched for KEGG pathways related to glycolysis/gluconeogenesis and RNA processing, respectively. Next, the most discriminative DEG between blastocysts that resulted or did not in pregnancy were selected by linear discriminant analysis. These eight putative biomarker genes were validated by modeling their expression in competent or noncompetent blastocysts through Bayesian logistic regression or neural networks and predicting embryo developmental fate in four external data sets consisting of in vitro-produced blastocysts (i) competent or not, or (ii) exposed or not to detrimental conditions during culture, and elongated conceptuses (iii) of different length, or (iv) developed in the uteri of high- or subfertile heifers. Predictions for each data set were more than 85% accurate, suggesting that these genes play a key role in embryo development and pregnancy establishment. In conclusion, this study integrated transcriptomic data from seven independent experiments to identify a small set of genes capable of predicting embryonic competence for survival.

Transcriptomic profiling of the bovine endosalpinx and endometrium to identify putative embryokines.

The objectives of the present study were to characterize the expression of genes encoding for cell signaling ligands in the bovine endosalpinx and endometrium and analyze spatial changes in gene expression. RNA sequencing was performed for the endosalpinx from the ampulla of the oviduct and endometrium from the upper and middle uterine horn and uterine body at day 2 after ovulation from ipsilateral and contralateral sides relative to the ovulatory ovary. Of the 17,827 unique mRNA transcripts mapped, 2,072 were affected by cranial-caudal position in the reproductive tract and 818 were affected by side (false discovery rate < 0.05). There were 334 genes encoding for cell signaling ligands, with 128 genes having greater than two transcripts per million on average. A total of 81 cell signaling ligand genes were affected by position and 24 were affected by side. A data set of the transcriptome of two to four cell embryos was used to identify cell signaling ligand genes that were highly expressed in the ampulla for which there was high expression of the receptor in the embryo. The most expressed ligand-receptor pairs were PSAP/SORT1, MIF/CXCR4, GPI/AMFR, and KITLG/KIT. These cell signaling ligands, as well as others whose gene is expressed in the endosalpinx and endometrium, may influence early embryonic development. Spatial changes throughout the reproductive tract highlight the distinctive expression profile of the oviduct versus the endometrium, including a set of the identified genes encoding for cell signaling ligands, and highlight the local influence of the ovary. The results also show the continuity of expression for large numbers of genes in the reproductive tract.NEW & NOTEWORTHY Examination of the transcriptome of the endosalpinx and endometrium revealed the degree to which gene expression in the reproductive tract varies spatially. The expression of genes encoding cell signaling molecules that could potentially regulate embryonic development was also identified.

Absence of a molecular circadian clock in the preimplantation embryo is a conserved characteristic in the mammal.

In brief: It is not known when a functional circadian clock is established in the developing embryo. Lack of expression of key genes involved in the clock mechanism is indicative that a functional circadian clock mechanism is absent in the mammalian preimplantation embryo through the blastocyst stage of development. Abstract: An embryonic circadian clock could conceivably organize cellular and developmental events temporally and in synchrony with other circadian rhythms in the mother. The hypothesis that a functional molecular clock exists in the preimplantation bovine, pig, human, and mouse embryo was tested by using publicly available RNAseq datasets to examine developmental changes in expression of the core genes responsible for the circadian clock - CLOCK, ARNTL, PER1, PER2, CRY1, and CRY2. In general, the transcript abundance of each gene decreased as development advanced to the blastocyst stage. The most notable exception was for CRY2, where transcript abundance was low and constant from the two-cell or four-cell to the blastocyst stage. Developmental patterns were generally the same for all species although there were some species-specific patterns such as an absence of PER1 expression in the pig, an increase in ARNTL expression at the four-cell stage in human, and an increase in expression of Clock and Per1 from the zygote to two-cell stage in the mouse. Analysis of intronic reads (indicative of embryonic transcription) for bovine embryos indicated an absence of embryonic transcription. Immunoreactive CRY1 was not detected in the bovine blastocyst. Results indicate that the preimplantation mammalian embryo lacks a functional intrinsic clock although specific components of the clock mechanism could conceivably play a role in other functions in the embryo.

Colony stimulating factor 2 protects the preimplantation bovine embryo from heat shock.

Heat stress can have severe deleterious effects on embryo development and survival. The present study evaluated whether CSF2 can protect the developmental competence of the bovine embryo following exposure to a heat shock of 41°C at the zygote and morula stages. In the first experiment, putative zygotes and 2-cell embryos were assigned to receive either 10 ng/ml CSF2 or vehicle, and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Heat shock reduced blastocyst development for embryos treated with vehicle but not for embryos cultured with CSF2. In the second experiment, day 5 embryos (morula) were treated with CSF2 or vehicle and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Temperature treatment did not affect development to the blastocyst stage and there was no effect of CSF2 treatment or the interaction. Results indicate that CSF2 can reduce the deleterious effects of heat shock at the zygote or two-cell stage when the embryo is transcriptionally inactive.

Progesterone-dependent and progesterone-independent modulation of luminal epithelial transcription to support pregnancy in cattle.

In cattle, starting 4-5 days after estrus, preimplantation embryonic development occurs in the confinement of the uterine lumen. Cells in the endometrial epithelial layer control the molecular traffic to and from the lumen and, thereby determine luminal composition. Starting early postestrus, endometrial function is regulated by sex steroids, but the effects of progesterone on luminal cells transcription have not been measured in vivo. The first objective was to determine the extent to which progesterone controls transcription in luminal epithelial cells 4 days (D4) after estrus. The second objective was to discover luminal transcripts that predict pregnancy outcomes when the effect of progesterone is controlled. Endometrial luminal epithelial cells were collected from embryo transfer recipients on D4 using a cytological brush and their transcriptome was determined by RNASeq. Pregnancy by embryo transfer was measured on D30 (25 pregnant and 18 nonpregnant). Progesterone concentration on D4 was associated positively (n = 182) and negatively (n = 58) with gene expression. Progesterone-modulated transcription indicated an increase in oxidative phosphorylation, biosynthetic activity, and proliferation of epithelial cells. When these effects of progesterone were controlled, different genes affected positively (n = 22) and negatively (n = 292) odds of pregnancy. These set of genes indicated that a receptive uterine environment was characterized by the inhibition of phosphoinositide signaling and innate immune system responses. A panel of 25 genes predicted the pregnancy outcome with sensitivity and specificity ranging from 64%-96% and 44%-83%, respectively. In conclusion, in the early diestrus, both progesterone-dependent and progesterone-independent mechanisms regulate luminal epithelial transcription associated with pregnancy outcomes in cattle.

Actions of WNT family member 5A to regulate characteristics of development of the bovine preimplantation embryo†.

WNT signaling is important for regulation of embryonic development. The most abundant WNT gene expressed in the bovine endometrium during the preimplantation period is WNT5A. One objective was to determine whether WNT5A regulates competence of the bovine preimplantation embryo to become a blastocyst and alters the number of cells in the inner cell mass and trophectoderm. A second objective was to delineate features of the cell-signaling mechanisms involved in WNT5A actions. WNT5A caused a concentration-dependent increase in the proportion of embryos developing to the blastocyst stage and in the number of inner cell mass cells in the resultant blastocysts. A concentration of 200 ng/mL was most effective, and a higher concentration of 400 ng/mL was not stimulatory. Bovine serum albumin in culture reduced the magnitude of effects of WNT5A on development to the blastocyst stage. WNT5A affected expression of 173 genes at the morula stage; all were upregulated by WNT5A. Many of the upregulated genes were associated with cell signaling. Actions of WNT5A on development to the blastocyst stage were suppressed by a Rho-associated coiled-coil kinase (ROCK) signaling inhibitor, suggesting that WNT5A acts through Ras homology gene family member A (RhoA)/ROCK signaling. Other experiments indicated that actions of WNT5A are independent of the canonical ß-catenin signaling pathway and RAC1/c-Jun N-terminal kinase (JNK) signaling. This is the first report outlining the actions of WNT5A to alter the development of the mammalian embryo. These findings provide insights into how embryokines regulate maternal-embryonic communication.

Actions of DKK1 on the preimplantation bovine embryo to affect pregnancy establishment, placental function, and postnatal phenotype†.

One mechanism by which the maternal environment regulates the early embryo is by secretion of cell-signaling molecules. One of these is dickkopf WNT signaling pathway inhibitor 1. Objectives were to (A) resolve discrepancies in the literature regarding effects of dickkopf WNT signaling pathway inhibitor 1 in the bovine embryo on development of trophectoderm and competence to establish pregnancy after embryo transfer and (B) determine whether there are long-term consequences of dickkopf WNT signaling pathway inhibitor 1 on placental function and postnatal phenotype. Embryos produced in vitro were cultured with vehicle or 100 ng/mL recombinant human dickkopf WNT signaling pathway inhibitor 1 from Days 5 to 7.5 of development (i.e., the morula and blastocyst stages of development). dickkopf WNT signaling pathway inhibitor 1 increased the number of cells positive for the trophectoderm marker CDX2 at Day 7.5 of development while having no effect on number of cells positive for the inner cell mass marker SOX2. There was no effect of dickkopf WNT signaling pathway inhibitor 1 on pregnancy or calving rate after transfer of blastocysts produced with Y-sorted semen to either lactating dairy cows or suckling beef cows. Treatment with dickkopf WNT signaling pathway inhibitor 1 at the morula-to-blastocyst stages programmed placental function, as measured by an effect of dickkopf WNT signaling pathway inhibitor 1 on plasma concentrations of pregnancy associated glycoproteins and placental lactogen at Day 160 of gestation (although not on other days examined). dickkopf WNT signaling pathway inhibitor 1 treatment also resulted in calves that were heavier at birth as compared to calves derived from control embryos. After birth, dickkopf WNT signaling pathway inhibitor 1 calves grew slower than controls. Results confirm that dickkopf WNT signaling pathway inhibitor 1 alters the developmental program of the bovine embryo to affect both prenatal and postnatal phenotypes.

Future of biomedical, agricultural, and biological systems research using domesticated animals.

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research.

Choline acts during preimplantation development of the bovine embryo to program postnatal growth and alter muscle DNA methylation.

The preimplantation period of embryonic development can be a key window for programming of postnatal development because extensive epigenetic remodeling occurs during this time. It was hypothesized that modification of one-carbon metabolism of the bovine embryo by addition of the methyl-donor choline to culture medium would change postnatal phenotype through epigenetic modification. Embryos produced in vitro were cultured with 1.8 mM choline chloride or control medium. Blastocysts were transferred into females and pregnancy outcomes and postnatal phenotype of the resultant calves determined. Exposure of embryos to choline increased gestation length and calf birth weight. Calves derived from choline-treated embryos were also heavier at weaning and had increased ratio of body weight to hip height than control calves. Choline altered muscle DNA methylation of calves 4 months after birth. A total of 670 of the 8149 CpG examined were differentially methylated, with the predominant effect of choline being hypomethylation. Among the genes associated with differentially methylated CpG were ribosomal RNAs and genes in AMPK, mTOR, integrin, and BEX2 canonical pathways and cellular functions involved in growth and proliferation. Results demonstrate that provision of the methyl-donor choline to the preimplantation embryo can alter its developmental program to increase gestation length, birth weight, and weaning weight and cause postnatal changes in muscle DNA methylation including those associated with genes related to anabolic processes and cellular growth. The importance of the nutritional status of the embryo with respect to one-carbon metabolism for ensuring health and well-being after birth is emphasized by these observations.

Physiological responses of Holstein calves and heifers carrying the SLICK1 allele to heat stress in California and Florida dairy farms.

Inheritance of the SLICK1 allele of the prolactin receptor gene improves thermotolerance of lactating Holstein cows under humid heat stress conditions. The aim of this study was to investigate whether pre- and postweaning Holstein heifers carrying the SLICK1 allele would show physiological responses indicative of higher tolerance to heat stress in high- and low-humidity climates. A total of 101 heifer calves of two age groups heterozygous for the SLICK1 allele and 103 wild-type half-siblings were evaluated during July 2020 in 3 dairy farms in central California and 2 in south Florida. Dry bulb temperature and relative humidity data were recorded during evaluation and used to calculate the temperature-humidity index (THI). Physiological measurements were obtained between 1600 and 1900 h in California, and 1200 and 1400 h in Florida and included rectal temperature, respiration rate, skin temperature, and sweating rate. Data were analyzed via Generalized Linear Mixed Models including the main effects of genotype, state, group, sire, farm within state, and interactions, with THI included as a covariate. The correlations between THI and dependent variables were analyzed via linear regression. The average 24-h THI was higher in Florida compared with California (90 vs. 72, respectively); the main driver of the higher THI in Florida was the high relative humidity (average 85.6% in Florida vs. 36.7% in California). In Florida, the rectal temperature of slick calves was 0.4°C lower than non-slick calves (39.5 ± 0.1 vs 39.9 ± 0.1°C); no differences were detected between slick and non-slick calves in California. Regardless of genotype, heifer calves in Florida had higher respiration rate, higher rectal and skin temperatures, and lower sweating rate than in California. This study is the first to evaluate physiological responses of calves carrying the SLICK1 allele under heat stress conditions in different climates. Our findings demonstrate that the presence of this allele is associated with lower rectal temperatures in pre- and post-weaning Holstein females. According to the physiological parameters evaluated, calves raised in Florida appeared to be under more severe heat stress; in those conditions, the SLICK1 allele was advantageous to confer thermotolerance as evidenced by lower rectal temperature in slick animals.

Effect of addition of ascorbate, dithiothreitol or a caspase-3 inhibitor to cryopreservation medium on post-thaw survival of bovine embryos produced in vitro.

Experiments were conducted to investigate whether supplementation of cryopreservation medium with ascorbate, dithiothreitol (DTT) or an inhibitor of caspase-3 (z-DEVD-fmk) could improve post-thaw survival of bovine embryos produced in vitro (IVP). For all experiments, embryos were harvested on day 7 after insemination and subjected to controlled-rate freezing in medium containing 1.5 M ethylene glycol and treatments as described below. In experiments 1-3, embryos were cryopreserved in freezing medium with ascorbate (0, 0.1, 0.3 or 0.5 mM), DTT (0, 50, 100 or 200 µM) and z-DEVD-fmk (0, 50, 100 or 200 µM), respectively. Post-thaw survival was assessed at 24, 48 and 72 h. For experiments 4-5, embryos were cryopreserved in freezing medium with or without 0.1 mM ascorbate. At 24 h post-thaw, embryo total cell number, DNA fragmentation and levels of reactive oxygen species (ROS) were evaluated. Embryos subjected to freezing and thawing in medium supplemented with 0.1 mM ascorbate had greater (p < .05) re-expansion rates at 24, 48 and 72 h and hatching rate at 72 h as compared to embryos not treated with ascorbate. Post-thaw cryosurvival was not affected by the addition of either DTT or z-DEVD-fmk to medium used for cryopreservation. Embryos cryopreserved in medium supplemented with 0.1 mM ascorbate had reduced (p < .001) levels of intracellular ROS and fewer (p < .001) cells with DNA fragmentation. In conclusion, post-thaw survival of bovine IVP embryos is enhanced by supplementation of freezing medium with ascorbate.

Regulation of gene expression in the bovine blastocyst by colony-stimulating factor 2 is disrupted by CRISPR/Cas9-mediated deletion of CSF2RA.

Colony-stimulating factor 2 (CSF2) functions in the reproductive tract to modulate the function of the preimplantation embryo. The ß subunit of the CSF2 receptor (CSF2RB) is not expressed in the embryo, and signal transduction is therefore different than for myeloid cells where the receptor is composed of α (CSF2RA) and ß subunits. Here, we produced embryos in which exons 5 and 6 of CSF2RA were disrupted using the CRISPR/Cas 9 system to test whether CSF2RA signaling was essential for actions of CSF2 in the bovine embryo. Wild-type and CSF2RA knockout embryos were treated with 10 ng/mL CSF2 or vehicle at day 5 of development. Blastocysts were harvested at day 8 to determine transcript abundance of 90 genes by real-time polymerase chain reaction (PCR). Responses in female blastocysts were examined separately from male blastocysts because actions of CSF2 are sex-dependent. For wild-type embryos, CSF2 altered expression of 10 genes in females and 20 in males. Only three genes were affected by CSF2 in a similar manner for both sexes. Disruption of CSF2RA prevented the effect of CSF2 on expression for 9 of 10 CSF2-regulated genes in females and 19 of 20 genes in males. The results confirm the importance of CSF2RA for regulation of gene expression by CSF2 in the blastocyst.

Importance of WNT-dependent signaling for derivation and maintenance of primed pluripotent bovine embryonic stem cells†.

The WNT signaling system plays an important but paradoxical role in the regulation of pluripotency. In the cow, IWR-1, which inhibits canonical WNT activation and has WNT-independent actions, promotes the derivation of primed pluripotent embryonic stem cells from the blastocyst. Here, we describe a series of experiments to determine whether derivation of embryonic stem cells could be generated by replacing IWR-1 with other inhibitors of WNT signaling. Results confirm the importance of inhibition of canonical WNT signaling for the establishment of pluripotent embryonic stem cells in cattle and indicate that the actions of IWR-1 can be mimicked by the WNT secretion inhibitor IWP2 but not by the tankyrase inhibitor XAV939 or WNT inhibitory protein dickkopf 1. The role of Janus kinase-mediated signaling pathways for the maintenance of pluripotency of embryonic stem cells was also evaluated. Maintenance of pluripotency of embryonic stem cells lines was blocked by a broad inhibitor of Janus kinase, even though the cells did not express phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Further studies with blastocysts indicated that IWR-1 blocks the activation of pSTAT3. A likely explanation is that IWR-1 blocks differentiation of embryonic stem cells into a pSTAT3+ lineage. In conclusion, results presented here indicate the importance of inhibition of WNT signaling for the derivation of pluripotent bovine embryonic stem cells, the role of Janus kinase signaling for maintenance of pluripotency, and the participation of IWR-1 in the inhibition of activation of STAT3.

Atlas of receptor genes expressed by the bovine morula and corresponding ligand-related genes expressed by uterine endometrium.

Regulation of the mammalian embryo involves cell-signaling molecules produced by the maternal oviduct and endometrium. Here, datasets on the transcriptome of the gestational Days 5 and 6 bovine morula and Day 5 maternal endometrium were examined to identify receptor genes expressed by the morula and expression of the corresponding ligand-related genes in the endometrium. A total of 175 receptor genes were identified in the morula, including 48 encoding for growth factors or WNT signaling molecules, 25 for cytokines and chemokines, 35 involved in juxtacrine and matricellular signaling and 25 encoding for receptors for small molecules. Some of the highly-expressed pairs of endometrial ligand and embryo receptor genes included MDK and its receptors ITGB1, SDC4 and LRP2, WNT5A (RYK), VEGFA (ITGB1), GPI (AMFR), and the hedgehog proteins IHH and DHH (HHIP). The most highly expressed receptors for small molecules were GPRC5C (retinoic acid receptor), PGRMC1 (progesterone), and CHRNB2 (acetylcholine). There were also 84 genes encoding for cell signaling ligands expressed by the morula, with the most highly expressed being GPI, AIMP1, TIMP1, IK, and CCN2. The atlas of receptor and ligand genes should prove useful for understanding details of the communication between the embryo and mother that underlies optimal embryonic development.

Uterine influences on conceptus development in fertility-classified animals.

A major unresolved issue is how the uterus influences infertility and subfertility in cattle. Serial embryo transfer was previously used to classify heifers as high-fertile (HF), subfertile (SF), or infertile (IF). To assess pregnancy loss, two in vivo-produced embryos were transferred into HF, SF, and IF heifers on day 7, and pregnancy outcome was assessed on day 17. Pregnancy rate was substantially higher in HF (71%) and SF (90%) than IF (20%) heifers. Elongating conceptuses were about twofold longer in HF than SF heifers. Transcriptional profiling detected relatively few differences in the endometrium of nonpregnant HF, SF, and IF heifers. In contrast, there was a substantial difference in the transcriptome response of the endometrium to pregnancy between HF and SF heifers. Considerable deficiencies in pregnancy-dependent biological pathways associated with extracellular matrix structure and organization as well as cell adhesion were found in the endometrium of SF animals. Distinct gene expression differences were also observed in conceptuses from HF and SF animals, with many of the genes decreased in SF conceptuses known to be embryonic lethal in mice due to defects in embryo and/or placental development. Analyses of biological pathways, key players, and ligand-receptor interactions based on transcriptome data divulged substantial evidence for dysregulation of conceptus-endometrial interactions in SF animals. These results support the ideas that the uterus impacts conceptus survival and programs conceptus development, and ripple effects of dysregulated conceptus-endometrial interactions elicit loss of the postelongation conceptus in SF cattle during the implantation period of pregnancy.

An improved method for specific-target preamplification PCR analysis of single blastocysts useful for embryo sexing and high-throughput gene expression analysis.

Gene expression analysis in preimplantation embryos has been used for answering fundamental questions related to development, prediction of pregnancy outcome, and other topics. Limited amounts of mRNA in preimplantation embryos hinders progress in studying the preimplantation embryo. Here, a method was developed involving direct synthesis and specific-target preamplification (STA) of cDNA for gene expression analysis in single blastocysts. Effective cell lysis and genomic DNA removal steps were incorporated into the method. In addition, conditions for real-time PCR of cDNA generated from these processes were improved. By using this system, reliable embryo sexing results based on expression of sex-chromosome linked genes was demonstrated. Calibration curve analysis of PCR results using the Fluidigm Biomark microfluidic platform (Fluidigm, South San Francisco, CA) was performed to evaluate 96 STA cDNA from single blastocysts. In total, 93.75% of the genes were validated. Robust amplification was detected even when STA cDNA from a single blastocyst was diluted 1,024-fold. Further analysis showed that within-assay variation increased when cycle threshold values exceeded 18. Overall, STA quantitative real-time PCR analysis was shown to be useful for analysis of gene expression of multiple specific targets in single blastocysts.

Effectiveness of tunnel ventilation as dairy cow housing in hot climates: rectal temperatures during heat stress and seasonal variation in milk yield.

Tunnel ventilation is an increasingly popular approach to mitigate the effects of heat stress on dairy cattle. Tunnel-ventilation barns use a bank of high-power fans to move air horizontally from one end of the barn to the other at cow level. The overall objective of the present experiments was to determine whether tunnel ventilation is superior to housing with fans and sprinklers with respect to rectal temperature during heat stress and seasonal variation in milk yield. In the first study, rectal temperatures were measured for 1097 lactating Holstein cows in six freestall barns with fans and sprinklers and 575 lactating Holsteins in four tunnel-ventilated freestall barns at a time point between 14:00 and 16:00 h during the months of June to August in Florida, USA. Rectal temperatures were lower for cows in tunnel-ventilation barns than sprinkler-and-fan barns when the tunnel-ventilation barns were built de novo but not when the tunnel-ventilation barns were produced by retrofitting a sprinkler-and-fan barn (interaction, P = 0.0129). In the second study, average daily milk yield in the first 90 days in milk was examined for 8470 lactating Holsteins housed in three sprinkler-and-fan barns and two tunnel-ventilation barns. Milk production for cows calving in cool weather (October to March) was greater (P < 0.0001) than for cows calving in hot weather (April to September). The seasonal reduction in milk yield was less for cows (P = 0.037) in tunnel-ventilation barns (3.5% decrease) than for cows in sprinkler-and-fan barns (5.8% decrease). With this difference in impact of heat stress, it was estimated at a dairy farm could invest up to a $332 more per cow space in a tunnel-ventilated barn than in a sprinkler-and-fan barn. It was concluded that housing cows in tunnel-ventilation barns can reduce the impact of heat stress on body temperature regulation and milk yield.