Alterations during the early stages of embryo development have been associated with long-term effects on the fetus, neonate, and adult, but this has not been investigated in horses. In recent years, intracytoplasmic sperm injection (ICSI) has gained in commercial popularity in the equine population. Research suggests an association between ICSI-produced embryos and placental malformations, but there exists little understanding of the physiology involved. Therefore, we aim to produce a complete transcriptomic analysis of chorioallantois and provide potential pathways that may be impacted following pregnancies associated with in vitro-produced equine embryos. To do so, seventeen warmblood mares were bred either naturally to produce in vivo-produced pregnancies that were carried by self (in vivo; n = 8) or with in vitro-produced pregnancies created via intracytoplasmic sperm injection (ICSI) that were transferred to a recipient (in vitro; n = 9). Mares were monitored throughout gestation to ensure the health of the pregnancy, and impending parturition was monitored for progress. Chorioallantois was collected immediately postpartum and placed in RNALater for future extraction. RNA was isolated using Trizol, and RNASeq was performed by Novogene, with 93.3 % total mapping and 40 million read depth. The false discovery rate (FDR) was set to <0.05. When comparing groups (in vivo vs. in vitro-produced embryos), 1589 genes were differentially expressed. This included an upregulation of 626 genes, alongside a downregulation of 963 genes. Impacted gene ontology included aspects of the central dogma of molecular biology, including ribosome biogenesis, RNA polymerase activity, and spliceosome function. Additional biological processes that were impacted included aspects of the immune system relating to auto-immunity and disordered antigen response, such as the IL-17 signaling pathway, rheumatoid arthritis, and lupus. Additionally, pathways relating to hypoxia and ribosome biogenesis were associated with in vitro-produced pregnancies. Overall, it appears that the in vitro production of pregnancies is associated with placental dysregulation during pregnancy, which may be related to poor fetal and neonatal outcomes that have been associated with ART in other species.

Transvaginal oocyte pickup (OPU) of the mare has become an increasingly common clinical procedure applicable to both academic and private breeding facilities around the world. This chapter discusses the processes involved in OPU. In order to identify a pre-ovulatory follicle for OPU, the mare's cycle must be monitored via transrectal ultrasonography and ovulation induction medications administered when she has a large follicle and uterine edema present. OPU sessions typically are performed at approximately 14-day intervals. The immature status of the oocytes collected results in less sensitivity to temperature alterations and the oocytes can be transported at room temperature in commercially available devices. If a bacterial endometritis is diagnosed, a vaginal lavage with 1 liter of saline is recommended prior to and after the OPU session; in addition antibiotics (such as a long-acting ceftiofur) should be administered.

Prostaglandins have important physiological roles in marine invertebrates, including larval development and reproduction. The prostaglandin E concentration fluctuates during the ovarian development of crustaceans. The biosynthetic pathway of prostaglandin, however, has not been well studied in portunid crabs, including in the mud crab, Scylla olivacea. In this study, the aim was to investigate the presence of prostaglandin E synthase (PGES), enzyme that catalyzes the terminal conversion in the prostaglandin E2 (PGE2) biosynthesis, and its gene expression in the central nervous system (CNS) and ovary during ovarian maturation of S. olivacea. cDNA sequence encoding PGES was cloned from the S. olivacea ovary. The PGES transcript of S. olivacea (Scyol-PGES) consists of 1258 nucleotides, which encodes for 420 amino acid PGES protein precursor. Investigation of gene expression by RT-PCR indicated that Scyol-PGES was detected in all organs studied. Based on in situ hybridization, Scyol-PGES was detected in the I to III stages for oocyte development of Stage 3 of ovarian development, and in the CNS, including the various neuronal clusters of the brain. In the ventral nerve cord, the Scyol-PGES gene was expressed in the neurons within the subesophageal, thoracic and abdominal ganglia. The Scyol-PGES gene expression as indicated by relative abundance of mRNA in the Stage 4 of ovarian development was greater than that at Stages 1 to 3 of ovarian development. This is the first report on PGES in the mud crab, S. olivacea, and its gene expression suggested the involvement of PGES in the ovarian development of this species.

Cryopreservation impairs oocyte quality, which may be associated with abnormal gene expression. Currently, alteration of mRNA levels in vitrified porcine oocytes has not been well characterized. The aim of this study was to analyze transcriptome profiles with RNA sequencing (RNA-seq) in porcine immature oocytes and their surrounding cumulus cells (CCs) after vitrification and in vitro maturation (IVM). There were 19 upregulated and 18 downregulated genes differentially expressed in vitrified oocytes, with no significant GO enrichment or KEGG pathway identified for these genes. In addition, CCs derived from vitrified oocytes had 40 significantly upregulated and 100 significantly downregulated genes. In total, 7 GO terms were significantly enriched in molecular function and biological process, and only MAPK signaling pathway reached significant enrichment based on KEGG analysis. Moreover, selected differentially expressed genes had similar expression patterns through comparison between results from qRT-PCR and RNA-Seq. In conclusion, our data provided detailed information on mRNA transcriptomes in porcine immature oocytes and CCs after vitrification and IVM, which offered now insights regarding reduced developmental potential of the vitrified oocytes.

Eleven pregnant pony mares (D270-326) were administered ceftiofur sodium intramuscularly at 2.2 mg/kg (n = 6) or 4.4 mg/kg (n = 5), once daily. Plasma was obtained prior to ceftiofur administration and at 0.5, 1, 2, 4, 8, 12, and 24 hr after administration. Eight pony mares were re-enrolled in the study at least 3 days from expected foaling to ensure steady-state concentrations of drug at the time of foaling. Mares were administered ceftiofur sodium (4.4 mg/kg, IM) daily until foaling. Parturition was induced using oxytocin 1 hr after ceftiofur sodium administration. Allantoic and amniotic fluid, plasma, and colostrum samples were collected at time of foaling. Serial foal plasma samples were obtained. Placental tissues were collected. Desfuroylceftiofur acetamide (DCA) concentrations were measured in samples by high-performance liquid chromatography (HPLC). Mean (±SD) peak serum concentrations of DCA were 3.97 ± 0.50 μg/ml (low dose) and 7.45 ± 1.05 μg/ml (high dose). Terminal half-life was significantly (p = .014) shorter after administration of the low dose (2.91 ± 0.59 hr) than after administration of the high dose (4.10 ± 0.72 hr). The mean serum concentration of DCA from mares at time of foaling was 7.96 ± 1.39 μg/ml. The mean DCA concentration in colostrum was 1.39 ± 0.70 μg/ml. DCA concentrations in allantoic fluid, amniotic fluid, placental tissues, and foal plasma were below the limit of quantification (<0.1 μg/ml) and below the minimum inhibitory concentration of ceftiofur against relevant pathogens. These results infer incomplete passage of DCA across fetal membranes after administration of ceftiofur sodium to normal pony mares.

We explore the hypothesis that the Eureka family of sub-km asteroids in the L5 region of Mars could have formed in a collision. We estimate the size distribution index from available information on family members; model the orbital dispersion of collisional fragments; and carry out a formal calculation of the collisional lifetime as a function of size. We find that, as initially conjectured by Rivkin et al. (2003), the collisional lifetime of objects the size of (5261) Eureka is at least a few Gyr, significantly longer than for similar-sized Main Belt asteroids. In contrast, the observed degree of orbital compactness is inconsistent with all but the least energetic family-forming collisions. Therefore, the family asteroids may be ejecta from a cratering event sometime in the past ∼ 1 Gyr if the orbits are gradually dispersed by gravitational diffusion and the Yarkovsky effect (Ćuk et al., 2015). The comparable sizes of the largest family members require either negligible target strength or a particular impact geometry under this scenario (Durda et al., 2007; Benavidez et al., 2012). Alternatively, the family may have formed by a series of YORP-induced fission events (Pravec et al., 2010). The shallow size distribution of the family is similar to that of small MBAs (Gladman et al., 2009) interpreted as due to the dominance of this mechanism for Eureka-family-sized asteroids (Jacobson et al., 2014). However, our population index estimate is likely a lower limit due to the small available number of family asteroids and observational incompleteness. Future searches for fainter family members, further observational characterisation of the known Trojans’ physical properties as well as orbital and rotational evolution modelling will help distinguish between different formation models.

Early pregnancy factor has been identified as a 10-kDa extracellular homolog of heat shock protein 10 (Hsp10). Hsp10 has been detected during early pregnancy in serum of mice, sheep, pigs, horses, cows, and humans by the rosette inhibition test. Hsp10 has also been associated with several neoplastic and autoimmune diseases. The goal of the present study was to determine if Hsp10 could be detected in the early equine embryo through the use of immunohistochemistry and quantitative real-time PCR. Additionally, analysis of systemically harvested peripheral blood mononuclear cells (PBMCs) from both pregnant and nonpregnant mares was evaluated to determine expression levels of HSP10. Embryos were collected from Quarter Horse mares by uterine lavage at either 8 or 25 days after ovulation. Collection and separation of PBMCs occurred on Day 8 for both pregnant and nonpregnant mares. Immunohistochemistry revealed cytoplasmic localization of HSP10 throughout the single layer of ectodermal cells forming the trophoblast in Day-8 embryos. Day-25 embryos demonstrated intense localization focally along the apical border of ectodermal cells forming the trophoblast layer of the developing chorion. There was no nuclear staining in either embryonic population. Quantitative real-time PCR detected the presence of mRNA for HSP10 in both 8- and 25-day equine embryos. Day-25 embryos exhibited an elevated degree of expression (P = 0.006) compared with the 8-day embryos for HSP10. Endometrial samples did not display any significant difference in degree of expression for HSP10 (P = 0.10). Finally, PBMCs from pregnant mares demonstrated elevated (P = 0.03) expression of HSP10 compared to the nonpregnant mares on Day 8 of the estrous cycle. This study confirmed the presence of HSP10 protein and mRNA expression of HSP10 in equine embryos at two maturation stages. Additionally, the presence of increased gene expression within PBMCs of pregnant mares suggests communication, possibly leading to necessary immunomodulatory effects between the embryo and mare.

REASONS FOR PERFORMING STUDY Systemic administration of ceftiofur crystalline free acid (CCFA) may be a potential treatment for infectious endometritis caused by Streptococcus equi ssp. zooepidemicus (S.  zooepidemicus) and other susceptible bacterial organisms in the mare. OBJECTIVE To determine if i.m. administration of CCFA at the label dose will exceed the minimum inhibitory concentration (MIC) of S.  zooepidemicus in the endometrium following single administration and multiple administration protocols. STUDY DESIGN Experimental pharmacokinetic study. METHODS Three mares (Group 1) were administered a single i.m. dose of CCFA (6.6 mg/kg bwt) and blood and endometrial biopsies were collected at selected intervals for 144 h. Six additional mares (Groups 2 and 3) received CCFA at times 0, 4, 11 and 18 days, and were sampled at predetermined times for 25 or 49 days, respectively. Plasma and tissue samples were analysed by high-pressure liquid chromatography with tandem mass spectrometry for desfuroylceftiofur acetamide concentration, which is a direct measure of all ceftofur and ceftiofur metabolites in the sample. RESULTS A mean plasma desfuroylceftiofur acetamide concentration of 0.367 ± 0.0162 μg/ml (mean ± s.e.) was detected at 96 h following administration. Mean endometrial tissue concentration was 0.510 ± 0.0418 μg/g at 96 h and exceeded the MIC for S.  zooepidemicus (0.25 μg/ml) throughout the 144 h monitoring period for Group 1. Mares in Groups 2 and 3, given multiple doses of CCFA, maintained plasma concentrations above the MIC for S.  zooepidemicus for 25 days. Endometrial tissue levels remained above the MIC at most data collection points for 25 days. CONCLUSIONS Ceftiofur crystalline free acid reaches appropriate endometrial tissue values to exceed the MIC of S.  zooepidemicus, a common cause of bacterial endometritis. Therefore, CCFA should be effective in the treatment of equine bacterial endometritis caused by S.  zooepidemicus and other susceptible bacterial pathogens in the mare.

Iron deficiency often results in nutritional disorder in fruit trees. Transcription factors play an important role in the regulation of iron uptake. In this study, we isolated an iron deficiency response transcription factor gene, MxFIT, from an iron-efficient apple genotype of Malus xiaojinensis. MxFIT encoded a basic helix-loop-helix protein and contained a 966 bp open reading frame. MxFIT protein was targeted to the nucleus in onion epidermal cells and showed strong transcriptional activation in yeast cells. Spatiotemporal expression analysis revealed that MxFIT was up-regulated in roots under iron deficiency at both mRNA and protein levels, while almost no expression was detected in leaves irrespective of iron supply. Ectopic expression of MxFIT resulted in enhanced iron deficiency responses in Arabidopsis under iron deficiency and stronger resistance to iron deficiency. Thus, MxFIT might be involved in iron uptake and plays an important role in iron deficiency response.

Equine clinicians rely on ovulation induction agents to provide a timed ovulation in mares for optimal breeding management. Numerous studies have been performed on the efficacy of human chorionic gonadotropin (hCG) to induce ovulation in the mare, but limited clinical data are available for the new deslorelin acetate product SucroMate. This study was designed to evaluate the efficacy of SucroMate (deslorelin) in comparison with hCG to induce ovulation. American Quarter horse mares (n = 256) presented to Colorado State University for breeding management were used in this study. Mares received either deslorelin or hCG when a follicle >= 35 mm was detected by transrectal ultrasound in the presence of uterine edema. Ultrasonographic examinations were subsequently performed once daily until ovulation was detected. Deslorelin was administered to 138 mares during168 estrous cycles, and hCG was given to 118 mares during 136 estrous cycles. Mares administered deslorelin had a similar (P < .05) higher ovulation rate (89.9%) within 48 hours following drug administration than mares administered hCG (82.8%). There are no effects of season or age on ovulation rates in either treatment group. Twenty-one mares administered deslorelin and 11 mares administered hCG were monitored by transrectal ultrasound every 6 hours to detect ovulation as part of a frozen semen management program. Average intervals from deslorelin or hCG administration to ovulation were 41.4 +/- 9.4 and 44.4 +/- 16.5 hours, respectively. Results of this study indicate that SucroMate is effective at inducing a timed ovulation in the mare. (C) 2012 Elsevier Inc. All rights reserved.

The objective of this study was to determine the pharmacokinetics of CCFA in mares with placentitis and evaluate the disposition of the drug in fetal fluids, fetal membranes, colostrum, and serum of foals. A secondary objective was to obtain pilot data regarding the efficacy of CCFA for improving foal survival in mares with placentitis. Twelve pregnant pony mares were enrolled in the study, inoculated with Streptococcus zooepidemicus, intracervically and assigned to one of three groups: CEFT (n = 3; administered CCFA only; 6.6 mg/kg, i.m., q96h); COMBO (n = 6; administered combination therapy of CCFA, altrenogest, and pentoxifylline); UNTREAT (n = 3, no treatment). Treatment was initiated at the onset of clinical signs. Concentrations of desfuroylceftiofur acetamide (DCA), the acetamide derivative of ceftiofur and desfuroylceftiofur metabolites, were measured using high-performance liquid chromatography. Maximum and minimum serum concentrations of DCA at steady state in treated mares were 2.40±0.40 μg/mL and 1.06±0.29 μg/mL, respectively. Concentration of DCA in colostrum was 1.51±0.60 μg/mL. DCA concentrations in placenta and fetal tissues were very low (median = 0.03 μg/mL) and below the minimum inhibitory concentration of relevant pathogens. DCA was not detected in amniotic fluid or foal serum. Treatment did not appear to improve foal survival (CEFT: 0/3; COMBO: 2/6; UNTREAT: 2/3). Bacteria were recovered from the uterus of most mares postpartum and from blood cultures of most foals regardless of treatment.

Water pollution from animal waste, and its influence on grazing animals, is a current concern regarding Mongolian grazing lands. We allocated 32 free-grazing lambs to four groups and provided each with water from a different source (upper stream, lower stream, well, and pond) for 49 days. We recorded the amount of water consumed by the lambs, as well as their body weight, white blood cell count, acute phase (haptoglobin) protein level, and fecal condition. We measured the chemical and biological qualities of the four types of water, and we detected enteropathogenic and enterohemorrhagic Escherichia coli in fecal samples by using a genetic approach. Pond water contained high levels of nitrogen and minerals, and well water contained high levels of bacteria. On day 15 of the experiment, the following parameters were the highest in lambs drinking water from the following sources: water intake (pond or lower stream), body weight gain (pond), WBC count (lower stream), haptoglobin concentration (well), and enteropathogenic E. coli infection rate (lower stream). Lambs given upper or lower stream water exhibited more severe diarrhea on day 15 of the experiment than before the experiment. Mongolian sheep seemed to adapt to chemically contaminated water: their productivity benefited the most from pond water, likely owing to its rich mineral content. Lambs that drank lower stream water showed increases in enteropathogenic E. coli infection, clinical diarrhea, and WBC count. Water intake was lowest in the lambs given well water, suggesting that they avoided drinking the water because of potential E. coli infection; they were thus at increased risk of negative health and production effects. Our study revealed the profound nature of the effects of water quality on livestock health and performance.