Factors influencing pregnancy loss between days 30 and 70 in a single cattle herd: A comprehensive analysis of sire, cow, and other contributing factors.

This retrospective study utilized a mixed-effects logistic model analysis to investigate variables associated with the probability of pregnancy loss (PL) between days 30 and 70 in a dataset comprising 9507 pregnancies from a single cattle herd over 10 years. The model incorporated fixed-effect variables including cow breed (Holstein, Crossbred, and Brown Swiss), parity (1st, 2nd, 3rd, and 4th or more), insemination seasons, insemination number (≤3 vs >3), estrus nature (spontaneous vs synchronized), postpartum problems, sire breed (Holstein, Fleckvieh, Brown Swiss, and Montbéliarde), zygote genotypic (pure vs crossbred), days in milk (DIM) at insemination, actual 305-day milk yield, and sire conception rate. Additionally, random effects included sire (n = 129), cow (n = 3463), and production years (n = 10). The results revealed that cows inseminated with Brown Swiss sires or sires with lower initial conception rates had higher PL rates. Biparous cows, cows with lower 305-day milk yield, cows inseminated later in DIM, cows receiving the 4th or subsequent insemination, and cows inseminated during winter or autumn had lower PL rates. The estrus type and zygote genotype did not significantly impact PL. The random effects of cow, sire, and production years were estimated at 0.230, 0.054, and 0.112, respectively. In conclusion, the study findings suggested that improving management practices for high-yielding cows, cows in early lactation stages, and cows exposed to thermal stress conditions, along with utilizing Brown Swiss cows and sires with high initial conception rates per insemination, could potentially decrease overall PL rates on the farm. Nonetheless, the results did not support the use of Crossbred cows, sires from different breeds, or specific sires to mitigate PL rates on the farm.

A single base pair duplication in the SLC33A1 gene is associated with fetal losses and neonatal lethality in Manech Tête Rousse dairy sheep.

We recently discovered that the Manech Tête Rousse (MTR) deficient homozygous haplotype 2 (MTRDHH2) probably carries a recessive lethal mutation in sheep. In this study, we fine-mapped this region through whole-genome sequencing of five MTRDHH2 heterozygous carriers and 95 non-carriers from various ovine breeds. We identified a single base pair duplication within the SLC33A1 gene, leading to a frameshift mutation and a premature stop codon (p.Arg246Alafs*3). SLC33A1 encodes a transmembrane transporter of acetyl-coenzyme A that is crucial for cellular metabolism. To investigate the lethality of this mutation in homozygous MTR sheep, we performed at-risk matings using artificial insemination (AI) between heterozygous SLC33A1 variant carriers (SLC33A1_dupG). Pregnancy was confirmed 15 days post-AI using a blood test measuring interferon Tau-stimulated MX1 gene expression. Ultrasonography between 45 and 60 days post-AI revealed a 12% reduction in AI success compared with safe matings, indicating embryonic/fetal loss. This was supported by the MX1 differential expression test suggesting fetal losses between 15 and 60 days of gestation. We also observed a 34.7% pre-weaning mortality rate in 49 lambs born from at-risk matings. Homozygous SLC33A1_dupG lambs accounted for 47% of this mortality, with deaths occurring mostly within the first 5 days without visible clinical signs. Therefore, appropriate management of SLC33A1_dupG with an allele frequency of 0.04 in the MTR selection scheme would help increase overall fertility and lamb survival.

Development of PCR-based genetic test for detection of TTF1 mutation causing abortion in Holstein Friesian cattle.

A stop-gain mutation (rs715966442; BTA11: 1,02,463,944 nucleotide position) in transcription termination factor, RNA polymerase I (TTF1) gene causes abortion in Holstein Friesian (HF) cattle. A PCR-restriction fragment length polymorphism (PCR-RFLP)-based genetic test has been developed and validated to screen the TTF1 mutation locus in HF cattle. The mutation locus was screened in 80 HF and HF crossbreds using the protocol, which revealed two animals as carriers of the mutant TTF1 allele. The test employed is cost-effective, rapid and precise and can be utilized as an effective tool for the screening of TTF1 mutation carriers in HF cattle population.

The role of genetic variability of the host on the resistance to Neospora caninum infection in cattle.

Neospora caninum is one of the most frequently diagnosed abortifacient pathogens in cattle. There is abundant genomic information about the parasite itself, but very little is known about the genetic variability of resistance in the most common intermediate host. The aim of this review was to compile all the available information about the genetic variability associated with the resistance to N. caninum both between and within cattle breeds. We systematically searched for published studies that investigated the influence of genetics of the host on the prevalence of N. caninum and risk of abortion. Beyond the potential confounding effects of feeding systems, management and animal density, some lines of evidence suggest that Holstein, the most popular breed for milk production, has a comparatively higher risk of abortion due to infections by N. caninum, whereas some beef breeds from Continental Europe seem to be more resistant. It is still not clear if different genetic mechanisms of resistance are involved in the two known routes of infection: postnatal ingestion of oocysts or transplacental transmission from the infected dam to the fetus. Genomic information associated with susceptibility to infection and risk of abortion in different cattle breeds is still scarce. The information reported here could be useful to identify new research alternatives and to define novel strategies to deal with this major problem of animal production.

Does inbreeding contribute to pregnancy loss in Thoroughbred horses?

BACKGROUND: Excessive inbreeding increases the probability of uncovering homozygous recessive genotypes and has been associated with an increased risk of retained placenta and lower semen quality. No genomic analysis has investigated the association between inbreeding levels and pregnancy loss. OBJECTIVES: To compare genetic inbreeding coefficients (F) of naturally occurring Thoroughbred Early Pregnancy Loss (EPLs), Mid and Late term Pregnancy Loss (MLPL) and Controls. The F value was hypothesised to be higher in cases of pregnancy loss (EPLs and MLPLs) than Controls. STUDY DESIGN: Observational case-control study. METHODS: Allantochorion and fetal DNA from EPL (n = 37, gestation age 14-65 days), MLPL (n = 94, gestational age 70 days-24 h post parturition) and Controls (n = 58) were genotyped on the Axiom Equine 670K SNP Genotyping Array. Inbreeding coefficients using Runs of Homozygosity (FROH) were calculated using PLINK software. ROHs were split into size categories to investigate the recency of inbreeding. RESULTS: MLPLs had significantly higher median number of ROH (188 interquartile range [IQR], 180.8-197.3), length of ROH (3.10, IQR 2.93-3.33), and total number of ROH (590.8, IQR 537.3-632.3), and FROH (0.26, IQR 0.24-0.28) when compared with the Controls and the EPLs (p < 0.05). There was no significant difference in any of the inbreeding indices between the EPLs and Controls. The MLPLs had a significantly higher proportion of long (>10 Mb) ROH (2.5%, IQR 1.6-3.6) than the Controls (1.7%, IQR 0.6-2.5), p = 0.001. No unique ROHs were found in the EPL or MLPL populations. MAIN LIMITATIONS: SNP-array data does not allow analysis of every base in the sequence. CONCLUSIONS: This first study of the effect of genomic inbreeding levels on pregnancy loss showed that inbreeding is a contributor to MLPL, but not EPL in the UK Thoroughbred population. Mating choices remain critical, because inbreeding may predispose to MLPL by increasing the risk of homozygosity for specific lethal allele(s).

Altered frequency and function of spleen CTLA-4+Tim-3+ T cells are associated with miscarriage†.

Normal pregnancy is associated with several immune adaptations in both systemic and local maternal-fetal interface to allow the growth of semi-allogeneic conceptus. A failure in maternal immune tolerance to the fetus may result in abnormal pregnancies, such as recurrent spontaneous abortion. The regulation of T-cell homeostasis during pregnancy has important implications for maternal tolerance and immunity. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) and T-cell immunoglobulin mucin-3 (Tim-3) are important negative immune regulatory molecules involved in viral persistence and tumor metastasis. Here we described the lower frequency of splenic T cells co-expressing CTLA-4 and Tim-3 accompanied by higher levels of proinflammatory but lower anti-inflammatory cytokines production in abortion-prone mouse model. Blockade of CTLA-4 and Tim-3 pathways leaded to the dysfunction of splenic T cells. By the higher expression during normal pregnancy, CTLA-4 and Tim-3 co-expression on splenic T cells linked to immunosuppressive phenotype. As the spleen is an important site for peripheral immune activation, our data suggest potential noninvasive biomarkers and therapeutic targets for miscarriage.

Notch1 is crucial for decidualization and maintaining the first pregnancy in the mouse†.

The endometrium undergoes a pregnancy-delivery-repair cycle multiple times during the reproductive lifespan in females. Decidualization is one of the critical events for the success of this essential process. We have previously reported that Notch1 is essential for artificial decidualization in mice. However, in a natural pregnancy, the deletion of Notch1 (PgrCre/+Notch1f/f, or Notch1d/d) only affects female fertility in the first 30 days of a 6-month fertility test, but not the later stages. In the present study, we undertook a closer evaluation at the first pregnancy of these mice to attempt to understand this puzzling phenomenon. We observed a large number of pregnancy losses in Notch1d/d mice in their first pregnancy, which led to the subfertility observed in the first 30 days of the fertility test. We then demonstrated that the initial pregnancy loss is a consequence of impaired decidualization. Furthermore, we identified a group of genes that contribute to Notch1 regulated decidualization in a natural pregnancy. Gene ontogeny analysis showed that these differentially expressed genes in the natural pregnancy are involved in cell-cell and cell-matrix interactions, different from genes that have been previously identified from the artificial decidualization model, which contribute to cell proliferation and apoptosis. In summary, we determined that Notch1 is essential for normal decidualization in the mouse uterus only in the first pregnancy but not in subsequent ones.

Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse.

The first 8 weeks of pregnancy is a critical time, with the majority of pregnancy losses occurring during this period. Abnormal chromosome number (aneuploidy) is a common finding in human miscarriage, yet is rarely reported in domestic animals. Equine early pregnancy loss (EPL) has no diagnosis in over 80% of cases. The aim of this study was to characterise aneuploidies associated with equine EPL. Genomic DNA from clinical cases of spontaneous miscarriage (EPLs; 14-65 days of gestation) and healthy control placentae (various gestational ages) were assessed using a high density genotyping array. Aneuploidy was detected in 12/55 EPLs (21.8%), and 0/15 healthy control placentae. Whole genome sequencing (30X) and digital droplet PCR (ddPCR) validated results. The majority of these aneuploidies have never been reported in live born equines, supporting their embryonic/fetal lethality. Aneuploidies were detected in both placental and fetal compartments. Rodents are currently used to study how maternal ageing impacts aneuploidy risk, however the differences in reproductive biology is a limitation of this model. We present the first evidence of aneuploidy in naturally occurring equine EPLs at a similar rate to human miscarriage. We therefore suggest the horse as an alternative to rodent models to study mechanisms resulting in aneuploid pregnancies.

Novel method for identification of the lethal mutation in bovine APAF1 gene and its preliminary prevalence in Polish Holstein-Friesian bulls.

The aim of the study was to develop a reliable and cost-effective method for detection of nonsense mutation in APAF1 gene causing lethal effect called HH1 (Holstein Haplotype1) and to evaluate its prevalence in a sample of Polish Holstein-Friesian bulls. One hundred seventy eight bulls born between 1996 and 2017 were included in the analysis. They were kept in four artificial insemination centers and have in the pedigree the known carrier of HH1. All bulls were diagnosed by novel PCR-SSCP technique. Specific amplicons of 261 bp APAF1 gene fragment were used to detect changes in single stranded conformation (SSCP) caused by nonsense mutation C/T responsible for HH1. Each new carrier was used to trace another potential carriers among their offspring available in Polish Holstein Bull Repository Database. Among 178 bulls, 85 HH1 carriers were found. Our results show that nonsense mutation in APAF1 gene is already transmitted and segregating in Polish Holstein-Friesian cattle and its frequency may increase if no action will be undertaken against actual carriers.

Genetic and genomic analysis of long insemination interval in Israeli dairy cattle as an indicator of early abortions.

One of the causes of observed low fertility is embryo loss after fertilization. Previous findings suggested that more than half of fertilizations result in embryo loss before pregnancy is detected. We proposed reinsemination between 49 and 100 d after the first insemination as an indicator trait for early abortion (EA) in dairy cattle based on the mean estrus interval of 21 d. This trait was compared with conception rate from first insemination and conception status, computed as the inverse of the number of inseminations to conception. Animal model variance components were estimated by REML, including parents and grandparents of cows with records. First-parity heritability for first insemination conception rate was 3%. In the multitrait analysis of parities 1 to 3 for putative EA, heritabilities ranged from 8.9% for first parity to 10.4% for second parity. All genetic correlations were >0.9, whereas all environmental correlations were <0.12. The variance component for the service sire effect for putative EA rate was less than half the variance component for conception rate. Thus, genetic control of the 2 traits is clearly different, and analysis of EA rate by a single-trait animal model is justified. Genetic evaluation for putative EA was computed using this model, including all first- through third-parity cows with freshening dates from January 1, 1985, through December 31, 2016, that either became pregnant on first insemination or were reinseminated between 49 and 100 d after the first insemination. All known parents and grandparents of cows with records were included in the analysis. The regression of the breeding value for non-abortion rate on the cows' birth year was 0.083%/yr. The genetic correlation between first-parity EA and conception status was 0.995. The genetic correlations between first-parity EA and milk, fat, and protein production were all negative, whereas the genetic correlation between EA and herd life was 0.33. Inclusion of putative EA in the selection index instead of conception status resulted in 10 to 20% greater genetic gain for both fertility traits. In a genome-wide association study based on 1,200 dairy bulls with reliabilities >50% for abortion rate genotyped for 41,000 markers, 6 markers were found with nominal probabilities of <10-12 to reject the null hypothesis of no effect on EA rate. The markers with the lowest probabilities for EA rate were also included among the markers with the lowest probabilities for female fertility, but not vice versa. The marker explaining the most variance for abortion rate is located within the ABCA9 gene, which is found within an ATP-binding cassette (ABC) genes cluster. The ABC family is the major class of primary active transporters in the placenta.

Characterization of abortion, stillbirth and non-viable foals homozygous for the Warmblood Fragile Foal Syndrome.

Warmblood fragile foal syndrome (WFFS) is a monogenetic defect with autosomal recessive inheritance. The WFFS homozygosity is non-compatible with extra-uterine life. Although as many as 15% of Warmblood horses are WFFS carriers, there has been little veterinary focus on this condition. The aim of this study was to determine outcomes and symptoms of clinical signs and pathological abnormalities during pregnancies when there were WFFS homozygous foetuses. Diagnostic material of 15 abortion or stillbirth cases with suspected diagnosis of WFFS was available for this study. Additionally, there were examinations in 37 cases where there were no indications of WFFS when submitted for routine diagnostic procedures. Foals in all cases were genotyped and external morphological defects were recorded. Amongst the 15 cases in which WFSS was suspected, there were 14 homozygous foetuses with the WFFS allele (WFFS/WFFS). Three heterozygous WFFS foetuses (N/WFFS) were detected in the cases submitted for routine diagnostic procedures. Of the 14 WFFS homozygous foetuses, 11 of mares had a gestation length of at least 320 days. Nine foals were born alive but died within a short time. Skin defects were obvious in 12 WFFS homozygous foals, and there was abnormal flexibility in the digital joints, flexed forelegs and incomplete closure of the abdominal wall in five, four, and one of the foals, respectively. In conclusion, the predominant manifestation of WFFS are death during the latter stages of gestation or live births with foals being non-viable. Losses in Warmblood horse breeding caused by WFFS are greater than previously assumed.

The Rho-Independent Transcription Terminator for the porA Gene Enhances Expression of the Major Outer Membrane Protein and Campylobacter jejuni Virulence in Abortion Induction.

Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. Its porA gene encodes the major outer membrane protein (MOMP) that is abundantly expressed and has important physiological functions, including a key role in systemic infection and abortion induction in pregnant animals. Despite the importance of porA in C. jejuni pathogenesis, mechanisms modulating its expression levels remain elusive. At the 3' end of the porA transcript, there is a Rho-independent transcription terminator (named T porA in this study). Whether T porA affects the expression and function of MOMP remains unknown and is investigated in this study. Green fluorescent protein (GFP) fusion constructs with the porA promoter at the 5' end and an intact T porA or no T porA at the 3' end of the gfp coding sequence revealed that both the transcript level of gfp and its fluorescence signals were more than 2-fold higher in the construct with T porA than in the one without T porA Real-time quantitative PCR (qRT-PCR) analysis of the porA mRNA and immunoblot detection of MOMP in C. jejuni showed that disruption of T porA significantly reduced the porA transcript level and the expression of MOMP. An mRNA decay assay demonstrated that disruption of T porA resulted in a shortened transcript half-life of the upstream gfp or porA gene, indicating that T porA enhances mRNA stability. In the guinea pig model, the C. jejuni construct with an interrupted T porA was significantly attenuated in abortion induction. Together, these results indicate that T porA enhances the expression level of MOMP by stabilizing its mRNA and influences the virulence of C. jejuni.

Missense mutation in SDE2 gene - new lethal defect transmitted into Polish Holstein-Friesian cattle.

The aim of the study was to find out whether carriers of new lethal mutation in SDE2 gene occur in the population of Polish Holstein-Friesian bulls. Eighty seven bulls were included in the analysis. Bulls were selected as having in the pedigree known carrier of SDE2 mutation (bull Mountain USAM000002070579). All bulls were diagnosed by PCR amplification of 524 bp fragment of SDE2 gene followed by digestion of Bcc I restriction enzyme. Heterozygotes (carriers) were confirmed by sequencing. Each new carrier was used to trace another potential carriers among its offspring available in Polish Holstein Bull Repository Database. Among 87 bulls, 50 new SDE2 carriers were found. The study has shown that mutation in SDE2 gene causing early embryo mortality is already transmitted to Polish Holstein-Friesian cattle. The results are sufficient to initiate the screening program to reveal new carriers and to avoid further spreading of SDE2 lethal mutation.

CXCL12 expression in aborted mouse uteri induced by IFN-γ: Potential anti-inflammatory effect involves in endometrial restoration after abortion in mice.

Anti-inflammation is a key process to restore tissue integrity and function. CXCL12 is a homeostasis chemokine, which plays a coordinating role in organogenesis, tumorigenesis and regeneration. In the present study we found that the uterus of abortion mice showed different histo-morphological changes with the development of abortion. The expression of chemokine CXCL12 and its receptor CXCR4 in abortion uterus showed a time-dependent pattern. Compared with normal pregnancy, the expression of CXCL12 and CXCR4 did not change in the uterus of GD7 abortion mice, but increased significantly in the uterus of GD8 and GD10 abortion mice. However, the expression of IFN-γ increased significantly in the uterus of GD7 abortion mice, while there was no significant change detected in GD8 aborted mice uterus. Our further data show that the expression of CXCL12 is not regulated by IFN-γ in endometrial stromal cell culture system in vitro. The treatment of CXCL12 significantly inhibits the expression of IFN-γ in in vitro cultured stromal cells and splenic monocytes. This suggests that CXCL12 may play an anti-inflammatory role in the uterus of abortion mice to promote the process of endometrial restoration after abortion, rather than participate in the process of abortion as a response molecule of IFN-γ.

Sire contribution to pregnancy loss and pregnancy-associated glycoprotein production in Nelore cows.

Pregnancy loss is a major contributing factor to reproductive inefficiency in both the beef and dairy industries. Sires can have a significant influence on the amount of pregnancy loss; however, this relationship is still poorly investigated. The primary objective of this study was to identify sires associated with high or low incidence of pregnancy loss (between d 30 and 100 of gestation) and investigate their effect on concentration of circulating pregnancy-associated glycoproteins (PAGs). Postpartum multiparous Nelore cows were inseminated artificially at a fixed time (FTAI, d 0) after synchronization of ovulation. A total of 736 cows were assigned randomly to be inseminated with semen from either of 6 Angus sires, whereas a separate subset of 492 cows were inseminated randomly with semen from either of 3 Nelore (n = 235) or either of 2 Angus sires (n = 257). Estrus expression was evaluated on d 0 using Estrotect Heat Detector patches. Blood samples were collected on d 30 of gestation for quantification of PAGs and pregnancy diagnosis was performed by ultrasound on d 30 and 100 after FTAI. Cows diagnosed pregnant at the first examination but not pregnant at the second were defined to have pregnancy loss. Overall pregnancy rate at d 30 was 54% (660/1,228) and pregnancy loss was 6.21% (41/660). Cows receiving semen from Nelore sires had greater (P < 0.001) pregnancy rate, greater (P = 0.014) pregnancy loss, and lesser (P = 0.002) PAG concentrations at d 30 of gestation compared with cows receiving Angus semen. Circulating PAG concentrations were lower (P = 0.008) in cows that had pregnancy loss (9.76 ± 0.25 vs. 7.41 ± 1.02 ng/mL). Angus sires were retrospectively classified according to percentage of pregnancy loss as either high pregnancy loss (mean of 7.25% or 67% of total) or low pregnancy loss (mean of 3.93% or 33% of total). Cows receiving semen from high pregnancy loss sires had 1.9 times greater (P = 0.123) rate of pregnancy loss and had lower (P = 0.059) PAG concentrations at d 30 of gestation compared with cows mated to low pregnancy loss sires. In summary, PAG concentrations reflected probability of pregnancy maintenance and were influenced by both sire and sire breed used at FTAI. Variation in the incidence of pregnancy loss was detected among sires that could not be predicted with standard semen fertility evaluations. Exploring the relationship of sire and PAG production might be promising to improve sire selection with regard to pregnancy loss.

Promoter variants of OAS1 gene are associated with reproductive performance and incidence of normal calving in cattle.

2',5'-oligoadenylate synthetase (OAS) are important components of an interferon-mediated antiviral pathway. But no polymorphisms have yet been identified and associated with reproduction and reproduction traits in the bovine OAS1 gene. The objective of this study was to detect and evaluate the effects of mutations in the promoter region of bovine OAS1 gene with production and reproduction traits in cattle. Two single nuclear polymorphisms were identified, including A to T transversion (rs715937117:A>T) and A to G transition (rs480985443:A>G) and subsequently genotyped in Sahiwal and Frieswal populations. Three haplotypes were constructed including H1 (A-A), H2 (T-A) and H3 (T-G), among them H2 was the most abundant in the Sahiwal (0.677) and Frieswal (0.838) population. The analysis revealed that genotypes of promoter region had significant effect (P ≤ 0.05) on age at first breeding, age at first calving, service period and pregnancy rate. The animals with H1H2 diplotype had higher age at first breeding and age at first calving compared to H2H2 genotyped animals. Further logistic regression analysis also revealed that the probability of normal calving was found more in H2H2 diplotype, as the odds ratios of H1H2 and H2H3 in comparison to H2H2 were 0.516 and 0.486, respectively. H2H2 diplotyped animals also exhibited higher milk production also in terms of total milk yield, 300 day milk yield and peak yield. These results outlined that the bovine OAS1 gene can be used as candidate genes for simultaneous improvement of both production and reproductive performance of cattle.

Detection of APAF1 mutation in Holstein cows and mummified foetuses in Japanese dairy herds.

Some of the highest genetic merit sires have been shown to harbour recessive mutations affecting fertility, which may spread rapidly in the population through AI. These disorders may result in abortion and decline in pregnancy per insemination in cows. This study was carried out on 240 Holstein-Friesian cows and 15 mummified foetuses. Blood and tissue samples were collected from the cows and mummified foetuses, respectively, for DNA extraction. Allele-specific PCR was designed for the detection of the cows and foetuses carrying the nonsense mutation (C/T) in apoptosis peptide activating factor 1 gene (APAF1). The mutant allele frequency of the APAF1 in carrier cows and mummified foetuses was calculated. Milk samples were taken from the carrier and non-carrier cows for progesterone assay. The allele-specific PCR reaction efficiently distinguished the C/T mutation in APAF1. Of 240 cows, seven cows (2.9%) were diagnosed to carry one copy of the mutant allele of APAF1. However, the carrier frequency was 33.3% in mummified foetuses (five of 15). The mutant allele frequency was 0.02 and 0.17 in the cows and mummified foetuses, respectively. Concentrations of progesterone did not differ between cows with APAF1 mutation and non-carrier cows during 45 days post-insemination. This study provided allele-specific PCR for the detection of APAF1 mutation in cows. Moreover, it reports the carrier and mutant allele frequencies of APAF1 in dairy cows and mummified foetuses in Japan.

De novo synthesis of sphingolipids is essential for decidualization in mice.

Sphingolipids play multiple roles in membrane structure, signal transduction, stress responses, neural development and immune reaction. The rate of de novo synthesis pathway of sphingolipids is regulated by two key enzymes, serine palmitoyltransferase (SPT), and ketoreductase (Kds). Here, we find that the mRNA levels of three subunits of the SPT holoenzyme (Sptlc1, Sptlc2, and Ssspta) are significantly up-regulated in mouse uterine stromal cells during decidualization. The expression of Kds, which reduces 3-keto-dihydrosphingosine to dihydrosphingosine, is co-localized with Sptlc1 in mouse uteri during early pregnancy. Moreover, l-Cycloserine, a specific inhibitor of SPT, can significantly decrease the weight and number of implantation sites, and impede the decidualization process in mouse uterine stromal cells, suggesting that blockage of de novo sphingolipid synthesis may cause defective decidualization and early pregnancy loss in mice. In addition, this study also shows progesterone (P4) can stimulate the expression of both Sptlc2 and Ssspta in mouse uterus. Therefore, our study shows that de novo synthesis of sphingolipids is necessary in implantation and plays a key role in decidualization of mouse.

Genome Report: Identification and Validation of Antigenic Proteins from Pajaroellobacter abortibovis Using De Novo Genome Sequence Assembly and Reverse Vaccinology.

Epizootic bovine abortion (EBA), or "foothill abortion," is the leading cause of beef cattle abortion in California and has also been reported in Nevada and Oregon. In the 1970s, the soft-shelled tick Ornithodoros coriaceus, or "pajaroello tick," was confirmed as the disease-transmitting vector. In 2005, a novel Deltaproteobacterium was discovered as the etiologic agent of EBA (aoEBA), recently named Pajaroellobacter abortibovis This organism cannot be grown in culture using traditional microbiological techniques; it can only be grown in experimentally-infected severe combined immunodeficient (SCID) mice. The objectives of this study were to perform a de novo genome assembly for P. abortibovis and identify and validate potential antigenic proteins as candidates for future recombinant vaccine development. DNA and RNA were extracted from spleen tissue collected from experimentally-infected SCID mice following exposure to P. abortibovis This combination of mouse and bacterial DNA was sequenced and aligned to the mouse genome. Mouse sequences were subtracted from the sequence pool and the remaining sequences were de novo assembled at 50x coverage into a 1.82 Mbp complete closed circular Deltaproteobacterial genome containing 2250 putative protein-coding sequences. Phylogenetic analysis of P. abortibovis predicts that this bacterium is most closely related to the organisms of the order Myxococcales, referred to as Myxobacteria. In silico prediction of vaccine candidates was performed using a reverse vaccinology approach resulting in the identification and ranking of the top 10 candidate proteins that are likely to be antigenic. Immunologic testing of these candidate proteins confirmed antigenicity of seven of the nine expressed protein candidates using serum from P. abortibovis immunized mice.

Placental abnormalities in equine pregnancies generated by SCNT from one donor horse.

Placental changes associated with SCNT have been described in several species, but little information is available in this area in the horse. We evaluated the ultrasonographic, gross, and histopathological characteristics of placentas from three successful and five unsuccessful equine SCNT pregnancies, established using cells from a single donor horse. Starting at approximately 6-month gestation, the pregnancies were monitored periodically using transrectal (TR) and transabdominal (TA) ultrasonography (US) to examine the placentas, fetal fluids, and fetuses. Of the five mares that aborted, one mare did so suddenly without any abnormal signs detected by US and four had enlarged umbilical vessels visible on TA-US before abortion. Placental edema (TR-US) and intravascular thrombi in the umbilical cords were seen (TA-US) in two of these four mares; one mare aborted shortly after acute placental separation was identified on TA-US. In three mares that delivered live foals, TA-US showed engorged allantoic vessels and enlarged umbilical vessels. Two of these mares had placental thickening visible on TR-US, interpreted as a sign of placentitis, that subsided after aggressive medical treatment. Seven of the eight placentas were submitted for gross and histopathological examinations after delivery. All placentas had some degree of edema, abnormally engorged allantoic vessels, and enlarged umbilical vessels. Placentitis, large allantoic vesicles, cystic pouches in the fetal part of the cord, and hemorrhages and thrombi in the umbilical vessels were detected only in placentas from mares that aborted. Equine pregnancies resulting from SCNT may be associated with placental pathologies that can be detected using ultrasonography. However, interpreting their severity is difficult. Although placental abnormalities have been observed in SCNT pregnancies in other species, to the best of our knowledge, placentitis has not been previously reported and may be an important complication of equine SCNT pregnancies, leading to pregnancy loss.