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Introduction: Targeted single nucleotide polymorphisms (SNPs) have been used in genomic prediction methodologies to enhance the accuracy of associated genetic transmitting abilities in Holstein cows. The objective of this study was to identify and validate SNPs associated with fertility traits impacting early embryo mortality. Methods: The mRNA sequencing data from day 16 normal (n = 9) and embryo mortality (n = 6) conceptuses from lactating multiparous Holstein cows were used to detect SNPs. The selection of specific genes with SNPs as preliminary candidates was based on associations with reproductive and fertility traits. Validation of candidate SNPs and genotype-to-phenotype analyses were conducted in a separate cohort of lactating primiparous Holstein cows (n = 500). After genotyping, candidate SNPs were filtered using a quality control pipeline via PLINK software. Continuous numeric and binary models from reproductive traits were evaluated using the mixed procedure for a generalized linear model-one way ANOVA or logistic regression, respectively. Results: Sixty-nine candidate SNPs were initially identified, but only 23 passed quality control procedures. Ultimately, the study incorporated 466 observations for statistical analysis after excluding animals with missing genotypes or phenotypes. Significant (p <0.05) associations with fertility traits were identified in seven of the 23 SNPs: DSC2 (cows with the A allele were older at first calving); SREBF1 and UBD (cows with the T or G alleles took longer to conceive); DECR1 and FASN (cows with the C allele were less likely to become pregnant at first artificial insemination); SREBF1 and BOLA-DMB (cows with the T allele were less likely to be pregnant at 150 days in milk). It was also determined that two candidate SNPs within the DSC2 gene were tag SNPs. Only DSC2 SNPs had an important allele substitution effect in cows with the G allele, which had a decreased age at first calving by 10 days. Discussion: Candidate SNPs found in this study could be used to develop genetic selection tools to improve fertility traits in dairy production systems. [ABSTRACT FROM AUTHOR]

Bovine viral diarrhea virus (BVDV) infections cause USD 1.5–2 billion in losses annually. Maternal BVDV after 150 days of gestation causes transient fetal infection (TI) in which the fetal immune response clears the virus. The impact of fetal TI BVDV infections on postnatal growth and white blood cell (WBC) methylome as an index of epigenetic modifications was examined by inoculating pregnant heifers with noncytopathic type 2 BVDV or media (sham-inoculated controls) on Day 175 of gestation to generate TI (n = 11) and control heifer calves (n = 12). Fetal infection in TI calves was confirmed by virus-neutralizing antibody titers at birth and control calves were seronegative. Both control and TI calves were negative for BVDV RNA in WBCs by RT-PCR. The mean weight of the TI calves was less than that of the controls (p < 0.05). DNA methyl seq analysis of WBC DNA demonstrated 2349 differentially methylated cytosines (p ≤ 0.05) including 1277 hypomethylated cytosines, 1072 hypermethylated cytosines, 84 differentially methylated regions based on CpGs in promoters, and 89 DMRs in islands of TI WBC DNA compared to controls. Fetal BVDV infection during late gestation resulted in epigenomic modifications predicted to affect fetal development and immune pathways, suggesting potential consequences for postnatal growth and health of TI cattle. [ABSTRACT FROM AUTHOR]

Fetal infection of bovine viral diarrhea virus (BVDV) before d 125 to 150 of gestation results in the birth of immunotolerant, persistently infected (PI) calves. Infection of BVDV during late gestation results in transient fetal infections (TI). Unvaccinated, yearling Hereford heifers (n = 25), seronegative for antibodies to BVDV1 and BVDV2, were bred by artificial insemination with X chromosome-bearing sperm from an Angus sire to examine the impact of TI on postnatal growth, estimated dry matter digestibility, blood glucose concentrations, and carcass characteristics. On d 175 of pregnancy, heifers were intranasally inoculated with either DMEM + 2% horse serum (sham control) to generate control calves or 4.0 log TCID50 noncytopathic type2 BVDV to generate TI calves. All sham-inoculated control dams remained seronegative, and all BVDV-inoculated dams seroconverted by d 14 post-inoculation. Sham-inoculated control dams (n = 12) and BVDV-inoculated dams (n = 12) gave birth to live calves. All control offspring were seronegative, and all TI offspring were seropositive for antibodies to type 2 BVDV at birth. All offspring were raised on pasture until weaning. At weaning, all calves were transported to our feedlot research facility, housed in one group feedlot pen, and transitioned to a high-energy concentrate-based diet until reaching an approximate BW of 600 kg. Upon arrival at the feedlot, all animals received a standard heifer growth implant, a modified live viral vaccine containing IBR-BRSV-PI3 and were dewormed. Heifer BW and jugular blood samples were collected every 28 d. On d 84 of the feeding period, titanium dioxide was added to the diet of 12, age-paired, individually fed, heifers (3 control and 3 TI heifers; approximately 1 yr of age) for 28 d and used to estimate dry matter digestibility. After approximately 280 d on feed heifers were transported to a USDAinspected abattoir and harvested. The TI heifers hadlighter birth weights (P < 0.03) and final BW (P < 0.04) when compared with control heifers. Average daily gain was greater (P < 0.01) in control compared with TI heifers. Blood glucose concentrations were similar between control and TI heifers at all sampling time points. Dry matter intake of individually fed heifers was similar across treatments. TI heifers had a 2.2% lesser (P < 0.05) dry matter digestibility and lighter (P < 0.01) hot carcass weights compared with controls. These data suggest that TI fetal BVDV infection negatively impacts growth throughout the feeding period, possibly by impacting gastrointestinal tract function. This research was supported by USDA-NIFA Grant # 2019-67015-29866. [ABSTRACT FROM AUTHOR]

The objective of this experiment was to examine the impact of dams infected with bovine viral diarrhea virus (BVDV) during late gestation on inflammatory blood biomarkers in their offspring. Fetal infection with BVDV before d 125 to 150 of gestation results in the birth of immunotolerant, persistently infected (PI) calves. Infection of BVDV during late gestation results in transient fetal infections (TI). It was hypothesized that offspring of dams transiently infected with BVDV on d 175 of gestation would have greater inflammatory blood biomarkers than control offspring. Unvaccinated, yearling Hereford heifers (n = 24), seronegative for antibodies to BVDV1 and BVDV2 were bred by artificial insemination with X chromosomebearing sperm from an Angus sire. On d 175 of pregnancy, heifers were intranasally inoculated with either Dulbecco’s Modified Eagle Medium + 2% horse serum (sham control) or 4.0 log TCID50 noncytopathic type 2 BVDV to generate control and transient infected calves, respectively. All BVDV-inoculated dams seroconverted by d 14 post-inoculation and all sham-inoculated control dams remained seronegative. Twelve, seronegative control heifer calves were born to the control dams, and 11 seropositive (to type 2 BVDV) TI heifer calves were born to BVDV-inoculated dams. All offspring were raised on pasture until weaning. At weaning, all calves were transported to our feedlot research facility, housed in one group feedlot pen, and transitioned to a high-energy concentrate-based diet until reaching an approximate body weight (BW) of 600 kg. Upon arrival, all animals received a standard heifer growth implant containing 200 mg of testosterone propionate and 20 mg of estradiol benzoate, a modified live viral vaccine containing IBR-BRSV-PI3, and were dewormed. Jugular blood samples were obtained every 28 d until harvest (280 d on feed) and analyzed for biomarkers of inflammation. Data were analyzed as a randomized block design with the MIXED procedures of SAS. There was a treatment x time interaction (P < 0.05) for plasma ceruloplasmin and the oxidized form of glutathione. On d 0 and 28 of the feeding period, all biomarkers were similar across treatments. By d 56 of the feeding period, ceruloplasmin (P < 0.03) and the oxidized form of glutathione (P < 0.01), indicators of chronic inflammation, were increased in plasma samples from TI heifers compared with controls. These parameters remained greater in TI heifers compared with controls throughout the feeding period. There were no treatment or treatment x time interactions for plasma interferon (INF)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and haptoglobin. These data suggest that TI heifers may be experiencing a greater level of oxidative stress later in the finishing period. [ABSTRACT FROM AUTHOR]

Pulmonary hypertension, characterized by elevated pulmonary arterial pressure (PAP) and right ventricular hypertrophy, is caused by decreased atmospheric oxygen at high altitude. We hypothesized that maternal undernutrition programs right ventricle gene expression and sensitivity to increasing PAP at high altitude (2,183 m). On day 30 of gestation, forty Angus X Gelbvieh cows received diets to induce either gain (Control) or loss of body weight (Restricted) until day 125 of gestation. On day 126 of gestation, Restricted cows were realimented to achieve the same body weight as Controls by day 250. Parturition occurred naturally. PAP, which ranged from 40 to 114 mmHg, was determined in 15-mo-old steers from Control or Restricted cows before necropsy. At necropsy, hearts were collected from steers, separated into right and left ventricles, atria, and septa and weighed. Ventricular thickness was recorded. Eight Affymetrix bovine microarrays were screened [four high PAP (two Control and two Restricted) and four low PAP (two Control and two Restricted)] with right ventricle mRNA. This analysis revealed that pentraxin-related protein, interferon-related developmental regulator, and peroxisome proliferator-activated receptor-[gamma] coactivator-1[alpha] were differentially expressed (P < 0.05) in steer right ventricle from high-PAP cows compared with low-PAP cows. Also, activation peptide and pancreas cationic trypsinogen, [alpha]-actin, similar to ubiquitin carboxylesterase, were differently expressed (P < 0.05) in steers from Restricted cows compared with those from Control cows. Upregulated genes in high-PAP right ventricle have been associated with pathological cardiac hypertrophy. It is concluded that right ventricle gene expression may be differentially programmed by maternal undernutrition in the fetus during early gestation and may be detrimental to health and longevity of offspring, particularly at high altitude. right ventricle; high altitude

Bovine viral diarrhea virus (BVDV) infection during early gestation results in persistently infected (PI) immunotolerant calves that are the primary reservoirs of the virus. Pathologies observed in PI cattle include congenital defects of the brain, heart, and bone as well as marked functional defects in their immune system. It was hypothesized that fetal BVDV infection alters T cell activation and signaling genes by epigenetic mechanisms. To test this, PI and control fetal splenic tissues were collected on day 245 of gestation, 170 days post maternal infection. DNA was isolated for reduced representation bisulfite sequencing, protein was isolated for proteomics, both were analyzed with appropriate bioinformatic methods. Within set parameters, 1951 hypermethylated and 691 hypomethylated DNA regions were identified in PI compared to control fetuses. Pathways associated with immune system, neural, cardiac, and bone development were associated with heavily methylated DNA. The proteomic analysis revealed 12 differentially expressed proteins in PI vs. control animals. Upregulated proteins were associated with protein processing, whereas downregulated proteins were associated with lymphocyte migration and development in PI compared to control fetal spleens. The epigenetic changes in DNA may explain the immune dysfunctions, abnormal bone formation, and brain and heart defects observed in PI animals. [ABSTRACT FROM AUTHOR]

Abstract Background The progression of implantation and placentation in ruminants is complex and is regulated by interplay between sex steroids and local signaling molecules, many of which have immune function. Chemokines and their receptors are pivotal factors in implantation and vascularization of the placenta. Based on known critical roles for chemokine receptor 4 (CXCR4) during early pregnancy in other species, we hypothesized that CXCR4 and its ligand CXCL12 would increase in the endometrium and conceptus in response to implantation in ewes. The objectives of the current study were to determine if CXCL12 and CXCR4 were upregulated in: endometrium from pregnant compared to non-pregnant ewes and in, conceptuses, cotyledons, caruncles and intercaruncular tissue. Methods Tissues were collected from sheep on Days 12, 13, 14, and 15 of either the estrous cycle or pregnancy and from pregnant ewes on Days 35 and 50. Blood samples from jugular and uterine vein were also collected on all days. Conceptuses were collected from mature ewes on Days 13, 15, 16, 17, 21 and 30 of gestation. Real time PCR was used to determine relative mRNA concentrations for CXCL12 and CXCR4 and Western blot analysis was employed to confirm protein concentration. Results Differences described are P < 0.05. In the endometrium, CXCR4 mRNA and protein was greater on Day 15 of pregnancy compared to the estrous cycle. CXCL12 and CXCR4 mRNA in conceptuses was greater on Days 21 and 30 compared to earlier days. CXCL12 mRNA was greater in cotyledons on Day 35 compared to Day 50. On Day 35 of gestation, CXCR4 was greater compared to Day 50 in caruncle and intercaruncular tissue. White blood cells obtained from jugular and uterine vein collection had the greatest mRNA concentration of CXCL12 on Day 35 of pregnancy. Conclusions A comprehensive analysis of CXCL12 and CXCR4 expression in fetal and maternal tissues during early pregnancy is reported with noteworthy differences occurring during implantation and placentation in sheep. We interpreted these data to mean that the CXCL12/CXCR4 pathway is activated during implantation and placentation in sheep and is likely playing a role in the communication between trophoblast cells and the maternal endometrium.

Abstract Background Isg15 covalently modifies murine endometrial proteins in response to early pregnancy. Isg15 can also be severed from targeted proteins by a specific protease called Ubp43 (Usp18). Mice lacking Ubp43 (null) form increased conjugated Isg15 in response to interferon. The Isg15 system has not been examined in chorioallantoic placenta (CP) or mesometrial (MM) components of implantation sites beyond 9.5 days post coitum (dpc). It was hypothesized that deletion of Ubp43 would cause disregulation of Isg15 in implantation sites, and that this would affect pregnancy rates. Methods Heterozygous (het) Ubp43 mice were mated and MM and CP implantation sites were collected on 12.5 and 17.5 days post-coitum (dpc). Results Free and conjugated Isg15 were greater on 12.5 versus 17.5 dpc in MM. Free and conjugated Isg15 were also present in CP, but did not differ due to genotype on 12.5 dpc. However, null CP had greater free and conjugated Isg15 when compared to het/wt on 17.5 dpc. Null progeny died in utero with fetal genotype ratios (wt:het:null) of 2:5:1 on 12.5 and 2:2:1 on 17.5 dpc. Implantation sites were disrupted within the junctional zone and spongiotrophoblast, contained less vasculature based on lectin B4 staining and contained greater Isg15 mRNA and VEGF protein in Ubp43 null when compared to wt placenta. Conclusion It is concluded that Isg15 and its conjugates are present in implantation sites during mid to late gestation and that deletion of Ubp43 causes an increase in free and conjugated Isg15 at the feto-maternal interface. Also, under mixed genetic background, deletion of Ubp43 results in fetal death.

Interferon-stimulated gene (ISG) 15, which is found to mediate antiviral responses, is upregulated within the endometrium in response to the developing embryo during early pregnancy, where the hinge region is found to contribute to the instability of rbovISG15. The models of bovISG15, site-directed mutant of rbovISG15 (Cys80ISG15) and Ovine (ov) ISG15 have confirmed that the hinge region between the two ubiquitin domains have destabilized rbovISG15 in solution.