The Influence of Preovulatory Estradiol on Uterine Transcriptomics and Proteomics Around Maternal Recognition of Pregnancy in Beef Cattle

Preovulatory estradiol has been reported to play a crucial role in pregnancy establishment and maintenance, but the mechanism by which estradiol exerts its effects has not been well characterized. Specifically, the interactions between the maternal uterine environment and the developing conceptus can greatly impact pregnancy success or loss. The objective of this dissertation is to determine the effects of preovulatory estradiol exposure on uterine and trophectoderm transcriptomes, and uterine luminal fluid (ULF) protein composition. Beef cows/heifers were synchronized, artificially inseminated (d 0), and grouped into either high (highE2) or low (lowE2) preovulatory estradiol. Uteri were flushed to collect d16 conceptuses either nonsurgically or following slaughter, and endometrial biopsies (n=29) were collected from the ipsilateral uterine horn. Real-Time PCR (RT-PCR) was performed on trophectoderm (TE; n=21) RNA to measure the relative abundance of IFNT, PTGS2, TM4SF1, C3, FGFR2, and GAPDH. Total cellular RNA was extracted from endometrium for RNA sequencing. ULF pools (n=28) for the following groups: highE2/noconceptus, highE2/conceptus, lowE2/noconceptus, and lowE2/conceptus were analyzed using a 2D LC-MS/MS based 8plex iTRAQ method. RT-PCR data were analyzed using the MIXED procedure in SAS. Transcript abundances in the endometrium were quantified using kallisto, differentially expressed genes (DEGs) were determined using DESeq2 (FDR2), and IPA was used for pathway analysis. Scaffold Q+ was used to quantitate peptide and protein identifications in the ULF. There were no differences in mRNA abundances in TE, but there were 432 DEGs among the highE2/conceptus versus lowE2/conceptus groups, 253 were downregulated (CR2, CDH4, TROAP, COL1A2) and 179 were upregulated (PRSS8, FABP3, IDO1, MUC13, CXCL10) in the highE2/conceptus group. There were 48 differentially expressed proteins (DEPs) among the highE2/conceptus and lowE2/conceptus groups (19 upregulated, 29 downregulated in the highE2/conceptus group), 6 of these were differentially expressed (FD

The Effects of Preovulatory Estradiol on the Uterine Environment and Conceptus Survival From Fertilization to Maternal Recognition of Pregnancy

Preovulatory estradiol has been reported to play a critical role in pregnancy establishment and embryonic survival, but the mechanism by which estradiol exerts its effects has not been well characterized. The objective of this thesis is to determine the effects of preovulatory estradiol on the uterine environment and conceptus survival from fertilization to maternal recognition of pregnancy. Beef cows/heifers were synchronized with the CO-Synch protocol and AIed (d 0). Cows were classified by estrus expression (estrus and no estrus), and uteri were flushed to collect d16 conceptuses nonsurgically (Rep 1; n = 29), or following slaughter (Rep 2; n = 37). Uterine luminal fluid (ULF) was analyzed for protein, glucose, and interferon tau (IFNT) concentrations. For replicate 1, total cellular RNA was extracted from blood leukocytes (d 16) to determine expression of interferon-stimulated genes (ISG): ISG-15, OAS-1, and MX2. For replicate 2, total cellular RNA was extracted from caruncular (CAR) and intercaruncular (INCAR) endometrial tissue to determine relative abundance of select glucose transporters (SLC2A1, SLC2A4, SLC2A5, and SLC5A1). There was no difference in conceptus recovery rate between estrus and no estrus cows (P = 0.20; 48% vs 29%) or between replicates (P = 0.46; 44% vs 33%). There were no differences between estrus and no estrus cows for ULF protein concentration (P = 0.36; 2222 ± 513 vs 1547 ± 525 mg/mL). There was no difference (P \u3e 0.20) in d 16 expression of ISG-15, OAS-1, or MX2 between estrus and no estrus cows, nor a difference between cows in which a conceptus was or was not recovered. In addition, there were no differences in IFNT concentrations in the ULF among estrus and no estrus cows (P = 0.42), nor a difference among cows that did and did not have a conceptus recovered from them (P = 0.71). Cows that exhibited estrus had greater glucose concentrations in ULF (P = 0.05; 51 ± 1.86 vs 45 ± 1.92 mg/dL) compared to no estrus cows, but there was no difference in protein concentration in the ULF (P = 0.36; 2222 ± 513 vs 1547 ± 525 mg/mL). Cows in which a conceptus was recovered had greater concentrations of protein in the ULF (P = 0.05; 2643 ± 585 mg/mL) compared to cows in which a conceptus was not recovered (1126 ± 463 mg/mL), glucose concentration was similar between groups (P = 0.29; 47 ± 2.12 vs 50 ± 1.70 mg/dL). For replicate 2, in both CAR and INCAR endometria, cows that exhibited estrus had greater abundance of SLC2A1 (P = 0.05) and SLC5A1 (P \u3c 0.04) mRNA. Presence of a conceptus tended to increase (P = 0.10) abundance of SLC5A1 mRNA in INCAR tissue, but had no effect (P \u3e 0.13) on abundance of SLC2A1 mRNA in either tissues or SLC5A1 mRNA in CAR tissue. In CAR tissue, cows from which a conceptus was recovered had decreased SLC2A4 mRNA abundance (P = 0.04), but there was no effect of estrus (P = 0.14) and no effect of estrus or conceptus in INCAR tissue. There was no difference in SLC2A5 mRNA abundance between estrus and no estrus cows (P \u3e 0.20), nor between conceptus and no conceptus cows (P \u3e 0.58) in CAR or INCAR tissue. In summary, conceptus recovery rates, IFNT, and protein concentration in ULF did not differ between cows that did or did not exhibit estrus, but ULF glucose content was greater in cows that exhibited estrus. There was no difference in ULF glucose concentration or IFNT between cows that did and did not have a conceptus, but ULF protein concentration was greater in cows from which a conceptus was recovered. Thus, there was no indication of increased conceptus survival to d 16 of pregnancy based on estrus expression, but ULF glucose and protein concentration changed based on estrus expression and conceptus presence

Proteomic Analysis of Epididymal and Ejaculated Sperm and Respective Fluids

Study Description: Ejaculated and epididymal semen was collected from mature Angus bulls (n = 9), and then centrifuged to separate sperm and fluid. Fluids were collected and sperm pellets were resuspended in a high ionic solution and vortexed to remove loosely attached proteins. Sperm samples were centrifuged, and the supernatant was collected. Samples collected for protein analysis were snap frozen in liquid nitrogen and stored at -80 ºC. Protein analysis was performed by liquid chromatography with tandem mass spectrometry (LCMS/MS). Yearling Angus cross bulls (n = 40) were used for sperm cultures. Ejaculated (n = 20) and epididymal (n = 20) sperm were collected, diluted and cultured in a commercial media at pH 5.8, 6.8 and 7.3, at 4 ºC and evaluated for motility and viability every 24 h until motility was below 20%. There was an effect of pH, time and pH by time interaction for motility and viability for both ejaculated and epididymal sperm (P ≤ 0.05). At 216 h of incubation epididymal sperm at pH 7.3 and ejaculated sperm at pH 6.8 dropped below 20% motility. Overall, in all samples, a total of 458 unique proteins were identified. It was identified that ejaculated fluid and ejaculated sperm had 178 and 298 proteins, respectively. Also, it was identified that epididymal fluid and epididymal sperm had 311 and 344 proteins, respectively. There were Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways significantly enriched (FDR \u3c 0.05) for ejaculated fluid (n = 8), epididymal fluid (n = 24), ejaculated sperm (n = 10), and epididymal sperm (n = 18). The most important KEGG pathway identified was the metabolic pathway. Within the metabolic pathway the glycolysis/gluconeogenesis, pentose phosphate, and glutathione metabolism pathways were significantly enriched among proteins only present in epididymal samples. Other proteins identified that may be related to epididymal sperm’s increased longevity were peroxidases and glutathione peroxidases for their antioxidant properties

Proteomic analyses identify differences between bovine epididymal and ejaculated spermatozoa that contribute to longevity

Sperm are stored for extended periods of time in the epididymis, but upon ejaculation motility is increased and lifespan is decreased. The objective of this study was to identify differences in proteins between epididymis and ejaculated samples that are associated with longevity. Ejaculated semen was collected from mature Angus bulls (n = 9); bulls were slaughtered and epididymal semen was collected. Epididymal and ejaculated semen were centrifuged to separate sperm and fluid. Fluids were removed and sperm pellets were resuspended in a high ionic solution and vortexed to remove loosely attached proteins. Sperm samples were centrifuged, and the supernatant was removed; both fluid and sperm samples were snap frozen in liquid nitrogen and stored at -80 oC. Protein analysis was performed by LCMS/MS. A different group of yearling Angus cross bulls (n = 40) were used for sperm cultures. Ejaculated (n = 20) and epididymal (n = 20) semen were diluted and cultured in a commercial media at pH 5.8, 6.8 and 7.3, at 4 oC. Sperm were evaluated for motility and viability every 24 h until motility was lower than 20%. There was an effect of pH, time and pH by time interaction for motility and viability for both ejaculated and epididymal sperm (P ≤ 0.05). At 216 h of incubation epididymal sperm at pH 7.3 and ejaculated sperm at pH 6.8 reached motility below 20%. A total of 458 unique proteins were identified; 178, 298, 311, and 344 proteins were identified in ejaculated fluid, ejaculated sperm, epididymal fluid and epididymal sperm, respectively. There were 8, 24, 10, and 18 significant KEGG pathways (FDR \u3c0.05) for ejaculated fluid, epididymal fluid, ejaculated sperm, and epididymal sperm, respectively. The metabolic pathway was identified as the most important KEGG pathway; glycolysis/gluconeogenesis, pentose phosphate, and glutathione metabolism pathways were significant among proteins only present in epididymal samples within the metabolic pathway. Other proteins identified that may be related to epididymal sperm\u27s increased longevity were peroxidases and glutathione peroxidases for their antioxidant properties. In summary, energy metabolism in the epididymis appears to be more glycolytic compared to ejaculated and epididymis sperm have a larger number of antioxidants available which may be helping to maintain sperm in a quiescent state. Epididymal sperm remained viable (membrane integrity) longer than ejaculated sperm when cultured at the same pH

Influence of estradiol on bovine trophectoderm and uterine gene transcripts around maternal recognition of pregnancy

Embryo survival and pregnancy success is increased among animals that exhibit estrus prior to fixed time-artificial insemination, but there are no differences in conceptus survival to d16. The objective of this study was to determine effects of preovulatory estradiol on uterine transcriptomes, select trophectoderm (TE) transcripts, and uterine luminal fluid proteins. Beef cows/heifers were synchronized, artificially inseminated (d0), and grouped into either high (highE2) or low (lowE2) preovulatory estradiol. Uteri were flushed (d16); conceptuses and endometrial biopsies (n = 29) were collected. RNA sequencing was performed on endometrium. Real-time polymerase chain reaction (RT-PCR) was performed on TE (n = 21) RNA to measure relative abundance of IFNT, PTGS2, TM4SF1, C3, FGFR2, and GAPDH. Uterine fluid was analyzed using 2D Liquid Chromatography with tandem mass spectrometry-based Isobaric tags for relative and absolute quantitation (iTRAQ) method. RT-PCR data were analyzed using the MIXED procedure in SAS. There were no differences in messenger RNA (mRNA) abundances in TE, but there were 432 differentially expressed genes (253 downregulated, 179 upregulated) in highE2/conceptus versus lowE2/conceptus groups. There were also 48 differentially expressed proteins (19 upregulated, 29 downregulated); 6 of these were differentially expressed (FDR \u3c 0.10) at the mRNA level. Similar pathways for mRNA and proteins included: calcium signaling, protein kinase A signaling, and corticotropin-releasing hormone signaling. These differences in uterine function may be preparing the conceptus for improved likelihood of survival after d16 among highE2 animals

Effects of preovulatory estradiol on uterine environment and conceptus survival from fertilization to maternal recognition of pregnancy

Preovulatory estradiol is known to impact embryo quality and survival. The objective of this study was to determine the effects of preovulatory estradiol on the uterine environment and conceptus survival through maternal recognition of pregnancy. Beef cows/heifers were AIed following induced ovulation. Cows were grouped into high and low preovulatory estradiol. Conceptuses were collected on day 16 nonsurgically (Rep 1; n = 20), or following slaughter (Rep 2; n = 29). Blood was collected to determine plasma glucose concentrations, and uterine luminal fluid (ULF) was analyzed for protein, glucose, and interferon tau (IFNT) concentrations. Total cellular RNA was extracted from caruncular (CAR) and intercaruncular (INCAR) endometrial tissue. There was no effect of preovulatory estradiol on conceptus recovery rate (P = 0.38) or on apoptosis rate in the trophectoderm (P = 0.64). Cows in which a conceptus was recovered had greater concentrations of protein in the ULF (P = 0.04). Animals with elevated preovulatory estradiol had greater endometrial abundance of SLC2A1 (P = 0.05) and SLC5A1 (P = 0.04) in both INCAR and CAR tissue. Presence of a conceptus also tended to increase (P = 0.10) abundance of SLC5A1 in INCAR. In CAR tissue, cows with a conceptus had decreased SLC2A4 abundance (P = 0.05). In summary, conceptus recovery rates, apoptosis in the trophectoderm, IFNT, glucose, and protein concentration in ULF did not differ between cows that did or did not have an increase in preovulatory estradiol concentrations. Thus, there is no indication of increased conceptus survival to day 16 of pregnancy based on estradiol concentrations

Differences in Relative Abundance of GnRH-I and GnRH-II in Granulosa Cells of Bovine Antral Follicles at Specific Stages of Follicular Development

Increased estradiol is the primary signal to initiate standing estrus, and cows that exhibit estrus prior to fixed-time artificial insemination have greater pregnancy rates than cows that do not. Therefore, understanding what factors may be present at the ovary that may regulate estradiol production is critical. A previous study reported that bovine follicles with greater follicular fluid concentrations of estradiol had decreased expression of GnRH-I and GnRH-II in granulosa cells (GC). The objective of this study was to characterize relative abundance of GnRH-I and -II mRNA within GC of follicles at specific stages of development. Beef cows were synchronized, and ovaries were collected at specific stages of follicular development [pre-selection (PRE), post-selection (POST), and post-selection 24 h after luteal regression (POST-PG)]. All surface follicles were classified as small (\u3c 5mm), medium (5 - 8mm), or large (\u3e 8mm) and aspirated to collect GC. Large follicles from each animal were kept separate and all other follicles were pooled by size within animal (n = 27, 27, and 18 for small, medium, and large). Total cellular RNA was extracted, and RT-PCR was performed for GnRH-I, GnRH-II, and GAPDH. Data were analyzed using the MIXED procedure of SAS. Across all follicles, GnRH-I and GnRH-II expression were not influenced by stage (P = 0.27) but were influenced by size (P \u3c 0.01). Smalls (4.55 ± 0.39 and 3.91 ± 0.44, respectively) had greater expression (P ≤ 0.01) of GnRH compared to mediums (0.83 ± 0.39 and 1.41 ± 0.44, respectively) and larges (0.52 ± 0.47 and 2.12 ± 0.54, respectively). There was also a stage by size interaction (P \u3c 0.01). POST (P \u3c 0.01) and POST-PG (P ≤ 0.08) smalls had or tended to have increased expression compared to PRE smalls, but PRE mediums had increased expression (P \u3c 0.03) compared to POST-PG mediums. When only the largest follicle for each animal was evaluated, stage of development influenced expression of GnRH-I (P = 0.03) but not GnRH-II (P = 0.91). For GnRH-I, PRE tended (P = 0.09; 2.29 ± 0.55) to have increased expression compared to POST (0.92 ± 0.55) and did have greater expression compared to POST-PG (P = 0.01; 0.11 ± 0.55). Thus, GnRH within antral follicles may be a key regulator of the follicle’s ability to produce estradiol

Effects of preovulatory estradiol on uterine environment and conceptus survival from fertilization to maternal recognition of pregnancy

Preovulatory estradiol is known to impact embryo quality and survival. The objective of this study was to determine the effects of preovulatory estradiol on the uterine environment and conceptus survival through maternal recognition of pregnancy. Beef cows/heifers were AIed following induced ovulation. Cows were grouped into high and low preovulatory estradiol. Conceptuses were collected on day 16 nonsurgically (Rep 1; n = 20), or following slaughter (Rep 2; n = 29). Blood was collected to determine plasma glucose concentrations, and uterine luminal fluid (ULF) was analyzed for protein, glucose, and interferon tau (IFNT) concentrations. Total cellular RNA was extracted from caruncular (CAR) and intercaruncular (INCAR) endometrial tissue. There was no effect of preovulatory estradiol on conceptus recovery rate (P = 0.38) or on apoptosis rate in the trophectoderm (P = 0.64). Cows in which a conceptus was recovered had greater concentrations of protein in the ULF (P = 0.04). Animals with elevated preovulatory estradiol had greater endometrial abundance of SLC2A1 (P = 0.05) and SLC5A1 (P = 0.04) in both INCAR and CAR tissue. Presence of a conceptus also tended to increase (P = 0.10) abundance of SLC5A1 in INCAR. In CAR tissue, cows with a conceptus had decreased SLC2A4 abundance (P = 0.05). In summary, conceptus recovery rates, apoptosis in the trophectoderm, IFNT, glucose, and protein concentration in ULF did not differ between cows that did or did not have an increase in preovulatory estradiol concentrations. Thus, there is no indication of increased conceptus survival to day 16 of pregnancy based on estradiol concentrations

Influence of microRNAs from Semen on Bovine Fertility

The objective of this study was to compare the miRNAs within sperm cells of bulls considered to have high and low fertility

Influence of Bovine Viral Diarrhea Virus Infection on Artificial Insemination Conception and Breeding Season Pregnancy Success in Vaccinated Beef Herds

Bovine Viral Diarrhea Virus (BVDV) causes reproductive economic losses in cattle. The objective of this study was to evaluate the influence of BVDV infection on reproductive success. Vaccinated cows (n = 370) and heifers (n = 528) from nine different herds were synchronized using the 7-day CO-Synch + CIDR protocol and were bred using fixed-time artificial insemination (FTAI). On d 28 following insemination, blood samples were collected and pregnancy status was determined. Non-pregnant animals were resynchronized and FTAI occurred a second time. In six herds, bulls were comingled with females beginning 10-15 d after the second AI. Final pregnancy status was determined 33-80 d following the first pregnancy diagnosis. Blood samples were tested for the presence of BVDV antigen using the IDEXX BVDV PI X2 Kit. Animals that tested positive were considered infected with BVDV at the time of blood collection. Herds were determined to be BVDV infected by the presence of at least one animal having a positive test for antigen (n = 4 infected herds, n = 5 non-infected herds). Statistical analyses were performed using the GLIMMIX procedure of SAS with herd as a random variable. Herds that had evidence of BVDV infection at d 28 following insemination had significantly decreased (P \u3c 0.01) first service AI conception rates compared to herds that had no evidence of infection (34 ± 2.3% vs. 54 ± 2.3%, respectively). Additionally, breeding season pregnancy rates were decreased (P \u3c 0.01) in BVDV infected herds compared to non-infected herds (69 ± 3.4% vs. 80 ± 3.6%, respectively). There was no significant effect of BVDV infection status on embryonic loss (P = 0.42) or percentage of animals which lost a pregnancy and rebred by the end of the breeding season (P = 0.63). In conclusion, BVDV infection in well vaccinated herds had a significant negative impact on both first service AI conception rate and overall breeding season pregnancy success