Early Pregnancy In Cattle Research Articles

Ultrasonographic diagnosis of early pregnancy in cattle using different ultrasound systems.

To evaluate the efficiency of different ultrasound devices in achieving an early diagnosis of pregnancy in dairy herds. A total of 1976 Holstein Friesian cows and heifers were artificially inseminated (AI) according to the herd manager's regime. Pregnancy diagnostics were performed between day26 and 35 after AI using six different types of ultrasound systems (linear vs. sector scanners). Manual rectal palpation between day45 and 60 after AI was used as the gold standard for pregnancy diagnostics. Sensitivity (SENS), specificity (SPEC), positive (PPV) and negative predictive value (NPV) and diagnostic accuracy (ACC) of the diagnostic measures were determined. Average SENS was 82% (range 67.7-95.2%) with a mean SPEC of 73% (range 50.0-81.0%). ACC was 78.2% with a minimum of 64.6% and a maximum of 89.4%, depending on the ultrasound system. The PPV (ratio of the number of pregnant cows with a positive examination result to the number of cows actually pregnant) was 80.8% (range 59.1-88.1%), whereas the NPV (defined as the ratio of the number of cows correctly diagnosed negative to the number of cows actually open) was 74.4% (72.3-91.9%). Significant differences for these parameters were found depending on the ultrasound system used (p≤0.01; Cramer's V. = 0.14). Regardless of the ultrasound device used, early pregnancy diagnostics between day26 and 35 show a moderate diagnostic efficiency. Comparing the accuracy of the different devices, there may be a significant influence of type and technical parameters. Even though ultrasound systems with mechanical sector probes are not as convenient to use as systems with linear probes, according to this study, sector scanners are a reasonable alternative.

2 CIRCULATING microRNA SIGNATURES ASSOCIATED WITH EARLY BOVINE PREGNANCY

High-producing dairy cattle are characterised by low fertility, which extends calving intervals and reduces annual milk production. Early pregnancy diagnosis before Day 21 of gestation can shorten calving intervals, improve milk production, and increase overall profits from modern dairy herds. At present, accurate diagnosis is only possible after the third week of pregnancy. Circulating microRNA (miRNA) have been proposed as diagnostic biomarkers of numerous human conditions such as cancer and diabetes. Moreover, distinct circulating miRNA profiles have been associated with mid to late stages of human pregnancy. The aim of this study was to determine whether specific circulating miRNA signatures that occur during early pregnancy in cattle could be used for diagnostic purposes. To do this, we have compared miRNA levels in plasma samples from cycling, nonpregnant (n = 24) and cycling, early pregnant (D16, n = 11; D24, n = 11) Holstein heifers using next-generation sequencing and PCR arrays. Using small-RNA sequencing on 46 individual samples, we generated 2.8 million miRNA reads, and up to 328 miRNAs were detected in each sample. There were no significant differences (false discovery rate <0.1) in miRNA levels between nonpregnant and pregnant (D16, D24) heifers. In addition, 12 pooled samples (n = 9 for nonpregnant, n = 3 for D24) were analysed for the expression of 176 miRNA using PCR arrays; 16 miRNA had differentially expressed levels (2- to 3-fold; false discovery rate <0.1) in pregnant heifers. The QPCR validation of miRNA candidates obtained from the high-throughput analyses identified 6 miRNA that were differentially expressed in pregnant heifers. These included bta-miR-26a, bta-miR-29c, bta-miR-138, and bta-miR-204, which were differentially expressed at D16, bta-miR-1249, which was differentially expressed at D24, and hsa-miR-4532, which was differentially expressed at both D16 and D24 of pregnancy (P < 0.05, fold-change ≤2.4). The miRNA miR-26a and miR-204 have been previously associated with early (D20) and late pregnancy in pig and human respectively. Furthermore, miR-1249 has been associated with the regulation of ovarian function, and miR-26a is involved in immune regulation, suggesting a potential role for this miRNA as an immuno-suppressor during early pregnancy. In summary, we identified a miRNA signature associated with early pregnancy in cattle. We are currently using next-generation sequencing to identify differentially expressed miRNA at other stages of pregnancy, including Day-60 heifers, and this data will also be presented.This study was funded by Zoetis and BBSRC.

Changes in expression of Th1 and Th2 cytokines in bovine peripheral blood mononuclear cells during early pregnancy

Conceptus-derived signals are involved in the maternal systemic immune regulation through blood circulation and lymph circulation by peripheral blood mononuclear cells (PBMC) in cattle. There exists a Th2-biased response in maternal uterus during early pregnancy in cattle. However, it is unclear that the changes in expression of Th1 and Th2 cytokines in PBMC during early pregnancy in cows. At present study, the expression of Th1 and Th2 cytokines in PBMC were explored at day 18 non-pregnant cows, day 18 and day 30 pregnant cows. Our results showed that IFN-?, was attenuated in PBMC, but TNF-a and IL-4 in PBMC were up-regulated during early pregnancy in cows. However, the member of the Th2 cytokines, including IL-4, IL-5, IL-6, IL-10 and IL-13, was augmented in day 30 pregnant PBMC. In conclusion, there existed a Th2-biased response in PBMC during early pregnancy, which was essential for successful pregnancy in cattle.

'Conceptualizing' the Endometrium: Identification of Conceptus-Derived Proteins During Early Pregnancy in Cattle.

The aim of this study was to identify conceptus-derived proteins, in addition to IFNT, that may facilitate pregnancy recognition in cattle. Analysis of the protein content of the uterine luminal fluid (ULF) from cyclic heifers on Day 16 by nano liquid chromatography tandem mass spectrometry identified 334 proteins. Comparison of these data with 299 proteins identified in the ULF of pregnant heifers on Day 16 identified 85 proteins only present in the ULF of pregnant heifers. Analysis of Day 16 conceptus-conditioned culture medium revealed the presence of 1005 proteins of which 30 proteins were unique to ULF from Day 16 pregnant heifers. Of these 30 proteins, 12 had mRNA expression values at least 2-fold higher in abundance (P < 0.05) in the conceptus compared to the endometrium (ARPC5L, CAPG, CKMT1, CSTB, HSPA8, HSPE1, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, TKT) as determined by RNA sequencing. In addition, genes that have a significant biological interaction with the proteins (ACO2, CKMT1, CSTB, EEF2, GDI1, GLB1, GPLD1, HNRNPA1, HNRNPA2B1, HNRNPF, HSPA8, HSPE1, IDH2, KRT75, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, PSMA4, PSMB5, PSMC4, SERPINA3, TKT) were differentially expressed in the endometrium of pregnant compared to cyclic heifers during the pregnancy recognition period (Days 16-18). These results indicate that 30 proteins unique to ULF from pregnant heifers and produced by short-term in vitro cultured Day 16 conceptuses could potentially be involved in facilitating the interactions between the conceptus and the endometrium during the pregnancy recognition period.

Amino acids in the uterine luminal fluid reflects the temporal changes in transporter expression in the endometrium and conceptus during early pregnancy in cattle.

In cattle, conceptus-maternal interactions are critical for the establishment and maintenance of pregnancy. A major component of this early interaction involves the transport of nutrients and secretion of key molecules by uterine epithelial cells to help support conceptus development during the peri-implantation period of pregnancy. Objectives were to: 1) analyze temporal changes in the amino acid (AA) content of uterine luminal fluid (ULF) during the bovine estrous cycle; 2) understand conceptus-induced alterations in AA content; 3) determine expression of AA transporters in the endometrium and conceptus; and 4) determine how these transporters are modulated by (Progesterone) P4. Concentrations of aspartic acid, arginine, glutamine, histidine, lysine, isoleucine, leucine, phenylalanine and tyrosine decreased on Day 16 of the estrous cycle but increased on Day 19 in pregnant heifers (P<0.05). Glutamic acid only increased in pregnant heifers on Day 19 (P<0.001). Asparagine concentrations were greater in ULF of cyclic compared to pregnant heifers on Day 7 (P<0.05) while valine concentrations were higher in pregnant heifers on Day 16 (P<0.05). Temporal changes in expression of the cationic AA transporters SLC7A1 SLC7A4 and SLC7A6 occurred in the endometrium during the estrous cycle/early pregnancy coordinate with changes in conceptus expression of SLC7A4, SLC7A2 and SLC7A1 (P<0.05). Only one acidic AA transporter (SLC1A5) increased in the endometrium while conceptus expression of SLC1A4 increased (P<0.05). The neutral AA transporters SLC38A2 and SLC7A5 increased in the endometrium in a temporal manner while conceptus expression of SLC38A7, SLC43A2, SLC38A11 and SLC7A8 also increased (P<0.05). P4 modified the expression of SLC1A1, -1A4, -1A5, -38A2, -38A4, -38A7, -43A2, -6A14, -7A1, -7A5 and -7A7 in the endometrium. Results demonstrate that temporal changes in AA in the ULF reflect changes in transporter expression in the endometrium and conceptus during early pregnancy in cattle, some of which are modified by P4.

Neospora caninum infection during early pregnancy in cattle: how the isolate influences infection dynamics, clinical outcome and peripheral and local immune responses

This work studies the influence of Neospora caninum intra-species diversity on abortion outcome, infection dynamics in terms of parasite dissemination and peripheral-local immune responses in pregnant cattle. Animals were intravenously inoculated at day 70 of pregnancy with 107 tachyzoites of two isolates showing marked differences in virulence in vitro and in pregnant mouse models: Nc-Spain7, a high virulence isolate, and Nc-Spain8, a low-to-moderate virulence isolate. After inoculation, pregnancy was monitored, and dams were culled when foetal death was detected. Foetal mortality occurred in all infected heifers between days 24 and 49 post-infection (pi), however, it was detected sooner in Nc-Spain7-infected animals (median day = 34) than those inoculated with Nc-Spain8 (median day = 41) with a trend towards significance (P < 0.11). Similar histological lesions were observed in placentomes and in most of the foetuses from the two infected groups. However, parasites were more frequently detected in the placenta and foetuses by PCR and in the foetal brain by immunohistochemistry in Nc-Spain7-infected animals. Specific antibodies were detected starting at day 13 post-infection in all infected cattle, with higher IgG levels in Nc-Spain7-infected group. IFN-γ and IL-4 profiles also varied between infected groups in PBMC stimulation assays. Infected animals showed significant increases in their cytokine mRNA levels (IFN-γ, IL-4, IL-10, IL-12p40 and TNF-α) in the caruncle at time of foetal death. Differences between the infected groups were also observed for cytokine profiles. These results demonstrate the influence of the N. caninum isolate on foetal death outcome, infection dynamics and immune responses in cattle.

Early pregnancy diagnosis in dairy cows using circulating nucleic acids

Early and accurate pregnancy diagnosis in dairy cattle is a prerequisite for successful herd management. However, most of the currently available methods allow an early diagnosis only approximately 30 days after insemination. Recently, circulating nucleic acids (CNAs) have been used successfully as biomarkers in prenatal diagnosis at different gestational stages in human and animals. Here we show that CNAs can also be used as markers for the detection of early pregnancy in cattle. Serum samples were collected from multiparous pregnant (N = 24) and nonpregnant (N = 16) dairy cows at different days after insemination (Days 0, 20, and 40). Isolated serum DNA was preprocessed using a modified serial analysis of gene expression technique, which generated concatemerized short sequence tags for downstream next generation sequencing. Bioinformatic analysis identified sequence tags specific for pregnant dairy cows at Day 20 after insemination. The identified CNA-tags originated from repetitive regions of the bovine genome. Tag sequences that showed increased hit counts per animal were used to design quantitative real-time polymerase chain reaction assays. These quantitative polymerase chain reaction assays were applied to CNA samples from matched pregnant (N = 12) and nonpregnant cows (N = 16) at different times after insemination (Day 0, 20, and 40). At Day 20 after insemination the quantities of the interspersed repeats Art2A and BovB were increased in the pregnant cows compared with the nonpregnant control cows (P < 0.05). The best performing CNA biomarker BovB yielded an area under the receiver operating characteristic curve of 0.76. At a defined cutoff value, the pregnant and the control groups can be distinguished with a sensitivity of 83% and specificity of 75%. These results suggest that CNAs can be used as biomarkers for the detection of early pregnancy in cattle.

Is cortisol a modulator of interferon tau action in the endometrium during early pregnancy in cattle?

Glucocorticoids (GCs) were recently found to be potent modulators of the secretion of uterine prostaglandins (PGs) in ruminants. The aim of the present study was to examine whether GCs may serve as a mediator/modulator of interferon-τ (IFNT) action during early pregnancy in cows. We examined whether IFNT affects cortisol output and expression of GC receptors (NR3C1) and 11β-hydroxysteroid dehydrogenases (enzymes responsible for GC conversion: HSD11B1 and HSD11B2) in bovine endometrium. Endometrial tissues were collected from cyclic and pregnant cows on Days 16-17. Endometrial stromal and epithelial cells were isolated from uteri from cyclic cows in the early luteal phase (Days 2-5). The mRNA and protein expressions of NR3C1, HSD11Bs in endometrial strips and cultured cells were analyzed. Endometrial slices and isolated cells were incubated with cortisone in the presence or absence of IFNT and HSD11B1 activity was evaluated. IFNT increased HSD11B1 activity in endometrial strips and both types of endometrial cells. IFNT influenced NR3C1 and HSD11Bs mRNA and protein expression in epithelial and stromal cells. Expressions of HSD11Bs and NR3C1 mRNA and protein in bovine endometrium were different on Days 16-17 of the estrous cycle compared with early pregnancy. Cortisol changed basal and IFNT-stimulated PGE2 secretion in the bovine endometrium. The overall results suggest that cortisol acts as modulator and/or mediator of IFNT actions in bovine uterus and that IFNT regulates PG secretion by up-regulating local cortisol, resulting in the maintenance of the corpus luteum during early pregnancy in cattle.

Fibroblast Growth Factor 2 Promotes Primitive Endoderm Development in Bovine Blastocyst Outgrowths1

Primitive endoderm (PE) is the second extraembryonic tissue to form during embryogenesis in mammals. The PE develops from pluripotent cells of the blastocyst inner cell mass. Experimental results described herein provide evidence that FGF2 stimulates PE development during bovine blastocyst development in vitro. Bovine blastocysts were cultured individually on a feeder layer-free, Matrigel-coated surface in the presence or absence of FGF2. A majority of blastocysts cultures formed outgrowths (76.8%) and the rate of outgrowth formation was not affected by FGF2 supplementation. However, supplementation with FGF2 increased the incidence of PE outgrowths on Days 13 and 15 after in vitro fertilization. Presumptive PE cultures contained cells with a phenotype distinct from trophectoderm (TE). Cell identity was validated by expression of GATA4 and GATA6 mRNA and transferrin protein, all markers of the PE lineage. Expression of GATA4 occurred coincident with blastocyst expansion and hatching. These cells did not express IFNT and CDX2 (TE lineage markers). Profiles of FGF receptor (FGFR) isoforms were distinct between PE and TE cultures. Specifically, FGFR1b and FGFR1c were the predominant FGFR transcripts in PE whereas FGFR2b transcripts were abundant in TE. Supplementation with FGF2 increased the mitotic index of PE but not TE. Moreover, FGF signaling appears important for initiation of PE formation in blastocysts, presumably by lineage committal from NANOG-positive epiblast cells, because chemical disruption of FGFR kinase activity with PD173074 reduces GATA4 expression and increases NANOG expression. Collectively, these results indicate that FGF2 and potentially other FGFs specify PE formation and mediate PE proliferation during early pregnancy in cattle.

Endometrial expression of progesterone-induced blocking factor and galectins-1, -3, -9, and -3 binding protein in the luteal phase and early pregnancy in cattle

Progesterone-induced blocking factor (PIBF) and galectins modulate the maternal immune response during pregnancy. We hypothesized that the relative transcript abundance of the above genes would be different during the luteal phase/early pregnancy and would be affected by progesterone supplementation. To further test this, hypothesis protein expression analyses were carried out to evaluate the abundance and localization of LGALS9 and PIBF. Following estrus synchronization, heifers were inseminated (n = 140) or not (n = 70). Half the heifers in each status (cyclic or potentially pregnant) were randomly assigned to receive a progesterone-releasing intravaginal device (PRID) on day 3 after estrus, which elevated progesterone concentrations from day 3.5 to 8 (P < 0.05), resulting in four treatment groups: cyclic and pregnant heifers, each with normal and high progesterone. After confirmation of pregnancy status in inseminated animals, uterine tissue was collected on days 5, 7, 13, or 16 of the luteal phase of the cycle/pregnancy. Gene and protein expression was determined using Q-RT-PCR and IHC, respectively, on 5 heifers per treatment per time point (i.e., 80 in total). Progesterone concentrations did not affect expression of any of the genes (P > 0.05). LGALS9 and LGALS3BP were expressed at low levels in both cyclic and pregnant endometria until day 13. On day 16, expression increased only in the pregnant heifers (P < 0.0001). LGALS1 and LGALS3 decreased on day 7 (P < 0.0001) and remained low until day 16. Pregnancy had no effect on the expression of LGALS1, LGALS3, and PIBF. Additionally, LGALS9 and PIBF proteins were expressed in distinct uterine cell types. These results indicate that the galectins may be involved in uterine receptivity and/or implantation in heifers.

Leukotrienes modulate secretion of progesterone and prostaglandins during the estrous cycle and early pregnancy in cattle: an in vivo study

Recently, we showed that leukotrienes (LTs) regulate ovarian cell function in vitro. The aim of this study was to examine the role of LTs in corpus luteum (CL) function during both the estrous cycle and early pregnancy in vivo. mRNA expression of LT receptors (BLT for LTB(4) and CYSLT for LTC(4)), and 5-lipoxygenase (5-LO) in CL tissue and their localization in the ovary were studied during the estrous cycle and early pregnancy. Moreover, concentrations of LTs (LTB(4) and C(4)) in the CL tissue and blood were measured. 5-LO and BLT mRNA expression increased on days 16-18 of the cycle, whereas CYSLT mRNA expression increased on days 16-18 of the pregnancy. The level of LTB(4) was evaluated during pregnancy compared with the level of LTC(4), which increased during CL regression. LT antagonists influenced the duration of the estrous cycle: the LTC(4) antagonist (azelastine) prolonged the luteal phase, whereas the LTB(4) antagonist (dapsone) caused earlier luteolysis in vivo. Dapsone decreased progesterone (P(4)) secretion and azelastine increased P(4) secretion during the estrous cycle. In summary, LT action in the bovine reproductive tract is dependent on LT type: LTB(4) is luteotropic during the estrous cycle and supports early pregnancy, whereas LTC(4) is luteolytic, regarded as undesirable in early pregnancy. LTs are produced/secreted in the CL tissue, influence prostaglandin function, and serve as important factors during the estrous cycle and early pregnancy in cattle.

Evaluation of models to induce low progesterone during the early luteal phase in cattle

Two experiments were designed to evaluate models for generation of low circulating progesterone concentrations during early pregnancy in cattle. In Experiment 1, 17 crossbred heifers (Bos taurus) were assigned to either prostaglandin F 2α (PGF 2α) administration on Days 3, 3.5, and 4 (PG3; n = 9) or to control (n = 8). Blood samples were collected from heifers from Days 1 to 9 for progesterone assay. Progesterone concentrations were decreased (P < 0.03) between 18 and 48 h after first PGF 2α treatment in heifers assigned to PG3 compared with that of controls. In Experiment 2, 39 crossbred heifers detected in estrus were inseminated (Day 0) and assigned to either (1) PGF 2α administration on Days 3, 3.5, and 4 (PG3; n = 10), (2) PGF 2α administration on Days 3, 3.5, 4, and 4.5 (PG4; n = 10), (3) Progesterone Releasing Intravaginal Device (PRID) insertion on Day 4.5 with PGF 2α administration on Days 5 and 6 (PRID + PGF 2α; n = 10), or (4) control (n = 9). Blood samples were collected daily until Day 15, and conceptus survival rate was determined at slaughter on Day 16. Progesterone concentrations during the sampling period in the PG3 and PG4 groups did not differ but were less than that of controls (P < 0.01). After an initial peak, progesterone concentrations in the PRID + PGF 2α group were similar to that of controls. More heifers in the PG4 group (6 of 10) had complete luteal regression than did those in the PG3 group (3 of 10). Conceptus survival rate on Day 16 did not differ between groups. There was a significant correlation between progesterone concentration on Days 5 and 6 and conceptus size on Day 16. In summary, treatment with PGF 2α on Days 3, 3.5, and 4 postestrus appeared to provide the best model to induce reduced circulating progesterone concentrations during the early luteal phase in cattle.

Effects of Interferon-Tau on Cortisol Production by the Bovine Endometrial Tissues.

Glucocorticoids (GCs) are well known as anti-inflammatory agents limiting the production of cytokines and prostaglandin (PG)s in various target organs. Access of GCs to GC receptor (GC-R) in target tissues is regulated by two 11β-hydroxysteroid dehydrogenases (11-HSD1 and 2). We previously demonstrated that cyclic bovine endometrium expresses 11-HSD1 mRNA, and converts biologically inactive cortisone into cortisol. Moreover, we have shown that cortisol suppressed PGF2α without affecting PGE2 production in the cyclic bovine endometrium. Interferon-τ (IFN-τ) produced by the trophoblast at the time of recognition of pregnancy acts on maintaining corpus luteum (CL) function. Furthermore, some previous study demonstrated that in ewes the plasma concentrations of cortisol increased in response to IFN-τ. Therefore, we suggest that cortisol acts as a luteoprotective factor by selectively suppressing luteolytic PGF2α production by the pregnant bovine endometrium as INF-τ mediator. The aim of the present study was to determine if IFN-τ affects cortisol production and secretion in bovine endometrial tissues during the early pregnancy comparing to the responsible days of the estrous cycle. In Exp. 1., GC-R, 11-HSD1 and 11-HSD2 expressions on the gene (mRNA) and protein levels in pregnant (Days 16-18 after insemination) and nonpregnant (Days 16-18 after ovulation) endometrial strips were measured by using real time RT-PCR and Western-Blot analysis, respectively. In Exp. 2. (tissue culture study), the endometrial explants were taken from cows on Days 16-18 of the cycle and pregnancy. Endometrial slices from both groups of cows were incubated for 12 h with cortisone (30 nM) and/or IFN-τ (30 ng/ml), and then 11-HSD1 activity was evaluated by measuring cortisol level. Moreover, the endometrial stromal and epithelial cells were isolated enzymaticaly from bovine uteri in the early luteal stage of estrous cycle (Exp. 3., cell culture study). The cells were seeded in 48-well cluster dishes and then were exposed to cortisone (30 nM) and/or IFN-τ (30 ng/ml). After incubation, the conditioned media were collected, and then 11-HSD1 activity was evaluated by measuring cortisol level. Endometrial GC-R and 11-HSD1 mRNA expressions were higher from pregnant than those from non-pregnant cows (P<0.05). Moreover, 11-HSD2 mRNA expression was lower in cyclic cows than that in pregnant cows. Protein expression for 11-HSD1 was significantly higher in the tissues from pregnant than those from non-pregnant cows (P<0.05). 30 ng/ml IFN-τ significantly increased 11-HSD1 activity and cortisol secretion in endometrial explants, and in both stromal and epithelial cells compare to control (P<0.05). The overall results suggest that the level of cortisol is locally regulated by INF-τ in the pregnant bovine endometrium by up-regulating 11-HSD1 expression and activity. Based on our previous and present findings, IFN-τ seems to suppress PGFα secretion by up-regulating cortisol activation, resulting in the maintenance of the CL during early pregnancy in cattle. Supported by Grants-in-Aid for Scientific Research from the Polish Ministry of Sciences and Higher Education (N308 3279 33) and Japan Society for the Promotion of Science (JSPS, No.18380166). MM, DJS and KO were supported by the Japanese-Polish Joint Research Project under the agreement between JSPS and Polish Academy of Sciences. (poster)

REVIEW ARTICLE: Control of Interferon‐Tau Expression During Early Pregnancy in Ruminants

Problem A type I interferon (IFN), termed IFN‐tau (τ), is responsible for the establishment and maintenance of early pregnancy in cattle and sheep. The control of IFNτ gene (IFNT) expression is not completely understood.Method of study This article will provide an overview of recent progress made in understanding the dynamic expression pattern of IFNT during pre‐ and peri‐attachment conceptus development.Results Several ubiquitous transcriptional regulators (Ets2 and AP1) and at least two trophectoderm factors (Cdx2 and Dlx3) control IFNT transcription during early pregnancy. Co‐activators (CBP/p300) are also involved in this process. At least two uterine‐derived factors (GM‐CSF and FGF2) stimulate IFN‐τ production in bovine trophectoderm, and multiple signaling pathways are functionally linked with IFNT expression.Conclusion Although understanding the regulation of IFNT expression is far from complete, considerable progress has been made in uncovering how uterine‐derived factors and key placental‐specific transcriptional regulators control IFNT expression.

Several fibroblast growth factors are expressed during pre-attachment bovine conceptus development and regulate interferon-tau expression from trophectoderm

The trophectoderm-derived factor interferon tau (IFNT) maintains the uterus in a pregnancy-receptive state in cattle and sheep. Fibroblast growth factors (FGFs) are implicated in regulating IFNT expression and potentially other critical events associated with early conceptus development in cattle. The overall objectives of this work were to identify the various FGFs and FGF receptors (FGFRs) expressed in elongating pre-attachment bovine conceptuses and determine if these FGFs regulate conceptus development and/or mediate IFNT production. In vitro-derived bovine blastocysts and in vivo-derived elongated conceptuses collected at day 17 of pregnancy express at least four FGFR subtypes (R1c, R2b, R3c, R4). In addition, transcripts for FGF1, 2, and 10 but not FGF7 are present in elongated bovine conceptuses. The expression pattern of FGF10 most closely resembled that of IFNT, with both transcripts remaining low in day 8 and day 11 conceptuses and increasing substantially in day 14 and day 17 conceptuses. Supplementation with recombinant FGF1, 2 or 10 increased IFNT mRNA levels in bovine trophectoderm cells and bovine blastocysts and increased IFNT protein concentrations in trophectoderm-conditioned medium. Blastocyst development was not affected by any of the FGFs. In summary, at least four FGFRs reside in pre- and peri-attachment bovine conceptuses. Moreover, conceptuses express at least three candidate FGFs during elongation, the time of peak IFNT expression. These findings provide new insight for how conceptus-derived factors such as FGF1, 2, and 10 may control IFNT expression during early pregnancy in cattle.

Is Interleukin 1α a Luteotropic or Luteolytic Agent in Cattle?

The genes for interleukin (IL)1α and its functional receptors are present in the bovine female reproductive tract during the estrous cycle. IL1α stimulates prostaglandin (PG) production in cultured bovine endometrial cells. The target of IL1α for stimulating both luteotropic PGE2 and luteolytic PGF2α production is the stromal cells. The stimulatory effect of IL1α on PG production is mediated by differently modulating of phospholipase A2 and expression of respective PG synthases in the stromal cells. Moreover, IL1α stimulates PG secretion in the steroidogenic cells of bovine corpus luteum (CL) in vitro without any effect on progesterone (P4) production. However, physiological meaning of these findings is unclear. To determine physiological role of IL1α, it's influence on PG secretion by the cultured bovine endometrial tissues obtained from the cyclic or early pregnant cows, and the effect of IL1α on PG, progesterone (P4) output and the CL lifespan in vivo were examined. In Exp. 1, effects of IL1α with/without an embryonic signal (interferone-τ, INFτ) on PG secretion by cultured endometrial tissues were examined. Explants of endometrium (30-40 mg) obtained from the cows at Day 16/17 of the estrous cycle (Group 1; n=7) or early pregnancy (Day 16/17 after insemination, Group 2; n=6) were stimulated for 18-h with: saline (control), TNFα (10 ng/ml), IL1α (10 ng/ml), IFNα(30 ng/ml) or IL1α combination with IFNτ. Concentrations of PGE2 and PGF2α in the culture medium were measured by EIA. IL1α alone stimulated secretion of luteotropic PGE2 in endometrial tissues of both Group 1 and Group 2 (more than 200% of the control, P<0.01). IL1α also stimulated luteolytic PGF2α output from endometrial explants, but only during the late luteal phase (150%, P<0.05). This stimulatory effect of IL1α on PGF2α secretion was inhibited by INFτ (P<0.05). Moreover, IFNτ augmented the stimulatory effect of IL1a on PGE2 secretion (P<0.05). In Exp. 2, the effects of IL1α administrated into the uterine lumen on PGF2α, PGE2 and P4 secretions, and on the lifespan of the CL were determined in vivo. Saline (5 ml, control; n=5) or IL1α at different doses: 0,001 (n=3); 0.01 (n=5); 0.1 (n=5); 1 (n= 5) and 10 μg (n=3) in 5 ml of saline were applied at Day 16 of the estrous cycle. P4, PGE2 and PGFM (stable metabolite of PGF2α) concentrations in peripheral blood were measured by EIA. IL1α at doses 0.1 and 1 μg stimulated P4 and PGE2 output (P<0.01), and simultaneously inhibited spontaneous luteolysis with prolonging the lifespan of CL (P<0.01). Although only the lowest dose of IL1α (0.001 μg) caused a temporal increase in PGFM concentration during the experiment (until 12-h after application; P<0.05), any doses of IL1α had effect on the CL lifespan. Concluding, IL1α may play some important autocrine or paracrine roles in regulating PG production in the endometrium and CL during the estrous cycle and early pregnancy in cattle. IL1α increased P4 and luteotropic PGE2 concentrations in blood plasma that may result in appropriate development and function of the bovine CL during the estrous cycle. These luteotropic effects of IL1α during pregnancy may support the early embryo development and implantation, and may play a supporting role in the maternal recognition of pregnancy in cattle. Supported by Grant 2P06K02529.

Fibroblast Growth Factor-2 Is Expressed by the Bovine Uterus and Stimulates Interferon-τ Production in Bovine Trophectoderm

Uterine-derived factors are essential for conceptus development and secretion of the maternal recognition-of-pregnancy factor, interferon-tau (IFNT), in ruminant species. The objectives of this study were to determine whether fibroblast growth factor-2 (FGF-2) is expressed in the bovine uterus during early pregnancy in cattle and to determine whether FGF-2 supplementation affects IFNT mRNA and protein abundance in bovine trophectoderm. FGF-2 mRNA was present in endometrium throughout the estrous cycle and was localized to the luminal and glandular endometrial epithelium at d 17-18 after estrus in pregnant and nonpregnant cows. Immunoreactive FGF-2 protein was detected within the endometrium and in the uterine lumen at d 17-18 after estrus, and concentrations did not differ based on pregnancy status. In a bovine trophectoderm cell line, CT-1, supplementation of medium with at least 1 ng/ml FGF-2 increased the incorporation of [(3)H]thymidine into DNA. Similarly, IFNT secretion from CT-1 cells increased after FGF-2 supplementation (1-100 ng/ml) for 72 h. Abundance of IFNT mRNA in CT-1 cells increased after 24 h exposure to 1, 10, or 100 ng/ml FGF-2. In bovine blastocysts, FGF-2 supplementation did not affect cell number after 72 h of culture but did stimulate IFNT protein concentrations in conditioned medium. In summary, FGF-2 is present in the uterine lumen during early pregnancy and increases IFNT mRNA and protein abundance in trophectoderm. The magnitude by which FGF-2 stimulates IFNT expression suggests that this uterine-derived factor plays an active role in regulating the establishment and maintenance of pregnancy in ruminants.

161 EFFECT OF MELOXICAM TREATMENT ON PREGNANCY RATES IN HOLSTEIN HEIFERS

We previously reported that administration of flunixin meglumine two times at a critical stage approaching pregnancy recognition associated with corpus luteum maintenance increased early embryo survival and pregnancy rate via an additive antiluteolytic effect with the conceptus (Guzeloglu et al. 2006 Reprod. Fertil. Dev. 18, 183). In this study, the objective was to determine if a single administration of meloxicam, a non-steroid anti-inflammatory drug (NSAID) with a longer half-life, could be used instead of flunixin meglumine to avoid repeated injections in heifers at a critical stage following insemination to increase pregnancy rate due to its inhibitory effect on PGF2� synthesis. Eighty-five 15-18-month-old Holstein heifers were synchronized; following insemination (Day 0), the heifers were randomly assigned to receive a subcutaneous meloxicam injection (0.5 mg kg-1; n = 37) in the afternoon of Day 15 or left untreated as controls (n = 48). Pregnancy rates were defined as the percentage of heifers diagnosed pregnant by ultrasound between Days 31 and 38 after AI. The effect of treatment on pregnancy rates was analyzed by chi-square test. Meloxicam treatment on Day 15 after insemination dramatically decreased the pregnancy rates in the heifers (25 of 48 (52%) in the control group vs. 9 of 37 (24.3%) in the meloxicam-treated group; P &amp;lt; 0.01). This result indicates that administration of meloxicam around a time associated with pregnancy recognition and corpus luteum maintenance was harmful to the pregnancy even though the drug is considered to be safe during pregnancy in cattle. It is concluded that direct or indirect effects of meloxicam on the uterine environment and/or embryo are not favorable at least during early pregnancy in cattle. How meloxicam exerted its detrimental effect on pregnancy warrants further investigation.

Regulation of endometrial prostaglandin F 2α synthesis during luteolysis and early pregnancy in cattle

Luteal regression is caused by a pulsatile release of prostaglandin (PG) F 2α from the uterus in the late luteal phase in most mammals including cattle. Although it has been proposed in ruminants that pulsatile PGF 2α secretion is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF 2α, the bovine endometrium may possess other mechanisms for initiation of luteolytic PGF 2α secretion. It has been recently demonstrated that tumor necrosis factor-α (TNF-α) stimulates PGF 2α output from bovine endometrial tissue not only during the follicular phase but also during the late luteal phase, suggesting that TNF-α is a factor in the initiation of luteolysis in cattle. Furthermore, our recent study has shown that IFN-τ suppresses the action of TNF-α on PGF 2α synthesis by the bovine endometrium in vitro, suggesting that IFN-τ plays a luteoprotective role by inhibiting TNF-α-induced PGF 2α production in early pregnancy. On the other hand, factors other than oxytocin or TNF-α have also been suggested to be involved in the regulation of PGF 2α synthesis by bovine endometrium. The purpose of this review is to summarize our current understanding of the endocrine mechanisms that regulate the timing and pattern of uterine PGF 2α secretion during the estrous cycle and early pregnancy.

Reproductive performance during early pregnancy in cattle with contrasting early pregnancy rates

AbstractCauses of variation amongst cattle within a herd in their ability to initiate and maintain pregnancy are largely unknown. An experimental animal resource has recently been established to understand the biology of early reproductive performance. This paper summarises the results achieved during the establishment phase and from several experiments aimed at determining the physiological basis of the difference between sub-herds of contrasting pregnancy rates on Day 60. Each of 155 contemporary yearling heifers received 2 in vitro-produced embryos on 6 separate occasions during a 26-month period. Sixty days after transfer, pregnancy and twinning rates were determined ultrasonically, pregnancies terminated and the process repeated. The interval between successive transfers was greater than 100 days. Heifers were ranked on their aggregate pregnancy rate performance after 6 rounds of transfer, and the highest (High) and lowest (Low) 25 were retained. Differences in reproductive performance during the establishment phase of the herd are reported. In addition, several subsequent experiments examined ovarian follicle turnover and progesterone levels during an oestrous cycle, early embryo development after either AI or embryo transfer, and protein, interferon tau and ubiquitin-cross-reactive protein levels in uterine luminal flushings.Pregnancy rates were 7-folder higher in the High sub-herd (76 vs. 11%), with much of this difference apparent by Day 25. The proportion of heifers observed in standing oestrus prior to embryo transfer and the interval from the end of synchronisation treatment to the onset of oestrus were similar in the sub-herds. Oestrous cycle length, ovarian follicular dynamics and progesterone profiles during the oestrous cycle were also similar. More conceptuses had elongated by Day 14 in the High sub-herd (67 vs. 14%, P&lt;0.05), which also tended to have a higher pregnancy rate after artificial insemination (52 vs. 29, P&lt;0.1). Total protein in flushings from the uterus was similar in the sub-herds on Day 14 and Day 17. Conceptuses in the High sub-herd were longer on Day 17 following embryo transfer (6.5 vs. 4.8, P&lt;0.05). Interferon-tau levels were higher in the High sub-herd (25.9 vs. 16.1, P&lt;0.01), although ubiquitin cross-reactive protein levels were also higher in the High sub-herd, but this difference just failed to reach significance. We conclude that: 1. Most of the difference in sub-herd pregnancy rate occurs within 3 weeks of ET; 2. Ovarian factors are unlikely to contribute to the difference; 3. Major differences occur after blastocyst hatching and probably depend upon a differing endometrial environment before Day 14; 4. Differences in the ability of the uterine milieu to stimulate the expression of interferon-tau may be responsible for the differences in pregnancy rate; 5. The two sub-herds are a unique experimental resource for understanding early pregnancy in cattle following either AI or ET.