In vitro reminiscence: uterine programming in vivo affects respective luminal epithelial cells function in vitro†.

In cattle, the endometrium during diestrus and early pregnancy displays cellular responses that are consequences of prior, transient stimuli. Goal was to establish a model to study cellular memory in the endometrium. The hypothesis is that stimuli given to endometrium in vivo are retained as a cellular memory that remains after bovine uterine epithelial cells (BUECs) are isolated, cultured, and further stimulated in vitro. Objectives were to measure BUEC proliferation/migration and responsiveness to recombinant bovine Interferon-tau (rbIFNT) in vitro: among cows that showed estrus (experiment 1 [Exp1]), cows that became or not pregnant to artificial insemination (Exp2), cows that received or not supplemental progesterone (P4; Exp3) and cows that received or not a COX-1/2 inhibitor (Exp4). Only cows that displayed estrus were included in studies. For all experiments endometrial cytology was collected 4 days after estrus, BUECs were cultured, propagated, and submitted to rbIFNT treatment and an in vitro scratch assay. In Exp1, different cows spontaneously grouped according to proliferative/migratory capacity and responsiveness to rbIFNT of their respective BUECs. In Exp2, BUECs from pregnant cows showed greater rbIFNT responsiveness and cellular proliferation. In Exp3, BUECs from cows supplemented with P4 presented inhibited proliferation and increased expression of RSAD2. In Exp4, Flunixin Meglumine modified rbIFNT responsiveness of BUECs in an IFN-signaling pathway-specific manner. In conclusion, physiological and pharmacological stimuli received by the endometrium in vivo were retained as cellular memory in BUECs, persisted in culture, and changed BUEC proliferation/migration and responsiveness to rbIFNT, which are characteristics associated with fertility in cattle.

FGF18 impairs blastocyst viability, DNA double-strand breaks and maternal recognition of pregnancy genes.

Embryonic mortality in cattle is high, reaching 10-40 % in vivo and 60-70 % in vitro. Death of embryos involves reduced expression of genes related to embryonic viability, inhibition of DNA repair and increased DNA damage. In follicular granulosa cells, FGF18 from the theca layer increases apoptosis and DNA damage, so we hypothesized that FGF18 may also affect the oocyte and contribute to early embryonic death. The aims of this study were to identify the effects of FGF18 on cumulus expansion, oocyte maturation and embryo development from cleavage to blastocyst stage using a conventional bovine in vitro embryo production system using ovaries of abattoir origin. Addition of FGF18 during in-vitro maturation did not affect FSH-induced cumulus expansion or rates of nuclear maturation. When FGF18 was present in the culture system, rates of cleavage were not affected however, blastocyst and expanded blastocyst development was substantially inhibited (P < 0.05), indicating a delay of blastulation. The number of phosphorylated histone H2AFX foci per nucleus, a marker of DNA damage, was higher in cleavage-stage embryos cultured with FGF18 than in those from control group (P < 0.05). Furthermore, FGF18 decreased accumulation of PTGS2 and IFNT2 mRNA in blastocysts. In conclusion, these novel findings suggest that FGF18 plays a role in the regulation of embryonic death during the early stages of development by impairing DNA double-strand break repair and expression of genes associated with embryo viability and maternal recognition of pregnancy during the progression from oocyte to expanded blastocysts.

Viperin (RSAD2) gene expression in peripheral blood mononuclear cells of pregnant crossbred beef cows is altered by Bos indicus genetics.

The expression of interferon (IFN) stimulated genes (ISGs) in lymphocytes has been used for pregnancy diagnosis in cattle. However, among-cow variability has yielded sub-optimal predictive accuracy. We hypothesized that the expression of ISGs (ISG15, OAS1, RSAD2, CLEC3B, and AKR1B1) in early pregnancy varies according to the proportion of Bos indicus (B. indicus) genetics on females. Multiparous cows were classified in three genetic groups, High Angus (HA; n = 45 [0-33% Brahman influence]), Angus-Brahman (AB; n = 30 [34-67%]), and High Brahman (HB; n = 19 [68-100%]) and submitted to a Select-Synch + CIDR protocol. Cows that displayed estrus (n = 94) were artificially inseminated (Day0; D0). On D19, blood samples were collected to obtain peripheral blood mononuclear cells (PBMC) and measure progesterone (P4) concentrations. On D30, pregnancy diagnosis was performed. The expression of RSAD2 in PBMC of pregnant cows was positively related to the proportion of B. indicus genetics of the groups, but not the expression of ISG15 and OAS1. In pregnant cows, the proportion of B. indicus genetics was negatively associated to circulating levels of P4 concentrations. The P4 concentrations were related positively with RSAD2 expression. ROC curve results determined that for cattle with B. indicus genetics lower than 67%, the CLEC3B and AKR1B1 combination was the most accurate option to predict the outcome of pregnancy. In cows with more than 68% of B. indicus genetics, RSAD2 provided the best accuracy. In conclusion, there is a relationship between the proportion of B. indicus genetics and the ISGs gene expression in PBMC during pregnancy.

Conjugated linoleic acid supplementation changes prostaglandin concentration ratio and alters the expression of genes involved in maternal-fetal recognition from bovine trophoblast cells in vitro.

Early embryonic mortality caused by maternal-fetal recognition failure in the three weeks after fertilization represents a major cause of reproductive inefficiency in the cattle industry. Modifying the amounts and ratios of prostaglandin (PG) F2α and PGE2 can benefit the establishment of pregnancy in cattle. Adding conjugated linoleic acid (CLA) to endometrial and fetal cells culture affects PG synthesis, but its effect on bovine trophoblast cells (CT-1) is unknown. The aim of this study was to determine the effects of CLA (a mixture of cis- and trans-9, 11- and -10,12-octadecadienoic acids) on PGE2 and PGF2α synthesis and the expression of transcripts involved with maternal-fetal recognition of bovine trophectoderm. Cultures of CT-1 were exposed to CLA for 24, 48 and 72 h. Transcript abundance was determined by qRT-PCR and hormone profiles were quantified by ELISA. The PGE2 and PGF2α concentrations were reduced in the culture medium of CLA-exposed CT-1 compared to that of unexposed cells. Furthermore, CLA supplementation increased the PGE2:PGF2α ratio in CT-1 and had a quadratic effect (P < 0.05) on the relative expression of MMP9, PTGES2, and PTGER4. The relative expression levels of PTGER4 were reduced (P < 0.05) in CT-1 cultured with 100 µM CLA than in the unsupplemented and 10 µM-CLA groups. Treatment of CT-1 with CLA decreased PGE2 and PGF2α synthesis but a biphasic effect of CLA was observed on the PGE2:PGF2α ratio and relative abundance of transcripts with 10 µM CLA providing maximal improvements in each endpoint. Our data suggest that CLA may influence eicosanoid metabolic process and extracellular matrix remodeling.

Expression profile of key genes involved in DNA repair mechanisms in bovine cumulus cells cultured with bovine serum albumin or fetal calf serum.

Cumulus cells from cumulus-oocyte complexes (COC) matured in vitro in serum-free medium show high incidence of apoptosis and DNA double-strand breaks (DSB). This study aimed to characterize the transcript expression profile of selected genes involved in DNA repair mechanisms in bovine cumulus cells cultured with bovine serum albumin (BSA) or fetal calf serum (FCS). Briefly, bovine cumulus-oocyte complexes were in vitro matured with either, 0.4% BSA or 10% FCS for 3, 6, 12 or 24 h. The total RNA of cumulus cells was used for real-time PCR analysis. Transcript abundance of XRCC6, XRCC5, DNAPK, GAAD45B, TP53BP1, RAD50, RAD52, ATM and BRCA2 target genes changed as the IVM proceeded (P < 0.05). However, an interaction between protein source (FCS or BSA) and time was not detected (P ≥ 0.05). Cumulus cells from COCs matured with BSA presented higher mRNA expression of two genes compared to FCS group: TP53BP1 at 6 h and BRCA1 at 3, 6, 12 and 24 h (P < 0.05). In summary, our results showed for the first time the expression profile of the key genes involved in DSB repair mechanisms in cumulus cells obtained from bovine COCs matured with FCS or BSA. The higher mRNA expression of BRCA1 and TP53BP1 and lower mRNA expression of TNFAIP6 suggests an increase in apoptosis rate and DNA damage in cumulus cells cultured in BSA-supplemented medium and may explain, at least to some extent, the reduced developmental potential of bovine oocytes matured in serum-free medium.

Endometrial receptivity in cattle: the mutual reprogramming paradigm.

Prior to implantation in cattle, the mucous medium contained in the uterine lumen serves as a working interface for molecular exchange and signaling between the lining endometrium and the embryo. The composition of this luminal fluid changes temporally according to the secretory and reabsorptive activities of the uterus and the embryo, which are under complex regulation. Via this interface, both the embryo and the endometrium reprogram each other's functions to support pregnancy continuation beyond the pre-implantation period. More specifically, the embryo receives elongation signals and the uterus receives anti-luteolytic stimuli. Here, characteristics of the luminal compartment as well as the regulation of its composition to determine the pregnancy outcome will be discussed.

Culture of endometrial epithelial cells collected by a cytological brush in vivo.

In cattle, mechanistic studies of endometrial function rely on cell lines or primary culture of cells harvested postmortem. Understanding the endometrial physiology in dairy cows is essential, because approximately 50% of pregnancies are lost in the first 3 wk of gestation for unknown reasons. The objective was to validate an in vivo, minimally invasive, and estrous cycle stage-specific method to obtain endometrial luminal epithelial cells for culture. The uterine body of 26 cows was sampled using a cytology brush (cytobrush) 4 d after estrus. The viability of cells was measured by flow cytometry (80% live cells) and epithelial identity was determined by anti-vimentin and anti-cytokeratin immunofluorescence and quantitative PCR for KRT18 and VIM. A pool of cells from 15 animals was passaged 4 times in culture until confluent and then treated with 0, 0.1, 1, or 10 ng/mL of recombinant bovine interferon-tau (rbIFN-τ). The relative expression of transcripts related to IFN-τ signaling (IFNAR1), early (IRF2) and late (ISG15, OAS1) response to IFN-τ stimulus, and other IFN-τ-stimulated genes (CCL8, CXCL10, and FABP3) was measured by quantitative PCR. The relative expression of KRT18 transcripts was similar across passages; the relative expression of VIM increased at passage 2, and IFNAR1 transcripts decreased in cultured compared with that in fresh cells. The relative expression of ISG15, OAS1, CCL8, and FABP3 increased in response to rbIFN-τ. In conclusion, culture of endometrial luminal cells collected by cytobrush was feasible, generating a monolayer enriched in epithelial cells, and therefore constitutes a novel model by which to study endometrial luminal epithelial cell function, including responses to IFN-τ.

Comparing the effect of estradiol benzoate and 17ß-estradiol plus progesterone on follicular turnover and development, and pregnancy outcomes in a timed artificial insemination protocol.

The objective of this study was to compare the efficiency of estradiol benzoate (EB) and 17ß-estradiol (E2) associated with progesterone (P4) in a fixed-time artificial insemination (FTAI) protocol. We hypothesized that E2+P4 induces an earlier emergence of a new follicular wave (NFW), improving pre-ovulatory follicle diameter and pregnancy rates to FTAI (P/FTAI). In Exp.1, on Day 0 (D0), all Bos indicus cows (n = 12/group) received an intravaginal P4 device and a dose of PGF2α analogue. On D0, females were randomly assigned to receive EB or E2+P4. On D8.5, P4 intravaginal devices were removed and a dose of PGF2α and EB were administered in all females followed by fixed-timed AI on D10. Between D0 and D10, the dominant follicular growth was determined by ovary ultrasonography exams. On D8.5 and D10 the percentage of color power-Doppler signals in the dominant follicular wall was evaluated. In Exp. 2, 467 females (2-year-old nulliparous [n = 76], primiparous [n = 92] and pluriparous [n = 299]) were subjected to the similar FTAI and assigned to be treated with EB (n = 243) or E2+P4 (n = 224). Pregnancy diagnosis was performed 30 days after FTAI by ultrasonography. The day to emergence of NFW was similar between treatments (EB: 3.7 ± 0.37 vs. E2+P4: 3.3 ± 0.3, P = 0.76). Females treated with E2+P4 presented greater (P = 0.06) follicular growth between the emergence and D9 (1.18 ± 0.07) than those treated with EB (0.97 ± 0.08). There was also a positive effect (P < 0.05) of E2+P4 on diameter of the dominant follicle on D9 (13.0 ± 0.6 vs. 10.9 ± 0.55) and blood perfusion of the follicle wall on D8.5 (49 vs. 40%). There was a treatment by parity category interaction effect on P/FTAI (P < 0.05). Treatment with E2+P4 was advantageous to P/FTAI of primiparous cows (E2+P4: 58% and EB: 30%). However, for nulliparous and pluriparous cows, P/FTAI was similar between treatments (∼50%). In conclusion, in a E2/P4-based protocol for FTAI, E2+P4 is as efficient as EB in inducing new follicular emergence within a similar day range, but it results similar or greater P/FTAI.

Comparison of estradiol benzoate doses for resynchronization of ovulation at 14 days after timed-AI in suckled beef cows.

We aimed to compare the effects of use of 1 or 2 mg estradiol benzoate (EB) associated with an intravaginal progesterone (P4) device for resynchronization of ovulation 14 days after timed-AI (TAI) in suckled beef cows. Nelore cows were submitted to a TAI (D0) and on D14, received an intravaginal P4 device and were randomly assigned to EB-1 group [n = 516] or EB-2 group [n = 510], which that received 1 or 2 mg EB, respectively. Also, cows had the ovaries scanned by ultrasound to detect an active CL on D14. On D22, devices were removed and structural luteolysis was detected by color-Doppler ultrasonography. In cows which underwent luteolysis, the resynchronization protocol was continued and they were submitted to second TAI on D24. Pregnancy diagnosis was performed 30-35 days after first or second TAI. A subgroup [n = 18-19/group] was submitted to daily ovarian ultrasound scanning from D14 to D22. Proportion of cows with an active CL on D14 did not differ (P > 0.1) between EB-1 and EB-2 groups. The proportion of cows with an active CL on D22 and pregnancy per AI (P/AI) at first TAI were greater (P ≤ 0.05) in EB-1 (55% and 51%) than in EB-2 group (48% and 42%). The P/AI at second TAI did not differ (P > 0.1) between EB-1 (47% [106/227]) and EB-2 group (42% [110/259]). Cumulative pregnancy rate was greater in EB-1 (73% [370/508]) than in EB-2 group (64% [322/502]). No difference (P > 0.1) was observed in the proportion of non-pregnant cows with a synchronized follicular wave emergence between EB-1 and EB-2 groups. In conclusion, treatment with either 1 or 2 mg EB associated with an intravaginal P4 device at D14 after TAI are efficient to synchronize a new follicular wave emergence. The decreased P/AI from first TAI observed in the group of cows receiving 2 mg indicates that this dose is not recommended for use in resynchronization programs initiated 14 days after TAI. The use of 1 mg EB associated with a P4 device provides an elevated cumulative pregnancy rate after two TAIs with an interval of 24 days.

Conceptus-modulated innate immune function during early pregnancy in ruminants: a review.

This review focuses on the innate immune events modulated by conceptus signaling during early pregnancy in ruminants. Interferon-tau (IFN-τ) plays a role in the recognition of pregnancy in ruminants, which involves more than the inhibition of luteolytic pulses of PGF2α to maintain corpus luteum function. For successful pregnancy establishment, the allogenic conceptus needs to prevent rejection by the female. Therefore, IFN-τ exerts paracrine and endocrine actions to regulate the innate immune system and prevent conceptus rejection. Additionally, other immune regulators work in parallel with IFN-τ, such as the pattern recognition receptors (PRR). These receptors are activated during viral and bacterial infections and in early pregnancy, but it remains unknown whether PPR expression and function are controlled by IFN-τ. Therefore, this review focuses on the main components of the innate immune response that are involved with early pregnancy and their importance to avoid conceptus rejection.

Feasibility and accuracy of using different methods to detect pregnancy by conceptus-stimulated genes in dairy cattle.

Development of new methods for early diagnosis of pregnancy can be important to increase the reproductive efficiency and profitability of dairy herds. The bovine conceptus secretes IFN-τ that stimulates the transcription of several genes in circulating immune cells and extrauterine tissues. The aims of this study were to evaluate the mRNA abundance for pregnancy predictability of a classic gene stimulated by IFN-τ (ISG15) and a novel potential pregnancy marker (LGALS3BP) in peripheral blood mononuclear cells (PBMC), total blood leukocytes (TBL) or milk leukocytes (TML), and cervical cells (CC) on d 20 after timed artificial insemination (TAI) in dairy cattle. Eighteen Holstein females (12 cows and 6 heifers) were submitted to an estrous synchronization protocol for TAI (d 0). On d 20 post-TAI, blood samples were collected from coccygeal vessels for isolation of PBMC and in Tempus Blood RNA tubes (Applied Biosystems) for TBL. Samples of CC were collected using a cytological brush, and the TML were isolated from milk samples collected before routine milking. Pregnancy diagnosis was performed on d 30 post-TAI using transrectal ultrasonography, and females were classified as pregnant (n = 8) or nonpregnant (n = 10). Total RNA was extracted and mRNA abundance of target genes (ISG15 and LGALS3BP) was quantified by reverse-transcription quantitative PCR and normalized in relation to reference genes. Data were analyzed by ANOVA using the MIXED procedure of SAS (SAS Institute Inc.). The mRNA abundance of ISG15 was greater in pregnant than in nonpregnant animals for PBMC, TBL, and CC. No difference was detected for TML based on pregnancy status. For LGALS3BP mRNA abundance, no difference was detected between pregnant and nonpregnant animals for PBMC, TBL, and TML, but a tendency for greater abundance in pregnant animals was observed for CC. The fold change for ISG15 in each pregnant cow related to the mean of nonpregnant animals was 2.73 ± 0.31, 3.40 ± 2.17, 1.64 ± 0.29, and 0.005 ± 0.002 for PBMC, CC, TBL, and TML, respectively. The fold change for LGALS3BP in each pregnant cow related to the mean of nonpregnant animals was 0.97 ± 0.38, 1.77 ± 0.39, 0.20 ± 0.08, and 0.70 ± 0.11 for PBMC, CC, TBL, and TML, respectively. The receiver operating characteristic curve analysis indicated that ISG15 abundance predicted pregnancy in PBMC (area under curve, AUC = 0.92) and CC (AUC = 0.77) but not in TBL (AUC = 0.72) or TML (AUC = 0.52). In conclusion, mRNA abundance for ISG15 in PBMC was the best predictor for pregnancy at d 20 post-TAI, whereas TBL and TML were not good predictors of pregnancy on d 20 post-TAI. The mRNA abundance of LGALS3BP was not associated with pregnancy status in any type of cell evaluated.

Early pregnancy-induced transcripts in peripheral blood immune cells in Bos indicus heifers.

Immune cells play a central role in early pregnancy establishment in cattle. We aimed to: (1) discover novel early-pregnancy-induced genes in peripheral blood mononuclear cells (PBMC); and (2) characterize the temporal pattern of early-pregnancy-induced transcription of select genes in PBMC and peripheral blood polymorphonuclear cells (PMN). Beef heifers were artificially inseminated on D0 and pregnancies were diagnosed on D28. On D10, 14, 16, 18, and 20, blood was collected for isolation of PBMC and PMN from heifers that were retrospectively classified as pregnant (P) or non-pregnant (NP). PBMC samples from D18 were submitted to RNAseq and 220 genes were differentially expressed between pregnant (P) and non-pregnant (NP) heifers. The temporal abundance of 20 transcripts was compared between P and NP, both in PBMC and PMN. In PBMC, pregnancy stimulated transcription of IFI6, RSAD2, IFI44, IFITM2, CLEC3B, OAS2, TNFSF13B, DMKN and LGALS3BP as early as D18. Expression of IFI44, RSAD2, OAS2, LGALS3BP, IFI6 and C1R in PMN was stimulated in the P group from D18. The novel early-pregnancy induced genes discovered in beef heifers will allow both the understanding of the role of immune cells during the pre-attachment period and the development of technologies to detect early pregnancies in beef cattle.

Applied use of interferon-tau stimulated genes expression in polymorphonuclear cells to detect pregnancy compared to other early predictors in beef cattle.

We aimed to evaluate the accuracy of Interferon-tau stimulated genes (ISG) abundance in peripheral blood polymorphonuclear cells (PMNs) on D20 after fixed-time artificial insemination (FTAI; D0) as a pregnancy diagnosis method against CL evaluation by Doppler ultrasonography and progesterone (P4) concentrations on D20, as well as Pregnancy Associated Glycoproteins (PAG) concentrations on D25. Additionally, we evaluated the potential of ISG abundance in PMNs as pregnancy loss predictors. Nelore heifers (n = 103) and cows (n = 144) underwent estrous synchronization and were artificially inseminated on D0. Pregnancy was diagnosed by B-mode ultrasonography on D30 and D70, and after the final diagnosis, females were classified in four groups: Pregnant; Non-pregnant; Functional CL on D20 but non-pregnant (CL-NP) and Pregnancy loss between D30 and D70 (PL). After determining cutoff values, the Sensitivity (SE), Specificity (SP), Positive Predictive Value (PPV), Negative Predictive Value (NPV) and Accuracy (ACC) were determined for each method. All methods were classified as significant (P < 0.05) predictors of pregnancy. Both ISG expression and PAG concentrations were greater (P < 0.05) in pregnant females than in non-pregnant and CL-NP females but did not differ (P > 0.05) from the PL group. ISG15 expression was greater (P < 0.05) in heifers than in cows, but this difference was not found in OAS1 expression and PAG concentrations. All the methods evaluated were proven to be adequate predictors of pregnancy, but greater accuracies were obtained through PAG concentrations and Doppler-US, due to the decreased number of false positive and false negative results.

Ultrasonography-accessed luteal size endpoint that most closely associates with circulating progesterone during the estrous cycle and early pregnancy in beef cows.

The aim was to evaluate the associations between circulating P4 concentrations, corpus luteum (CL) size (diameter, area or volume) and blood perfusion (BP) in cows. In Experiment 1, Pearson's correlations (P < 0.05) with P4 concentrations were observed during CL development (D8) for total area (TA; r = 0.76), luteal area (ACL; r = 0.72), total and luteal diameter (TD and DCL respectively; r = 0.46). During mid-late diestrus, there was a positive correlation (P < 0.05) only at D15 with TA and ACL (r > 0.60), TD, total volume (TV) and luteal volume (VCL; r > 0.434). During luteal regression, the correlation was only observed at D18 for ACL (r = 0.478) and D20 with several variables. In Experiment 2, CL weight and ACL had the greatest correlation with P4 (r > 0.6). In Experiment 3, TA and ACL were the variables that were most closely correlated with serum P4 concentrations at D7 in recipient cows. Correlation coefficients were greater for luteal measurements when there were compact compared with cavitary CLs. In Experiment 4, there was no correlation (P > 0.05) between P4 and any of the variables measured on D4 and D7 in recipient cows detected in estrus. On D18 to D20, all CL characteristics were correlated (P < 0.05) with plasma P4, and luteal BP and BP area were more closely (P < 0.05) correlated than ACL. In conclusion, CL perimeter area measurements had the greatest association with luteal function during CL development; whereas for BP there was a greater correlation with P4 than luteal size during luteolysis.

Reproductive performance of Bos indicus beef cows treated with different doses of equine chorionic gonadotropin at the end of a progesterone-estrogen based protocol for fixed-time artificial insemination.

Two experiments were performed to evaluate the reproductive performance of zebu beef cows treated with different doses of eCG at the end of a progesterone (P4)/estrogen based protocol for timed artificial insemination (TAI). In Experiment 1, suckling Bos indicus Nelore cows (n = 261) received, on day 0, a progesterone (P4) intravaginal device (PD) and an injection of 1 mg estradiol benzoate (EB). On day 8, the PD was removed, 500 µg of cloprostenol was injected, and cows were assigned to one of the following groups: Control (no treatment), 300 (300 IU of eCG), 600 (600 IU of eCG), and 900 (900 IU of eCG). On day 9, all cows received 1 mg EB and TAI performed 54-56 h after cloprostenol injection. A pregnancy diagnosis was done by ultrasound scanning 40 days after TAI, and the number of fetuses and calves was recorded at pregnancy diagnosis and at birth. More cows treated with eCG displayed estrus within 48 h after removal of the PD (42.3% vs. 11.6%, P < 0.01), and ovulated more than one follicle (42%, 58/138 vs. 1.8%, 1/54; P < 0.01). This effect on ovulation rate was dose dependent (P < 0.05). The pregnancy rate was affected only by cow parity (primiparous, 25.3% vs. multiparous, 48.9%; P < 0.01). Twin pregnancy was higher (P < 0.01) in cows treated with eCG (42%, 58/138) than controls (0%, 0/54). However, few cows (33.3%) were able to keep both fetuses intact until birth. For evaluation of ovarian characteristics by B-mode and Doppler ultrasonography, 43 Nelore cows were submitted In Experiment 2 to the same four groups described in Experiment 1. Although no difference (P > 0.1) was observed for size and blood perfusion in the pre-ovulatory follicles, corpus luteum was larger and with greater blood perfusion (P < 0.05) in eCG-treated cows. In conclusion, eCG increased the number of double/multiple ovulations in a dose-dependent manner, induced larger and more vascularized corpora lutea, but did not affect the fertility of cyclic or anestrous cows. Although eCG results in twin pregnancies, most of cows underwet embryo/fetus loss and birth a single calf.

Use of Doppler ultrasonography in embryo transfer programs: feasibility and field results.

The intensive use of Doppler ultrasonography in several studies in the last decade allowed the characterization of vascular perfusion and the estimation of function of the reproductive organs and tissues along the estrous cycle and pregnancy in cattle. We aim to discuss the possibility of using Doppler imaging and to explore the potential of its inclusion in reproductive programs in cattle industry. Recent studies in dairy and beef cows indicated a high accuracy and sensitivity when Doppler ultrasonography is used to evaluate corpus luteum function and to diagnosis pregnancy between days 20 and 22. Moreover, resynchronization programs starting 5 to 7 days after timed embryo transfer (FTET) coupled with early pregnancy diagnosis were developed for beef cattle, and have been implemented in commercial embryo transfer programs. These strategies allow a reduction in the interval between two FTET from ≈ 40 to 24 days and may improve the gains in reproductive efficiency when compared to traditional programs than begin resynchronization after the pregnancy diagnosis at 30 days. A second alternative to use Doppler imaging is the evaluation of luteal blood perfusion at the time of embryo transfer for selection of recipients with greater receptivity potential. This may increases fertility in FTET, as embryos would not be transferred to females with non-functional CL, and in cases with recipients surplus, females with higher receptivity would be prioritized.