ABSTRACT
The aim of this study was to determine the relationship between observed estrous-related behavior, activity clusters (AC; detected by automatic activity monitor), endocrine profiles, and ovulation time. Twenty-one cows in estrus (after 2 cloprostenol treatments, 11 d apart) and 12 nonsynchronized cows, to establish Heatime (SCR Engineers Ltd., Netanya, Israel) herd baseline activity, were enrolled. Cows had Heatime monitors applied 3 wk before the trial to establish their own baseline activity level. Cows in standing estrus had ultrasonography and phlebotomy carried out every 4 h to determine dominant follicle size, endocrine profiles, and ovulation time. After ovulation, these procedures were repeated once on d 3 to 6. Heatime alerted estrus in 90% of cows, and incorrectly alerted 17% of AC. The mean±SEM duration for standing estrus was 9±1 and 13±1 h for estrous-related behavior. Estrous-related behavior began after the start of the proestrous estradiol-17ß (E2) increase (59±6.5 h). Cows with longer durations of raised proestrous E2 had longer intervals from its onset to the start of standing estrus and AC. The AC duration increased with longer durations of estrous-related behavior. Higher peak E2 occurred with longer standing estrus and estrous-related behavior. As E2 concentration decreased after the peak, 90% of cows still had estrous-related behavior. Duration of estrous-related behavior increased with higher average E2 concentration during the last 8 h before the start of the LH surge. During this surge 90% of cows had all of their standing estrus. As yields increased, so did the magnitude of the preovulatory FSH surges. Higher surges occurred with shorter standing estrus and estrous-related behavior. Cows with shorter LH surges had longer standing estrus. Peak LH preceded the AC peak (6.6±0.8 h). Duration of overlap between the AC start and the LH surge end ranged between 0 and 14 h; 1 cow had none. No association was found between the AC characteristics with the E2, LH, or FSH profiles. In conclusion, the relationship between the timing of the E2 increase and estrous activity may be mediated by other factors (GnRH surge). Estrous-related behavior, but not endocrine profiles, was related to AC duration. Timing of standing estrus during the LH surge ensures that mating allows sperm maturation before ovulation. Based on the interval from the start of an AC to ovulation (27±1 h), the optimum time to artificial insemination is, on average, between 9 and 15 h after the AC start.
Subject(s)
Cattle/physiology , Estrous Cycle , Gonadal Steroid Hormones/blood , Gonadotropins, Pituitary/blood , Motor Activity , Accelerometry/veterinary , Animals , Female , Ovulation , Random AllocationABSTRACT
The aims of the present study were to investigate (1) the risk factors that influence the achievement of reproductive targets postpartum (pp) and (2) the key factors that influence pregnancy rate following first artificial insemination (AI) in dairy cows. Ninety-eight Holstein-Friesian pp cows were blood sampled from wk 1 to 4 pp for hematology and biochemistry. Reproductive tract health was assessed weekly by ultrasonography and vaginal mucus scoring. Body condition score (BCS), lameness score, and milk yield were assessed every 2 wk. Milk samples for progesterone assay were collected twice weekly and on d 4, 5, and 7 after AI. Risk factors associated with achieving reproductive targets depended on (1) increased metabolic activity of the liver (increased glutamate dehydrogenase at calving and increased γ-glutamyl transpeptidase in wk 4), (2) a competent immune system (increased neutrophils in wk 1; decreased α1-acid glycoprotein in wk 1, 2, and 3), (3) an endocrine system that was capable of responding by producing sufficient triiodothyronine in wk 2 and increased insulin-like growth factor I in wk 3 and 4, (4) a lower negative energy balance status (decreased nonesterified fatty acid concentration in wk 1; decreased ß-hydroxybutyrate concentration in wk 2; BCS loss between calving and d 28 pp <0.5), (5) good reproductive tract health [normal uterine scan at d 45 pp; clear vaginal mucus discharge at first ovulation and at d 45 pp; resumed ovarian cyclicity by the end of the voluntary waiting period (≥ d 35 pp)], and (6) adequate diet (to ensure increased glutathione peroxidase in wk 2 and 3 and increased magnesium in wk 4). Risk factors that increased the odds of a successful first AI were previous ovulation(s) (odds ratio=3.17 per ovulation), BCS >2.5 at AI (odds ratio=3.01), and clear vaginal mucus (score=0) compared with purulent mucus (score >0) 4 d after first AI (odds ratio=2.99). In conclusion, this study identified key risk factors in the early pp period that give a higher probability of cows achieving their reproductive targets and of having a first-AI pregnancy.
Subject(s)
Fertility , Reproduction , 3-Hydroxybutyric Acid/metabolism , Animals , Cattle , Fatty Acids, Nonesterified/metabolism , Female , Insemination, Artificial/veterinary , Insulin/blood , Insulin-Like Growth Factor I/metabolism , Lactation , Limit of Detection , Milk/metabolism , Ovulation , Postpartum Period/metabolism , Pregnancy , Progesterone/analysis , Risk Factors , Thyroxine/blood , Triiodothyronine/blood , gamma-Glutamyltransferase/metabolismABSTRACT
We hypothesised that the expression pattern of members of the fibroblast growth factor (FGF) family would be altered in the endometrium as the oestrous cycle/early pregnancy progressed associated with changes in the expression pattern of their receptors in the developing embryo/conceptus. Expression of FGF1 and FGF10 transcript variants 1 and 2 increased significantly as the oestrous cycle/early pregnancy progressed. Neither progesterone (P4) supplementation nor pregnancy status significantly affected the expression of any of the FGF ligands studied. However, there was a significant interaction between day, pregnancy and P4 status on FGF2 expression (P<0.05) and a significant interaction between P4 status and day on FGF10_tv2 expression. FGF10 protein was localised in the luminal and glandular epithelium as well as the stroma but was not detected in the myometrium. By RNA sequencing, the expression of FGF ligands in the developing embryo/conceptus was found to be minimal. The expression of FGF receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, FGFRL1 and FRS3 was significantly affected by the stage of conceptus development. Interestingly, the expression of FGFR1 and FGFR4 was higher during early embryo development (days 7-13, P<0.05) but decreased on day 16 (P<0.05) while FGFR2 (P<0.001) expression was similar from day 7 through to day 13, with a significant increase by day 16 (P<0.05) that was maintained until day 19 (P>0.05). In conclusion, these data demonstrate that FGF ligands are primarily expressed by the endometrium and their modulation throughout the luteal phase of the oestrous cycle/early pregnancy are associated with alterations in the expression of their receptors in the embryo/conceptus.
Subject(s)
Blastocyst/metabolism , Endometrium/metabolism , Fibroblast Growth Factors/metabolism , Receptors, Fibroblast Growth Factor/metabolism , Animals , Cattle , Estrous Cycle/metabolism , Female , Fibroblast Growth Factors/genetics , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gestational Age , Immunohistochemistry , Pregnancy , Receptors, Fibroblast Growth Factor/genetics , Reproductive Techniques, Assisted , Time FactorsABSTRACT
The aims of this study were to 1) identify the earliest transcriptional response of the bovine endometrium to the presence of the conceptus (using RNAseq), 2) investigate if these genes are regulated by interferon tau (IFNT) in vivo, and 3) determine if they are predictive of the pregnancy status of postpartum dairy cows. RNAseq identified 459 differentially expressed genes (DEGs) between pregnant and cyclic endometria on day 16. Quantitative real-time PCR analysis of selected genes revealed PARP12, ZNFX1, HERC6, IFI16, RNF213, and DDX58 expression increased in pregnant compared with cyclic endometria on day 16 and were directly upregulated by intrauterine infusion of IFNT in vivo for 2 h (P < 0.05). On day 13 following estrous endometrial expression of nine genes increased [ARHGAP1, MGC127874, LIMS2, TBC1D1, FBXL7, C25H16orf71, LOC507810, ZSWIM4, and one novel gene (ENSBTAT00000050193)] and seven genes decreased (SERBP1, SRGAP2, AL7A1, TBK1, F2RL2, MGC128929, and WBSCR17; P < 0.05) in pregnant compared with cyclic heifers. Of these DEGs, significant differences in expression between pregnant and cyclic endometria were maintained on day 16 for F2RL2, LIMS2, LOC507810, MGC127874, TBC1D1, WBSCR17, and ZSWIM4 (P < 0.05) both their expression was not directly regulated by IFNT in vivo. Analysis of the expression of selected interferon-stimulated genes in blood samples from postpartum dairy cows revealed a significant increase (P < 0.05) in expression of ZXFX1, PARP12, SAMD9, and HERC6 on day 18 following artificial insemination in cows subsequently confirmed pregnant compared with cyclic controls. In conclusion, RNAseq identified a number of novel pregnancy-associated genes in the endometrium of cattle during early pregnancy that are not regulated by IFNT in vivo. In addition, a number of genes that are directly regulated by short term exposure to IFNT in vivo are differentially expressed on day 18 following estrus detection in the blood of postpartum dairy cows depending on their pregnancy status.
Subject(s)
Endometrium/metabolism , Interferon Type I/metabolism , Pregnancy Proteins/metabolism , Pregnancy, Animal/genetics , Pregnancy, Animal/metabolism , Adaptor Proteins, Signal Transducing/blood , Adaptor Proteins, Signal Transducing/genetics , Animals , Calibration , Cattle , Estrous Cycle/genetics , Female , Fetus/metabolism , Gene Expression Profiling , Gene Expression Regulation , Gene Regulatory Networks/genetics , Interferon Type I/genetics , Oligonucleotide Array Sequence Analysis , Pregnancy , Pregnancy Proteins/genetics , Pregnancy, Animal/blood , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, RNAABSTRACT
Pregnancy per insemination is a major determinant of reproductive efficiency in cattle and is affected by concentrations of progesterone (P4) during early pregnancy. The relationship between pregnancy per artificial insemination (P/AI) and early luteal concentrations of P4, and repeatability of concentrations of P4 was examined on d 4, 5, 6, and 7 (day of standing estrus=d 0) in 118 Holstein Friesian heifers following 2 rounds of AI to 1 high-fertility sire. Repeatability estimates (R(e)) for P/AI were established following 4 rounds of AI. We found a linear and quadratic relationship between P/AI and concentrations of P4 on d 4 to 7 after estrus, as well as a linear and quadratic relationship between P/AI and the change in concentration of P4 from d 4 to 7 and from d 5 to 7. Optimum concentrations of P4 to maximize probability of P/AI were 2.5, 4.0, 5.0, 5.2, and 3.5 ng/mL for d 4, 5, 6, and 7, and the change from d 4 to 7, respectively. Repeatability of P/AI following 4 rounds of AI was low (R(e)=0.07). Repeatability estimates for concentrations of P4 from cycle to cycle indicated low repeatability between d 4 (R(e)=0.05) and 7 (R(e)=0.20). These data indicated the importance of P4 in the early luteal phase for pregnancy survival, but also demonstrated that high concentrations of P4 on these days have a deleterious effect on embryo viability. Early luteal (d 4 to 5) concentrations of P4 were a reasonable predictor of concentrations on d 7 and could be used as a diagnostic tool to identify animals at risk of subsequent embryo loss.
Subject(s)
Corpus Luteum Maintenance/physiology , Insemination, Artificial/veterinary , Progesterone/physiology , Animals , Cattle , Corpus Luteum/physiology , Estrus Detection/methods , Estrus Synchronization/methods , Estrus Synchronization/physiology , Female , Insemination, Artificial/physiology , Pregnancy , Progesterone/bloodABSTRACT
The aim was to investigate 1) the relationship between the physical activity index created for each cow by activity monitoring systems and the identification of the preovulatory follicular phase, and 2) the influence of various production, health, and cow factors on the relationship between physical activity and estrous behavior. Eighty-nine spring calving cows, on pasture, were monitored during the breeding season using the neck-mounted estrous activity monitor Heatime (SCR Engineers Ltd., Netanya, Israel). Milk samples were collected twice weekly for progesterone assay to characterize resumption of reproductive activity. Reproductive tract health was assessed weekly by ultrasonography and vaginal mucus scoring. Body condition score and milk yield were assessed every 2 wk. Heatime identified 72% of preovulatory follicular phases from which 145 inseminations resulted in 69 conceptions; 32% of activity clusters were associated with high-progesterone states (i.e., false positives). By inclusion of a 6 to 8-h duration threshold and maintaining the borderline peak activity threshold, this was improved to 87.5% with 21.3% false positives. Mean (± standard error of the mean) peak activity and cluster duration (19.3±0.53 and 10.8±0.38, respectively) were highest for the second or subsequent preovulatory follicular phases followed, in descending order, by those during first preovulatory follicular phases (14.8±2.13 and 8.4±1.4, respectively) and high progesterone states (8.0±0.47 and 3.0±0.42, respectively). The odds of an activity cluster being in a preovulatory follicular phase rather than a high-progesterone phase improved by 29% for every 1-unit increase in peak activity and by 91% for every 2-h increase in duration. The probability of an activity cluster detecting a preovulatory follicular phase was improved if it was a second or subsequent follicular phase, if body condition score was 0.25 units higher, if milk yield was 10 kg lower, and uterine infection was absent. Conception rate was influenced by insemination on the same day (52%) or day after a cluster (34.3%); inseminations were carried out using the a.m.-p.m. rule. Advances in the development of more accurate automatic monitoring of the preovulatory follicular phase will aid the timing of insemination and, thus, improve conception rates.
Subject(s)
Estrus Detection/methods , Animals , Cattle , Dairying/methods , Female , Milk/chemistry , Monitoring, Physiologic/veterinary , Motor Activity/physiology , Postpartum Period/physiology , Pregnancy , Progesterone/analysis , Time FactorsABSTRACT
In the study presented, the hypothesis that ligands of the insulin-like growth factor (IGF) family, as well as their binding proteins (BPs), are temporally regulated and are altered by elevated progesterone (P4) and/or pregnancy was tested. Heifers detected in standing estrus following synchronization (n = 210, day 0), were artificially inseminated (n = 140) or left as noninseminated cyclic controls (n = 70). On day 3, half of each group were randomly assigned to receive a P4-releasing intravaginal device resulting in four treatment groups, pregnant and cyclic heifers with high and normal P4 concentrations on either day 5, 7, 13, or 16 of the estrous cycle/early pregnancy. Quantitative real-time PCR and immunohistochemistry were performed on endometrial homogenate and uterine cross sections to measure transcript abundance and protein localization respectively (n = 5 per treatment per time point). No effect of day, treatment, or their interactions was observed for IGF2, IGFBP4, and -5 (P > 0.05). IGF1 and IGFBP6 expression decreased, while IGF1R and IGFBP2 expression increased (P < 0.05) as the days of the cycle or pregnancy progressed. The expression of IGFBP2 was increased by elevated P4; in addition, localization of both IGF2 and IGFBP2 protein in the luminal and superficial glandular epithelium displayed a P4 × day interaction. In conclusion it is proposed that decreased IGF1 and IGFBP6 expression with a coordinate increase in IGF1R and IGFBP2 as the estrous cycle/early pregnancy progresses, along with other factors, are required to establish a uterine environment that promotes the growth and development of the conceptus prior to implantation. In addition, the increased protein abundance of both IGF2 and IGFBP2 observed in heifers supplemented with P4 contributes to the enhanced conceptus elongation observed in this model.
Subject(s)
Cattle/genetics , Endometrium/metabolism , Gene Expression , Pregnancy, Animal/genetics , Progesterone/pharmacology , Animals , Estrous Cycle/drug effects , Estrous Cycle/genetics , Female , Immunohistochemistry , Insulin-Like Growth Factor Binding Protein 2/genetics , Insulin-Like Growth Factor Binding Protein 2/metabolism , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Insulin-Like Growth Factor II/genetics , Insulin-Like Growth Factor II/metabolism , Pregnancy , Pregnancy, Animal/metabolismABSTRACT
The cervix and its secretions undergo biochemical and physical changes under the differential influences of estrogen and progesterone. These include changes in the glycoprotein profile of the endocervix and its secretions. A comprehensive survey of such changes in cervical epithelium and cervical secretions was performed on bovine samples throughout the periestrous period. Cervical tissue samples and swabs were collected from synchronized beef heifers that were slaughtered 1) 12 h after controlled intravaginal progesterone-releasing device (CIDR) removal, 2) 24 h after CIDR removal, 3) at the onset of estrus, 4) 12 h after the onset of estrus, 5) 48 h after the onset of estrus, and 6) 7 d after the onset of estrus. Histological staining with hematoxylin and eosin, periodic acid Schiff, Alcian blue, and high-iron diamine was carried out to map overall patterns of stored glycoproteins and tissue structure. Biotinylated lectins were also used to detect the presence and distribution of a range of saccharide structures. The activities of ß-galactosidase, α-L-fucosidase, ß-N-acetyl-hexosaminidase, and sialidase were measured in cervical swabs using specific substrates. The epithelial layer of the cervix exhibited dynamic changes in cellular hypertrophy and amounts of stored glycoprotein. The greatest content of neutral and acidic mucins was observed 48 h after onset of estrus (P < 0.05). Sialylated mucins predominated at the bases of cervical folds, whereas sulfated mucins were more abundant (P < 0.05) at their apices. The stained area of core mucin glycans changed (P < 0.05) in association with follicular versus luteal phases, whereas terminal glycans changed (P < 0.05) mainly at the time of estrus and shortly thereafter. The greatest activity of ß-galactosidase and sialidase was observed 12 h after onset of estrus, whereas ß-hexosaminidase and α-fucosidase peaked at the luteal time point (P < 0.05). Taken together, we suggest that the well-known changes in the endocervix and its secretions that are associated with the physiological modulation of sperm transport and function of the cervical barrier are, in part, driven by glycosylation changes.
Subject(s)
Cattle/physiology , Cervix Uteri/metabolism , Estrus/physiology , Glycoproteins/metabolism , Glycoside Hydrolases/metabolism , Animals , Epithelium/metabolism , Female , Glycoproteins/genetics , Glycoside Hydrolases/genetics , Lectins/metabolism , Mucins/physiology , Protein BindingABSTRACT
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.
Subject(s)
Carrier Proteins/metabolism , Endometrium/metabolism , Galectins/metabolism , Luteal Phase/metabolism , Pregnancy Proteins/metabolism , Animals , Carrier Proteins/genetics , Cattle , Endometrium/cytology , Endothelial Cells/cytology , Endothelial Cells/metabolism , Female , Galectins/genetics , Gene Expression Regulation , Pregnancy , Pregnancy Proteins/genetics , Protein Transport , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
This study sought to determine the earliest response of the bovine uterine endometrium to the presence of the conceptus at key developmental stages of early pregnancy. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7, and 13 postestrus, but the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). Of these 514 genes, MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD9, EIF4E, and IFIT2 increased to the greatest extent in pregnant endometria (>8-fold log2 fold change increase). The expression of OXTR, Bt.643 (unofficial symbol), and KCNMA1 was reduced the most, but short-term treatment with recombinant ovine interferon tau (IFNT) in vitro or in vivo did not alter their expression. In vivo intrauterine infusion of IFNT induced the expression of EIF4E, IFIT2, IFI44, ISG20, MX2, RSAD2, SAMD9, and USP18. These results revealed for the first time that changes that occur in the endometrial transcriptome are independent of the presence of a conceptus until pregnancy recognition. The differentially expressed genes (including MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD, and EIF4E) are a consequence of IFNT production by the conceptus. The identified genes represent known and novel early markers of conceptus development and/or return to cyclicity and may be useful to identify the earliest stage at which the endometrial response to the conceptus is detectable.
Subject(s)
Endometrium/metabolism , Gene Expression Regulation , Interferon Type I/metabolism , Pregnancy Proteins/metabolism , Pregnancy, Animal/metabolism , Animals , Cattle , Female , Fibroblasts/metabolism , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , PregnancyABSTRACT
The oestrous cycle in cattle lasts for 18-24 days. It consists of a luteal phase (14-18 days) and a follicular phase (4-6 days). During the cycle there are generally two (dairy cows) or three (heifers and beef cows) waves of ovarian follicle growth. Each wave of follicle growth consists of a period of emergence of a cohort of follicles, selection of a dominant follicle and either atresia or ovulation of the dominant follicle. These waves of follicle growth, initially established during the early pre-pubertal period of development occur throughout the entire cycle, with only the dominant follicle (DF) of the final wave coinciding with the follicular phase that undergoes final maturation and ovulation. Ovarian functions (follicle growth, ovulation, luteinisation and luteolysis) are regulated by the endocrine hormones of the hypothalamus (gonadotrophin-releasing hormone), anterior pituitary (follicle-stimulating hormone and luteinising hormone), ovaries (progesterone, oestradiol and inhibins) and the uterus (prostaglandin F2α). In postpartum cows resumption of regular oestrous cycles (in addition to uterine involution) is fundamental for re-establishment of pregnancy.
Subject(s)
Cattle/physiology , Corpus Luteum/physiology , Estrous Cycle/physiology , Ovarian Follicle/physiology , Animals , Female , Pregnancy , Sexual Behavior, Animal/physiologyABSTRACT
In cattle, elevated concentrations of circulating progesterone (P4) in the immediate postconception period are associated with advanced conceptus development, while low P4 is implicated as a causative factor in low pregnancy rates observed in dairy cows. This study aimed to: 1) describe the transcriptional changes that occur in the bovine endometrium during the estrous cycle, 2) determine how elevated P4 affects these changes, 3) identify if low P4 alters the expression of these genes, and 4) assess the impact that low P4 has on conceptus development. Relatively few differences occurred in endometrial gene expression during the early luteal phase of the estrous cycle (Day 5 vs. 7), but comparison of endometria from more distant stages of the luteal phase (Day 7 vs. 13) revealed large transcriptional changes, which were significantly altered by exogenous supplementation of P4. Induction of low circulating P4 altered the normal temporal changes in gene expression, and these changes were coordinate with a delay in the down-regulation of the PGR from the LE and GE. Altered endometrial gene expression induced by low P4 was associated with a reduced capacity of the uterus to support conceptus development after embryo transfer on Day 7. In conclusion, the present study provides clear evidence that the temporal changes in the transcriptome of the endometrium of cyclic heifers are sensitive to circulating P4 concentrations in the first few days after estrus. Under low P4 conditions, a suboptimal uterine environment with reduced ability to support conceptus elongation is observed.
Subject(s)
Embryonic Development , Endometrium/metabolism , Estrous Cycle/metabolism , Gene Expression Profiling , Progesterone/blood , Animals , Cattle , Dinoprost/pharmacology , Down-Regulation , Embryo Transfer , Embryo, Mammalian , Estrous Cycle/genetics , Female , Gene Expression/drug effects , Osmolar Concentration , Pregnancy , Principal Component Analysis , Receptors, Progesterone/metabolism , Time FactorsABSTRACT
The aim of the present study was to compare the hormonal and metabolic characteristics and endometrial gene expression profiles in beef heifers yielding either a viable or degenerate embryo on Day 7 after insemination as a means to explain differences in embryo survival. Oestrus was synchronised in cross-bred beef heifers (n = 145) using a controlled internal drug release (CIDR)-prostaglandin protocol. Heifers (n = 102) detected in standing oestrus (within 24-48 h after CIDR removal) were inseminated 12-18 h after detection of oestrus (Day 0) with frozen-thawed semen from a single ejaculate of a bull with proven fertility. Blood samples were collected from Day 4 to Day 7 after oestrus to measure progesterone (on Days 4, 5 and 7), insulin and insulin-like growth factor (IGF)-I (on Days 4 and 6) and urea (on Day 7) concentrations. All animals were killed on Day 7. Uterine pH was determined at the time of death. Animals from which an embryo was recovered were classified as either having a viable embryo (morula/blastocyst stage; n = 32) or a retarded embryo (arrested at the two- to 16-cell stage; n = 19). In addition, 14 single-celled unfertilised oocytes were recovered, giving an overall recovery rate of 64%. There was no significant difference in the blood parameters determined or uterine pH at the time of death between heifers with either a viable or retarded embryo. The relative abundance of nine transcripts (i.e. MOGAT1, PFKB2, LYZ2, SVS8, UHRF1, PTGES, AGPAT4, DGKA and HGPD) of 53 tested in the endometrial tissue differed between heifers with a viable or retarded embryo. Both LYZ2 and UHRF1 are associated with regulation of the immune system; PFKFB2 is a mediator in glycolysis; MOGAT, AGPAT4 and DGKA belong to the triglyceride synthesis pathway; and PTGES and HGPD belong to the prostaglandin pathway. Both these metabolic pathways are important for early embryonic development. In conclusion, retarded embryo development in the present study was not related to serum progesterone, IGF-I, insulin or urea concentrations, nor to uterine pH at the time of death. However, altered expression of genes involved in the prostaglandin and triglyceride pathways, as well as two genes that are closely associated with the regulation of immunity, in the endometrium may indicate a uterine component in the retardation of embryo development in these beef heifers.
Subject(s)
Embryonic Development/physiology , Endometrium/metabolism , Gene Expression , Analysis of Variance , Animals , Area Under Curve , Cattle , Estrus/metabolism , Estrus Synchronization/metabolism , Female , Insemination, Artificial , Insulin/blood , Insulin-Like Growth Factor I/metabolism , Progesterone/blood , Radioimmunoassay , Reverse Transcriptase Polymerase Chain Reaction , Urea/bloodABSTRACT
To investigate the effects of pregnancy or post-ovulatory progesterone (P(4)) supplementation on the expression of oestrogen and P(4) receptors (ESRs and PGRs) in the bovine uterus, heifers (n=263) were randomly assigned to the following treatments: i) cyclic, normal P(4); ii) cyclic, high P(4); iii) pregnant, normal P(4); and iv) pregnant, high P(4) on days 5, 7, 13 and 16 of pregnancy/oestrous cycle. Elevated P(4) was achieved through P(4)-releasing intravaginal device insertion on day 3 after oestrus, resulting in increased concentrations from day 3.5 to 8 (P<0.05) in the high groups than in the normal groups. Irrespective of treatment, PGR and ESR1 mRNA expressions were highest on days 5 and 7 and decreased on day 13 (P<0.05), while ESR2 mRNA expression increased on day 7 (P<0.05) and similar levels were maintained within the normal P(4) groups subsequently. Expression in the high P(4) groups decreased on day 13 (P<0.05). PGR-AB and PGR-B protein expressions were high in the luminal and superficial glands on days 5 and 7, but by day 13, expression had declined to very low or undetectable levels and high P(4) concentration tended to decrease or decreased significantly (P<0.05) the expression in these regions on days 5 and 7. ESR1 protein expression was high, with no treatment effect. ESR2 protein was also highly expressed, with no clear effect of treatment. In conclusion, early post-ovulatory P(4) supplementation advances the disappearance of PGR protein from the luminal epithelium on days 5 and 7, and decreases ESR2 mRNA expression during the mid-luteal phase, but has no effect on PGR or ESR1 mRNA expression.
Subject(s)
Pregnancy, Animal/blood , Progesterone/blood , RNA, Messenger/biosynthesis , Receptors, Estrogen/biosynthesis , Receptors, Progesterone/biosynthesis , Uterus/metabolism , Animals , Cattle , Down-Regulation , Estrogen Receptor alpha/biosynthesis , Estrogen Receptor beta/biosynthesis , Estrous Cycle , Female , Immunohistochemistry , Pregnancy , Proteins/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The objective of this study was to determine the temporal and spatial expression patterns of genes encoding transporters, as well as selected secreted proteins that may be regulated by progesterone (P4) and/or the presence of the conceptus in the bovine endometrium. Estrus-synchronized beef heifers were randomly assigned to either: 1) pregnant, high P4; 2) pregnant, normal P4; 3) cyclic, high P4; or 4) cyclic, normal P4. Uteri were collected on days 5, 7, 13, and 16 of the estrous cycle or pregnancy. Localization of mRNAs for ANPEP, CTGF, LPL, LTF, and SLC5A1 in the uteri was determined by radioactive in situ hybridization, and expression quantified in the endometria by quantitative real-time PCR. ANPEP localized to luminal (LE) and superficial glandular (sGE) epithelia of all heifers on days 5 and 7 only. SLC5A1 mRNA was detected in the LE and sGE on days 13 and 16 in all heifers, and expression increased on day 16 in pregnant groups. CTGF localized weakly to the LE and GE on days 5 and 7 but increased on days 13 and 16 with an increase (P < 0.05) in CTGF expression in high P4 (day 7) and pregnant heifers (day 16). Both LPL and LTF localized to the GE only on days 5 and 7. In conclusion we have characterized the temporal expression pattern of these genes and modulation of their transcript abundance by P4 (CTGF, LPL) and/or the conceptus (CTGF, SLC5A1) likely modifies the uterine microenvironment, enhancing histotroph composition and contributing to advanced conceptus elongation.
Subject(s)
Endometrium/drug effects , Endometrium/metabolism , Gene Expression Regulation/drug effects , Membrane Transport Proteins/genetics , Pregnancy Proteins/metabolism , Pregnancy, Animal/genetics , Progesterone/pharmacology , Animals , CD13 Antigens/genetics , CD13 Antigens/metabolism , Cattle , Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Female , Lactoferrin/genetics , Lactoferrin/metabolism , Lipoprotein Lipase/genetics , Lipoprotein Lipase/metabolism , Membrane Transport Proteins/metabolism , Pregnancy , Pregnancy Proteins/genetics , Pregnancy, Animal/drug effects , Progesterone/administration & dosage , RNA Transport/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sodium-Glucose Transporter 1/genetics , Sodium-Glucose Transporter 1/metabolismABSTRACT
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(2alpha) (PGF(2alpha)) 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 48h after first PGF(2alpha) 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(2alpha) administration on Days 3, 3.5, and 4 (PG3; n=10), (2) PGF(2alpha) 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(2alpha) administration on Days 5 and 6 (PRID+PGF(2alpha); 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(2alpha) 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(2alpha) 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.
Subject(s)
Cattle/physiology , Luteal Phase/blood , Progesterone/blood , Administration, Intravaginal , Animals , Corpus Luteum/anatomy & histology , Dinoprost/administration & dosage , Female , Luteolysis , Organ Size , Pregnancy , Progesterone/administration & dosageABSTRACT
The postovulatory rise in circulating progesterone (P4) concentrations is associated with increased pregnancy success in beef and dairy cattle. Our study objective was to determine how elevated P4 alters endometrial gene expression to advance conceptus development. Synchronized heifers were inseminated (Day 0) and randomly assigned to pregnant high P4 or to pregnant normal P4. All high P4 groups received a P4-release intravaginal device on Day 3 after insemination that increased P4 concentrations up to Day 7 (P < 0.05). Tissue was collected on Day 5, 7, 13, or 16 of pregnancy, and endometrial gene expression was analyzed using the bovine Affymetrix (Santa Clara, CA) microarrays. Microarray analyses demonstrated that the largest number of P4-regulated genes coincided with the day when the P4 profiles were different for the longest period. Genes with the largest fold change increase (such as DGAT2 and MSTN [also known as GDF8]) were associated with triglyceride synthesis and glucose transport, which can be utilized as an energy source for the developing embryo. Temporal changes occurred at different stages of early pregnancy, with the greatest difference occurring between well-separated stages of conceptus development. Validation of a number of genes by quantitative real-time PCR indicated that P4 supplementation advances endometrial gene expression by altering the time (FABP, DGAT2, and MSTN) or duration (CRYGS) of expression pattern for genes that contribute to the composition of histotroph.
Subject(s)
Embryonic Development , Endometrium/metabolism , Pregnancy, Animal/metabolism , Progesterone/metabolism , Animals , Cattle , Female , Gene Expression/drug effects , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Pregnancy , Progesterone/administration & dosage , Time FactorsABSTRACT
The steroid hormone progesterone (P(4)) plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating P(4) in the immediate post-conception period have been associated with an advancement of conceptus elongation, an associated increase in interferon-tau production and higher pregnancy rates in cattle. Using in vitro and in vivo models and approximately 8500 bovine oocytes across six experiments, the aim of this study was to establish the route through which P(4) affects bovine embryo development in vitro and in vivo. mRNA for P(4) receptors was present at all stages of embryo development raising the possibility of a direct effect of P(4) on the embryo. Exposure to P(4) in vitro in the absence or presence of oviduct epithelial cells did not affect the proportion of embryos developing to the blastocyst stage, blastocyst cell number or the relative abundance of selected transcripts in the blastocyst. Furthermore, exposure to P(4) in vitro did not affect post-hatching elongation of the embryo following transfer to synchronized recipients and recovery on Day 14. By contrast, transfer of in vitro derived blastocysts to a uterine environment previously primed by elevated P(4) resulted in a fourfold increase in conceptus length on Day 14. These data provide clear evidence to support the hypothesis that P(4)-induced changes in the uterine environment are responsible for the advancement in conceptus elongation reported previously in cattle and that, interestingly, the embryo does not need to be present during the period of high P(4) in order to exhibit advanced elongation.
Subject(s)
Cattle , Embryo, Mammalian/drug effects , Endometrium/drug effects , Endometrium/physiology , Pregnancy, Animal , Progesterone/pharmacology , Animals , Cattle/embryology , Cattle/genetics , Cattle/metabolism , Cattle/physiology , Cells, Cultured , Embryo Transfer , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Embryo, Mammalian/physiology , Embryonic Development/drug effects , Embryonic Development/genetics , Endometrium/metabolism , Female , Gene Expression Regulation, Developmental/physiology , Pregnancy , Pregnancy, Animal/blood , Pregnancy, Animal/genetics , Pregnancy, Animal/physiology , Progesterone/blood , Progesterone/metabolism , Progesterone/physiology , RNA, Messenger/analysis , Receptors, Progesterone/genetics , Receptors, Progesterone/metabolism , Signal Transduction/physiologyABSTRACT
Progesterone is essential for establishment and maintenance of pregnancy in mammals. The objective of this study was to examine the effect of elevating progesterone during the different physiological stages of early embryo development on embryo survival. Estrus was synchronized in cross-bred beef heifers (n=197, approximately 2-years old) and they were inseminated 12-18h after estrus onset (=Day 0). Inseminated heifers were randomly assigned to 1 of 3 treatments: (1) Control, n=69; (2) progesterone supplementation using a Controlled Internal Drug Release Device (CIDR) from Day 3 to 6.5, n=64; or (3) progesterone supplementation using a CIDR from Day 4.5 to 8, n=64. Body condition (BCS) and locomotion scores (scale of 1-5) were recorded for all animals. Animals with a locomotion score >/=4 (very lame) were excluded. Embryo survival rate was determined at slaughter on Day 25. Conceptus length and weight were recorded and the corpus luteum (CL) of all pregnant animals was dissected and weighed. Supplementation with exogenous progesterone increased (P<0.05) peripheral progesterone concentrations, but did not affect embryo survival rate compared with controls. Mean CL weight, conceptus length and conceptus weight were not different between treatments. There was a positive relationship (P<0.04) between the increase in progesterone concentrations from Days 3 to 6.5 and embryo survival rate in treated heifers and a similar trend existed between the increase from Days 4.5 to 8 (P<0.06). There was also a positive relationship (P<0.05) between the progesterone concentration on Day 6.5 and the embryo survival rate in treated heifers. A direct correlation was seen between locomotion score and embryo survival rate, with higher (P<0.05) early embryo survival rates in heifers with a lower locomotion score. In conclusion, supplementation with progesterone at different stages of early embryo development increased peripheral progesterone concentration and resulted in a positive association between changes in progesterone concentration during the early luteal phase and embryo survival rate. Supplementation with progesterone had no effect on either CL weight or conceptus size in pregnant animals. Lameness had a significant negative effect on early embryo survival.