Bovine Estrous Cycle Research Articles

Synchronization of the bovine estrous cycle: Current and emerging opportunities

Controlling the bovine estrous cycle is a necessity if performing fixed-time artificial insemination or embryo transfer. Addition­ally, irrespective of the genetic objectives driving the decision to use reproductive technologies, the use of synchronization protocols often has beneficial effects on the overall reproduc­tive performance and commercial production output of a beef cattle operation. This proceedings paper provides a general review of the conceptual goals and challenges associated with synchronization of estrus, and directs readers to resources on current protocols and emerging opportunities in the field.

Uterine Fluid Extracellular Vesicles Proteome Is Altered During the Estrous Cycle

Uterine environment is tightly and finely regulated via various signaling pathways mediated through endocrine, exocrine, autocrine, juxtacrine, and paracrine mechanisms. In utero signaling processes are paramount for normal and abnormal physiology which involves cell to cell, cells to gametes, cells to embryo, and even interkingdom communications due to presence of uterine microbiota. Extracellular vesicles (EVs) in the uterine fluid (UF) and their cargo components are known to be mediators of in utero signaling and communications. Interestingly, the changes in UF-EV proteome during the bovine estrous cycle and the effects of these differentially enriched proteins on embryo development are yet to be fully discovered. In this study, shotgun quantitative proteomics-based mass spectrometry was employed to compare UF-EV proteomes at day 0, 7, and 16 of the estrous cycle to understand the estrous cycle-dependent dynamics. Furthermore, different phase UF-EVs were supplemented in embryo cultures to evaluate their impact on embryo development. One hundred fifty-nine UF-EV proteins were differentially enriched at different time points indicating the UF-EV proteome is cycle-dependent. Overall, many identified pathways are important for normal uterine functions, early embryo development, and its nutritional needs, such as antioxidant activity, cell morphology and cycle, cellular homeostasis, cell adhesion, and carbohydrate metabolic process. Furthermore, the luteal phase UF-EVs supplementation increased invitro blastocyst rates from 25.0 ± 5.9% to 41.0 ± 4.0% (p ≤ 0.05). Our findings highlight the importance of bovine UF-EV in uterine communications throughout the estrous cycle. Interestingly, comparison of hormone-synchronized EV proteomes to natural cycle UF-EVs indicated shift of signaling. Finally, UF-EVs can be used to improve embryo production invitro.

NR3C1 and glucocorticoid-regulatory genes mRNA and protein expression in the endometrium and ampulla during the bovine estrous cycle

The bovine reproductive tract exhibits changes during the estrous cycle modulated by the interplay of steroid hormones. Glucocorticoids can be detrimental when stress-induced but are relevant at baseline levels for appropriate reproductive function. Here, an analysis of quantitative real-time PCR was performed to study the bovine glucocorticoid-related baseline gene transcription in endometrial and ampullar tissue samples derived from three time points of the estrous cycle, stage I (Days 1–4), stage III (Days 11–17) and stage IV (Days 18–20). Our results revealed expression differences during stages, as expression observed in the ampulla was higher during the post-ovulatory phase (stage I), including the glucocorticoid receptor NR3C1, and some of its regulators, involved in glucocorticoid availability (HSD11B1 and HSD11B2) and transcriptional actions (FKBP4 and FKBP5). In contrast, in the endometrium, higher expression of the steroid receptors was observed during the late luteal phase (stage III), including ESR1, ESR2, PGRMC1 and PGRMC2, and HSD11B1 expression decreased, while HSD11B2 increased. Moreover, at protein level, FKBP4 was higher expressed during the late luteal phase, and NR3C1 during the pre-ovulatory phase (stage IV). These results suggest that tight regulation of the glucocorticoid activity is promoted in the ampulla, when reproductive events are taking place, including oocyte maturation. Moreover, most expression changes in the endometrium were observed during the late luteal phase, and may be related to the embryonic maternal recognition. In conclusion, the glucocorticoid regulation changes across the estrous cycle and may be playing a role on the reproductive events occurring in the bovine ampulla and endometrium.

The mRNA expression of the three major described cold-inducible proteins, including CIRBP, differs in the bovine endometrium and ampulla during the estrous cycle

The cold-inducible proteins (CIPs) are essential for post-transcriptional gene regulation playing diverse tissue-specific roles in maintaining normal cellular function and morphogenesis. The potential implications of CIPs in reproductive events raise questions about their role in the physiology of the bovine reproductive tract. However, the expression changes of CIPs during the bovine estrous cycle have not been studied so far. Here, we hypothesized that the bovine estrous cycle could affect the mRNA expression of the CIPs and other candidate transcripts in the reproductive tract. This study aimed to examine estrous cycle-dependent mRNA expression patterns in the bovine endometrium and ampulla of three of the major described CIPs (CIRBP, RBM3, SRSF5), a set of inflammatory cytokines (IL-10, IL-18, IL-1β), and other candidate genes (IL-10RA, IL-10RB, BCL2, NLRP3, STAT1, STAT3, STAT5A, STAT6). Endometrial and ampullar tissues were assessed by RT-qPCR. Additionally, the mRNA expression levels were correlated among them and with follicular progesterone and estradiol concentrations. The transcript levels of CIPs increased in the endometrium during stage III (Days 11–17) compared to stage I (Days 1–4) and IV (Days 18–20). In the ampulla, the mRNA expression of CIRBP increased during the late luteal phase (stage III), but no differences in the expression of other CIPs were observed. This study expands the current knowledge regarding mRNA expression in the endometrium and oviductal ampulla of cycling heifers, focusing mainly on the CIPs. A better understanding of the mechanisms within the uterus and oviduct during the estrous cycle is crucial to improving the fertility rate.

Study of progesterone level during first trimester of pregnancy in cows.

Measurement of progesterone is an indirect method for pregnancy diagnosis in many livestock species including cattle, buffaloes, sheep, and goats. Conception extends the life of the corpus luteum (CL) by preventing the luteolytic mechanism from being triggered, thus prolonging and maintaining its functional characteristics, ensuring continued high progesterone levels (Spencer, et.al., 1995). (Shemesh et al., 1973) proposed that the difference in peripheral plasma progesterone levels between pregnant and non pregnant cows, 19 days after insemination, can form the basis for a very early pregnancy test. (Laing and Heap 1972) first documented this in milk to diagnose cows in early pregnancy Progesterone maintains the uterine endometrium in a state which supports embryonic development, implantation, and foetoplacental development. Progesterone concentrations vary with the stage of the estrous cycle which makes it one of the most commonly studied reproductive hormones in bovine ruminants for pregnancy detection and ovarian activity (Kaneko,et.al.,1994). Studies in the bovine estrous cycle indicate that the milk or serum progesterone concentrations reach a maximum value 13-14 days after estrus, and if the animal is pregnant, these continue to remain elevated up to day 21 after fertilization (Parkinson, et al.,1994) . These high levels of progesterone in serum or milk between days 18 and 24 after insemination form the basis of establishment of pregnancy in cattle (Sasser, and Ruder,1987; Shemesh,1973). Interferon-???? exerts its antiluteolytic effect by inhibiting the endometrial expression of oxytocin receptors, through which oxytocin stimulates pulsatile PGF2???? release (Wolf,2003). Although low progesterone concentrations at 18 to 24 days after breeding can accurately predict non pregnancy, high progesterone concentrations during this period are not the specific indicators of pregnancy due to variations among cows in duration of the estrous cycle as well as the incidence of early or late embryonic mortality. The advantages of progesterone assay for pregnancy diagnosis include noninvasive collection of milk sample and the feasibility to conduct the test on the farm using commercial cow-side milk progesterone test kits [Pennington,1985 ; Gowan,1982 ; Wimpy,et.al.,1986), though the sensitivity gets compromised to some extent with these assay kits.
 Key Word: progesterone, pregnancy, cows

Modelling Oscillatory Patterns in the Bovine Estrous Cycle with Boolean Delay Equations

Boolean delay equations (BDEs), with their relatively simple and intuitive mode of modelling, have been used in many research areas including, for example, climate dynamics and earthquake propagation. Their application to biological systems has been scarce and limited to the molecular level. Here, we derive and present two BDE models. One is directly derived from a previously published ordinary differential equation (ODE) model for the bovine estrous cycle, whereas the second model includes a modification of a particular biological mechanism. We not only compare the simulation results from the BDE models with the trajectories of the ODE model, but also validate the BDE models with two additional numerical experiments. One experiment induces a switch in the oscillatory pattern upon changes in the model parameters, and the other simulates the administration of a hormone that is known to shift the estrous cycle in time. The models presented here are the first BDE models for hormonal oscillators, and the first BDE models for drug administration. Even though automatic parameter estimation still remains challenging, our results support the role of BDEs as a framework for the systematic modelling of complex biological oscillators.

36 Analysis of miRNA content of oviduct and uterine extracellular vesicles across the bovine estrous cycle

With the aim of investigating the possible hormonal regulatory effect of the oestrous cycle on miRNA content in the extracellular vesicles (EVs) of bovine oviducal and uterine fluids (OF, UF), we performed a bioinformatic analysis of these miRNAs, their target genes, and their biological pathways. Reproductive tracts were collected from slaughtered heifers and selected according to their corpus luteum morphology, corresponding to the 4 stages of the oestrous cycle (n=5 per stage; S1: days 1 to 4, S2: days 5–10, S3: days 11–17, S4: days 18–20) and transported to the laboratory on ice. EVs were obtained by size exclusion chromatography (PURE-EVs-Hansa Biomed) from the flushing of 1.2mL and 2.5mL of OF and UF, respectively. To concentrate the EVs, they were ultracentrifuged and suspended in 100µL of PBS. Total RNA extraction was obtained from 70µL of the previous pellet, using miRNeasy Mini Kit (Qiagen). Then, 100 to 200ng of the obtained RNA was reverse transcribed using miScript II RT Kit (Qiagen). MicroRNA (miRNA) expression profiling was done by primer-based real-time quantitative PCR of 383 mature miRNA sequences. Possible miRNA target genes and their biological pathways were predicted using the miRWalk database. Among EV miRNAs in OF, bta-miR-130a, bta-miR-382, and bta-miR-1291 were the most abundant at all stages of the oestrous cycle, displaying a significantly progressive increase from stages 1 to 4 (P<0.05). In UF, bta-miR-17-5p, bta-miR-206, bta-miR-22-5p, bta-miR-502a, and bta-miR-503-3p were the most abundant at all stages of the cycle, showing greater differences between S1 and S3 (P<0.05). Other miRNAs were exclusively present in a specific stage of the oestrous cycle in OF: bta-miR-21-5p (S1), bta-miR-146a (S2), bta-miR-128 (S3), and bta-miR-147 (S4). In UF, bta-miR-218 (S1), bta-miR-208b (S2), bta-miR-340 (S3), and bta-miR-335 (S4) were found. Table 1 presents some of these miRNAs, their predicted target genes, and functional pathways. In conclusion, this study highlights the effect of the oestrous cycle on miRNAs contained in the EVs of OF and UF. These miRNAs are related to relevant biological pathways implicated in oviduct and uterus modulation across the cycle, but they may also prepare those organs for embryo/conceptus presence and development. Table 1. Micro (mi)RNAs of oviductal (OF) and uterine fluid (UF) extracellular vesicles (EVs), their target genes, and biological pathways Reproductive fluid miRNAs Target genes Target pathways OF bta-miR-130a BMPR2, SMAD5, SMAD4 BMP signalling bta-miR-1291 SLC2A1 Glucagon signalling bta-miR-21–5p LIF Pluripotency stem cells regulation UF bta-miR-17-5p STAT3 Prolactin signalling bta-miR-206 ESR1 Oestrogen signalling bta-miR-340 HRAS Ras/MAPK/ERK signalling (embryo implantation) This research was funded by MINECO-Spain AGL2015-70140-R, PID2019-111641RB-I00, RTI2018-093548-B-I00; SENESCYT-Ecuador (YNC); FAPESP-Brazil 2017/20339-3 (CLVL), 2014/22887-0 (JCS), 2019/04981-2 (RM); CNPq-Brazil 304276/2018-9, 420152/2018-0 (CLVL).

The effect of basic fibroblast growth factor 2 on the bovine corpus luteum depends on the stage of the estrous cycle and modulates prostaglandin F2α action

The roles of fibroblast growth factor 2 (FGF2) in the corpus luteum (CL) function and its modulatory effect on prostaglandin (PG) F2α during the bovine estrous cycle were studied using the following design of in vivo and in vitro experiments: (1) effects of FGF2 and FGF receptor 1 inhibitor (PD173074) on bovine CL function in the early (PGF2α-resistant) and mid (PGF2α-responsive) luteal stage in vivo, (2) the modulatory effect of FGF2 on PGF2α action during the luteal phase in vivo and (3) effects of FGF2 and PD173074 on bovine CL secretory function in vitro. Cows were treated by injection into the CL with: (1) saline (control), (2) FGF2, (3) PD173074, (4) FGF2 followed by intramuscular (i.m.) PGF2α, (5) PD173074 followed by i.m. PGF2α and (6) i.m. PGF2α as a positive control. For in vitro experiments, CL explants were treated with the aforementioned factors. Progesterone (P4) concentrations of blood samples or culture media were determined by radioimmunoassay. Relative mRNA expressions of the genes involved in angiogenesis and steroidogenesis were determined by quantitative real-time PCR. Although FGF2 treatment on day 4 of the estrous cycle did not change the cycle length, FGF2 with PGF2α decreased the P4 concentrations observed during the estrous cycle compared to the control group (P < 0.001). Moreover, FGF2 treatment on day 10 prolonged CL function as indicated by a significantly greater concentration of P4 on day 21 compared to the control group. In the in vitro study, FGF2 decreased cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase (HSD3B1) mRNA expression (P < 0.01) and decreased P4 production in the early-stage CL (P < 0.001). However, FGF2 + PGF2α or PGF2α alone resulted in an elevation of steroidogenic acute regulatory protein and CYP11A1 mRNA expression and P4 secretion in the early-stage CL (P < 0.01). In the mid-luteal phase, FGF2 upregulated CYP11A1 and HSD3B1 mRNA expression (P < 0.01), while FGF2 + PGF2α increased only HSD3B1 mRNA expression (P < 0.001). In conclusion, FGF2 seems to play a modulatory role in CL development or luteolysis, differentially regulating steroidogenesis and angiogenic factors as well as PGF2α actions.

Treatment with prostaglandin F2α and an intravaginal progesterone insert promotes follicular maturity in advance of gonadotropin-releasing hormone among postpartum beef cows

An experiment was designed to evaluate treatments to promote ovarian follicular maturity in advance of administration of exogenous gonadotropin-releasing hormone (GnRH; 100 μg gonadorelin) for control of the bovine estrous cycle. We hypothesized prostaglandin F2α (PGF2α; 500 μg cloprostenol) followed by an intravaginal progesterone-releasing insert (CIDR; 1.38 g progesterone) would induce greater follicle size and serum estradiol at the time of GnRH administration. Postpartum cows (n = 194) in two locations were assigned to one of five treatments based on age, days postpartum, and body condition score. Cows in Treatment 1 were treated with the standard 7-d CO-Synch + CIDR protocol: administration of GnRH and CIDR insertion on Day −10, and administration of PGF2α and CIDR removal on Day −3. Treatments 2–5 were designed in a 2 × 2 factorial arrangement, with Treatment 1 included as an additional reference. On Day −17, cows in Treatments 2–5 received a CIDR insert, either with (Treatments 2 and 3) or without (Treatments 4 and 5) administration of PGF2α at CIDR insertion. On Day −10, all cows were administered GnRH, and CIDR inserts were either removed (Treatments 2 and 4) or remained in place until Day −3 (Treatments 3 and 5). Treatment with PGF2α and CIDR in advance of GnRH (Treatments 2 and 3) resulted in increased diameter of the largest ovarian follicle (P < 0.001) and increased serum concentrations of estradiol (P < 0.0005) on Day −10. In addition, variation among cows in CL status (no CL vs. a single CL vs. multiple CL) on Day −3 tended to be decreased (P = 0.08), with cows more likely to have a single CL rather than no CL or multiple CL. Lastly, the proportion of cows expressing estrus prior to fixed-time artificial insemination tended (P = 0.08) to be improved. Results support the hypothesis that administration of PGF2α and treatment with a CIDR for 7 days prior to GnRH promotes follicular maturity in advance of GnRH administration and may provide an approach by which to enhance response of postpartum beef cows to GnRH-based estrus synchronization programs.

Model-based exploration of the impact of glucose metabolism on the estrous cycle dynamics in dairy cows

BackgroundNutrition plays a crucial role in regulating reproductive hormones and follicular development in cattle. This is visible particularly during the time of negative energy balance at the onset of milk production after calving. Here, elongated periods of anovulation have been observed, resulting from alterations in luteinizing hormone concentrations, likely caused by lower glucose and insulin concentrations in the blood. The mechanisms that result in a reduced fertility are not completely understood, although a close relationship to the glucose-insulin metabolism is widely supported.ResultsFollowing this idea, we developed a mathematical model of the hormonal network combining reproductive hormones and hormones that are coupled to the glucose compartments within the body of the cow. The model is built on ordinary differential equations and relies on previously introduced models on the bovine estrous cycle and the glucose-insulin dynamics. Necessary modifications and coupling mechanisms are thoroughly discussed. Depending on the composition and the amount of feed, in particular the glucose content in the dry matter, the model quantifies reproductive hormones and follicular development over time. Simulation results for different nutritional regimes in lactating and non-lactating dairy cows are examined and compared with experimental studies. The simulations describe realistically the effects of nutritional glucose supply on the ovulatory cycle of dairy cattle.ConclusionsThe mathematical model enables the user to explore the relationship between nutrition and reproduction by running simulations and performing parameter studies. Regarding its applicability, this work is an early attempt towards developing in silico feeding strategies and may eventually help to refine and reduce animal experiments.ReviewersThis article was reviewed by John McNamara and Tin Pang (nominated by Martin Lercher).

Deciphering the oviductal extracellular vesicles content across the estrous cycle: implications for the gametes-oviduct interactions and the environment of the potential embryo

BackgroundThe success of early reproductive events depends on an appropriate communication between gametes/embryos and the oviduct. Extracellular vesicles (EVs) contained in oviductal secretions have been suggested as new players in mediating this crucial cross-talk by transferring their cargo (proteins, mRNA and small ncRNA) from cell to cell. However, little is known about the oviductal EVs (oEVS) composition and their implications in the reproductive success. The aim of the study was to determine the oEVs content at protein, mRNA and small RNA level and to examine whether the oEVs content is under the hormonal influence of the estrous cycle.ResultsWe identified the presence of oEVs, exosomes and microvesicles, in the bovine oviductal fluid at different stages of the estrous cycle (postovulatory-stage, early luteal phase, late luteal phase and pre-ovulatory stage) and demonstrated that their composition is under hormonal regulation. RNA-sequencing identified 903 differentially expressed transcripts (FDR < 0.001) in oEVs across the estrous cycle. Moreover, small RNA-Seq identified the presence of different types of ncRNAs (miRNAs, rRNA fragments, tRNA fragments, snRNA, snoRNA, and other ncRNAs), which were partially also under hormonal influence. Major differences were found between post-ovulatory and the rest of the stages analyzed for mRNAs. Interesting miRNAs identified in oEVs and showing differential abundance among stages, miR-34c and miR-449a, have been associated with defective cilia in the oviduct and infertility. Furthermore, functional annotation of the differentially abundant mRNAs identified functions related to exosome/vesicles, cilia expression, embryo development and many transcripts encoding ribosomal proteins. Moreover, the analysis of oEVs protein content also revealed changes across the estrous cycle. Mass spectrometry identified 336 clusters of proteins in oEVs, of which 170 were differentially abundant across the estrous cycle (p-value< 0.05, ratio < 0.5 or ratio > 2). Our data revealed proteins related to early embryo development and gamete-oviduct interactions as well as numerous ribosomal proteins.ConclusionsOur study provides with the first molecular signature of oEVs across the bovine estrous cycle, revealing marked differences between post- and pre-ovulatory stages. Our findings contribute to a better understanding of the potential role of oEVs as modulators of gamete/embryo-maternal interactions and their implications for the reproductive success.

MicroRNA-424/503 cluster members regulate bovine granulosa cell proliferation and cell cycle progression by targeting SMAD7 gene through activin signalling pathway

BackgroundThe granulosa cells are indispensable for follicular development and its function is orchestrated by several genes, which in turn posttranscriptionally regulated by microRNAs (miRNA). In our previous study, the miRRNA-424/503 cluster was found to be highly abundant in bovine granulosa cells (bGCs) of preovulatory dominant follicle compared to subordinate counterpart at day 19 of the bovine estrous cycle. Other study also indicated the involvement of miR-424/503 cluster in tumour cell resistance to apoptosis suggesting this miRNA cluster may involve in cell survival. However, the role of miR-424/503 cluster in granulosa cell function remains elusive Therefore, this study aimed to investigate the role of miRNA-424/503 cluster in bGCs function using microRNA gain- and loss-of-function approaches.ResultsThe role of miR-424/503 cluster members in granulosa cell function was investigated by overexpressing or inhibiting its activity in vitro cultured granulosa cells using miR-424/503 mimic or inhibitor, respectively. Luciferase reporter assay showed that SMAD7 and ACVR2A are the direct targets of the miRNA-424/503 cluster members. In line with this, overexpression of miRNA-424/503 cluster members using its mimic and inhibition of its activity by its inhibitor reduced and increased, respectively the expression of SMAD7 and ACVR2A. Furthermore, flow cytometric analysis indicated that overexpression of miRNA-424/503 cluster members enhanced bGCs proliferation by promoting G1- to S- phase cell cycle transition. Modulation of miRNA-424/503 cluster members tended to increase phosphorylation of SMAD2/3 in the Activin signalling pathway. Moreover, sequence specific knockdown of SMAD7, the target gene of miRNA-424/503 cluster members, using small interfering RNA also revealed similar phenotypic and molecular alterations observed when miRNA-424/503 cluster members were overexpressed. Similarly, to get more insight about the role of miRNA-424/503 cluster members in activin signalling pathway, granulosa cells were treated with activin A. Activin A treatment increased cell proliferation and downregulation of both miRNA-424/503 members and its target gene, indicated the presence of negative feedback loop between activin A and the expression of miRNA-424/503.ConclusionThis study suggests that the miRNA-424/503 cluster members are involved in regulating bovine granulosa cell proliferation and cell cycle progression. Further, miRNA-424/503 cluster members target the SMAD7 and ACVR2A genes which are involved in the activin signalling pathway.

Development of an Endocrine Profiles Based Mathematical Model for the Estrous Cycle in Indian Crossbred CowsDevelopment of an Endocrine Profiles Based Mathematical Model for the Estrous Cycle in Indian Crossbred Cows

Declined fertility of the high-yielding dairy cows reared under intensive condition of management is of great concern not only for India but for the whole world. The bovine estrous cycle, particularly the estrus, is tightly controlled by the complex interplay of various organs and hormones. Mathematical modeling of the bovine estrous cycle could help in understanding the dynamics of this complex biological system. The authors, therefore, developed a mechanistic mathematical model for the estrous cycle of Indian crossbred cows that exclusively includes the key hormones that interact to control these processes. The authors used the data generated by them for approximately 163 estrous cycles having 1793 readings related to different hormones like LH, FSH, progesterone, estrogen, etc., under the control of the hypothalamo-pituitary-gonadal (ovarian) axis. The developed model was capable enough to generate successive estrous cycles of 21-day length. The model contains seven differential equations and 34 parameters. By simulating the developed model, a set of equations and parameters that were obtained were found to describe vividly the system consistent with empirical knowledge. In conclusion, the authors for the first time developed a mechanistic mathematical model of the estrous cycle for the Indian crossbred cows and it can be used for predicting hormonal inter milieu of the complex biological system without spending much time and money towards laboratory estimation. The developed model may also be used as a basis for more elaborate models that can predict the effects of external manipulations and genetic differences in Indian bovines. Keywords: Indian crossbred cow, hormones, differential equations, systems biology Cite this Article Mondal M, Karunakaran M, Ghosh MK et al. Development of an Endocrine Profiles Based Mathematical Model for The Estrous Cycle in Indian Crossbred Cows. Research & Reviews: Journal of Veterinary Science and Technology . 2015; 4(3): 35–38p.

Differences in the bovine milk whey proteome between early pregnancy and the estrous cycle

Current bovine pregnancy detection methods are not reliable until at least day 28 post artificial insemination (AI). The bovine estrous cycle is approximately 21 days; consequently, producers miss an opportunity to rebreed at the next estrous event. Therefore, commercial interest exists for the discovery of novel biomarkers of pregnancy which could reliably detect pregnancy status at or before day 21 of pregnancy. The objective of the present study was to use liquid chromatography tandem mass spectrometry (LC-MS/MS) to perform a global, label-free, proteomics study on (i) milk whey and (ii) extracellular vesicle (EV) enriched milk whey samples, from day 21 of pregnancy, compared with day 21 of the estrous cycle, in order to identify potential protein biomarkers of early pregnancy. The estrous cycles of 10 dairy cows were synchronized, they went through one (control) estrous cycle and these cows were artificially inseminated during the following estrus. These cows were confirmed pregnant by ultrasound scanning. Milk whey samples were collected on day 21 of the estrous cycle and on day 21 post AI. Milk whey samples and EV enriched milk whey samples were analyzed by LC-MS/MS and subsequent analyzes of the label-free quantitative data was performed in MaxQuant and Perseus. Four proteins (APOB, SPADH1, PLIN2 and LPO) were differentially expressed between the proteomes of milk whey from day 21 of pregnancy and day 21 of the estrous cycle (P < 0.05). Ten proteins (PIGR, PGD, QSOX1, MUC1, SRPRA, MD2, GAPDH, FOLR1, GPRC5B and HHIPL2) were differentially expressed between the proteomes of EV enriched milk whey from day 21 of pregnancy and day 21 of the estrous cycle (P < 0.05). These proteins are potential milk whey biomarkers of early pregnancy.

41 Regulation of preovulatory estradiol and its impacts throughout the bovine estrous cycle.

41 Regulation of preovulatory estradiol and its impacts throughout the bovine estrous cycle.

Gene expression profiling of upregulated mRNAs in granulosa cells of bovine ovulatory follicles following stimulation with hCG

BackgroundOvulation and luteinization of follicles are complex biological processes initiated by the preovulatory luteinizing hormone surge. The objective of this study was to identify genes that are differentially expressed in bovine granulosa cells (GC) of ovulatory follicles.MethodsGranulosa cells were collected during the first follicular wave of the bovine estrous cycle from dominant follicles (DF) and from ovulatory follicles (OF) obtained 24 h following injection of human chorionic gonadotropin (hCG). A granulosa cell subtracted cDNA library (OF-DF) was generated using suppression subtractive hybridization and screened.ResultsDetection of genes known to be upregulated in bovine GC during ovulation, such as ADAMTS1, CAV1, EGR1, MMP1, PLAT, PLA2G4A, PTGES, PTGS2, RGS2, TIMP1, TNFAIP6 and VNN2 validated the physiological model and analytical techniques used. For a subset of genes that were identified for the first time, gene expression profiles were further compared by semiquantitative RT-PCR in follicles obtained at different developmental stages. Results confirmed an induction or upregulation of the respective mRNAs in GC of OF 24 h after hCG-injection compared with those of DF for the following genes: ADAMTS9, ARAF, CAPN2, CRISPLD2, FKBP5, GFPT2, KIT, KITLG, L3MBLT3, MRO, NUDT10, NUDT11, P4HA3, POSTN, PSAP, RBP1, SAT1, SDC4, TIMP2, TNC and USP53. In bovine GC, CRISPLD2 and POSTN mRNA were found as full-length transcript whereas L3MBLT3 mRNA was alternatively spliced resulting in a truncated protein missing the carboxy-terminal end amino acids, 774KNSHNEL780. Conversely, L3MBLT3 is expressed as a full-length mRNA in a bovine endometrial cell line. The 774KNSHNEL780 sequence is well conserved in all mammalian species and follows a SAM domain known to confer protein/protein interactions, which suggest a key function for these amino acids in the epigenetic control of gene expression.ConclusionsWe conclude that we have identified novel genes that are upregulated by hCG in bovine GC of OF, thereby providing novel insight into peri-ovulatory regulation of genes that contribute to ovulation and/or luteinization processes.

Progesterone-releasing devices for cattle estrus induction and synchronization: Device optimization to anticipate shorter treatment durations and new device developments

Synchronization programs using progesterone-releasing intravaginal devices that allow for fixed time artificial insemination are still finding increasing application in bovine reproduction. This practice is useful for rationalizing livestock management because an increased number of cows can be inseminated in one session without the need for estrus detection. Although much of the innovation related to the design and development of intravaginal devices for use in cattle took place in the previous century, progress in understanding the physiology of the bovine estrous cycle resulted in shorter treatment durations, a trend which is still continuing. In this competitive market, with little functional differentiation between the existing devices, the shorter treatment duration prompted for optimization of the progesterone content in the device, as the cost of the drug significantly contributes to the price per unit. For CIDR® a reduction of the progesterone content of about 30 per cent was realized. Price reduction remained an important target for further device development. Next to reduction of progesterone content, cheaper and easier to process materials like polyethylene vinyl acetate (EVA) copolymers have been explored to replace the commonly used silicone elastomers. The reengineering effort of CIDR® demonstrated that knowledge of release kinetics and insight into gradual depletion patterns in the device is critical for optimization of drug content without compromising performance (blood levels). More recent publications related to the use of alternative polymers like EVA and polyisoprene (IP) indicated encouraging results regarding further reduction of progesterone content. The use of EVA seems most promising, because it is in principle a low-cost polymer available in many grades and this thermoplastic polymer can be processed easily by means of commonly used techniques like injection molding and extrusion. The use of thermoplastic polymers, however, requires insight into the physical-chemical phenomena related to drug dissolution and re-crystallization taking place in the polymer during processing at high temperatures. These aspects, which may critically affect product stability, are often overlooked and are prompting to cover some of the background in this review. Finally, two different innovative approaches are discussed, one related to programable electronic devices for tailored simultaneous drug release and the other is a flexible drug-loaded helix, which is retained very well in several species without causing the usual inflammatory response.

Validation of a mathematical model of the bovine estrous cycle for cows with different estrous cycle characteristics

A recently developed mechanistic mathematical model of the bovine estrous cycle was parameterized to fit empirical data sets collected during one estrous cycle of 31 individual cows, with the main objective to further validate the model. The a priori criteria for validation were (1) the resulting model can simulate the measured data correctly (i.e. goodness of fit), and (2) this is achieved without needing extreme, probably non-physiological parameter values. We used a least squares optimization procedure to identify parameter configurations for the mathematical model to fit the empirical in vivo measurements of follicle and corpus luteum sizes, and the plasma concentrations of progesterone, estradiol, FSH and LH for each cow. The model was capable of accommodating normal variation in estrous cycle characteristics of individual cows. With the parameter sets estimated for the individual cows, the model behavior changed for 21 cows, with improved fit of the simulated output curves for 18 of these 21 cows. Moreover, the number of follicular waves was predicted correctly for 18 of the 25 two-wave and three-wave cows, without extreme parameter value changes. Estimation of specific parameters confirmed results of previous model simulations indicating that parameters involved in luteolytic signaling are very important for regulation of general estrous cycle characteristics, and are likely responsible for differences in estrous cycle characteristics between cows.

418 Corpus luteum blood flow correlation with circulating progesterone during the bovine estrous cycle

418 Corpus luteum blood flow correlation with circulating progesterone during the bovine estrous cycle

Complementing ODE-Based System Analysis Using Boolean Networks Derived from an Euler-Like Transformation.

In this paper, we present a systematic transition scheme for a large class of ordinary differential equations (ODEs) into Boolean networks. Our transition scheme can be applied to any system of ODEs whose right hand sides can be written as sums and products of monotone functions. It performs an Euler-like step which uses the signs of the right hand sides to obtain the Boolean update functions for every variable of the corresponding discrete model. The discrete model can, on one hand, be considered as another representation of the biological system or, alternatively, it can be used to further the analysis of the original ODE model. Since the generic transformation method does not guarantee any property conservation, a subsequent validation step is required. Depending on the purpose of the model this step can be based on experimental data or ODE simulations and characteristics. Analysis of the resulting Boolean model, both on its own and in comparison with the ODE model, then allows to investigate system properties not accessible in a purely continuous setting. The method is exemplarily applied to a previously published model of the bovine estrous cycle, which leads to new insights regarding the regulation among the components, and also indicates strongly that the system is tailored to generate stable oscillations.