Browsing by Author "Bruemmer, Jason, advisor"
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Item Open Access Contraception vaccination for mares and its effects on cyclicity and estrous behavior(Colorado State University. Libraries, 2019) Reisenauer, Ashley, author; Bruemmer, Jason, advisor; Coleman, Stephen, committee member; Eckery, Douglas, committee member; Bouma, Jerry, committee memberOverpopulation is an issue for wild horses due to limited forage and decreasing water sources. Sterilizing mares without surgical ovariectomy would be cost effective and safer. There are currently no vaccines that cause permanent sterility in mares. Bone Morphogenetic Protein 15 (BMP-15) and Growth Differentiation Factor 9 (GDF-9) are oocyte-specific proteins involved in every stage of follicular development from primordial activation through ovulation. This study investigated the effects of a combination vaccine consisting of these oocyte-specific growth factors, GDF-9 and BMP-15, on mare cyclicity and estrous behavior. We hypothesized that immunization against the combination of these two factors would result in no ovarian cyclicity. Mature, fertile Quarter Horse type mares (n=10/group) each of which had successfully carried a foal within the last 24 months were used. The experiment was conducted from February through September 2018. All mares were vaccinated a total of 5 times starting at week 0, continuing at week 6, 12, 18, and 24. Ten mares received the vaccine consisting of both peptides (GDF-9; SEYFKQFLFPQNEC and BMP-15; QAGSMGSEVLGPSREREGPESNQC) and adjuvant (Seppic Montanide™ Pet Gel A), while control mares received adjuvant alone. All vaccinations were administered IM. Ovarian activity and ovulations were recorded by trans-rectal ultrasonography at least once a week and estrous behaviors were evaluated three days a week by interacting individually with a stallion on a tease rail. Follicle diameters were recorded according to measurements and estrous behavior scored on a 6-point scale (0 = hostile toward stallion – 5 = actively seeking stallion with associated behaviors). Jugular blood samples were collected prior to each weekly palpation, and serum was aspirated for further investigation of the progesterone levels. All control mares cycled normally with ovulations associated with estrus at approximately 3-week intervals. None of the 10 treated mares ovulated or grew a follicle larger than 20 mm during the 8-month experimental period. Mixed estrous behaviors were noted in a few mares throughout the study. Low progesterone levels in serum samples confirmed these findings and are associated with the presence and/or lack of detected corpra lutea. Future research will focus on the active duration of the vaccination to determine the length of effectiveness. This vaccination could serve as a long-term contraceptive in wild horse herd populations.Item Open Access Effects of pregnancy status on organic anion transporters and prostaglandin receptors in equine endometrium during maternal recognition of pregnancy(Colorado State University. Libraries, 2010) Cleys, Ellane R., author; Bruemmer, Jason, advisor; Hansen, Thomas, committee member; Denniston, David, committee member; Bouma, Gerrit, committee memberTo view the abstract, please see the full text of the document.Item Open Access Evaluating the equine endometrial transcriptome during maternal recognition of pregnancy(Colorado State University. Libraries, 2018) Klohonatz, Kristin, author; Bruemmer, Jason, advisor; Coleman, Stephen, committee member; Bouma, Gerrit, committee member; Hess, Ann, committee member; Thomas, Milton, committee memberTo view the abstract, please see the full text of the document.Item Open Access Immunoreactivity of anti pZP antibodies from the serum of SpayVac vaccinated mares to equine zona protein(Colorado State University. Libraries, 2014) Mask, Tracy Ann, author; Bruemmer, Jason, advisor; Bouma, Gerrit, committee member; Kane, Albert, committee member; Ransom, Jason, committee memberImmunocontraception with the porcine zona pellucida (pZP) antigen is a well-published means of wild horse contraception. There are three pZP vaccines currently proposed for use in horses, Zonastat-H®, PZP-22, and SpayVac®. Despite abundant research concerning the safety and contraceptive efficacy of pZP vaccines in numerous species, the contraceptive mechanisms of the pZP antigen remain unclear and have not been investigated thoroughly for SpayVac. We investigated the immunoreactivity of anti pZP antibodies from the serum of SpayVac vaccinated mares to equine zona protein using Western blot and immunohistochemical techniques. These techniques were first applied using a bovine model because bovine oocytes are more easily obtained in large quantities relative to equine oocytes. Once the procedure was validated, equine samples were utilized. Western blot analysis revealed immunoreactivity of anti pZP antibodies that were produced in response to SpayVac vaccination to protein isolated from mature equine oocytes, equine zona pellucidae, equine follicular tissue, and equine ovarian stromal tissue. Immunohistochemical analysis identified the location of binding of anti pZP antibodies to the zona pellucida of mature oocytes isolated from Graafian follicles as well as the zona pellucida of immature oocytes in ovarian tissue. Western blot and immunohistochemical analyses also indicate high specificity of anti pZP antibodies for equine zona protein and predominant affinity for zona protein 3. Collectively, results suggest a model where anti pZP antibodies produced in response to SpayVac vaccination are immunoreactive to equine zona protein in vitro. If available in the follicular fluid and able to permeate the ovary following SpayVac vaccination, anti pZP antibodies may act on not only mature oocytes, but also oocytes of growing follicles in vivo. The results of this study lend insight into the infertility observed following SpayVac vaccination, and may also help explain the long-term ovarian effects following pZP vaccination reported by other studies.Item Open Access Regulatory microRNA delivered to stallion spermatozoa during epididymal maturation(Colorado State University. Libraries, 2016) Twenter, Hannah, author; Bruemmer, Jason, advisor; Bass, Luke, committee member; Bouma, Gerrit, committee member; Coleman, Stephen, committee memberStallion spermatozoa are produced in the seminiferous tubules of the testis. After spermatogenesis, spermatozoa migrate through the seminiferous tubules to the rete testis then efferent ducts which converge to form a single duct within the caput of the epididymis. The epididymis is a convoluted tubule with region-specific luminal profiles. The epididymis consists of three commonly descried regions; caput, corpus, and cauda. Each region performs distinct functions in epididymal maturation of spermatozoa. The caput is responsible for concentration of spermatozoa by reabsorbing excess fluid from the epididymal lumen. The corpus is where majority of maturation occurs as spermatozoa gain motility and shed their cytoplasmic droplet. The cauda primarily serves as a storage site for the spermatozoa until ejaculation or spontaneous emission. All regions of the epididymis are lined with multiple epithelial cell types, each with different functions to provide the ideal luminal environment for the maturation. These epithelial cells also create apical blebs containing small, membranous vesicles named epididymosomes. The apical blebs are released from the apical surface via apocrine secretion and will disintegrate in the lumen, releasing epididymosomes. Epididymosomes transport proteins from the epithelium to the spermatozoa in the lumen. They also contain microRNAs (miRNAs). MicroRNAs are small, non-coding post-transcriptional regulators of mRNAs and can interfere with mRNA transcription through translational repression or degradation. We hypothesize that epididymosomes also transfer miRNA from the epididymal epithelium to spermatozoa. Quantitative real-time polymerase chain reaction was used to determine the miRNA profile of epididymal tissue from the caput and cauda, epididymal spermtozoa from the caput and cauda, and epididymosomes from the caput, proximal corpus, distal corpus, and cauda. Our focus turned to 33 newly-acquired miRNAs with expression specific to the spermatozoa located within the cauda as these are fully mature cells. Comparing the miRNAs present in each sample, 11 miRNAs had a distinct path from epididymal tissue to epididymosomes to spermatozoa, suggesting that miRNAs are transported to spermatozoa from the epididymal epithelium via epididymosomes. Pathway analysis was performed using DIANA tools on the 33 miRNA with expression on in caudal spermatozoa using an a posteriori method with predicted pathways considered significant with P≤0.05. Fifty-one predicted pathways were statistically significant. Some of those predicted pathways suggest a role in cell motility and viability, while others influenced factors in the oocyte or embryo. By developing a better understanding of the mechanisms behind the processes that regulate sperm maturation as well as the roles in embryogenesis better diagnostics for infertility in the horse may be generated.Item Open Access Role of α/β hydroxylase domain containing protein 2 in stallion sperm activation(Colorado State University. Libraries, 2019) McQuagge, Matthew, author; Bruemmer, Jason, advisor; Pinedo, Pablo, committee member; Graham, James, committee member; Winger, Quinton, committee memberCell signaling pathways involved in stallion sperm activation are not completely understood. Furthermore, failure of equine in vitro fertilization is commonly attributed to an inability to successfully capacitate sperm. Sperm activation describes the process by which sperm undergo capacitation, hyperactivation, and acrosome reaction in preparation for interaction with an oocyte. 2-arachidonoylglycerol (2AG) is found in the human sperm membrane and prevents calcium influx through the CatSper channel. The α/β hydroxylase domain containing protein 2 (ABHD2) is also found in the human sperm membrane and functions as a progesterone receptor. When progesterone binds to ABHD2, it removes 2AG from the membrane allowing CatSper to open, which leads to calcium entrance into the cell, resulting in sperm activation. It is unclear if this mechanism holds true in stallion. Experiments were conducted to test the hypothesis that progesterone causes sperm activation through interaction with ABHD2 by 1) determining whether the ABHD2 receptor exists on stallion spermatozoa, 2) determining if progesterone binds to ABHD2 on stallion spermatozoa and 3) demonstrating the role of ABHD2 in sperm activation through correlations between ABHD2 and hyperactivation and/or acrosome reaction. Immunoblotting identified ABHD2 protein in stallion sperm and immunocytochemistry (ICC) localized the receptor to the tail region of stallion spermatozoa. Immunocytochemistry also demonstrated that ABHD2 binds progesterone by restricting fluorescence exhibited by ABHD2 when incubated with progesterone. Stallion sperm were evaluated for hyperactivation with computer assisted sperm analysis (CASA) following incubation in capacitation medium with either 1) an endogenous activator of sperm; 3 µM progesterone (P4), 2) a positive pharmacological stimulator of hyperactivation not associated with ABHD2; procaine or 3) a known ABHD2-action inhibitor methyl arachidonyl flourophosphatnate (MAFP). MAFP is a serine hydroxylase inhibitor and functions by preventing the removal of 2AG caused by exposure of ABHD2 to progesterone and, thus, limits hyperactivation. Flow cytometry was used to measure the acrosomal status of treated sperm as a subset of the hyperactivation measurements. When MAFP was administered prior to treatment with either P4 and/or procaine the hyperactive movement was inhibited (p < 0.05) in the presence of P4 but did not affect procaine induced activity. Results were similar for all ejaculates. The reduced hyperactivation of sperm when incubated with both progesterone and MAFP illustrates a potential connection between ABHD2 and CatSper. No change in acrosomal status was discovered through incubation with P4, procaine, or MAFP. These results indicate 1) that ABHD2 is present on stallion sperm, 2) that progesterone binds to ABHD2 and 3) that progesterone has the potential for causing hyperactivation but does not affect the acrosome reaction.Item Open Access The effects of immunization against bone morphogenetic protein-15 and growth differentiation factor-9 on ovarian function in the mare(Colorado State University. Libraries, 2017) Davis, Kelli, author; Bruemmer, Jason, advisor; Eckery, Douglas, committee member; Bouma, Gerrit, committee member; Pinedo, Pablo, committee memberThe Bureau of Land Management (BLM) claims the current wild horse and burro population is nearly three times what the rangeland can support. Unmanaged, the horse population doubles every four years, which is detrimental for wild horses, wildlife, and rangeland. The BLM is investigating means of population control for these horses. Currently, no contraceptive vaccine exists capable of inducing permanent sterility in mares. This project serves as the first half of a two-year study investigating the effect of vaccination against Bone Morphogenetic Protein 15 (BMP-15) and Growth Differentiation Factor 9 (GDF-9) on follicular growth and ovulation rates in mares. In mice, rats, sheep, cattle, humans, and deer, these growth-factors have been shown to be critical regulators of follicular development and ovulation rate. Mutations involving expression of either BMP-15 or GDF-9 either increase ovulation rates or induce sterility in investigated species, indicating their importance in fertility and their potential value as a target for contraceptive use. Since the role of these growth factors has been proven to be critical for normal follicular development in other species, we hypothesize that vaccination against BMP-15 and/or GDF-9 will prevent ovulation and/or accelerate the depletion of the oocyte reserve in the horse. For this project, 30 mares were randomly assigned to one of three groups (n=10/group). Mares were vaccinated with either BMP-15 or GDF-9 peptides conjugated to keyhole limpet hemocyanin (KLH) and Seppic Montanide™ Pet Gel A polymeric adjuvant, or a control of phosphate buffered saline and adjuvant. The horses received a primary vaccination and three booster injections at weeks 0, 6, 12, and 18. Mares were evaluated three days a week during the breeding season for follicular size and date of ovulation via transrectal ultrasonography. Abnormal ovulations and follicular development were noted. Estrous behavior and sexual receptiveness to a stallion were evaluated using a six-point teasing scale during a rail teasing technique with a stallion three times a week. In order to determine individual antibody responses to the immunizations, blood samples were collected every two weeks, with sera from the samples used for enzyme-linked immunosorbent assay (ELISA). Evaluation of antibody responses demonstrated the majority of BMP-15 treatment mares elicited a consistently high response to the BMP-15 vaccine. However, only two mares in the GDF-9 treatment group demonstrated a consistently high antibody response to the GDF-9 treatment. No difference in ovulation rate (P=0.66) was noted in the GDF-9 group when compared to controls (10.8 and 10.0 ovulations respectively). However, the number of ovulations in the BMP-15 group was decreased (P=0.02; 4.9 ovulations) compared to the control group. Both treatment groups demonstrated differences in the average size of ovulatory follicles (P<0.001) when compared to controls. On average the last recorded size of ovulatory follicles prior to ovulation measured 21.3 mm, 27.8mm, and 35.7mm in the BMP-15, GDF-9 and control groups respectively. Upon evaluation of teasing records, the both the BMP-15 and GDF-9 vaccinated mares displayed estrus behavior less frequently than controls following the second vaccination (P=0.05 and 0.03 respectively). This indicates altered estrous behavior in both BMP-15 and GDF-9 vaccinated mares. In the first year of this study, vaccination against BMP-15 successfully altered ovarian function in the mare by decreasing the ovulation rate and the size of ovulatory follicles. This altered ovarian function was also indicated by an alteration in estrous cycle behaviors in BMP-15 treated mares. Although GDF-9 did not appear to alter ovulation rate, the decrease in average size of follicles at ovulation and altered estrous behavior indicates further research is required to determine if greater effects are observed in subsequent years. Overall, altered ovarian function in both the BMP-15 and GDF-9 groups shows promise that vaccination against these growth factors could potentially serve as a contraceptive for use in controlling wild horse populations.Item Open Access The role of interferon-tau (IFNT) in luteal gene expression, steroidogenesis, and luteal lifespan in the ewe(Colorado State University. Libraries, 2009) Bott, Rebecca Clark, author; Bruemmer, Jason, advisor; Niswender, Gordon, advisorInterferon-tau (IFNT) was evaluated for endocrine actions on the corpus luteum (CL). The hypothesis was that infusion of IFNT would increase luteal expression of interferon-stimulated gene (ISG)-15, and the length of time for ewes to return to estrus. Osmotic pumps containing 200 μg IFNT or BSA (n=12 each) were connected to the uterine vein of non-pregnant ewes 10 days post-estrus. Messenger RNA encoding ISG15 was elevated in CL from pregnant and IFNT-infused ewes (P<0.05) compared to nonpregnant and BSA-treated ewes, respectively. Luteal mRNA encoding ISG15 from ewes treated with IFNT was greater than in ewes treated with BSA (P<0.05). Serum concentrations of progesterone were not different in ewes that received infusions of BSA or IFNT. Progesterone decreased by six hours (P<0.05) in ewes that received BSA+PGF or IFNT+PGF, but did not differ in ewes that received infusions of IFNT +/- PGF at 8, 10, or 12 hours after PGF. There were no differences in prostaglandin E synthase (PGES) or prostaglandin F synthase (PGFS), or in prostaglandin dehydrogenase (PGDH), steroidogenic acute regulatory protein (StAR), peripheral type benzodiazepine receptor (PBR), cytochrome P450 side chain cleavage enzyme (CYP-11A), or 3μ-hydroxysteroid dehydrogenase (3μ-HSD). Seven day infusion of IFNT during the time frame of maternal recognition of pregnancy resulted in 20% of IFNT-treated ewes returning to estrus by d19 compared to 100% of BSA-treated ewes (P<0.01). In conclusion IFNT acts systemically, alters gene expression in the corpus luteum, and decreases the number of ewes returning to estrus by d19.