Browsing by Author "Pinedo, Pablo, committee member"
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Item Open Access Attitudes and competencies of third year veterinary students towards their role as an animal welfare advocate and attitudes towards pain and pain mitigation practices in beef and dairy cattle in the United States by veterinarians and producers(Colorado State University. Libraries, 2019) Johnstone, Ellizabeth Charlotte Spencer, author; Edwards-Callaway, Lily, advisor; Pinedo, Pablo, committee member; Ahola, Jason, committee member; Stuart, Amy K., committee memberAlthough leading veterinary organizations emphasize the importance of animal welfare knowledge, there exists a gap in current veterinary student animal welfare education and training. A survey instrument was created to assess third-year Doctor of Veterinary Medicine (DVM) student knowledge of key animal welfare topics, opinions regarding the inclusion of welfare education in the veterinary curriculum, and views on veterinarian responsibilities as advocates. In Spring 2018, Colorado State University added a required animal welfare course to the DVM curriculum. Pre- and post-course paper surveys were distributed to the third-year students enrolled in the animal welfare. One hundred thirty one completed pre-course surveys were collected and 125 completed post-course surveys were collected. Of the pre and post-course surveys collected, 61 were paired with identification codes and utilized for statistical comparison. Results indicated that the course led students to view the inclusion of an animal welfare course in the veterinary curriculum more favorably (p=0.009) and improved their confidence in conducting research on animal welfare topics (p<0.001). The course did not change students' sense of responsibility towards welfare advocacy. Associations were not found between attitudes towards these issues and demographic variables of home community, respondent gender, and track selection (p>0.06). Veterinarians were consistently ranked by students as the most influential member of a community in matters of animal welfare. Future research on the lack of veterinary student knowledge of animal welfare should be done on a national scale to facilitate strategic development of mandatory animal welfare courses in veterinary curricula. Future research should be designed to gain knowledge regarding DVM students' opinions and attitudes regarding effective methods of incorporating animal welfare education into their professional training. The objective of the second survey project was to evaluate the current pain management practices and opinions towards pain management in cattle of beef and dairy veterinarians and producers in the United States. Pain management strategies in livestock have evolved in the last few decades but a variety of obstacles continue to limit improvements in the use of pain mitigation by members of the cattle industries. One such obstacle is the lack of FDA-approved analgesic drugs for use in cattle in the United States which offers limited pain management options to cattle veterinarians and producers. An on-line survey was developed to investigate current use of pain mitigation by cattle veterinarians and producers. The survey was distributed electronically to multiple listservs in Summer 2018 (BEEF Magazine, American Association of Bovine Practitioners, National Milk Producers Federation Farmers Assuring Responsible Management evaluators, Dairy Moms and Dairy Girls Facebook groups; N=46,577). A total of 1,187 (2.5%) surveys were received; 41.9% of respondents identified as producers, 47.9% as veterinarians, and 10.2% as both. Multivariate logistic regression was used for analysis. Veterinarians (OR [95% CI] = 10.2 [7.21-14.4]) and producer-veterinarians (OR [95% CI] = 3.30 [2.02-5.39]) had significantly greater odds of using analgesia than producers in all cattle ages. Summary data suggest that analgesic use changed with cattle age; 57.6% of respondents used pain management in calves <2 months of age, while 71.6% of respondents used pain management in cattle more than 12 months of age. Respondents agreed that "cattle benefit from receiving analgesic drugs" (76.6%) and that "US/USDA/FDA regulations limit my ability to use analgesic drugs in cattle" (64.01%). Fifty-eight percent of respondents indicated their use of pain management had increased in the last 10 years. Data identify impediments to improving pain management practices in cattle. Results indicate the need for education and communication between veterinarians and producers on the necessity of pain management.Item Open Access Dairy heifer habituation to the milking routine: stress in the primiparous cow and its impacts on behavior and production(Colorado State University. Libraries, 2023) Kness, Danielle, author; Grandin, Temple, advisor; Pinedo, Pablo, committee member; Edwards-Callaway, Lily, committee member; Garry, Franklyn, committee memberThe transitional period surrounding parturition and onset of lactation is undoubtedly a stressful time in the life of a dairy cow. This is especially true for primiparous cows, who have no previous experience to the milking routine and must become accustomed to increased contact with human caretakers as well as the host of novel sights, sounds, smells and sensations in the milking parlor. Behaviors stemming from acute stress have the potential to increase risk of injury to parlor employees, who must be located close to the cows in order to perform their duties. Even so, the specific changes in cow behavior in the parlor over the course of the first lactation are not well documented, presenting a challenge to farm managers who wish to train employees in primiparous cow management. The main focus of this thesis is to present current research on this topic, as well as present new research regarding specific, daily changes in primiparous cow behavior during the first lactation. Chapter one is a review of the current literature regarding sources of stress in first-lactation heifers and their impacts on various aspects of cow behavior and production, as well as on worker safety and wellbeing. The roles of precision livestock farming technologies on modern dairy farms are also discussed in this chapter, as well as the potential of these technologies for dairy cow welfare management and research. The objective of chapter two is to describe the dynamics of milking unit kick-off in primiparous and multiparous cows during the first three months of lactation. Data were collected from 199 primiparous (PRI) and 670 multiparous (MUL) cows who calved between August and November of 2020. From 3 days in milk (DIM) until 90 DIM, data were downloaded daily for each cow from the farm's software program. The main variables of interest were parity category and milking machine kick-offs (KO), which were reported by the milking system when an abrupt interruption in the milk flow occurred. KO events were used in our analysis as a proxy for habituation to the milking routine, and were analyzed by DIM. We found that proportions of KO were greater in PRI than in MUL throughout the monitoring period, and that when analyzed by DIM, first-lactation cows showed a non-linear trend of kick-offs. This indicated that changes in behaviors displayed during the habituation process are not linear, but instead are more complex. Chapter 3 is an analysis of additional data that were collected during the study presented in chapter 2. Study participants were the same, but our goal in this chapter was to investigate any possible relationships between rates of machine kick off, daily changes in milk yield, and occurrence of mastitis during early lactation. Cows with varying frequencies of machine kick-offs were categorized into quartiles. Quartiles were then analyzed for potential interactions with milk yield and mastitis occurrence. Overall, we found no differences in milk yield between KO quartiles, but both primiparous and multiparous cows in the quartile with the highest KO rates had higher rates of mastitis.Item Open Access Developing an integrated livestock-human infectious disease management framework for the dairy farm environment(Colorado State University. Libraries, 2023) Fathke, Robert, author; Rao, Sangeeta, advisor; Pinedo, Pablo, committee member; Reynolds, Stephen, committee member; Duncan, Colleen, committee memberThis research aimed to develop a framework integrating cattle and human infectious disease prevention in the dairy farm environment. Infectious disease dynamics on dairy farms can be complex, with various factors impacting cattle and human health. The advent of the COVID-19 pandemic reminded the world of the complexities of disease dynamics and prevention. Biosecurity is key to infectious disease prevention on dairy farm settings, but preventive efforts might not focus on both cattle and human health. Those trained in veterinary medicine may be especially suited to help bridge this animal-human gap on dairy farms, as these professionals understand disease dynamics and may be trusted to serve in this capacity. Infectious disease risk assessment tools for dairy farms might not fully integrate human health. Developing more integrated risk assessment tools first requires a greater understanding of existing tools and dairy farmer knowledge, attitudes, and practices regarding cattle and human infectious disease prevention. The research described biosecurity, biosafety, and identified potential areas of overlap to create a foundational integrated animal-human infectious disease prevention model. A systematic literature review was conducted on animal producer knowledge, attitudes, and practices regarding personal protective equipment for zoonotic disease prevention. Current biosecurity and biosafety assessment surveys and tools applicable to dairy farm environment were also assessed for structure, content, and degree of human health integration. Assessment of the survey questions and score report from one of these tools included obtaining feedback from a small sample of Front Range Colorado dairy producers. A knowledge, attitudes, and practices questionnaire including elements of cattle and human infectious disease prevention was developed, and data was collected from 50 personnel, including workers and supervisors, across six Front Range Colorado dairy farms. This work found that the word "biosecurity" has many definitions that can vary by profession setting. Many elements of efforts aimed at preventing animal diseases can also be effective in preventing human diseases. Personal protective equipment is an example of such an element. Systematically reviewing literature on personal protective equipment knowledge, attitudes, and practices revealed that animal producers often fail to use preventive measures and may not always perceive zoonoses as a threat. Assessment of existing infectious disease risk assessment tools revealed that none fully and directly integrated human infectious disease prevention. Producer feedback on one tool focusing on cattle health provided valuable feedback on tool design and helped shape recommendations for developing integrated tools. Construction of the integrated knowledge, attitudes, and practices questionnaire was a novel approach to creating a research tool that integrates animal and human infectious disease prevention. Results revealed strengths and weaknesses in knowledge, attitudes, and practices regarding zoonotic disease prevention and helped identify elements that can be addressed to develop a shared understanding between dairy farm supervisors and workers.Item Open Access Development of a bison-specific embryo culture system through targeted supplementation of media with stage-specific growth factors(Colorado State University. Libraries, 2024) Acevedo Barriga, Carolina, author; Barfield, Jennifer P., advisor; Dawit, Tesfaye, committee member; Pinedo, Pablo, committee member; McCue, Patrick, committee member; Yuan, Ye, committee memberIn vitro embryo production (IVP) offers a practical genetic exchange method for bison herds, eliminating the need for live animal transport and reducing stress on the animals. While successful in cattle, IVP efficiency is lower in bison. This study aims to enhance bison embryo quantity and quality by supplementing IVP media with stage-specific growth factors. Thirteen growth factor receptors (GFRs) were screened in bison and bovine embryos, with six GFRs falling within acceptable ranges. Abattoir-sourced oocytes were used for IVP (4 replicates). GFR expression, notably IGFR2, BMPR2, FGFR1, and EGFR1, peaked in bison embryos at the zygote and 8-16 cell stages, with higher IL6 expression at the morula stage. Bovine embryos displayed highest expression of BMPR2, EGFR, and IGFR1 at zygote and 8-16 cell stages, and of FGFR1 and IGFR2 at zygote stages. Corresponding growth factors were incorporated into bison culture media based on GFR expression. Treatment evaluations, included EGF, IGF1, IGF2, IL6, BMP2, FGF1, and a combination of all GFs to bison culture media. Results revealed that the addition of BMP2 resulted in a decrease in cleavage rates. Notably, EGF, IGF1, and IGF2 enhanced blastocyst rates, with IGF1 significantly higher than the control. Subjective embryo qualitative evaluation showed an upward trend in the number of high quality expanded blastocysts, and lipid content decreased (IGF1) while cell count increased (IGF1, IL6, BMP2, FGF1) with growth factor supplementation. In conclusion, supplementing IVP media with GFs, particularly IGF1 at 50 ng/mL, significantly improved both quantity and quality of bison embryos. Application of IVP theology has advance the use of assisted reproductive technologies for bison, potentially benefiting other species.Item Open Access Establishment and characterization of day 30 equine chorionic girdle and allantochorion cell lines(Colorado State University. Libraries, 2019) Salman, Saleh M., author; Bruemmer, Jason E., advisor; Bouma, Gerrit, advisor; Magee, Christianne, committee member; Pinedo, Pablo, committee member; Winger, Quinton, committee memberEstablishing cell lines is a good model for experimental applications to study molecular mechanisms and cell-specific gene expression. A human resistant telomerase reverse transcriptase (hTERT) lentivirus was utilized to establish stable equine embryonic cell lines. Equids have a diffuse epitheliochorial placenta, where the invasive trophoblast is represented by the chorionic girdle (CG) and the noninvasive trophoblast are the allantochorion (AC). Embryonic CG cells are unique to horses compared to other farm animals' embryos. The CG cells are the predecessor of endometrial cups (EC) that differentiate, proliferate, and invade the endometrium by day 38 of pregnancy, yet morphologically, both have similar characteristics supporting the fetal origin for EC. The CG cells have a crucial role in equine chorionic gonadotropin (eCG) production and maintenance of pregnancy during the first trimester. This study has three objectives: 1) establishing a stable cell line from day 30 CG cells and AC using lentivirus encoding hTERT; 2) Characterization of day 30 CG cells and AC cell morphology and expression of eCG alpha (eCGα) and beta (eCGβ) subunits, major histocompatibility complex class II (MHC II), and Kisspeptin receptor (KISS1R) in CG and AC cells; 3) investigating eCG protein production in vitro from day 30 CG and AC cells. Reverse transcriptase (RT) PCR was used to study gene expression in cells and radioimmunoassay (RIA) was used to investigate protein presence in the media. We established a hygromycin-resistant day 30 CG and AC cell lines that express eCGα, eCGβ, and hTERT and confirmed using RT-PCR yielding the predicted bands. The cell lines were maintained for 16 passages, 10 of which were cultured after the lentiviral infection steps. Also, we characterized CG cells as fast-growing, large, binucleated, and epithelioid, and AC cells as rapid-growing showing smaller, squamous, mononucleate, epithelioid, and elongated fibroblastic cells. The RT-PCR results showed eCGα and eCGβ subunits are expressed by both day 30 CG and AC cells, but MHC II and KISS1R genes were not expressed in either of cells. Moreover, RIA results showed that day 30 CG cells did produce eCG protein in vitro earlier than what previous literature has shown. However, day 30 AC cells did not produce eCG protein in vitro, and both CG and AC cell lines stopped secreting eCG in the media after the lentiviral infection.Item Open Access Impact of low-level tannin supplementation on enteric methane emissions, estimated nitrogen excretion, oxidative stress, and animal performance in organic dairy heifers(Colorado State University. Libraries, 2023) Schilling, Ashley, author; Stackhouse-Lawson, Kim, advisor; Place, Sara, committee member; Pinedo, Pablo, committee member; Velez, Juan, committee member; Moore-Foster, Rhyannon, committee memberHeightened attention and concern regarding the role of anthropogenic greenhouse gas (GHG) emissions in climate change has challenged every industry to reduce their environmental impact. In cattle production systems, the importance of feeding the growing human population while minimizing environmental impacts has been given significant attention throughout the 21st century (Steinfeld et al. 2006; Golub et al., 2012; Eisler et al. 2014). In 2020, the United States dairy industry was responsible for approximately 1.4% of total anthropogenic GHG emissions (EPA, 2021). The GHGs with the largest global warming potential (GWP) equivalents in dairy cattle production systems are nitrous oxide (N2O) and methane (CH4) (Rotz et al., 2021). The use of tannins as a feed additive in cattle production systems has been explored as a GHG mitigation strategy given their potential to reduce enteric CH4 and reactive-nitrogen (N) emissions, while also benefiting animal health. Tannins are secondary components of plants comprised of phenolic compounds of diverse molecular weights and of variable complexity (Place et al., 2011). They are classified into two major classes: 1) hydrolysable and 2) condensed tannins and exhibit variable affects depending on their class, concentration/purity, dose, type, and other factors such as animal species, animal physiological state, and diet composition (Makkar 2003; Aboagye and Beauchemin, 2019). When fed to ruminants, such as dairy cattle (Bos taurus), tannins act as rumen modifiers by altering protein and carbohydrate degradation in the rumen. Moreover, tannins have demonstrated anti-microbial, anti-parasitic, antioxidant, anti-inflammatory, and anti-viral effects in animals and the ability to serve as a bloat control mechanism (Mangan, 1988; Jones et al., 1971, Min et al., 2005). Since tannins target rumen microbial populations that assist in fiber degradation, unintended consequences can include reductions in feed intake, digestibility, and rate of BW gain when tannins are supplemented at concentrations greater than 55 g condensed tannins/kg dry matter (DM) (Min et al., 2003). Therefore, the objective of this study was to determine the impact of low-level tannin (< 0.30 g/kg DMI) supplementation on enteric CH4 emissions, estimated N excretion, oxidative stress, and performance in organic Holstein heifers. Heifers (n=20) were supplemented with Silvafeed® ByPro, a Schinopsis lorentzii condensed tannin product, at increasing levels as recommended by the manufacturer: 0% (CON), 0.075% (LOW), 0.15% (MED), and 0.30% (HIG) of dry matter intake (DMI). Based on animal success to a 28 d acclimation period, 20 certified organic Holstein heifers (BW = 219 ± 17 kg) were randomly assigned into one of the four treatment groups and stratified based on initial body weight (i.e., a completely randomized design). A 7 d pretrial gas analysis was performed prior to study initiation to account for individual animal emission differences. Daily, heifers were supplemented with one kg of sweet feed and tannin in accordance with the assigned treatment in individual feeding stanchions for 45 d and fed a basal total mixed ration (TMR) diet through four SmartFeed Pro intake measurement bunk systems (C-Lock Inc., Rapid City, SD) which allowed for measurement of individual animal feed intake. Additionally, CH4 and carbon dioxide (CO2) production was measured using one GreenFeed automated head chamber system (AHCS, C-Lock Inc., Rapid City, SD) for the entirety of the study. Statistical analysis was conducted in R© (R Core Team, 2021, v. 4.1.2). Data were analyzed as a completely randomized design with animal (n=20) as the experimental unit, using the Type III ANOVA procedure. Post-hoc pairwise comparisons for dependent variables by treatment were performed using the least squared means procedure with the Tukey HSD adjustment applied. Daily CH4 production ranged from 136.5 to 140.1 g CH4/hd/d between treatments. No significant difference was observed between treatments for daily CH4 production (P=0.95), CO2 production (P=0.95), CH4 as a percent of gross energy (GE) intake (Ym; P=0.87), CH4 yield (MY; g CH4/kg DMI; P=0.80), and CH4 emission intensity (EI; g CH4/kg of BW gain; P=0.70). Similarly, a treatment effect was not observed for DMI (kg/d; P=0.92), average daily gain (ADG; kg BW gain/d; P=0.53), or feed efficiency (G:F; kg of BW gain/kg of DMI; P=0.42). Nitrogen intake ranged from 195 to 214 g/d among treatments (P=0.93). No significant difference was observed among treatments for fecal output (P=0.98), fecal N (FN; P=0.98), fecal neutral detergent fiber (NDF; P=0.33), or fecal acid detergent fiber (ADF; P=0.30). Estimated urine nitrogen (UN) (P=0.77), FN:UN (P=0.93), and N excretion (P=0.86) did not differ among treatments when estimated using methodologies described by Kohn (2005) (Table 5). Similarly, estimated UN (P=0.66), FN:UN (P=0.94), and N excretion (P=0.72) did not differ among treatments when estimated using methodologies described by Reed (2015). Moreover, no significant difference was observed among treatments for serum parameters, blood urea nitrogen (BUN; P=0.99) or creatinine (P=0.20), the common oxidative stress biomarker malondialdehyde (MDA; P=0.63), or antioxidant enzyme biomarkers superoxide dismutase (SOD; P=0.26) and reduced glutathione (GSH; P=0.19). Ultimately, the results of this study would not indicate that low-level tannin supplementation alters CH4 emissions, estimated N excretion, oxidative stress, or animal performance in organic dairy heifers.Item Open Access Innate and adaptive immune responses after bovine viral diarrhea virus infection in the fetal thymus(Colorado State University. Libraries, 2018) Knapek, Katie, author; Mathiason, Candace, advisor; Hansen, Thomas R., advisor; Kendall, Lon V., committee member; Pinedo, Pablo, committee memberBovine viral diarrhea virus (BVDV) infection of bovine fetuses in the first 125 days of pregnancy results in persistently infected (PI) cattle and transiently infected (TI) cattle become infected, but have a developed immune system enabling them to clear the virus. PI cattle are the main source of BVDV infection in populations causing significant economic losses to the industry worldwide. The mechanisms that lead to this "immunotolerant" state are not well defined. Following maternal inoculation with BVDV on day 75 of gestation, fetal infection was determined by presence of viral mRNA. Fetal infection occurred by day 89 of gestation with a peak at day 97 and a significant 10-fold decrease in viral titer at days 192 and 245 of gestation. BVDV was never completely eliminated in the fetus and persisted at lower, but significant levels. We hypothesized that there is dysgenesis of the fetal thymus, inhibiting functional innate and adaptive immune responses following maternal infection with BVDV. Total RNA was extracted from the thymus of uninfected control and BVDV-infected fetuses at days 89, 97, 190, and 245 of gestation. Genes representing the innate immune system, T and B cell receptor signaling, and the antigen processing and presentation in both the MHC I and MHC II pathways were explored using RT-qPCR. There were significant increases (P ≤ 0.05) observed in all innate genes analyzed (RIG-I, IRF7, NF-kB, IFNβ, and ISG15) in TI fetuses, with minimal responses in PI fetuses. There were significant decreases (P ≤ 0.05) of antigen presentation genes and adaptive immune system genes LMP2, TAP1, β2M, CD8a, CD8b, CIITA, CD4, IFI16, CXCL16, CXCR6, and CD79b in day 190 PI fetuses. Persistent BVDV infection may initiate fetal adaptive immune responses, which are not fully activated over time because of "immunotolerance" caused by impaired antigen processing. Longer-term consequences of fetal PI remain to be determined in context of postnatal impaired immune responses to secondary infections.Item Open Access Investigation of potential advancements in bovine assisted reproductive technologies(Colorado State University. Libraries, 2023) LaVelle, Gerica Anne Helton, author; Barfield, Jennifer, advisor; Tesfaye, Dawit, advisor; Cairo, Betsy, committee member; Pinedo, Pablo, committee memberThe assisted reproductive technologies (ART) area of research within bovine encompasses a diverse group of technologies including artificial insemination, cryopreservation of gametes and embryos, in vitro fertilization, induction of multiple ovulations, sex determination of sperm and embryos, and more. These techniques have been studied thoroughly within the last forty years, yet there is a plethora of knowledge to still be discovered on a molecular basis extending to longitudinal effects of ART interference generationally. One large sector of ART research in the bovine industry is to gain information that will aide in the advancement of current protocols and practices to increase productivity and cost effectiveness. This includes improving cryopreservation of gametes, creating higher in vitro embryo production yield, reducing costs, and developing easier practices for technicians with hopes of providing a more efficient system, from both a labor and fiscal standpoint. The following dissertation contributes specifically to this research sector by providing novel information regarding cryopreservation and capacitation timing of bovine spermatozoa treated with cholesterol prior to cryopreservation, embryo production rates when such spermatozoa are used within an in vitro fertilization (IVF) system, and the use and metabolomic components of spent media from bovine embryo culture. The first chapter of this dissertation will provide background information on the complex sperm capacitation and acrosome reaction process, and methods on how to assess and modify the capacitation process via molecular processes, both pre- and post-cryopreservation. This lays the foundation for the research presented here, which focuses on the loading of bovine spermatozoa with cholesterol prior to cryopreservation, and what effect this treatment has on the cells post-thaw in relation to capacitation timing analyzed using flow cytometry within the second chapter. The first chapter also provides background to the fertilization process and in vitro production of embryos which is examined by using cholesterol-loaded spermatozoa for IVF presented in the third chapter. Additionally, contextual information is explained regarding the type of culture systems within IVF, where embryos are individually cultured or in groups. This information is required for the fourth chapter which includes experimentation with spent culture media and the study of the metabolomic components of media exposed to group embryo culture. Pre-loading bovine sperm with cholesterol prior to freezing is known to increase cryosurvival, though the timing of capacitation in these sperm has not yet been evaluated. The objective of the study performed in chapter two was to determine if there is a potential delay in capacitation timing in these sperm due to the increased cholesterol content. Flow cytometric evaluation was utilized to assess viability, and stain technology to assess acrosome intactness (Propidium Iodide/FITC-PNA), intracellular calcium levels (Propidium Iodide/FLUO 3-AM) and membrane fluidity (Merocyanine 540/YO-PRO-1). Through this investigation it was determined that treatment of sperm with cholesterol-loaded cyclodextrin (CLC) (2 mg/mL) increased cryosurvival and viability over time, though longer time to capacitate was required compared with non-treated sperm. Treatment with CLC also significantly decreased membrane fluidity in sperm (P<.05). by 1 hour, resulting in increased intracellular calcium and increased acrosome reaction, and consequently viability loss by 3 hours. Taken together, these results determined sperm pre-loaded with cholesterol display significantly improved post-thaw viability but require longer to capacitate which may hinder fertilization capacity and/or require adjustments to timing of in vitro fertilization. Given the delayed capacitation timing of sperm treated with CLC, it was then investigated if CLC-treated sperm could be successful in IVF to determine if the additional retained cholesterol hindered fertilization ability and consequently, embryo production. The research summarized in chapter three examines the fertilization ability of CLC-treated sperm and subsequent in vitro embryo development by offsetting sperm preparation timing in order to adjust for delayed capacitation due to the increased cholesterol content of CLC-treated sperm. Additionally, treatment with methyl-β-cyclodextrin (MβCD) was utilized as a capacitation inducing agent by causing an efflux of cholesterol from the sperm plasma membranes during the sperm preparation incubation period. Bovine sperm pre-loaded with cholesterol via CLC prior to cryopreservation did not result in significant difference in cleavage or blastocyst formation (P>0.05) when sperm preparations were completed 90 minutes prior to co-incubation to adjust for delayed capacitation timing. There was significant decline (P<0.05) in embryo cleavage percentage in both control and CLC-treated sperm groups when MβCD was used as a capacitation inducer at 2 mg/ml. The findings presented in chapter three demonstrate that bovine sperm treated with CLC resulted in a nonsignificant difference in embryo production when used in IVF, even when delayed capacitation timing is adjusted for in sperm preparation. This concludes that the fertilization ability of the spermatozoa treated with CLC was not impaired, and that the most important application of CLC-treated sperm is not within in vitro fertilization examination, but rather, within in vivo models utilizing AI synchronization timing adjusted for delayed capacitation. Here the benefit of improved cryosurvival rates can be best utilized, potentially resulting in the ultimate goal of increased fertility. One of the most widely pursued research avenues to improve embryo development is advancements in culture media and conditions. Many attempts have been made to optimize in vitro production of embryos by component supplementation and recipe adjustment. An additional culture condition with a dramatic impact on bovine preimplantation embryogenesis is whether embryos are cultured individually or in groups, where increased embryo production is observed in group culture conditions. These items together, have created an active area of research which analyzes culture media after exposure to embryo culture to examine the artifacts secreted by embryos including various amino acids, proteins, and other metabolites that are considered to be embryotrophic, or positive signals from companion embryos. Chapter four experiments include culturing embryos both individually and in groups with spent culture media from a group culture donor cycle with embryo production analyzed via cleavage and blastocyst formation with time-lapse imaging. Similar cleavage percentages between control and spent media were observed, however, an increase in blastocyst formation was observed in grouped embryos compared with single embryos. No difference (P>.05) was observed in the timing of developmental events in embryos cultured in control or spent media within individual culture. Analysis of the metabolomic profile of spent culture media with gas chromatography-mass spectrometry (GC-MS) was also completed, suggesting potential embryotrophic factors in the spent media. These findings conclude that bovine embryos progress in a more timely and developmentally differentiated degree when cultured within groups, and artifacts left within the spent culture media were not sufficient to result in significant increase in embryo production within spent media culture.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 Time lost to disease in dairy cattle: associations between two consecutive lactations(Colorado State University. Libraries, 2017) Bacigalupo Sanguesa, Paola, author; Garry, Franklyn B., advisor; McConnel, Craig S., advisor; Lombard, Jason E., committee member; Pinedo, Pablo, committee memberIn the dairy industry, individual diseases and their effects are normally studied independently. However, in many cases the diseases are all related. The diverse effects of these diseases provide the foundation for creating a measure that incorporates morbidity and removal (death or culling) measures and evaluates the impact diseases can have during lactation. This summary health measure is called the disease-adjusted lactation (DALact) and it represents time lost due to disease and injury. The DALact is a time-based summary measure that represents a new approach to assess the impact of diseases in a lactation. The objective of this study was to evaluate the association between time lost due to specific diseases and total time lost due to diseases in two consecutive lactations using the DALact. Health and removal (culling and death) data were obtained from a Colorado dairy with approximately 1,400 lactating cows. A total of 803 cows in their second or greater lactation that calved, were sold, or died from July 1, 2015, through June 30, 2016, were selected. Health data were collected from Dairy Comp 305 for each most recently completed lactation and from the previous lactation. Health events included calving injury, displaced abomasum, diarrhea, hypocalcemia, ketosis, lameness, mastitis, metritis, musculoskeletal injuries, pneumonia, and retained placenta. All cow-level data were imported into SAS® for validation, calculation of DALact and modeling. The DALact was calculated by adding the Days Lost due to Premature Death or Culling (DLRD) and the Days Lost due to Illness (DLI). DLRD was calculated as the difference between the average completed lactation days in milk for that herd and the days in milk at culling or death. The DLI was the product of the number of cases multiplied by previously established disability weights and estimated disease durations (days) for a specific disease. The PROC GLM procedure was used to model the association of DALacts between the 2 consecutive lactations. A p-value of ≤0.05 was considered significant. Positive significant associations (P<0.001) were found between the DALact of the previous lactation and the current lactation for lameness and mastitis. The total DALact of the previous lactation was significantly associated (P<0.001) with the total DALact of the current lactation. Significant associations (P<0.001) were also found between the mastitis and lameness DALact of the precious lactation with the total DALact of the current lactation. Identification of diseases and reasons for removal that significantly affect time lost during two consecutive lactations will help producers focus management and preventive measures on diseases having the greatest impact on future productivity and wellbeing.Item Embargo Transposable elements and early pregnancy in the horse(Colorado State University. Libraries, 2024) Southerland, Sierra L., author; Coleman, Stephen, advisor; Bruemmer, Jason, committee member; Fedorka, Carleigh, committee member; Pinedo, Pablo, committee member; Chicco, Adam, committee memberUnderstanding the molecular events and physiological dynamics in the endometrium during early pregnancy is crucial for improving our understanding of reproductive outcomes in horses. Much of the work investigating early pregnancy in the horse has focused on maternal recognition of pregnancy (MRP). This critical signal, which initiates the pathways and regulatory changes to help make the endometrium receptive to pregnancy, remains elusive in the horse. Transposable elements (TEs), once overlooked components of the genome, have been implicated in regulating gene expression during critical stages of embryo and placental development. The current study investigated the expression of TEs during the early phase of pregnancy post-ovulation. The hypothesis tested was that pregnancy status would impact the expression patterns of TE-derived transcripts in the endometrium around the time of maternal recognition. It was anticipated that the TE transcripts would increase in the endometrium of pregnant mares either in response to or as a part of the maternal recognition signal. RNA sequencing data was generated from endometrial biopsies at days 9, 11, and 13 in pregnant and non-pregnant mares. Bioinformatics analyses identified distinct patterns of TE expression across the duration of early pregnancy. Contrary to expectations, these findings did not reveal the accumulation of TE transcripts in the pregnant endometrium compared to the non-pregnant endometrium during early pregnancy. Instead, there was a significant decrease in the number of differentially expressed transcripts over the time points studied. These results challenge the initial assumption and suggest that the regulation of TE transcriptional activity during early pregnancy may be more complex than previously thought, highlighting the need for further research into the roles of TEs in equine reproductive physiology. An alternative explanation for the results of this analysis is that maternal recognition of pregnancy may involve the downregulation of transcripts expressed from TEs, potentially serving as a switch to prevent luteolysis and maintain pregnancy. This study underscores TEs as potential biomarkers and regulators in equine reproduction, providing insights into endometrial processes and offering avenues for improving fertility outcomes and breeding efficiency in horses.