Animal & Avian Sciences Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2741
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Item OVARIAN STROMAL CELLS IMPROVE SURVIVAL, BUT NOT GROWTH, IN PRE- AND EARLY ANTRAL FELINE FOLLICLES(2024) Marks, Batsheva Naomi; Keefer, Carol; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ovarian stromal cells act as crucial support and regulators for in vivo folliculogenesis; however, less is known about their effect on in vitro grown follicles. The objective of this study was to investigate the impact of ovarian stromal cell co-culture or conditioned medium (CM) on survival and development of cat pre-, early, and antral follicles in vitro. Ovaries were obtained from cats older than six months (n = 3), then enzymatically digested to release stromal cells. The ovarian stromal cells were allowed to grow to confluency in a T75 flask, before being cryopreserved for long term storage in liquid nitrogen. Cells were thawed one week prior to follicular culture onset, and passaged once before CM collection. CM was subsequently removed 24 - 48 hours after feeding, and stored at -80C until used. Ovarian follicles were mechanically isolated from cats older than six months (n = 23 cats, 155 follicles), encapsulated in 0.5% alginate hydrogel. The isolated follicles were then divided into five treatment groups (control, ovarian stromal cell co-culture, 20% CM, 50% CM, and 100% CM in Endothelial Cell Growth Medium), and classified based on initial diameter as preantral (224.4 + 4.7 m), early antral (394.8 + 7.4 m), or antral (592.2 + 18.8 m). Culture subsequently lasted for 13 days, and survival and growth of the follicles were evaluated on Days 0, 4, 6, 8, 11 and 13. At the end of culture, follicles were assessed via qRT-PCR for expression of CYP19A, FSHR, and GDP9 to further quantify development. Statistical analysis was done in R software. Follicles in 100% CM had higher survival up to Day 11 of culture as compared to other treatment groups (Cox proportional hazards model, p < 0.01). Initial stage also influenced survival, with antral follicle survival significantly lower than that of pre- and early antral follicles (p < 0.0001). However, no differences in growth were detected across the treatment groups, nor across initial size classifications (Kruskal-Wallis test, p > 0.05). Post culture qRT-PCR analysis of the three selected genes showed upregulation of CYP19A in 50% CM follicles compared to the control (ANOVA, p < 0.05). However, there were no differences in CYP19A expression between the control and other treatment groups, or in GDF9 and FSHR expression among culture groups (p > 0.05). In summary, the findings demonstrated that conditioned medium collected from primary culture of ovarian stromal cells improves in vitro survival and modulates CYP19A expression of isolated cat follicles. Further research to identify paracrine factors present in conditioned medium will elucidate the roles of ovarian stromal cells pertaining to follicle survival during in vitro folliculogenesis.Item Population Declines and Genetic Variation: Effects of Serial Bottlenecks(2015) Callicrate, Taylor Eilers; Song, Jiuzhou; Fleischer, Robert C; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Islands foster unique biodiversity, yet also present biogeographic limitations that impose increased risk for population extinction through demographic and genetic constraints and decreased probability of surviving a catastrophe. Of particular interest, especially with regard to endangered species, is the genetic response of insular species to severe population declines or translocations. Both types of events, considered population bottlenecks, are expected to reduce genetic variation, and correspondingly, adaptive potential. For these reasons, it is important to understand how bottlenecks interact with insular population dynamics to affect genetic diversity. I used a combination of a laboratory model experiment and population genetics study of an in situ bottleneck in an endangered species to investigate how quantitative and molecular genetic variation are affected during bottlenecks. I used a laboratory animal model (red flour beetle, Tribolium castaneum) to compare how quantitative genetic variation is affected if a serial bottleneck occurs in a novel versus familiar environment. The experiment was designed to model a founder event or translocation to a new island with a novel environment. I found that phenotypic and additive variance for a quantitative trait were larger following a bottleneck occurring in the novel environment, suggesting that the novel environment could improve adaptive potential in bottlenecked populations. Next, I used molecular genetic markers to assess variation and signatures of selection in the Laysan finch (Telespiza cantans), a Hawaiian honeycreeper endemic to a small Northwestern Hawaiian island. Laysan finches experienced a major bottleneck on Laysan in the early 20th century, followed by a translocation and series of founder events as populations were established on the islets of Pearl and Hermes Reef (PHR) in the 1960s – 70s. I found that, contrary to expectation, bottlenecked Laysan finch populations did not show declines in genetic variation and were not differentiated as a result of genetic drift. These results are potentially caused by insular demographic dynamics. I identified loci with extreme differentiation between modern populations, potentially indicating genomic signals of selection. These regions could be important for adaptation to the novel environment on PHR and are candidates for future study.Item CHARACTERIZATION, ENRICHMENT, AND IN VITRO CULTURE OF SPERMATOGONIAL STEM CELLS IN THE DOMESTIC CAT: A MODEL FOR RARE AND ENDANGERED FELIDS(2014) Vansandt, Lindsey Marie; Keefer, Carol L; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Spermatogenesis is a highly prolific process in which millions of spermatozoa are produced daily. Spermatogonial stem cells (SSCs), the adult stem cell population of the testis, sustain this process by providing a constant source of new progenitor cells. The ability of this stem cell population to self-renew makes it a promising alternative to spermatozoa for genetic preservation of rare and endangered animals. While innovative advances in SSC technologies have been made in the mouse, there is a paucity of information concerning felid SSCs. Therefore, the overall objective of the dissertation was to develop SSC technology in the domestic cat (Felis catus) as a model for rare and endangered felids. In the first study, mRNA transcripts for six SSC marker genes (THY1, GPR125, GFRalpha1, PLZF, UCHL1, and OCT4) were identified in cat testes. Localization within the appropriate in situ niche was confirmed by immunohistochemistry for three of the markers (PLZF, UCHL1, and OCT4). The expression pattern of these markers was conserved in the cheetah (Acinonyx jubatus) and Amur leopard (Panthera pardus orientalis), validating the cat as an appropriate felid model. In Study 2, we explored two techniques to enrich cat testis cells for SSCs. We found that the efficiency of enrichment depends on age of the donor and that prepubertal testes are the preferred source for differential plating. Magnetic-activated cell sorting did not achieve any level of enrichment for cat SSCs, likely due to unsuitability of the antibody. The final study modified the traditional mouse SSC culture system for use in the cat. A clear effect of feeder cell type was demonstrated, with mouse endothelial C166 cells supporting a significantly higher number of germ cell colonies as compared to STO cells or primary cat fetal fibroblasts. Identity of germ cell colonies was confirmed by co-expression of UCHL1, PLZF, and OCT4. During subculture, colonies maintained SSC marker co-expression and displayed alkaline phosphatase activity. At the time of writing, cells had been maintained for 78 days in vitro. Together, these studies provide the groundwork towards application of SSC technology in management of rare and endangered felid populations.