College of Agriculture & Natural Resources

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    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.
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    Investigating genetic and health factors related to AA amyloidosis prevalence in captive cheetahs (Acinonyx jubatus): implications for population management
    (2014) Franklin, Ashley Danielle; Porter, Tom E; Crosier, Adrienne E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Systemic amyloid A (AA) amyloidosis is an increasingly important cause of morbidity and mortality among captive cheetahs, yet wild cheetahs are virtually unaffected, suggesting the phenomenon is a result of the captive condition. The self-aggregating AA protein responsible for this disease, is a byproduct of serum amyloid A (SAA) protein degradation, an acute phase protein highly upregulated during inflammation. The objective of this study was to identify the relationship between genetics, stress, and inflammation with serum concentrations of the SAA protein and the incidence of AA amyloidosis in captive cheetahs. Fecal and serum samples collected from cheetahs held at the Smithsonian (NZP-SCBI) and Cheetah Conservation Fund (CCF) facilities, as well as wild, free-ranging cheetahs, were examined. Enzyme-linked immunosorbent assays were used to measure SAA protein and proinflammatory cytokine concentrations in serum samples and cortisol concentrations in feces. Additionally, cheetahs were genotyped for the SAA1A-97delG single nucleotide polymorphism (SNP) in the promoter region of the SAA1 gene. This study was the first to demonstrate that serum concentrations of the SAA protein in cheetahs are affected by the SAA1A-97delG SNP (P=0.0453). However, the high prevalence of AA amyloidosis observed among captive cheetahs is not attributable to genetic differences at this locus, but rather appears to be related to stress and/or inflammation, as captive cheetahs at NZP-SCBI have significantly higher SAA protein concentrations in serum compared to captive cheetahs at CCF, regardless of genotype (P=0.0003). Captive cheetahs at NZP-SCBI show levels of stress (fecal cortisol concentrations) greater than their captive counterparts at CCF in Namibia. Interestingly, wild cheetahs and captive cheetahs at CCF in Namibia had significantly higher proinflammatory cytokine concentrations (TNF-α and IL-1β) in serum compared to cheetahs at NZP-SCBI (P<0.0001). It is possible that chronic stress may be suppressing the production of proinflammatory cytokines in the NZP-SCBI cheetah population. Controlling the currently high SAA protein concentrations associated with AA amyloidosis is the best strategy to decreasing the diseases prevalence among captive cheetahs. Promoting management practices that reduce stress could help re-establish proper immune system homeostasis and mitigate the overproduction of SAA protein, decreasing the probability of developing AA amyloidosis.