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chorionic gonadotropin and bovine luteinizing hormone. J. Anim. Sci 1988;66;719-726. 8. Grieger DM, Reeves JJ. Active immunization of beef heifers against luteinizing hormone: II. Evaluation of conjugation technique on antigenicity of LH. J. Anim. Sci 1990;68;3755-3764. 9. Ginther OJ. Sexual behavior. In: Reproductive Biology of the Mare, Basic and Applied Aspects, 2nd edn, Ginther OJ (Ed.), Cross Plains, WI: Equiservices, 1992a, pp. 75-104.
31721 Optimization of Culture Conditions for Equine Umbilical Cord Blood Derived Stem Cells S.A. Reed* and S.E. Johnson, University of Florida, Gainesville, FL, USA
INTRODUCTION Equine umbilical cord blood derived stem cells (eUCB) are naı¨ve cells with great potential as a therapeutic aid. Similar to stem cells derived from bone marrow and adipose tissue, UCB stem cells readily differentiate into a variety of cell types, including osteogenic and chondrogenic cells.1-5 Current stem cell therapies in horses involve the autologous injection of bone marrow derived stem cells or adipose derived stem cells.6,7 However, these populations may not be optimal for therapeutic uses given their adult nature. More naı¨ve stem cells may provide a more malleable population that can more readily contribute to repair and regeneration of injured tissues. One of the challenges of in vitro stem cell research is expansion and maintenance of a malleable population of stem cells throughout the duration of culture. Thus, the objective of this work was to identify a set of culture conditions that optimized the maintenance of the naı¨ve eUCB stem cell.
MATERIALS AND METHODS Umbilical cord blood (UCB) was collected from Thoroughbred mares (n ¼ 3) at the time of foaling and stem cells were isolated as previously described.3 Equine UCB stem cells were cultured in growth media (GM), GM + FGF2, GM + Tpo/Flt/Kit or conditioned media (CM). Four substrata (uncoated plastic, gelatin, collagen, and fibronectin) were chosen from current human and mouse literature. Cell number was recorded daily for four days. Population doubling times were calculated as N ¼ (N0)(2 t/dt) where N is the final cell number, N0 is the initial cell number, t is time in hours and dt is doubling time. Doubling time data was analyzed by ANOVA using the GLM procedure of SAS. Total RNA was isolated and RT-PCR performed for Oct4, Nanog, Sox2, Klf4, and cMyc. GAPDH was included as a control. Transcript
expression was analyzed by logistic regression using the LOGISTIC procedure in SAS.
RESULTS Equine UCB stem cells cultured in basal media supplemented with 10% fetal bovine serum (GM) exhibited fibroblast-like morphology typical of UCB stem cells. Supplementation of GM with FGF2 did not alter this morphology. A combination of Tpo, Flt3 and c-kit, or CM resulted in a morphology resembling replicative senescence. Changes in cell number over a four-day culture period were measured and population doubling times (PDT) determined. GM and GM + FGF maintained shorter doubling times than other cultures. GM+ Flt/Tpo/Kit and CM were discontinued due to morphological changes, a prolonged PDT, and cell death. Equine UCB stem cells were seeded at equal densities on gelatin, fibronectin, collagen and uncoated surfaces. Determination of PDT indicated that eUCB stem cells on coated surfaces proliferated faster than controls on uncoated tissue plasticware. Changes in PDT were independent of the type of matrix used. PDT was increased in cells cultured in GM + FGF compared to growth media controls (p < 0.0001). The interaction of media and substrata types demonstrated that cells cultured on any type of matrix in control media have significantly faster doubling times than their respective cultures in media supplemented with FGF2. Cells cultured on collagen, fibronectin, and gelatin matrices in control media exhibited no differences in growth kinetics. Furthermore, population doubling times during continuous culture were determined at passages three, six, and nine. Time in culture significantly increased PDT from passage three through passage nine (p < 0.0001 for all interactions). To determine the presence of stem cell markers, total RNA was isolated from eUCB and analyzed for the expression of Oct4, nanog, Sox2, Klf4 and c-myc mRNA. Similar to mouse embryonic stem cells, Oct4, nanog, Sox2, Klf4 and c-myc amplicons were present in eUCB. Oct4 expression was maintained over time in culture regardless of substrata or media, however nanog and Sox2 mRNA decline with serial passage.
DISCUSSION Initial culture strategies for equine UCB stem cells involved growth on uncoated tissue plasticware surfaces, but no work has assessed the effects of various substrata or media on the prolonged culture of these cells.3,5 Use of eUCB stem cells as injury repair aids likely will require an initial expansion in culture to provide sufficient numbers of cells. Culture on protein matrices aids the proliferation of eUCB without jeopardizing the expression of pluripotency markers. Collagen, fibronectin, and gelatin matrices
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decreased population doubling time at all time points examined. However, supplementation of medium with FGF2 was detrimental to doubling time, regardless of the substrate used. Serial passage resulted in increases in PDT and replicative senescence. Additionally, expression of nanog and Sox2 mRNA declined with time in culture, indicating a maturation of cell type. Keywords: Umbilical cord blood; Stem cells; Equine
REFERENCES 1. Kisiday JD, Kopesky PW, Evans CH, Grodzinsky AJ, McIlwraith CW, Frisbie, DD. Evaluation of adult equine bone marrow- and adipose-derived progenitor cell chondrogenesis in hydrogel cultures. J Orthop Res 2008;26:322-31. 2. Arnhold SJ, Goletz I, Klein H, Stumpf G, Beluche LA, Rohde C, Addicks K, Litzke LF. Isolation and characterization of bone marrow-derived equine mesenchymal stem cells. Am J Vet Res 2007;68:1095105. 3. Reed SA, Johnson SE. Equine umbilical cord blood contains a population of stem cells that express Oct4 and differentiate into mesodermal and endodermal cell types. J Cell Physiol 208;215:329-36. 4. Fortier LA, Nixon AJ, Williams J, Cable CS. Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells. Am J Vet Res 1998;59:1182-7. 5. Koch TG, Heerkens T, Thomsen PD, Betts DH. Isolation of mesenchymal stem cells from equine umbilical cord blood. BMC Biotechnol: 2007;7:26. 6. Taylor SE, Smith RK, Clegg PD. Mesenchymal stem cell therapy in equine musculoskeletal disease: scientific fact or clinical fiction? Equine Vet J 2007;39:172-80. 7. Richardson LE, Dudhia J, Clegg PD, Smith R. Stem cells in veterinary medicine–attempts at regenerating equine tendon after injury. Trends Biotechnol 2007;25:409-16.
31714 Stallion Spermatozoal Motility Parameters- an Examination of Intra and Inter-Stallion Variability Over a Breeding Season on a Commerical Texas Ranch A.L. Garcia,*1 H.A. Brady,1 M.A. Ballou,1 D.D Varner,2 C.C. Love,2 and G. Blodgett3, 1Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX, USA, 2College of Veterinary Medicine, Texas A&M University, College Station, TX, USA, 36666 Ranch, Burnett Farms, Guthrie, TX, USA
INTRODUCTION Spermatozoal motility is a parameter used to investigate the fertility of a stallion.1 Distinct seasonal changes have been
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noted in stallion semen evaluation.2 The need for an objective approach resulted in the development of CASA, (Computer Assisted Sperm Analysis) which is useful in determining fertility and sub fertility of a stallion.3 The IVOS (Integrated Visual Optics System) by Hamilton Thorne allows the user to observe the spermatozoa in real-time, recording several important motility parameters. Considerable variation in semen quality is noted over the year, notably in the breeding season, so multiple collections and evaluations are necessary to obtain an accurate view of a stallion’s fertility potential.2 This study involved 21 stallions being evaluated every other day using computerized techniques for the entirety of a six-month breeding season at a working breeding facility. The goal of the study was to analyze motility and collection parameters to evaluate both intra- and inter-stallion variation over a breeding season.
MATERIALS AND METHODS Twenty one Quarter Horse stallions at the 6666 Ranch in Guthrie Texas were evaluated over an entire breeding season (February-July 2008). Stallions age ranged from 4-18 years of age (mean ¼ 9.8 y). Most stallions were collected every other day using a Missouri (Nasco, Ft. Atldnson, WI) artificial vagina and a phantom. Libido was tracked by recording mount attempts for each collection. Ejaculates were analyzed immediately upon collection. The ejaculate was filtered, and the gel-free fraction was extended with INRA 96 (IMV Technologies, L’Aigle, France) in a 1:3 ration dilution supplemented with Timentin (1 mg/mL) (GlaxoSmithKline, Philadelphia). Concentration was determined by a NucleoCounter SP1000 (Chemometic, Allerød, Denmark). For motility analysis, a 6 ml sample was pipetted on a Leja (Leja, Nieuw-Vennep, The Netherlands) chamber slide for IVOS analysis (IVOS; Hamilton Thorne, Beverly, USA). Mean values for path velocity (VAP), mean straight-line velocity (VSL), mean curvilinear velocity (VCL), amplitude of head displacement (ALH), flagellar beat frequency (BCF), linearity (LIN), straightness (STR) were recorded. Counts were recorded for % Motile, % Progressive, % Rapid Progressive, % Moderately Progressive, % Slow Progressive.
RESULTS AND DISCUSSION Statistical analysis was performed on both NucleoCounter and IVOS data (Table 1).4 Statistical means were performed on each motility parameter, sperm count, and mount attempt using SAS software (SAS Institute, Inc. Cary, NC, USA). The standard deviation of the means was deduced and used to define the coefficient of variation (CV). (CV ¼ the mean standard deviations/mean).