Abstracts � Vol 29, No 5 (2009) 331

decreased population doubling time at all time points ex-amined. However, supplementation of medium withFGF2 was detrimental to doubling time, regardless of thesubstrate used. Serial passage resulted in increases in PDTand replicative senescence. Additionally, expression ofnanog and Sox2 mRNA declined with time in culture, indi-cating 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-de-

rived progenitor cell chondrogenesis in hydrogel cultures. J OrthopRes 2008;26:322-31.

2. Arnhold SJ, Goletz I, Klein H, Stumpf G, Beluche LA, Rohde C, Ad-

dicks K, Litzke LF. Isolation and characterization of bone marrow-de-

rived equine mesenchymal stem cells. Am J Vet Res 2007;68:1095-105.

3. Reed SA, Johnson SE. Equine umbilical cord blood contains a popula-

tion 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 chondro-

cytic 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 mesen-

chymal stem cells from equine umbilical cord blood. BMC Biotechnol:

2007;7:26.

6. Taylor SE, Smith RK, Clegg PD. Mesenchymal stem cell therapy inequine 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. TrendsBiotechnol 2007;25:409-16.

31714 Stallion SpermatozoalMotility Parameters- an Examinationof Intra and Inter-Stallion VariabilityOver a Breeding Season ona Commerical Texas RanchA.L. Garcia,*1 H.A. Brady,1 M.A. Ballou,1 D.D Varner,2

C.C. Love,2 and G. Blodgett3, 1Department of Animaland Food Sciences, Texas Tech University, Lubbock, TX,USA, 2College of Veterinary Medicine, Texas A&MUniversity, College Station, TX, USA, 36666 Ranch,Burnett Farms, Guthrie, TX, USA

INTRODUCTIONSpermatozoal motility is a parameter used to investigate thefertility of a stallion.1 Distinct seasonal changes have been

noted in stallion semen evaluation.2 The need for an objec-tive approach resulted in the development of CASA, (Com-puter Assisted Sperm Analysis) which is useful indetermining fertility and sub fertility of a stallion.3 TheIVOS (Integrated Visual Optics System) by HamiltonThorne allows the user to observe the spermatozoa inreal-time, recording several important motility parameters.Considerable variation in semen quality is noted over theyear, notably in the breeding season, so multiple collectionsand evaluations are necessary to obtain an accurate view ofa stallion’s fertility potential.2 This study involved 21stallions being evaluated every other day using computer-ized techniques for the entirety of a six-month breedingseason at a working breeding facility. The goal of the studywas to analyze motility and collection parameters to evalu-ate both intra- and inter-stallion variation over a breedingseason.

MATERIALS AND METHODSTwenty one Quarter Horse stallions at the 6666 Ranch inGuthrie Texas were evaluated over an entire breeding sea-son (February-July 2008). Stallions age ranged from 4-18years of age (mean¼ 9.8 y). Most stallions were collectedevery other day using a Missouri (Nasco, Ft. Atldnson,WI) artificial vagina and a phantom. Libido was trackedby recording mount attempts for each collection. Ejacu-lates were analyzed immediately upon collection. Theejaculate was filtered, and the gel-free fraction was ex-tended with INRA 96 (IMV Technologies, L’Aigle,France) in a 1:3 ration dilution supplemented with Time-ntin (1 mg/mL) (GlaxoSmithKline, Philadelphia). Con-centration was determined by a NucleoCounter SP1000(Chemometic, Allerød, Denmark). For motility analysis,a 6 ml sample was pipetted on a Leja (Leja, Nieuw-Ven-nep, The Netherlands) chamber slide for IVOS analysis(IVOS; Hamilton Thorne, Beverly, USA). Mean valuesfor path velocity (VAP), mean straight-line velocity(VSL), mean curvilinear velocity (VCL), amplitude ofhead displacement (ALH), flagellar beat frequency(BCF), linearity (LIN), straightness (STR) were recorded.Counts were recorded for % Motile, % Progressive, %Rapid Progressive, % Moderately Progressive, % Slow Pro-gressive.

RESULTS AND DISCUSSIONStatistical analysis was performed on both NucleoCounterand IVOS data (Table 1).4 Statistical means were per-formed on each motility parameter, sperm count, andmount attempt using SAS software (SAS Institute, Inc.Cary, NC, USA). The standard deviation of the meanswas deduced and used to define the coefficient of variation(CV). (CV¼ the mean standard deviations/mean).

Table 1. Mean Coefficient of Variation for Inter andIntra-Stallion Motility Parameters 1

2ParametersInter- StallionMean CV

Intra-StallionMean CV

Gel Free Vol (ml) 29.9 24.6Concentration (mil) 29.12 37.67Total Sperm (bil) 25.3 27.1% Motile 20.6 8.56% Progressive 24.1 11.9VAP (ms) 13.6 15.6VSL (ms) 15.2 15.5VCL (msm/s) 13.7 14.1ALH (mm) 15.4 22.1BCF (Hz) 7.6 5.71STR% 9.6 13.9LIN% 11.2 8.0Mounts 47.5 36.91 n¼ 21 stallions2 parameters taken from February 2, 2008 through July 7, 2008.

332 Abstracts � Vol 29, No 5 (2009)

Analysis of the mean CV revealed relatively high levels ofvariability both between stallions and inter-stallion data.Highest intra-stallion variation parameters were moder-ately progressive sperm (cv¼ 42.7), total sperm concentra-tion (cv¼ 37.7), ALH (cv¼ 22.1), and slowly progressivesperm (cv¼ 37.9). In contrast, highest inter-stallion varia-tion included mount attempts (cv¼ 47.5), % moderatelyprogressive sperm (cv¼ 30.5), and % slowly progressivesperm (cv¼ 29.95).

The least variable parameters (intra-stallion) variationwere BCF (cv¼ 5.71), % LIN (cv¼ 8.04), % motilesperm(cv¼ 8.56). Conversely, the least variable values (in-ter-stallion) were BCF (cv¼ 7.6), % STR (cv¼ 9.56), and% LIN (11.2).

Overall, motility parameters were highly variable be-tween and within stallions. Current analysis is being con-ducted to examine correlations of these parameters tofertility in both on-the-farm artificial insemination and incool-shipped inseminations.

REFERENCES

1. Jasko DJ. Evaluation of Stallion Semen. Vet Clin North Am Equine

Pract 1992 Apr;8(1):129-48.2. Janett F, Thun R, Niederer K, Burger D, Hassig M. Seasonal Changes

in Semen Quality and Freezability in the Warmblood Stallion. Therio-

genology 2003;60(3):453-61.

3. Katila T. In Vitro Evaluation of Frozen-Thawed Stallion Semen: A Re-view. Acta Vet Scand. 2001;42(2):199-217.

4. Copyright � [2008] SAS Institute Inc. SAS and all other SAS Institute

Inc. product or service names are registered trademarks or trademarks

of SAS Institute Inc., Cary, NC, USA.

31856 Response to CortisolStimulation Tests in Old vs. YoungStandardbred MaresN.R. Liburt,* K.H. McKeever, and E.K. Wunderlich,Equine Science Center, Department of Animal Science,Rutgers, The State University of New Jersey, NewBrunswick, NJ, USA

INTRODUCTIONPrevious work has documented aging-induced changes inendocrine function1,2 with a suppression of the cortisoland vasopressin responses to exercise as well as a disruptionof the glucose and insulin response following exercise inStandardbred mares. Twelve wks of exercise training didnot reverse the effect of age on the cortisol response to acuteexertion.2 The present study used a series of endocrine stim-ulation tests to examine the hypothesis that the age-inducedalteration in cortisol concentration is due to differences inthe response of the hypothalamic-pituitary-adrenal axis(HPAA) in old horses compared to younger horses.

MATERIALS AND METHODSEight unfit, healthy, Standardbred mares were used in thestudy (4 young (Y), 6� 1 yrs and 4 old (O), 21.5� 2.5 yrs;mean� SD). Mares were housed in 2-acre fields at theRutgers University and were offered ~ 4 kg of commer-cially available grain ration divided into two feedings. Grasshay, salt and water were provided ad libitum. Each mare re-ceived four stimulation tests [Adrenocorticotropin Hor-mone Stim (ACTH), Dexamethasone Suppression(DEX), Combined Dexamethasone Suppression/ACTHStim (DEX/ACTH) and Control (CON)] in a random-ized, crossover design with one week between each test.Feeding schedule was not altered during the tests. In alltests, a jugular catheter was placed using sterile techniquesand lidocaine anesthesia, and baseline blood samples(30 mL) were collected at -0.5 and 0 hrs. For the DEXtests, mares received 0.04 mg/kg dexamethasone at1700, with blood samples obtained at 0.5, 1, 2, 4, 6, 15,16, 17, 18 and 19 hrs. For DEX/ACTH, dexamethasonewas administered at 0900 (0.04 mg/kg) and samplesdrawn at 0.5, 1, 2 and 4 hrs. After the 4 hr blood sample,0.20 mg ACTH was administered, with additional samplesdrawn at 0.5, 1, 2, 4 hrs post-ACTH. Samples were alsodrawn at 24 and 28 hrs after the beginning of the test.For ACTH, 0.20 mg ACTH was administered at 0900,with samples at 0.5, 1, 2, 4, 6, and 24. For CON, a doseof 10 mL normal saline was given at 0900, with samplingat 0.5, 1, 2, 4, 6, and 24 hrs. Plasma glucose was measuredin duplicate using a glucose analyzer (Model 2300, YellowSprings Instruments, Yellow Springs, OH). Plasma cortisolwas measured using a commercially available