Table 1Mean (� SD) values for percentage of total motility (%TM), percentage of progressive motility (%PM), average path velocity (VAP) in mm/sec, straight linearvelocity (VSL) in mm/sec, percentage of rapid sperm (%RAP), hyperactive sperm (%HIP) and percentage of plasmamembrane integrity (%PMI) in G1, G2 and G3,obtained at 0 hour (M0) and 24 hours after cooled (M1)

Groups TM (%) PM (%) VAP (mm/s) VSL (mm/s) RAP (%) HIP (%) PMI (%)

G1 (M 0) 76.0 � 9.9a 32.6 � 8.6a 125.0 � 14.5a 228.8 � 24.6a 67.9 � 12.2a 4.9 � 4.7 63.5 � 16.2G2 (M 0) 83.3 � 4.8b 43.0 � 8.5b 134.0 � 17.8b 238.4 � 27.1b 75.8 � 6.7b 5.4 � 3.6 70.8 � 13.2G3 (M 0) 83.9 � 6.4b 43.7 � 6.8b 139.8 � 12.4ab 249.0 � 23.2ab 78.3 � 7.3b 5.4 � 3.6 66.2 � 19.9G1 (M 24) 30.0 � 22.0a 8.1 � 5.8a 108.4 � 25.2 210.5 � 22.0 23.0 � 19.3a 4.0 � 5.1 30.0 � 22.0a

G2 (M 24) 67.7 � 19.0b 29.5 � 13.4b 103.7 � 23.3 199.5 � 33.1 54.6 � 22.9b 1.9 � 2.3 67.7 � 19.0b

G3 (M 24) 73.0 � 13.9b 26.7 � 12.6b 112.4 � 23.2 218.4 � 40.5 60.9 � 19.3b 9.6 � 19.9 73.0 � 13.9b

Lowercase letters indicate difference in the same column (P < 0.05) at the same timeG1 (Control group), G2 (Equipure group) e G3 (Percoll group).

6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518510

viability of stallion cooled semen, and are efficient forselection without sperm hyperactivation, however equi-pure show better sperm recovery.

Study of testicular thermoregulation efficiency installion with different ages

C. Ramires-Neto, G.A. Monteiro, D.J.Z. Delfiol,E.G. Fioratti, J.A. Dell`aqua, Jr., F.O. Papa, andM.A. AlvarengaSchool of Veterinary Medicine and Animal Science, SãoPaulo State University, Botucatu, Brazil

In horses, spermatogenesis normally occurs at an averageintratesticular temperature of 35�C; therefore, mechanismsfor testicular thermoregulation are essential. Measuringthe heat radiated by the scrotum using thermography isa non-invasive method used to evaluate the superficialscrotal temperature and the effectiveness of testicularthermoregulation. The objective of this study was todetermine the efficiency of testicular thermoregulation inhorses of different ages. For this a total of 15 healthyQuarter Horse stallions without anatomical or functionalalterations to their reproductive tracts and with goodsperm quality were used and divided into three groups: YS(n ¼ 5, stallions between 2 and 3 years of age), AS (n ¼ 5,stallions between 5 and 15 years) and OS (n¼ 5, stallions 17years of age or greater). The animals were previouslyconditioned in a shady environment with ambienttemperature between 30 and 32oC and the rectaltemperature (RT, oC) measured using a dry bulb ther-mometer and the scrotal surface temperature (SST, oC) andbody surface temperature (BST, oC) using an infrared ther-mographer (Infra CamTM, FLIR Systems Inc.) in two stages:when the animals were conditioned in a shady environ-ment (M1) and after 1 hour of sun exposure (M2). Therewere no differences (P > 0.05) between YS, AS and OS in RTat M0 (YS ¼ 37.74 � 0.23; AS ¼ 37.90 � 0.19; OS ¼ 37.52 �0.36), in RT at M1 (YS ¼ 38.26 � 0.68; AS ¼ 38.46 � 0.59;OS ¼ 38.72 � 0.62), in BSC at M0 (YS ¼ 36.02 � 1.34; AS ¼37.22� 0.50; OS¼ 36.76� 0.83), in BSC at M1 (YS¼ 37.10�0.62; AS ¼ 38.54 � 0.63; OS ¼ 37.72 � 0.84), in SST at M0(YS ¼ 34.12 � 0.85; AS ¼ 32.22 � 0.59; OS ¼ 34.54 � 0.49),in SST at M1 (YS ¼ 34.52 � 0.73; AS ¼ 35.06 � 0,89; OS ¼35.14 � 0.68). The results of this study demonstratethat normal stallions in different age groups can maintaina consistent testicular temperature even under

environmental heat stress conditions, thereby demon-strating the efficiency of testicular thermoregulationmechanisms independent of the age, since no differencewere observed between groups in SST temperature in theshade (M0), and after one hour of sun exposure (M1). It canbe observed also that the testicular temperature in horses is3-4�C below body temperature. In addition, the use ofa digital infrared thermograph to measure the heat radi-ated by the scrotum has proven to be a powerful tool forcharacterising scrotal surface temperatures. This tool alsohas potential for use as a complementary examinationtechnique in andrological evaluations.

Effect of addition of soybean lecithin to Botu-Crio� onsperm parameters and fertility rates of frozen equinesemen

A.S. Rocha, I. Martin, G.A. Monteiro, P.N. Guasti,Y.F.R. Sancler-Silva, and F.O. PapaDepartment of Animal Reproduction and VeterinaryRadiology – FMVZ/ UNESP, Botucatu, SP, Brazil

The technique of artificial insemination using equine frozensemen obtained substantial progress in the last decade. Thesemen extenders are composed by substances thatpromote the preservation of plasmamembrane integrity bystabilizing of the pH, neutralization of toxic productsproduced by the sperm, protection against thermal shock,maintenance of electrolyte balance and osmotic potential,inhibition of bacterial growth and providing energy. Milkand egg yolk are commonly found on the freezingextenders to assist in stabilizing of the membranes duringfreezing and thawing. In addition, studies using soybeanlecithin have been developed in order to optimize thequality of the cryopreserved semen, since membraneintegrity is still low for sperm after thawing in extenderscomposed by egg yolk. In an attempt to improve the indicesrelated to sperm membrane integrity and, consequently,fertility, the aim of this study was to evaluate the associa-tion between soybean lecithin lipids and egg yolk lipids inequine semen cryopreservation. For cryopreservation, eachejaculate from three “bad freezers” stallions was mixed 1:1with Botu-Semen� (Botupharma / ME Ltda., SP, Brazil) andsplit into three aliquots that were centrifuged at 600xg for10 minutes. The sperm pellets were resuspended withBotu-Crio� (Botupharma / ME Ltda., Botucatu, SP, Brazil)(Control Group), Botu-Crio� plus soybean lecithin (Group

6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518 511

1) and with Botu-Crio� plus soy phosphatidylcholine(Group 2), to a concentration of 200x106 viable sperma-tozoa per mL. The following motility parameters wereevaluated by CASA (HTMdIVOS 12, Hamilton ThorneResearch, USA): total motility (MT, %), progressive motility(MP, %), linear velocity (VSL, mm/s), curvilinear velocity(VCL, mm/s), average path velocity (VAP, mm/s) and fastsperm cells (RAP, %). The plasma membrane integrity (MI,%) was evaluated using the fluorescent probes carboxy-fluorescein diacetate and propidium iodide (CFDA/PI). Thesamples were frozenf on the automated machineTK�4000C (TK Tecnologia em Congelação Ltda, Uberaba,MG, Brazil). Also, the sperm parameters and plasmamembrane integrity were evaluated after thawing ina water bath at 46�C for 20 seconds. For fertility tests 46mares were inseminated at the tip of the uterine horn.Fifteen of them were inseminated using the control group,fifteen using the group 1 and sixteen inseminations usingthe group 2. The insemination dose was 600x106 viablespermatozoa. The values (mean � standard deviation)obtained in the post-thawing evaluation of control group,group 1 and group 2 were, respectively: MT(%): 69.16 �9.28; 64.63 � 11.05; 68.47 � 7.92; MP(%): 25.00 � 5.43;23.26 � 5.63; 26.16 � 5.50; VAP(mm/s): 100.89 � 14.72;102.47 � 13.09; 101.26 � 13.78; VSL(mm/s): 75.16 � 8.47;76.21 � 8.07; 76.00 � 8.80; VCL(m/s): 182.74 � 22.18;186.11 � 19.52; 182.95 � 21.32; RAP(%): 53.16 � 12.77;50.00 � 14.29; 53.16 � 13.28; MI(%): 39.37 � 8.82; 39.95 �9.52; 41.63 � 9.24. The pregnancy rates relating to thecontrol group, group 1 and group 2, were respectively:46.7%, 13.3% and 37.5%. There was no statistical differencebetween the groups in relation to sperm parameters andpregnancy rates. Thus, the association between soybeanlecithin lipids with egg yolk lipids on cryopreserved semenof “bad freezers” stallions did not provide an increase insperm parameters and fertility rates.

Effect of time interval from insemination to ovulationon pregnancy rates of mares inseminated with jackasssemen diluted and cooled at 5�C for 12 hours

R. Rossi, J.M. Silva Filho, M.S. Palhares, E. Ribeiro,C.L.A. Silva, and M.P. RoscoeVeterinary School, Federal University of Minas Gerais(UFMG), Brazil

Numerous factors influence pregnancy rates of mares bredby artificial insemination (AI). These include the type(fresh, cooled or frozen-thawed) and viability of semen, thenumber of spermatozoa per insemination dose and thenumber and time of inseminations relative to ovulation.The most important single factor to achieve good fertility isto reduce the time from AI to ovulation. Because the lengthof oestrus and the time of ovulation relative to the onset ofoestrus differ between mares and cycles, careful moni-toring of mares and timing of AI is required. AI three times/week is less laborious and allows avoiding work on week-ends compared to AI every other day. The viability of semenand time of insemination (AI-ovulation interval), includinginseminations three times/week on fixed days have beenstudied for horses but not for donkeys. The objective of the

present study was to determine the effect of time ofinsemination relative to ovulation and inseminations threetimes/week on pregnancy rates of mares inseminated withjackass semen diluted and cooled at 5 �C for 12 hours. Theresults of 183 cycles of 141 mares were grouped, in accor-dancewith the AI to ovulation interval (24P¼ 24 hours pre-ovulation, 58 cycles; 48P ¼ 48 hours pre-ovulation, 44cycles; 48PP ¼ 48 hours pre-ovulation and on the day ofovulation, 71 cycles; 72PP ¼ 72 hours pre-ovulation and onthe day of ovulation, 10 cycles). The mares were examinedby transrectal palpation and teasing every day, andinseminated every Tuesday, Thursday and Saturday afterdetection of a 3.0 to 3.5 cm diameter follicle, until ovula-tion. The semen of five Pêga jackasses was diluted in skimmilk-glucose [1] or egg yolk-glycine [2] extender andcooled at 5�C for 12 hours, with an insemination dose of400x106 motile spermatozoa after final dilution beforecooling. Conception rates/cycle per cycle were compared byChi square test. The extenders had no effect (P > 0.05) onfertility. Pregnancy rates for the first cycle were 50.0% (24/48), 48.3% (14/29), 54.9% (28/51) and 66.7% (4/6) and thepregnancy rates/cycles were 46.6% (27/58), 45.5% (20/44),50.7% (36/71) and 60.0% (6/10), respectively for 24P, 48P,48PP and 72PP. The pregnancy rates for all AI-ovulationintervals were similar. The results indicate that jackasssperm survival allows to inseminate mares on fixed daysthree times per week.

Acknowledgments

This study received financial support from the Foundationfor Research in Minas Gerais State (FAPEMIG/CAPES) andwas supported by Mr. Renato Resende and Vau Farm.

References

[1] Foote RH. Reproduction in. Domestic Animals 2002;37:61-3.[2] Kenney RM, et al. Hastings: Society for Theriogenology Conference;

1983. 100.

Effect of centrifugation and addition of catalase and/orTrolox� on motility and viability of cooled stallionspermatozoa

C. Sabatini, M. Montella, D. Panzani, F. Camillo, andA. RotaDipartimento di Clinica Veterinaria, Università di Pisa, vialivornese lato monte, 56122 S. Piero a Grado, Pisa, Italy

During cooled preservation, reactive oxygen species maycause damage to spermatozoa. The aim of this study was toevaluate the effects of addition of catalase and/or Trolox toa semen extender (INRA82) and of two methods of dilution(final concentration of 8% or 20% of seminal plasma) onstallion sperm motility and viability after 24 or 48 hours ofcooled-storage. Seventeen ejaculates collected from sixmature, fertile stallions, were each split in 8 aliquots, onefor each treatment (4 extenders x 2 dilutions). The testedextenders were INRA82 (CONTR), INRA82 with 200 IU/mlcatalase (CAT) added, INRA82 with 200 mM Trolox (TROL)added and INRA82 with 100 IU/ml catalase and 100 mMTrolox (CA-TR) added. Samples were either diluted at