Table 1Mean values of total motility (TM), progressive motility (PM), rapid cells (RAP) and plasma membrane integrity (PMI) analyzed at 0 (0h), 24 (24h) and 48hours (48h)

0h 24h 48h

C G1 G2 G3 C G1 G2 G3

TM (%) 76 � 9 50 � 21 63 � 16 62 � 18 62 � 16 33 � 21a 49 � 17b 49 � 15b 50 � 21b

PM (%) 37 � 9 25 � 17 34 � 15 35 � 16 34 � 15 15 � 13 24 � 14 24 � 14 25 � 15RAP (%) 68 � 11 42 � 22 54 � 18 55 � 19 54 � 18 27 � 19a 43 � 17b 42 � 19b 43 � 20b

PMI (%) 56 � 13 53 � 14 60 � 11 60 � 9 59 � 11 41 � 13a 56 � 9b 55 � 11b 53 � 12b

a,b Values in the same row with different superscript differ significantly (P < 0.05).

6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518494

substrates. LC has a powerful antioxidant effect by reducingthe availability of lipids for peroxidation and increasingantioxidant enzyme activity such as superoxide dismutaseand glutathione peroxidase. Moreover, carnitines ensure theoperation of oxidative pathways by reducing acetyl-CoAlevels, and provide acetyl groups for sperm motility.Despite these findings, there are no reports about the use ofLC and AC in cooling extenders for equine semen. The aimof this study was to evaluate the effect of these substanceson cooled sperm viability. Two ejaculates of 10 stallionseach were used. After collection, the ejaculates were eval-uated for volume, motility and concentration, and thendiluted with skim milk-based extender (Botu-Semen) toa final concentration of 50x106 spermatozoa/mL. Ejaculateswere divided into 4 groups: Control (no LC/AC), G1(0,05mM/mL of LC+0,05mM/mL of AC), G2 (0,1mM/mL ofLC+0,1mM/mL of AC) and G3 (0,15mM/mL of LC+0,15mM/mL of AC). Thereafter, the samples were kept at 5�C for 24and 48 hours. Spermatozoal variables evaluated were:movement kinetics (Hamilton Thorne CASA), and plasmamembrane integrity by epifluorescence microscopy withthe fluorescent probes carboxyfluoresceine and propidiumiodide. Evaluations were performed in fresh semen (0h),and at 24 hours (24h) and 48 hours (48h) of cooling.Statistical analyses were performed by ANOVA followed byTukey test and the level of significance was set at P < 0.05.After 48hoursof storage treatedgroupsyielded significantlyhigher percentages of TM, RAP and PMI than the control(P < 0.05) while at 0h and 24h no significant differencesoccurred. There was no statistical difference betweentreatedgroups at any time. In conclusion, the combinationofL-carnitine and acetyl-L-carnitine enhanced the mainte-nance of spermmotility and provided a better protection ofthe plasma membrane after 48 hours of storage at 5�C.

Preliminary results using a new container for cooledstallion semen transport

A.C. Maciel 1,2, F.S. Castro 1,2, A. Pires Neves 1,R.C. Mattos 1,2, and S. Fiala Rechsteiner 1,21 PPGMAE – Post-graduation program in AnimalMedicine: Equine, UFRGS, Porto Alegre, RS, Brazil,2 HISTOREP - Department of Morphology UFPel, Pelotas,RS, Brazil

Since nearly all horse breed registries authorize the use ofartificial insemination, shipped cooled semen is used ona large scale in many countries. Nowadays there aredifferent types of containers to transport semen, rangingfrom sophisticated devices tomore simple styrofoamboxes.

This study aimed to verify the effectiveness of a thermalpolyethylene bag and to compare it with other sementransport devices during storage for 8 hours. Six ejaculatesfrom three fertility-proven stallions were used. The semenwas diluted 1:1 with skim milk and split in 4 aliquots. Thesemen was analyzed at 0 h (control group) and at 8 h in 3different chilling devices which were Equitainer (EG), Botu-flex (BG) and thermal polyethylene bag (TBG). Spermmotility (%) and vigor (0-5) were evaluated with a lightmicroscope by an experienced technician. Sperm concen-tration was measured using a SpermCue photometer.Membrane integrity was tested by CFDA/PI staining and themembrane functionality by HOST. Statistical analysis wasperformed by ANOVA and the comparison of means wasperformed using Least Significant Difference (LSD). Freshsemen (CG) had better motility (64%) and vigor (2.2) whencompared to all cooled aliquots. However, there was nodifference in the same parameters between the cooled-stored semen in each container (EG 39% and 1.6; BG 37% and1.5; TBG 35% and 1.5). Functionality of the membrane didnot differ between semen aliquots. Membrane integrity inTBG (69%) was the same as in the CG (61%) but TBG and CGwere different (P< 0.05) fromEG (73%) and BG (72%). Use ofa thermal polyethylene bag for the transportation of stallionsemen did not harm sperm quality when compared withother types of container. In addition, this bag seems to bea competitive alternative for cooled semen transport due tothe lowcost and the size of a large envelope. Since therewasno damage in the evaluated characteristics of semen storedin the thermal polyethylene bag, this alternative containercan be recommended for equine cooled semen transport forup to 8 hours.

Acknowledgments

The authors kindly acknowledge Luciana Meirelles Richerfor suggestions on revision.

In Vitro culture of equine spermatogonial cells ina suspension cell system

I. Martin 1, C.C. Macedo 1, G.A. Monteiro 1, D.P. Leme 2,F.C. Landim-Alvarenga 1, and F.O. Papa 1

1Department of Animal Reproduction – College ofVeterinary Medicine and Animal Science – Unesp –

Botucatu, Brazil, 2 Department of Animal Science andRural Development, UFSC – Florianópolis, Brazil

Spermatogenesis is one of the most complex and longestprocesses of sequential cell proliferation and differentiation

6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518 495

in the body. The study of spermatogenesis and spermato-gonial stem cells (SSC) may result in the discovery of newknowledge about the biology of these and other types ofadults stem cells (SC). The aim of the present study was todevelop a method to culture spermatogonial cells collectedfrom adult horses (between 3 and 4 years) in a suspensioncell system and to induce cells differentiation and sper-matogenesis in vitro. T testicles were cut in small piecesand remained under digestion solution (5 mg/mL Collage-nase Type I, and 5 mg/mL Hyaluronidase) for 1 h and 30min at 37oC. After digestion, the samples were passedthrough a 40 mm filter and then centrifuged to remove thedigestion solution and washed in HBSS. Viability of cellswas checked with Trypan Blue staining. Meanwhile, a 24-weel culture plate was prepared with 300 mL of 1.5%agarose gel in each well forming a thin layer of agar. Thecollected cells were diluted in the culture medium that wascomposed by MEM a, 10% KSR, 50 mg/mL gentamicin, 3.0mg/mL amphotericin B, 80 mL/mL ITS, 50 UI/L rFSH, 1 mmol/Lof testosterone and equine pituitary extract (EPE). Duringthe first week 3.3x10�7 M of retinoic acid and 3.3x10�7 M ofretinol was added to the medium. Half of the medium ineach well was changed once a week. The culture systemwas incubated with 5% CO2 in air at 32oC. After 56 days inculture it was possible to see the formation of clusters ofspermatogonia and the presence of many cells above theagar layer. Immunofluorescence was used to characterizethe presence of spermatid and Sertoli cells. The cells werefixed with 4% paraformaldehyde and permeabilized withsaponine for 20 min at room temperature. Then, the cellswere incubated with the primary antibody for protamineand for SHBG (rabbit polyclonal antibodies), diluted 1:50,for 18h at 4oC. Next step was the incubation with thesecondary antibody conjugated with Alexa-Fluor 555diluted 1:100, for 1 h at room temperature. Nuclear coun-terstaining was done with DAPI. The slides were evaluatedunder a fluorescent microscope at magnification of 20x and40x. Cells fluorescent for protamine showing the formationof spermatid cells under in vitro culture could be observed.The presence of cells positive for SHBG shows the main-tenance of Sertoli cell viability under our culture condi-tions. Other studies need to be done to check the fertility ofthese spermatids.

Acknowledgments

FAPESP for financial support.

Effects of lactoferrin supplementation to milk- andcaseinate-based extenders on sperm motility,membrane integrity and nitric oxide levels of cooledstallion semen

H.S. Martins 1, M.R. Souza 2, C.F.A.M. Penna 2, G.C. daSilva 3, S.F. Côrtes 3, R. Stahlberg 4, M.F. Brito 1, and M.A.Lagares 1

1 Department of Veterinary Clinic and Surgery, VeterinarySchool, Federal University of Minas Gerais, Av. AntonioCarlos 6627, CEP:31270-901, 2Department of Technologyand Inspection of Animal Origin Products, Veterinary

School, Federal University of Minas Gerais, Av. AntonioCarlos 6627, CEP:31270-901, 3Department ofPharmacology, Institut of Biological Science, FederalUniversity of Minas Gerais, Av. Antonio Carlos 6627,CEP:31270-901, 4 Faculty of Veterinany Medicine, PUCMinas Betim, R. do Rosário 1081, Bairro Angola, Betim,CEP: 32604-115, MG, Brasil

Stallion sperm are subjected to oxidative damage duringcooling and particularly during storage. Lactoferrin (Lf) ispresent in the seminal plasma of various mammalianspecies and appears to represent a major component of thesperm-coating antigens that protect sperm. It displaysbacteriostatic activity and prevents the formation ofhydroxyl radicals. The aim of this study was to determinethe effects of Lf supplementation to milk- and sodiumcaseinate-based semen extenders on sperm motility,membrane integrity and nitric oxide (NO) levels of cooledequine semen. Semen from five stallions was collected withan artificial vagina. The ejaculates were divided intosamples according to the different cooling extenders: C1)Kenney extender; C2) 0.6% caseinate; C3) 0.6% caseinate +Lf 200 mg/mL; C4) 0.6% caseinate + Lf 500 mg/mL; and C5)0.6% caseinate + Lf 1000 mg/mL. The samples were cooled to5�C (0.27�C/min). After 24h, the percentage of sperm withtotal and progressive motility, membrane integrity and theNO levels was determined. Total and progressive motilitywas assessed using a computer-assisted sperm (CASA)analysis system (Sperm Class Analyser, SCA� 2005 VS 4.0.0Microptik S.L., Barcelona, Spain). Sperm membrane integ-rity was evaluated by fluorescent microscopy with car-boxifluorescein and propidium iodide staining (CFDA/PI).To verify the possible antioxidant effect of Lf in differentconcentrations, NO levels were measured (mM/x106sperm)using spectrophotometry with Griess reagent. Means andstandard deviations of the evaluated datawere analyzed byANOVA using the SAS program. A probability of P < 0.05was considered significant. There was no significantdifference in the percentage of motile sperm (C1:50.7 �22.0, C2:47.8 � 20.9, C3:44.7 � 22.8, C4:42.4 � 22.0 andC5:45.3 � 10.8) or progressively motile sperm (C1:11.7 �7.6, C2:9.9 � 16.0, C3:10.1 � 13.5, C4:15.4 � 22.3 andC5:9.5 � 7.3), or sperm with intact membranes (C1:41.7 �18.5, C2:30.7 � 14.1, C3:27.3 � 15.6, C4:24.2 � 18.2 andC5:32.9 � 10.5) or NO levels [fresh semen (control): 8.6 �1.9; C1: 90.8 � 95.1, C2: 119.3 � 139.4, C3: 43.3 � 37.7, C4:18.7 � 37.3, C5: 75,8 � 85,3 mM/106 sperm] with regard totreatment. These results corroborate with the observationsin many studies reporting the lack of positive effect ofantioxidant addition to skim-milk-based extenders. Thefact that seminal plasma was not removed might be thereason why the effect of Lf was not significant. The testedextenders seemed to protect stallion sperm during cooling;nevertheless, the addition of Lf to skim-milk- andcaseinate-based extenders without the removal of seminalplasma did not improve the characteristics of stallionsperm during cooled storage.

Acknowledgments

FAPEMIG, CAPES and CNPq for financial support.