6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518498

with significantly lower sperm numbers than with dummymount semen collection.

The effect of platelet-rich plasma on endometrial pro-inflammatory cytokines in susceptible mares followingsemen deposition

E.S. Metcalf 1, K. Scoggin 2, and M.H.T. Troedsson 2

1Honahlee, PC, 14005 SW Tooze Rd, Sherwood,2 University of Kentucky Lexington, KY 40546-0099

Mares susceptible to persistent mating-induced endome-tritis (PMIE) are a source of substantial economic ineffi-ciency. Recent studies report a significant postinsemination up-regulation of endometrial anti-inflam-matory cytokines IL-10 and IL-Ra in normal versussusceptible mares, as well as significantly increased andprolonged expression of IL-1b, IL-6, IL-8, and IL1Ra insusceptible mares following intrauterine inoculation withE.coli [1]. Furthermore, nitric oxide is suspected to playa significant role in uterine clearance, because increasedendometrial expression of inducible nitric oxide synthetase(iNOS) has been reported in susceptible mares followinginsemination [2]. This project was developed to investigatethe expression of inflammatory biomarkers of uterinebiopsies obtained from susceptible mares following intra-uterine PRP (platelet-rich plasma) infusion. Plateletscontain alpha-granules that, upon activation, secretebioactive molecules with regenerative capacity in the formof growth factors, cytokines, and chemokines. Barrenmareswith a history of PMIE (n ¼ 9) underwent daily ultraso-nographic examination of the reproductive tract per rectumwhile in estrus. All mares underwent an untreated (no PRP)cycle followed by a treated (PRP) cycle. When a dominantfollicle >35 mm in diameter and evidence of endometrialedema were detected, 1.0 mg deslorelin acetate(SucromateR Bioniche Animal Health Pullman, WA) wasadministered intramuscularly. On the treatment cycle, 180ml of citrated whole blood was obtained for PRP processingin a specialized blood fraction separating centrifuge system(Angel� Cytomedix, Inc. Gaithersburg, MD). The resultantPRP was brought to a final volume of 10 ml with platelet-poor plasma and infused into the uterus. Mares wereinseminated with >200 million motile sperm 24-36 hoursfollowing deslorelin administration. An endometrial biopsyobtained the following day was preserved in RNAlaterR

(Sigma Inc., St Louis, MO) for reverse transcription poly-merase chain reaction (RT–PCR) analysis [1] for mRNAexpression of inflammatory biomarkers IL-1b, IL1-Ra, TNFa,IL-6, IL-8, IL-10, and iNOS. Data were analyzed using SAS(SAS Institute, Cory NC) with Wilcoxon signed-rank test(non-parametric). Comparisons were made between pre-treatment and post-treatment groups. Significance was setto P < 0.05. In susceptible mares treated with an intra-uterine infusion of PRP, the mRNA expression of IL-1b, IL-6,IL-8 and iNOS was significantly down-regulated (P < 0.05)when compared with untreated mares. All other cytokineswere similar in treated and untreated mares. Therefore,following semen deposition, PRP influences the mRNAexpression of endometrial pro-inflammatory cytokines andiNOS in mares susceptible to PMIE.

References

[1] Woodward E, et al. Endometrial cytokine expression in mares withdifferent resistance to persistent breeding induced endometritis(PBIE) at multiple time points after insemination. Biol. Reprod; 2011.SSR (Accepted).

[2] Alghamdi AS, et al. Nitric oxide levels and nitric oxide synthaseexpression in uterine samples from mares susceptible and resistantto persistent breeding-induced endometritis. AJRI 2005;53:230-7.

Comparison of caspase activity between cryopreservedejaculated sperm and epididymal sperm in stallions

G.A. Monteiro, C.P. Freitas-Dell'Aqua, P.N. Guasti, F.P.Hartwig, F.P. Lisboa, R.R.D. Maziero, J.A. Dell`Aqua, Jr., andF.O. PapaDepartment of Animal Reproduction and VeterinaryRadiology, School of Veterinary Medicine and AnimalScience, UNESP, Botucatu, SP 18610-970, Brazil

The development of a reliable technique to freeze epidid-ymal semen would provide a unique opportunity topreserve valuable genetic material from an unexpectedlyloss of stallions. Phenomena similar to apoptosis wereidentified by sperm cell analysis. These phenomena inflictdifferent degrees of damage on spermatozoa structures thatare important for longevity. The aim of this work was tocompare the apoptotic indices of sperm obtained fromejaculate (G1) and sperm from cauda epididymis (G2). ForG1, two ejaculates from each of seven stallions werecollected and then subjected to cryopreservation usinga BotuCrio� extender. One week after the last semencollection, the stallions underwent bilateral orchiectomy.Sperm from cauda epididymis was harvested immediatelyafter castration (G2) by retrograde flushing the caudalportion of the epididymis using a skimmilk-based extender(BotuSêmenTM1). The recovered sperm was then cry-opreserved using BotuCrio� extender. The sperm motilityparameters were analyzed by CASA (HTMdIVOS 12, Ham-ilton Thorne), and apoptosis was estimated using epifluor-escence microscopy for caspase activity (FITC-VAD-FMK,G7462, Promega). The sampleswere evaluated immediately(0 h) and 8 hours (8 h) after thawing. The sperm parametersof the G1 vs G2 samples at 0 h were 62.3 � 12.9a vs 72.6 �8.4a, 31.6� 9.2a vs 35.3� 10.32a and 49.3� 14.33a vs 59.7�13.59a, at 8 h, the results were 26.0 � 21.6b vs 54.7 � 12.2a,6.1�6.4bvs 17.4�8.54a and13.7�14.85bvs37.6�14.15a fortotal motility, progressive motility and percentage of rapidcells, respectively. Evaluation of the caspase activity in theG1 and G2 samples yielded 25.7 � 4.71a vs 27.6 � 2.51a,13.14� 3.72a vs 13.0� 3.51a and 34.6� 6.29a vs 36.6� 3.10a

at 0 h and 5.86� 2.67b vs 12.9� 2.54a, 9.14�1.57b vs 13.6�4.04a and 55.7 � 5.88a vs 49.1 � 2.91b at 8 h for thepercentage of viable cells, percentage of viable cells withactivated caspase and percentage of dead cells, respectively,after thawing.At0 h, nodifferences in the spermparameterswere observed between groups, but statistical differenceswere observed in the sperm motility parameters betweenthe treatment groups after 8 h. For caspase analysis, nodifferencewas found at 0 h between the groups. However, 8h after thawing, a smaller percentage of viable cells wereobserved in the ejaculated sperm, as compared to the

6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518 499

epididymal sperm.Moreover, the percentageof dead cells inthe G2 sample was lower than in the ejaculated sperm. Onthe basis of these results, we can conclude that frozen-thawed cauda epididymal sperm has similar or highermotion parameters than ejaculated sperm after thawing. Inaddition, incubating the sperm at 20oC for 8 h after thawingresulted in highermotion parameters and a lower apoptoticstatus of the epididymal sperm. This finding suggests thatepididymal sperm are more resistant to the cold shockcaused by cryopreservation.

Acknowledgments

FAPESP for financial support and Botupharma for donationof BotuSêmen� and BotuCrio� extender.

Freezing stallion semen: Trial of an extender made withlow density lipoproteins (LDL) from chicken egg yolk

D. Moreno 1, D. Bencharif 1, L. Amirat-Briand 1,S. Destrumelle 1, M. Anton 2, P. Barriere 3, andD. Tainturier 11 Laboratory of Biotechnology and Pathology ofReproduction, ONIRIS: The National Veterinary School ofNantes, France, 2 Laboratory of Biopolymers InteractionsAssemblies, Unit Interfaces and Dispersed Systems, INRA,Nantes, France, 3Department of Reproductive Pathology,Mother and Child, CHU Hôtel Dieux, Nantes, France

The aim of this study was to determine the best concen-tration of low-density lipoproteins (LDL) in a semenextender to improve the percentage of motile spermatozoain equine sperm after freezing and thawing in comparisonwith standard extenders. Ten extenders were compared:one containing 2% egg yolk (EY), eight containing differentconcentrations of LDL (0.25, 0.50, 0.75,1, 2, 3, 4, and 5%) andmilk protein-based INRA-96 extender; all extenders con-tained 2.5% glycerol. Fourteen ejaculates were collectedfrom four different stallions. The first dilution was madewith equal parts at +37�C, centrifuged (600 x g/10min), andre-suspended in the corresponding extenders to obtaina final sperm concentration of 100 x 106 /ml. The resultingmixture was cooled at +4�C for 1 hour, then packed intofour 0.5 ml straws before being left for a further 30 minutesat +4�C. Finally, the straws were frozen in nitrogen vapours4 cm over liquid nitrogen for 10 minutes before beingimmersed in liquid nitrogen at -196�C and stored. Twostraws per extender and per ejaculate were thawed ina water bath at +37�C for 30 seconds. The contents of eachstraw were recovered into a cryotube and placed in a waterbath at +37�C for 10 minutes before being examined withan image analyser (CEROS 12). The extenders made with 2and 3% LDL gave superior spermatozoal motility rates(35.5% and 35.3% respectively) compared with the EYextender (33.3%) (P > 0.05) and INRA-96 extender (22.1%)(P< 0.05). The last two strawswere thawed to perform fourin-vitro fertility tests. The hypoosmotic test to assess theintegrity of the plasma membrane; the best results wereobtained with the 0.5%, 2%, and 3% LDL extenders and eggyolk (31.3%, 32.3%, 32.4%, and 31.3% respectively) (P> 0.05).

The FITC/PSA test was used to verify acrosome integrity; thebest results were obtained with the 0.5%, 0.75%, and 3% LDLand INRA-96 extenders, but the difference was not statis-tically significant in comparison with the 2% LDL extender(85.8%, 85.0%, 84.7%, 84.8%, and 84.0% respectively). Theacridine orange test was used to assess DNA integrity; therewas no significant difference between the variousextenders, the DNA was preserved in 98.0% of the sper-matozoa. Finally, spermatozoal morphology was examinedusing Spermac STAIN; 78.0% of the spermatozoa did notpresent any anomaly in the 0.25% and 2% LDL extenders. Forthe others extenders the results were among 74.0 and77.0%. In conclusion, 2% LDL extender gave the best post-thaw percentage of motile spermatozoa. The results of thein-vitro fertility tests were also superior for this extender.

Comparison of two freezing extenders for stallionspermatozoa: Caceres and Botu-crio�

A. Morillo Rodriguez 1, T. Pessanha Guimaraes 2,M. Graça Lopes 2, A. Rocha 2, and F.J. Peña 1

1 Laboratory of Equine Reproduction, Veterinary TeachingHospital, Faculty of Veterinary Medicine, University ofExtremadura, Avd. de la Universidad s/n, 10003 Cáceres,Spain, 2 ICBAS and UNID, University of Oporto, Oporto,Portugal

Freezing technology is used in the equine industry world-wide. This technology allows international trade of semenand permits the conservation of the genetics. The use offrozen semen has increased in the last decades since studbooks have allowed the use of artificial insemination. Themain problem of the technique is the large inter-individualvariability, which is also present in sperm survival afterfreezing and thawing procedures, which prevents itsstandardization. Such differences could be genetics inorigin, and stallions are usually selected for reproductionbased only on their performance and phenotype. The aim ofthis work is to compare the outcome of cryopreservation ofstallion spermatozoa using recently developed extenders,Caceres extender developed in our laboratory (University ofExtremadura, Cáceres, Spain patent pending) and thecommercial freezing medium Botu-Crio�. Using these twomedia, three ejaculates from 2 stallions were split andsimultaneously frozen in Botu-crio� and the extenderCaceres. The ejaculates were collected using a Missourimodel artificial vagina. The filtered ejaculate was extended1:1 (v/v) with INRA 96 (IMV, L'Aigle, France), split into twosubsamples and centrifuged at 600g for 10 min. The spermpellets were re-extended in the different freezing mediaCáceres and Botu-crio� to a final concentration of 100x106

spermatozoa/mL. The spermatozoa were slowly cooled to 4C within 1 h, loaded in 0.5 mL plastic straws and frozenhorizontally in racks placed 4 cm above the surface of liquidN2 for 10 min, after which they were directly plunged inliquid N2 for storage. After at least 4 weeks of storage,straws were thawed in a water bath at 37 C for 30 s, foranalyses. Samples were evaluated for motility using CASAsystem (ISAS� Proiforser Valencia Spain). The parametersevaluated with this system were, total motility (TM) linear