Developmental competence and lipid droplet features of maturing bovine oocytes are affected by saturated and not by unsaturated fatty acids

Poster Session I (Student Competition; odd numbered posters)

Abstract P1

Developmental competence and lipid droplet features of maturing bovine oocytes are affected by saturated and not by unsaturated fatty acids

H Aardema, PLAM Vos, BAJ Roelen, HM Knijn and BM Gadella Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Fertility in high producing dairy cows has declined over the last decades. An increased serum and follicular fluid concentration of non- esterified fatty acids (NEFA), due to body fat mobilisation in the early post-partum period, has been postulated as a cause for this fertility decline (Leroy 2005: Reproduction 130, 485). Therefore effects of unsaturated (oleic acid) and saturated (palmitic acid) NEFA during maturation of cumulus oocyte complexes (COCs) on developmental competence and lipid droplet (LD) features in the oocyte were examined in this study. COCs from 3 to 8 mm follicles of slaughter- house ovaries were cultured in control maturation medium (TCM199) and medium containing oleic or palmitic acid (100lM NEFA bound to 0.1% BSA), based on follicular fluid concentrations early post- partum. After 23 h of maturation, COCs were fertilized (500 per group) and cultured to score embryonic development, or fixed (60 per group) for LD staining with C1-BODIPY500/510 C12. Embryonic development was impaired after COCs were matured in presence of palmitic acid (18 ± 1.4% blastocysts) compared to the control (23 ± 1.6%) and oleic acid group (23 ± 1.6%). Moreover, the average LD size (inlm²) in MIIstage oocytes matured in the presence of palmitic acid (2.16 ± 0.24) was smaller compared to oleic acid (2.91 ± 0.30), but not different from standard medium (2.81 ± 0.37).

Whether the effect of palmitic acid on lipid droplet features in the oocyte is correlated with reduced developmental capacity of COCs is under current research.

Abstract P3

Leptin mRNA is Present in Bovine Ejaculated Spermatozoa

A Abavisani¹, A Baghbanzadeh², P Shayan², P Tajik², H Dehghani³and M Bahreini⁴

1Department of Animal Sciences, University of Tehran, Iran;

2Veterinary Faculty, Tehran, Iran;³Veterinary Faculty, Mashhad, Iran;⁴Country Breeding Center, Karaj, Iran

Leptin, hormonal product of the ob gene, is involved in energy metabolism and reproduction. Expression of leptin and its receptor in some reproductive organs suggest that in addition to endocrine effects of leptin, it has paracrine/autocrine effects on reproduction. Evidence, such as expression and secretion of leptin in human and pig spermatozoa, have pointed to a direct role of leptin in the physiologic functions of spermatozoa. The present study was the first to investigate the presence of the leptin mRNA transcript in ejaculated spermatozoa of Holstein cattle using RT-PCR analysis. Total RNA was extracted from sperm cells by TRizol procedure, and used to construct cDNA.

The PCR with spermatozoal cDNA and outer leptin primer pairs resulted in amplification of the expected product. To confirm the first results, RT-PCR products were amplified with nested PCR using inner leptin primer pairs. Both outer and inner primer pairs, which were located in exon 2 and exon 3 RNA specific, gave the expected PCR amplified products of 441 bp and 384 bp respectively, and ruled out the possibility of DNA contamination. The presence of leptin mRNA in the ejaculated spermatozoa of bulls suggests that leptin might act on physiological processes of bovine spermatozoa through autocrine/

DNA Fragmentation in Fresh and Frozen-thawed Stallion Spermatozoa

O Anglada, E Taberner, A Pen˜a and J Miro´

Facultat Veterina`ria. Universitat Auto`noma de Barcelona, Spain Sperm DNA fragmentation (sDF) seems to be a cause of infertility.

The aim of this study was to evaluate the level of sDF in fresh semen of several stallions and its evolution in frozen-thawed semen.

28 ejaculates were collected from 5 stallions. Semen was immediately diluted 1 : 5 in pre-warmed (37C) Kenney extender in 50-ml Corning tubes. Semen samples were centrifuged at 660·g, 15 min, 20C. The supernatant was removed and the pellet resuspended in Gent diluent to obtain a final concentration of 200·10⁶viable sperm/ml. Semen was packaged in 0.5-ml straws and frozen in a programmable liquid nitrogen freezer. Frozen semen was thawed in a water bath at 37C for 30 s. Aliquots of 25ll of extended semen before centrifugation and frozen/thawed semen were used to evaluate DNA fragmentation using the Sperm-Halomax kit. The slides were stained by propidium iodide in Vectashield mounting medium H-1000 and 200 sperm cells/sample were evaluated under fluorescence microscopy. No significant differ- ences in sDF were observed between fresh semen samples (14.6 ± 1.7%) and frozen-thawed semen samples (17.2 ± 1.5%).

No inter-individual differences were observed. The only significant differences in sDF were observed, by the General Linear Model (SAS 9.1), between two stallions in fresh semen (23.1 ± 3.3% vs 9.7 ± 1.5%, p < 0.05). However, a high intra-individual variability coefficient was noted. These results suggest that the freezing process does not affect DNA fragmentation and that there is variability between ejaculates of the same stallion.

Abstract P7

Effect of Porcine Oviductal Fluid on Spermatozoa-zona pellucida Binding

K Avile´s-Lo´pez, FA Garcı´a-Va´zquez and C Mata´s

Dpto de Fisiologı´a, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain

Porcine IVP has not been fully developed because of several problems including polyspermy after IVF. In vivo the sperm cell is probably modified by the oviduct epithelium that adsorbs proteins from the sperm surface and secretes glycoproteins with an unknown function in sperm-ZP binding. The aim of the study was to evaluate the effect of porcine oviductal fluid (POF) protein on binding spermatozoa-ZP using ejaculated (EJ) and epididymal (EP) sperm under different treatments. EJ and EP spermatozoa were divided into 3 groups:1) spermatozoa without any treatment (control=C); 2) washed through Percollgradient (P); 3) washed through Percolland incubated with POF protein (50lg/ml) for 30 min (P-POF). IVM oocytes (n = 1135) were inseminated in TALP medium for 30 min using different sperm groups. Finally, the oocytes were fixed and Hoechst stained to evaluate the spermatozoa bound under epifluorescence microscope. Six assays were evaluated. The results were analysed by two-way ANOVA. Data showed that i) the binding to ZP is higher using epididymal sperm than ejaculated (32.98 vs 17.74 respectively, p < 0.001); ii) binding decreased significantly when epididymal sperm was incubated with POF (25.36) vs other groups (C = 37.14, p = 36.44, p < 0.05); and iii) the ejaculated sperm binding results were similar in all experimental groups (C = 22.87, p = 15.73 and P-FOP=14.62, p < 0.118). In conclusion sperm source as well as sperm treatment affect the binding on ZP. Protein POF decreased the binding of sperm in our studied conditions.(Supported by Seneca 08752/PI/08 and AGL2006-03495.)

Abstract P9

Relationship between NEFA Plasma Concentrations and Fertility in Synchronized Dairy Cows

OG Balogh, H Fe´bel, Cs Hatvani and G Ga´bor

Research Institute for Animal Breeding and Nutrition, Herceghalom, Hungary

H-F cows were put on Provsynch regimen (Day 0 PG, Day 14 PG, Day 26 GnRH, Day 33 PG, Day 35 GnRH, Day 36 AI) 30–36 days post- partum (PP) in July (summer, n = 10) and in December (winter, n = 10) 2008. During the treatment period, blood samples were collected once a week. Since the 5^thday post-insemination (PI) rectal ultrasound examinations were carried out once a week (uterus and ovaries). Pregnancy diagnosis was completed 30 day PI. Samples were assayed for plasma NEFA, BHB, FRAP (ferric reduction ability) and serumb-carotene concentrations. Plasma NEFA and BHB concentra- tions were higher in summer than in winter (p < 0.001) and NEFA values exceeded the normal range. Both of them increased periodically in summer followed the hormonal treatments, while values of all other parameters were not found over the normal range. None of the cows were pregnant in summer and 8 of the 10 cows had irregular luteal forms (ILF) on their ovaries. In winter the plasma NEFA concentra- tion had an elongated peak between 49 and 62 days PP. In winter 6 of 10 cows were pregnant, and 4 cows had ILF’s on their ovaries. Based on these results it looks that NEFA plasma concentration may influence the conception rate and frequency of ILF’s. Increased NEFA concentration in summer was probably caused by the lower food intake (high ambient temperature) and NEFA peaks followed the hormonal treatments may associated with the heat signs. The elongated NEFA peak in winter was probably resulting from the high individual daily milk production (41 kg/day/cow), which was signif- icantly higher, than in summer (36.4 kg).

Abstract P11

Improving the Use of Sex-sorted Ram Spermatozoa for In vitro Fertilisation (IVF)

KH Beilby, SP de Graaf and CG Grupen

The Faculty of Veterinary Science, The University of Sydney, Australia The current protocol for sex-sorting ram sperm results in a dilute end product. This is problematic when sorted sperm are used for IVF as the obligatory removal of cryopreservation medium vastly reduces cell numbers. As such, this study aimed to increase the number of viable, sex-sorted sperm available for IVF by increasing the pre-freeze concentration of sorted sperm and comparing and refining common sperm preparation methods. Semen was collected from 2 rams, sorted, and frozen at concentrations of 20, 40 or 80·10⁶ sperm/ml.

Continuous (80%, 90%) and discontinuous (40/80%, 45/90%) Pure- sperm (PS) gradients, ‘migration-sedimentation’, ‘swim-up’ and

‘washing’ methods were compared. Cleavage and blastocyst rates were determined to assess the function and fertility of non-sorted and sorted sperm using an insemination concentration of 5·10⁵motile sperm/ml.

A continuous 80% PS gradient (500·g, 10 min) produced the highest motile sperm concentration (6.2 ± 0.2 sperm/ml). There was no difference in embryo development between sorted and non-sorted sperm. However, there was a difference in cleavage rate between rams (p = 0.001). In conclusion, a similar fertile sub-population of sperm can be recovered from samples with either low or high initial concentrations. Furthermore, this study has shown that sorted sperm perform equally well as non-sorted sperm in vitro. This supports existingin vivoevidence that sex-sorted ram sperm are of comparable fertility to non-sorted sperm.

Abstract P13

Immuno-histochemical Insulin Receptor Staining in Bovine Follicles

P Bossaert¹, JLMR Leroy², P Bols², G Opsomer¹and H De Cock²

1Faculty of Veterinary Medicine, Ghent;²Faculty of Biomedical, Pharmaceutical and Veterinary Sciences, Antwerp, Belgium

Insulin stimulates mammal granulosa cell (GC) function and follicular growth. In cows, low insulin blood levels increase the risk of anoestrus.

In women, low GC insulin receptor (IR) expression is associated with ovulation disorders. Our aim was to develop a staining technique suitable for evaluating IR expression on the bovine GC membrane.

Paraffin slides of bovine slaughterhouse ovaries were made. Following deparaffinization, antigen retrieval, peroxidase quenching and protein blocking, slides were incubated with mouse IR-antibodies, enzyme- labeled goat anti-mouse antibodies and a substrate chromogen in an automatic staining device. A brownish-red color indicated presence of IR. Follicles were morphologically classified as pre-antral (PAF), early antral (EAF; n = 73), dominant (DF; n = 15) and subordinate (SF; n = 23). Using a software pack, color intensity (INT) was quantified within 4 histologically defined ovarian regions: GC, internal (TI) and external (TE) theca layers and stroma (STR). INT was analyzed in a univariate linear model with ‘region’ and ‘follicle type’ as fixed factors. There was a ‘follicle type * region’ interaction (p = 0.005). In PAF, no staining signal was present. In all other follicle types, INT was higher in GC than in any other region (p < 0.0001). In EAF, INT was stronger in TI than TE (p = 0.0018).

In SF and DF, INT was higher in TI than STR (p < 0.05). The appearance of IR on GC when follicles enter the antral stage suggests that the acquisition of IR is a key event in the process of follicular growth.

Abstract P15

Detection and Localization of GLUTs 1, 2, 3 and 5 in Donkey Spermatozoa

D Bucci¹, C Vallorani¹, A Contri², A Carluccio², G Isani¹, C Tamanini¹, G Galeati¹and M Spinaci¹

1Veterinary Faculty, Bologna, Italy;²Veterinary Faculty, Teramo, Italy Hexose transporters have been studied in male gametes of several species, such as boar, stallion, dog, human, rat and bull, but information on donkey spermatozoa is lacking. This preliminary study was aimed at determining the distribution of GLUTs 1, 2, 3 and 5 in donkey spermatozoa by both indirect immunofluoresence and western blotting to better understand their hexose metabolism and to highlight possible differences with other species. GLUT 1 showed a reactive band at approximately 50 kDa and was localized in the outer apical part of acrosomal membrane (AM) and in the tail, with a faint signal in the midpiece; GLUT 2 presented a 45 kDa reactive band, a diffuse AM, positive tail, and an evident equatorial line. GLUT 3 showed a very intense band at approximately 65 kDa and a strong fluorescent signal in the tail, with a faint reaction in the midpiece, equatorial line and outer apical acrosomal region. GLUT 5, the fructose transporter, presented an immunoreactive band at 65 kDa and was localized in the membrane of both principal piece of the tail and apical AM. In conclusion, we localized for the first time GLUTs 1, 2, 3 and 5 in donkey spermatozoa, determining both their peculiar distribution and molecular weight. Further studies are necessary to better understand the metabolism of donkey spermatozoa and its application in reproductive technologies. (Work supported by a Bologna University grant (ex 60%))

Effect of Heparin on Pig In vitro Fertilization

S Ca´novas, I Monde´jar, R Romar, M Avile´s and P Coy Department of Physiology, Veterinary Faculty, Murcia, Spain Participation of the glycosaminoglycan heparin on the pre-fertilization zona pellucida hardening mechanism has been shown to improvein vitro fertilization efficiency in pigs (Coyet al., 2008, PNAS 105:15809–15814).

On the other hand, it was suggested that heparin did not supportin vitro penetration of pig oocytes (Wanget al., 1991, J Reprod fertil 93:491–496) although it is an active component for capacitation in cattle (Parrish et al., 1989, Biol Reprod 40:1020–1025). Since heparin-like glycosami- noglycans are present in the porcine oviduct (Tienthaiet al., 2000, Reprod Dom Anim 35:167–170), this study was designed to clarify the role of heparin on different steps during pig in vitro fertilization. In vitro matured pig oocytes (n = 221) were fertilized in TALP medium containing or lacking (control) 1lg/ml heparin. A gamete co-incubation time of 15 min and a sperm concentration of 1·105 cells/ml were used.

At 18-h post-insemination, mean number of sperm bound to ZP, mean number of sperm inside the oocytes, and percentages of penetration and monospermy were assessed. The results showed that heparin did not affect the ability of the sperm to bind zona pellucida (43.8 ± 3.6 vs 39.84 ± 2.7), but it decreased their ability to penetrate it (87.7 ± 3.1%

vs 28.97 ± 4.40%, p < 0.001). Since hyperactivation of capacitated spermatozoa is necessary to penetrate the zona (Suarez, 2008, Hum Reprod Update 14:647–657), the possibility that heparin can induce capacitation but blocks hyperactivation in pig is proposed, although other hypotheses such as failures in acrosome reaction cannot be discarded.(Granted by MEC/FEDER, AGL2006-03495/GAN.)

Abstract P19

Freezability of Pie´train Boar Ejaculates Evaluated by Sperm Kinematics

I Casas, M Briz, S Sancho, E Pinart, E Bussalleu, M Yeste, A Fa`brega, M Puigmule´, E Garcia and S Bonet

Biotechnology of Animal and Human Reproduction (TechnoSperm), University of Girona, Spain

Pie´train is a well-known paternal line used as the terminal boar in many crossbreeding systems, mainly due to its excellent food conversion and low fat indexes. Semen banking conserves valuable genetic stock for breeding. The present study compares changes in kinematic parameters in refrigerated (17C) and frozen/thawed sperm, with the objective of describing the freezability of the Pie´train breed. Sperm-rich fractions of 180 ejaculates collected from 44 mature Pie´train boars were diluted 1 : 5 (v:v), transported and stored at 17C in our laboratory until cryopres- ervation using the Westendorf method. Eight kinematic parameters were analyzed in refrigerated semen and post-thawing (30 min, 37C), using CASA software. Values were compared using one-way ANOVA and significance was set at p < 0.01. 37.2% of the cryopreserved ejaculates displayed over 40% of progressive motility 30 min post-thawing. Data analysis revealed that all motility parameters decreased in Pie´train spermatozoa after freezing/thawing: -22.6% total motility; -22.3%

progressive motility; -9.1% circular trips; -18.1lm/s VCL; -6.2lm/s VSL; -14.5lm/s VAP; -0.2lm ALH, and -1.4 Hz BCF.

Abstract P21

Pro/acrosin Expression in Epididymal Spermatozoa from Fertile Boars

A Fabrega, M Puigmule´, M Briz, S Sancho, E Bussalleu, M Yeste, I Casas, E Garcia, S Bonet, and E Pinart

Biotechnology of Animal and Human Reproduction, University of Girona, Spain

experiment was to study the expression of pro/acrosin along epidid- ymis in order to characterize the changes associated to its maturation.

Epididymal samples were obtained by cannulation of six epididymal regions of post-pubertal fertile boars (proximal and distal caput;

proximal and distal corpus; proximal and distal cauda). Undiluted samples collected from each region were divided into two Eppendorf tubes. One tube was centrifuged at 13000·gfor 30 min at 4C, and supernatant was centrifuged again during 5 min to obtain the sperm- free epididymal fluid. The other tube was washed twice with PBS at 600·gfor 10 min at 4C to obtain the sperm fraction. Epididymal fluid and sperm samples of each region were analyzed by SDS-PAGE and Western Blot, differentiating four pro/acrosin isoforms. Both spermatozoa and fluid samples showed a similar pattern: in the proximal caput only the proacrosin band (54–49 kDa) was detected;

the a-acrosin band (47–42 kDa) appeared in distal caput, whereas ß- (37–32 kDa) andc-acrosin bands (32–27 kDa) appeared in prox- imal corpus and their expression increased progressively throughout the cauda. These results indicated that in the boar the maturation of proacrosin into acrosin isoforms begins in the caput and becomes progressively more intense throughout the corpus and, specially, the cauda regions.

Abstract P23

The Effect of the Local Administration of FSH and/or Misoprostol on Cervical Penetration in the Ewe during the Peri-Ovulatory Period

L Falchi, M Taema, M Khalid and RJ Scaramuzzi The Royal Veterinary College, Hatfield, UK

Trans-cervical intrauterine insemination in the sheep is limited by the convoluted anatomy of the cervix, making passage of an insemination catheter into the uterine lumen almost impossible. The aim of the study was to investigate the effect of intra-cervical administration of FSH, Misoprostol (a PGE1 analogue) or a combination of the two on cervical penetration 54 h after sponge removal. Forty ewes were synchronised using progestagen sponges and were randomly assigned to four groups of 10: control; Misoprostol; FSH; Misoprostol plus FSH. Misoprostol (1 mg) was administered twice at 46 and 50 h after sponge removal while FSH (2 mg) was administered once 24 h after sponge removal. The hormones were prepared in 0.5 ml of 50% gum acacia in saline and deposited into the cervix as far cranial as possible.

The depth of cervical penetration (mm) was measured 54 h after sponge removal using a modified cattle artificial insemination pipette.

The experiment was performed in two replicates and the data were analysed using one wayANOVA. No significant differences in the depth of cervical penetration were found between the control group (mean=

14.5 ± 3.3) and the FSH (mean= 17.7 ± 2.2), Misoprostol (mean=

21.3 ± 3.5), and the Misoprostol plus FSH (mean= 19.2 ± 3.0) treated groups (p > 0.05). Misoprostol, FSH, or the combination treatment were not effective in dilating the cervix 54 h after sponge removal, the time at which artificial insemination produces the highest fertility rates in sheep.

Abstract P25

The Expression of Oxytocin Receptor, cPhospolipase A

and Cyclooxygenase 2 in the Cervix of Ewes during the Oestrous Cycle

L Falchi, K Perry, M Khalid and R J Scaramuzzi The Royal Veterinary College, Hatfield, UK

In the cervix of the ewe at oestrus, there is a degree of natural relaxation mediated by locally synthesised cervical prostaglandins.

Oxytocin, binding to its receptor (OTR), may stimulate the synthesis of prostaglandin E2 (PGE2) through the activation of cytosolic phos- pholipase A (cPLA), mobilizing the substrate arachidonic acid (AA),

COX-2 in the cervix of ewes during the oestrous cycle. Cervical tissue was collected from 25 Welsh ewes at three stages of the oestrous cycle:

on day 8 (n = 8; Luteal), day 0 (n = 8; pre-LH surge), day 1 (n = 9;

post-LH surge). The expression of OTR, cPLA2 and COX-2 was analysed by Western Blotting. The data were analysed by mixed model

ANOVA. The expression of the three proteins was low during the luteal phase but increased during the pre-LH surge stage for COX-2 (p < 0.05) and later in the post-LH surge stage, for OTR and cPLA2

(both p < 0.05). There was an increase in cPLA2expression from the pre-LH surge to the post-LH surge stages (p < 0.05) but this pattern was not observed for OTR and COX-2. It is hypothesised that during oestrus oxytocin through its receptor stimulates cervical synthesis of PGE2 increasing cPLA2 and the release of AA substrate from membrane phospholipids for conversion to PGE2by COX-2, leading to cervical relaxation.

Abstract P27

TNFa and IFNc Receptors Presence in the Equine Corpus Luteum and their Ligands Effect on Luteal Cells Viability

A Galva˜o¹, D Skarzynski², K Lukasik², D Ramilo¹, A Tramontano³, A Mollo³, L Mateus¹and G Ferreira-Dias¹

1C.I.I.S.A., Faculty of Veterinary Medicine, TULisbon, Portugal;

2Institute of Animal Reproductive and Food Research of PAS, Olsztyn, Poland;³Faculty of Veterinary Medicine, Padua, Italy

The aim of the present study was to show the presence and expression pattern of the receptors TNFRI, TNFRII and IFNRI on equine CL and the effect of their ligands on equine luteal cells viability, across the luteal phase. Samples from different luteal phases (early CL, n = 5;

mid-CL, n = 5; late CL, n = 5) were used in immunohistochemistry and real time PCR for qualitative and quantitative receptors expres- sion assessment. To understand the role of cytokines on cell viability, isolated luteal cells from the same luteal phases (n = 5 mares/phase) were plated in 96-well cell culture plates and stimulated for 24 h with:

(i) no exogenous factor (Control); (ii) TNFa (10 ng/ml); (iii) TNFa+INFc(10 ng/ml) and (vi) INFc(10 ng/ml). All receptors were present in large luteal cells and endothelial cells. Increased staining for TNFRII was observed in mid-CL, comparing with early-CL (p < 0.05) and late-CL (p < 0.001). TNFRI and IFNR1 stained equally for all luteal structures. Nevertheless, gene expression for TNFRI, TNFRII and IFNRI was higher in early-CL (p < 0.05).

Concerningin vitroTNFaand INFceffects on luteal cells, there was a reduction on cell viability during late-CL for isolated stimulations (p < 0.05), with an evident synergy between both cytokines (p < 0.001). These results suggest that cytokines might be involved in luteal regression in the mare.

Abstract P29

The Effect of Cytokines on Equine Luteal Production of Nitric Oxide and Angiogenic Activity and Luteal Cells Viability

A Galva˜o¹, D Skarzynski², K Lukasik², L Valente¹, D Ramilo¹, L Mateus¹and G Ferreira-Dias¹

1C.I.I.S.A., Faculty of Veterinary Medicine, TULisbon, Portugal;

2Institute of Animal Reproductive and Food Research of PAS, Olsztyn, Poland

The aim of this study was to evaluate the effect of different cytokines (FasL, TNFa and IFNc) on Nitric Oxide (NO) production and angiogenic factor(s) that can stimulate in vitro endothelial cell proliferation (BAEC), by equine luteal cells. Also, the influence of these factors on luteal cell viability was assessed. Luteal cells were isolated from mid luteal phase CL (Mid-CL; n = 6), in order to understand how luteal maintenance and regression are determined.

After dose optimization (FasL at 1; 10 and 50 ng/ml), luteal cells were exposed for 24 h to: (i) media without cytokines – control; or with (ii) FasL (10 ng/ml), (iii) FasL+IFNc(10 ng/ml), (iv) FasL+IFNc+TN-

Fa(10 ng/ml). The cytokine FasL (10 ng/ml) significantly reduced NO output (p < 0.05), while combination of all studied cytokines increased it (p < 0.05). Although FasL (10 ng/ml) and FasL+IFNc (10 ng/ml) stimulated endothelial cell proliferation (p < 0.05), the combination of FasL+IFNc+TNFa (10 ng/ml) had the opposite effect (p < 0.05). Luteal cell viability was reduced under the influence of FasL+IFNc (p < 0.001) and the association of all cytokines (p < 0.01). An increase in cytokines FasL+IFNc+TNFa Nitric Oxide mediated production may be responsible for inhibition of endothelial cells proliferation and luteal cells viability. These data suggest that, in mid-CL cytokines may play a role on luteal regression in the mare.

Abstract P31

Expression of Cytokines and their Receptors in the Equine Endometrium during the Estrous Cycle

A Galva˜o¹, D Ramilo¹, L Mateus¹, A Szo´stek², D Skarzynski²and G Ferreira-Dias¹

1C.I.I.S.A., Faculty of Veterinary Medicine, Technical University of Lisbon, Portugal;²Institute of Animal Reproductive and Food Research of PAS, Olsztyn, Poland

In the endometrium, locally produced cytokines play a physiologic role on uterine activity throughout the estrous cycle. The aim of this study was to (i) identify cytokine receptor sites and (ii) quantify the expression of the same cytokines (tumor necrosis factor – TNFa, interferon-gamma – IFNc, Fas Ligand – FasL) and their receptors (TNFa-R1 and R2; IFNc-R1; Fas) in the mare¢s endometrium throughout the estrous cycle. Tissue was obtained from 20 cyclic mares on the follicular phase (FP, n = 5), and early (Early-LP, n = 5), mid (Mid-LP; n = 5) and late luteal phases (Late-LP, n = 5).

Cytokine receptor localization was done by immunohistochemistry, while relative quantification of gene expression for cytokines and their receptors was accomplished by Real Time PCR. In all phases, cytokines receptors were always present in epithelia and glandular cells. However, Fas stain was either slight or absent. TNFaexpression decreased in the Late-LP (p < 0.05), while its receptor increased in Mid-LP (p < 0.05). IFNc increased in Late-LP (p < 0.05), but its receptor expression did not change. FasL expression was the lowest in the FP and Mid-LP (p < 0.05) and Fas was constant throughout the cycle. Cytokine and their receptors expression changes in the mare’s endometrium during the estrous cycle suggest an important (auto/

paracrine) role of these cytokines in the regulation of endometrial function, mainly both at epithelial cells and endometrial glands locations.

Abstract P33

Dynamics of Boar Sperm Morphology during Co-incubation with Epididymal Explants

E Garcia-Bonavila, M Yeste, S Bonet, E Pinart, S Sancho, E Bussalleu, I Casas, A Fa`brega and M Briz

Biotechnology of Animal and Human Reproduction, Department of Biology-INTEA, University of Girona, Spain

Previous reports have shown that co-culturing of epididymal sperma- tozoa with epididymal cell monolayers induces the migration of cytoplasmic droplet in boars. The present study sought to determine the effects of co-incubating ejaculated spermatozoa of boars with epididymal explants, using LLC-PK1 cell monolayers and media without explants as positive and negative controls, respectively.

Epididymides were processed separating into caput, corpus and cauda, with 200 fragments per region in each co-incubation. Sperm samples were obtained from Pie´train boars presenting a high percentage of cytoplasmic droplets, the co-incubation media being Tyrode’s medium with BSA and EGTA. After 2, 4, 6 and 24 h of co-incubation, sperm motility, morphology, and viability, and acrosome and mitochondrial sheath integrity were assessed. Data were analysed with a repeated measures ANOVA and a post-hoc Scheffe´’s test. After 24 h of

increased in caput (61.5 ± 3.3) and corpus (66.4 ± 3.5) treatments, but neither in cauda, negative nor positive controls, when comparing with negative control at 0 h (52.6 ± 2.6). Moreover, a significant decrease in the percentage of sperm cells with proximal cytoplasm droplets was also observed in caput and corpus, but not in the other treatments at the same time point. In conclusion, epididymal explants from caput and corpus can induce the migration of cytoplasmic droplet after co-incubation with boar spermatozoa.

Abstract P35

Modulation of Telomerase Activity in Bovine Embryos using Cytoplasmatic Plasmid Injection

W Garrels, WA Kues, U Baulain and H Niemann

Institute of Farm Animal Genetics, Friedrich Loeffler Institute, Federal Research Institute of Animal Health, Mariensee, Germany

Telomeres are repetitive, non-coding sequences at the end of chromo- somes that shorten with each cell division. They play an important role in ageing and affect the regeneration capacity of cells. The holoenzyme telomerase, composed of two components, telomerase reverse trans- criptase and telomerase RNA component (TERC), rebuilds telomeres.

In the present study the TERC component of human telomerase was expressed in bovine embryos. DNA expression constructs encoding human TERC and GFP were injected into a total of 528 bovine zygotes. GFP fluorescence was used to identify successfully injected embryos. We obtained 45 blastocysts with green fluorescence. Injected and control embryos were culturedin vitroup to the blastocyst stage and the impact on early embryonic development were assayed.

Blastocysts were either used for PCR analysis to examine TERC expression or for qFISH to monitor telomere length. Control groups were analysed for the endogenous levels of bovine TERC expression.

The results proofed, that human TERC could be expressed in blastocysts after cytoplasmic injection of plasmids into bovine zygotes.

Control groups showed endogenous TERC level to be upregulated in morulae and blastocysts. The expression of the hTERC component caused a significant extension of telomere length in early embryonic stages. The ectopic expression of telomerase components in bovine embryos may pave new approaches for generating stem cells and for the development of novel regenerative therapies.

Abstract P37

Hoechst 33342 Staining Conditions Affect the Efficiency of Sex-sorting Stallion Sperm

Z Gibb¹, LHA Morris², WMC Maxwell¹, G Evans¹and CG Grupen¹

1Faculty of Veterinary Science, The University of Sydney, Sydney, Australia;²EquiBreed NZ Ltd., Cambridge, New Zealand

One of the factors limiting the efficiency of sex-sorting stallion sperm is the use of an opaque diluent (Kenney’s Modified Tyrode’s diluent;

KMT) during preparation of sperm for sorting. In previous studies, sperm have been stained with Hoechst 33342 (H33342) using a wide range of incubation times (30–90 min) and H33342 doses (0.045–

0.09 mM). The aim of this study was to develop an optimal protocol for the staining of stallion sperm with H33342 before sex-sorting.

Three ejaculates from each of three pony stallions were collected and split into six aliquots. The aliquots were diluted in a Modified Tyrode’s diluent containing skim milk (SM-KMT) or 1% bovine serum albumin (BSA-KMT, a clear diluent) and stained with 0.045, 0.0675 or 0.09 mM H33342. Sperm were assessed for resolution into X- and Y-chromosome bearing populations (split efficiency; SE), percentage unstained (US) and motility characteristics (using computer assisted sperm analysis) after 30, 45, 60 and 90 min of incubation at 34C.

Incubation in BSA-KMT resulted in a significantly higher SE and lower US compared to SM-KMT (SE 0.17 vs 0.08, US of approx 32 vs 41%). The highest SE and lowest US were observed after 45 min of

53.5%), the use of a clear diluent allowed a significant reduction in staining time, improved resolution and reduced the percentage of unstained sperm. The findings highlight how relatively small differ- ences in H33342 staining procedures dramatically influence the efficiency of stallion sperm sex-sorting. SM-KMT was superior to BSA-KMT in the maintenance of sperm motility, but BSA-KMT allowed stallion sperm to be sorted more effectively than SM-KMT.

Abstract P39

Impact of Storage on Bicarbonate-induced Motility in Liquid-preserved Boar Semen

H Henning¹, AM Petrunkina^1,2, RAP Harrison³and D Waberski¹

1University of Veterinary Medicine, Hannover, Germany;²University of Cambridge UK;³Great Shelford, Cambridge, UK

Stimulation of boar sperm motility by bicarbonate combined with cluster analysis was used to investigate whether the response of sperm subpopulations is altered by storage length. Three normospermic ejaculates each of seven boars were diluted in BTS and stored at 17C.

After 12, 24, and 72 h samples were washed through Percoll and incubated at 38C in parallel in Ca²⁺-free variants of a Tyrode¢s medium with bicarbonate (at 5% CO2, Tyrbic) or without (Tyrcontrol).

After 10 min, motility parameters were assessed with a CASA-system and membrane integrity was analysed on a flow cytometer. Although membrane integrity of the washed samples was always >82%, total motility in Tyrbicdeclined from 81.5% at 12 h to 43.5% at 72 h of storage (p < 0.001). Within the motile population of spermatozoa, five groups of spermatozoa with different motility characteristics were identified. The distribution of spermatozoa between these groups differed between Tyrbicand Tyrcontrolat any given time (p < 0.001).

Regardless of storage, bicarbonate generally induced comparable increases in a ‘fast linear’ moving sperm population in Tyrbic(means of 66.4% at 12 h and 63.9% at 72 h); however, in one boar, storage resulted in a marked reduction in inducability of the ‘fast linear’ sperm population from 71.8% at 12 h to 49.3% at 24 h and 44.0% at 72 h;

this was not accompanied by a decline in standard semen parameters.

In conclusion, responsiveness to bicarbonate in the motile sperm population is maintained during semen storage in a boar-specific manner.

Abstract P41

The Effect of Age at First Calving on Subsequent Production in Dairy Cattle

M Hostens, T Caluwaerts, B Van Ranst and G Opsomer Faculty of Veterinary Medicine, Ghent, Belgium

Data collection and interpretation has become an important tool in modern dairy industry. In a preliminary study using a recently automated tool developed at the Ghent University in cooperation with a herd health management software company (Uniform-Agri, Assen, The Netherlands) to monitor parameters for milk production and fertility, the effect of age at first calving on subsequent production was analyzed. Data concerning dairy heifers calving between 1990 and 2005 were collected from 994 herds situated in Belgium (n = 5), Denmark (n = 1), The Netherlands (n = 976) and The UK (n = 12). All heifers (n = 309930) were allocated to a specific class according to their age at first calving: younger (Y) (<700 days, n = 10917), normal (N) (700–760 days, n = 108149) and older (O) (>760 days, n = 190864). The O heifers had the highest (p < 0.001) total milk production during their first lactation (7940 ± 3269 kg) followed by the N (7523 ± 2729 kg) and the Y heifers (7308 ± 2931 kg). Heifers that started their first lactation around the generally advised age of 24 months (N) calved on average 3.54 ± 1.91 times which significantly differed from both the O (3.29 ± 1.86) and the Y (3.46 ± 1.88) heifers (p < 0.001). The cumulative 305 day milk production during their

had a higher cumulative 305 day milk production than the O heifers (25689 ± 16629 kg) (p < 0.001). These preliminary results open perspectives to analyze the effect of age at first calving on subsequent production in more detail.

Abstract P43

The Effect of Marine Algae Supplementation in High Yielding Dairy Cows during Transition on Metabolic Parameters in the Serum and Follicular Fluid Early Post-partum

M Hostens¹, B Vlaeminck², V Fievez²and G Opsomer¹

1Department of Obstetrics, Reproduction and Herd Health, Faculty of Veterinary Medicine, Ghent, Belgium;²Laboratory for Animal Nutrition and Animal Product Quality, Faculty of Bioscience Engineering, Melle, Belgium

Sixteen Holstein cows were assigned to 2 groups to evaluate the effect of feeding marine algae (ALG) during early lactation on milk production characteristics and the profiles of metabolic parameters non-esterified fatty acids, b-hydroxy-butyrate (BHB), glucose, urea andc-glutamyl transferase (GGT) in the serum and/or follicular fluid.

All cows received a corn- and grass silage-based Total Mixed Ration (TMR). In the diet of the ALG group, 2 kg of the balanced concentrate was replaced by an iso-energetic concentrate containing ALG (11% on product basis). ALG increased milk yield (p = 0.057), whereas milk fat yield (p = 0.005) and milk fat content (p = 0.011) decreased. Protein yield (p = 0.465) was not affected whereas a tendency for reduced milk protein content (p = 0.094) was observed.

Serum urea levels were higher in the ALG group (p = 0.02) next to a tendency for increased BHB and GGT and decreased glucose levels.

Follicular fluid BHB was significantly increased in the ALG group (p = 0.005). Other indicators for a negative energy balance in follicular fluid did not differ between groups. These findings indicate that supplementation with poly unsaturated fatty acids during transition in high yielding dairy cows, can lead to a decrease in milk fat production and an increase in milk production, which may worsen the cows’ energy status as measured in the serum as well as in the follicular fluid.

Abstract P45

Origin of ZP1 and ZP4 in Golden Hamster (Mesocricetus auratus) Ovaries

MJ Izquierdo-Rico¹, L Gimeno², C Jime´nez-Cervantes³and M Avile´s¹

1Department of Cell Biology and Histology, School of Medicine, University of Murcia, Spain;²Department of Immunology, Virgen de la Arrixaca Hospital, Murcia, Spain;³Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Spain The zona pellucida (ZP) surrounds mammalian oocytes and plays a key role in the gamete interaction. Recently, our group has described the presence of ZP1 and ZP4 proteins in hamster ZP. Addressing the unresolved issue of the origin of ZP, we assessed the expression of ZP1 and ZP4 by means of in situ hybridization (ISH). Female hamsters (n = 3) obtained at random stages of their oestrous cycles were sacrified and ovaries (n = 6) were processed for ISH on 5lm- paraffin sections. ZP1 and ZP4 sequences used as ISH probes were amplified from ovaries total mRNA using RT-PCR method. Antisense and sense ZP1 and ZP4 digoxigenin (DIG)-UTP labelled probes were generated and sections were hybridized with them. Probe detection was accomplished using an anti-DIG alkaline phosphatase-conjugated antibody and the staining was observed under the microscope. The images showed a specific staining only in the oocyte. Particularly, primordial and small primary follicles revealed strong labeling. In addition, ZP signal decreased in the more mature ovarian follicles.

Accordingly, the signal was very weak or absent in the later developmental stage of ovarian follicles (Graafian follicle). On the other hand,ZP1andZP4gene expression was absent in granulosa cell population. Results of this study revealed that the expression ofZP1 andZP4is exclusively restricted to the oocyte.

Abstract P47

Developmental Competence of Bovine Oocytes is Subject to their Follicular Environment

D Janowski¹, D Tesfaye², H Alm¹and H Torner¹

1Research Institute for the Biology of Farm Animals, Dummerstorf, Germany;²Institute of Animal Science, University of Bonn, Germany The aim of the study was to investigate whether oocytes with a different developmental competence show differences concerning the degree of apoptosis in their granulosa cells (GC) or in the gene expression pattern of their follicular environment [GC and cumulus cells (CC)]. It has been hypothesized that there is a correlation between the appearance of light atretic GC in the follicle and an increased developmental competence of the oocyte. Single aspiration of follicles (3–5 mm) allowed identification of the follicular origin from oocytes.

Oocytes were grouped for their developmental competence with regard to glucose-6-phosphate dehydrogenase (G6PDH) activity indicated by brilliant cresyl blue (BCB) staining. The blastocyst rate at day 8 after IVF was significantly higher in the BCB⁺group (19.3%) than in the BCB^- group (7.4%). Apoptosis in GC was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). The degree of apoptosis in GC from BCB⁺oocytes was increased in contrast to the BCB group (17.0% vs 11.0%; p < 0.05).

Gene expression profiles analysed by cDNA microarrays showed various differences in transcript enrichment between GC and CC, especially in the BCB group for catenin (cytoskeleton) and neurotrimin (cellular adhesion). In conclusion the results of the study demonstrate that the developmental competence of oocytes is related to the apoptotic level and the gene expression pattern of their follicular cell environment.

Abstract P49

Fast Cooling Rates from 17 to 5C do not Impair Frozen-thawed Boar Sperm Quality

JD Juarez, M Herna´ndez, JM Va´zquez, EA Martı´nez and J Roca Veterinary Science, University of Murcia, Spain

Frozen boar sperm requires a long cooling period (2–3 h) from 17C to 5C. In order to shorten the cooling period, we evaluated the effects of 4 different cooling rates from 17 to 5C on semen quality. Five centrifuged pooled sperm rich fractions (17C) from 4 boars were diluted in Tris-egg yolk, divided in 4 aliquots and cooled to 5C at 12.5C/min (usual) or at 7.5, 2.5 and 1.5C/min. After glycerol addition at 5C, the aliquots were packaged in 0.5-straws and frozen at 40C/min. Sperm quality, assessed according to the percentages of motile (computer-assisted sperm analysis) and viable spermatozoa (plasma membrane and acrosome integrity cytometrically assessed by a triple fluorescent stain), was recorded in 5 steps during the cryopres- ervation process: in the semen pool before and after centrifugation, and in the 4 aliquots at 5C and after thawing and holding at 37C for 30 and 150 min. Data were analyzed using a mixed modelANOVAwith cooling rate and cryopreservation step as fixed effects. Interaction cooling rate x cryopreservation step was not significant (p > 0.05).

The cryopreservation step influences (p < 0.01) sperm motility and viability. However, the percentage of motile and viable sperm did not differ (p > 0.05) among cooling rates in any of the 5 cryopreservation steps. In conclusion, the freezing process for boar sperm can be successfully shortened by using fast cooling rates from 17 to 5C.

(Founded by AGL2005-00760/GAN, Madrid and GERM (04543/07), Murcia, Spain)

Comparing of Response to Ovsynch Protocol in Ovular and Anovular Lactating Dairy Cows

E Karakaya¹, G Yilmazbas¹, A Keskin¹, V Mutis²and A Gumen¹

1Faculty of Veterinary Medicine, Bursa, Turkey;²TARFAS, Bursa, Turkey

The Ovsynch protocol is a timed artifical insemination program using GnRH and PGF2ain which ovulation was synchronized within an 8-h period. Previous studies showed that the Ovsynch protocol synchronized ovulation in both ovular and anovular cows but pregnancy rate (PR) was lower in anovular than ovular cows. The aims of this study were (1) to detect the maximum follicle size in anovular cows, and (2) to compare response to Ovsynch protocol between ovular and anovular cows. GnRH (100 mcg, i.m., Buserelin acetate) and PGF2a(500 mcg, i.m., Cloprostenol) were used in an Ovsynch protocol (GnRH-7d-PGF2a-56h-GnRH-18h-AI). Cows were examined twice, 7 d apart, to determine cyclicity by ultrasonography.

Cows (n = 253) were divided into two groups, ovular (presence of CL, n = 182; mean DIM 122) or anovular (no CL, n = 71; mean DIM 121). More anovular cows (73%, 52/71) had large follicles (greater than 15 mm in diameter) and 27% of cows had smaller follicles (9–

14 mm in diameter). Maximal follicle size at AI was similar between ovular (15.9 ± 2.2 mm) and anovular cows (15.5 ± 2.4 mm). Ovula- tion rate to first GnRH of Ovsynch was 57% (104/182) in ovular and 97% (69/71) in anovular cows. Synchronization rate (ovulatory response to second GnRH injection) was similar in ovular (88%, 161/182) and anovular (83%, 59/71) cows. The PR was similar in ovular (39.5%, 72/182) and anovular (30%, 22/71) cows. Thus, PR in anovular cows was similar to ovular cows, in contrast to previous studies.

Abstract P53

Follicular Responses of the Right vs Left Ovaries to eCG Treatment in Shall Iranian Ewes

H Kermani moakhar and H Kohram

Department of Animal Science, Faculty College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

The present study considers right vs left ovarian responses to different doses of eCG treatment in the ewe. Estrous cycles of 50 adult Shall Iranian ewes were synchronized whit a CIDR during the breeding period. At the time of CIDR removal, 450 (n = 10), 550 (n = 10), 650 (n = 10), 750 (n = 10) and 850 (n = 10) IU of eCG (Intervet, Boxmeer, Netherlands) was administered to all ewes. The number of medium and large follicles (‡3 mm, LF) in the right and left ovaries was monitored by transrectal ultrasonography on the day of estrus. All data were analyzed by GLM procedure of SAS. The mean number of LF on the right (2.78 ± 0.14) was higher (p < 0.001) in comparison to the left (1.72 ± 0.14) ovaries in all treatment groups combined.

Also, the mean number of LF in the right was higher (p < 0.05) than that of the left within each treatment group. For subsequent statistical analysis, all ewes (n = 50) were divided into 3 groups. Following superovulation, group A ewes had at least one extra follicle in the right ovary than that of the left at estrus. Group B had the opposite position and group C had equal LF in the ovaries. There were more ewes in group A (n = 32; 64%; p < 0.001) than group B (n = 7, 14%) and group C (n = 11, 22%). Thus, all doses of eCG resulted in a greater number of LF (‡3 mm) in the right ovaries of Shall ewes at estrus.

Factors Influencing Dystocia in the Double Muscled Belgian Blue Breed

I Kolkman^1,2, G Hoflack², D Lips¹and G Opsomer²

1Department Agro- and Biotechnology, KaHo Sint-Lieven, Sint Niklaas, Belgium;²Faculty of Veterinary Medicine, Ghent University, Depart- ment of Reproduction, Obstetrics and Herd Health, Merelbeke, Belgium In order to identify selection parameters towards a natural calving population within the double muscled Belgian Blue (BB) breed, the cow’s pelvic height (PH) and the calf’s hip width (HW), as well as the two principal determinants of feto-pelvic disproportion, were deter- mined and compared. In some cases, although theoretically impossible (PH–HW = -2.4 cm on average), calves were delivered naturally.

Hypotheses to clarify these rare cases were sought in adaptations in pelvic dimensions around parturition (PH increased 1.4 cm;

p < 0.001) and the compressibility of the calf (on average 10% which represents up to 2.9 cm). These two factors combined can explain almost all the cases (1.4 + 2.9 > 2.4 cm on average). To investigate another hypothesis, the influence of the shape of the pelvic inlet, comparison was made between the PH and the pelvic diagonal height (left oblique diameter [ODL] and right oblique diameter [ODR]) in 87 BB cows measured by a Rice pelvimeter. Overall, a significant difference was shown between PH and ODL and ODR respectively (PH – ODL: -0.43 cm; PH – ODR: -0.45 cm; p < 0.001), but no dissimilarity was noticed between the ODL and ODR. In individual cows this can be a world of difference in the decision for a natural delivery. The elliptic shape of the pelvic inlet might also have an influence on calving ability and can be a possible explanation for these rare individual cases where the calf is born naturally even though this is theoretically impossible.

Abstract P57

Trans-10 cis-12 Conjugated Linoleic Acid (CLA) reduces Fatty Acid (FA) Content of Bovine Matured Oocytes

M Lapa¹, CC Marques¹, SP Alves¹, MI Vasques¹, MC Baptista¹, I Carvalhais², AEM Horta¹, RJB Bessa³and RM Pereira¹

1INRB Santare´m;²Ac¸ores University;³FMV Lisboa, Portugal Our previous results showed that CLA decreases in vitro embryo cytoplasmic lipid deposition, improving blastocysts cryosurvival.

However, this action has never been tested in oocytes. We aimed to examine CLA effects on oocyte maturation, lipid profile and cryosur- vival. Abattoir-derived oocytes were matured in TCM199 + 10%

serum + 10lg/mL FSH + 100lM GSH supplemented or not with 100lM CLA during 22–24 h. Matured oocytes (MO) were vitrified (MO n = 313, MOCLA n = 321, 7 sessions). Denuded oocytes were subjected either to FA analysis (immature-IO n = 343, MO n = 348, MOCLA n = 341) or maturation rate evaluation (nuclear staining, MO n = 116, MOCLA n = 116). FA profiles in IO and MO, cumulus cells (CC) and culture media (CM) suplemented or not with CLA (4 samples each) were analysed. FA was determined by gas chromatography. Data were analyzed using ANOVA /LSD. Oocyte maturation or cryosurvival was not affected by CLA. Total FA (TFA, lg/oocyte) of IO and MO were higher (p < 0.04) than in MOCLA although FA profiles were similar. Total polyunsaturated FA (PUFA) were superior (p < 0.001) in CM than in oocytes or CC independently of CLA presence or oocyte maturation status. Differences (p£0.004) on PUFA, C18:2n-6, C18:3n-3, C20:3n-6 and C20:5n-3 were identified among groups. Results suggest that CLA did not influence oocyte maturation or cryosurvival. However, CLA interfered on lipid metabolism, reducing oocyte TFA during maturation without modi- fying FA profile, independently of CM composition.

Abstract P59

Use of Ascorbic Acid for Freezing of Canine Semen of Fertile and Sub-fertile Dogs

BV Lopes, VH Chirinea, AAP Derussi, GHM Araujo, F Arduı´no and MD Lopes

Veterinary Faculty, UNESP-BOTUCATU, Sa˜o Paulo, Brazil Spermatozoa are susceptible to Reactive Oxygen Species (ROS) damage because their membrane has high concentration of polyun- saturated fatty acid. The use of antioxidants could diminish this negative effect of ROS. The aim of this study was to evaluate post- thaw quality of frozen dog semen processed with diluents containing different concentrations of ascorbic acid (AA). After thawing, sperm motility and membrane integrity were evaluated. Semen samples were collected using manual stimulation, from 3 dogs with low sperm count (>20·10⁶sperm/ml) and 3 with normal sperm count. The samples were divided in four aliquots, which were processed for cryopreserva- tion. After centrifugation, the sperm pellets were diluted to a final concentration of 30·10⁶ spermatozoa /ml with TRIS-fructose-egg yolk extender containing: no AA (control group) and AA concentra- tions of 1.0, 1.5 or 3.0 mM. Spermatozoa motility was evaluated using CASA (HTM – IVOS 12, Hamilton Thorne Research, Beverly, MA) and membrane integrity using propidium iodide and CFDA. Results were analyzed using ANOVA and tukey’s test. In dogs with normal concentration of spermatozoa no statistical difference (p > 0.05) was observed between groups. In dogs with oligozoospermy, membrane integrity was better (p = 0.0004) in the group with 1.0 mM AA in comparison with control group, although they had lower motility. It can be concluded that the use of AA at tested concentrations, had no positive effect on frozen/thawed semen from dogs with normospermy.

Contrarily, in dogs with oligozoospermy a membrane protection effect can be seen, when a concentration of 1.0 mM AA was used. We believe that AA protected sperm from lipid peroxidation, especially in dogs with fertility problems, probably because they are more susceptive to lipid peroxidation.

Abstract P61

Integrin Receptors on Canine Peripheral and Endometrial Lymphocytes

P Moretti¹, D Groppetti², M Aralla¹, ME Gelain³, S Comazzi³, F Cremonesi²and S Arrighi¹

1VSA;²SCV;³DIPAV Departments, Faculty of Veterinary Medicine, Milan, Italy

The role of immune cells in the endometrium is a matter of considerable interest in recent years. Integrin (ITG) adhesion mole- cules play a critical role in peripheral blood lymphocyte (PBL) migration and their recruitment into the endometrium. Immunoendo- crine functions of endometrial stromal cells were reported to vary in fertile vs infertile women. It was hypothesized that the expression of a4 andb1-ITG subunits on PBL might be used as clinical marker to assess endometrial receptivity. This work investigated the expression of a4-ITG subunit on PBL compared with endometrial ITG immunode- tection in healthy bitches during the different estrous phases (n = 12), pregnancy (n = 5) and in bitches affected by uterine diseases (Cystic Endometrial Hyperplasia, Hydrometra, Pyometra: n = 9). Bitches underwent clinical examination and careful monitoring of their reproductive status by vaginal cytology and P4 blood-assay. Uterine samples were taken during routine OHE and processed for immun- odetection of endometriala4-ITG subunit by canine CD49d antibody.

Comparison was made with flow cytometry on respective peripheral blood samples. Results showed a higher but non-significant ITG expression in the PBLs of the pathological group in comparison with healthy bitches, without correlation with endometrial immunodetec- tion. In pregnant bitches ITG expression significantly increased in the peripartum, both in PBLs and endometrial cells. It can be hypothe- sized thata4-ITG plays a key role both in uterine diseases and during pregnancy, especially in the peripartum.

Abstract P63

Sperm Chromatin Fragmentation in Cryopreserved Semen of Brahman Bulls Measured with Toluidine Blue Stain

H Nava-Trujillo¹, A Quintero-Moreno¹, G Finol¹, J Rubio¹and M Hidalgo²

1Laboratorio de Andrologı´a, Facultad de Ciencias Veterinarias, Universidad del Zulia, Maracaibo, Venezuela;²Animal Reproduction Group, University of Cordoba, Spain

Sperm chromatin fragmentation (SCF) is an important feature that can affect the reproductive potential of bulls. Toluidine blue (TB) is a nuclear dye used to evaluate sperm chromatin by detecting the absence or rupture of disulfide bonds; thus, stained nuclei indicate altered sperm chromatin. The objectives of this study were: (1) determinate the percent of SCF in cryopreserved semen of Brahman bulls, (2) correlate SCF results with sperm viability (SV) and sperm motility (SM). Three ejaculates from 6 Brahman bulls were evaluated after thawing. The SCF was evaluated on semen smears stained with TB (sperm with normal chromatin, stained light blue or green; sperm with SCF, stained dark blue or violet). SV was determined by eosin-nigrosin stain and SM was measured under light microscopy. Cryopreserved semen of Brahman bulls had a low SCF (4.17 ± 0.40%) and showed a negative correlation with SM (r= -0.48337, p = 0.0421) and SV (r= -0.43104, p = 0.0087). TB is a practical and economic dye to study the SCF in bull spermatozoa. However, the methods on slides have the inherent limits of repeatibility dictated by dye equilibrium variations and by a limited number of cells, which can be reasonably scored. Further studies to compare TB with other SCF methods are necessary.

Abstract P65

Lipid Peroxidation, Assessed with BODIPY-C11, Increases after Cryopreservation of Stallion Spermatozoa, is Stallion-dependent and is Related to Apoptotic-like Changes

C Ortega Ferrusola¹, L Gonza´lez Ferna´ndez², JM Morrell³, C Salazar Sandoval¹, B Macı´as Garcı´a¹, H Rodrı´guez-Martinez³, JA Tapia²and FJ Pen˜a¹

1Veterinary Teaching Hospital, Laboratory of Spermatology, and²Cell Physiology Research Group, University of Extremadura, Ca´ceres, Spain;³Division of Reproduction, Faculty of Veterinary Medicine and Animal Sciences, SLU, Uppsala, Sweden

Lipid peroxidation (LPO) of stallion spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in susceptibility to LPO and its possible relationship with freezeability.

Innate levels of LPO were very low in fresh spermatozoa but increased after thawing, a change which was largely stallion-dependent. The level of lipid peroxidation in fresh spermatozoa was not correlated with that of thawed spermatozoa. Negative correlations existed between lipid peroxidation and intact membranes post-thaw (r= -0.789, p < 0.001), and also between lipid peroxidation and spermatozoa with high mitochondrial membrane potential post-thaw (r= -0.689 p < 0.001). LPO was also highly and significantly correlated with caspase activity. The correlation between caspase activity in ethidium positive cells and LPO wasr= 0.772, p < 0.001. LPO is unlikely to represent,per se, a sign of cryopreservation-induced injury, but it is apparently capable of triggering ‘apoptotic-like changes’ that could result in the sub-lethal cryodamage often seen among surviving spermatozoa.

Inhibition of the Mitochondrial Permeability Transition Pore Reduces ‘Apoptosis like’ Changes during

Cryopreservation of Stallion Spermatozoa

C Ortega Ferrusola¹, L Gonza´lez Ferna´ndez², C Salazar Sandoval¹, B Macı´as Garcı´a¹, H Rodrı´guez Martı´nez³, JA Tapia²and FJ Pen˜a¹

1Veterinary Teaching Hospital, Laboratory of Spermatology, and

2Department of Physiology, Faculty of Veterinary Medicine, University of Extremadura, Ca´ceres, Spain;³Division of Reproduction, Faculty of Veterinary Medicine and Animal Sciences, SLU, Uppsala, Sweden In order to evaluate to what extent the changes that occur during cryopreservation involve the mitochondrial permeability transition pore (PT–pore), a specific inhibitor was used while cryopreserving stallion spermatozoa. Four ejaculates from 7 stallions were frozen using a standard protocol. Each ejaculate was split in three subsamples. The first one was supplemented with bongkrekic acid (BA, 2.5lM) and the second one with 5lM BA. The third subsample served as control. BA significantly reduced the percentage of spermatozoa depicting active caspases after thawing, reduced the percentage of spermatozoa with increased membrane integrity and increased the mitochondrial membrane potential of thawed stallion spermatozoa. Sperm motility was clearly reduced as a result of the treatment. It is concluded that the mitochondrial pathway of apoptosis seems important as a factor explaining the sub-lethal damage of equine spermatozoa after freezing and thawing. Also, the motility of spermatozoa in the equine species largely depends on mitochondrial ATP produced by oxidative phosphorylation.

Abstract P69

Susceptibility of Ejaculated Stallion Spermatozoa to Induced Oxidative Stress

C Ortega Ferrusola¹, A Johannisson², FJ Pen˜a¹, H Rodriguez- Martinez²and JM Morrell²

1Veterinary Teaching Hospital, Laboratory of Spermatology University of Extremadura, Ca´ceres, Spain;²Division of Reproduction, Faculty of Veterinary Medicine and Animal Sciences, SLU, Uppsala, Sweden The susceptibility of ejaculated stallion spermatozoa to lipid peroxida- tion (LPO) maintained in 3 different commercial extenders or in homologous seminal plasma (SP) was studied in 4 ejaculates of 3 stallions. Using SOFe²⁺as pro-oxidant, the susceptibility for LPO was flow-cyometrically assessed using Bodipy–C11Each ejaculate was split and aliquots extended with (i) INRA, Kenney´s or Equipro extenders, before a single layer centrifugation (SLC) was issued in Androcoll-E, where the selected spermatozoa were-suspended in the above mentioned extenders, without (ii) or with addition of 50%-SP (iii).

Sperm motility was monitored using a Qualisperm^TM motility analyser. While sperm motility varied among treatments and stallions and was best in the SLC-selected spermatozoa; the responses to induced-LPO (Bodipy –C11+-cells) were very low and variable (from 1.09±0.87% to 3.77±0.93%) among stallions, treatments and concentrations of SOFe²⁺. Removal of SP by the SLC procedure did not significantly increase the susceptibility of stallion spermatozoa to induced LPO, calling for further studies to unravel the roles of SP and extenders in preventing induction of LPO in equine semen.

(Funded by SSH, Stockholm and INIA, Spain).

Mitofusin2 and Actin Determination and Localization in Boar Spermatozoa during ‘In vitro Capacitation’ and Further Acrosome Reaction

L Ramio´-Lluch¹, D Bucci², JM Ferna´ndez-Novell³and JE Rodriguez-Gil¹

1Department Animal Medicine & Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, Bellaterra, Spain;

2Department Morfofisiologia Veterinaria e Produzioni Animali (DIMORFIPA), Universita` di Bologna, Ozzano Emilia, Bologna, Italia;³Department Biochemistry & Molecular Biology, University of Barcelona, Barcelona, Spain

The main aim of this study was the observation of changes in two sperm proteins that are relationated with both the spatial distribution and the functional control of mitochondria, actin and mitofusin2. For this purpose, boar sperm from fresh ejaculates were subjected to an

‘in vitro’capacitation (IVC) and further progesterone-induced, acro- some reaction (IVAR) by incubation at 39C in a 5% CO2atmosphere for 4 h in an specific capacitation medium. Sperm aliquots were taken at 0 h and 4 h of IVC and 5 min and 60 min after acrosome reaction.

Our results show the presence of mitofusin2 in sperm for the first time, both through Western blot and immunofluorescence analyses. Regard- ing the overall expression, actin did not undergo changes during all the incubation period. Mitofusin2 progressively diminished after IVAR.

Actin immunocytochemistry showed that a subequatorial band appeared after 4 h of IVC and after 60 min of IVAR the connecting piece showed a strong increase in fluorescence. Moreover, IVC and IVAR induced specific changes in the mitofusin2 location into the midpiece. We can conclude that IVC and IVAR produce changes in these two proteins, what would be related to the regulation of mitochondrial activity during these processes.

Abstract P73

Relationship between Follicle Size and Factors Secreted by Porcine Oocytes and Cumulus Cells during Maturation In vitro

R Romaguera¹and C Grupen²

1Faculty of Veterinary, Barcelona, Spain;²Faculty of Veterinary Science, University of Sydney, Camden, Australia

In gilts, the developmental potential of oocytes from small follicles is poor compared with those from large follicles. Supplementation of maturation medium with oocyte-secreted factors has been found to improve the developmental potential of bovine and murine oocytes.

The objective of this study was to examine the relationship between follicle size (small: 3–4 mm, large: 5–8 mm) and the factors secreted by oocytes and cumulus cells that may influence porcine oocyte matura- tion. Groups of 30 cumulus-oocyte complexes (COCs) from small follicles were transferred to 150ll droplets (experiment 1) or 500ll wells (experiment 2) of maturation medium with 75 denuded oocytes (DOs) or COCs from small or large follicles, respectively. After 44 h, oocytes were activated and transferred to droplets of Porcine Zygote-3 medium for 7 d. Each experiment was replicated at least 4 times. In experiment 1, there was no difference in cumulus expansion between the groups at 24 h or 44 h or in embryo development following treatment with or without DOs. In experiment 2, co-culture with COCs from large follicles increased the cumulus expansion index of small follicle-derived COCs (1.76 vs 2.45; p < 0.05), but did not affect embryo development. In conclusion, the effect of factors secreted by cumulus cells differs in COCs recovered from small and large follicles.

Under the co-culture conditions used in this study, oocyte secreted factors did not exert a beneficial effect on the developmental competence of porcine oocytes.