Journal für Reproduktionsmedizin und Endokrinologie – Journal of Reproductive Medicine and Endocrinology –

Andrologie • Embryologie & Biologie • Endokrinologie • Ethik & Recht • Genetik Gynäkologie • Kontrazeption • Psychosomatik • Reproduktionsmedizin • Urologie

50th Annual Conference of Physiology and Pathology of Reproduction and 42th Mutual Conference of Veterinary and Human Reproductive Medicine Munich, 15th–17th February, 2017 J. Reproduktionsmed. Endokrinol 2017; 14 (1), 24

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Offizielles Organ: AGRBM, BRZ, DVR, DGA, DGGEF, DGRM, D·I·R, EFA, OEGRM, SRBM/DGE

Indexed in EMBASE/Excerpta Medica/Scopus Krause & Pachernegg GmbH, Verlag für Medizin und Wirtschaft, A-3003 Gablitz FERRING-Symposium digitaler DVR 2021 Mission possible – personalisierte Medizin in der Reproduktionsmedizin Was kann die personalisierte Kinderwunschbehandlung in der Praxis leisten? Freuen Sie sich auf eine spannende Diskussion auf Basis aktueller Studiendaten. SAVE THE DATE 02.10.2021 Programm 12.30 – 13.20Uhr Chair: Prof. Dr. med. univ. Georg Griesinger, M.Sc.

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Wir freuen uns auf Sie! 50th Annual Conference of Physiology and Pathology of Reproduction and 42th Mutual Conference of Veterinary and Human Reproductive Medicine Munich, 15th–17th February, 2017 Zentrum für Translationale Reproduktionsmedizin, Ludwig-Maximilians-University, Munich Abstracts* DGRM-Abstracts 01 02 or after fetotomy represented risks for mater- nal recovery. The fertility rate after fetotomy New Serum Parameters for Detec- Fetotomy in Dromedary Camels: was generally encouraging. tion of Uterine Puerperal Distur- an Evaluation of 50 Cases bances in Dairy Cows A. Ali1,2, R. Derar1,2, F. Alsobayil1, A. Alsamri1 A. Abultdinova1, I. Dzakupov1, J. Roth2, A. Wehrend3, 1Department of Veterinary Medicine, College of Agri- 03 M. Sickinger3 culture and Veterinary Medicine, Qassim University, 1Department of Veterinary Medicine, Faculty of Ve- Qassim, Saudi Arabia; 2Department of Theriogenol- Effect of the Ovarian Superstimu- terinary Medicine and Technology of Animal Hus- ogy, Faculty of Veterinary Medicine, Assiut Universi- lation and Ovum Pick-up Pro- bandry, S. Seifullin Kazakh Agro Technical University, ty, Assiut, Egypt cedure on the Fertility of Donor Astana, Kazakhstan; 2Institute for Veterinary Physio- Heifers logy; 3Clinic for Obstetrics, Gynaecology and Andro- The objectives of this study were to investi- logy with Veterinary Ambulance, Justus Liebig Uni- gate the cases of camel dystocia in which fe- A. Ali1, M. Gilles2, A. Lange2, P. Klocke2, P.S. Glatzel2 versity Giessen, Germany totomy was indicated, the related complica- 1Department of Theriogenology, Faculty of Veterinary tions and the associated risks for maternal Medicine, Assiut University, Egypt; 2Clinic of Repro- Impairment of puerperal fertility may be a mortality and fertility. Fifty female drome- duction, Free University-Berlin, Germany severe problem in some dairy herds. With- dary camels were handled with fetotomy due in the last years, several diagnostic possibili- to uncorrectable dystocia. The complications The aim of this study was to assess the effect ties were developed and validated. The aim during and after fetotomy and post-operative of ovarian superstimulation and ovum pick- of this study was to evaluate the applicability fertility rates were recorded. Logistic regres- up on the fertility of donor heifers. Twen- of blood serum parameters substance P (SP) sion was performed to identify risk factors for ty Frisian heifers (12 treated and 10 control) and vasoactive intestinal polypeptide (VIP) the dependent variables of maternal mortali- were used in this study. The treated group re- for their use in early diagnostics of uterine ty and fertility after fetotomy. The independ- ceived 2000 IU eCG on day 10 and 25 mg involution disturbances. Blood serum sam- ent variables were parity, duration of dysto- PGF2 on day 12. All follicles > 9 mm were ples of 86 dairy cows taken within the fi rst cia, presence of foetal emphysema, number transvaginally aspirated on day 14. One week 20 days post partum were examined using of cuts, duration of fetotomy and occurrence after follicle aspiration, these heifers re- commercially available ELISA test kits. Ani- of complications during or after fetotomy. ceived 25 mg PGF2. Two days later, they mals were divided into two groups (healthy Common indications of fetotomy were head were artifi cially inseminated. Heifers of the or diseased) depending on the results of clin- and neck deviations alone or with carpal or control group were artifi cially inseminated ical and gynecological examination. Statis- shoulder fl exions (54%) and breech presen- on a PGF2-induced estrus. Pregnancy di- tical analysis consisted of the timely chang- tation (14%). Complications included tear- agnosis was performed using ultrasonogra- es in blood serum levels as well as the group ing of the soft tissue of the birth canal (24%), phy one month after insemination. Non-con- comparison of healthy cows and cows with uterine prolapse (6%) and retained placenta ceived animals were re-inseminated. The re- uterine disease. Blood serum concentrations (4%). Maternal mortality occurred in 26% of sults showed that the total number of folli- for SP increased statistically signifi cant with- the cases. A signifi cant association was de- cles > 9 mm in diameter existed on day 14 in the fi rst 20 days after calving (P < 0.04). tected between maternal deaths and dura- were 143 (14.3 follicles/animal). The num- There was no statistically signifi cant differ- tion of dystocia (odds ratio = 4.67, P = 0.03), ber of aspirated follicles were 122 (12.2 aspi- ence between the groups. Concerning VIP, presence of foetal emphysema (odds ratio = rated follicles/animal). The number of the re- neither for the timely course nor for the group 3.93, P = 0.04) and occurrence of complica- covered oocytes was 36 (3.6 oocytes/animal). comparison a signifi cant difference could be tions during or after fetotomy (odds ratio = The conception rates after the fi rst, second shown. SP represents an accredited biomark- 8.9, P = 0.003). Post-operative fertility was and third insemination were 60%, 80%, and er for pain in cattle. An increase of substance 62.2%. None of the estimated factors showed 90% in the treated group. The correspond- P within the fi rst 20 days post partum sug- a signifi cant relationship with post-operative ing rates in the control group were 70% and gests the presence of persisting pain presum- fertility. In conclusion, postural abnormali- 100% after the fi rst and second insemination. ably due to uterine involution processes. Nev- ties constituted the most common indication The difference between groups was not sig- ertheless, both serum parameters do not seem of fetotomy in dromedary camels. The dura- nifi cant. In conclusion, ovarian superstimu- suitable as indicators for the presence of uter- tion of dystocia, presence of foetal emphyse- lation and ovum pick-up procedure did not ine involution disorders. ma and occurrence of complications during negatively affect the fertility of donor heifers.

*Supporting Organisations: Deutsche Veterinärmedizinische Gesellschaft (DVG) and Deutsche Gesellschaft für Reproduktionsmedizin (DGRM) With permission of Wiley, the abstracts of this conference will be jointly published in the Journal of Reproduction of Domestic Animals (RDA) and the Journal of Reproductive Medicine and Endocrinology (JRE). Peer-reviewed and compiled by the scientifi c committee, Index of authors (only primary authors) see page 39.

2 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) For personal use only. Not to be reproduced without permission of Krause & Pachernegg GmbH. DGRM-Abstracts

04 CART is a recently described neuropeptide that treatment of spermatozoa with NAADP with anorectic activity, which appears to play resulted in a loss of the acrosomal vesicle. Vaginal and Cervical Tumors in an important role in the appetite regulation Furthermore, it was found that the NAADP Dromedary Camels and energy homeostasis. There is growing antagonist trans-Ned-19 signifi cantly reduc- evi dence that energy homeostasis factors such es acrosome reaction events in response to 1 1 1 1 F. Alsobayil , A. Ali , R. Derar , M. Tharwat , as leptin and ghrelin may play a role in re- the natural ligand Zona pellucida. Impor- 2 3 A. Fathy , M. Khodeir production and may be involved in pathogen- tantly, applying a broad concentration range 1 Department of Veterinary Medicine, College of esis of some reproductive disorders such as of NAADP two narrow bell-shaped dose-re- 2 Agriculture and Veterinary Medicine; Department endometriosis. Accordingly, the present study sponse-curves with maxima in the nanomo- of Pathology, College of Medicine, Qassim Universi- was aimed at investigation whether CART lar and low micromolar concentration range 3 ty, Saudi Arabia: Department of Surgery, Faculty of may be expressed in uterine tissues and whe- were registered. Moreover, quantifying loss Veterinary Medicine, Assiut University, Egypt ther this expression may be present in hetero- of the acrosomal vesicle in TPC1 null sperm Female dromedary camels (n = 1621) were topic endometrial tissues in the murine mod- upon application of different NAADP con- examined for failure of conception. The re- el of endometriosis. The study was performed centrations, responsiveness to low micromo- productive system was evaluated using trans- on 8-week-old inbred C57BL/6 female mice. lar NAADP concentrations was abolished. rectal palpation, ultrasonography and ex- Uterine fragments were transplanted into ab- Our study shows that NAADP mediates acro- ploration of the vagina. Tissue overgrowths, dominal wall of syngeneic recipients un- somal secretion in mouse spermatozoa with- 2+ which partially or completely surrounding der general anesthesia. Following 2, 4 and 8 out additional extracellular Ca , and that the vaginal lumen or the cervix were detect- weeks of observation the ectopic endometrial TPC1 is expressed in spermatozoa from mice DGRM-Abstracts ed in 8 cases (incidence, 0.005%). A com- foci were excised and processed for immuno- and humans. The fi nding that two convergent mon history of post-mating vaginal bleed- histochemical examinations. Tissues were NAADP-dependent pathways with non-over- ing of these females was noticed. The over- fi xed by immersion in 4% buffered formal- lapping activation and self-inactivation pro- grown tissue masses bled easily upon palpa- dehyde, and, following dehydratation, were fi les for distinct NAADP concentrations op- tion. All females were multipara and aged embedded in parafi n wax. The specifi city test erate in driving acrosomal exocytosis sup- between 9 and 13 years. Vaginal specimens performed for the CART antibody included: ports the concept that both NAADP-gated were taken for histopathology. Blood sam- negative control, where the antibodies were cascades match local NAADP concentrations ples were obtained for hematology and bio- replaced by normal rabbit serum (Vector La- with the effl ux of acrosomal calcium. chemistry. Microscopically, vaginal adeno- boratories; Burlingame, CA, USA) at the re- carcinoma (n = 5), vaginal leiomyoma (n = 2) spective dilution. Positive control was carried 07 and cervical adenocarcinoma (n = 1) were out for the specifi c tissue, as recommended by identifi ed. By ultrasound, these tumors were the producer (for our research we used mice Microscopical Investigations homogenous and echogenic, but sometimes stomach). Immunoperoxidase staining with on the Early Development of with multiple hypo-echogenic cavities. In CART-specifi c antibodies showed that this Binucleate Trophoblast Giant Cells peptide is constitutively expressed in endome- one case, metastasis was observed in the re- in the Bovine Placenta gional and mesenteric lymph nodes and liver. trial epithelial cells in normal eutopic uterus Compared to healthy controls (n = 15), cam- and heterotopic endometrioid cysts. This sug- J. Attiger, A. Boos, K. Klisch els with tumors showed signifi cant increases gests that CART may play a part in regulation Institute of Veterinary Anatomy, Vetsuisse-Faculty, of lymphocytes and monocytes and decreas- of murine reproductive system, however, the University of Zurich, Switzerland es in erythrocytes, hemoglobin and packed possible role of this neuropeptide in the repro- cell volume. Blood chemistry of camels with duction remains to be elucidated. Bovine binucleate trophoblast giant cells tumors revealed signifi cant decreases in the (TGCs) play an important role for the trans- total protein, albumin, calcium, phosphorus 06 port of fetal mediators into the maternal tis- and magnesium and increases in globulin and sue. Functional and morphological charac- alkaline phosphatase. The serum activity of Two-Pore Channel Protein 1 con- teristics of the TGCs are quite well known, creatine kinase, aspertate aminotransferase tributes to NAADP triggered Acro- but their origin is yet still discussed. Differ- ent authors hypothesized whether TGCs de- and gamma glutamyl transferase did not dif- some Reaction in Spermatozoa fer signifi cantly compared to controls. In velop from stem cells (SCs) within the tro- conclusion, this primary report described the E. Arlt1, L. Arndt1, A. Breit1, G. Wennemuth2, M. Biel3, phectoderm or whether they can arise from prevalence, types, gross and microscopic ap- C. Wahl3, T. Gudermann1, N. Klugbauer4, I. Boekhoff1 any uninucleate trophoblast cell (UTC). With- pearances and changes in the hemogram and 1Walther-Straub-Institute, Ludwig-Maximilians-Uni- in the latter, generally accepted theory, a ba- blood chemistry of female dromedary cam- versity, Munich; 2Institute for Anatomy, University of sally located, mononuclear cell without con- els affected with vaginal and cervical tumors. Duisburg-Essen; 3Department of Pharmacy, Ludwig- tact to the feto-maternal interface would rep- Further investigations are needed to identify Maximilians-University, Munich; 4Institute for Experi- resent a transient cell (TC) between UTC and the associated risk factors. mental and Clinical Pharmacology and Toxicology, TGC. So far no evidentiary images either for Albert-Ludwigs-University, Freiburg, Germany the existence of such TCs or for the presence of SCs have been shown. The aim of the pre- The sperm acrosome reaction, an all-or-none sented study is to illustrate basally located, 05 secretion process, mainly follows the prin- potential progenitor cells microscopically and ciples of the conserved process of Calcium- to defi ne the sequence of their development to Expression of Cocaine and Am- regulated exocytosis. However, the function- mature TGCs more detailed. Placentomal tis- phetamine Regulated Transcript al relationship between the formation of hun- sue of 6 pregnant cows in different gestation- (CART) in Eutopic and Ectopic En- dreds of fusion and the mobilization of cal- al stages has been examined for basally locat- dometrium in Mice cium from the acrosomal organelle has only ed, mononuclear cells and TGCs either in se- been partially defi ned. Hence, the second rial sections (light and transmission electron O. Aniołek1, I. Janiuk2,3, J. Olkowska-Truchanowicz3, messenger NAADP, promoting effl ux of cal- microscopy (TEM), n = 3), in single sections R. Maksym3, B. Ziarkiewicz-Wróblewska3, cium from lysosome-like compartments, (TEM, n = 2) or in automatic serial section- J. Malejczyk3, Z. Gajewski1 and one of its potential targets, the two-pore ing (serial block face scanning electron mi- 1Department of Large Animal Diseases with Clinic, channel TPC1, were analyzed for its func- croscopy (SBF-SEM, n = 1). These investiga- VRC and CBB, Faculty of Veterinary Medicine, WULS tional involvement in triggering acrosome re- tions revealed the occurrence of basally locat- – SGGW; 2Institute of Health Sciences, Siedlce Uni- action using a TPCN1-gen defi cient mouse ed, mononuclear cells without apical contact versity of Natural Sciences and Humanities, Siedlce; strain. The present manuscript documents to the feto-maternal interface. To differentiate 3Center of Biostructure Research, Medical University that TPC1 and NAADP-binding site show a whether these cells are SCs or TCs, further in- of Warsaw, Poland co-localization at the acrosomal region, and vestigations will be needed. Additionally, for

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 3 DGRM-Abstracts

the fi rst time TGCs with contact to the base- Introduction Syndecan-1 (Sdc-1) belongs played a change of the expression of selected ment membrane could be shown by TEM. to a family of transmembrane heparansulfate marker genes, like CYP19A1, FSHR, RGS2 Further morphological investigations will be or chondroitinsulfate binding proteoglycans. and VNN2. The present study aims in identi- done to approach the roles of these newly re- Sdcs have been linked to diverse functions fying the mechanisms of the density derived vealed trophectodermal cell stages. in human and mice, e.g. wound healing, im- effects performing a genome-wide mRNA mune response, angiogenesis, cell prolifera- microarray transcriptome analysis. Data re- tion and migration. Regarding the prepara- vealed that the expression of 1,510 anno- 08 tion of implantation of an embryo, an upregu- tated genes (represented by 1,575 transcript Necroptosis – an Unexplored lation of Sdc-1 during secretory phase was clusters) were differentially regulated com- found in human endometrium. Furthermore, paring both culture conditions (fold change Form of Cell Death in the Ovary a decreased expression in 1st trimester preg- > 1.5, p < 0.05, FDR < 0.05). Of these, near- K. Bagnjuk1, J. Blohberger1, A. Tiefenbacher1, nancies was shown to be a possible prognos- ly two-thirds were up- and one-third down- L. Kunz1, D. Mayr2, A. Mayerhofer1 tic factor for low birth weight and preterm regulated. Among the top up-regulated genes 1Biomedical Center (BMC), Cell Biology, Anatomy III, birth as well as low placental levels of Sdc-1 VNN2 and RGS2 could be identifi ed, as well Ludwig-Maximilians-University, Planegg; 2Patholo- were expressed in preeclamptic females. Due as HBA or LOXL2. Besides important key gisches Institut, Ludwig-Maximilians-University, to legal restrictions concerning human preg- genes of folliculogenesis (e.g. CYP19A1 and Munich, Germany nancy, we performed a detailed analysis of FSHR) we could newly identify TXNIP or metabolic and reproductive factors in Sdc-1 XDH as tremendously down-regulated. In-

DGRM-Abstracts A previous study in human IVF-derived knock out (ko) mice. genuity pathway analysis identifi ed “AMPK granulosa cells 1 (GCs) [Blohberger et al., Material and Methods Phenotypically, fe- signaling” and “cAMP-mediated signaling” Cell Death Dis 2015] led to the discovery of male and male Sdc-1 (ko) mice seemed to be as some of the most affected pathways. Main a form of cell death, termed necroptosis (pro- smaller compared to C57BL/6 wildtype (wt) putative upstream regulators were TGFB1 grammed necrosis) in the ovary. This type of mice. We could prove this lower body weight and VEGF, both factors involved in angio- cell death involves RIP1/3 and MLKL and from birth to adolescence in more than 100 genic processes. From this data we hypothe- can be prevented by necrostatin-1 and ne- offsprings of each line. Lower body weight size that specifi c cell-cell interactions induce crosulfonamid. In cultured human primary might be based upon deviated metabolic fea- an early post-LH stage in GC cultured at high GCs it occurs spontaneously and in the ova- tures. Therefore, corticosterone, insulin and density including transformations necessary ry, it may be involved, among others, in the leptin levels were determined in 6 weeks and to promote angiogenesis. demise of large follicles. While it was linked 6 months old animals. Corticosterone was re- to a splice-variant of AChE (AChE-R), the duced in 6 months old female and male Sdc-1 11 mechanism and the regulation of necroptosis ko mice compared to wt. On the contrary, of GCs are, however, not known. Human IVF- leptin was increased in adult ko mice of both Infl ammatory Changes in Dogs derived GCs are notoriously heterogeneous. sexes. Regarding pregnancy parameters, we with Azoospermia – a Normal To be able to study the mechanisms involved found more implantation sites in ko females Finding? in induction and execution of GC-necropto- on day 6 post mating, but a signifi cantly high- sis, we explored the suitability of a human er number of dead pups during the fi rst week C. Behrens Mathiesen, H. Körber, S. Goericke-Pesch GC tumor cell-line, KGN. It is thought that after birth. Section for Veterinary Reproduction and Obstetrics, these cells stem from follicu lar GCs, to which University of Copenhagen, Denmark they are closely related. Indeed as shown by Conclusion In summary, Sdc-1 ko mice PCR, KGN express typical GC markers, in- show a striking reproductive infl uence ac- Azoospermia is a common, yet not well-un- cluding FSH-receptors and aromatase. Fur- cording to the absence of this surface recep- derstood problem in male dogs with a gener- thermore, life cell imaging showed that with- tor that might also be associated with meta- ally poor prognosis. Testicular biopsies could out renewal of medium, KGN die within 72– bolic changes and therewith possibly lower provide deeper insights necessary for under- 120 h. Morphological signs of the cell death appetite. standing of the etiology. Ten dogs with con- suggested necrotic cell death, including a bal- fi rmed azoospermia were included; blood looning and cell burst. A blocker of apoptosis 10 samples for hormone analysis (LH, testoster- (zVAD-fmk) did not prevent this process, but one, T, and estradiol, E2) and karyotyping, necrostatin-1 effectively did. Further evidence Transcriptome Analysis demon- semen samples for alkaline phosphatase in for necroptosis as the form of cell death is de- strates a Luteinization-like Dif- seminal plasma and bacteriology, and testic- rived from immunoblotting. Results show ferentiation of Bovine Granulo- ular biopsies for histology were collected. To that phosphorylation of MLKL at Ser358 oc- sa Cells Cultured at High Plating assess the stage of spermatogenesis, the most curs when KGN die. Thus KGN can undergo developed germ cells observed were evaluat- necroptosis and are a suitable model to iden- Density ed in 50 round tubules. Additionally, 6 testicu- tify the mechanism of GCs-necroptosis. We A. Baufeld1, D. Koczan2, J. Vanselow1 lar infl ammatory parameters (thickness of ba- conclude that further studies in KGN may 1Institute of Reproductive Biology, Leibniz Institute sal membrane, presence of fi brosis, immune lead to insights into the role of necroptosis of for Farm Animal Biology, Dummerstorf; 2Institute cells, shrinkage of lumen, abnormal mitotic GCs in health and disease. for Immunology, University of Rostock, Rostock, patterns, vacuoles) were assessed. To iden- Grants: Supported by DFG, 20 MA1080/19-2 Germany tify the percentage distribution of the three compartments (seminiferous tubules, STC, During folliculogenesis the pre-ovulatory LH lymph and blood vessels, VC, and interstitial 09 surge leads to dramatic changes of the follicle tissue, IC), a morphometric evaluation of the including alterations of the gene expression testicular tissue was performed individually Are Leptin and Corticosterone profi le. To identify underlying mechanisms for each left and right testis and compared to responsible for Metabolic and Re- cell culture models are essential tools. In pre- normospermic control dogs (n = 5). 7/10 dogs productive Modifi cations in the vious studies it has been shown that bovine sired successfully before with a mean of 2.3 Syndecan-1 Knock out Mouse? granulosa cells (GC) mimic a process of early ± 1.5 years since the last successful mating. luteinization if the cells were cultured at a high Testicles were smaller (n = 5) or in the low- D. Baston-Büst1, C. Gougoula2, S. Böddeker1, plating density. Therefore, GC were cultured er reference range (n = 4) in andrological ex- W. Benten2, J. Krüssel1, A. Bielfeld2 serum-free with FSH, IGF-1 and androstene- amination. LH, T and E2 were within the ref- 1Department of OB/GYN and REI (UniKiD), Medical dione at normal and high density i.e. 1.0×105 erence ranges. Infl ammatory changes were Center, University of Düsseldorf, 2Central Unit for and 10.0×105 cells per well, respectively. GC present in 9/10 dogs with generalized auto- Animal Research and Animal Welfare Affairs (ZETT), cultured at high density switches from estra- immune orchitis in 6 dogs – 4 with early ar- Heinrich-Heine-University, Düsseldorf, Germany diol- to progesterone-producing cells and dis- rest (Sertoli-cell only or spermatogonia) and

4 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts

2 with late arrest of spermatogenesis. 3 oth- olus capreolus) is an interesting animal model Methods Testicular volume was reduced er dogs with late arrest had focal infl ammato- due to its embryonic dormancy phenomenon. correspondingly to the vascular volume as as- ry changes. IC and VC were increased, STC Here, we evaluated the effects of culture time sessed by micro-CT. Stereology was used to decreased compared to normospermic dogs; on in vitro oocyte maturation (IVM) in the roe assess the smaller vessels which are not ac- no difference was found between sides. Our deer. Ovaries were collected from hunted ani- cessible by micro-CT and revealed the reduc- results show that infl ammatory changes are mals between September and October. A to- tion of capil lary length, volume and surface common in dogs with acquired azoospermia. tal of 237 oocytes were obtained by slicing area. Stereological investigation of Leydig from 7 animals. Retrieved oocytes were clas- cell number in testes was similar in WT and sifi ed morphologically according to 5 catego- KO and remained unaffected by age. How- 12 ries: I (> 4 layers of compact cumulus cells ever, Leydig cell size was signifi cantly low- PTPIP51 – Localisation, Expres- [CC], clear and even cytoplasm), II (< 4 lay- er in KOs in all age groups. In line with this ers CC, cytoplasm with coarser appearance), fi nding, serum testosterone levels were sig- sion and Interactions in Relation III (1–2 layers CC, irregular cytoplasm), IV nifi cantly lower in KOs, whereas intratesticu- to Sperm Motility (denuded) and V (expanded or degenerated). lar testosterone was unchanged between WT and KO. D. Bepler1, C. Tag1, K. Steger2, H.C. Schuppe2, After either 20 h or 24 h IVM (oocyte quali- M. Wimmer1 ties I–III), oocytes were denuded, fi xed and Results Our data suggest a link between re- 1Justus-Liebig-University, Giessen; 2UKGM, Germany stained for nuclear evaluation. Fisher’s exact duced microvascular density, Leydig cells, test was used for meiotic status comparison testos terone reduction and male infertility. Protein tyrosine-phosphatase-interacting pro- (p < 0.05). The total number of oocytes/an- This study calls for specifi c treatment of male DGRM-Abstracts tein (PTPIP51) was identifi ed as a substrate of imal was 33.9 ± 3.5. Oocyte qualities I and infertility induced by microvascular damage PTP1B and TcPTP. The human protein is cod- II were scarce (0.3 ± 0.2 and 1.9 ± 0.6/ani- through hypercholesterolemia and arterio- ed at chromosome 15 (15q15.1). In its struc- mal). Qualities III and IV were more abun- sclerosis. ture there are several binding domains to gen- dant (12.6 ± 1.5 and 18.1 ± 2.9/animal). Simi- erate multi-enzyme complexes. Many inter- lar number of oocytes reached the MII stage action partners of PTPIP51 were found – en- after 20 h (33.3% [15/45]) and 24 h (43.1% zymes, receptors, proteins of mitosis, small [25/58]). Several oocytes remained between 15 GTPases and motor proteins. Due to these the MI and TI stage after 20 h (37.7% [17/45] Membrane Bound Progesterone interactions signaling pathways are regulat- or 24 h (31.0% [18/58]). Some oocytes did not Receptors in the Bovine Uterine ed and modulated. Hereby cell motility is in- resume meiosis after 20 h (8.9% [4/45] or 24 fl uenced. The interaction of PTPIP51 is con- h 5.1% [3/58]). The results show that roe deer Wall and Placentome trolled by phosphorylation state of its tyrosine oocytes as their counterparts from domestic N.M. Beyer1, M.K. Kowalik2, M.P. Kowalewski1, A. or serine residues. PTPIP51 is connected to ruminants are able to reach the meta phase II Boos1 MAP kinase pathway by Raf1 directly and by in vitro. However, a considerable number of 1Institute of Veterinary Anatomy, Vetsuisse Faculty, 14-3-3 proteins indirectly. Because of this Erk oocytes remain in earlier stages after standard University of Zurich, Switzerland; 2Institute of Ani- is activated and so cell motility is increased. IVM (BO-HEPES-IVM, IVF Bioscience). mal Reproduction and Food Research, Polish Acade- Erk1/2 stimulates sperm motility. Newer stud- These preliminary studies represent a promis- my of Sciences, Olsztyn, Poland ies prove the interaction of PTPIP51 and axo- ing start for the use of ARTs in this species. nemal dynein. In sperm fl agella dynein is as- As a hormone of pregnancy, progesterone sociated with microtubules and it is consid- 14 (P4) is known to interact with its well-in- ered as effector molecule of motility. Sperm vestigated nuclear receptors (PGR). Howev- motility is induced by variation of calcium ho- Testicular Microvasculature and er, it can also exert its effects by binding to meostasis and modifi ed by PKA activity. As Leydig cell function in the ApoE- membrane-bound P4 receptors, which me- an interaction partner of PKA PTPIP51 is in- /-/LDL Receptor-/-Arteriosclerosis diate rapid, non-genomic actions. Two of volved in the regulation of calcium level. That these membrane-bound receptors are the pro- is why we ask which role does PTPIP51 play Mouse Model gesterone receptor membrane component- in human sperm in terms of motility. For this D. Beyer1, K. Steinfeld1,2, C. Mühlfeld3, A. Mietens1, (PGRMC-) 1 and -2. The distribution pat- purpose immunohistochemical analyses of lo- T. Linn4, W. Weidner2, G. Schuler5, R. Middendorff1 terns of these 2 receptors have been exam- calization and expression profi les of PTPIP51 1Institute of Anatomy and Cell Biology; ²Department ined in different female reproductive tissues, and its interacting partners for sperm motility of Urology, Pediatric Urology and Andrology, Gies- but little is known about their presence in the were realized. Therefor we tested both, ejac- sen; ³Institute of Anatomy, Hanover; 4Medical Clinic bovine uterine wall and placentome during ulates with normozoospermia and such which and Policlinic, Giessen; 5Clinic for Veterinary Obstet- pregnancy, especially in mid and late stages. were classifi ed as dysfunctional by spermio- rics, Gynecology and Andrology of Large and Small Therefore, the aim of this study was, to dem- gram. Additionally the effect of one specifi c Animals, Giessen, Germany onstrate the cellular localisation, as well as modulator of PTPIP51 was assayed concern- mRNA and protein expression of PGRMC-1 ing sperm motility. This pharmaceutical de- Introduction Arteriosclerosis affects many and -2 in the bovine uterine wall and placen- veloped by our cooperation partners changes vascular beds. However, there is virtually no tomes during the entire period of pregnancy the interaction profi le of PTPIP51 and accel- information on arteriosclerosis-based chang- at the protein and mRNA level, by immuno- erates sperm motility. es in testicular vasculature and in how far dis- histochemistry and qPCR, respectively. Tis- turbances of the local vascular system of the sues from 45 cows (n = 5 per each month of testis may result in testicular malfunction. pregnancy) were used. The strongest pos- 13 We used the Apolipoprotein E (ApoE)/Low itive reaction in the uterine wall was found In vitro maturation of Roe Deer Density Lipoprotein (LDL) receptor defi cient in the luminal epithelial cells, the glandular mouse model (KO), in which disturbed sper- epithelial cells, and also in the endothelium Oocytes matogenesis was previously described by us. of blood vessels. The staining was less pro- S. Bernal-Ulloa, B. Drews, A. Rudolf-Vegas, V. van This could be due to a reduction of the capil- nounced in the smooth muscular and stromal der Weidjen, V. Milojevic, S. Ulbrich lary network, an impaired testosterone syn- cells. In the placentomes, both proteins were Animal Physiology, Institute of Agricultural Sciences, thesis, a reduced Leydig cell number or an in- detected in the maternal part (stroma, epithe- ETH Zurich, Switzerland suffi cient transport between Leydig cells and lial and endothelial cells) as well as in the en- the Inter-Leydig cell capillaries. Against this dothelium of fetal blood vessels. PGRMC-2 Assisted reproductive technologies (ARTs) background, we analysed to what extent po- was additionally expressed in the fetal cho- application in wild animals is a big challenge tential changes of testicular microvasculature rionic epithelium. Staining intensities and due to the variability among species and the are associated with changes of Leydig cell mRNA expression levels did not vary greatly limited research access. The roe deer (Capre- function. for both receptors throughout gestation. The

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 5 DGRM-Abstracts

expression and distribution patterns of sig- Introduction Oxidative stress in sperm is trol group of non-biopsied embryos was nals, however, suggest that PGRMCs may be inducing DNA damage. Oocytes can repair stored under the same conditions. The gross involved in the non-genomic action of P4 in paternal damaged DNA to a certain extent. morphological quality was determined before bovine uterine wall and placentomes during The ability of the oocyte to repair compro- and after LP and further assessed with live- pregnancy. mised paternal DNA is probably dependent dead staining. Embryos were again cultured from different factors. Brilliant Cresyl Blue for 48h to determine re-expansion and hatch- (BCB) stain indicates the activity of the glu- ing rates. The live dead cell ratio was signifi - 16 cose-6-phosphate dehydrogenase (G6PDH) cantly lower in embryos out of the group with and has been used as marker of oocyte quali- BSA compared to those out of the group with Effect of Intravaginal Prosta- ty. We hypothesized that oocytes with a high FBS and FBS with 1mg/ml BSA (BSA:FBS glandin E2 Application in Farrow- G6PDH activity (BCB-) fail to repair dam- P < 0.05; BSA:FBS with 1 mg/ml BSA ing Sows During Parturition aged sperm and consequently have a lower P < 0.05). Embryos from the group 1 mg/ml competence to support embryo development BSA (biopsied and non-biopsied) have simi- R. Bill, H. Nathues, A. Grahofer after fertilization with oxidatively stressed lar re-expansion rates (50.0% vs 66.7%), but Clinic for Swine, Vetsuisse Faculty, University of sperm. different hatching rates (12.5% vs 33.3%), as Bern, Switzerland Methods To investigate this, bovine sperm well as embryos out of group with 10 mg/ml BSA (biopsied and non-biopsied; re-expan- The duration of birth is an important parame- were incubated with or without 100 µM H2O2 sion rates: 57.1% vs 46.7%; hatching rates: ter linked to piglets’ survival and sow’s health for 1 h at 38° C and were used to fertilize in DGRM-Abstracts 14.3% vs 26.7%). Biopsied embryos out of in the context of parturition. Therefore, pro- vitro matured bovine oocytes which were se- group with FBS (25% and 50%) show simi- longations are frequently treated with oxy- lected using BCB stain. Oocytes incubated in lar re-expansion rates (100.0% vs 80.0%) and tocin – a potent uterotonic agent – although PBS served as control. Presumptive zygotes hatching rates (88.9% vs 70.0%). Similar re- several undesirable side effects such as high- were cultured in vitro and blastocyst rate on sults were obtained with non-biopsied em- er incidence of umbilical cord lesions, meco- day 6 and 7 was determined in 7 IVF cycles. bryos out of group with FBS (25% and 50%; nium staining and weak piglets have been de- Fertilization of control oocytes with control re-expansion rates: 70.0% vs 80.0%; hatch- scribed. In human medicine, Prostaglandin sperm yielded in signifi cant higher blasto- ing rates: 50.0% vs 50.0%). Results show for E2 (PGE2) is used instead, because less side cyst rates (d6: 12 blastocysts/120 oocytes, the fi rst time that LP could be an alternative effects have been observed. The aim of this d7: 34 blastocysts/135 oocytes) than fertiliza- to cryopreservation. pilot study was to test, whether 2 mg PGE2 tion of BCB-oocytes with control sperm (d6: applied as intravaginal gel after the birth of 10 blastocysts/167 oocytes, d7: 22 blasto- the fourth piglet infl uences the birth process. cysts/180 oocytes). After fertilization of con- 19 Overall, 3 randomly selected sows in a pig trol oocytes with H2O2 exposed sperm, the herd were treated with PGE2-Gel (group T) blastocyst rates on d6 were reduced by 32.6 Expression of MIF, DDT and Zinc and 3 other sows were treated with a place- ± 31.8% (10 blastocysts/204 oocytes) and Transporters in the Stallion’s bo gel (group C). The total duration of birth on d7 by 58.7 ± 21.1% (23 blastocysts/233 Reproductive Tract (time between fi rst piglet and last placenta) oocytes), in comparison to fertilization with and the piglet interval were recorded, and control sperm. When BCB-oocytes were fer- S.A. Bramer1, H. Henning2, T.A. Stout2, C. Klein1 1 each piglet was scored for meconium staining tilized with H2O2 exposed sperm, blastocyst College of Veterinary Medicine, University of Cal- and vitality. In group T the duration of birth rate was signifi cantly lower by 94.8 ± 13.8% gary, Canada; 2Department of Equine Sciences, Uni- was 386 min (average of 20.0 piglets per lit- on d6 (1 blastocyst/299 oocytes) and 87.1 ± versity of Utrecht, The Netherlands ter) compared to 439 min in group C (average 19.36% on d7 (7 blastocysts/352 oocytes) than after fertilization with control sperm. Zinc is a trace element that has received par- of 14.3 piglets per litter). The piglet to piglet ticular attention for its role in male fertili- interval was 10.2 min in group T compared to Results In conclusion BCB-oocytes have a ty. Within sperm cells, zinc is localized pri- 14.3 min in group C. No or only slight meco- lower competence to support embryo devel- marily to the tail where it regulates the oxida- nium staining was observed in 53% and 35% in vitro opment after fertilization with H2O2 tion status of the sulfhydryl groups located in of piglets in group T, and in 70% and 30% exposed sperm, which indicates a possible in- the outer dense fi bers, and thereby indirectly of the piglets in group C. Severe meconi- volvement of G6PDH activity in oocyte com- modulates sperm motility. During epididymal um staining was only found in 12% of pig- petence to repair damaged sperm. transit, the zinc content of the sperm tail is re- lets in group T. Moreover, 10% of piglets in duced by a mechanism involving macrophage group T showed an oedematous and haemor- 18 migration inhibitory factor (MIF), secreted by rhagic umbilical cord, lethargy and anoxia. the epididymal epithelium. Using real-time This study describes the impact of PGE2 gel Liquid Preservation of Bovine RT-PCR and immunohistochemical stain- on the birth process in sows. The duration ing the expression of MIF, D-dopachrome of birth and the piglet interval tended to de- Embryos as an Alternative to Cryopreservation tautomerase (DDT; a functional homolog of crease, whereas umbilical cord lesions, fetal MIF) and selected zinc transporters was ex- distress and anoxia increased. Further investi- N. Blad-Stahl, F. Kotarski, C. Wrenzycki amined in the equine testis and epididymis. gations should focus on the optimal dosage of Clinic for Veterinary Obstetrics, Gynecology and An- The effect of zinc on sperm motility was also PGE2 in order to evaluate the use of this drug drology, Chair for Molecular Reproductive Medicine, assessed. MIF expression was highest in the in farrowing sows. Justus-Liebig-University, Giessen, Germany head of the epididymis, where an accumu- lation of intensely stained material could be At present, the only reliable possibility to seen engulfi ng the sperm cells. DDT staining 17 store biopsied embryos is cryopreservation intensity and pattern was similar across the with lower pregnancy after thawing. The aim High Glucose-6-phosphat-Dehy- different regions of the epididymis; immuno- of the present study is to develop an effi cient reactive material was visible in the lumen of drogenase Activity in Oocytes is procedure that is able to preserve biopsied, in the epididymis, with the largest amount as- Related to low Embryo Develop- vitro produced bovine embryos for up to sev- sociated with the tail region of spermatozoa. mental Rates after In vitro fertili- en days under hypothermic conditions. Among the zinc transporters, SLC39A5 dis- zation with Oxidatively Stressed Bovine IVP-derived day 6 morulae were played a distinctive expression pattern with Sperm bio psied, followed by liquid preservation signifi cantly higher abundance in the caput (LP) in TCM plus either BSA (1 mg/ml and epididymis than any other tissue investigated. L. Bittner, C. Herrera, S. Wyck, H. Bollwein 10 mg/ml), FBS (25% and 50%) or BSA with Both addition of free, extracellular zinc to, Clinic of Reproductive Medicine, Vetsuisse Faculty, FBS (25% FBS and 50% FBS with 1 mg/ ml and intracellular chelation of zinc in, ejacu- University of Zurich, Switzerland BSA each) for seven days at 0–4°C. A con- lated sperm signifi cantly reduced sperm mo-

6 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts tility in a dose and time dependent manner, 21 formation of iPS cells with embryos is pos- whereas viability was unaffected. We con- sible. However, embryos and stem cells have clude that zinc plays a role in regulating mo- ROS-Generating NOX Enzymes totally different requirements concerning the tility of stallion sperm, while the MIF expres- and H2O2 in Human Ovary and culture media. The aim of this study was to sion pattern in the epididymis suggests a cen- Granulosa Cells fi nd optimal culture conditions for both cell tral role for MIF in depleting zinc from the types by testing the development of porcine sperm tail during epididymal maturation. T. Buck1, C. Herrmann1, A. Tiefenbacher1, parthenogenetic embryos in different cul- J. Blohberger1, D. Berg2, U. Berg2, L. Kunz3, ture media. Porcine parthenogenetic em- A. Mayerhofer1 bryos were cultured in different media mix- 20 1Biomedical Center, Cell Biology, Anatomy III, tures of the standard embryo culture medium Ludwig-Maximilians-University, Planegg; 2A.R.T., (PZM- 3) and stem cell media beginning at Method for the Indirect Detection Bogenhausen, Munich; 3Division of Neurobiology, day 3 (4–8 cell) or day 4 (early morula) after of Interferon-τ by Using an In vitro Department of Biology II, Ludwig-Maximilians-Uni- activation (1.0kV/cm for 45 µs, followed by Leukocyte stimulation test versity Planegg, Germany incubation with 2 mM 6-DMAP for 3 h). If not otherwise indicated, total embryo number 1 1 The mammalian ovary generates reactive Y. Brockelmann , K. Mense², M. Jung², M. Schmicke used per medium mixture was 74–110, subdi- 1 oxygen species (ROS). However, the roles University of Veterinary Medicine Hanover, Clinic vided into 3–4 repeats. It was shown, that the 2 of ROS and the mechanism of ROS-genera- for Cattle, Endocrinology Laboratory, Institute for blastocyst rate from day 3 embryos in PZM-3 tion in the ovary remain poorly understood. the Reproduction of Farm Animals Schönow, Ber- (n = 12, 305 embryos) was signifi cantly high- DGRM-Abstracts Physiological levels of ROS modulate signal nau, Germany er (41.0%) than in both stem cell media (ES transduction and hormone signaling, while medium 1.3%, ciPS medium 3.7%). A mix- Interferon-τ (IFN-τ) is the signal of preg- its accumulation leads to oxidative stress. ture of PZM-3 with 25% or 50% stem cell nancy recognition produced by the concep- Human granulosa cells (GC) express two medium improved the blastocyst rate (16.2– tus around day 16 of early pregnancy in cat- members of NADPH oxidase (NOX) family, 25.0%). The results for the day 4 embry- tle. Because circulating concentrations of which produce ROS, namely NOX4/5. The os were different. Blastocyst rate in PZM-3 IFN-τ are extremely low, the measurement present study was aimed to further character- (34.7%, n = 10, 251 embryos) was between of Interferon-stimulated genes (ISG) as re- ize the expression of NOX members in the the rates of ES medium (39.0%) and ciPS sponse in leukocytes is an alternative meas- ovary and in GC, to determine their contri- medium (34.1%). Developmental rates in ure. The aim was to establish a simple and in- bution to ROS generation, and to obtain in- media mixtures (40.5–46.4%) were even bet- direct method to detect IFN-τ by using a leu- sights into their regulation. We studied IVF- ter than in PZM-3. It can be concluded that kocyte stimulation test. Leukocytes were iso- derived GC and human ovarian sections. RT- the development of parthenogenetic embryos lated from a non-pregnant donor cow. Plasma PCR studies confi rmed the expression of in pure stem cell medium is possible begin- samples from early pregnant (p) heifers on NOX4/5 and further identifi ed DUOX1/2 but ning on day 4 after activation, however me- day 18 (n = 4) and from the animals during not NOX1-3 in GC and human ovarian sec- dium-specifi c differences have to be consid- a non-pregnant cycle (np, n = 5) were avail- tions. Immunohistochemistry/immunocyto- ered. For porcine parthenogenetic embryos able from a previous project [Meyerholz et chemistry showed pre-adsorbable NOX4 in beginning on day 3, a mixture of PZM-3 and al. 2015]. The leukocytes were incubated in isolated GC and in GC of ovarian follicles. stem cell medium is recommended. vitro in 12-well plates with plasma samples Cultured GC produce ROS, especially H2O2 from either pregnant or non-pregnant animals as determined by fl uorometric assays. Super- for 2 h and 37°C in Iscove-medium. Plasma oxide was not detected. To study the contri- from a non-pregnant cow served as negative bution of NOX4 to ROS produced by GC, we 23 control and this plasma spiked with 0.06 ng employed the NOX4-blocker GKT137831. It Investigations of some Life Quali- recombinant bovine IFN-τ was used as posi- signifi cantly reduced ROS/H2O2 production tive control. After the incubation, mRNA was by about 50% without altering cell morpho- ty Parameters during early Neona- extracted and the relative expression of ISG logy and viability. As isolated/cultured GC tal Period in Low Environmental (MX1, MX2, ISG15, OASX1) in leukocytes stem from large antral follicles, this may in- Temperature induced born Lambs was measured by using quantitative real-time dicate that NOX4 is signifi cantly contributing PCR. Due to small number of samples only to the ROS environment of the follicle. Fur- N. Cetin1, S. Sendag1, I. Tasal2, A. Wehrend3 descriptive statistics was performed. The thermore, our qPCR data suggest that the ad- 1Clinic for Veterinary Obstetrics and Gynecology, mRNA expression of ISG in the leukocyte dition of follicle-stimulating hormone (FSH) Yuzuncu Yil University, Van; 2Clinic for Veterinary Ob- stimulation test with plasma samples of preg- and human chorionic gonadotropin (hCG) to stetrics and Gynecology, Mehmet Akif Ersoy Univer- nant heifers was numerically higher than the the GC elevated mRNA levels of NOX5 and sity, Burdur, Turkey; 3Clinic for Veterinary Obstetrics, cycle-control samples (mean ± SD; MX1 p = DUOX1/2 but not of NOX4. Thus, gonado- Gynecology and Andrology, Justus Liebig University, 21.5 ± 10.3 vs np = 11.8 ± 6.4; MX2 p = 33.8 tropins may be involved in the regulation of Giessen, Germany ± 28.7 vs np = 13.1 ± 8.6; ISG15 p = 53.1 ± ovarian ROS production. 49.5 vs np = 35.4 ± 14.7; OASX1 p = 23.2 ± The purpose of this study was to investiga- Grants: Supported by DFG MA1080/26-1; tion of some life quality parameters during 13.7 vs np = 11.8 ± 5.7). The ISG mRNA ex- KU 1082/7-1 pression in leukocytes from samples of preg- early neonatal period in low environmen- nant heifers was comparable to the ISG ex- tal temperature induced born lambs. The an- pression of the positive-control. In conclu- 22 imal material was consisted of 20 pregnant sion, the incubation in vitro of leukocytes Morkaraman sheep housed in Research and with plasma samples from pregnant cows al- Effect of Stem Cell Media on the Application Farm in University of Yuzun- lows the indirect detection of IFN-τ in ear- Development of Porcine Partheno- cu Yil Van-Turkey. Parturition of 10 sheep ly pregnancy. (Group I) was induced by corticosteroids at genetic Embryos the 141th day of gestation. Ten sheep with B. Burchardt, A. Lucas-Hahn, P. Hassel, M. Ziegler, spontaneous parturition constituted the con- H. Niemann trol group (Group II). Some blood parame- Institute of Farm Animal Genetics, Friedrich-Loeffl er- ters (blood gases and glucose), body tempera- Institut, Mariensee, Germany tures and birth weights of these lambs who were born in extremely low environmental Since induced pluripotent stem cells (iPS) temperature (day: 3–4˚C, night –5 to –1˚C) can be artifi cially produced from farm ani- were recorded in the fi rst week of birth. Dif- mals, the generation of animals for disease ferences between the life qualities of these models and xenotransplantation by chimera lambs were determined by comparing all pa-

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 7 DGRM-Abstracts

rameters. Only blood PO2 value was differ- the basis for further investigations on steroid fungus) associated with repeat breeder buffa- ent between Group I and Group II in the fi rst driven mechanisms behind cyclic functional los and cows. This work was carried out on 24 hours (p < 0.05). However these differ- changes in the oviduct epithelium. 120 cervico-vaginal and uterine swabs col- ences did not lead to any advantage or dis- lected from 50 buffalos and 70 cows suffer- advantage on the life qualities. Another inter- ing from repeat breeding. Bacteriological ex- esting fi nding is that the birth weights of all 25 amination revealed that 200 microbial agents lambs did not have any difference. Individual Evaluation of Selected Parame- were identifi ed and isolated from the collect- changes in the body temperatures did not lead ed samples. The results showed that 60%, to unfavourable effects. Results of our study ters of Ram Semen, including Mo- 20%, 12.5% and 7.5% of the isolates were show that lambs whose birth induced by exo- tile Sperm Organelle Morpholo- single bacterial, mixed bacterial, single fun- genous hormone in 141th day of pregnancy; gy (MSOME), after Thawing and gal and mixed bacterial and fungal, respec- up to –5˚C low environmental temperatures Stimulation with Pentoxifylline tively. The most prevalent aerobic isolates didnt lead to negative effect in their life quali- were E. coli (11.9%), Trueperella pyogenes ty. Morkaraman lamb’s neonatal life quali- S. Dabrowski1, J. Szlendak1, S. Gizinski1, R. Faundez1, (9.4%). The most common mixed bacteri- ty, gestation period and genetic resistance Ç. Çebi2, M. Domino1, B. Pawlin´ski1, Z. Gajewski1 al isolates were E. coli + Trueperella pyo- against the environment need further investi- 1Department of Large Animal Diseases with Clinic, genes + Proteus spp (22%) and Staphylococ- gation for a better understanding. VRC and CBB, Faculty of Veterinary Medicine, WULS cus aureus + Corynebacterium bovis (20%). – SGGW, Warsaw, Poland; 2 Veteriner Fakültesi, Har- The most common fungal isolates were As- DGRM-Abstracts 24 ran University, Turkey pergillus spp (24%) and Candida (24%). E. coli and Aspergillus spp (33.3%) were most- Mimicking Estrous Cycle Stages Artifi cial insemination is a widely used meth- ly isolated as a mixed bacterial and fungal in- in vitro: Impact of Estradiol and od in livestock breeding. In case of sheep, re- fection. After Sensitivity test, the most ac- sults are insuffi cient, what is caused by ana- tive antibiotics were Enrofl uxacin, Oxytetra- Progesterone on the Transcriptom- tomic structure of female reproductive organs, ic Profi le of Porcine Oviduct Epi- cycline, Gentamicin and Nalidixic acid. Most as well as semen conservation problems. It bacterial isolates were resistant to Neomycin, thelial Cells has been proven, that successful insemination Erythromycin and Ampicillin. Sensitivity test is signifi cantly dependent on spermatozo- 1 1,2 3 revealed that most bacterial isolates, in this S. Chen , S. Palma-Vera , B. Kempisty , on’s functional activity. Among these sperm 3 1 1 study, were highly sensitive to Enerofl uxacin, M. Rucinski , A. Vernunft , J. Schoen motility is particularly important. It can be 1 Oxytetracycline, Gentamicin and Nalidixic Institute of Reproductive Biology, Leibniz Institute stimulated by some activators, such as pen- for Farm Animal Biology (FBN), Dummerstorf; 2In- acid and resistant to Neomycin, Erythromy- toxifylline. The aim of the study was to deter- cin and Ampicillin. stitute of Veterinary Biochemistry, Department of mine the effect of pentoxifylline on selected Veterinary Medicine, Freie Universität Berlin, Ger- sperm kinetics parameters and motile sperm 3 many; Department of Histology and Embryology, organelle morphology (MSOME) of thawed Department of Anatomy, Poznan University of Medi- semen from wrzosówka breed rams. In the 27 cal Science, Poznan, Poland study, 18 healthy rams were used. For sperm Relationship between Color Flow Cyclic changes in the oviduct epithelium are motility assessment, the Sperm Class Ana- Doppler Sonographic Assessment known to be predominantly regulated by Es- lyser CASA system (Microptic S.L, Spain) was used. Furthermore, samples were taken of Hemorrhagic Anovulatory Fol- tradiol (E2) and Progesterone (P4). Thus, the licles (HAFs) and of the Follicle on present study aimed to examine in vitro, the to MSOME (Leica DMi8 DIC inverted mi- transcriptomic impact of these hormones on croscope) at 6600 magnifi cation. The results the Day before Ovulation in Mares showed that pentoxifylline in concentrations highly differentiated porcine oviduct epithe- 1 1 1 of 1 µg/ ml, 10 µg/ml, 100 µg/ml have a posi- D. Domanska , M. Trela , K. Skierbiszewska , lial cells cultured at the air-liquid interphase 2 1 3 1 tive effect on straight-line veloci ty (VSL); M. Masko , M. Domino , A. Wehrend , Z. Gajewski (ALI-POEC). Diestrus (D, 10 days) and es- 1 linearity (LIN) and straightness (STR). Pen- Department of Large Animal Diseases with Clinic, trus (E, 2.5 days) were sequentially simulated VRC and CBB, Faculty of Veterinary Medicine; 2De- in ALI-POEC (5 donor animals) by basolat- toxifylline at the concentration of 1 µg/ml showed a major effect, however there were partment of Animal Breeding, Faculty of Animal eral application of physiological levels of E2 Science, WULS – SGGW, Warsaw, Poland; 3Klinik für and/or P4. Three groups were included: E2 no signifi cant differences between the tested concentrations of pentoxifylline. The same Veterinärmedizin, Klinik für Geburtshilfe, Gynäkolo- group (D: 10 pg/ml E2; E: 50 pg/ml E2), P4 gie und Andrologie der Groß- und Kleintiere mit group (D: 35 ng/ml P4; E: 0.5 ng/ml P4) and was true for MSOME. In conclusion the posi- tive effect on ram sperm motility and a lack Tierärztlicher Ambulanz der Justus-Liebig-Univer- E2+P4 group (D: 10 pg/ml E2 and 35 ng/ml sität, Giessen, Germany P4; E: 50 pg/ml E2 and 0.5 ng/ml P4). Cul- of negative effect on morphology suggested tures were harvested and processed for his- that pentoxifylline can be utilized to improve The wall of hemorrhagic anovulatory folli- tology and microarray analysis after each the effectiveness of artifi cial insemination in cles (HAFs) developed well-vascularized lu- simulated phase. In vivo-like morphological various breeds of the sheep. teal tissue as indicated by echotexture and changes could only be observed in the P4 and color Doppler signals. The aim of this study E2+P4 groups, whose transcriptional profi les 26 was to assess the relationships between HAFs were further studied. Gene expression pat- and normal follicle vascularity evaluated by terns of simulated estrus and diestrus stages Sensitivity of different Micro bial Color Doppler ultrasonography (USG) in were clearly distinct in both groups as dem- Isolates to Selected Antimicro- mares. The ultrasound exami nations were car- onstrated by principal component analysis. bials in vitro in Repeat Breeder ried out on 15 Warmblood mares at -2 and -1 Comparing estrus versus diestrus stage 166 days before and 1 and 2 days after ovulation. genes were up and 35 were down regulated Cows and Buffalos in Upper Egypt Follicles and HAFs size was determined by in the E2+P4 group; 75 genes were up and 18 D. Refaat1,2, A.A. Abdel-Rahman3 measurement of the maximal cross-section- genes were down regulated in the P4 group. 1Department of Veterinary Medicine, College of Agri- al area of follicles (MCSF) and blood supply In concordance with in vivo studies top regu- culture and Veterinary Medicine, Qassim University, by the maximum colored area of the lutein- lated genes were CDC20, CDC20B, OVGP1 Saudi Arabia; 2Department of Theriogenology, Fac- ized tissue (MCAL) from Doppler ultrasound and PGR. Comparison of P4 estrous stage ulty of Veterinary Medicine, Assiut University; 3Ani- images. There were no signifi cant differenc- with E2+P4 estrous stage showed only 9 up mal Health Research Institute Alexandria Laborato- es in MCSF (P > 0.05) between days -2 and (top regulated NANOG, CDC20, CDC20B) ry, Egypt -1 before ovulation in both groups of mares and 8 down regulated genes. P4 and E2+P4 with normal ovulation and with formation diestrous stages did not exhibit any differ- The present study aimed to investigate the HAFs. Whereas at day -1, MCAL signifi cant entially expressed genes. Our study builds microbial and mixed infections (bacterial and decreased (P = 0.0004) in cyclic mares with

8 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts normal ovulation and increased (P = 0.003) 29 case of unselected oocytes belonging to the in mare with HAFs formation. The increase same cohort of oocytes used in the ICSI and of the blood supply of the HAFs (MCAL) TRV2 Ion Channels in Human Tes- IMSI procedures. In the study 329 oocytes corresponded with vascularization at the api- ticular Peritubular Cells were used and 178 of them were selected for cal area on day -1. The lack of vasculariza- sperm microinjection. Morphological assess- 1 2 3 tion at -1 day (normal follicles) was related K. Eubler , J.U. Schwarzer , F.M. Köhn , ment of oocytes by differential interference 1 to constant MCAL decreases. HAFs formed A. Mayerhofer contrast (DIC) was done with an inverted mi- 1 from viable preovulatory follicles that did not Biomedical Center (BMC), Cell Biology, Anatomy III, croscopy (Leica DMi8, Germany). Birefrin- 2 differ from ovulatory follicles in diameter or Ludwig-Maximilians-University, Planegg; Androlo- gence of zona pellucida (ZP) and the meiot- 3 gray-scale echotexture. Color Doppler USG gy-Center, Munich; Andrologicum, Munich, Germany ic spindle visualization (MSV) was assessed may be used to estimate the follicle growth, In man, several layers of peritubular cells form by a Nikon Ti microscope with OCTAX Po- maturation and ovulation better than standard the wall of seminiferous tubules. They con- larAIDE™ system. The median value of ZP USG due to more an accurately determina- tract and thus transport immotile sperm. They birefringence was 7.9 and 83.5% of oocytes tion of follicles aging. Therefore, Color Dop- also secrete numerous factors, which are in- showed high birefringence (HB) over the pler USG provides more quantitative infor- volved in the regulation of testicular function, median. The MS was observed in 66.7% of mation than the standard USG in phases of including immunological activities. A culture the oocytes, in this 99.5% in HB oocytes the estrous cycle and ovulation. method enables us to study human testicular (p < 0.01). The index of birefringence for co- peritubular cells (HTPCs) in detail. A previ- hort (BIC) was also calculated. Within the ous proteomic study identifi ed a member of same cohort, the ZP birefringence results for DGRM-Abstracts the transient receptor potential family of ion unselected oocytes are closely correlated with 28 channels, TRPV2. TRPs are involved in ther- the fertilization rate (r = 0.27, p < 0.05) , em- mo-, osmo-, and mechanosensation, in pain bryo quality at the days 2 (r = 0.30, p < 0.05) The Pacemakers and Conduction perception and in infl ammation. TRPV2 was and 3 (r = 0.36, p < 0.02) and the clinical Pathways of Myoelectric Activity previously described in immune cells and can pregnancy rate (CPR) obtained for selected oocytes after ICSI (r = 0.28, p < 0.05) and Patterns in the Porcine Uterus be activated by hypoosmolarity and mechani- cal stress. The identifi cation of TRPV2 in IMSI (r = 0.32, p < 0.05) groups. A similar M. Domino, B. Pawlinski, Z. Gajewski smooth muscle-like cells of the testis implies correlation was found for IDK. The birefrin- Department of Large Animal Diseases with Clinic, a role of this channel in peritubular cell func- gence values for ZP, MSV and BIC in con- VRC and CBB, Faculty of Veterinary Medicine, WULS tions. Expression in HTPCs was confi rmed junction with oocyte morphology in unselect- – SGGW, Warsaw, Poland by RT-PCR and sequencing. Expression lev- ed oocytes may provide additional prognostic els (qPCR) decreased in advanced passages. parameter in assisted reproduction programs The myoelectrical activity is a result of volt- Western blotting showed a single band cor- in which the number of oocytes for IVF is re- age- and time-dependent changes in mem- responding to the size of TRPV2. Using the stricted by the law as in the case of Poland. brane ionic permeability and may be detect- same antibody, TRPV2 was identifi ed in tes- ed directly in myometrium by electromyo- ticular peritubular cells in vivo by immuno- 31 graphy (EMG). The ICLC (Interstitial Ca- histochemistry. Functionality of this non-se- jal-Like Cells) contained in myometrium are lective cation-channel was examined in HT- Sperm-induced Neutrophil Extra- suspected to display characteristic patterns PCs by monitoring intracellular Ca2+ levels cellular Trap (NET) Formation in of rhythmical activity and to have dual func- using Fluoforte. Activators (CBD/ O-1821) the Bovine System tions as pacemakers and conduction path- increased levels of intracellular Ca2+. They ways for the active propagation of electrical also evoked a cation-current in patch clamp T. Fichtner1,2, F. Kotarski2, T. Muñoz-Caro1, J. Stöhr2, slow waves. The aim of the study was to de- studies. First results of prolonged exposure of C. Hermosilla1, C. Wrenzycki2, A. Taubert1 scribe relations between uterine EMG activi- HTPCs to a specifi c activator (O-1821) indi- 1Institut für Parasitologie; 2Klinik für Geburtshilfe, ty and ICLC occurrence in pig in vivo model. cate that it elevates expression of infl ammato- Gynäkologie und Andrologie der Groß- und Klein- The spontaneous uterine myoelectrical activ- ry cytokines on mRNA level. This may sug- tiere, Lehrstuhl für Molekulare Reproduktionsmedi- ity in the 10 non-pregnant, mature sows dur- gest involvement of TRPV2 in the immune zin, Justus-Liebig-University, Giessen, Germany ing diestrus was recorded by the combination regulation of the human testis, but addition- of three electrodes connected to transmitter al studies are required to elucidate the role of In cattle the natural site of semen deposition used in large animals. The typical pattern of this channel in the human testis in health and is the vagina. Besides phagocytosis and se- rhythmic electrical activity was recorded dur- disease. cretion of immune modulators, polymorpho- ing 3 weeks of registration. Mean amplitude nuclear neutrophils (PMNs) are able to form (A), mean root mean square (RMS), duration Grants: Supported in part by DFG „Neutrophil Extracellular Traps“ (NETs). of electrical activity (D), duration of paus- MA1080/27-1 These are web-like structures, mainly com- es between activities (P), number of excita- posed of chromatin. Bovine PMN were iso- tions (N) were analyzed regarding to differ- 30 lated via Ficoll gradient centrifugation. Fro- ent topographic regions. In parallel immuno- zen/thawed sperm cell suspension (SCS) of fl uorescent studies, the distribution of specif- Relationship between Oocyte bulls with proven fertility were used. The ic c-kit/CD 117 receptors (display on ICLC Morphology, Zona pellucida and visu alization and identifi cation of NETs was surface) in the same topographic regions of meiotic Spindle Birefringence in achieved by SEM or via fl uorescence micros- porcine reproductive tract was determined. unselected and selected Oocytes copy analysis, respectively. For NET induc- The signifi cant strong correlation (Pearson‘s tion, PMN and SCS were co-cultured for dif- r) between ICLC density and EMG parame- for ICSI and IMSI ferent time points (0, 15, 30, 45, 60, 120, 180 ters such as duration of electrical activity (D) R. Faundez1,2, K. Zareba1, M. Petrajtis-Golobow1, min). NET induction of sperm and superna- (r = –0.85, P = 0.03) and duration of paus- S. Dabrowski1,2, Z. Gajewski1 tant alone was also measured. Zymosan was es (P) (r = –0.81, P = 0.05) in uterine horn 1Department of Large Animal Diseases with Clinic, used to induce the formation of NETs in bo- tip were demonstrated. No signifi cant corre- VRC and CBB, Faculty of Veterinary Medicine, WULS vine PMN as positive control. Quantifi cation lations in any of the parameters studied were – SGGW; 2InviMed Fertility Clinics Warsaw, Poland of NETs formation was performed by spec- observed for the uterine body and horns. The trofl uorometric analyses using an automated electrophysiological studies indicate that Birefringence of oocyte structures assessed plate monochrome reader (Varioskan Flash; ICLC in the horn tip myometrium may par- by polarizing microscopy correlates with its Thermo Scientifi c). Data were analyzed for ticipate in the regulation of slow waves dura- quality and the developmental potential of the statistical signifi cance employing a one way tions and frequency similar to ICC in gastro- embryo.The aim of the study was investigate Anova followed by a Tukey test (p  0.05). intestinal tract. whether a similar correlation exists in the SEM as well as fl uorescence microscopy

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 9 DGRM-Abstracts

analyses revealed that the exposure of bovine rine and intracrine regulation by a local sup- A decrease in reproductive function has be- PMN to frozen/thawed bovine SCS trigger ply of sulfated and free steroid hormones in- come apparent in men with advancing age. the formation of NETs. After quantifi cation, side the human seminiferous epithelium. The decline in spermatogenesis may be at- the following fl uorescence intensities (FI in tributable to an impaired capacity of the cells arbitrary units, AU) were obtained for PMN building the spermatogonial stem cell (SSC) plus SCS: 10.0 ± 0.6, PMN plus sperm cells: 33 niche to provide an appropriate environment 3.2 ± 0.5 and PMN plus supernatant (extend- in older males. Human testicular peritubular er plus minimum amount of seminal plasma): Scent Detection as a Diagnostic cells (HTPCs) form the wall of the seminifer- 9.3 ± 0.6. After incubation for different time Tool for Mastitis Pathogens: Proof ous tubules and are emerging as an important points, FI were as follows; 7.2 ± 0.5, 7.7 ± of Principle component of the SSC niche. It is so far un- 0.4, 8.1 ± 0.4, 8.4 ± 0.8, 10.8 ± 0.7, 9.7 ± 0.6, known, whether they are affected by age and 9.6 ± 0.7 for 0, 15, 30, 45, 60, 120, 180 min. C. Fischer-Tenhagen1, V. Theby1, V. Krömker2, whether they contribute to aging of the SSC Sperm alone led to signifi cantly reduced fl u- W. Heuwieser1 niche in man. To characterize HTPCs and the orescence intensities suggesting that the ex- 1Clinic of Animal Reproduction, Freie Universität Ber- associated secretome – also known as the se- tender and the remaining seminal plasma are lin; 2Institiut of Microbiology, Hanover University of nescence-associated secretory phenotype affecting NET formation to a higher extent. Applied Science and Arts, Germany (SASP) – we analyzed in vitro-aged HTPCs A signifi cant increase in FI was seen until 60 (i.e. cells from advanced passages) with pre- Introduction Mastitis therapy generally in- min of incubation indicating that NET forma- senescent (early passages) from 8 individual cludes antibiotic treatment. For the prudent DGRM-Abstracts tion might be fi nished at that time point. men with respect to their quantitative protein use of antibiotic drugs it is essential to iden- profi les by nano-LC-MS/MS. Among more tify the causing pathogen. The gold standard than 3,000 proteins identifi ed, we detected for pathogen diagnostic in milk samples is extensive age-associated changes in the cel- still microbiological culturing on blood agar. 32 lular proteome and the secretome of HTPCs. As logistics and methods are time consuming Our analysis revealed proteins considerably Examination and Biological a diagnose-based start of therapy is delayed altered in abundance in senescent HTPCs, Signifi cance of the Sulfatase Path- by at least 24 h or based on best guess. For which are yet undescribed in this context. bacterial growth the plate has to be incubated way in the Human Testis Furthermore, common hallmarks of aging for at least 24 h, which causes either a delay 1 1 2 2 could be confi rmed, thus emphasizing the va- D. Fietz , K. Hartmann , K. Bakhaus , J. Bennien , in start of therapy or leads to a “best guess” 1 3 4 2 lidity of our approach. The quantitative se- B. Wapelhorst , S. Kliesch , W. Weidner , J. Geyer , choice of antibiotic. Thus a just in time meth- 1 cretome alterations indicate a rearrangement M. Bergmann od for bacterial diagnostic would be advan- 1 of the paracrine network of the testis, in gen- Institute of Veterinary Anatomy, Histology and Em- tageous. Odor was used as a diagnostic indi- 2 eral, and specifi cally consequences related to bryology; Institute of Pharmacology and Toxicology, cator already in ancient centuries. Nowadays 3 the communication of peritubular cells with Justus-Liebig-University, Giessen; Department of electronic noses have proofed to be accurate adjacent SSCs. Hence, changes in secreted Clinical Andrology, Centre for Reproductive Medicine in identifying specifi c odors (e.g. tuberculo- 4 factors may cause an age-related decline in and Andrology, Münster; Department of Urology, sis). Attempts to use this method for diagno- SSC-niche function, underlining the impor- UKGM Giessen, Germany sis of mastitis pathogens were not yet suc- tance of the SASP for neighboring cells and cessful. Dogs have a sensitive olfactory sys- Large amounts of sulfated steroid hormones maintenance of homeostasis. Our data shall tem and are trainable. They are not only used are produced within the human testis. As improve understanding of aging in HTPCs for detection of drugs and explosives, but to shown previously in breast tissue and placen- and delineate the consequences for male re- identify diseases as cancer or diabetes. The ta, these might not only be produced for ex- productive aging. cretion, but may also be re-activated in tar- hypothesis of this study was that dogs can get cells by steroid sulfatase (STS). This pro- discriminate Staph aureus in milk samples of cess is called sulfatase pathway and may play cows suffering from clinical mastitis. 35 a pivotal role in para- and/or intracrine regu- Methods Ten dogs of various breeds and Assessment of Anti-Müllerian sexes were trained to 1.) discriminate Staph lation of testicular cells by creating a local Hormone in Elderly Mares in Rela- steroid hormone supply. This requires a fa- aureus on agar plates and 2.) discriminate cilitated transport via uptake carriers and ef- Staph aureus in milk samples spiked with the tion to Fertility pathogen. At the end of the training the dogs fl ux transporters as these hydrophilic sulfates N. Fouché1, V. Gerber1, R. M. Bruckmaier2, B. Wespi1, were tested if they can discriminate Staph au- cannot pass the cell membrane by diffusion. Y. Zander3, H. Sieme4, D. Burger1 reus in mastitis samples. Dogs could identify Moreover, blood-testis barrier formation in 1Swiss Institute of Equine Medicine, University of Staph aureus in the head space of agar plates the testis requires a transport through Sertoli Bern and Agroscope; 2Veterinary Physiology, Vet- against fi ve other pathogens with an accuracy cells (SCs) to reach germ cells (GCs). SCs are suisse-Faculty, University of Bern, Switzerland; of 97.2%. The accuracy for identifying this therefore expected to play a key role as gate- 3Landgestüt Marbach, Gomadingen; 4Clinic for Hors- pathogen in mastitis milk was 89%. keepers for sulfatase pathway in human semi- es – Unit for Reproductive Medicine, University of niferous epithelium. We analyzed the mRNA Conclusion In this study we demonstrated Veterinary Medicine Hanover, Germany and protein expression of uptake carriers and that Staph aureus can be identifi ed by means effl ux transporters like Organic Anion Trans- of a specifi c odor. The process of ageing is accompanied by porting Polypeptides (OATP2B1, OATP3A1) changes in female hormonal regulation and and Multidrug Resistance-related Proteins in reproductive activity. A hormone that has (MRP1, MRP4) in testicular tissue as well as 34 been shown to be helpful in predicting ovari- cultured SCs (FS1, HSEC). Additionally, ex- an reserve in women is Anti-Müllerian hor- pression pattern of STS as well as sulfonating Quantitative Poteomics Reveals mone (AMH). The relationship between enzymes (SULTs) were analyzed. OATP2B1, Cellular Senescence of Human AMH and fertility has so far not been evalu- OATP3A1, and STS were detected in SCs as Testicular Peritubular Cells ated in mares. The experiments were per- well as GCs, whereas MRP1 was only ex- formed in two public stud farms during the pressed in SCs, and SULT1E1 only in Leydig F. Flenkenthaler1, N. Schmid2, T. Fröhlich1, physiological breeding season from April to cells, respectively. By transcellular transport K. Dietrich2, J.U. Schwarzer3, A. Mayerhofer2, August. A gynecological examination was of [H3]DHEAS in HSEC, we showed a func- G. J. Arnold1 performed according to a standard protocol. tional transport of sulfated steroids in vitro. 1Laboratory for Functional Genome Analysis LAFU- Insemination protocols included the use of Our data indicate a possible steroid synthesis GA, Gene Center; 2Biomedical Center, Cell Biology, fresh, cooled-transported or frozen semen. via sulfatase pathway in Sertoli cells in vivo Anatomy III, Ludwig-Maximilians-University, Munich; Confi rmation of pregnancy was made by ul- and in vitro. This may contribute to a parac- 3Andrologie-Centrum-München, Germany trasonographic examination on day 14 to 20

10 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts and it was recorded whether or not the mare showed Ki67 staining in nuclei and confi rm- 38 was pregnant and if the mare foaled the fol- ing that DID/PKH 26-labeled MSC under- lowing year. The pregnancy rate per cycle and went in vivo multiplication in transplantation Characterization of Prolactin Re- per season was calculated. A total of 44 hors- places. The Ki67 expression was signifi cant ceptor (PRL R) Expression in the es were included in the study. Mares were higher (P < 0.0001) in DID (35.40 ± 4.89) Cat Mammary Gland Tissue aged between 12 and 21 years with a mean and PKH26 (34.81 ± 5.49) positive MSC age of 16.3 ± 2.6 (SD) years. Results for with respect to DID/PKH 26 negative cells S. Gizinski1, M. Domino1, J. Olszewski1, A. Rodo2, AMH ranged between 0.01 and 2.40 ng/ml (13.16 ± 1.94) of surrounding tissue. There Z. Gajewski1 (0.74 ± 0.50 ng/ml). AMH concentrations was no signifi cant differences between Ki67 1Department of Large Animal Diseases with Clinic, were signifi cantly associated with fertility. in both marked-types of MSC (P > 0.158). VRC and CBB, Faculty of Veterinary Medicine; 2De- Lower AMH concentrations were found in partment of Pathology and Veterinary Diagnostics, mares that were positively scanned for early Faculty of Veterinary Medicine, WULS – SGGW, pregnancy (p = 0.036) and looking at AMH 37 Warsaw, Poland in 2 categories (50th percentile), mares with Idiopathic Male Infertility: The Prolactin (PRL) has been fi rst recognized as lower AMH levels had a higher pregnancy Role of Sperm Epigenetics and a hormone that plays an important role in rate per cycle than mares with higher plas- breast cancer initiation and development in ma AMH concentrations (p = 0.033). Foal- Nucleosome Preservation rodents, and, at least partly, in humans. In un- ing rate was not associated with previous 1 1 S. Gies , N. Oeztuerk¹, K. Ni , N. Rogenhofer², affected tissues PRL affects several physio- AMH concentrations (p = 0.869). These re- 1 DGRM-Abstracts K. Steger¹, C. Wrenzycki³, U. Schagdarsurengin logical processes, such as mammary develop- sults suggest a novel role of AMH as a prog- 1 Department of Urology, Pediatric Urology and An- ment, and is suspected to infl uence on mam- nostic marker in equine fertility. drology, Section Molecular Andrology, Justus-Liebig- mary cancer, promote cancer progression and University Giessen; ²Division of Gynecological Endo- acts by binding to a specifi c membrane re- 36 crinology and Reproductive Medicine, Department ceptor – prolactin receptor (PRL R). The ob- of Gynecology and Obstetrics, Clinical Centre of Lud- jective of the current study was to character- Evidence of Mesenchymal Stem wig-Maximilians-University, Munich; ³Clinic for Ob- ize the expression of the PRL R in the feline Cells Proliferation after Transplan- stetrics, Gynecology and Andrology of Large and mammary tumors based upon immunofl uo- tation into the Muscle Layer of Small Animals, Faculty of Veterinary Medicine, Jus- rescence (IF). Samples were collected from Uterine Cervix in Swine tus-Liebig-University Giessen, Germany 48 mature queens during radical mastecto- my with normal (N), dysplastic (D) and neo- 1 1 1 The components of a sperm epigenome are Z. Gajewski , M. Dabrowski , J. Olszewski , plastic (A–C, adenocarcinoma; A, adenoma) 3 1 2 unique and tailored for successful fertiliza- A. Wehrend , B. Pawlinski , A. Burdzinska , mammary tissue and fi xed for IF. The sample 1 tion and proper embryo development. Ab- M. Domino sections were stained with HE, labeled with 1 errations in these components are associat- Department of Large Animal Diseases with Clinic, primary (anti-PRLR) and fl uorescent second- ed with idiopathic male infertility. In hu- VRC and CBB, Faculty of Veterinary Medicine, ary antibodies linked with 7-AAD, then im- 2 man sperm cells a 97% nucleosome-to-prota- WULS – SGGW Warsaw; Department of Immunol- aged using light and confocal microscopy mine exchange occurs to compact and protect ogy, Transplantology and Internal Diseases, Medi- and scanning cytometry respectively. The ep- 3 the paternal DNA. In our previous work we cal University of Warsaw, Poland; Klinik für Veteri- ithelial cell cytoplasm was immuno-positive found that the majority of sperm nucleo- närmedizin, Klinik für Geburtshilfe, Gynäkologie und while the stroma cells were mostly negative. somes occurred in repetitive DNA elements Andrologie der Groß- und Kleintiere mit Tierärztli- No differences in PRLR expression (mean% like LINEs and SINEs. LINE-1 is a retro- cher Ambulanz der Justus-Liebig-Universität Gies- ± SEM) were observed between A–C (22.13 transposon that makes up to 17% of the hu- sen, Germany ± 4.10), A (24.01 ± 7.30), D (20.59 ± 7.67) man genome and therefore it can be used as and N (19.00 ± 3.69) groups, although a low Mesenchymal stem cells (MSC), during in a surrogate for global DNA methylation lev- PRLR expression (5.52 ± 0.86) was observed vitro expansion, undergo a progressive loss of els. DNA methyltransferases (DNMTs) regu- in 10% normal (N), 10% dysplastic (D) and proliferative potential that leads to the senes- late methylation maintenance (DNMT1) and 25% neoplastic (19% A–C; 6% A) samples. cent state, associated with a reduction effi ca- de novo methylation (DNMT3A/3B). The A moderate PRLR expression (25.37 ± 1.46) cy in the treatment of injured tissues. The aim aim of this study was to analyse the LINE-1 was observed in 13% normal (N), 2% dys- was to evidence the proliferation of MSC after methylation and DNMT1/3A mRNA lev- plastic (D) and 21% neoplastic (15% A–C; transplantation into the muscle layer of uter- els (median ± SEM) in spermatozoa of sub- 6% A) samples whereas an overexpression ine cervix in swine. Ten Polish Landrace fe- fertile patients, who underwent ART, and of PRLR (50.42 ± 2.61) was demonstrated in male pigs, weighing 90–120 kg, have under- in healthy donors. LINE-1 methylation was 2% normal (N), 2% dysplastic (D) and 14% gone general inhalational anesthesia (GIA) determined by pyrosequencing. Regarding neoplastic (10% A–C; 4% A). Results point during which 40 ml of red bone marrow were both groups there were no signifi cant differ- that PRL may prove a more important factor gathered from the head of the humerus. The ences in the three investigated LINE-1 CpG in mammary gland carcinogenesis that it was MSC from collected bone marrow were iso- sites (donors: 63% ± 1.08/45% ± 0.97/49% supposed so far. lated and cultivated in vitro during 3 weeks ± 1.14, n = 19; patients: 63% ± 0.98/44% ± as well as labeled with PKH 26 and DID. 0.61/49% ± 0.75; n = 35, p > 0.05). How- The MSC suspension was transplanted (GIA, ever, there were in both groups individu- lapa rotomy) into the muscle layer of the cer- als with strongly varying LINE-1 methyla- 39 vix and after 2 weeks the cervix was collected tion levels (± 25%). With RT-qPCR the Purinergic Receptors in Human and fi xed for immunofl uorescence (IF). Im- DNMT1/3A mRNA levels in sperm cells Testicular Peritubular Cells – munolabeling of Ki67, a marker of prolifer- were determined. These levels were signif- ating cells expressed in all active phases of icantly higher in donors (DNMT1/3A: 1.49 Involved in Male Infertility? the cell cycle (G1, S, G2) were performed. ± 0.2/1.96 ± 0.30; n = 100) compared to pa- L. Glashauser1, K. Dietrich1, A. Tiefenbacher1, The sample sections were labeled with pri- tients (DNMT1/3A: 1.05 ± 0.08/0.47 ± 0.10; J. U. Schwarzer2, F. M. Köhn3, A. Mayerhofer1 mary (anti-Ki67) and fl uorescent secondary n = 48; p = 0.008/p < 0.0001, Mann-Whitney 1Biomedical Center, Anatomy III – Cell Biology, antibodies linked with Hoechst, then imaged U-test). Our study demonstrates that DNMT1 Luwig-Maximilians-University, Planegg-Martinsried; using confocal microscopy and scanning and as well as DNMT3A mRNA levels are sig- 2Andrologie Centrum München, Munich; 3Andrologi- analysis system for fl uorescence respectively. nifi cantly reduced in subfertile patients. In cum München, Munich, Germany Living MSC were found in all translantation contrast, the preliminary methylation data in places where accounted (mean % ± SEM) for LINE-1s show no difference regarding glob- Human testicular peritubular cells (HTPCs) DID 6.64 ± 2.30 and PKH 26 3.90 ± 1.31 of al methylation between fertile and subfertile are the smooth muscle-like cells forming the the cells. Cells positive for DID or PKH 26 men. wall of seminiferous tubules. They may, e.g.

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 11 DGRM-Abstracts

via cytokine secretion, be involved in infl am- tems might rely on genetic selection to de- sidered being ‘not restricted’. Though to the matory processes in the testis often observed velop animals more resistant against specifi c high prevalence of hypospadias in humans in male infertility. Accumulating mast cells in diseases. The aim of this study was to investi- (1 in 200) and the known association with and near the tubular wall further illustrate on- gate potential differences in myometrial con- other congenital abnormalities such as atre- going testicular infl ammation. Beyond that, tractility in vitro examining 35 heifers, which sia ani, it is assumed that this condition ap- mast cells are origin of the ubiquitous pu- had been genetically selected for favorable pears more frequently in pigs than currently rinergic agonist ATP. To explore potential ac- (Q; n = 18) and unfavorable (q; n = 17) pa- described in literature. Therefore, further in- tions of extracellular ATP on purinergic re- rental chromosome 18 haplotypes associated vestigations should aim on examining a pos- ceptors of HTPCs, a series of studies was per- with milk somatic cell score. They received sible genetic background. In the present case, formed. intramammary challenge with 500 CFU E. c. the pedigree of the boar will be analysed and Cultured HTPCs were found to express a va- 24 h (n = 12; Q: n = 6; q = 6) or with 10,000 male siblings will be clinical investigated. In riety of purinoceptor subtypes on mRNA lev- CFU S. a. 96 h (n = 23; Q: n = 12; q: n = 11) addition, a monitoring for this urethral disor- el, among them P2X7, which is known for before slaughter. Tissue was collected on day der of the offsprings will be conducted at the contribution to infl ammatory signaling cas- 39 ± 4 post partum. Four longitudinal myo- slaughterhouse. Together with genetic analy- cades. P2X7 expression in HTPCs was veri- metrial samples per animal were suspend- ses a better understanding of this abnormality fi ed at the cellular level in cultured cells ed in an organ bath containing Krebs solu- will be achieved. and in human testicular sections. Its distri- tion (KS) and air treated with 95% O2 and bution correlated with the degree of fi brot- 5% CO2. The spontaneous (SC) and induced DGRM-Abstracts ic remodel ing in the wall of seminiferous tu- (IC; through natural PGF2 analogue [PGF], bules and strong expression was detected in PGE2, cefapirin, or negative controls [KS]) 42 the immediate vicinity of mast cells. Moni- contractile performance during 30 min in- toring of cultured HTPCs loaded with a calci- tervals (T) were examined over a 3 h period. Assessment of Chilling Injury in um-sensitive fl uorescent dye revealed that the The area under the curve (AUC), mean (MA), Boar Spermatozoa Using a Com- minimal (minA) and maximal (maxA) ampli- potent P2X7 agonist BzATP induced tran- petitive Oviduct Binding Assay sient elevations of intracellular Ca2+ concen- tudes were calculated for every T. Q-animals trations. Pre-incubation of cells with a specif- displayed higher values of minA (P < 0.05) B. Grünther1, H. Henning2, D. Waberski1 ic P2X7 antagonist (AZ11645373) reduced than q-animals during T1 and T2 (SC). No 1Unit for Reproductive Medicine of Clinics, University the magnitude of the BzATP-evoked Ca2+ re- differences in SC between S. aureus/E. coli- of Veterinary Medicine Hanover, Foundation, Germa- lease. Stimulation of HTPCs with BzATP or groups were found. There were no differenc- ny; 2Dep. of Equine Sciences, Faculty of Veterinary ATP increased mRNA levels of infl amma- es among the stimulating substances (PGF, Medicine, Utrecht University, The Netherlands tion-associated genes. PGE2 or cefapirin) compared to KS. Strips originating from Q-animals showed high- Sperm binding to the oviduct epithelium is Taken together, these observations support er minA when stimulated with 10-7 mmol/L an important step on the route to fertiliza- the notion that P2X(7)-mediated actions in cefapirin and 10-7 mmol/L PGE2 than q-an- tion. Testing the sperm’s ability of binding to HTPCs have a say in promoting testicular in- imals. Further investigations are required to fresh (non-cultured) oviduct explants collect- fl ammation. However, it is important to fur- elucidate these differences between Q/q ani- ed from slaughter house tissue may therefore ther characterize the other identifi ed P2 sub- mals. indicate the functional competence of sper- types and to determine possible interactions matozoa. The aim was to test the sensitivity that may also participate in infl ammatory of a standardized competitive oviduct bind- processes. ing assay for the assessment of chilling inju- Grants: Supported by DFG MA 1080/23-1. 41 ry in liquid preserved boar spermatozoa. In- dividual ejaculates (n = 9 boars) were split Hypospadias in a Crossbred Boar and sperm labeled with either MitoTracker® ® A. Grahofer, R. Bill, H. Nathues Green or MitoTracker Red. Subsequently, 40 Clinic for Swine, Vetsuisse Faculty, University of samples were diluted in AndroStar Plus ex- Spontaneous and Induced Myo- Bern, Switzerland tender and stored at 17°C or 5°C, respective- ly. After 24 h and 48 h storage, differential- metrial Contractility In vitro in Congenital abnormalities, cover a broad ly stained semen samples stored at 17°C or Genetically Selected Heifers Post- range of different conditions, occur relative- 5°C were co-incubated for 15 min with the partum after Experimentally In- ly frequently in swine, especially compared same oviduct explant under capacitating con- duced Mastitis with Escherichia to other domestic animals. Hypospadias, a ditions. The binding index (BI), i.e. the num- 2 coli (E. c.) and Staphylococcus congenital anomaly of the urogenital tract ber of sperm bound per mm , and the ratio of characterized by abnormal position of the ex- red to green sperm were calculated. In gen- aureus (S. a.) ternal urethral meatus on the male phallus, is eral, BI differed highly between explants L. Górriz-Martín1, M. Meyerholz2, L. Rohmeier4, rarely described in boars. The present report (16.1–332.9; 36 explants from 14 sows). In J. Safar1, L. Zambo1, M. Hoedemaker1, describes a distal hypospadias in an 8 months Experiment 1 (n = 3), the BI after 48 h was C. Kühn3, W. Petzl4, H. Zerbe4, S. Engelmann5,6, old crossbred intact Pietrain x Duroc boar. higher for sperm stored at 17°C (160 ± 106) H. J. Schuberth2, A. C. Bajcsy1 Until the desired andrological examination, compared to sperm stored at 5°C (72 ± 50; 1Clinic for Cattle; 2Immunology Unit, University of the boar was perceived as healthy in the herd p < 0.05) corresponding to a binding ratio of Veterinary Medicine, Hanover; 3Leibniz Institute for and showed a normal performance. General 69% to 31%. In Experiment 2, a slower cool- Farm Animal Biology, Dummerstorf; 4Clinic for Rumi- physical examination revealed no abnormali- ing regime and AndroStar Plus 5 extender nants, Ludwig-Maximilians-University, Munich; 5Mi- ties. The andrological examination supple- were used (n = 6). The ratio of bound sperm crobial Proteomics, Helmholtz Centre for Infection mented with ultrasound revealed no abnor- was less different for samples stored at 17°C Research, Braunschweig; 6Institute for Microbiology, malities of the genital tract apart from the dis- (54.6%) and 5°C (45.4%) after 24 h storage; Technical University, Braunschweig, Germany tal hypospadias. Subsequently, the boar was the BI did not differ. The binding index mod- exposed to a sow showing signs of oestrus, erately correlated with the amount of sperm Myometrial contractility is of crucial impor- in order to assess the ability to mount and to with intact plasma and acrosome membrane tance for an unimpaired uterine involution ejaculate. The boar showed a normal mating (r = 0.53, p < 0.05), but neither with motili- postpartum. Puerperal metritis and endome- behaviour and the semen quality parame ters ty, nor the responsiveness of sperm to bicar- tritis are common pathological states after were within the normal ranges. Taking these bonate as assessed by a calcium infl ux assay. calving in some cases demanding veterinary results together with the abnormality of the In conclusion, a competitive oviduct explant assistance and seriously affecting the repro- urethra in the anterior part of the penis into assay sensitively detects chilling injury in liq- ductive performance. Future breeding sys- account, the fertility of the boar was con- uid preserved boar spermatozoa.

12 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts

43 Urospermia (US) may result from retard- ty (PM) were assessed using computer assist- ed closure of the urinary bladder neck dur- ed sperm analysis (AndroVision®, Minitüb, Impact of a Maternal Diabetes on ing semen emission due to local infl amma- Germany). Evaluation of semen viability Embryonic DNA Methylation in tion or insuffi cient -adrenergic stimulation was performed using bromophenol-nigrosine Rabbit Blastocyst of the smooth muscle layers. By this urine staining. For cryopreservation, a commercial passes the bladder neck when pre-secretion semen extender (Andromed®, Minitüb, Ger- K. J. Grybel1, E. Canon2, J. Knelangen1, D. and the sperm rich fraction are reaching the many) was used. After 24 h, samples were Kradolfer3, M. Pendzialek1, M. Schindler1, J. Gürke1, pelvic urethra. In the present case US is sus- thawed and examined microscopically af- T. Blachère4, M. Godet4, R. Obeid5, S. Ulbrich3, pected to be the result of a bilateral ectopic ter 3, 5 and 10 min. In native samples, only B. Fischer1, V. Duranthon2, A. Navarrete Santos1 ureter (EU), a congenital malformation, with the TP had a signifi cant infl uence on viabil- 1Department of Anatomy and Cell Biology, Faculty the ureteral orifi ce terminating intravesical- ity of sperm cells (p < 0.0001). The number of Medicine, Martin Luther University, Halle, Ger- ly near the bladder neck or extravesically at of live sperm cells already decreased 10 min. many; 2INRA, UMR, Department of the Reproduc- the prostate, deferent duct, or urethra. Case after collection. The TP had a signifi cant ef- tive Biology, Jouy-en-Josas, France; 3Institute of report: A 4-year-old Entlebucher Moun- fect on semen pH (p < 0.0001). After 10 min. Agricultural Sciences, Zurich, Switzerland; 4Stem- tain Dog was presented for breeding sound- a reduction of the pH could be shown at both cell and Brain Research Institute, Bron, and Universi- ness evaluation due to an unsuccessful mat- temperatures. In frozen-thawed samples, ty of Lyon, Université Lyon I, France; 5Department of ing 5 months ago. At the age of 2 years an temperature during semen evaluation had Clinical Chemistry, University Hospital of the Saar- abnormal course of both ureters in the area a signifi cant effect on PM (p = 0.053). Pro- land, Homburg, Germany of the bladder neck had been diagnosed so- gressive motility was better in samples exam- DGRM-Abstracts nographically. As the fi nding was not classi- ined at 20°C. In conclusion, handling temper- In early pregnancy maternal diabetes leads to fi ed as serious, the dog had been approved for ature was identifi ed as factor infl uencing na- delay in embryo development and changes in breeding. The current andrological examina- tive goat semen assessment, while tempera- nutritional and hormonal signals of the uter- tion revealed both testes and epididymides of ture during semen handling had a signifi cant ine environment. The current study focuses adequate size and shape. The collected ejacu- effect on PM after thawing. on consequences of maternal diabetes on em- late showed normal volume and total sperm bryonic tissue formation and its DNA methy- count, but severe defi ciencies in sperm motil- lation. ity, plasma and acrosome membrane integri- Therefore we investigated the expression ty and sperm morphology. The most striking 46 and promoter methylation of epiblast line- macroscopic fi nding was a yellowish appear- age specifi er Oct4 in 6 day old rabbit blasto- ance showing continuously increasing inten- Enzymes and Transporters in- cysts at early gastrulation stage. The expres- sity in the watery prostatic secretion. Odor volved in the Process of Estro- sion of Oct4 was higher in epiblast of diabet- and pH-value revealed admixture of urine. gen Glucuronidation are Precise- ic blastocysts, accompanied by upregulation A repeat ultrasound examination confi rmed ly Regulated across the Estrous of Nanog and Sox2. Specifi c methylation of a bilateral intramural EU entering the uri- Cycle and during Early Pregnancy the POU5F1 (Oct4) promoter region was in- nary bladder about 1 cm cranial of the vesi- vestigated by bisulfi te sequencing. The Oct4 co-urethral transition with the opening point- in Pigs promoter was hypomethylated in hypoblasts ing caudally. This fi nding may not indicate a A.K. Hankele1, T. Esatbeyoglu2, S. Bauersachs1, and trophoblasts of diabetic rabbits, implying causal connection between EU and US per S. Sterk3, S.E. Ulbrich1 also a mark of delay in differentiation. The se. Nevertheless the latter may be a function- 1Animal Physiology, Institute of Agricultural Sci- global DNA methylation of male and female al indicator of a urine leakage possibly ex- ences, ETH Zurich, Switzerland; 2Institute of Human blastocysts from diabetic and healthy rab- isting as additional congenital abnormality in Nutrition and Food Science, Kiel University, Germa- bits was examined, employing embryoblast the caudal vesical/cranial urethral area. ny; 3RIKILT Wageningen UR, The Netherlands and trophoblast tissue separately, using LU- minometric Methylation Assay (LUMA). No Estrogen metabolism includes the endoge- signifi cant changes in global DNA methyla- nous formation of inactive estrogen conju- tion were observed. Furthermore we verifi ed gates that are proposed to function as a res- that concentrations of methyl group donor S- 45 ervoir for active estrogens during estrous cy- adenosyl methionine (SAM) and the product Factors Affecting Semen Evalua- cle and pregnancy. Based on our study in of the methylation reactions S-adenosylho- tion of Native and Frozen-thawed gilts where estradiol-17 (E2) was orally ap- mocysteine (SAH) were changed in diabetic plied (1000 µg E2/kg body weight/day) and pregnancy. SAM and SAH were measured Goat Semen estrogen metabolites were analyzed in plas- by use of a modifi ed liquid chromatogra- K. Hahn1, A. Wehrend2 ma, bile, heart muscle and endometrium by phy-tandem mass spectrometry in rabbit 1Clinic for Horses, Medicine and Reproduction, GC-MS, we have good evidence that the glu- blood plasma collected at the day 6 post coi- Ludwig-Maximilians-University, Munich; 2Clinic for curonides are the most abundant conjugates tum. Our data showed that maternal diabetes Obstetrics, Gynecology and Andrology of Large and in pigs. To elucidate the potential availabil- mellitus affects the Oct4 promoter methyla- Small Animals with Ambulatory Service, Justus- ity of estrogen glucuronides in the endome- tion in a specifi c way with consequences for Liebig-University Giessen, Germany trium during estrous cycle and early preg- Oct4 gene transcription and embryo develop- nancy, we analyzed the mRNA expression ment. Alteration of Oct4 methylation was not Parameters collected during semen evalua- of enzymes (UGTs, GUSB) and transport- caused by global methylation changes. tion are the basis to establish the suitability of ers (MRPs, OATPs) possibly involved in glu- an ejaculate. Therefore, the conditions must curonide metabolism in the endometrium on be kept as constant as possible. The aim of days 0, 6, 10, 12, 14, and 18 of estrous cycle 44 this study was to evaluate possible factors af- as well as on days 10, 12 and 14 of pregnan- fecting semen analysis of native and frozen- cy. UDP-glucuronsyltransferases UGT1A1, Urospermia in an Entlebucher thawed goat semen. A total of 20 ejaculates UGT1A6 and UGT1A10 displayed mini- Mountain Dog with ectopic ure- from 5 healthy peacock goat bucks were ob- mal expression on day 6 of estrous cycle and ters – A case report tained using an artifi cial vagina. After a mac- maximal expression during late luteal phase. roscopic evaluation 2 aliquots were assessed UGT2B31 however was only minimally ex- A.R. Günzel-Apel1,2, K. Wolf1,2, A. Baumeister1,2, at different handling temperatures (20°C and pressed already during estrous. Beta-glucu- J.P. Bach2, S. Hungerbühler2 37°C) and time points (TP) (3, 10, 15 and 30 ronidase in turn showed highest expression 1Unit for Reproductive Medicine of Clinics; 2Small min. after collection). The pH of each sample at estrous and proestrous and low expression Animal Clinic, University of Veterinary Medicine was evaluated with indicator-paper every 10 during diestrous indicating a negative regula- Hanover, Germany min. for 1 h. Density and progressive motili- tion by progesterone. The mRNA expression

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 13 DGRM-Abstracts

of the infl ux-transporter OATP2A1 signifi - 48 The character and pathogenesis of subclini- cantly increased from day 0–6 and decreased cal endometritis (sE) in dairy cows is not ful- again by day 10, while effl ux-transporters Study of the Impact of Somatic ly understood which makes it diffi cult to set (MRP1, MRP2, MDR1) displayed minimal Cell Nuclear Transfer (SCNT) on a gold standard for its diagnosis. Uterine cy- expression during early diestrous. Pregnant Transcriptome of Early Bovine Em- tology and histopathology are mainly applied pigs displayed differential expression of the bryos as diagnostic devices. A new approach to un- respective enzymes and transporters mainly derstand the pathogenesis of sE and to facil- at day 12. Their precise regulation both dur- M. Heininen-Brown1,3, A. Graf1, A. Wünsch2, itate its diagnosis is the examination of uter- ing estrous cycle and pregnancy indicates a V. Zakhartchenko2, S. Mallok1, S. Krebs1, E. Wolf1,2, ine secretions (uS). A newly developed de- participation of estrogen glucuronides in the H. Blum1 vice is used for the collection of uterine local availability of estrogens to fi ne-tune en- 1Laboratory for Functional Genome Analysis (LAF- secretion samples containing a highly absor- dometrial function. UGA); 2Chair for Molecular Animal Breeding and bent Merocel®-swab. During sampling slight Biotechnology; 3Graduate School of Quantitative haemorrhages may occur in the endometri- Biosciences Munich (QBM), Gene Center, Ludwig- um. The aims of this study are (1) to quan- Maximilians-University, Munich, Germany tify the blood contamination and (2) to dis- play its infl uence on the detected IL-8 con- 47 RNA sequencing (RNA-seq) of bovine centration in uS samples. Whole blood sam- oocytes and different stages of in vitro pro- Peri-Conceptional Overweight ples of ten cows were collected. 100 µl of DGRM-Abstracts duced (IVP) embryos (4-cell, 8-cell, 16-cell, each blood sample were affi liated to the swab in Male and Female Mice causes blastocyst) revealed major embryonic ge- material and processed identically to the uS Sex-Specifi c Transcriptome Chang- nome activation (EGA) at the 8-cell stage samples. A serial dilution of the blood sam- es in Blastocysts which could [Graf et al., PNAS 2014; 4139–44]. The pre- ples was established with phosphate buffer- sent study investigated the impact of cloning ed saline. Of each cow 12 dilution stages of result from differential expressed by somatic nuclear transfer on the transcrip- sncRNA in Spermatozoa of Obese blood samples were examined photometrical- tome profi les of corresponding stages of bo- ly at 570 nm. Mean values of optical density Males vine SCNT embryos. Three pools of 10 em- at each dilution stage were used for calcula- bryos per stage were analyzed. Lysed embry- 1 2 2 3 tion of a type curve of blood dilution. To eval- K. Hedegger , S. Krebs , A. Graf , S. Kunzelmann , os were used to generate and amplify cDNA 2 1,2 1 uate the infl uence of blood contamination on H. Blum , E. Wolf , M. Dahlhoff with the Ovation RNAseq v2 kit ( NuGEN). 1 the presence of chemokines, IL-8 concentra- Institute of Molecular Animal Breeding and Bio- Barcoded sequencing libraries (NuGEN rap- 2 tions of uS and plasma samples of 30 cows technology; Laboratory for Functional Genome id library kit) were sequenced on an Illumi- 3 were determined using AlphaLISA. The im- Analysis; Gene Center, Ludwig-Maximilians-Univer- na Genome Analyzer IIx (80-base single-end sity, Munich, Germany pact of IL-8 originating from blood contami- reads; 20 million per library). Reads were nation on IL-8 concentration in uS samples Human epidemiological studies show that the mapped to version bostau7 of the bovine ref- was calculated. The percentage of blood con- offspring‘s risk to develop obesity in later life erence genome using TopHat2. RNA-seq tamination in uS samples is estimated by its is strongly associated with maternal obesity. data of IVF embryos were re-analysed ac- optical density using a polynomial function. In a previous study, we showed that a peri- cordingly, and differentially abundant tran- Mean values of IL-8 concentration in blood conceptional obesogenic exposure of female scripts (DAT) were identifi ed using DESeq2. contaminated uS and plasma samples were mice is suffi cient to induce sex-specifi c ab- The number of DAT between SCNT and IVP 420 pg/ml and 47 pg/ml, respectively. Con- normal health outcomes in the offspring. In embryos was lowest at the 4-cell stage (266), sidering the percentage of blood contamina- adult male offspring, we found overweight, highest at the 8-cell stage (2841), decreased tion in uS samples and the IL-8 concentration insulin resistance, hyperleptinemia, hyper- at the 16-cell stage (1724) and even further in plasma, the IL-8 concentration in pure uS uricemia and hepatic steatosis, but not in fe- at the blastocyst stage (816). At the 8-cell can be calculated. male offspring. In our current study, we in- stage, most DAT in SCNT embryos were de- vestigated the transcriptome of the early em- creased in abundance, suggesting that nuclear bryo in utero of mice after maternal or pa- reprogramming lacks behind major EGA in 50 ternal pre-conceptional obesogenic exposure. IVP embryos. Analysis of de novo transcrip- Microarray analysis of blastocysts from tion (identifi ed by intron sequence contain- Relationship between Uterine obese fathers, obese mothers and lean parents ing reads above the threshold level observed Contractility and Gene Expression revealed that male embryos from one over- in oocytes) at the 8-cell stage revealed acti- vation of 3548 genes in IVP, but only 1732 of Hormonal Receptors in Puer- weight parent, regardless of father or moth- peral Uteri with and without Me- er, had several upregulated genes compared genes in SCNT embryos. At the 16-cell stage, to control embryos of lean parents. In female SCNT embryos activated a higher number of tritis genes (2772) than IVP embryos (1740). In blastocysts from obese fathers, 48 genes were 1 1 2 summary reprogramming in SCNT embry- M. Heppelmann , J. Volland , C. Pfarrer , downregulated, whereas female blastocysts 3 4 5 os caused a delayed onset of transcription of M. Kietzmann , W. Bäumer , S. Merbach , from overweight mothers only revealed a 6 7 1 multiple genes, including important regula- H. A. Schoon , O. Wellnitz , M. Schmicke , moderate differential gene expression. To ad- 1 8 tors of early development such as NANOG, M. Hoedemaker , H. Bollwein dress the underlying molecular mechanisms 1 2 3 FOSL1, MTA2, and PDCD2. Clinic for Cattle; Institute of Anatomy; Department of the paternal derived transcriptome chang- of Pharmacology, Toxicology and Pharmacy, Univer- es of the early embryo we investigated the sity of Veterinary Medicine, Foundation, Hanover, sncRNA pattern in spermatozoa by next gen- Germany; 4Department of Molecular Biomedical Sci- eration sequencing. We found several differ- 49 ences, North Carolina State University Raleigh, USA; entially expressed microRNAs and piRNAs 5 Quantifi cation of Blood Con- Chemical and Veterinary Investigation Offi ce West- in the spermatozoa of obese males compared phalia, Arnsberg; 6Institute of Pathology, Faculty of to lean males. Our data indicate that over- tamination and its Infl uence on Veterinary Medicine, Leipzig, Germany; 7Veterinary weight of mothers and fathers during the IL-8 Concentration in Uterine Se- Physiology, Vetsuisse Faculty, University of Bern, Po- peri-conceptional period may lead to sex-de- cretion Samples sieux, Switzerland; 8Clinic of Reproductive Medicine, pendent transcriptome changes already in the Vetsuisse Faculty, University of Zurich, Switzerland blastocyst stage. Additionally, we found that A. Helfrich1, H.-D. Reichenbach2, H. Zerbe1, F Weber1 the spermatozoa of obese male mice have a 1Clinic for Ruminants, Ludwig-Maximilians-Universi- The aim of this study was to investigate the different sncRNA pattern, these RNAs may ty, Munich; 2Institute for Animal Breeding, Bavarian relationship between uterine contractility and act as early regulators of the embryonic tran- State Research Center for Agriculture, Freising, Ger- gene expression of hormonal receptors (es- scriptome. many trogen receptor  [ER], progesterone re-

14 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts ceptor [PR], PGF2 receptor [FP] and oxy- viously for equine embryos. The DNA in the (20 ± 5%) in GHR-/- compared to GHR+/- tocin receptor [OTR]) in puerperal uteri with blastocoel fl uid was amplifi ed by PCR us- and GHR+/+ pigs (11 ± 2%). When correct- and without metritis. Therefore, 13 puerper- ing primers to detect the bovine Y-encoded ed for body weight, liver and kidney weights al uteri of cows showing clinical signs of a testis-specifi c protein (TSPY) and 1715 bo- of GHR-/- pigs were reduced (74 ± 6% and metritis (n = 7) or no metritis (n = 6) were vine satellite (b1715Sat). Collapsed or intact 76 ± 12% of GHR+/+ pigs), while relative extracorporeally perfused after euthaniza- blastocysts were vitrifi ed and warmed using brain weight was almost doubled (190 ± 29% tion. Measurement of uterine contractility the open pulled straw (OPS) method. After of GHR+/+ pigs). Mating of a GHR-/- boar was performed by using four piezoelectric warming, they were cultured in vitro for 24 h with a GHR-/- sow (age 8 months) resulted crystals (Sonometrics Corp., London, Ontar- and observed under the stereomicroscope to in a litter of 6 healthy piglets. Their birth- io, Canada), which were implanted into the detect re-expansion. The DNA in the blasto- weight (808 ± 103 g) was markedly smaller myometrium of the great curvature of the for- coel fl uid from 11 out of 11 blastocysts was compared to GHR-/- piglets from GHR+/- × merly pregnant horn in a longitudinal direc- successfully amplifi ed by PCR (5 males and GHR+/- matings (1331 ± 298 g). In summa- tion. After 1 hour of equilibration, a bolus of 6 females). After vitrifi cation, warming and ry, GHR defi ciency in pigs results in reduced oxytocin (5 IU) and PGF2 (2.5 mg Dino- in vitro culture for 24 h, 19/20 intact blasto- body weight gain associated with dispropor- prost) were administered with a time interval cysts and 11/11 collapsed embryos survived. tionate changes in organ growth, but does not of 30 minutes via the perfusion system into Although these results are preliminary, we generally impair fertility. the uterine arteries. The infusion of oxytocin demonstrate that the DNA in the blasto- induced shortenings of 2 distances between coel fl uid can be amplifi ed by PCR to diag- crystals in uteri with metritis and of one dis- nose the sex of bovine IVP embryos. Moreo- DGRM-Abstracts tance in healthy uteri (P < 0.05). In all distanc- ver, all collapsed embryos survived vitrifi ca- es (n = 3) uteri with metritis showed a high- tion, showing that blastocoel fl uid collection 53 er shortening after stimulation with oxytocin does not impair their viability. Further exper- than healthy uteri (P < 0.05). After applica- iments with a larger number of embryos will Pregnancy-associated Glyco- tion of PGF2 no changes of crystal distanc- describe the effi ciency of this methodology. proteins in Cows with retained es were noticed in healthy uteri (P > 0.05). Fetal Membranes In uteri with metritis one distance exhibit- ed a shortening and a widening (P < 0.05). R. Hooshmandabbasi1,2, H. Zerbe3, A. Boos1, Myometrial gene expression of FP and OTR 52 K. Klisch1 was higher (P < 0.05) in metritis uteri than in 1Institute of Veterinary Anatomy, Vetsuisse Faculty, healthy uteri. In summary, PGF2 had no ef- Impaired Weight Gain and Dis- University of Zurich, Switzerland; 2Department of Ba- fect on uterine contractility in puerperal uteri proportionate Organ Growth, but sic Sciences, Faculty of Veterinary Medicine, Univer- without and with metritis. In contrast an in- Preserved Fertility in Growth Hor- sity of Tehran, Iran; 3Clinic for Ruminants, Ludwig- fusion of oxytocin induced a contraction of mone Receptor Defi cient Pigs Maximilians-University, Munich, Germany the uteri. Similar to the gene expression of FP and OTR myometrial contractility was higher (Laron syndrome) Retained fetal membranes (RFM) are a com- mon problem in bovine reproduction and se- in cows with a metritis. 1 1 1 1,2 A. Hinrichs , B. Kessler , M. Kurome , B. Rathkolb , verely impair reproductive health in cows. 3 4 4 5 W. Blum , A. Scholz , M. Bernau , A. Blutke , Associated with a timely release of fetal 1 1,2 M. Dahlhoff , E. Wolf membranes are cellular changes in the mater- 1 Gene Center and Center for Innovative Medi- nal and fetal parts of the placenta. In a mature cal Models (CiMM), Ludwig-Maximilians-Universi- placenta the number of binucleate tro phoblast 51 2 ty, Munich; German Center for Diabetes Research giant cells (BNC) declines prior to parturi- 3 Genetic Testing of Bovine (DZD), Helmholtz Zentrum Munich, Neuherberg; Uni- tion, while in RFM the number remains high. Embryos Using Blastocoel Fluid versity Children‘s Hospital, University of Giessen; Since the BNC produce Pregnancy Associat- 4 5 as a Source of DNA Livestock Center of the Veterinary Faculty; Institute ed Glycoproteins (PAGs) and release these of Veterinary Pathology at the Centre for Clinical into the maternal organism, we hypothesized 1,2 1 1 1 C. Herrera , S. Wyck , L. Bittner , F. Janett , Veterinary Medicine, Ludwig-Maximilians-Universi- that differences of PAG concentrations in 1 H. Bollwein ty, Munich, Germany maternal blood could be used as a dia gnostic 1 2 Clinic of Reproductive Medicine; Center for Clini- tool for RFM in cattle. cal Studies, Vetsuisse Faculty, University of Zurich, Laron syndrome is a rare, autosomal reces- Switzerland sive disorder in humans caused by loss-of- A radioimmunoassay was used to measure function mutations of the growth hormone PAG concentrations in serum samples from Preimplantation Genetic Diagnosis (PGD) of receptor (GHR) gene. To generate a large cows with RFM (n = 20) and from controls embryos is a well-established technique that animal model of GHR defi ciency, we used (n = 68). The numerical density of immu- allows the detection of genes associated to CRISPR/Cas9 in porcine zygotes to mutate nostained PAG-positive BNC was counted in certain diseases or traits. PGD involves ob- exon 3 of the GHR gene.Two heterozygous paraffi n sections of placentomes, that were taining a few cells from an embryo, which founder sows (GHR+/-) with a 1-bp or 7-bp taken at parturition from cows with RFM will then be used for PCR. Recently, Palini insertion in GHR exon 3 (causing a reading (n = 20) and from controls (n = 20). At par- et al. [Reprod Biomed Online 2013; 26: frame shift) were mated with wild-type (Ger- turition mean PAG blood concentrations in 603–10] were able to diagnose the sex hu- man Landrace) boars to obtain heterozygous cows with RFM were slightly higher than man embryos using only the blastocoel fl u- F1 pigs, which were intercrossed to produce in controls (3038.09 vs. 2476.50 ng/ml), but id as a source of DNA. We have also test- GHR-/- pigs. The birthweight of GHR-/- pig- differences were not signifi cant. The densi- ed this technology using equine embryos, lets did not differ from GHR+/- or GHR+/+ ty of PAG-positive BNC in fetal tissue was successfully diagnosing the sex of in vit- littermates. GHR-/- animalsshowed a marked higher in RFM, compared to controls (39.5 ro produced (IVP) and in vivo derived em- reduction in serum IGF1 levels (12.2 ± 9.1 vs vs. 26.5 BNC/mm2, p = 0.02). Our study re- bryos. Therefore, the aim of our work was to 70.9 ± 38.2 ng/ml in GHR+/- and GHR+/+ veals that PAG concentrations in maternal study the blastocoel fl uid of IVP bovine em- pigs determined by RIA). Postnatal growth blood do not differ signifi cantly between bryos, as a possible source of DNA for ge- failure became obvious at the age of 3 weeks. cows with RFM and controls. The number of netic testing and the post-freezing viability At the age of 6 months, body weight of GHR- PAG-positive BNC in RFM was higher than of the embryos after blastocoel fl uid collec- /- pigs (37.7 ± 6.4 kg) was 60% reduced com- in controls. Thus the high PAG concentra- tion. Bovine embryos were produced in vit- pared to GHR+/+ (89.2 ± 15 kg) and GHR+/- tions in maternal blood of cows with RFM ro using slaughterhouse oocytes and some pigs (92.2 ± 13.1 kg). Dual energy X-ray do not result from a “mass exodus” of BNC of the blastocysts were collapsed, by aspi- absorptiometry (DXA) revealed a mark- from the fetal into the maternal placenta pri- rating the blastocoel fl uid as described pre- edly increased percentage of total body fat or to parturition.

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54 in most mammals, up to 6 months in bats or had an infl uence on wound healing: Signifi - even 9 years in some Australian snakes. To cantly more severe rather than moderate/light Camel whey Protein as Anti- date sperm reservoir formation in animals has wound healing disorders could be found if oxidant and Anti-infl ammatory been well documented but the existence of a the Caesarean section took more than 2 hours Ameliorate the Testicular Damage human sperm reservoir has not yet been de- (Fisher: p = 0.022, OR = 6.65). In future a in Streptozotocin-induced Dia- scribed. Using newly established live cell im- larger sample size may point out additional aging technology the aim of our study was signifi cant associations of infl uencing factors betic mice to investigate the human sperm oviduct inter- with wound healing disorders after Caesari- H. El-Din M. Omar1, L. Sayed1, A. Abd El-Rahim2, action under near in vivo conditions. All hu- an section. M. Mahmoud3, G. Badr4 man tubal samples were obtained with ethi- 1Lab.of Physiology, Zoology Dept., Faculty of Sci- cal approval and informed consent. Oviducts ence; 2Dairy Dept., Faculty of Agriculture, Assiut Uni- from 25 patients undergoing ovariohysterec- versity; 3Food Science and Nutrition Dept., National tomy for benign pathologies were co-incu- 57 Research Center, Dokki, Cairo; 4Lab. of Immunolo- bated with fresh or frozen/thawed sperm. Our gy and Molecular Physiology, Zoology Dept., Facul- fi ndings showed that human sperm stay mo- Trueperella Pyogenes isolated ty of Science, Assiut University, Egypt and Deanship tile for up to 4 days in the oviduct. They bind from a Cow Developing Clinical of Scientifi c Research, King Saud University, Riyadh, exclusively to the oviductal secretory cells Endometritis Expressed mRNA of at specifi c sites of the tubal epithelium. This Saudi Arabia Fimbrial Subunits and induced a DGRM-Abstracts binding is competitive and transient in nature Induction of diabetes using STZ in experi- lasting between 1 and 90 seconds with sperm Pro-Infl ammatory Response in Bo- mental animals caused deformation of nor- being observed to change the angle at which vine Endometrial Epithelial Cells mal histological seminiferous tubular struc- they are bound. This is in strong contrast to and Peripheral Blood Mononu- ture mediated by infl ammation and oxida- sperm binding in mammals where there is a clear Cells tive stress. Camel whey protein (CWP) can permanent binding to the cilia until ovulation modulate immune functions and improves with no competition and no preference for M. Ibrahim1, S. Peter1, K. Wagener2,3, M. Drillich2, the normal infl ammatory process in diabetic specifi c binding sites in the isthmus. M. Ehling-Schulz3, R. Einspanier1, C. Gabler1 models. The present study aimed to explore Our results imply that the human sperm res- 1Institute of Veterinary Biochemistry, Freie Univer- the benefi cial role of CWP against STZ in- ervoir is unique in regard to binding sites and sität Berlin, Germany; 2University Clinic for Rumi- duced testicular abnormalities in diabetic binding mechanisms. Sugar-mediated bind- nants; 3Institute of Microbiology, Vetmeduni Vien- mice. Forty-fi ve adult male mice weighing ing might be replaced by more transient mo- na, Austria 25–30 g were divided into 3 groups (15 mice lecular mechanisms in humans. This knowl- st After parturition, different bacterial species each). The 1 group served as control while edge is pivotal in improving the results of as- the other 2 groups were injected with STZ colonize the bovine uterus. Some bacteria sisted reproductive technologies (ART) in can induce a uterine disease, especially T. (60 mg/kg b.w, i.p., 3 successive doses) for male infertility. induction of diabetes. Then after 2 weeks one pyo genes is associated with clinical endome- of the diabetic groups was treated with CWP tritis (CE). Virulence of invading bacteria and (100 mg/kg b.w. for 4 weeks). The diabet- host innate immunity will determine the abil- ic mice showed a reduction in insulin levels, ity of a cow to defend against T. pyogenes. an increase in blood glucose levels and cy- 56 This study investigated a T. pyogenes strain (TP2) isolated from the uterus of a healthy tokines (IL-2, IL-1B, IL-4 IL-6, and TNF-), Assessment of Predisposing Fac- alterations in oxidative stress markers (LPO, dairy cow on day 15 pp, which showed signs CP, NO, GSH, CAT, and SOD), and structur- tors for Wound Healing Disorders of CE on day 21 pp. Aims of this study were al damage in the seminiferous tubules. Sup- after Caesarian Section in Cows to evaluate the transcription of bacterial viru- lence factors crucial for colonizing host cells plementation of diabetic mice with CWP al- J. Humbs, W. Petzl, A. Rieger, H. Zerbe most normalized the blood glucose and insu- and to investigate the effect of this strain on Clinic for Ruminants, Ludwig-Maximilians-University, a pro-infl ammatory response in bovine endo- lin levels and inhibited the elevation of pro- Munich, Germany infl ammatory cytokines and oxidative stress metrial epithelial cells and peripheral blood markers in the testes accompanied by an im- Wound healing disorders are a common com- mononuclear cells (PBMC) in vitro. TP2 was provement in the histological structure of plication after Caesarian section and may re- grown in BHI broth, at 6, 12 and 24h, bac- the seminiferous tubules. In conclusion, the late to different techniques of surgery and lo- teria were harvested, total RNA was extract- supplementation of diabetic mice with CWP cal anesthesia. The aim of the present study ed and subjected to RT-qPCR. Epithelial cells ameliorate the testicular damage infl icted by was to evaluate additional predisposing fac- and PBMC did not survive a co-culture with diabetes possibly by a mechanism suppress- tors. We included 39 cows (aged 1.5–9.9 TP2 longer than 24 h. Endometrial epithelial ing oxidative stress and infl ammation. years), which underwent a Caesarian sec- cells (n = 5) and PBMC (n = 3) were co-cul- tion between August 2014 and March 2016 tured up to 8 h with alive TP2 at a multiplici- at the Clinic for Ruminants, Ludwig-Maxi- ty of infection of 1 and 0.1, respectively. To- milians-University, Munich. In total, 65 vari- tal RNA was extracted from endometrial cells 55 ables (e.g. concurrent diseases, suture tech- and from PBMC and subjected to RT-qPCR. niques, duration of surgery, presence of The mRNA expression of fi mA, fi mC, fi mE, Tubal Sperm Storage in Humans and fi mG was observed in TP2. Epithelial versus Animals – The Reservoir swelling, edema or fl uctuation) were record- ed. Statistical analyses were carried out by cells responded to TP2 by increased mRNA Redefi ned chi-square test (exact Fisher) and odds ratios expression of CXCL3, CXCL5, IL1A, IL6, and IL8 after 8 h compared with controls. B. Hughes, S. Kölle (OR) and relative risks (RR) were calculat- ed. We could confi rm, that a “fi rm” palpato- PBMC co-cultured with TP2 showed high- School of Medicine, University College Dublin, er transcription of PTGS2 and IL8 after 2 h, Ireland ry clinical fi nding of the incision site related to signifi cantly more severe wound healing CXCL3 after 4 h, and IL1A after 6 h com- Upon entering the oviduct, the spermatozoa disorders than moderate or light ones (Fish- pared with controls. In conclusion, this T. py- of most animals bind to the cilia of the ovi- er: p = 0.009). However, we could not con- ogenes strain showed pathogenic characteris- ductal epithelium forming what is commonly fi rm that infi ltration of local anesthesia in the tics, which induced the innate immunity by referred to as the sperm reservoir. This spe- incision site caused signifi cantly more wound increasing the pro-infl ammatory responses in cies specifi c, sugar mediated binding main- healing than paravertebral techniques (Fish- endometrial epithelial cells and PBMC. tains the viability and fertilizing capacity of er: p = 0.669, OR = 6.57). On the other hand Grants: Supported by DFG (GA 1077/5-1) sperm in the female genital tract for 3–4 days we could show, that the duration of surgery and Erasmus Mundus.

16 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts

58 dismutase (SOD), catalase (CAT) and glu- 61 tathione (GSH) content. Histopathologically, PRID Treatment of Anoestrous testes of mice treated with CP loaded AgNPs Fetal Ultrasonographic Measure- Milk Cows with Follicles Growth showed congestion, interstitial edemas well ments near Parturition as Predic- up to the Deviation Stage as degeneration of spermatogenic cells with tors of the Time of Birth in the Do- increase in the number of apoptotic cells in mestic Cat T. Janowski, M. Barczykowski, A. Nowicki, the seminiferous tubules. The prior adminis- W. Baran´ski, M. Rudowska, S. Zdun´czyk tration of RA (0.5 mg/kg/twice weekly, for 2 R. Keiser, I. M. Reichler, O. Balogh Department of Animal Reproduction with Clinic, Uni- weeks) before CP-loaded AgNPs by 24 hours Clinic of Reproductive Medicine, Vetsuisse Faculty, versity of Warmia and Mazury in Olsztyn, Poland signifi cantly reduced the elevation in LPO University of Zurich, Switzerland and NO levels. However, it reduced the effect This paper presents the results of progester- of CP-loaded AgNPs on antioxidant levels. In the cat, accuracy of parturition day predic- one based treatment of anovulatory anoes- The administration of RA before CP-load- tion by ultrasonographic measurement of fe- trous cows with follicles growth up to the de- ed AgNPs injection improved the histologi- tal structures is decreasing towards the end viation stage. The case defi nition of anoes- cal pictures and reduced the number of apop- of pregnancy. Our goal was to determine fe- trous was no detected oestrus during 60–80 totic cells. In conclusion, RT is a potent an- tal biparietal, abdominal and eye diameter days postpartum and the presence of small tioxidant, against CP-loaded AgNPs induced (BPD, AD and ED, respectively) by ultra- follicles (< 9 mm) on ovaries without cor- testicular oxidative damage. Further study sonography within 5 days of delivery to pre- pus luteum. The study was performed on must be done to evaluate this regime in can- dict parturition date. Twenty-fi ve pregnancies DGRM-Abstracts 47 cows from one commercial herd select- cer chemotherapy. in 24 queens of different breeds were exam- ed by transrectal palpation and ultrasound. ined. BPD, AD and ED measurements were Cows were randomly assigned to one of two taken once, on the day of parturition in 13 groups: 1 (n = 31) treated with progesterone 60 queens presented at our clinic due to dysto- insert (PRID Delta, Ceva) or 2 (n = 16) left cia. Eleven queens were examined on 62–63 untreated. Clinical response and reproductive Can post-thaw Motility of Canine days after the fi rst mating and 48–72 hours dates were recorded for 120 days after treat- Semen be Predicted by the Hypo- later if birth did not occur. Two pregnancies ment by rectal palpation and ultrasound. The osmotic Swelling Test? were excluded because of elective Cesarean oestrus rate (51.7% vs 19.5%) and total preg- section. Linear regression analysis was used nancy rate (87.1% vs 18.8%) were higher in S. Karger, B. Geiser, M. Grau, O. Burfeind, for the association of BPD, AD and ED with group 1 than in control cows (P < 0.05). Also W. Heuwieser, S. Arlt days before birth (DBB). Fetal ultrasono- treatment-conception interval 115.8 ± 34.6 Clinic for Animal Reproduction, Freie Universität Ber- graphic measurements varied among and vs. 234.7 ± 21.9 was shorter in treated cows lin, Germany within litters. There was a signifi cant, moder- (P < 0.05). In contrast the number of insemi- Integrity of sperm cell membranes is consid- ate relationship between BPD, DBB and lit- nations per conception (2.6 vs 1.7) was better ered to be crucial for survival during cryo- ter size. While BPD increased with time clos- in control group. Although, in general there is preservation because cells are exposed to er to birth, litter size was inversely related to no recommendation for hormonal treatment considerable osmotic challenges. Objective BPD. Linear regression showed a low agree- of anoestrous cows follicular development ar- of this study was to examine relationships be- ment between AD and DBB, which was not rested in its early stage, our results suggest tween Hypoosmotic swelling (HOS) test re- infl uenced by litter size. Fetal ED length and that PRID protocol can be used with moder- sults and standard semen parameters before width were not associated with time to par- ate success rate in these cases. freezing and after thawing. Semen of 35 pri- turition. Using the linear regression equation vate owned male dogs of different breeds was of Beccaglia et al. (2008) for BPD measure- ments within the last fi ve days before parturi- 59 collected and analyzed. Standard semen pa- rameters as well as the HOS test were eval- tion resulted in less accurate birth prediction Retinoic Acid Modulates Testi cular uated in fresh and frozen-thawed semen (40% within 1 day and 53% within 2 days) samples. For cryopreservation CaniPRO™ than it was previously reported for earlier Oxidative Damage induced by pregnancy stages. In conclusion, fetal ultra- Cisplatin-loaded Silver Nanoparti- Freeze A&B and for thawing CaniPRO™ Culture Medium (MOFA) were used. For sonographic BPD and AD have low accura- cles in Mice grading as good quality, frozen-thawed se- cy for the estimation of the day of birth when measured in the last 5 days of pregnancy. Fe- 1 2 3 men had to show at least 50% progressive A. Abd el Ghaffar , N. Nafady , H. El-Din M. Omar , tal ED measured shortly before birth is not 1 4 3 motile sperm cells and less or equal than 20% N. Mohamed , M. Sayed , E. Ahmed suitable for estimation of delivery date in the 1 morphologi cal abnormal sperm cells. Cryo- Department of Pathology and Clinical Pathology, cat. Faculty of Veterinary Medicine; 2Department of preservation caused a decrease in all stand- Botancy, Faculty of Science;3Department of Zoology, ard semen parameters as well as in the results Faculty of Science, Assiut University; 4Department of of the HOS test (P < 0.01). In fresh semen, 62 Chemistery, Animal Health Resaerch Institute, Assiut HOS test results were correlated to progres- Laboratory, Egypt sive motility (ρ = 0.51), viability (ρ= 0.50) Metoclopramide Administration and normal morphology (ρ = 0.46). In fro- Changes Milk Composition in Oxidative stress has been proven to be in- zen-thawed semen correlations between HOS volved in cisplatin- (CP-) induced toxicity. test and progressive motility (ρ = 0.67) and Lactating Bitches Silver nanoparticles modulate ATP-Binding viability (ρ = 0.86) were found. Fresh semen S. Keller1, N. Sprenger2, S. Austin2, B. Wichert3, Cassette transporter (ABC transporter) ac- HOS test results were correlated with post- A. Liesegang3, I. Reichler1, O. Balogh1 tivity and enhance chemotherapy in multid- thaw viability (ρ = 0.50), normal morpholo- 1Clinic of Reproductive Medicine, Vetsuisse Faculty, rug resistant cancer. The present study was gy (ρ = 0.55) and HOS test results (ρ = 0.43). University of Zurich; 2Nestle Research Center, Nest- designed to evaluate the antioxidant activ- Out of 35 ejaculates, 14 were classifi ed show- ec S.A., Lausanne; 3Institute of Animal Nutrition, ity of Retinoic Acid (RA) against CP-load- ing good semen quality. For testing predict- Vetsuisse Faculty, University of Zurich, Switzerland ed Silver Nanoparticles induced testicular ability of individual freezability, results of oxidative damage in mice. Our data indicat- fresh semen parameters and HOS test results Hypo- or agalactia in the bitch negatively ed signifi cant increases in lipid peroxides of good and poor semen quality were com- affects puppy growth rate and results in in- (LPO) and Nitric Oxide (NO) levels in tes- pared. Statistical analysis demonstrated that creased pup morbidity and mortality. Meto- tes of mice treated with CP- loaded AgNPs it is not possible to predict post-thaw motil- clopramide (MC) is recommended to stimu- (2 mg/kg/week, for 2 weeks) that was asso- ity using standard semen parameters such late milk production through increases in se- ciated with a signifi cant reduction in the ac- as progressive motility (ρ = 0.22), viability rum prolactin (PRL), but this has not been tivity of the antioxidant enzymes superoxide (ρ = 0.33) or the HOS test results (ρ = 0.35). confi rmed in the dog. While direct assess-

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 17 DGRM-Abstracts

ment of milk yield is not possible, monitor- histologically investigated and divided into 65 ing milk energy and nutrient content may neonates, juveniles and adults. Within sem- give an indication on the effects of MC on ca- iniferous cords/tubules of newborn and ju- Expression of PTGS2 and PGFS/ nine milk composition and yield. Lactose is venile harbour porpoises only spermatogo- PTGFR during Recrudescence the major determinant of milk volume by its nia and Sertoli cells could be observed. Tes- of Canine Spermatogenesis fol- osmotic actions, and its production is stimu- tes of adult males showed special character- lowing Downregulation with an lated by PRL through increased glucose up- istics during the course of the year. Analysis take and availability of -lactalbumin [Ben- further revealed that in sexually active males Azagly-nafarelin Implant Jonathan et al., 2006]. We hypothesize that different stages of spermatogenesis could be H. Körber, S. Goericke-Pesch MC stimulates milk production by increas- detected. Results of the present study give Section for Veterinary Reproduction and Obstetrics, ing milk lactose. Ten control and ten treated fi rst insights into the regular postnatal repro- University of Copenhagen, Denmark (MC at 0.2 mg/kg PO q6h for 6 days) healthy ductive biology of male harbour porpoises in bitches were included in the study. Body German waters. This analysis is the baseline Cyclooxygenases (PTGS) and Prostaglandin- weight, age, parity and litter size was similar data for the identifi cation of alterations in tes- F2 synthase (PGFS) are considered to play between the groups. Milk was collected on ticular development and spermatogenesis in an important role for male fertility. PTGS2 Day 0 (10–24 h after parturition and before this wild life species due to human activites and PGFS are expressed in Leydig cells (LC) start of treatment), and on Day 4 and 6. Day and changing environmental factors. In ongo- and arachidonic acid has a stimulatory ef- 0 samples, and the combined Day 4+6 sam- ing studies, the cellular processes of season- fect on LC-derived testosterone production.

DGRM-Abstracts ples were analyzed for gross energy (GE) by al changes in adult males will be investigat- Consequently, they are considered to be in- bomb calorimetry, dry matter (DM) by drying ed immunohistochemically with established volved in regulation of Steroid acute regula- till constant weight, and lactose by high per- Sertoli cell and germ cell markers. tory (StAR) protein. Application of a slow re- formance anion exchange chromatography. lease GnRH agonist implant results in a ful- Data was analyzed by independent and paired ly reversible downregulation of testicular en- t-test, Mann-Whitney-U test and Wilcoxon 64 docrine (and germinative) function including signed rank test. In the whole population and Prolonging Survival Time of the whole steroidogenic apparatus with StAR being the bottle neck. To further investigate in each group, milk lactose (g/kg) on whole Spermatozoa in the Female Geni- milk, GE (J/g) on DM, and lactose/DM sig- the role of PTGS2 and PGFS on steroidogen- nifi cantly increased from Day 0 to Day 4+6. tal Tract – Novel Insights esis, testicular samples were obtained from Milk DM did not signifi cantly change over S. Kölle1, R. Leeb2, M. Trottmann2 previously downregulated mature male Bea- time. Milk composition was similar between 1School of Medicine, University College Dublin, gle dogs during different times of testicular the groups on Day 0. On Day 4+6, lactose/ Ireland; 2School of Medicine, University of Munich, recrudescence (week 0, 3, 6, 9, 12) after im- DM was signifi cantly higher in the treated Germany plant removal. Untreated adult healthy dogs than in the control group, which may indicate (CG, n = 5) and juvenile dogs (JG, n = 3) higher milk yield in the treated dogs based on Successful fertilization only occurs if sperm served as controls. Expression of PTGS2, the osmotic properties of lactose. successfully survive in the female geni- PGFS and PTGFR on mRNA level was iden- tal tract till ovulation takes place. Thus, the tifi ed in all samples, with, however, no sig- 63 aim of our study was to prolong survival time nifi cant differences between groups in whole of spermatozoa in the bovine female geni- testicular homogenates. Specifi c immuno- Seasonal Effects on Testicular tal tract. For this purpose, we investigated positive staining against PTGS was found in Development and Spermatogene- sperm survival time before and after addition the cytoplasm of LC and Sertoli cells (SC). For LC, staining intensity was weak in week sis in Harbour Porpoises from the of specifi c supplements under in vitro condi- tions (37 oC, sperm-TALP). The effectivity 0 and strong in week 3; no difference was North and Baltic Sea of successful candidates was then evaluated found between week 6 to 12 and CG. Where- T. Kesselring1, S. Viquerat1, M. Langeheine2, under near in vivo conditions using live cell as SC cytoplasm was most intensely stained A. Gröne3, L. I. Jsseldijk3, P. Wohlsein4, U. Siebert1, imaging in the oviduct. The following sup- at the basal membrane, it was equally stained R. Brehm2 plements were evaluated: a) protein extracts from week 3 onwards. In JG, strong staining 1Institute for Terrestrial and Aquatic Wildlife from bovine oviductal cells of cyclic cows of LC and SC (adluminal) was found. Identi- Research; 2Institute for Anatomy, University of and heifers, b) protein extracts from bovine fi ed differences in LC-PTGS protein expres- Veterinary Medicine Hanover Foundation, Hano- oviductal cells by from cyclic cows after co- sion at downregulation and during recrudes- ver, Germany; 3Veterinary Pathology Diagnostic Cen- incubation with sperm and c) L-Arginine (0.1 cence correlate with StAR protein expression tre, Utrecht University, Utrecht, The Netherlands; mM, 1 mM, 10 mM, 15 mM, 30 mM). Our and indicate a need for investigation of LC/ 4Department of Pathology, University of Veterinary studies showed that protein extracts from ovi- interstitial tissue only to verify possible dif- Medi cine Hanover, Foundation, Germany ductal cells (0.15 µg/ml) from cyclic cows ferences on mRNA level. were able to increase the number of motile The harbour porpoise (Phocoena phocoena) sperm over 6 hours up to 25% as compared 66 is a seasonal breeding marine mammal that to control sperm incubated in sperm-TALP can be found native in the German North and without supplement. Contrary there was no The possible Role of the SIRT1 Baltic Sea. The subpopulation from the cen- signifi cant effect when using the protein ex- Enzyme in Oxidative Stress and tral Baltic Sea was listed as critically endan- tracts of heifers or after co-incubation with Spindle Assembly in Bovine gered by the IUCN Red List. Like all mam- spermatozoa. L-Arginine (1 mM) was able Oocytes – A large Animal Model mals, harbour porpoises undergo distinct to increase the number of motile sperm up to phases towards sexual maturity. However, 20% and over a time interval of 8 hours under for Reproductive Ageing knowledge about male reproductive biolo- in vitro conditions. Live cell imaging showed P. Kordowitzki1,2,3, K. Hadeler1, P. Aldag1, A. Lucas- gy such as postnatal testicular development, that this also occurred under near in vivo con- Hahn1, L. Wu2, D. Sinclair2,3, H. Niemann1 seminiferous tubule differentiation, age of ditions without affecting physiological be- 1Institute of Farm Animal Genetics, Friedrich- initiation of spermatogenesis and morpho- havior of sperm in the female genital tract. Loeffl er-Institut, Mariensee, Germany; 2Laborato- logical changes associated with regression Our results show that addition of arginine and ry for Ageing Research, School of Medical Science, and re-initiation of spermatogenesis as well supplementation of proteins secreted from University of New South Wales, Sydney, Australia; as seasonal changes in testicular activity is oviductal cells prolong sperm survival time 3Department of Genetics, Harvard Medical School, scarce. To gain more insights in initial signs in the female genital tract. This knowledge is Boston, USA of testicular maturity, testes of male harbour pivotal for reducing sperm concentrations in porpoises of various ages that became strand- straws and increasing commercial success in Chromosomal defects and oxidative stress ed or bycaught in the years 1998–2016 were artifi cial insemination. become much more prevalent with advanc-

18 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts ing maternal age and are considered the ma- dium pyruvate. Both aliquots were equili- (P < 0.05) only SERBP1 mRNA, but P4 in jor factors responsible for the increased in- brated for 24 hours before freezing. Frozen the dose of 10-5M increased (P < 0.05) the cidence of miscarriage and birth defects sperm samples were thawed, and examined expression of this gene. Other factors did not in women over 35 year of age. However, immediately after thawing (0 h) as well as affect (P > 0.05) the gene expression. These mecha nisms that modulate the meiotic ap- after 3, 6, 12, and 24 h incubation at 37 °C. results suggest that steroids may regulate paratus remain to be discovered. The SIRT1 By using computer assisted sperm analy- PGRMC1, SERBP1 and mPR mRNA ex- enzyme deacetylates a spectrum of transcrip- sis (CASA) system and fl ow cytometric as- pression in the bovine myometrial cells the tional regulators and can be activated by res- says respectively, the percentages of rapid- day and dose-dependent effect and this way veratrol. Therefore, the goal of our large an- ly motile sperm (RMS), plasma membrane may infl uence on the myometrium function imal model was to examine, how the SIRT1 and acrosome intact sperm (PMAI), sperm during bovine estrous cycle. enzyme infl uences oxidative stress and with high mitochondrial membrane poten- spindle assembly in bovine oocytes from do- tial (HMMP), and sperm with a high degree nors of different age. COCs of prepubertal of DNA fragmentation (%DFI) were deter- (5–6 months old), adult cows (2nd lactation) mined. Sperm diluted with the extender con- and aged cows (10th lactation) were collected taining sodium pyruvate showed higher lev- by Ovum Pick-up twice a week. Medium for els of RMS, PMAI and HMMP and low- 69 IVM and IVF was supplemented with 2 µM er %DFI values at all time points after thaw- Resveratrol® (Sigma-Aldrich, Buchs, Swit- ing (P < 0.001) compared to sperm frozen in A Comparison of Diagnostic zerland). Standard lab protocol was used as an extender without sodium pyruvate. The Methods used for the Identifi - DGRM-Abstracts control. SIRT1 activity was measured by us- results of this study show that the addition cation of coagulase-negative ing a fl uorometric SIRT1 activator kit (Bio- of sodium pyruvate to the semen extend- Staphylococci isolated from Cows Vision, Milpitas, USA). The spindle assem- er improves not only the viability, but also bly of oocytes was analyzed by fl uorescence the DNA integrity of cryopreserved bovine with Mastitis immunostaining under a confocal micro- sperm. Therefore, in an ongoing study we in- J. Król1, J. Twardo´n2, A. Wanecka1, J. Mrowiec2, scope (LSM510, Zeiss, Germany). Reactive vestigate the effect of sodium pyruvate on A. Bartoszewicz2, A. Korzeniowska-Kowal3, oxygen species (ROS) levels were assessed fertility of bovine sperm. A. Tobiasz3 in oocytes by using Dihydroethidium (Ther- 1Department of Pathology; 2Department of Reproduc- mo Fisher, Bonn, Germany). In total 238 tion and Clinic of Farm Animals, Wroclaw University COCs of all 3 donor groups were analyzed. of Environmental and Life Sciences; 3Department of A 4-fold increase of SIRT1 enzyme activity 68 Immunology of Infectious Diseases, Ludwik Hirszfeld (4 independent replicates) could be detected Effect of Steroids and Luteotropic Institute of Immunology and Experimental Therapy, in oocytes of resveratrol treated groups com- Polish Academy of Sciences, Wrocław, Poland pared to the ones out of the control groups. and Luteolytic Factors on Mem- A high frequency of spindle defects and brane Progesterone Receptors In many countries, members of the genus chromosome disorganization were observed mRNA Expression in the Bovine Staphylococcus have now become the lead- in the oocytes of the untreated aged donor ing cause of intramammary infections (IMI) group (35.5 ± 8.7%) and a lower frequency Myometrial Cells in cows. Apart from the major pathogen in the resveratrol treated aged donor group K. Krajza, M. Kowalik, J. Kotwica S. aureus, coagulase-negative staphylococ- (9.6 ± 3.8%, respectively 4 independent rep- Institute of Animal Reproduction and Food Research, ci (CNS) are also more and more frequent- licates). The ROS levels (12 independent Polish Academy of Sciences, Olsztyn, Poland ly isolated. In spite of the well-recognized replicates) were signifi cantly lower (121 ± pathogenic potential of many CNS species 34 FIU) in the resveratrol treated samples We have found the variable expression of and their marked heterogeneity, they are fre- than in those of the control group (865 ± 45 mRNA and protein for progesterone recep- quently not identifi ed at the species level by FIU, p  0.05). In conclusion, these results tor membrane component (PGRMC) 1 and routine laboratory procedures. The aim of the indicate that resveratrol is able to infl uence 2, PGRMC1 binding partner – SERBP1 (ser- present work was to compare various diag- SIRT1 enzyme activity, the redox status and pine 1 mRNA binding protein) and mem- nostic methods used for the identifi cation of spindle formation in bovine oocytes of dif- brane progestin receptors (mPR) ,  and  CNS isolated from cows with IMI. The study ferent maternal age. In this regard, SIRT1 in bovine myometrium during the estrous cy- was performed on 120 isolates of CNS re- could be a marker for reproductive ageing cle, but the regulatory mechanism of mRNA covered from milk samples collected in the and useful for further understanding of hu- expression of these receptors in myometrium years 2013–2016 in three dairy farms in Low- man infertility caused by ageing. is not clear. The aim of the present study was er Silesia, Poland. The bacteria isolated were to examine the effect of P4 (10-7;10-6;10- tentatively identifi ed as CNS by colony mor- 5M), estradiol (E2; 10-10;10-9;10-8 M), P4 phology, Gram staining, catalase activity and together with E2 (10-6M P4/10-9M E2; 10- the coagulase test. Then, 3 independent diag- 5M P4/10-8M E2) and oxytocin (OT;10-7M), nostic procedures were carried out: pheno- 67 prostaglandin (PG) E2 (10-6M), PGF2 (10- typic identifi cation (ID 32 STAPH system, Improvement of DNA Integrity in 6M), tumor necrosis factor  (TNF; 10-4M) bioMérieux), genotypic identifi cation (se- Cryopreserved Bovine Sperm by and arachidonic acid (AA;10-5M, positive quence analysis of the 16S rRNA gene and, if control) on gene expression for: PGRMC1, necessary, dnaJ gene) and MALDI-TOF-MS Sodium Pyruvate PGRMC2, mPR,  and  in the bovine myo- analysis (Bruker Daltonics, Germany). Of the 5 F. Korkmaz, M. Siuda, E. Malama, H. Bollwein metrial cells (2.5 × 10 /ml; n = 5) from days methods used, the most accurate turned out to Clinic of Reproductive Medicine, Vetsuisse Faculty, 6–10 and 11–16 of the estrous cycle. After be the DNA-based ones. However, sequenc- University of Zurich, Switzerland 6h, the concentration of PGF2 and PGE2 ing of the 16S rRNA gene alone allowed for in medium and mRNA expression of stud- an unequivocal identifi cation of only 70% This study aimed to assess effects of addition ied genes were determined by EIA and Real isolates. The remaining strains were suc- of the carbon source and antioxidant sodium Time PCR method, respectively. Obtained cessfully assigned to the species level by se- pyruvate on sperm quality of cryopreserved data indicate that E2 (10-8M) stimulated quence analysis of the dnaJ gene. The pheno- bovine sperm with special attention to DNA (P < 0.05) expression of PGRMC1 mRNA, typic identifi cation and MALDI-TOF proce- integrity. For this purpose, in each of 23 Sim- and P4 (10-5M) and P4 together with E2 in- dure showed a lower diagnostic performance. mental AI bulls three ejaculates were collect- creased (P < 0.05) mPR mRNA in the cells Results of our study suggest the necessity of ed. In a split sample design ejaculates were from 6–10 days of the estrous cycle. In the upgrading the existing diagnostic procedures diluted by using a TRIS egg yolk extender cells from 11–16 days of the estrous cycle, and databases for CNS isolated from masti- without and with the addition of 5 mM so- P4 (10-6 M) and E2 (10-10M) decreased tis in cows.

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 19 DGRM-Abstracts

70 RNA sequencing (RNA-seq) of pools of bo- cut from gels, decoloured and identifi ed by vine embryos revealed major embryonic ge- mass spectrometry using MALDI TOF tech- Production of Porcine Embryonic nome activation at the 8-cell stage [Graf et nique. The results of identifi cation were com- Chimeras by Morula Aggregation al., PNAS 2014; 4139–44]. The present study pared with Swiss-Prot database. The analy sis addressed the questions i) how heterogene- of gel pictures showed 1,393 detected spots. 1,2 1 1 2 M. Kurome , B. Kessler , K. Simmet , K. Nakano , ous the transcriptome profi les of early embry- Out of them 59 spots were signifi cantly dif- 2 1,2 H. Nagashima , E. Wolf onic cells are; and ii) if expression profi ling ferent between examined samples. 32 spots 1 Chair for Molecular Animal Breeding and Biotech- provides evidence for early differentiation expressed higher intensity of staining in pre- nology and Center for Innovative Medical Models events. Using single-cell RNA-seq, we deter- mature delivery than in control samples while (CiMM), Ludwig-Maximilians-University, Munich, mined the transcriptome profi les of 161 blas- 27 spots showed opposite relationship. Nine 2 Germany; Meiji University International Institute for tomeres from in vitro produced bovine em- spots which differed signifi cantly were iden- Bio-Resource Research, Kawasaki, Japan bryos at Day 2 (n = 6) and Day 3 (n = 8) post tifi ed. Three of them expressed higher inten- Chimeras have been proven as an invaluable fertilization. After removal of the zona pel- sity of staining in premature delivery than in experimental tool for studying mechanisms lucida, blastomeres were separated mechani- controls (Dedicator of cytokinesis protein 1, 2+ 2+ of embryonic development, as well as for cally in Ca - and Mg -free PBS and lysed Metallothionein-2, Guanylyl cyclase-activat- evaluating the status of pluripotent stem cells. individually in buffer A of Prelude Dilute Ly- ing protein 1) and 6 showed opposite rela- To optimize a chimera system based on mor- sis Module (NuGen). RNA-seq libraries were tionship (Epithelial-stromal interaction pro- ula aggregation using in vitro produced (IVP) prepared using the Single Cells RNA Bar- tein 1, Serum albumin, Tyrosine--tRNA li- DGRM-Abstracts pig embryos, the effect of decompaction on coding and Sequencing (SCRB-Seq) pro- gase, cytoplasmic [EC:6.1.1.1], Protein production effi ciency and developmental po- tocol (http://dx.doi.org/10.1101/003236). chibby homolog 3, Leukemia inhibitory fac- tential was investigated. Furthermore, the The libraries were sequenced on an Illumina tor receptor, Adenosylhomocysteinase 3 generation of presumptive tetraploid chime- HiSeq 1500 (50 nt, paired-end reads; 45000 [EC:3.3.1.1]). In conclusion, further research ras was attempted. Compacted morulae de- unique molecular identifi er [UMI] reads on with higher number of patients is necessary rived from IVP parthenotes were used for the average per library). After normalization in order to confi rm the usefulness of saliva as experiments. Decompaction was performed with DESeq2, expression profi les were clus- the source of biomarkers of different altera- by incubation in Ca2+/Mg2+-free DPBS con- tered using R package SC3 (http://dx.doi. tions including premature delivery. taining 0.1 mM EDTA-2Na. After that, the org/10.1101/036558). Three clusters of cells zonae pellucidae were removed and the in- were revealed: CI: 43 Day 2, 28 Day 3 cells; tercellular junctions were further loosen ed CII: 31 Day 3 cells; and CIII: 59 Day 3 cells. by pipetting. The embryos were treated with The order of these clusters was determined 73 by computing their distances. In CI, enriched phytohemagglutinin (150 µg/ml) and pairs MRI Imaging of Placenta in Feline of embryos were cultured individually. A gene ontology (GO) terms were “DNA meta- pair forming a single blastocyst after 3 days bolic process” and “Organelle localization”. K. Lorenc, K. Skierbiszewska, K. Siewruk, T. Jasinski, of culture was assessed as complete chime- In CII, but not in CIII, the most enriched Z Gajewski ra, monitored with the PrimoVision system. GO term was “RNA processing”. Our anal- Department of Large Animal Diseases with Clinic, Presumptive tetraploid embryos were gener- ysis suggests developmental heterogeneity in VRC and CBB, Faculty of Veterinary Medicine, WULS ated by electrofusion of 2-cell embryos. Most Day 3 embryos, with some delayed cells hav- – SGGW, Warsaw, Poland aggregations from compacted morulae result- ing a transcript profi le of Day 2 cells. Two ed in two separated blastocysts, while decom- additional distinct clusters of cells in Day 3 Magnetic resonance imaging (MRI) has a su- paction of morulae signifi cantly improved embryos may indicate fi rst differentiation perior soft-tissue contrast compared to other the formation of chimeric blastocysts (3.1%, events. radiological imaging modalities and its struc- 1/32 vs. 70.5%, 31/44; P < 0.05). Decompac- tural and functional applications have led tion treatment did not affect development to to a signifi cant increase in performed MRI blastocyst (95.5%, 84/88). Presumptive tetra- scans worldwide. Magnetic resonance imag- ploid embryos treated by decompaction effi - 72 ing is considered to be safe and is not asso- ciently formed complete chimeric blastocysts ciated with risk. Nevertheless, it should be as well (85.7%, 6/7). These results indicate Preliminary Studies on Salivary used prudently only when use is expected that decompaction treatment markedly im- Protein Profi le in Human Physio- to answer a relevant clinical question. Mag- proves the formation of porcine embryonic logical Pregnancy and Complicat- netic resonance imaging serves as a prob- chimeras by morula aggregation. lem-solving examination in instances where ed by Premature Delivery the US fi ndings are equivocal or where ad- Grants: Funded by the German Research 1 2 3 ditional information is needed. Animal mod- Council – Transregio Collaborative Research M. Lopucki , J. Wawrzykowski , T. Geca , 3 2 2 el provides a convenient way to optimize MR Center 127 “Xenotransplantation”. A. Miturski , M. Franczyk , M. Kankofer 11st Chair and Clinic for Oncologic Gynaecology and protocol that is particularly important in fe- Gynaecology, Medical University in Lublin; 2Depart- tus and placenta diagnostics. The aim of the ment of Biochemistry, University of Life Sciences in study was to evaluate structure and function- Lublin; 3Chair and Clinic for Obstetrics and Pathology al parameters in feline placenta on a 3.0T MR of Pregnancy, Medical University in Lublin, Poland scanner. MRI examination was performed in 71 pregnant female cats prior to elective ova- Easy obtainable and reasonable biomarkers riohysterectomy. Anesthetized cat was posi- Single-cell RNA Sequencing of of different physiological and pathological tioned feet fi rst, prone on the MR table. Pro- Bovine Day 2 and Day 3 Embry- states are crucial for appropriate diagnosis. tocols were validated with MR imaging on os reveals Developmental Hetero- The hypothesis was stated that the analysis Discovery MR750w 3.0T. MRI examination geneity and Early Differentiation of salivary proteomic profi le may allow for can provide additional information about pla- Events the selection of possible biomarkers of pre- centa function complimentary to US imag- mature delivery in humans. Saliva samples ing. However feline model is challenging due I. Lavagi1,3, S. Krebs1, K. Simmet2, V. Zakhartchenko2, were collected around 30 weeks of pregnan- to a small FOV what increases scan time, dis- E. Wolf1,2, H. Blum1 cy from 8 patients with diagnosis of prema- tortion, and noise level. On the other hand, 1Laboratory for Functional Genome Analysis ture delivery and 8 controls with physiologi- the model is particularly useful for the specif- (LAFUGA); 2Chair for Molecular Animal Breeding and cal course of pregnancy. Samples were sub- ic study of the diagnostic methods used in hu- Biotechnology; 3Graduate School of Quantitative Bio- jected to 2D electrophoresis. Statistical mans as well as MRI is superior method for sciences Munich (QBM), Gene Center, Ludwig-Maxi- analysis of electrophoretic gels allowed for pregnancy pathologies evaluation in domestic milians-University, Munich, Germany the selection of spots of interest, which were cats in veterinary medicine.

20 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts

74 Two studies were performed to investigate the of E. coli and S. aureus. Vitamin D3 had no effects of bovine mastitis and endometritis on effect on the immune response of udder tis- Long-term Exposure of Boar the corpus luteum (CL). In the fi rst study, on sue in vitro. The epigenetic modifi ers SAHA Spermatozoa to Seminal Plasma Day 9 of the estrous cycle (Day 1 = ovula- and S2101 signifi cantly blocked the upregu- during In Vitro-Storage and Sub- tion), 14 lactating cows received once 200 µg lation of CXCL8, TNF, S100A9 and LAP sequent Thermic Stress may affect E. coli LPS (dissolved in 10 mL NaCL; n = 8) (P < 0.05). However, the pathogen induced or 10 mL saline (n = 6) into one quarter of the upregulation of the IL10 was not affected af- Sperm Quality mammary gland. In the second study, estrous ter treatment with SAHA and S2101. Tran- A. Luther1, H. Henning2, D. Waberski1 cycles with and without intrauterine infusions script abundances for CXCL8 were validat- 1Unit for Reproductive Medicine of Clinics, Univer- of 9 mL phosphate buffered saline with LPS ed by ELISA for IL8 and a functional chemo- sity of Veterinary Medicine Hanover, Germany; 2De- (3 µg/kg) every 6 h from 12 h before until 9 d taxis assay. In conclusion these data show partment of Equine Sciences, Faculty of Veterinary after ovulation were examined in 8 heifers. In the potential of epigenetic modifi ers to block Medicine, Utrecht University, The Netherlands both studies, blood sampling and ultrasonog- overshooting infl ammation in the udder. Thus raphy of the ovaries were performed on Days epigenetic modifi ers may serve in future as High dilution of boar semen is associated 1, 4, 8, 9, 10, 12, 15, 18, and afterwards eve- immune modulators in treatment and prophy- with a reduction of seminal plasma (SP). This ry 2 d until ovulation. Biopsies of the CL for laxis of clinical mastitis. might restrict the reduction of sperm numbers RT-qPCR were taken 24 h before and 6 h after Grants: Funded by Deutsche Forschungsge- in semen doses. The aim of this study was to intramammary LPS, and on Day 6 of cycles meinschaft (PE 1495/2-1). examine the effect of reduced amounts of SP with and without intrauterine LPS. Repeated DGRM-Abstracts in semen doses with regular and low sperm measures ANOVA was performed. Intramam- numbers on sperm quality. Centrifuged se- mary LPS caused severe local infl ammation, 77 men from individual boars (n = 8) was used an increase (P < 0.05) in plasma levels of cor- The Population of Small RNAs in to produce semen doses (100 ml) in Belts- tisol and haptoglobin, but no change in those Cryopreserved Semen of Fertile ville Thawing Solution with standardized of PGF2 metabolites (PGFM) and proges- amounts of autologous SP (0.5%, 10%) and terone (P4), luteal size (LTA), and luteal blood and Sub-fertile Bulls 9 9 sperm numbers (1.0 × 10 , 1.8 × 10 ). Semen fl ow (LBF). Furthermore, mRNA abundance E. Malama1, S. Bauersachs2, J. Bick2, F. Janett1, doses were stored at 17°C for 144 h. Motili- of TNF was increased (P < 0.05), whereas H. Bollwein1 ty assessment with CASA revealed no differ- those of TLR4 and steroidogenic STAR and 1Clinic of Reproductive Medicine, Vetsuisse Facul- ence between samples until 72 h storage. At 3HSD remained similar. In contrast, intra- ty, University of Zurich; 2Group of Animal Physiology, 144 h, sperm motility was dramatically re- uterine LPS induced subclinical endometritis Institute of Agricultural Sciences, ETH Zurich, Swit- duced (P < 0.001) in samples of half of the and premature luteolysis, which was charac- zerland boars (n = 4) with 1.0 × 109 sperm in pres- terized by increased PGFM and decreased P4, ence of 10 % SP (19 ± 9%) when compared LTA and LBF (each P < 0.05). Furthermore, The present study was a preliminary attempt to 0.5% SP (70 ± 7%). In samples from the intrauterine LPS increased mRNA expression to characterize the population of small RNA other boars, motility was higher with 10% of TLR4, decreased STAR and 3HSD (each molecules in cryopreserved bovine semen, SP (84 ± 6% vs 77 ± 4%; P < 0.01). Samples P < 0.05), and had no effect on TNF. In sum- collected from bulls of diverse fertility sta- with 10% SP contained fewer membrane in- mary, intramammary LPS induced systemic tus. For this purpose, single ejaculates from tact sperm (P < 0.05) irrespective of the ab- infl ammatory reactions and alterations in lu- 10 mature bulls serving as sperm donors in solute sperm number. After incubation with teal mRNA but no lysis of the CL, whereas a commercial semen production center were additional thermic stress (38°C for 300 min) intrauterine LPS induced premature luteolysis used. The corrected fertility index (FI) based at 72 h storage, motility and curvilinear ve- without clinical signs of infl ammation. on the non-return rates of more than 300 AI locity were lower (P < 0.05) in samples with was used for the classifi cation of the bulls in 10% SP compared to samples with 0.5% SP two groups: a) fertile bulls, with FI > mean without major boar differences. In doses with 76 FI+SD of the center’s bull population, and b) 1.8 × 109 sperm, motility was 60 ± 19% (10% Epigenetic Modifi ers alter the Im- sub-fertile bulls, with FI  mean FI-SD of the SP) vs 74 ± 23% (0.5% SP), while in doses center’s bull population. From each one of with 1.0 × 109 sperm, motility was 44 ± 7% mune Response in udder Tissues the 10 frozen-thawed ejaculates, total RNA ® (10% SP) vs 67 ± 5% (0.5% SP). In addition, L. Macias, H. Zerbe, W. Petzl was extracted applying the standard TRIzol the percentage of membrane intact sperm with Clinic for Ruminants with Ambulatory and Herd (Thermo Fisher Scientifi c, Switzerland) pro- high mitochondrial transmembrane potential Health Services, Ludwig-Maximilians-University, tocol followed by DNase treatment. Small was lower (P < 0.05) in samples with 10% SP. Munich, Germany RNA libraries were prepared with the NEXT- In conclusion, long-term exposition of sperm fl ex Small RNA-Seq Kit v2 (Bioo Scientifi c, to 10% SP during storage infl uences sperm Mastitis accounts for large parts of antimicro- USA) and sequenced using the Illumina high- quality in a boar-specifi c manner. Under ther- bial drug use in dairy production worldwide. throughput sequencing technology. After ex- mic stress conditions in vitro the presence of Due to the imminent normative to reduce the cluding sequences of rRNA, tRNA and 7SL 10% SP is detrimental to boar sperm quality. use of antimicrobial drugs in livestock, new RNA, the analysis of the data revealed 1750 ways for therapy and prophylaxis of masti- unique small RNA sequences (with mini- 75 tis are needed. Recently epigenetic regulation mum length of 16 nucleotides) in total, with of infl ammation by chromatin modifi cations miRNA and piRNA being the most abundant Lipopolysaccharide-induced has increasingly drawn attention. Currently classes of RNA molecules. Unique sequenc- Mastitis and Endometritis have some epigenetic modifi ers have already been es with > 100 reads were mapped to species- different Effects on the Bovine approved for the use in humans, however lit- specifi c precursors in the miRBase 20.0 us- Corpus Luteum tle is known about their actions in the bo- ing BLAST. Hierarchical cluster analysis vine system. The aim of our study was to in- showed signifi cant differences between fer- J. Lüttgenau1, B. Lingemann1, O. Wellnitz1,2, vestigate whether three epigenetic modifi ers tile and sub-fertile bulls regarding the fre- D. Kradolfer3, A.K. Hankele3, M. Schmicke4, S.E. (Vitamin D3, SAHA and S2101) infl uence quency of the 43 most abundant RNA mole- Ulbrich3, R.M. Bruckmaier2, H. Bollwein1 the initial immune response towards masti- cules (P < 0.05). Concluding, next generation 1Clinic of Reproductive Medicine, Vetsuisse Faculty, tis pathogens in bovine udder tissue. Tissue sequencing revealed a wide variety of small University of Zurich; 2Veterinary Physiology, Vetsui- explants of the teat cistern and udder paren- RNA molecules in cryopreserved bovine se- sse Faculty, University of Bern; 3Animal Physiology, chyma were collected from 21 cows and were men. The results of this study further suggest- Institute of Agricultural Sciences, ETH Zurich, Swit- preincubated in the absence and presence of ed a differentiation in the population of small zerland; 4Clinic for Cattle, University of Veterinary epigenetic modifi ers. Subsequently the tissue RNAs in sperm collected from bulls with dif- Medicine Hanover, Germany was stimulated with heat inactivated particles ferent fertility.

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 21 DGRM-Abstracts

78 individuals, impaired fertility, reduced vitali- syndrome and single giant cells. Germ cell ty, weaker growth and worse performance sloughing was observed in a few tubules. Impaired Glucose Tolerance in could appear. The aim of this study was to es- There was complete absence of sperm in Newborn Piglets Exposed to Mild timate the relationship and the inbreeding co- the epididymides. Tubules with intratubu- Hyperglycemia in utero effi cient based on pedigree analy sis of mares lar lymphocytic infi ltrations showed loss of participating in stationary performance tests. smooth muscle actin, indicating a mechani- A. Martins1,2, E. Streckel2, C. Braun-Reichart2, Pedigree data from stationary riding perfor- cal and functional alteration of the tubular B. Kessler2, A. Bähr2, P. Peugnet2, A. Blutke3, mance tests of 1007 mares obtained in Pol- wall. The staining patterns of the epithelial B. Rathkolb3, R. Wanke3, M. Hrabe de Angelis4, ish training stations in 2007–2012 were com- tight junction proteins claudin3/-11 and the E. Wolf1,2, S. Renner2 bined and analyzed using INBREED proce- gap junction protein con nexin43 were dis- 1MWM Biomodels, Tiefenbach; 2Institute of dure of the SAS package and a self-written continuous suggesting an alteration of the Molecular Animal Breeding and Biotechnology, Gene software. The group included mares of the BTB at these locations, and vimentin identi- Center; 3Institute of Veterinary Pathology, Ludwig- following breeds: Polish Warmblood (sp) fi ed somatic Sertoli cells. These fi ndings in- Maximilians-University; 4Institute of Experimental (n = 469), Malopolska breed (mlp) (n = 224) dicate the possibility of an autoimmune or- Genetics, Helmholtz-Centre, Munich, Germany and Wielkopolska breed (wlkp) (n = 314). The chitis leading to azoospermia. For the fi rst mean inbreed coeffi cients and the mean relat- time an impairment of the canine BTB and Elevated blood glucose during pregnancy is edness between breeds, within breeds and be- the tubular wall associated with a putative detrimental for the fetus as it increases the tween training stations were determined in autoimmune orchitis could be demonstrated risk to develop metabolic diseases as obesity DGRM-Abstracts the population of studied mares. Mean rela- by using immunohistochemistry. or diabetes in adulthood. However, effects of tionships and inbreeding coeffi cient within maternal diabetes and underlying mechanisms the entire population of analyzed mares were are diffi cult to study in humans and have 0.59% and 0.38%, respectively. An inbreed- not been fully clarifi ed by animal models. ing coeffi cient over 10% was achieved for 4 Here, the effects of maternal hyperglycemia 81 mares undergoing stationary tests (3 mares of on neonatal offspring are investigated using Wielkopolska and 1 of Malopolska breeds). INSC93S transgenic pigs exhibiting reduced Establishment of a Reliable In total, 76% of the mares were not inbred. In glucose tolerance and insulin secretion as well Method to Generate Bovine conclusion, the inbreeding coeffi cient in the as mild hyperglycemia. INSC93S transgenic Embryos via Intracytoplasmic population of half-bred mares is maintained (tg, n = 3) and wild-type (wt, n = 9) sows were at a low level. The obtained mean values of Sperm Injection (ICSI) artifi cially inseminated. Non-pregnant (np) wt relationship and inbreeding coeffi cients can sows served as controls. Fasting blood glucose L. Maurer, J. Stöhr, F. Kotarski, C. Wrenzycki show the correctness of the method of selec- was monitored weekly throughout pregnancy. Clinic for Veterinary Obstetrics, Gynecology and An- tion of stallions. Within the 3rd trimester hyperinsulinemic-eu- drology, Giessen, Germany glycemic clamps and mixed-meal glucose tol- Intracytoplasmic sperm injection (ICSI) is a erance tests were performed. At birth, wt pig- successful method of assisted reproduction lets born to wt sows (wt/wt, n = 18) and wt 80 technology to treat male factor infertility. In piglets born to tg sows (wt/tg, n = 13) under- addition to its clinical usefulness, ICSI is also went an oral glucose tolerance test before fi rst Impairment of the Blood-testis a valuable research tool for studying funda- colostrum intake. As in humans, pregnant (p) Barrier in a Beagle Dog Showing mental aspects of gamete interaction during wt sows showed a signifi cantly reduced insu- Intratubular Lymphocytic Infi ltra- fertilization. Oocyte activation is a prerequi- lin sensitivity (–35%) compared to np-wt sows tion site for decondensation of the sperm head (p = 0.028), while insulin sensitivity of preg- 1 1 1 and formation of the male pronucleus regard- nant INSC93S transgenic sows was reduced to C. Matschurat , K. Rode , J. Heinrich , 1 1 2 2 less of whether the spermatozoon enters the the same extent. Glucose tolerance was near- M. Langeheine , D. Walter , K. Wolf , C. Urhausen , 2 1 oocyte by injection or by fertilization. In cat- ly, however not fully (p = 0.018) sustained in A.R. Günzel-Apel , R. Brehm 1 tle, additional oocyte activation is necessary wt-p vs wt-np sows due to increased insulin Department of Anatomy, University of Veterinary 2 for embryonic development after ICSI. How- secretion while pregnant INSC93S transgen- Medicine Hanover; Unit for Reproductive Medicine ever, oocyte activation might also increase ic sows did not meet the increased insulin de- of Clinics – Small Animal Clinic, University of Veteri- the number of parthenotes. The objective of mand (p = 0.04). Fasting hyperglycemia in nary Medicine Hanover, Germany the present work was to establish a reliable tg-p sows did not deteriorate throughout preg- Impairment of blood-testis barrier (BTB) in- method to generate bovine embryos via ICSI. nancy. Newborn wt/wt piglets revealed unal- tegrity has been observed in infl ammation, Oocytes were matured for 22 h in TCM199 tered body and organ weights compared to wt/ infection, trauma and experimental auto- supplemented with BSA and eCG/hCG. ICSI tg piglets but reduced oral glucose tolerance immune orchitis, which is inducible in ro- was performed after denudation of cumu- and alterations in lipid metabolism. Metabo- dents. In the present study, an initially fertile lus-oocyte-complexes. After ICSI, the oo- lomics analyses will give further insight into 2-year-old Beagle dog presented a decline in cytes were activated by incubation in 5µM the offspring’s metabolism after exposure to total sperm number to azoospermia within ionomycin for 5 min and subsequently cul- mild hyperglycemia in utero. 5 months, verifi ed by twice monthly semen tered in TALP. The oocytes were then incu- analyses. The dog was clinically healthy bated in 2mM 6-DMAP for 3 h. Sperm-in- 79 and showed normal thyroid function. Bacte- jected and control oocytes which were only riological semen examination, hematologi- activated or sham injected and activated (par- Relationships between Coeffi cient cal and serum biochemical analyses resulted thenogenetic controls) were cultured in SOF and Inbreeding level for Polish in physiological fi ndings. To assay the caus- medium. After 18 h of culture embryos were Warmblood Mares Participating in es of azoospermia, the dog was fi nally cas- stained with Hoechst 33342 to visualize pro- trated. Histological examination of haema- nuclear formation. First results show a cor- Stationary Performance Tests toxylin and eosin stained testicular sections rect pronucleus formation (2PN/2PB) rate of M. Masko1, M. Domino2, T. Jasinski2, Z. Gajewski2 and immunostaining of smooth muscle ac- 20.8% (27/129) in the ICSI group. In the con- 1Department of Animal Breeding, Faculty of Animal tin, claudin3, claudin11, connexin43 and vi- trol groups, rates of chemical activation (CA) Science; 2Department of Large Animal Diseases with mentin were conducted to elucidate a pos- or CA and sham-injection (SHAM+CA) in- Clinic, VRC and CBB, Faculty of Veterinary Medicine, sible impairment of the BTB. Histological dicated by the generation of haploid embry- WULS – SGGW, Warsaw, Poland examination revealed focal interstitial and os (1PN/2PB) were 51.7% (56/115, CA) and intratubular lymphocytic infi ltrations, an ar- 46.7% (43/91, SHAM+CA). These data show Inbreeding has a negative effect on the repro- rest of spermatogenesis at different levels, that bovine embryos can be generated via ductive characteristics of animals. For inbred semi niferous tubules with Sertoli cell only ICSI in a reasonable number.

22 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts

82 ic merit model” involved Holstein heifers with ferences between groups were detected con- low (LFH) and high fertility (HFH) index and cerning day of calving p.i. (Q: d278.6 ± 3.1 Bisphenol A Induced Oxidative heifers from the Montbéliarde (MBD) breed. vs. q: d275.7 ± 7.8), day p.p. when uterus Damage in the Rat Testis: A possi- Providing the microenvironment for oocyte, was detectable in the pelvic cavity via tran- ble Cause for Decline in Steroido- sperm and early embryo, oviduct fl uid (OF) srectal palpation (Q: d19 ± 6.7 vs. q: d20 ± genesis plays a pivotal role for fertilization and early 3.8), incidence of retained fetal membranes embryo development. We performed a holis- (P > 0.1) or uterine weight at day of slaugh- A. Meki1, T. Essa2, S. Abd Elghaffar3 tic proteome analysis of OF samples from am- ter (Q: 0.669 ± 0.095 kg vs. q: 0.622 ± 0.123 1Department of Biochemistry, Faculty of Medicine, pulla and isthmic regions of both models us- kg). However, Q-heifers showed reduced in- Assiut University; 2Departments of Anatomy, Faculty ing nano-LC-MS/MS analysis and a label-free cidence of metritis grade I and II compared to of Medicine, Alexandria University; 3Department of quantifi cation approach. Substantial quantita- q-heifers (P = 0.006). The causal relationship Pathology and Clinical Pathology, Faculty of Veteri- tive differences among the OF proteomes were between genotype and immune competence nary Medicine, Assiut University, Egypt detected. Among the 2,240 proteins identifi ed, needs further investigation. However, genet- a total of 216 differed signifi cantly in pairwise ic selection might be profi table for breeding Bisphenol A (BPA), an estrogenic chemi- comparisons. The majority of proteome dif- cows with better periparturient clinical per- cal, has been shown to adversely affect hu- ferences was found in isthmus OF samples of formance. man health and fertility; however, the under- MBD vs. LFH (52 proteins), MBD vs. HFH lying mechanisms remain unknown. The pre- (47), and MH vs. Lact (19). Evaluation of this 85 sent study was done to investigate the effect dataset using bioinformatic tools revealed that DGRM-Abstracts of BPA on pre-pubertal male reproductive or- affected proteins are predominantly assigned The Impact of Nutritional Status gans by histological examination and the bio- to the GO terms translation, immune response chemical assays of hormones and oxidative on Histotroph Composition dur- and cytoskeletal protein binding. Therefore, ing the Periconceptional Period in stress markers. Forty male albino rats were the dataset comprises a pool of proteome divided equally into 4 groups. GI was served changes associated with fertility, supporting Mares as control and was injected SC with the vehi- the assumption that immune processes are in- 1 1 2 2 cle (DMSO); GII, GIII and GIV were inject- V. Milojevic , A. Rudolf Vegas , L. Wimel , C. Dubois , volved in early embryonic loss. M. Robles2, P. Chavatte-Palmer2, K. Giller1, S.E. ed SC with BPA in a dose of 11.4, 57.1 and 1 1 114.2 mg/kg b.w/ day, respectively. The se- Ulbrich , B. Drews 1Institute of Agricultural Sciences, Animal Physiol- verity of the BPA effects observed was dose 84 2 1198 dependent, SC injection of BPA (57.1 and ogy, ETH Zurich, Switzerland; INRA, UMR Biolo- 114.2 mg/kg b.w/day) signifi cantly decreased Genetically Selected Cows differ gie du Développement et Reproduction, Jouy en Jo- the levels of testosterone and LH in compari- in Periparturient Clinical Perfor- sas, France son with controls and signifi cantly increased mance Embryo development in the horse is unique the oxidative stress markers in the testis. The 1 2 3 with respect to its prolonged preimplanta- histopathological changes observed in the M. Meyerholz , L. Rohmeier , L. Gorriz Martin , 4 1 1 tion period. Prior to attachment, the uterine testis treated with BPA demonstrated its po- A. Heimes , T. Eickhoff , A. Hülsebusch , 5,6 3 4 luminal fl uid or so-called histotroph is the tentials to induce cytotoxic and endocrine S. Engelmann , M. Hoedemaker , H.M. Seyfert , 4 2 2 1 embryo’s sole source of nutrients. It can be disrupting effects on the spermatogenic, Ser- C. Kühn , W. Petzl , H. Zerbe , H.J. Schuberth 1 directly altered by nutrition and may serve toli and Leydig cells. The amount of sperm Immunology Unit, University of Veterinary Medicine 2 for the identifi cation of metabolic biomark- in the epididymis was decreased in GIII and Hanover; Clinic for Ruminants, Ludwig-Maximilians- 3 ers during the periconceptional period. We there were no sperm in the epididymis in University, Munich; Clinic for Cattle, University of 4 aimed at determining the infl uence of mater- GIV. These results indicate that exposure to Veterinary Medicine Hanover; Leibniz Institute for 5 nal nutritional status on histotroph composi- BPA induces oxidative damage in the rat tes- Farm Animal Biology, Dummerstorf; Institute for Mi- crobiology, Technical University Braunschweig; 6Mi- tion. Ten mares were selected from a herd of tis and impairs steroidogenesis which may broodmares at the research station in Cham- lead to male infertility. crobial Proteomics, Helmholtz Centre for Infection Research, Braunschweig, Germany beret, France, and assigned to 2 groups (n = 5/ group) based on body condition score (BCS, 83 High incidences of periparturient diseas- INRA-HN-IE 1997 scale) and subcutaneous es in dairy cows have a negative impact on fat thickness (FT) (FT_BCS = [FT*BCS]/ Impact of Metabolic Status and economic effi ciency, frequency of antibiot- [FT+BCS]), as introduced by [Superchi et Genetic Merit for Fertility on ic treatment and animal welfare. Genetic se- al., 2014]). The normal and overweight group Proteomic Composition of Bovine lection for disease resistance is a sustainable had a FT_BCS of 0.87–1.07 and 1.09–1.26, tool for improving dairy farming. The aim respectively (P = 0.01). Plasma and uterine Oviduct Fluid of our study was to evaluate uterine involu- fl uid were collected on day 7–8 after ovula- K. Meyer1, T. Fröhlich1, M. Kösters1, E. Riedel1, tion and diseases (incidence and severity) in tion. In both, obesity related hormones (lep- S. Fritz2, P. Salvetti2, P. Lonergan3, E. Wolf1, Holstein heifers having genetically been se- tin, adiponectin, insulin) were determined by G. Arnold1 lected via single nucleotide polymorphism RIA. LC-MS was used to characterize the 1Laboratory for Functional Genome Analysis typing for alternative parental chromosome metabolic profi le and GC-MS for the fatty LAFUGA, Gene Center, Ludwig-Maximilians-Univer- 18 haplotypes associated with favourable acid content. There was no effect of FT_BCS sity, Munich, Germany; 2Allice, Paris, Franc; 3School (Q) or unfavourable (q) udder health. Heif- on hormones. There was a trend (P = 0.07) of Agriculture and Food Science, University College ers (n = 36, 18Q/18q) originating from dif- towards higher fatty acid concentration in Dublin, Belfi eld, Dublin, Ireland ferent German farms were kept in individual plasma of animals with a higher FT_BCS, pens at the Clinic for Cattle in Hanover and with conjugated linoleic acid being signifi - Over the last decades, a decrease of dairy cow supervised from day (d) 259 post insemina- cantly different (2.9 ± 0.5 vs 3.5 ± 0.3 µg/g, fertility, comprising early embryonic loss, has tion (p.i.), around calving until slaughter on P  0.05). Plasma analysis showed differen- been observed. To study the effects of meta- d39 ± 4 postpartum (p.p.). Health status was tial abundance (P  0.05) of 34 metabolites bolic status as well as genetic merit for fertil- monitored daily, rectal palpation and trans- involved in e.g. energy metabolism, cell sign- ity of dairy cattle on oocytes, embryos, and rectal ultrasonography was performed once aling and energy storage, compared to 17 dif- their maternal environment, two animal mod- a week p.p. In case of disease, animals were ferentially metabolites in histotroph, with an els have been created by the EU consortium treated according to good veterinary practice. overlap of 2. Our results suggest that nutri- FECUND. The “metabolic disturbance mod- Occurrence of retained fetal membranes, me- tional status impacts plasma and histotroph el” included lactating cows (Lact) that were tritis grade I and II were recorded. Compar- composition via different pathways. FT_BCS compared with cows dried off after calving isons between groups were performed using effects on the histotroph in which the embryo (Dry) and maiden heifers (MH). The “genet- Chi-squared test and unpaired t-test. No dif- develops demand further investigation.

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 23 DGRM-Abstracts

86 of the bacterial count in commercially pro- ed after freeze-drying, sperm chromatin of cessed boar semen. In total 224 fresh boar se- freeze-dried sperm was found to be large- Motility of Stallion Epididymal men samples collected and processed at an ly intact (DFI-values: 5–8%). Sperm freeze- Sperm after 48 hours of Cooled AI Station were analyzed. The number of live dried in TRIS+ without supplements exhibit- Storage using two different Ex- bacteria was determined by using fl ow cy- ed rapid changes in chromatin structure dur- tenders tometry 15 minutes after staining with SYBR ing dried storage at 37°C, reaching DFI val- Green I and Propidium Iodide. In the fi rst ues of 68 ± 33 and 95 ± 5% after 2 and 4 S. Neuhauser1,5, P. Gösele1,5, A. Deutsch1, part of the study 111 fresh boar semen sam- weeks storage, respectively. Also, sperm D. Mehnert2, T. Kosjak3, A. Kaul4, C. Lischer5, ples were spiked with pure cultures with de- freeze-dried in skim milk extender or TRIS+ J. Handler1,5 fi ned numbers of bacteria commonly detect- supplemented with glucose/albumin were 1Pferdezentrum Bad Saarow, FU Berlin; 2Tierarzt- ed in boar ejaculates (Staphylococcus, Strep- found to have DFI-values of 83−65% after 1 praxis Ottersberg; 3Pferdeklinik München-Riem; tococcus, Aeromonas, Pseudomonas, Pro- month storage. However, if freeze-drying was 4Tierarztpraxis Letschin; 5Klinik für Pferde, FU Ber- teus, Klebsiella, E. coli) and analyzed on the done in TRIS+ supplemented with disaccha- lin, Germany day of sperm collection. A strong correlation rides with or without albumin DFI-values re- between the measured amounts and the ex- mained 9−11%. Taken together, sperm chro- Collection of epididymal sperm can be a last pected numbers could be observed (p < 0.01, matin structure can be preserved during long- chance to preserve genetic material of valu- Pearson correlation coeffi cient = 0.97). In the term dried storage at elevated temperatures, able breeding stallions. Twenty epididy- second part of the study 113 fresh boar semen after freeze-drying with non-reducing sugars

DGRM-Abstracts mides were collected after routine castration samples were analyzed. On the day of collec- such as sucrose or trehalose. of macroscopically normal gonads from ten tion the samples were analyzed via fl ow cy- healthy, 2.5 years old stallions of different tometry and the serial dilution cultures for breeds. Sperm from the cauda epididymidis the Most Probable Number (MPN) technique 89 were collected and diluted to 25×106 sperm/ were prepared and subsequently assessed Mammary Squamous-Cell Carci- ml with Equi Plus (group 1) or Gent extender after 48 hours of incubation. The bacterial (group 2) for chilled semen (Minitüb, Tiefen- counts determined with both methods were noma (SCC) in the Mare bach, Germany). Total motility (TMOT, %), only moderately correlated (p < 0.01, Spear- 1 1 1 progressive motility (PMOT, %), curviline- J. Olszewski D. Domanska , M. Domino , man‘s rho = 0.28) with higher values detect- 1 1 1 ar velocity (VCL, µm/s), average lateral head B. Pawlinski , M. Petrajtis-Golobow , K. Górski , ed by the MPN method (MPN: 145,923 ± 2 1 displacement (ALH, µm) and beat cross fre- A. Wehrend , Z. Gajewski 300,921 bacteria/ml; fl ow cytometry: 37,549 1Department of Large Animal Diseases with Clin- quency (BCF, Hz) were evaluated. Differenc- ± 59,538 bacteria/ml). In summary fl ow cy- es were evaluated by Wilcoxon signed rank ic, VRC and CBB, Faculty of Veterinary Medicine, tometry is a suitable and fast method to de- WULS – SGGW, Warsaw, Poland; 2Klinik für Veteri- test and p-value < 0.05 was considered sig- termine bacterial count in boar semen. Dif- nifi cant. In group 1, TMOT (61 [19–87] and närmedizin, Klinik für Geburtshilfe, Gynäkologie und ferences in the results of both techniques may Andrologie der Groß- und Kleintiere mit Tierärztli- 54 [24–79]), PMOT (57 [15–86] and 50 [19– be related to the time of measurement. While 78]), and VCL (145.2 [119.8–174.1] and cher Ambulanz der Justus-Liebig-University Gies- with the fl ow cytometric method the bacteri- sen, Germany 143.4 [122.1–160.1], p > 0.05) did not dif- al count is measured directly after collection fer between H0 and H48, respectively, while of semen, the classical microbiological cul- Mammary tumors are common in dogs and ALH increased [3.2 (2.6-3.8) and 3.5 (3.0- ture techniques are evaluated 48 hours later. cats but are very rare in other domestic ani- 4.0), p < 0.05] and BCF decreased (37.9 mals, especially in mares with the exception [33.1–43.0] and 32.2 [27.3–37.5], p < 0.05). of a few reported cases of carcinomas. The In group 2 TMOT (24 [7–76] and 21 [6– 88 aim of this case report is to describe the clin- 48]), PMOT (16 [2–61] and 13 [2–40]), and Preservation of Sperm Chromatin ical, cytological and microscopic fi ndings of BCF (32.5 [27.3–36.7] and 30.9 [27.7–34.4] a mammary squamous-cell carcinoma (SCC) for H0 and H48, respectively) did not differ during Dried Storage in an 18-year-old, grey, thoroughbred mare. A among time points while VCL (165.4 [118.5– 1 1,2 1 3 clinical examination was conducted 10 days 182.0] and 137.4 [94.6–168.1]) and ALH H. Oldenhof , K. Narten , J. Bigalk , W.F. Wolkers , 1 after parturition, demonstrating cachexia, de- (3.9 [3.1–4.5] and 3.4 [2.8–4.0]) were high- H. Sieme 1 pression and dehydration associated with the er at H0 than at H48, respectively. At H0, all Clinic for Horses, Unit for Reproductive Medicine, 2 SCC. The right halves of the mammary gland motion characteristics differed between the 2 University of Veterinary Medicine Hanover; Nation- 3 demonstrated a fi rm enlargement and puru- groups, whereas at H48, TMOT and PMOT al Stud Lower Saxony, Celle; Institute of Multiphase Processes, Leibniz University Hanover, Germany lent discharge. Oedema of the ventral abdo- differed between the two groups, but none of men and right hind limbs was present. The the kinematic parameters. While motility did Sperm chromatin structure and level of con- metastasis to the regional lymph nodes, vul- not change over 48 hours of cooled storage densation determine accessibility for dam- va and vestibule vaginae were observed and using either extender, kinematic values de- age, and hence success of fertilization and locally aggressive biological behavior of the creased except from ALH in group 1, which development. Subjecting sperm to preserva- tumor was confi rmed. Cytological evaluation increased. Concerning motility, the egg yolk tion protocols and storage may cause oxi- of the mammary secretion revealed the preva- containing extender appeared not ideal. dative stress and DNA damage. The aim of lence of degenerated neutrophils, macrophag- the current study was to evaluate stallion es, giant cells and no bacteria. Part of the sperm chromatin structural stability, for di- cells demonstrated pyknosis and karyolysis, 87 luted semen maintained at 37°C as well as the others indicated polymorphism, anisoka- Development of a Flow Cyto- after freeze-drying and dried storage. Sperm riosis and atypia. After euthanasia, samples metry Assay to Assess Bacterial chromatin structure was evaluated using vari- were collected from mammary tissue and vul- ous assays on the same samples. Chromatin va and were fi xed for histology and immuno- Count in Boar Semen structure of sperm diluted in skim milk ex- fl uorescence (IF). The sample sections were C. Oehler1, F. Janett1, R. Gugelmann2, S. Schmitt3, tender rapidly degraded during storage at stained with HE, labeled with antibodies (an- E. Malama1, M. Siuda1, H. Bollwein1 37°C. DNA fragmentation index (DFI) val- ti-prolactine receptor [PRL R]), anti-neprily- 1Clinic of Reproductive Medicine, Vetsuisse-Faculty, ues increased from 11 ± 3 to 77 ± 11% after sin (CD10, anti-vascular endothelial growth University of Zurich; 2SUISAG, Sempach; 3Institute of 24 h storage. An increase in DFI-values cor- factor [VEGF]) and linked with Hoechst, then Veterinary Bacteriology, Vetsuisse-Faculty, University related with a decrease in halo sizes observed imaged using light and confocal microsco- of Zurich, Switzerland with the sperm chromatin dispersion assay, py. SCC was confi rmed histologically. CD10 and an increase in comet tail length observed and VEGF reactivity were localized on either The aim of the study was to develop a new with single cell gel electrophoresis. Despite stromal and neoplastic cells bodies. Epitheli- fl ow cytometry assay for the determination that no membrane intact sperm were recover- al cell cytoplasm was PRL R immuno-posi-

24 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts tive, while stroma cells were mostly negative. are largely unknown. Therefore, the aim of ter gonadotropin injections. After that period Semi-quantitative research is needed to evalu- our study was to elucidate the effects of in- the sows were slaughtered, electrodes were ate differences between SCC and unaffected fl ammation on sperm and oocyte transport checked for their position and the reproduc- equine mammary gland tissue. and gameto-maternal interaction. For this tive system gently fl ushed for embryo collec- purpose, oviducts revealing mild (n = 35), tion. Examination of reproduction tract car- 90 moderate (n = 30) and severe (n = 22) infl am- ried out post mortem indicated that telemetry mation were removed from cows immediate- recording system and material (bipolar elec- Testicular Biopsy as a Diagnostic ly after slaughter. Specimens were investigat- trodes) did not have a negative infl uence on Tool in Sub-fertile Bulls ed using live cell imaging, stereomicrosco- related tissues. The duration of activity peri- py, histomorphology and histochemistry. Our ods was signifi cant higher in the isthmus and 1 2 2 3 H. Otzen , K. Rode , R. Brehm , E. Hasenpusch , studies showed that 70% of cows affected by ampulla during early pregnancy than those 4 5 4 3 C. Wesenauer , J.H. Osmers , M. Kasten , T. Röpke , uterine infl ammation exhibited infection in detected in estrus phase (P < 0.0001). In the 1 1 1 C. Hettel , M. Heppelmann , Á.C. Bajcsy the oviducts. Moderate and severe infl amma- uterus, no signifi cant difference (P > 0.05) 1 Clinic for Cattle, University of Veterinary Medicine tion caused a signifi cant increase in the thick- was observed in the mean duration activi- 2 Hanover; Institute of Anatomy, University of Veteri- ness of tubal folds (Anova post hoc Dunnett`s ty during estrus and early pregnancy peri- 3 nary Medicine Hanover; Rinderzucht Schleswig-Hol- test, p < 0.05). Severe infl ammation was char- ods. Analysis of oviduct and uterus electro- 4 5 stein eG; RinderAllianz GmbH; Rinderproduktion acterized by luminal accumulations of glyco- myography (EMG) activity in sows in estrous Berlin-Brandenburg GmbH, Germany proteins, increased apoptosis, loss of tight phase as well as early stages of pregnancy ex- Genomic selection in cattle resulted in the junctions, and shedding of epithelial cells. plicitly suggests that telemetry makes it pos- DGRM-Abstracts need for Artifi cial Insemination centers to Particle transport speed (PTS) was 31.3% (± sible to obtain results in vivo enabling assess- supply their breeders with semen of young 19.2 µm/s) reduced in oviducts with salpingi- ment of myoelectrical activity of parts of re- bulls, which are often just yearlings. If such tis as compared to controls. The coeffi cient productive system which were the focus of a young bull fails to provide satisfactory se- of variation of the PTS was 11.5 times high- this study. men a great interest arises to assess possi- er in infl amed oviducts as compared to the ble testicular defects. Clinical and ultrasound controls (Mann-Whitney-U-test, p < 0.05). examination can provide some information Spermatozoa were stuck in mucus preventing regarding the testicular status. Histological them to form a sperm reservoir resulting in 93 evaluation of the testicular parenchyma adds reduced survival time and loss of fertilizing further information. However, it should be capacity. Our results imply that infl ammation The Evaluation of Placental Func- without detrimental effects on sperm produc- of the oviduct has a major nega tive impact on tion in fi rst 105 days of Gestation tion to allow future sperm harvesting. Prelim- bovine reproductive success by compromis- using 17beta-estradiol and PAG ing oocyte and embryo transport and by re- inary results of the evaluation of possible det- Concentrations in Dairy Cattle rimental effects of testicular biopsy in bulls ducing survival time and fertilizing capacity were presented. Biopsies were collected from of sperm. Thus, novel concepts for improving M. Petrajtis-Gołobów1, M. Domino1, B. Pawlin´ski1, 8 young sub-fertile bulls of different breeds success rates of assisted reproduction have to R. Faundez1, N. Melo De Sousa2, J.F. Beckers3, (13–27 months old), housed and biopsied at implement the use of bio markers for diagno- A. Wehrend4, Z. Gajewski1 their respective home station. At the puncture sis and specifi c medications for treatment of 1Department of Large Animal Diseases with Clin- site a local anesthesia was administered sub- salpingitis. ic, VRC and CBB, Faculty of Veterinary Medicine, cutaneously and an introducer needle was in- WULS – SGGW; 2Department of Immunology, Trans- serted percutaneously into the testis. Through plantology and Internal Diseases, Medical University this needle a 14 gauge biopsy instrument was of Warsaw, Poland; 3Physiol. of Reprod., FVM, ULG, introduced and a biopsy was collected. Sam- 92 Liege, Belgium; 4Klinik für Veterinärmedizin, Klinik ples were fi xed in Bouin’s solution, embed- für Geburtshilfe, Gynäkologie und Andrologie der ded in paraffi n and stained with Hematoxy- Uterine and Oviduct Activity Groß- und Kleintiere mit Tierärztlicher Ambulanz der lin-Eosin. Later, testes from the bulls were During Early Pregnancy in Sows Justus-Liebig-University Giessen, Germany retrieved from abattoir and prepared for his- Registered by Telemetry Method tological evaluation. Bulls mounted within 1 The pregnancy estrogen level of fetal origin week after biopsy again and produced fur- B. Pawlinski1, M. Domino1, A. Wehrend2, is much higher than that of ovarian origin, ther semen. In one bull the appearance of free Z. Gajewski1 which makes it possible to use this parame- fl uid within the scrotum was reported. With- 1Department of Large Animal Diseases with Clin- ter for assessing the status of the embryo and in the testis of this bull a hematoma was evi- ic, VRC and CBB, Faculty of Veterinary Medicine, the placenta. It was also demonstrated that dent after slaughter. In other bulls the biopsy WULS – SGGW, Warsaw, Poland; 2Klinik für Veteri- the concentration of PAG’s, secreted by the sites were only visible as a scar on the tunica närmedizin, Klinik für Geburtshilfe, Gynäkologie und trophoblast cells is proportional to the mass vaginalis or small adhesions between tunica Andrologie der Groß- und Kleintiere mit Tierärztli- of the placenta and therefore can be an in- vaginalis and testis. To conclude, a 14-gauge cher Ambulanz der Justus-Liebig-University Gies- direct method of the function of trophoblast testicular biopsy for evaluation of spermato- sen, Germany assessment in the fi rst trimester of pregnan- genesis can be performed in bulls with little cy. The data analyzed were derived from 50 adverse effects on testicular integrity. This The aim of the present study was to record the multiparous dairy cows. Animals were di- project is funded by the FBF. myoelectric activity of isthmus and ampul- vided into 3 groups: pregnant (n = 19), non- la parts of oviduct and uterine horn in sows pregnant (n = 11) and late embryonic mor- during the estrous phase and early pregnancy tality (n = 10). Transrectal ultrasonographic 91 period via telemetry recording system. A to- examination, blood and urine samples were Bovine Salpingitis – Effects on tal of 8 non-pregnant pigs surgically fi tted by obtained 105 days of pregnancy. RIA meth- Oviductal Microarchitecture and TL10M3-D70-EEE implants positioned be- od was used to determine PAG concentra- tween the abdominal muscles, and 3 silicone tion in blood, milk and urine and to deter- Fertility electrodes sutured on the left oviduct (isth- mine E2 concentration in plasma. In the per- L. Owhor, S. Kölle mus and ampulla) and the ipsilateral uter- formed analysis, a statistical signifi cant pos- School of Medicine, University College Dublin, Ire- ine horn and the bioelectrical activity of or- itive correlation (Sr + = 0.73) between E2 in land gans was recorded. The blood samples were blood and PAG concentration in plasma was collected in order to monitor P4 and LH lev- found. There was no signifi cant correlation The oviduct is pivotal for successful repro- els. The estrous cycle was synchronized with between E2 concentrations and PAG in other duction. The effects of infl ammatory altera- eCG and hCG and animals were subject to materials, milk and urine of P and NP group. tions in the oviduct on reproductive success artifi cial insemination (AI) 24 and 48 hrs af- Confi rmed correlation of those 2 factors with

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 25 DGRM-Abstracts

placenta function is also evident in the LEM 95 In the bovine placenta, intimate feto-mater- group. The existence of statistically signifi - nal contact is restricted to multiple discrete cant differences between the groups was con- The Testicular Expression of placentomes, which are composed of the fetal fi rmed. The same results were obtained be- KATNB1 during Human Spermato- cotyledon and the maternal caruncle. Weak- tween PAG concentration in the groups of LE genesis ly invasive binucleated trophoblast giant and HE in blood, milk and urine. The great- cells (TGCs) permanently fuse with individ- est variations in the concentrations of PAG C. Pleuger1, D. Fietz1, K. Hartmann1, W. Weidner2, ual caruncular epithelial cells to form short- between groups NE and HE were meas- S. Kliesch3, M. O`Bryan4 , A. Dorresteijn5, lived trinucleated feto-maternal hybrid cells. ured from blood (p < 0.0001), then urine M. Bergmann1 TGCs are continuously regenerated from (p < 0.001) and in milk (p < 0.01). Based on 1Institute of Veterinary Anatomy, Histology and Em- uninucleated trophoblast cells (UTCs). Aim obtained results in 40% of cows diagnosed bryology, Justus-Liebig-University Giessen; 2 Depart- of this work was to identify transcripts which with LEM late embryonic mortality were ob- ment of Urology and Andrology, University Hospital are associated with TGC differentiation. To served disorder of the secretory function of Giessen; 3Centre for Reproductive Medicine and An- this end, UTCs and TGCs were obtained from the placenta resulting in impaired secretion of drology, Department of Clinical Andrology, University 3 bovine placentas (days 118–130 of gesta- PAG molecules and E2. Hospital Münster, Germany; 4Department of Anato- tion) using an optimized fl uorescence activat- my and Developmental Biology, Monash University, ed cell sorting procedure. For transcriptome Melbourne, Victoria, Australia; 5Institute for General analysis, total RNA from UTC and TGC Zoology and Developmental Biology, Justus-Liebig pools was labeled and hybridized to Affyme-

DGRM-Abstracts University, Giessen, Germany trix Bovine Gene 1.0 ST Arrays. Lists of dif- 94 ferentially expressed genes (DEGs, n = 2452) Katanin, a microtubule-severing protein, con- and genes that were expressed only in UTCs Expression of CBE1 (ciliated bron- sists of an enzymatic p60 (KATNA1) and a (n = 682) or TGCs (n = 780) were derived chial epithelium 1) in Human regulatory p80 subunit (KATNB1). A mis- from the microarray data sets and subjected sense mutation in the highly conserved Spermatogenesis to gene ontology (GO) term enrichment ana- WD40 domain of the Katnb1 gene causes de- 1 1 1 2 lysis using DAVID software. Our analyses re- C. Pleuger , D. Fietz , K. Hartmann , H.-C. Schuppe , fects in spermatogenesis (Taily mouse). Ho- 2 3 4 5 vealed that DEGs were most signifi cantly as- W. Weidner , S. Kliesch , M. Baker , M. O´Bryan , mozygous Taily males are infertile due to a 1 sociated with biological processes including M. Bergmann decreased sperm production, sperms with ab- 1 “intracellular signaling cascade”, “extracel- Institute for Veterinary Anatomy, Histology and Em- normal head shape and an absence of pro- 2 lular matrix organization” and “regulation of bryology, Justus-Liebig-University; Department of gressive motility. This phenotype is similar cell migration”. Genes that were expressed Urology, Pediatric Urology and Andrology, Universi- to the human oligoasthenoteratozoo spermia 3 only in UTCs did not reveal remarkable GO ty Hospital Giessen; Department of Clinical Androl- syndrome (OAT). Therefore, we analyzed terms or pathways. Interestingly, however, of ogy, Centre for Reproductive Medicine and Androlo- the expression pattern of the regulatory p80 4 780 genes expressed only in TGCs, 110 genes gy, University Hospital Münster, Germany; School of subunit during human spermatogenesis on were associated with the GO term “G-protein Environmental and Life Science, University of New- mRNA (RT-PCR, RT-qPCR and in situ hy- 5 coupled receptor protein signaling pathway” castle; Monash University, Department of Anatomy bridization) and protein level (immunohis- and 72 genes with the GO term “olfactory re- and Developmental Biology, Clayton, Victoria, Aus- tochemistry). We observed that KATNB1 ceptor activity”. This suggests that chemotax- tralia mRNA is exclusively expressed in germ is mediated by olfactory receptors may be im- cells. In normal spermatogenesis, mRNA is CBE1 (ciliated bronchial epithelium 1), portant for TGCs. localized in pachytene primary spermato- which was described related to motile (brain, cytes. KATNB1 is quantitatively reduced in Grants: This work was supported by a grant lung, testis) and sensory ciliated cells (kid- maturation arrests at level of primary sper- from the Deutsche Forschungsgemeinschaft ney, heart), is a largely uncharacterized pro- matocytes and spermatogonia. The KATNB1 (DFG). tein. Expression databases show that CBE1 is protein was detected in type B spermatogonia highly testis-enriched in amniotes. To analyse and co-localized with Golgin A2 in the Golgi the precise cellular localization in the human complex of pachytene spermatocytes. Addi- testis, we performed immunohistochemistry, tionally, KATNB1 is co-localized with peri- in situ hybridization and RT-(q)PCR to de- centrin in the cleaving centrosome immedi- 97 tect CBE1 protein and mRNA in testicular ately before the fi rst meiotic division (stage biopsies showing normal and impaired sper- Testicular Volumetry using a 5). The p80 subunit was also detected in ear- matogenesis such as an arrest at the level of Caliper, two-dimensional (2D)- , ly round spermatids in the dictyosome above spermatocytes and round spermatids. In nor- the acrosomal cap. The expression and locali- and three-dimensional (3D)-So- mal spermatogenesis, CBE1 mRNA is ex- zation of KATNB1 support its role in spindle nography to determine Daily pressed in pachytene primary spermatocytes formation as well as in formation of micro- Sperm Output (DSO) in the Stal- and the protein is localized within the fl agel- tubule-based structures during spermiogene- lum of elongating spermatids from step 5 on- lion sis. These data are consistent with the demon- wards. Immune-electron microscopy showed 1,2 1,2 1,2 strated role of KATNB1 in mouse spermato- S. Pricking , K. Spilker , G. Martinsson , CBE1 clearly associated with microtubules 3 4 1 genesis and suggest the strong conservation D. Burger , H. Bollwein , H. Sieme in the fl agellum and at a lower level at the 1 of function even between distantly related Clinic for Horses – Unit for Reproductive Medicine, manchette. The mRNA expression is signifi - 2 species. University of Veterinary Medicine Hanover; National cantly reduced in samples with an arrest of Stud Lower Saxony, Celle, Germany; 3Swiss Institute spermatogenesis at level of round spermatids of Equine Medicine ISME, University of Berne and compared to normal spermatogenesis. Oligo- Agroscope, Avenches; 4Clinic of Reproductive Medi- zoospermic patients with immotile sperma- cine, Vetsuisse Faculty, University of Zürich, Zürich, tozoa and severe hypospermatogenesis show 96 Switzerland reduction of mRNA expression and almost immuno-negative spermatids indicating an Comparative Transcriptome Ana- Measurement of testes size is an important important role of CBE1 in the development lysis of Bovine Uninucleated part of the stallion breeding soundness eval- of sperm motility. Further studies in testicu- Tropho blast Cells and Trophoblast uation since testes size is associated with lar biopsies showing impaired spermatogen- Giant Cells sperm production. The number of sperm esis and ejaculates of patients with diverse produced by the stallion may impact fertili- clinical manifestations will clarify the possi- M. Polei, R. Fürbass ty and, thus, the number of mares that a stal- ble causal relationship of CBE1 to male in- Leibniz Institute of Farm Animal Biology, Dum- lion can breed. Thus, accurate prediction of fertility. merstorf, Germany daily sperm output (DSO) is thought to pro-

26 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts vide meaningful information for reproductive mal spermatogenesis (Nsp, n = 10) by immu- reductions performed days 30–35 of gesta- management of breeding stallions. Measur- nohistochemistry and immunofl uorescence tion showed success rates of 76%, while this ing the size of the testes can be performed ei- using antibodies against CD11, CD68 (M1), number decreased to 62% if done days 36−40 ther by traditional orchidometry using a cali - CD163, CD206 (M2), CD1c, CD11c (mDc1), and 38% for days 40–49. For mares young- per to measure scrotal dimensions (length, CD11c, CD141 (mDC2) and CD123, CD303, er than 7 years, the TUA effi cacy was not af- height, width) or by measuring testicular di- CD304 (pDC). Transcripts encoding relevant fected by the day of gestation. In contrast, for mensions using 2D-ultrasound imaging. Es- chemokines (CCL15, CCL2, CCL5, CCL18, older mares TUAs were less successful with timation of DSO is calculated based on to- CCL22) and cytokines (TGF-, IL-6) were increasing gestation age. Mares aged 8−15, tal testicular volume (TTV) derived from tes- evaluated by qRT-PCR from cryopreserved showed a signifi cant decrease in the success ticular dimensions (4/3 [L/2] [W/2] [H/2]) tissue samples. rate if done after day 35 of gestation. In the and in conjunct with a formula for maximal Results Compared to Nsp we found high group of mares older than 15 years, perform- (0.024[VolL+VolR]–0.76) and minimal ex- numbers of M1 and M2-polarized macro- ing TUA after day 40 resulted in total preg- pected DSO (0.024[VolL+VolR]–1.26). We phages as well as pDCs and mDCs in GCNIS nancy loss. hypothesize, that 3D testicular sonography and seminoma. We also detected an upregu- allows a more precise measurement of tes- lation of chemokines (CCL2, CCL5, CCL18, ticular volume than caliper and 2D. Testic- CCL22) and cytokines (TGF-, IL-6), but a ular dimensions from 52 breeding stallions downregulation of CCL15 chemokine. 100 were obtained by caliper and 2D. 3D-scans Conclusion Our data suggest that in con- were analyzed by VOCAL-software to deter- Effect of Exogenous Estradiol DGRM-Abstracts mine testicular volume. Values for DSO were trast to Nsp, tumor tissue shows a high ex- Benzoate Administration on collected from semen records. TTV showed pression of immune cell attracting chemokine pattern which differs from other tumours and Uterine Blood Flow in Postpartum signifi cant correlations between methods Dairy Cows by Transrectal Doppler (3D vs 2D, r = 0,75; p < 0.001, 3D vs caliper cytokines being important for the recruitment r = 0,63; p < 0.001, 2D vs caliper r = 0,56; and differentiation of immune cells. Most in- Ultrasonography terestingly, the downregulation of CCL15 im- p < 0.001). Correlation of TTV and DSO was 1,2 2 2 mune cell attracting chemokine in testis neo- M.S. Rawy , D.R. Ibrahim , G.A. Megahed , high for 2D (r = 0,639; p < 0.001), and for 1 1 plasia might indicate a functional importance G. Kitahara , T. Osawa 3D (r = 0,604; p < 0.001), and moderate for 1 of CCL15 in normal testis tissue, which still Laboratory of Theriogenology, Faculty of Agricul- the caliper technique (r = 0,46; p < 0.01). So- 2 has to be evaluated. ture, University of Miyazak, Japan; Department of nographic testicular volumetry measures are Theriogenology, Faculty of Veterinary Medicine, As- more accurate and can be done quicker than siut University, Egypt the caliper technique; furthermore, the paren- chyma can be directly visualized, and specifi c 99 The objective of this study was to quantify parenchymal landmarks can be better paced the uterine blood fl ow after estradiol benzo- by 3D vs 2D. Effi cacy of Transvaginal Ultra- ate administration in dairy cows by transrec- sound-guided Twin Reduction in tal Doppler sonography. Six Holstein-Frie- the Mare is Dependent on Age sian cows were subjected for daily examina- tion for 10 successive days starting from 4th 98 and Day of Gestation week post partum (PP). All cows were exam- 1 1 1 2 ined clinically for vaginal mucous scoring, Neoplasia in Human Testis: Evi- J. Rau , D. Tiedemann , J. Sielhorst , G. Martinsson , D. Burger3, H. Sieme1 endometrial cytology and Doppler transrectal dence of Functional Polarization 1Clinic for Horses – Unit for Reproductive Medi- ultrasonography to quantify the blood fl ow to of Macrophages and Dendritic cine, University of Veterinary Medicine, Hanover; the uterine arteries ipsilateral and contralater- 2 3 al to the previously pregnant uterine horn. At Cells Na tional Stud Celle, Germany; Swiss Institute of rd Equine Medicine ISME, University of Berne and the 3 day of experiment, healthy cows ad- 1 1 2 1 D. Püschl , B. Klein , S. Indumathy , D. Fietz , Agroscope, Avenches, Switzerland ministered with intramuscular injection of 10 S. Kliesch3, M. Hedger2, K. Loveland2,4, mg Estradiol benzoate. Blood samples were H.-C. Schuppe5, M. Bergmann1 In mares, advanced twin pregnancies result in collected for the assessment of estradiol-17 ¹Department of Veterinary Anatomy, Histology and abortion, stillbirth or delivery of non-viable (E2) concentrations. At each examination, Embryology, Justus-Liebig-University, Giessen, Ger- or severely compromised foals in most of the pulsatility index (PI), resistance index (RI), many; 2Centre for Reproductive Health, Hudson In- cases. Moreover mares frequently suffer from time average maximum velocity (TAMAX), stitute for Medical Research, Clayton, Victoria, dystocia and fetal membrane retention caus- blood fl ow volume (BFV) and uterine arter- Australia; 3Centre of Reproductive Medicine and An- ing low fertility in the following breeding ies diameter were recorded. The PI and RI drology, University of Muenster, Germany; 4School season. Therefore reducing twin pregnancies indices decreased while the TAMAX veloc- of Clinical Sciences, Monash University, Clayton, to a singleton pregnancy is recommended. ity, BFV and diameter of ipsilateral and con- Victoria, Australia; 5Department of Urology, Pediat- Twin reduction is more successful the earlier tralateral uterine arteries increased signifi - ric Urology and Andrology, Justus-Liebig-University, it is performed in gestation. Transvaginal ul- cantly in response to E2 treatment. There was Giessen, Germany trasound-guided aspiration (TUA) of amnio- a high correlation between both the ipsilateral tic fl uid is the method of choice to obtain a and contralateral uterine arteries in all studied Introduction In human testicular germ cell singleton pregnancy before the fetal phase if parameters (R = 0.860, p < 0.0001, R = 0.922, neoplasia, i.e. GCNIS and seminoma, infi l- twin pregnancies persist after day 25 of ges- p < 0.0001, R = 0.651, p < 0.0001, R = 0.879, trating immune cells are frequently found. tation. During 2 following breeding seasons, p < 0.0001, R = 0.861, p < 0.0001 for the PI, These immune cells have previously been 58 TUAs were performed between day 30 RI, TAMAX, BFV and uterine arteries diam- identifi ed as lymphocytes (T- and B-cells), and 49 of gestation in 3–21 year old mares. eter, respectively). It can be concluded that macrophages and dendritic cells. We ana- Success was defi ned as a singleton pregnan- the higher levels of estrogen may be responsi- lysed the subpopulation of macrophages (M) cy diagnosed 4 weeks after TUA. The aim ble for the higher time average maximum and and dendritic cells (DC) including respective of the present study was to evaluate the ef- blood fl ow velocities, increased dia meters cytokine and chemokine expression in order fects of the mare's age and the day of ges- and decreased pulsatility and resistance indi- to further insights in the regulation of tumour tation on outcomes. A singleton pregnancy ces of the uterine arteries during the repro- growth. was found for 67% of the cases after TUA. ductive cycle of the non-pregnant dairy cows. Material/Method We evaluated Bouin- Success rates were 91% in case mares were fi xed, paraffi n-embedded tissue samples from younger than 7 years, whereas this decreased seminoma (n = 10), preinvasive germ cell down to 72% and 44% for mares aged 8−15 neo plasia in situ (GCNIS, n = 10) and nor- and older than 15 years, respectively. Twin

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 27 DGRM-Abstracts

101 102 expressed in a cell, PGRA acts as an inhibi tor of PGRB, thereby reducing the effects of P4. Effect of Stage and Quality of Variation of Genomic estimat- Therefore the aim of this study was to check Biopsied Bovine Embryos ob- ed Breeding Values within Geno- if the promoters of both isoforms can be me- tained by Repeated Superovula- typed Full-sibs of Fleckvieh bo- thylated and to determine the percentage of tion on Developmental Rates vine Embryos the methylation of each receptor isoform. The studied tissues were corpora lutea (CL) and after Conventional Freezing M. Reichenbach1, K. Simmet2, S. Jung1, R. Fries3, endometrial slices from cows on days 1–5, M. Reichenbach1, K. Simmet2, S. Jung1, R. Fries3, J. Scherzer1, T. Grupp1, E. Pimentel4, R. Emmerling4, 6–10, 11–16, and 17–20 of the estrous cycle. J. Scherzer1, T. Grupp1, E. Wolf2, H.D. Reichenbach4 K.U. Götz4, E. Wolf2, H.D. Reichenbach4 Isolated genomic DNA was bisulfi te convert- 1Bayern-Genetik GmbH, Grub; 2Chair for Molecular 1Bayern-Genetik GmbH, Grub; 2Chair for Molecular ed and amplifi ed using methyl specifi c PCR Animal Breeding and Biotechnology, Ludwig-Maxi- Animal Breeding and Biotechnology, Ludwig-Maxi- with primers for methylated and unmethylat- milians-University, Munich; 3Chair for Animal Breed- milians-University, Munich; 3Chair for Animal Breed- ed sequences. Determination of the percent- ing, Technische Universität München, Freising; 4Ba- ing, Technische Universität München, Freising; 4Ba- age of the methylation was carried out using varian State Research Center for Agriculture (LfL), varian State Research Center for Agriculture (LfL), HpaII and Mspl, a pair of restriction enzymes Institute for Animal Breeding, Grub, Germany Institute for Animal Breeding, Grub, Germany with different sensitivity to methylation at a specifi c locus. Agarose gel electrophoresis of Procedures for freezing biopsied bovine em- In cattle, the interest of genotyping embry- methyl specifi c PCR showed partial methyla-

DGRM-Abstracts bryos are of critical importance for breeding os for selection on the basis of their genomic tion of PGRA and PGRB promoters in CL programs involving preimplantation embryo estimated breeding values increased in many and endometrium during the estrous cycle. genetic screening. The aim of this study was countries in the last years. The genomic to- The percentage of methylation was higher for to evaluate the effect of embryo stage and tal merit index (gTMI) represents the overall PGRA than for PGRB promoter in CL about quality on post-thaw in vitro development of breeding value of the embryo. ET and OPU/ 2.5–3 times (P < 0.001–0.0001) and 0.7–2.5 biopsied embryos conventionally frozen in IVP enable to provide more embryos for se- times in endometrium (P < 0.05–0.01) during 1.5 M EG. All embryos were collected from lection, and now available embryo genotyp- the estrous cycle. Obtained data may indicate one Simmental cow repeatedly superovulat- ing tools allow to select the best candidate that higher promoter methylation of PGRA ed (n = 7) at intervals of 5–8 weeks. Brief- before the transfer to recipients. In the pre- isoform can be a mechanism for regulation of ly, the animal received a PRID-alpha® at a sent study the variation of gTMI within full- PGRA inhibitory activity against PGRB and random stage of the estrous cycle (d0) and sibs morulae and blastocysts recovered on this way infl uence the regulation the P4 ac- was superstimulated with FSH (Pluset®) ad- d 7 from the uterus of 2 FSH-superstimulat- tion in CL and endometrium. ministered twice daily in decreasing doses ed German Fleckvieh donor cows inseminat- from d 5–8. Cloprostenol (Estrumate®) was ed with elite bulls was examined. Embryos adminis tered on d7/d8 and PRID® removed of two donors (D1, 9 embryos; D2, 11 em- 104 on d 8. Fixed-time AI was performed 36, 48 bryos) were biopsied in isotonic NaCl solu- Prostaglandin E2 (PGE2) is a Tes- ® tion supplemented with 1.5% PVP without and 60 h after PRID removal. Embryos were ticular Peritubular Cell-derived collected by fl ushing 7 d after 1st AI. Mor- BSA (biopsy medium) by separating 5–10 ulae (M), early blastocysts (EB) and blasto- blastomeres with a microblade to provide Factor involved in Human Testicu- cysts (B) evaluated by morphological criteria suffi cient DNA for amplifi cation and subse- lar Homeostasis quent embryo genotyping without compro- (IETS) as grade 1 (G1) and 2 (G2), were bio- V. Rey-Ares1, S. Rossi1,2, K. Dietrich1, F. M. Köhn3, psied with a microblade in order to separate mising their developmental potential. Each 4 1 2 biopsy was transferred directly into the as- J. U. Schwarzer , H. Welter , M. Frungieri , 5–10 blastomeres to provide suffi cient DNA A. Mayerhofer1 for analysis. Biopsied embryos were cultivat- say tube containing 2.5 µl TE-buffer. Ampli- 1 fi ed DNA (REPLI-g® Mini Kit, Qiagen) was Biomedical Center (BMC), Cell Biology, Anatomy III, ed for 24 h in SOF+ Estrus cow serum (ECS) Ludwig-Maximilian-University, Planegg, Germany; to blastocysts before freezing. After thaw- sent to GeneControl GmbH (Grub, Germa- 2 ny) for SNP analysis using Illumina 50K chip Instituto de Biología y Medicina Experimental, Con- ing, the embryos were cultivated for 48 h in sejo Nacional de Investigaciones Científi cas y Téc- SOF+ECS to evaluate re-expansion and de- technology. The gTMI from dam/sire were 3 111/125 and 85/107 for D1 and D2, respec- nicas, Buenos Aires, Argentina; Andrologicum, Mu- velopment. Data were analyzed with a gen- nich; 4Andrology-Center, Munich, Germany eral linear model (Proc GLM, SAS). A total tively. Call rates within the embryos ranged of 142 (x ̄ = 20.3) ova/embryos were recover- for D1 from 0.874–0.939 (x ̄ = 0.898) and In the testis, smooth muscle-like peritubu- ed of which 120 (x ̄ = 17.1) were fertilized D2 from 0.858–0.967 (x ̄ = 0.915). The gT- lar cells build the wall of seminiferous tu- and 107 (x ̄ = 15.3) evaluated as viable. From MIs of the embryos ranged from 110–120 bules. Transport of immotile sperm to the the 80 biopsied embryos, 33 were M (20 G1 and 89–104 within D1 and D2, respectively. rete testis has been regarded as their ma- and 13 G2), 27 EB G1 and 20 B G1. Frozen- Results demonstrate that bovine d7 morulae jor role. We have established a culture pro- thawed biopsied G1 M, EB and B had simi- and blastocysts can be successfully screened cedure for human testicular peritubular cells lar development (80.0%, 85.2% and 65.0%; and selected for breeding on the basis of their (HTPCs) and a subsequent proteome-analy- p > 0.10). However G2 M resulted in low- genomic estimated breeding values. sis of HTPCs has revealed among others cy- er development (7.7%; p < 0.001) compared clooxygenase 1 (COX1) and 2 PGE-synthas- to G1 M. Results indicate that conventional 103 es (PTGES2 and 3). This implies that HTPCs freezing is suitable for biopsied G1 M, EB are a source of PGE2, which consequent- and B. Methylation of Progesterone ly may be a physiological factor in the hu- Receptor Isoform A and B Promot- man testis. Western blots confi rmed that HT- ers in the Reproductive System of PCs express COX1 and ELISA-measure- ments showed that they constitutively secrete Cows PGE2. Indomethacin, a prototype non-ste- R. Rekawiecki, M.K. Kowalik, J. Kotwica roidal anti-infl ammatory drug (NSAID), sig- Institute of Animal Reproduction and Food Research, nifi cantly reduced PGE2 synthesis. Immu- Polish Academy of Sciences, Olsztyn, Poland nohistochemistry (IHC) showed COX1 and specifi c receptors for PGE2, EP1–4 in hu- Progesterone receptor (PGR) isoform B man testicular samples. EP1, EP2 and EP4 (PGRB) acts as a potent activator of genes were revealed in HTPCs in vivo and in vitro that are dependent on progesterone (P4), by RT-PCR and/or IHC. Results of collagen whereas isoform A (PGRA) is a weak acti- gel contraction assay indicate that PGE2 does vator of these genes. When both isoforms are not acutely contract HTPCs, yet qPCR stud-

28 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts ies showed that it increased the mRNA lev- infection (p.i.). Whether the initial lower bac- licles present in the ovaries and that it is a rele- els for the contractility proteins, smooth mus- terial load after 12 h in Q-animals relates to vant marker of ovarian reserve and thought to cle actin (SMA) and calponin in a concentra- an improved resistance against pathogens will be a predictor of ovarian response in humans tion-depended manner. HTPCs, like Sertoli be investigated in follow-up studies. In con- like other parameters e.g. FSH an antral folli- cells, produce glial derived neurotrophic fac- clusion, genetic selection might be an oppor- cle count. It may be a useful marker to predict tor (GDNF), which regulates the renewal of tunity for breeding cows with improved re- treatment outcome. To fi nd a predictive mod- spermatogonial stem cells (SSCs). PGE2 in- sistance towards mastitis pathogens. el of the treatment outcome of GnRH antago- creased GDNF mRNA and GNDF levels in nist stimulated ICSI cycles we analyzed in the culture medium. Thus, HTPCs are sourc- this retrospective single-center study stimu- es of PGE2, which may contribute to testic- 106 lated E2 and AMH levels of 249 women with ular homeostasis. This includes maintenance a mean age of 34.06 years undergoing ICSI of the contractile phenotype of peritubu- Interferon-τ induced Gene Expres- treatment in our clinic. Different dynamic lar cells and, via GDNF, a fi ne tuning of the sion in Hepatocytes during Early parameters were assessed during the treat- SSC niche. Blockage of PG-production e.g. Pregnancy in Angus Heifers ment as markers of success and compared by NSAIDs, steroids or other substances may to the hormone parameters. AMH level was thus have unwanted testicular side effects. B. Ruhmann1, K. Giller2, A. Hankele2, S.E. Ulbrich2, strongly correlated to the number of follicles 1 Grants: DFG MA1080/25-1/DAAD M. Schmicke (rs = 0.39, p < 0.001) and isolated oocytes 1Clinic for Cattle, Endocrinology Laboratory, Univer- (rs = 0.70, p < 0.001) and immature oocytes 2 sity of Veterinary Medicine Hanover, Germany; Ani- (rs = 0.27, p < 0.001). It also correlates with DGRM-Abstracts mal Physiology, Institute of Agricultural Sciences, the pregnancy rates (rs = 0.21, p < 0.05). Lin- 105 ETH Zurich, Switzerland ear regression between AMH, E2 levels and pregnancy rate revealed no stronger correla- Divergent Genotype in Holstein The conceptus-signal Interferon- (IFN) in- tion. Interestingly AMH correlates signifi - duces specifi c hepatic gene expression (ISG) cantly to formation of multiple PN (rs = 0.27, Heifers infl uences initial Staphylo- in heifers. The aim of this study was to con- coccus aureus Shedding after Ex- p < 0.001). No correlation could be found be- fi rm the ISG induction in vivo on day 18 of tween basal E2 and dynamic parameters. This perimentally induced Mastitis pregnancy and to investigate if hepatocytes demonstrates AMH measurement predicts re- react to IFN. Specifi cally, it should be con- 1 2 2 1 sponse of patients and is also correlated to the L. Rohmeier , S. Jander , M. Koy , L. Macias , fi rmed in vitro if primary bovine hepatocytes 2 3,4 5 treatment outcome and other dynamic param- M. Meyerholz , S. Engelmann , M. Hoedemaker , showed an increase of ISG mRNA expression 6 2 6 1 eters during stimulation and embryo culture. C. Kühn , H.J. Schuberth , H.M. Seyfert , H. Zerbe , after stimulation with IFN. Angus heifers 1 The correlation of AMH and stimulated E2 W. Petzl (n = 13) were cycle synchronized. The day 1 revealed the potential predictive capacity of Clinic for Ruminants, Ludwig-Maximilinans-Univer- of ovulation (day 0) was defi ned by ovarian 2 AMH to reduce OHHS. sity, Munich; Immunology Unit, University for Ve- ultrasonography and verifi ed by the absence terinary Medicine, Hanover; 3Microbial Proteomics, of progesterone (P4). The heifers were artifi - Helmholtz Centre for Infection Research, Braun- cially inseminated either with sperm or with schweig; 4Institute for Microbiology, Technical Uni- seminal plasma as a mock control. Blood versity Braunschweig; 5Clinic for Cattle, University of samples for pregnancy detection were taken 108 Veterinary Medicine Hanover; 6Leibniz Institute for on day 18, 21 and 24 after insemination, and Cell type-specifi c Analysis of Farm Animal Biology, Dummerstorf, Germany the concentration of P4 and pregnancy asso- ciated glycoproteins was determined. A liver Endometrial Gene Expression in Staphylococcus aureus (S. aureus) is the the Mare During the Time of Initial most important pathogen causing chronic in- biopsy was taken on day 18 for qPCR analy- tramammary infections and subclinical mas- sis and immunohistochemistry. Primary bo- Recognition of Pregnancy vine hepatocytes were isolated and incubat- titis. Besides its large economic impact it also 1,3 2 1 1 ed with 200 ng IFN in vitro for 2 hours. The I. Scaravaggi , N. Borel , R. Romer , I. Imboden , raises concerns over the use of and resist- 1 3 3 3 ISG in the liver increased in pregnant versus H. Bollwein , S. Zeng , S.E. Ulbrich , S. Bauersachs ance towards antimicrobial drugs as well as 1Clinic of Reproductive Medicine; 2Institute of Vet- animal welfare. Specifi c breeding programs non-pregnant heifers (p vs np; relative abun- dance in CT = ISG 15: 22.2 ± 2.6 vs 3.8 ± erinary Pathology, Vetsuisse-Faculty, University of seem to be a promising tool for improving an- 3 1.1; MX 1: 39.6 ± 8.9 vs 6.6 ± 1.9; MX 2: 0.9 Zurich; Animal Physiology, Institute of Agricultural imal resistance against production diseases. Sciences, ETH Zurich, Switzerland The aim of the study was to evaluate differ- ± 0.1 vs 0.2 ± 0.3; OAS 1: 71.3 ± 15.2 vs 29.9 ences in the bacterial load after experimen- ± 8.3). In vitro the primary bovine hepato- The results of endometrial gene expression tal S. aureus challenge in divergent geno- cytes showed an increased mRNA expression studies during the time of conceptus migra- types. Holstein Friesian heifers were selected of ISG after IFN stimulation (p < 0.0001) tion (days 8 to 16) did not provide clear con- for favourable (Q) and unfavourable (q) pa- (IFN vs control; CT= ISG 15: 465.1 ± 20.7 clusions on possible mechanisms of maternal rental chromosome 18 haplotypes regarding vs 27.7 ± 2.2; MX 1: 442.4 ± 13.2 vs 50.6 recognition of pregnancy (MRP) in the mare. somatic cell count, used as surrogate trait for ± 5.9; MX 2: 75.6 ± 5.3 vs2.6 ± 0.2; OAS This fi nding called for a deeper analysis of mastitis susceptibility. The animals (n = 24; 1: 180.7 ± 6.4 vs 30.8 ± 3.1). Furthermore cell type-specifi c gene expression in the en- Q = 12, q = 12) were kept in individual pens OAS 1 was localized in hepatocytes. In con- dometrium in response to the equine embryo, at the Clinic for Cattle in Hanover. On day clusion the fi ndings confi rm that IFN reach- which could be the key for improved under- 36 ± 3 post partum the heifers received an es the liver in early pregnancy of cattle and standing of the mechanism of MRP. In a pi- intramammary S. aureus challenge in both the results show in vitro as well as in vivo that lot study we used Laser Capture Microdissec- hindquarters. The following 96h bacterial re- bovine hepatocytes react to IFN. tion (LCM) to collect samples for the luminal covery was assessed in quarter milk samples epithelium (LE), glandular epithelium (GE), every 12h by plate count. No effects on bacte- and stroma from endometrial biopsies taken rial shedding have been detected between the 107 from 5 mares on day 12 of pregnancy and day hindquarters after using one way ANOVA and Predictive Role of AMH in ICSI 12 of the estrous cycle, respectively. RNA Tukey post test (Q: P = 0.094; q: P = 0.14). was isolated in order to perform RNA se- However, using an unpaired t-test, q-heifers Treatments quencing. The preparation and sequencing of showed a signifi cant > 10 fold higher shed- N. Saymé, T. Krebs RNA-Seq libraries was successful for about ding of S. aureus than Q-heifers 12h p.i. (Q: Team Kinderwunsch Hanover, Germany 2 thirds of the libraries. Interestingly, data 1.02 logCFU/ml ± 0.32 vs q: 2.2 logCFU/ml analysis showed that gene expression differ- ± 0.27; P < 0.01). This could no longer been It is already known that serum AMH level re- ences are greater between cell types than be- observed at time points 24 h until 96 h post fl ects the number of antral and pre-antral fol- tween pregnant and cyclic state. Differen-

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 29 DGRM-Abstracts

tial gene expression between pregnant and 110 from cows were obtained from a local abat- cyclic state was mainly found in the LE but toir and cumulus-oocyte complexes were ma- also in GE and stroma. The comparison with Histochemical Investigation of the tured for 22 hours in TCM-199 supplement- a previous RNA-Seq data set for whole biop- Epididymal Duct Basal Cells of ed with BSA and eCG/hCG. ICSI was per- sy samples derived from day 12 of pregnan- the Domestic Rabbit (Oryctolagus formed in 10 µl droplets TCM-199 using fro- cy revealed the specifi c origin of the observed cuniculus domesticus) zen-thawed semen, which was stained with gene expression differences. Furthermore, we MitoTracker® Green FM to visualize the mid- found that a number of genes are specifi cal- B. Schick, R. Kenngott piece. A single sperm was immobilized by ly differentially expressed (DE) in only 1 cell Institute of Anatomy, Histology and Embryology, Uni- the application of several piezo impulses and type. Some genes not DE when looking at versity of Veterinary Medicine, Munich, Germany then injected into a matured oocyte, also us- the whole biopsy were DE in 1 cell type and ing electric pulses to draw a hole in the zona showed similar levels in the other cell types The epididymis contains a diversity of func- pellucida, open the oolemm and to activate in comparison of pregnancy and cyclic stage. tions including the concentration, maturation the oocyte. After the ICSI procedure, the in- These results support our hypothesis of the and storage of sperm. To collect more infor- jected oocytes were cultured for 18 hours in existence of a specifi c response in different mation about the different cell types we ex- SOF medium. To identify the success of ac- endometrial cell types in the mare and call for amined the morphology of the epididymis of tivation and pronuclear formation they were further studies. the rabbit. Special attention is given to the ba- stained with Hoechst 33342. Parthenoge- sal cells. To my knowledge little information Grants: This study was supported by the netic (sham-injected) controls were includ-

DGRM-Abstracts regarding presentation and variation of rab- foundation “Forschung für das Pferd”. ed. First results show a paternal pronuclear bit basal cells is available. Epididymes from formation rate of 23.5% (16/68) in the ICSI three rabbits of different ages were used. group. No pronuclear formation could be ob- They were removed during orchidectomy in a served (0/65) after sham-injection. In conclu- veterinarian clinic in Munich, Germany, dis- sion, equine sperm cells can decondense and 109 sected into small pieces and fi xed in Bouin’s form paternal pronuclei in bovine oocytes af- solution. After embedding according to rou- ter ICSI. Improving Cryopreservation Suc- tine protocols we prepared serial sections cess of Shipped Boar Semen (5 µm) of the epididymes. They were inves-

1,2 1 2 1 tigated using overview stainings like Hema- 112 J. Schaefer , M. Jung , D. Waberski , M. Schulze toxylin-Eosin and Goldner and immunohis- 1Institute for the Reproduction of Farm Animals A Nonhuman Primate Model for 2 tochemical stainings for keratins (K5, 14, 18) Schoenow, Bernau; Unit for Reproductive Medicine and claudins (Cl 1,3). The investigated kerat- the Study of Testicular Peritubu- of Clinics, Clinic for Pigs and Small Ruminants, Uni- ins produced a specifi c expression pattern in lar Cells versity of Veterinary Medicine Hanover, Germany the different cell populations, particularly in 1 2 1 the basal cells. These cells showed a marked N. Schmid , F. Flenkenthaler , K. Dietrich , The demand for cryopreserved boar semen 2 2 3 1 positive staining with K5 and K14. Cl 1 was T. Fröhlich , G. J. Arnold , R. Behr , A. Mayerhofer for export or gene banking is constantly in- 1Biomedical Center, Anatomy III – Cell Biology, creasing. To offer the opportunity of semen localized along the entire length of the later- al plasma membranes between adjacent prin- Ludwig-Maximilians-University; Planegg-Martins- freezing also to small boar studs, a procedure ried; 2Laboratory for Functional Genome Analysis allowing cryopreservation of pre-diluted se- cipal cells as well as at the interface between principal and basal cells. Cl 3 was localized LAFUGA, Gene Center, Ludwig-Maximilians-Univer- men shipped over night was established. Us- sity, Munich; 3Plattform Degenerative Erkrankungen, ing a modular study design, different holding apically at the epithelial border in all regions of the rabbit epididymis. The investigation Deutsches Primatenzentrum GmbH, Leibniz-Institut and equilibration times, extenders and addi- für Primatenforschung, Göttingen, Germany tives that may help to sustain the integrity provides accurate description of the different of spermatozoa during and after cryopreser- cell types of rabbit epididymal duct and en- Human testicular peritubular cells (HTPCs) vation were compared. Normospermic ejac- vironment. Cl 1 seems not only to be local- are smooth-muscle-1 like cells, which form ulates of 42 Pietrain boars were pre-dilut- ized in the realm of tight junctions. It appears the wall of the seminiferous tubules and are ed 1+1 (v/v) with BTS extender and shipped along the entire length between adjacent epi- being associated with the transport of immo- over night to the laboratory at IFN Schönow. thelial cells and basal cells and might play a tile sperm. New data indicate that these cells Quality control post-thaw (PT) included as- role as an adhesion molecule to adhere prin- play important roles and are involved in male sessment of motility (CASA; 10, 30 and 120 cipal cells to basal cells? (in)fertility in a complex way. For example, min at 38°C), mitochondrial activity (Rhoda- by producing GDNF (glial cell line derived min123/PI), membrane status (FITC-PNA, 111 neurotrophic factor) they may contribute to -PSA/PI) and DNA integrity (SCSA) using the spermatogonial stem cell niche. Whether fl ow cytometry. Total sperm motility at 10 Intracytoplasmic Injection of they age and whether their functions may de- min PT was signifi cantly (P < 0.05) high- Equine Sperm Induces Paternal cay is not known. To be able to examine such er for 2h than for 24h of a post-arrival hold- Pronuclear Formation in Bovine a possibility and to control confounding infl u- ing time at 17°C (36.5 ± 11.2 % vs 30.7 ± ences (lifestyle, age, nutrition and the medi- 13.2 %). There was no difference between 2, Oocytes cal history of patients), we turned to a rele- 4, 24 and 48 h of equilibration at 5°C. Motil- M. Schlipf, C. Wrenzycki vant primate model, the marmoset monkey ity was signifi cantly (P < 0.0001) higher in Clinic for Veterinary Obstetrics, Gynecology and An- (Callithrix jacchus). Marmoset monkeys are extenders based on lactose (L) or trehalose drology, Chair for Molecular Reproductive Medicine, nonhuman primates used as model organisms ® (T) compared to Androstar CryoPlus (43.9 Giessen, Germany in reproductive research and share many bi- ± 10.1 % and 41.7 ± 6.9 % vs 27.1 ± 6.4 %). ologically relevant aspects with humans. We Contrary to literature, addition of 100, 500 When oocyte availability is limited, interspe- successfully isolated monkey testicular per- and 1000 mM zinc sulfate heptahydrate to ei- cies intracytoplasmic sperm injection (ICSI) itubular cells (MKTPCs) from young adult ther cooling, freezing or thawing extender did is a useful method to study early events of animals. MKTPCs, like their counterpart not alter motility. Flow cytometry parameters fertilization. It was shown that equine sperm in vivo, express characteristic markers, e.g. were not infl uenced by any freezing modifi - are capable to induce pronuclear formation in smooth muscle-actin and the androgen recep- cation. In conclusion, for highest PT sperm matured oocytes of different species such as tor, but lack markers for Leydig cells (LH- quality and for best practicability we recom- cat, swine and cow [Rodriguez et al., Reprod receptor) and Sertoli cells (FSH-receptor). A mend 2h of post-arrival holding time at 17°C, Fertil Dev 2014; 27: 95]. The objective of the LC-MS/MS pilot study revealed that the se- 4h of equilibration at 5°C and the use of lac- present work was to study male pronuclear creted and cellular proteins of MKTPCs con- tose extender for cryopreservation of pre-di- formation after interspecies ICSI of equine siderably overlap with the ones of HTPCs. luted shipped boar semen. sperm into matured bovine oocytes. Ovaries This supports the relevance of the marmoset

30 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts monkey as a translational model. We there- Sulfonated steroids (s-St) have been tradi- by the loggers and also sent telemetrically to fore obtained MKTPCs also from old animals tionally regarded as inactive metabolites but a monitor. Bitches were defi ned to be healthy and are in the process of studying them. It is are increasingly recognized as a pool of pre- if the total count of leucocytes was not high- becoming clear from increased cell size and cursors for the local production of active ster- er than 12.0 x 1000/µl in blood samples tak- increased -galactosidase activity that these oids in specifi c target cells. Moreover, direct en on day 3 and 7. In total, 8 out of 20 bitch- cells have aged. We expect detailed insights effects without preceding hydrolysis have es were defi ned as healthy in the fi rst 3 days into ageing related changes of testicular peri- been observed. We use the boar as a model to after parturition. The other animals had to be tubular cells from the ongoing studies involv- study the still widely unknown s-St physiolo- excluded because of C-section (n = 5), leu- ing MKTPCs and HTPCs. gy as it exhibits an unusually broad s-St spec- cocytosis (n = 5), drug administration (n = 1) Grants: Support: DFG MA1080/27-1 trum primarily of testicular origin. Previous and mastitis (n = 1). Mean body temperatures attempts to characterize s-St metabolism in were (mean ± SD) 38.9°C ± 0.40 on day 0 boars mainly applying GC/MS or immunoas- p.p. (end of birth until end of day of partu- 113 says were limited by high expenses, low sen- rition), 38.9°C ± 0.44 on day 1 p.p., 38.9 °C sitivity, low specifi city or the lack of meth- ± 0.35 on day 2 p.p., 38,7°C ± 0.31 on day The Luteal Capacity of Bovine ods. The use of LC-MS/MS in ster oidomics 3 p.p., 38,8°C ± 0.30 on day 4 p.p., 38.6°C Corpora lutea is Simultaneously enables the analysis of free and intact sulfat- ± 0.35 on day 5 p.p., 38.5°C ± 0,27 on day Infl uenced by their Location, their ed steroids with highest specifi city and good 6 p.p. and 38.4°C ± 0.34 on day 7 p. p., re- local Relationship to the Prede- sensitivity, making possible the simultaneous spectively. Within the fi rst 3 days 3 out of cessor, and the Ovulatory Folli- measurement of numerous analytes in high- the 8 healthy bitches showed temperatures DGRM-Abstracts er numbers of samples. Profi les (6 hours, 20 above 39.5°C. We conclude that temperature cle‘s Lifespan min intervals) were established for sulfonat- of bitches in the fi rst days p.p. does not differ J. Schneebeli ed 5-androstene-3, 17-diol (ADS), an- from those of healthy dogs in general, while Summaprada, Switzerland drosterone (AS), dehydroepiandrosterone the appearance of short of febrile episodes (DHEAS), epiandrosterone (EAS), epites- seems to be physiological. With the aim to better understand the infl u- tosterone (ETS), estrone (E1S), estradiol- ence of spontaneously varying pre-oestrus 17 (E2S), pregnenolone (P5S), 17OH- conditions on the secretory capacity of the pregnenolone (OHP5S) and free testosterone 116 later Corpus luteum (CL), ovarian function (T) in 4 unstimulated and 4 hCG-stimulated was studied between d-12 and d12 (d1 = oes- boars. Moreover, single measurements were Assessing Infl uence of Barn Cli- trus) in Swiss Brown dairy cattle (daily pro- performed in testicular efferent and afferent mate on Reproductive Perfor- gesterone-RIA in peripheral blood; ovary pal- blood to differentiate between testicular vs. mance of AI boars using Big Data pation at intervals of 1–2 days; 393 datasets). extratesticular origin. Highest concentrations The fi ndings revealed a basically superior lu- were found for EA-S, followed by ETS, ADS Management teal activity of right-sided corpora lutea (r- and DHEAS, which generally exhibited high- M. Schulze1, J. Schäfer1, R. Bortfeldt1, M. Jung1, CL) as compared to their left-sided counter- er levels than E1S and AS. Lowest concentra- C. Ammon2 parts (l-CL). This asymmetry, being evident tions were found for E2S, P5S and OHP5S. 1Institute for the Reproduction of Farm Animals when CLs do not have a direct fully active Sulfonated T, dihydrotestosterone and cho- Schoenow; 2Leibniz Institute for Agricultural Engi- predecessor (fi rst normal oestrus cycle after lesterol also included in the analytical profi le neering Potsdam-Bornim, Department of Engineering parturition), proved to be by far less obvious were undetectable. Profi les of all quantifi a- for Livestock Management, Germany in regularly cycling cows in which, all in all, ble s-St showed simultaneous wave-like pat- just the regression of r-CLs was delayed. The terns virtually synchronous to spontaneous or Barn climate fl uctuations, especially tem- local relationship between consecutive CLs hCG-stimulated T pulses. peratures above 25°C and high air humidity was found to infl uence the P4 secretion; the cause depressions of sperm output and qual- luteal capacity of successors in ipsilateral po- ity that vary boar specifi c. To predict crit- sition being signifi cantly impaired. The me- 115 ical production dates caused by this, for 12 dian lifespan of ovulatory follicles (OF) was month barn climate data (temperature [T], equal on both ovaries, and no effect of the de- What is the Body Temperature air humidity [H], air pressure [P], brightness veloping OF on the activity of a concomitant Measured by Ingestible Tempera- [B]) of 3 barns in one boar stud in south of CL could be seen. Yet, the OF‘s lifespan, and ture Loggers in Healthy Bitches Germany were recorded using data loggers especially the time the vesicle is exposed to (MSR145, MSR Electronics GmbH, Switzer- an ipsilateral CL, are factors which infl uence after Parturition? land). Additionally, production and sperma- the ensuing luteinisation. In conclusion, a ba- L. Schulze, W. Heuwieser, S. Arlt tological data of raw semen as well as sperm ® sic difference in activity of bovine l-CLs and Clinic for Animal Reproduction, Freie Universität Ber- kinetics (SpermVision Automorph, Minitüb r-CLs seems to be blurred by local effects of lin, Germany GmbH, Germany) were documented. During the CL on OFs that happen to develop on the measurement period, 9,145 climate data re- same ovary. As a result, CLs indirectly inter- The fi rst days after parturition are character- cords per barn, 11,590 ejaculate data records fere with the activity of ipsilateral successors. ized by many claims raised by nursing the of 304 AI boars and 67,344 kinetic data re- puppies, uterine involution, milk production cords were collected. Data analysis was per- and hormonal changes. Some authors con- formed with SAS statistics, calculating line- 114 sider body temperatures higher than 39.5°C ar mixed models and spearman correlations. Simultaneous long-term Profi les in this period to be normal, others don’t. The The production parameter weekday, ejacula- objective of this study was to identify the tion interval, breed and age of boar showed of Sulfonated Androgens, Estro- physiological ranges of body temperature signifi cant (P < 0.0001) impact on output and gens and Progestogens in postpu- in the fi rst 7 days p.p.. This method enables motility. The course of seasons translated into bertal Boars measured by liquid the collection of continuous data with a re- quarters and its effect on sperm output and Chromatography-tandem Mass duced potential bias due to the handling of the kinetic parameter velocity and distance Spectrometry (LC-MS/MS) animals and less yielding of stress. The study average path (VAP, DAP) could be demon- was performed with 20 private-owned bitches strated clearly (P = 0.0021 and P = 0.0122). G. Schuler1, A. Sánchez-Guijo2, S.A. Wudy2 of different breeds. During each experiment In detail, barn climate (T, H and T × H) and 1Veterinary Clinic for Obstetrics, Gynecology and An- the bitch daily swallowed temperature log- weekday infl uenced sperm kinetics VAP and drology; 2Steroid Research and Mass Spectrometry gers (ANIPILL, BodyCap, Hérouville Saint- DAP (P < 0.0001). An effect of P and B on Unit, Center of Child and Adolescent Medicine, Jus- Clair, France) for 7 days. Core body temper- sperm output and quality could not be shown. tus-Liebig-University, Giessen, Germany ature was measured every 15 minutes, stored Morphologic defects correlated negatively

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 31 DGRM-Abstracts

(rs = –0.4; P < 0.0001) with sperm motility. 118 The aim of this study was to compare the In summary, we were able to verify the in- use of two different hand-powered vacuum fl uence of barn climate, boar breed, age of Time lapse Technology for Embryo pumps and the suspension by the hind legs boar and production proceeding on quanti- Selection at the Pronuclear Stage within the scope of resuscitating newborn ty and quality of ejaculates. Continuous ac- in Countries where the Culture of calves after dystocia. The present study rep- quisition of climate, production and sperma- Supernumerary Embryos is limit- resents the fi rst summary based on fi ndings tological data as well as sperm kinetics is the from examinations performed under fi eld foundation of developing a prediction model, ed: Where is the Benefi t? conditions. A total of 60 calves (born with which can help to cope with times of sperm L. Sela, S. Mittenzwei, N. Rogenhofer, S. Mahner, dystocia) from different farms were exam- depression. C.J. Thaler, V. von Schönfeldt ined. They were randomly divided into three Grants: Supported by FBF Division of Human Gynaecologic Endocrinology and groups with 20 calves per group. To remove Reproductive Medicine, Department of Gynaecology mucus and fetal fl uid from the respirato- and Obstetrics, Ludwig-Maximilians-University, Mu- ry tract of calves, following operations were 117 nich, Germany performed: Group I: HK-Beatmungspumpe (Rheintechnik), Group II: Calf Aspirator and Bovine Cystic Ovary Disease: How Tripronucleate embryos (3PN) after intra- Resuscitator (McCulloch Medical), Group Does it Affect Tubal Functional In- cytoplasmatic sperm injection (ICSI) have III: Only method of suspension by the hind tegrity and Gameto-Maternal In- a low developmental potential, giving rise legs of the calf. After these procedures in the DGRM-Abstracts to poor-quality embryos which are associat- groups, the vitality (Time to Sternal Recum- teraction? ed with signifi cantly decreased implantation, bency, T-SR) of the calves and the incidence D. Scully, S. Kölle pregnancy and life birth rates. Accordingly of diseases like diarrhea, pneumonia and om- School of Medicine, University College Dublin, Ire- the transfer of 3PN embryos should be avoid- phalitis in the fi rst weeks of life were record- land ed making accurate identifi cation of such em- ed. After delivery, the girth of the muzzle of bryos highly desirable. In the present study 55 calves was also measured. Considering the Cystic ovary disease (COD) is a major fac- we analysed whether time-lapse technology study results, there was no signifi cant differ- tor contributing to poor reproductive effi cien- can be used to properly identify 3PN embry- ence of the T-SR between the groups. How- cy of lactating dairy cows. However, the pre- os that would have been erroneously classi- ever the calves that were treated with a hand- cise effects of follicular and luteal cysts on fi ed as normal if looking at them at a specif- powered vacuum pump in tendency showed oviductal function are still unknown. As the ic time interval. We performed a retrospec- a shorter T-SR (p = 0.07). In conclusion, the oviduct is pivotal for gamete transport, fer- tive time-lapse movie evaluation from pa- present study was not able to show that the tilisation and early embryonic development, tients undergoing ICSI from September 2013 use of hand-powered vacuum pumps is supe- the aim of our study was to analyse the effects to December 2013. Eighteen of these patients rior to the suspension by the hind legs. of ovarian cysts on the functional integrity of had at least 1 3PN embryo and were therefore the oviduct using light microscopy, stereo- included in the subsequent study. Different microscopy, histochemistry, scanning elec- times of appearance of the individual pronu- 120 tron microscopy and quantitative and quali- clei of 29 3PN embryos were analysed using Effect of eCG Administration on tative live cell imaging. Our results showed the EmbryoScopeTM as a time-lapse imag- that 90% of the bovine female tracts collect- ing device. Other analysed parameters were Day 6 post partum in Cows on Se- ed in the abattoir revealed COD on the right fi rst division, number of cells on day 3, com- rum Concentration of Estradiol ovary. The ampullar epithelium from cows paction and blastulation. In 48.3% of all 3PN M. Sharifabad1, S. Oschidari1, C. Schürmann2, with luteal cysts revealed a constant predomi- embryos the appearance of all 3 pronuclei oc- A. Wehrend2 nance of ciliated cells lacking the ability to curred at the same time. In contrast, in 51.7% 1 2 rd Islamic Azad University of Varamin, Iran; Clinic for form secretory cells required for embryo nu- the 3 pronucleus appeared later than the oth- Obstetrics, Gynaecology and Andrology with Veteri- trition after fertilization. Synthesis of glyco- er 2 (more than one hour later in 44.8% and nary Ambulance, Justus-Liebig-University, Giessen, proteins and acidic mucopolysaccharides was more than 5 hours later in 10.3%). Of all 3PN Germany increased within the ciliated cells of the am- embryos, 9 were subjected to further culture, pulla. Whereas the attachment of the cumu- 44.4% of them until day 3 and 55.6% of them In modern dairy industry, the amount of lus-oocyte-complex to the ampullar epitheli- until day 5. Only 33.3% presented a regu- postpartum anestrous has increased in the um and the formation of the sperm reservoir lar fi rst cleavage and had 8 or more cells on last decades. In recent studies a positive in- in the isthmus was not negatively affected day 3. From the embryos cultured until day fl uence of eCG administration could be de- in cows with ovarian cysts, ciliary beat fre- 5, only 20% showed signs of compaction and tected on early resumption of ovarian activi- quency and particle transport speed were in- began to blastulate. These fi ndings may en- ty, resulted in an improved reproductive per- creased in the tubal ampulla as compared to courage other centres under restrictive legis- formance by day 6 post partum [Rostami et controls. Our results imply that ovarian cysts lation to introduce time-lapse technology into al., 2011, Vojgani et al., 2013]. These studies impair formation of secretory cells in the am- their laboratory routine, since this technique revealed enhancing ovarian follicle growth pulla. Ciliated cells do increase their secreto- can also be used for early embryo selection. and early ovulation in post partum cows but ry activity but are not able to synthesize gly- An example is the accurate identifi cation of did not provide any estradiol serum-level in cogen which is essential for successful preg- 3PN embryos, avoiding further culture or cows. The aim of this study was to collect nancy. At the same time the transport of the even transfer of these embryos. data on estradiol serum levels on day 6 post early embryo is compromised. Thus, suc- partum and after eCG administration on day cessful therapeutic strategies for treatment of 10 post partum. 63 dairy and beef cows were ovarian cysts include not only successful in- 119 included in this study. The animals were di- duction of ovulation and fertilization, but op- vided into two groups, healthy and dystocia/ timizing the conditions for the early embryo Comparison of different Reanima- puerperial disorders. These groups were fur- in the oviduct. tion Methods in New born Calves ther divided into 2 subgroups; animals treat- after Dystocia under Field Condi- ed with eCG and control. Blood serum sam- tions ples were taken on day 6 post partum fol- lowed by an injection of 480 I.E. eCG or S. Sendag1, J. Homberg2, A. Wehrend2 nothing respectively. On day 10, a second se- 1Clinic for Veterinary Obstetrics and Gynecology, rum sample was taken. The estradiol concen- Yuzuncu Yil University, Van, Turkey; 2Clinic for Veteri- trations were determined by radio-immono- nary Obstetrics, Gynecology and Andrology, Justus- assay. There was no statistically signifi cant Liebig-University, Giessen, Germany increase in the estradiol-concentration ob-

32 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts served in any group. Although the literature 122 123 showed that the reproductive performance of dairy cattle, including an enhanced folli- Evaluation of Equine Cervical Morphokinetics of Preimplanta- cle growth to a dominant stage, could be im- Function Based on the Histologi- tion Mouse Embryo Development proved by administration of eCG, no rise in cal Evidences of Smooth Muscle using Time Lapse Monitoring of estradiol concentration could be detected in Tissue Embryo Development (TLMED) this study. The cause of the discrepancy of the study results and the literature is unclear. Pos- K. Skierbiszewska1, M. Domino1, J. Olszewski1, G. Sokolowski1, R. Faundez2, J. Ostrowski1, sible is that in contrast to the results found in D. Domanska1, A. Wehrend2, Z. Gajewski1 M. Gajewska1, Z. Sandowska-Markiewicz1, the literature, either no effect of eCG could be 1Department of Large Animal Diseases with Clinic, A. Niwinska2, U Wojcik-Trechcinska1, achieved or the eCG effects are not mediat- VRC and CBB, Faculty of Veterinary Medicine, M. Woszczynski1, A. Tyl-Bielicka1, M. Borowa- ed by the stimulation of the follicular growth. WULS – SGGW, Warsaw, Poland; 2Klinik für Veteri- Chmielak1, S. Dabrowski2, K. Pysniak1, Z. Gajewski2 närmedizin, Klinik für Geburtshilfe, Gynäkologie und 1Department of Genetics, The Maria Sklodowska – Andrologie der Groß- und Kleintiere mit Tierärztli- Curie Oncology Center and Institute; 2Department 121 cher Ambulanz der Justus-Liebig-Universität Gies- of Large Animal Diseases with Clinic, VRC and CBB, sen, Germany Faculty of Veterinary Medicine, WULS – SGGW, Serum Level of Neuropeptides in Warsaw, Poland Cows with Torsio uteri Proper cervical contractility and relaxa-

tion is a major factor in mare’s fertility and The purpose of this study was to deter- DGRM-Abstracts M. Sickinger1, J. Roth2, A. Wehrend1 uterine defense mechanisms. Histological- mine the preimplantation developmental 1Clinic for Obstetrics, Gynaecology and Androlo- ly, the cervix consists mostly of connective potential of embryos from different mouse gy with Veterinary Ambulance; 2Institute for Veteri- tissue, smooth muscle and extracellular ma- strains, using TLMED technique. A to- nary Physiology, Justus-Liebig-University, Giessen, trix. Smooth muscles and Cajal-like inter- tal of 457 embryos were collected from Germany stitial cells (ICLC) are responsible for con- 80 female mice, 10 strains: 129S1/SvW, tractility. Cervical disorders are predisposing AKR/W, C57BL/6W, C57BL/10W, C3H/W, Introduction Torsio uteri intra partum is a factors for fl uid accumulation and contamina- C3Hwad/W, CBAmut/W, A.CA/W, BN/aW, common reason for dystocia in dairy cows. tion of the uterus, might impair fertilization BALB/cW. Mice were superovulated, mat- This disease resembles the displacement of as well as infl uence pregnancy and parturi- ed and euthanized according to the stand- the abomasum as both diseases are associated tion. The purpose of this study was to demon- ard procedure. Only 301 morphological nor- with a torsion of a hollow organ. Due to the strate ICLC in the equine cervix to comple- mal embryos (zygotes, 2–3 cell) were ana- formerly shown changes in the neuropeptic ment the knowledge on mare’s reproductive lyzed. Embryos were cultured in CSC me- content of the abomasal wall, the topic of this tract conditions with detailed information re- dium containing 10% SSS for 96–120 hours study was to examine the neuropeptide serum garding cervical function in diestrus. Fresh in three gas atmosphere incubator contain- levels of cows suffering from torsio uteri. whole thickness samples of the uterine body ing 6% CO2, 5% O2, and 89% N2. The mor- Materials and Methods Blood samples of and cervix were collected from 30 slaugh- phokinetic assessment was performed using 20 cows with torsio uteri, 36 healthy controls tered mature mares with no signs of repro- the Primo Vision Time Lapse Embryo Moni- and 15 cows intra partum without torsio uteri ductive tract pathologies. The samples were toring System. From C3H/W, C3Hwad/W, were examined and neuropeptic contents for stained with hematoxylin eosin (HE), Mas- 129S1/SvW, and C57BL/6W it was obtained substance P (SP) and vasoactive intestinal son’s Trichrome and immunofl uorescent la- respectively 63% (24/38), 72% (28/39), 66% polypeptide (VIP) were measured by means beling (IF) with primary (anti-vimentine and (21/32) and 87% (28/32) of blastocysts with of commercially available ELISA kits. For all anti-CD117) and secondary antibodies (con- a cleavage average time over 1189 and 1243, cows Interleukin 1 (Il 1) levels were deter- juncted with AF488 and AF647) and imaged 986, 1399, 1418 min from 3rd and 2nd cleav- mined and compared between groups. by using light and confocal microscopy, re- age respectively. These strains showed the Results Signifi cant differences between spectively. Cells with morphologic and im- highest preimplantation developmental po- groups could be shown for the SP concentra- munologic phenotypes similar to the cells of tential. C57BL/10W, CBAmut/W and BN/ tions in cows with torsio uteri and cows calv- Cajal of the gastrointestinal tract were iden- aW are characterized respectively by average ing without torsio uteri (P < 0.0001). SP se- tifi ed inside of the uterine body and cervix. timing of 3rd cleavage – respectively: 1418, rum levels in cows sub partu were marked- The CD117 and vimentine positive cells were 681, 1255 min and by lower n, % and quali- ly higher than in those suffering from torsio localized in the muscle layers as fusiform, tri- ty of embryos. The blastocysts rate was 71% uteri. Comparing the healthy controls with angular or starlike-cells with dendritic pro- (12/17), 23% (7/30), 61% (34/56). AKR/W, cows during calving also resulted in signifi - cesses, which formed a cellular network and BALB/cW and A.CA/W embryos showed cantly higher SP levels in cows sub partu were classifi ed as ICLC. The fusiform ICLC deeply compromised embryo developmental (P = 0.002). Concerning VIP and Il 1 no sig- were the predominant cell type in the corpus showing respectively: 2% (1/26), 0% (0/22), nifi cant differences between the groups could uteri. Vimentin reactivity was mainly local- 0% (0/9) and timing of third cleavage over be detected. ized within the cell processes, and CD117 has 2061, 527 and 1861 min. Embryos from dif- Discussion and Conclusion Remarkable a patchy pattern in the cell body. In conclu- ferent mouse strains showed clear differenc- amounts of SP are released into the blood. sion, the double IF labeling protocol was op- es in their developmental potential and tim- SP being a biomarker and mediator for pain timized with respect to ICLC identifi cation in ing. The time of 3rd cleavage is the princi- suggests the hypothesis that cows with torsio equine reproductive tract. pal morphokinetics parameter for prediction uteri do not experience as much pain as cows of developmental and implantation potential that are calving without dystocia. A possi- of tested mice strains. ble reason could be the lack of opening of the cervix in cows with uterine torsion. Further research is needed to proof this assumption.

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 33 DGRM-Abstracts

124 tion. Therefore additional stimuli are needed -ER and -ER independently from proges- which may also initiate parthenogenetic de- terone administration. The mRNA expres- Assessment of the Thyroid Hor- velopment. The aim of this study was to vali- sion (mean ± SD) was signifi cant higher be- mone Status During Late Preg- date the paternal contribution to embryonic fore (-ER 0.23 ± 0.098; -ER 0.03 ± 0.021) nancy and Early Lactation development employing the analyses of neu- than after (-ER 0.19 ± 0.292; -ER 0.02 ± ronatin (NNAT) mRNA expression, a pater- 0.031) treatment both for -ER (P < 0.0001) L. Steinhoff1, M. Meyerholz2, H.J. Schuberth2, nally expressed gene, via RT-PCR. Cumulus- and -ER (P = 0.016). Seven day progester- M. Schmicke1 oocyte-complexes were isolated from abat- one administration affected oestrogen endo- 1Clinic for Cattle, Endocrinology Laboratory, Univer- toir-derived ovaries. After 24 h of maturation, metrial receptors expression the major -ER sity of Veterinary Medicine Hanover; 2University of fertilization was realized using a standard and the second one -ER. The results confi rm Veterinary Medicine, Immunology Unit, Germany protocol [Parrish et al. 1995]. Presumptive the inhibitory effect of progesterone on estro- zygotes were cultured in SOFaa for 8 days. gen receptors expression. Thyroid hormones are important regulators To generate parthenogenetic blastocysts, ma- of metabolic adaption during pregnancy and tured oocytes were activated by incubation in in early lactation. However, less data is avail- 127 5 µM ionomycin for 5 minutes and in 1.9 mM able from modern high-yielding dairy cows. 6-dimethylaminopurine for 4 h. Afterwards, Diversity of DQA2 Gene in Rela- The aim of the study was to assess thyroid treated oocytes were cultured in SOFaa for 8 hormones (Triiodothyronine [T3], Thyroxine tion to Somatic Cell Count (SCC) days. RT-PCR was performed for six embry- [T4]) and Thyroid stimulating hormone of Milk and Subclinical Mastitis DGRM-Abstracts os stemming from the IVF-group and three [TSH] during transition from late pregnan- Occurrence in Low-input Dairy embryos out of the chemical activated group. cy to early lactation as well as to examine, NNAT mRNA was expressed in fi ve blasto- Sheep Farms whether basic thyroid ultrasound is feasible cysts out of the IVF-group, one blastocyst did 1,2 2 1 in cows and may refl ect the thyroid hormone N. Tzanidakis , C.N. Brozos , N. Voutzourakis , not show NNAT mRNA expression suggest- 1 3 1 status. Holstein Friesian heifers (n = 12) were A. Stefanakis , E. Malama , A. Zdragkas , ing that it was derived from parthenogene- 4 1 2 sampled twice weekly from day 259 ± 2 after J. Hickford , S. Sotiraki , E. Kiossis sis. NNAT mRNA could not be detected in all 1 artifi cial insemination to day 34 ± 2 after par- Veterinary Research Institute, Hellenic Agricultural blastocysts derived from chemical activation 2 turition and T3, T4 as well as TSH were an- Organization Demeter, Thessaloniki; Clinic of Farm indicating that only the maternal genome was alyzed. At the end of the clinical puerperium Animals, Faculty of Veterinary Medicine, School of present. These results clearly demonstrate an ultrasonographic examination of the thy- Health Sciences, Aristotle University of Thessaloniki, that NNAT mRNA expression is an appropri- 3 roid gland was conducted. A subsequent dis- Greece; Clinic of Reproductive Medicine, Vet suisse ate method to prove the paternal participation 4 section of the animals allowed the determina- Faculty, University of Zurich, Switzerland; Gene- in early bovine embryo development. tion of thyroid size, weight and volume which Marker Laboratory, Faculty of Agriculture and Life were correlated to the ultrasonografi cally de- Sciences, Lincoln University, New Zealand tected thickness of the thyroid gland. Concen- 126 This study aimed to explore the diversity of trations of T3 and T4 signifi cantly decreased the ovine major histocompatibility complex with a nadir during the fi rst and second week The Effect of Progesterone on the (MHC) DQA2 gene in relation to Somat- postpartum (p < 0.05) while TSH remained Endometrial Expression of Estro- ic Cell Count (SCC) of milk and subclini- constant (p > 0.05), excepting a peak around gen mRNA in the Dairy Cows cal mastitis occurrence in low-input dairy calving. Interestingly, the highest inter-in- sheep farms, with regard to different biot- dividual variance for T3, T4 and TSH was M. Trela, M. Domino, J. Olszewski, D. Domanska, ic (age class of ewes, lactation month) and detected at time of parturition. Neither the M. Kaczmarek, Z. Gajewski abiotic (management [MS] and milking sys- thickness of the thyroid gland assessed ultra- Department of Large Animal Diseases with Clinic, tem [MLS]) risk factors. Samplings were car- sonographically nor the data measured dur- VRC and CBB, Faculty of Veterinary Medicine, ried out in 10 extensively and 10 semi-in- ing dissection showed signifi cant correla- WULS – SGGW, Warsaw, Poland tensively managed low-input dairy sheep tions with hormone concentrations (p > 0.05). Estrogen acts through endometrial steroid re- fl ocks, with three milking systems: milk- Moreover the thyroid gland altered its shape ceptors (oestrogen -ER and Estrogen -ER), ing parlour machine (MPM), portable milk- if the cow changed the position of the head. infl uence the timing of upregulation in proper ing machine (PM) and hand milking (HM). This derogated the usefulness of this method. as well as failure luteolysis. This study exa- Over 2 consecutive lactations, milk samples The low thyroid hormone levels after calving mined the expression patterns of steroid re- (n = 9,624) were collected monthly in each can be related to the negative energy balance ceptors (-ER, -ER) in the bovine endome- fl ock, from 20 ewes (10 primi- and 10 multi- postpartum. Moreover, an important effect of trium during intravaginal progesterone treat- parous ewes). SCC was assessed in milk sam- thyroid hormones on the individual capacity ment in dairy cows. Uterine biopsy samples ples with pH  6.00, using the FossomaticTM for metabolic adaption around calving is sug- were collected over the proestrus before in- system (FOSS®, Denmark) and samples with gested. travaginal progesterone administration (pro- SCC  400 × 103 cells/ml (n = 2,541) were gesterone insert, 1.38 g, 7 days) and after 7 processed with bacteriological examination. days from 25 HF nonlactating cows. From Blood samples were collected from the stud- 125 clippings from endometrium full RNA was ied ewes (n = 800) onto FTA Whatmann™ Assessment of Successful Ferti- isolated using columnary method. Complete Cards. Following DNA extraction from the lization in Bovine Blastocysts via RNA was then undergone a reverse transcrip- FTA cards, PCR-SSCP analysis of DQA2 tion reaction using oligo T primers to re- was performed to ascertain genotype. Only 2 Neuronatin Transcript Analyses ceive mRNA transcripted onto cDNA. cDNA alleles were found in > 10% of the blood sam- J. Stöhr, L. Maurer, B. Zimmer, C. Wrenzycki was then get multiplied using cyclic polime- ples: alleles B1 („0602“) and K („0301“), that Clinic for Veterinary Obstetrics, Gynecology and An- rase reaction using paired starters (ESR-- were identifi ed in 215 and 244 sheep respec- drology, Chair for Molecular Reproductive Medicine, GAPDH and ESR--GAPDH) specifi ed for tively, with an occurrence of 0.139 and 0.158. Justus-Liebig-University, Giessen, Germany each examined sequence using QuantStudio The occurrence of any of the alleles did not Real-Time PCR system. Progress of the re- differ between ewes with SCC < 400 × 103 Oocyte activation is a prerequisite for decon- action of polimerisation of complimentary and SCC  400 × 103 cells/ml of milk or be- densation of the sperm head and formation strands for each sequence was registered on tween different MSs, MLSs, age class of of the male pronucleus regardless of wheth- a diagram of growth of the fl uorescence in- ewes or lactation months. MHC DQA2 allele er the spermatozoon enters the oocyte by in- tensity that occurs during attaching a marked B1 („0602“) occurred with a lower frequency jection or by fertilization. In several species nucleotide to the matrix. Signifi cant differ- in ewes with subclinical mastitis (p < 0.05). including cattle the injection procedure it- ences (P < 0.0001) in gene expression in the To our knowledge, this is the fi rst report ex- self is insuffi cient to induce oocyte activa- endometrial samples were found between ploring the effect of DQA2 gene on subclin-

34 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts ical mastitis of sheep reared under low-input the number of antral follicles of an individ- sperm function is infl uenced by taste signal- management systems. ual (ovarian reserve). Recently it was pub- ing components. Combining complementa- lished that high AMH levels could be pos- ry molecular, cellular and reproductive biol- itively related to fertility in cattle. In this ogy approaches we found that the 2 subunits 128 study we aimed to prove, whether AMH of the umami taste receptor dimer (Tas1r1/ Embryo-Maternal Interaction in levels of Holstein-Friesian heifers could be Tas1r3) as well as some members of the Tas2r Roe Deer used as a predictive and independent bio- family of bitter receptors and components of marker for their subsequent fertility as dairy their downstream signaling cascade, such as V. van der Weijden, A. Rudolf Vegas, B. Drews, cows. Therefore 1043 plasma samples and Gustducin and TRPM5, are expressed during S.E. Ulbrich data were collected from post puberal heif- late phases of spermatogenesis as well as in Animal Physiology, Institute of Agricultural Sciences, ers of 5 farms and correlated to their fertili- mature spermatozoa. Moreover, employing ETH Zurich, Switzerland ty parameters. Plasma samples were analyzed a Tas1r1-defi cient mCherry reporter mouse with an ELISA-Kit (DSL-10-144400, Beck- strain, we found that Tas1r1 gene deletion Embryonic development is a complex, well- man Coulter, USA) for their AMH concen- resulted in spermatogenic abnormalities, in- orchestrated process taking place in differ- tration. Overall, the studied heifers had 0.493 cluding an increase in the rate of apoptosis of ent compartments of the female reproduc- ± 0.3 ng/ml AMH in the plasma. We fi rstly developing germ cells. Furthermore, a signifi - tive tract. Maternal recognition of pregnancy checked possible but unknown impact factors cant increase in spontaneous acrosomal re- (MRP) is considered a critical event for em- on the individual AMH levels of the heifers. action was observed in Tas1r1 null mutant bryo survival and establishment of pregnan- We found no signifi cant effects of the farm, sperm which was accompanied by an elevat- DGRM-Abstracts cy. The embryo continuously faces chang- age, weight, height, BCS, progesterone lev- ed level of the second messenger cAMP and es of its microenvironment, the histotroph, els and pregnancy status at sampling or their an increase in cytosolic Calcium. Since pro- which contains different constituents impor- later milk yield, fat and protein on the AMH duction and maturation but also sperm’s fer- tant for embryo survival and development. levels (r < 0.1, p > 0.1). For 672 heifers fertil- tilizing competence strongly depend on their We aim at unravelling how the embryo of roe ity parameters of their fi rst lactation could be ability to detect diverse chemical ligands in deer, a mono-oestrous seasonal species, sig- acquired and analyzed. We found no correla- their microenvironments, it will be essential nals its presence. Therefore, samples from tions between the AMH levels and the heif- to uncover the physiological function of taste the endometrium of hunted roe deer during ers’ age of fi rst breeding or calving, services receptors for male germ cell’s ability to ade- the period of diapause and the reactivation of per conception as heifer or cow, days to fi rst quately respond to distinct external stimuli in embryonic growth were examined (n = 5 for service post-partum, days open to pregnancy their surrounding milieu. endometrium of embryos < 1 mm, 1–2 mm, or conception rates (r < 0.1, p > 0.1). Our re- > 2 mm, and n = 3 for elongated embryos, sults support the notion that AMH levels of 131 sampled between early November until end post puberal heifers could be considered as of December). This is comparable to the time mainly independent of environmental and Comparative Study of Automatic period when MRP takes place in other rumi- individual factors at the time of blood sam- nants. A targeted approach was used to de- and Manual Thermogram Analy- pling. However, our results showed that a sin- sis of Bovine Udders with induced termine the differential mRNA expression of gle assessment of the ovarian reserve in heif- genes known to be involved in MRP of cattle, ers by AMH plasma concentrations, meas- E. coli-Mastitis pig, and sheep. Genes involved in the clas- ured by a human ELISA kit is not a suitable sical IFN-signalling, e.g. IRF2, ISG15, and S. Watz¹, W. Petzl¹, H. Zerbe¹, W. Schröter², parameter or biomarker to predict fertility in T. Landgraf², M. Metzner¹ MX1, conceptus elongation, e.g. AKR1B1, dairy cattle. HSD11B1, PTGS2, PTGES, and PTGER2, ¹Clinic for Ruminants with Ambulatory and Herd implantation, e.g. MUC1, ESR1, and PGR, Grants: FBF Bonn is gratefully acknowl- Health Services, Ludwig-Maximilians-University, and amino acid and glucose transporters, e.g. edged for funding this study. Munich; ²Fraunhofer Institute for Transportation and SLC1A5, SLC2A1, SLC5A1, SLC7A2, and Infrastructure Systems IVI, Dresden, Germany SLC15A3 were analysed by qPCR. Our fi rst 130 Infrared thermography is a simple and nonin- preliminary data show that not even minute vasive method to measure changes in bovine amounts of IFN seem to be secreted by the Functional Implication of Taste Re- ceptors and Gustatory Signaling udder surface temperature and thus a help- embryo, since upregulation of IFN-stimulat- ful tool for early mastitis detection. However, ed genes in the maternal endometrium, as ob- Molecules for Male Reproduction the manual analysis of thermograms is very served in other ruminants is lacking. In con- 1 1 1 2 time-consuming. The aim of this study was clusion, it remains to be shown which other A. Wartenberg , L. Fahr , D. Meyer , A. Voigt , U. 3 1 1 1 to compare whether the automatic segmenta- signals, e.g. oestrogens, may play a role in Boehm , T. Gudermann , W. Meyerhof , I. Boekhoff 1 tion and evaluation of thermal images gains MRP in roe deer. The endometrial response Walther-Straub-Institute, Ludwig-Maximilians-Uni- 2 comparable results. Five healthy Holstein- to signals from the embryo may provide a versity, Munich; German Institute of Nutrition, Pots- 3 Friesian cows were challenged with E. coli conclusive fi ngerprint. dam-Rehbrücke; Department of Pharmacology and Toxicology, University of Saarland, Homburg, Ger- into the right hind quarter. In each case, acute many mastitis was induced. Thermograms were 129 taken every two hours in a period of 24 hours Male fertility is characterized by a sequential before and after challenge. The images were Relations between Plasma Anti- process which can be subdivided into 3 sub- interpreted manually, using a polygon-tool. Muellerian-Hormone (AMH) Con- steps: The proliferation and differentiation of Automatic image analysis based on Active centrations of Holstein-Friesian male germ cells in the seminiferous tubules Shape Model approach was implemented by Heifers and their later Fertility as of the testis, the travelling of motile sperm Fraunhofer Institute. For each image, values through the different segments of the epididy- for average and maximum udder tempera ture Dairy Cow mal duct, and fi nally, the long lasting journey were calculated and compared by boxplot- A. Vernunft1, A. Boldt2, T. Viergutz1, J.M. Weitzel1 of sperm in the female genital tract which al- analysis. Manually determined temperatures 1Institute of Reproductive Biology; Leibniz Institute lows sperm-egg fusion. The recent observa- were higher than automatically analyzed val- for Farm Animal Biology, Dummerstorf; 2Institute of tion of an expression of taste receptors and ues (median = 0.80K; lower quartile [q1] = Livestock Farming, Mecklenburg-Vorpommern Re- coupled downstream signaling components, 0.76K; upper quartile [q3] = 0.87K). Both search Centre for Agriculture and Fisheries, Germany such as the taste G protein -Gustducin and methods detected peak values for maximum the cation channel TRPM5 in non-taste tis- udder temperature 13 and 15 hours post in- Plasma levels of Anti-Muellerian Hormone sue led to the question whether gustatory fectionem (p.i.): 2.20K (13 h p.i.) and 2.19K (AMH), produced only from granulosa cells, signaling molecules are also expressed in the (15 h p.i.) difference from median maxi- refl ect the total number of oocytes and also male reproductive system and whether proper mum temperature (37.44°C; q1 = 37.27°C;

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 35 DGRM-Abstracts

q3 = 37.61°C) for manual analysis, as well Reproductive dysfunction observed in lactat- tion and the expression of the GC-responsive as 2.33K (13 h p.i.) and 2.08K (15 h p.i.) for ing cows has been attributed to metabolic ad- target genes, including PLZF and FKBP5. automatic segmentation (median 36.38°C; aptation which is needed to ensure energy de- The GR antagonist RU486 blocked these

q1 = 36.19°C; q3 = 36.61°C). It is presumed mand for lactation. This is especially critical actions indicating that GR is fully active in that higher temperatures in manual analysis during the post partum period of negative en- these cells. Further qPCR screening studies occur due to inclusion of warmer regions, ergy balance marked by high rates of oxida- of HTPCs revealed that Dex infl uenced the e.g. udder-thigh cleft, whereas automatic seg- tive stress; however, conception is highly de- expression of known and formerly reported mentation leaves these regions out. Still, tem- sirable. Between 6 and 30% of fi rst-lactat- candidate genes associated with HTPC func- perature curves for each method show simi- ing and especially multiparous cows devel- tion, amongst others, smooth muscle actin, lar progresses. Thus, automatic segmentation op a cystic ovarian follicle (COF) during the calponin, cyclooxygenase 2 and angiotensin of infrared images is a quick and promising early post-partum period. Cystic ovarian fol- receptor type I. Thus, our studies demons trate approach to enhance early mastitis detection. licles interfere with normal ovarian activity that HTPCs express functional GRs, which and thus prevent conception and prolong in- are linked to functions of peritubular cells. ter-calving interval. In the present study we GC-signaling thus likely plays an important 132 developed a technique to induce COFs by ul- role in the normal adult testis. It remains to trasound-guided follicle injection. We inject- be shown, if this system is altered in states of Maturational Potential of Feline ed different cyclooxygenase inhibitors, such infertility and if elevated GC levels, as asso- Oocytes with different Morpho- as indomethacin and fl unixin at different time ciated with stress or medication, may impair

DGRM-Abstracts logical Characteristics points relative to the (expected) ovulation in male fertility. different concentrations. Ovulation is suc- Grants: Supported in part by DFG T. Weiser1,2, J. Zahmel1, K. Jewgenow1 cessfully inhibited by injection of 50 µM in- MA1080/25-1 1Department of Reproductive Biology, IZW Berlin; domethacin 16 hours post-GnRH with 100% 2TU Munich, Germany effi ciency. The remaining non-ovulated fol- Since many felid species living in the wild licular structure resembles those of a COF. are threatened from extinction, assisted re- We compared these COFs with classical de- 135 veloping corpus lutea at days 1 and 5 after productive techniques (ART), such as in vitro Transient Oligo-Astheno-Terato- maturation (IVM) and fertilization (IVF) gain the (expected) ovulation. We measured gene more importance to support reproduction of expression patterns of marker genes of the zoospermia in a 3-year old captive cats. Due to its similarities to many cyclooxy genase pathway as well as endo- German Shorthair Pointer: A Case wild living species the domestic cat (felis ca- crine parameters (such as PGE2 and 8-iso- report tus) serves as a model for all feline species. PGF2). Our data indicate that PGE2 is mas- 1,2 1,2 1,2 Ideally oocytes with optimal quality, defi ned sively down-regulated after injection of indo- K. Wolf , E. Schröder , A. Baumeister , 1,2 1,2 by homogenous almost black ooplasm and methacin, thus preventing an essential step F. Schirmer , A.R. Günzel-Apel 1 2 intact cumulus cell layer, are used for ART. for proper ovulation. Unit of Reproductive Medicine of Clinics; Small But since oocytes are not always suffi cient Grants: This work has been supported by Animal Clinic, University of Veterinary Medicine in number, maturational potential of oocytes the German Research Foundation (DFG). Hanover, Germany with poorer quality is of interest. Oocytes Acquired infertility may develop occasional- were matured in vitro in 3 different groups: ly, but in many cases the causal background Oocytes with optimal quality as positive con- remains obscure. Oligo-astheno-teratozoo- trol (control; n = 149), oocytes with grey, un- 134 spermia is a condition including concurrent even or patchy ooplasm but intact cumulus Expression of the Nuclear Gluco- oligozoospermia, asthenozoospermia and ter- cell layer (oopl-; n = 242) and oocytes with corticoid Receptor in Peritubular atozoospermia and may end up with azoo- partially missing or damaged cumulus lay- spermia. ers which had a high quality ooplasm (cum-; Cells of the Human Testis and Ac- n = 122). After IVM (M199 based medi- tivation of Target Genes by Dexa- Case report: A 3-year-old German Short- hair Pointer of 37 kg body weight, which had um, 5.5% CO2, 38.5 °C) the nuclear stage of methasone the oocytes was determined with propidium a history of 11 successful breedings, was pre- iodide staining. Maturation rate was defi ned H. Welter1, C. Herrmann1, F. M. Köhn2, sented for semen freezing. The accompany- as relation of MII oocytes to the total number J. U. Schwarzer3, A. Mayerhofer1 ing breeding soundness evaluation revealed of oocytes per group. The control (75.84%) 1Biomedical Center (BMC), Cell Biology, Anatomy III, a generalized pyodermia also affecting the and the cum- (73.39%) group showed simi- Ludwig-Maximilian-University, Planegg; 2Andrologi- scrotum. At morphological examination of larly high maturation rates. In contrast, the cum, Munich; 3Andrology-Center, Munich, Germany the genital tract increased scrotal temperature maturation of oopl- oocytes was signifi cantly and pain as well as scrotal lesions and slight- The receptors for glucocorticoids (GCs) lower (38.91%). In the cum-group, the num- ly swollen scrotal lymph nodes were detect- were described in the testis, among oth- ber of parthenogenetic cleavage (13 of 122) ed. The general condition and libido were un- ers in peritubular cells, i.e. the cells form- after maturation was strikingly high. To fur- disturbed. Microscopic semen analysis yield- ing the wall of seminiferous tubules. Peritu- 6 ther evaluate the developmental potential of ed a total sperm count of 315 × 10 , 40% pro- bular cells are emerging as important players the oocytes IVF was performed. First fer- gressively motile spermatozoa, 29% dead of human testicular functions. They are con- tilization results confi rm the trends being spermatozoa and 94% morphologically ab- tractile and transport sperm, are involved in achieved during IVM. A strong selection of normal spermatozoa indicating disturbed tes- immunoregulation of the testis and contrib- oocytes for ART with even dark cytoplasm as ticular and epididymal function probably due ute to the spermatogonial stem cell niche. a quality marker is important, whereas full in- to periorchitis. In the sperm rich fraction a Whether GCs are involved in these functions tactness of cumulus is negligible. low content of Staphylococcus pseudinterme- is not known. After confi rming in testicular dius, -hemolytic streptococci, -hemolytic biopsies of healthy men that peritubular my- streptococci and coagulase-negative staphy- oid cells are immunopositive for the nuclear lococci were identifi ed. Complete hemato- 133 glucocorticoid receptor (GR), we explored its logic analy sis showed no devia tions. Regard- Characterization of Cystic ovarian expression in cultured human testicular peri- ing pyodermia a 4-week oral therapy with ce- Follicles in Dairy Cows and Heifers tubular cells (HTPCs). We detected mRNA phalexin was performed accompanied by a of GRalpha, but not beta, and protein of GR 1-week anti-infl ammatory therapy with car- J.M. Weitzel, T. Viergutz, C. Plinski, A. Vernunft in these cells. Stimulation with the synthet- prodyl and scrotal cooling directed against Institute of Reproductive Biology, Leibniz Institute for ic GC dexa methasone (Dex; 1 µM) resulted periorchitis. At control semen analysis 9 Farm Animal Biology, Dummerstorf, Germany in a ligand-dependent GR nuclear transloca- weeks after the end of treatment all ejaculate

36 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts parameters were within the reference ranges. The aim of the study was to evaluate the ef- The morphology of spermatozoa and the per- This case report demonstrates that early dia- fectiveness of a herd health management pro- centage of live and dead spermatozoa were gnosis of testicular and epididymal dysfunc- gram (HHMP) in dairy cattle herd. The study assessed microscopically by Diff-Quick stain tion and specifi c etiological treatment even in was conducted during 2012–2015 in the herd and nigrosin/eosin stain, respectively. Activ- case of severe semen alterations may lead to in northeastern Poland. In the experi ment ity of glutathione peroxidase (GSH-Px) and complete restauration of fertility. were 211 of HF breed cattle. Control group total antioxidant capacity (TAC) in sperma- was a herd in 2011 (without HHMP). HHMP tozoa were determined spectrophotometri- was conducted by the method with a use of cally. The concentration of spermatozoa, the 136 Agranova computer program which had fol- most motility parameters determined (MOT, lowing functions: recording and analyzing PMOT, VSL, BCF, RAPID) and the percent- Functional Characterization of a data, creating statistics, evaluating fi nan- age of spermatozoa with normal morpholo- Calcium Sensor in a Transgenic cial losses due to reproduction failures, re- gy and of live spermatozoa increased signifi - Pig Model minding about certain events (examinations, cantly (p < 0.05) after 60 days of supplemen- calvings, drying off, estrus, injections etc.). tation. There was an increase (p < 0.05) in A. Wünsch1, P. Kameritsch2, E. Kemter1, M. Kurome1, 1 1 1 Veteri narian did following rectal ultrasound GSH-Px-activity and TAC in spermatozoa. B. Kessler , E. Wolf , N. Klymiuk examinations once a month in all cows: post In conclusion, supplementation with seleni- 1Chair for Molecular Animal Breeding and Biotech- 2 partum (10; 30; 60 ± 5 d.), pregnancy detec- um and Vitamin E for a period of 60 days im- nology; Walter-Brendel-Centre of Experimental tion (30 and 60 ± 5 d.), not inseminated > 90 prove the quality of semen and enhance the Medicine, Ludwig-Maximilians-University, Munich, days post partum, cows not in calf inseminat- antioxidant status of semen in dog with low- DGRM-Abstracts Germany ed  3 times, heifers at the age of > 400 days. ered fertility. More studies on a larger num- Calcium plays a crucial role as second mes- Each examinations were followed by thera- ber of animals are needed to confi rm these re- senger in many physiological and pathologi- py, estrus/ovulation synchronization proto- sults. cal processes such as oocyte fertilization, neu- col or decision to cull the cow was made. Ef- rotransmitter release, muscle contraction and fectiveness of the therapy was evaluated dur- endothelial cell activation, which are charac- ing next visitation. Comparison of reproduc- terized by an increase of the intracellular cal- tive parameters between the control group of 139 2+ the study (2011) and the last year (2015) re- cium concentration. Intracellular Ca chang- EP3 Correlates with Poor Progno- es can be monitored by loading cells with cal- vealed improvement of intercalving period cium sensitive fl uorescent dyes such as Fura- (IP) (468 vs 424 d.), age of fi rst calving (945 sis of Endometrial Carcinoma Pa- 2, but their usage is often compromised by vs 782 d.), insemination index (2.1 vs 1.5 AI/ tients reproducibility, toxic cleavage products and pregnancy), culling index (0.19 vs 0.05) to- J. Zhu1, V. von Schönfeldt2, C. Kuhn1, S. Mahner1, leakage. Therefore, we generated transgenic gether with increased mean annual milk yield U. Jeschke1 pigs carrying the genetically encoded cal- (6639 vs. 8984 liters/cow). Economic evalua- 1Department of Gynecology and Obstetrics; 2Division cium sensor Case12 under the control of tion showed decrease in mean costs of ex- of Gynecological Endocrinology and Reproductive the ubiquitously active CAG promoter for a tended IP of 64.7%. Results indicate the ef- Medicine, Department of Gynecology and Obstetrics, stable transgene expression. Case12, a fusion fectiveness of used HHMP as a method of im- Ludwig-Maximilians-University, Munich, Germany protein of calmodulin, M13 and green fl uo- proving the profi tability of milk production. rescent protein, changes conformation after Background The underlying mechanisms Ca2+ binding, resulting in an increase of the leading to endometrial carcinoma remain un- green fl uorescence signal. After somatic cell known to date. The expression of prostaglan- nuclear transfer using a mix of stably trans- 138 din E2 (PGE2) receptor 3 (EP3) is decreased fected kidney cells 8 living Case12 transgenic Effect of Selenium and Vitamin E in colon and prostate cancer. Furthermore, piglets were born. After expression analysis EP3 reduces tumor cell proliferation, migra- via epifl uorescence microscopy and Western Supplementation on Semen Qual- tion and invasion. The aim of this investiga- Blot using fi broblasts and endothelial cells ity, Glutathione Peroxidase Activ- tion was to analyze the expression of EP3 in isolated from fi ve different founders, best ex- ity, and Total Antioxidant Capacity endometrial cancer and its effect on clinical pressing founder cells were used for further in Dogs with Lowered Fertility features and patients’ prognosis. studies. Functionality of Case12 transgenic Materials and Methods We retrospective- endothelial cells could be shown by treatment S. Zdunczyk1, A. Domosławska1, M. Kankofer2, T. 1 2 ly analyzed 140 endometrial carcinoma pa- with ATP, resulting in a fast increase followed Janowski , M. Franczyk tients. Routinely processed tissue samples by a decrease of the fl uorescence signal, what 1Department of Animal Reproduction with Clinic, 2 were immunohistochemically stained using is the typical picture seen after ATP treat- University of Warmia and Mazury in Olsztyn; De- anti-EP3 antibodies. Specifi c staining was ment. Furthermore we used Case12 cells to partment of Animal Biochemistry and Physiology, analyzed by designating a percentage of posi- show endothelial activation during rejection Faculty of Veterinary Medicine, University of Life tive glandular epithelial cells to each sample. processes in xenotransplantation, suggesting Sciences in Lublin, Poland that cells from Case12 transgenic pigs could Results Detailed medical records of 140 be used for monitoring changes of intracellu- Twenty clinically healthy dogs with poor se- endometrial cancer patients including age, lar Ca2+ concentration during (patho-)physio- men quality (low total sperm count and per- stage of disease and WHO gradings were logical processes. centage of progressive forward motility, in- available. EP3 staining differed signifi cant- creased percentage of morphologically ab- ly according to WHO tumor grading: Tumors normal sperm) and lowered fertility were used graded G3 displayed the highest expression, 137 in this study. Lowered fertility was defi ned as while tumors graded G1 displayed the lowest low pregnancy rate and/or smaller than ex- expression (p = 0.011). No differences were The Effects of a Herd Health pected litter size. Ten dogs were supplement- evident regarding the histological classifi ca- Management Program on Select- ed daily by selenium (0.6 mg/kg organic se- tion and FIGO stages. Grouped according to ed Reproductive parameters in a lenium yeast) and Vitamin E (5 mg/kg) per lymph node or organ metastasis or relapse, Dairy Herd in Poland os for 60 days. The control group consisted the staining intensity also was showed no on 10 males without supplementation. Semen signifi cant differences. Using ROC curves, K. Zalewski1, W. Zalewski2, Z Gajewski1 was collected by manual manipulation from a cut-off of 77.5% was identifi ed defi ning 1Department of Large Animal Diseases with Clinic, all dogs on day 0, 30, 60 and 90. The sperm low (< 77.5% positive glandular epithelial VRC and CBB, Faculty of Veterinary Medicine, concentration and motility parameters were cells) and high (> 77.5% positive cells) EP3 WULS – SGGW, Warsaw; 2Private Veterinary Prac- evaluated using computer assisted sperm ana- expression. Kaplan-Meier analysis indicat- tice VET.COM.EU, Stare Babice, Poland lyzer (Hamilton Thorne, version IVOS 12.3). ed, that the overall survival (OS) of patients

J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) 37 DGRM-Abstracts

with a high EP3 expression in their respec- system was 35 days, in AD system no de- ments demonstrated the biological activity tive tumors was poorer (8.19 vs 14.83 years, vice had to be changed during the trial pe- of elongated mucins against histone-mediat- p = 0.004) as was the progression-free surviv- riod. In conclusion, both systems are appli- ed cytotoxicity. Thus, polysialylated mucins al (PFS) (7.88 vs 14.13 years, p = 0.011). A cable of distinguishing “estrus” and “no es- may represent a novel element to counteract multivariate analysis showed EP3 expression trus” in dairy cows. However, the novel MV pathological processes mediated by extracel- as independent prognostic marker for PFS system showed no advantage over the estab- lular histones. (p = 0.042) besides age, stage and recurrence. lished commercially available AD system. Discussion Here we demonstrate for the The project was supported by Schaumann fi rst time, that EP3 expression in glandular Foundation. 142 epithelial cells increases with the malignan- Sonographic Characterization cy of tumor stage and correlates with poor patients’ prognosis. These results are contra- 141 of Changes on Bovine Placen- dictory to previous studies and indicate that ta, Uterine Wall and Foetal Mem- EP3 might act in a cell and tissue type specif- In vitro polysialylated Cervical branes during Pregnancy ic manner and is a potential therapeutical tar- Mucins Counteract the Cytotoxici- get in endometrial cancer. ty of Extracellular Histones D. Zoller, S. Peterson, U. Bleul Clinic of Reproductive Medicine, Department of K. Zlatina1,2, C. E. Galuska1,2, T. Tharmalingam3, Farm Animals, Vetsuisse-Faculty University Zurich, DGRM-Abstracts 140 G. Prem2, W. F. Vann4, J. Vionnet3, C. J. Reid3, Switzerland M.E. Gallagher3, F. A. Carrington3, S. L. Hassett3, Comparison of two Methods for S. D. Carrington3, S.P. Galuska1,2 The objectives of this study were to describe Automated Heat Detection in Cat- 1Department of Reproductive Biology, Leibniz Insti- the sonographic anatomy of placenta, uterine wall and foetal membranes and to determine tle tute for Farm Animal Biology (FBN), Dummerstorf; 2Institute of Biochemistry, Faculty of Medicine, Jus- measurable changes during normal pregnan- L. Zimmermann, R. Martin, F. Weber, O. Dietrich, tus-Liebig-University, Giessen, Germany; 3UCD Vet- cy in cows. Transrectal examinations were R. Wagner, H. Zerbe erinary Sciences Centre, University College Dublin, regularly performed using an 8 MHz linear Clinic for Ruminants, Ludwig-Maximilians-University, Belfi eld, Dublin, Ireland; 4Food and Drug Administra- transducer between week 6 and 43 of preg- Munich, Germany tion, Silver Spring, MD, USA nancy. Size and appearance of different struc- tures such as the endometrium, myometrium, Heat detection is one of the most important Neutrophil extracellular traps (NET) can be height and width of placentomes, the thick- management factors on dairy farms. To assist formed during the invasion of pathogens to ness of the uterine wall with adherent cho- the farmer on modern dairies, different auto- arrest and kill bacteria and fungi. However, rion leave (CTUP) and the echogenicity of mated systems for heat detection are availa- numerous studies demonstrated that various the foetal fl uids were assessed. Measure- ble. Two methods investigated in this study diseases are directly related to this meshwork ment of uterine wall and placentomes were were based on a continuous automatic analy- of DNA, histones and antimicrobial factors. done close to the cervix, in the uterine cor- sis of locomotion activity in a freestall barn Furthermore, the female reproductive system pus, in the middle and close to the tip of the on a 24/7-basis. A new heat detection sys- responds to the insemination with a severe in- pregnant and non-pregnant uterine horn. The tem involving machine vision (MV) was vasion of neutrophils and NETosis. The bind- height and the width of placentomes in the compared with a commercial activity detec- ing of sperm to released NET could be asso- pregnant uterine horn showed a good cor- tion system (AD). The MV system combined ciated with a decreased fertilization. The se- relation with the gestation period (height high-resolution GigE-cameras and 2D matrix men included DNases destroying the DNA- r = 0.78, width r = 0.83, both p < 0.0001). codes for marking individual cows. Proges- meshwork but the extracellular histones are The size of placentomes increased steadily terone determination in skimmed milk (EIA) still present representing the major cytotox- until week 27 of gestation followed by a fl at- was used as a reference for both methods. The ic component of NET during these process- tening of the growth curve until week 31 of study was carried out in a 75 cow dairy farm es. Recent studies demonstrated that polySia gestation reaching a plateau until week 43. In over 6 months. 173 heat periods of 53 cows chains attached to N-glycans of NCAM and the non-pregnant horn smaller placentomes and 149 non-estrus periods (diestrus or ear- ST8SiaII are present on the surface of sperms were found compared to the pregnant horn ly pregnancy) until day 28 were detected by in ejaculates. These polysialylated proteins (p < 0.01). Placentomes in the area of the tip progesterone profi les. The median increase in may counteract the cytotoxicity of released of the pregnant horn were signifi cant small- moving activity in relation to a activity av- histones. We wanted to combine the capaci- er (height and width) than those in the mid- erage of the preceding 10 days was 111.6% ty of sialic acid polymers to counteract his- dle of the horn, corpus or close to the cer- by MV and 121.8% by AD. From a practical tone mediated cytotoxicity with the possibil- vix. Endometrium, myometrium and CTUP point of view, the MV system showed a sen- ity to reduce adhesion of neutrophils using showed no measurable alterations throughout sitivity of 80.3% and a specifi city of 45.6%. mucins. To this end, we examined mucins for pregnancy. While the echogenicity of the al- The AD system however showed a sensitivity their capability to serve as a target for bacteri- lantoic fl uid has not altered during pregnan- of 84.4% and a specifi city of 66.4%. The me- al polysialyltransferases. Intriguingly, bovine cy the amniotic fl uid became more echogenic dian of the required time to mount and main- cervical mucins can be effi ciently elongated (p < 0.0001). Ultrasonographic monitoring of tain the devices were 38.4 minutes for mark- by bacterial polySiaT in vitro. The resulting placentae may be a valuable diagnostic tool ers in MV system and 2 minutes for the AD polymers can comprise of more than 50 sial- for the characterization of the placenta in bo- system. The median marker durability in MV ic acid residues and succeeding cell experi- vine pregnancies.

38 J REPRODUKTIONSMED ENDOKRINOL_ONLINE 2017; 14 (Supplementum 1) DGRM-Abstracts

Index of authors (only primary authors)

A G L S Abd el Ghaff ar A...... 17 Gajewski Z...... 11 Lavagi I...... 20 Saymé N...... 29 Abultdinova A...... 2 Gies S...... 11 Lopucki M...... 20 Scaravaggi I...... 29 Ali A...... 2 Gizinski S...... 11 Lorenc K...... 20 Schaefer J...... 30 Alsobayil F...... 3 Glashauser L...... 11 Luther A...... 21 Schick B...... 30 Aniołek O...... 3 Górriz-Martín L...... 12 Lüttgenau J...... 21 Schlipf M...... 30 Arlt E...... 3 Grahofer A...... 12 Schmid N...... 30 Attiger J...... 3 Grünther B...... 12 M Schneebeli J...... 31 Grybel K. J...... 13 Macias L...... 21 Schuler G...... 31 B Günzel-Apel A. R...... 13 Malama E...... 21 Schulze L...... 31 DGRM-Abstracts Bagnjuk K...... 4 Martins A...... 22 Schulze M...... 31 Baston-Büst D...... 4 Masko M...... 22 Scully D...... 32 Baufeld A...... 4 H Matschurat C...... 22 Sela L...... 32 Behrens Mathiesen C...... 4 Hahn K...... 13 Maurer L...... 22 Sendag S...... 32 Bepler D...... 5 Hankele A. K...... 13 Meki A...... 23 Sharifabad M...... 32 Bernal-Ulloa S...... 5 Hedegger K...... 14 Meyerholz M...... 23 Sickinger M...... 33 Beyer D...... 5 Heininen-Brown M...... 14 Meyer K...... 23 Skierbiszewska K...... 33 Beyer N. M...... 5 Helfrich A...... 14 Milojevic V...... 23 Sokolowski G...... 33 Bill R...... 6 Heppelmann M...... 14 Steinhoff L...... 34 Bittner L...... 6 Herrera C...... 15 N Stöhr J...... 34 Blad-Stahl N...... 6 Hinrichs A...... 15 Neuhauser S...... 24 Bramer S.A...... 6 Hooshmandabbasi R...... 15 T Brockelmann Y...... 7 Hughes B...... 16 O Trela M...... 34 Buck T...... 7 Humbs J...... 16 Oehler C...... 24 Tzanidakis N...... 34 Burchardt B...... 7 Oldenhof H...... 24 Olszewski J...... 24 V C I Otzen H...... 25 van der Weijden V...... 35 Cetin N...... 7 Ibrahim M...... 16 Owhor L...... 25 Chen S...... 8 Vernunft A...... 35 P W D J Pawlinski B...... 25 Dabrowski S...... 8 Janowski T...... 17 Petrajtis-Gołobów M...... 25 Wartenberg A...... 35 Domanska D...... 8 Pleuger C...... 26 Watz S...... 35 Domino M...... 9 Polei M...... 26 Weiser T...... 36 K Pricking S...... 26 Weitzel J.M...... 36 Welter H...... 36 E Karger S...... 17 Püschl D...... 27 Wolf K...... 36 El-Din M. Omar H...... 16 Keiser R...... 17 Wünsch A...... 37 Eubler K...... 9 Keller S...... 17 R Kesselring T...... 18 Rau J. 27 F Kölle S...... 18 Rawy M.S...... 27 Z Faundez R...... 9 Körber H...... 18 Refaat D...... 8 Zalewski K...... 37 Fichtner T...... 9 Kordowitzki P...... 18 Reichenbach M...... 28 Zdunczyk S...... 37 Fietz D...... 10 Korkmaz F...... 19 Rekawiecki R...... 28 Zhu J. 37 Fischer-Tenhagen C...... 10 Krajza K...... 19 Rey-Ares V...... 28 Zimmermann L...... 38 Flenkenthaler F...... 10 Król J...... 19 Rohmeier L...... 29 Zlatina K...... 38 Fouché N...... 10 Kurome M...... 20 Ruhmann B...... 29 Zoller D...... 38

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