56th Annual Conference of Physiology and Pathology of Reproduction and 48th Joint Conference of Veterinary and Human Reproductive Medicine

General Theme: „United Excellence – Reproductive Research in Animals and Men 2023“

1st–3rd March, 2023 Lecture Hall of the Medical Faculty of the University Münster

Congress Organizer: Stefan Schlatt; University Münster, Germany

Co-organizers: Christine Wrenzycki, Daniela Fietz; University Gießen, Germany

Welcome Note to all participants of the DVG Conference 2023

Dear colleagues,

It is a great pleasure to invite you to the 56th Annual Meeting for Physiology and Patho­logy of Reproduction and the 48th Joint Meeting of Veterinary and Human Reproductive Medicine. The Deutsche Veterinärmedizini­sche Gesellschaft (DVG) and the German Society for Reproductive Medicine (DGRM) have once more organized a meeting, which brings together basic scientists and clinicians in reproduction research from veterinary and human medicine. I am very thankful that a highly motivated group of colleagues joined the program and local organizing committees and has helped to generate a highly exciting meeting. It was our primary goal to combine outstanding people and exciting topics from veterinary and human medicine and we have therefore selected the theme „United Excellence – Reproductive Research in Animals and Men 2023“ for this year’s meeting.

With financial support from the DFG, we were able to invite outstanding national and international speakers presenting a broad array of basic and clinical science. The many excellent contributions from submitted abstracts will provide a basis for exciting short talks and posters. I am thankful for all contributors submitting their scientific work as abstracts and for the committee members who reviewed the submissions. I am delighted that this year we can get together in person on site and look forward to an exciting event with 11 keynotes, 21 short communications and approx. 60 posters.

This meeting is for the first time organized by the Westfalian-Wilhelms-University Münster. The slogan of the University is „wissen.leben“. It is therefore timely that this meeting bringing together scientific disciplines dealing with profound aspects of life is now coming to Münster. We will gather on the medical campus next to the towers of the University Hospital pictured below. The University Münster does not have a veterinary faculty rendering it for most of the DVG participants a novel place to visit and for the local participants an unusual opportunity for exchange and making new experiences and friends. Münster is an extremely vibrant and growing city, which has much to offer in terms of history, culture and architecture. „Münster. Wissenschaft und Lebensart“ is the official profile of the city. When you are on site, you will best hire a bicycle to get around town, as this is the preferred mode of eco-friendly transportation in a city dominated by education and research. Expect a highly exciting meeting and take some extra time to visit our beautiful town.

I am very excited and look forward to welcoming you in Münster.

Stefan Schlatt (on behalf of Profs. Christine Wrenzycki and Daniela Fietz and the organizing Societies and committees)

Abstracts*

*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 scientific committee.

Full set Talks

01

Saturated fatty acids in the follicular fluid are indispensable for the endocrine regulation of the ovarian cycle and estradiol production

Gesättigte Fettsäuren in der Follikelflüssigkeit sind für die endokrine Regulierung des Eierstockzyklus und die Östradiolproduktion unerlässlich

V. S. Baddela, M. Michaelis, J. Vanselow

Institute of Reproductive Biology, Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany

The ovarian cycle is regulated by the follicle-stimulating hormone (FSH) and luteinizing hormone (LH), whose actions are mediated via respective receptors in granulosa (GC) and theca cells of ovarian follicles to stimulate steroidogenesis. GCs are the principal centers of 17-?-estradiol (E2) production, essential for manifesting estrous symptoms in animals but also for secondary sexual characteristics in humans. It has been shown that metabolic conditions characterized by increased concentrations of non-esterified fatty acids (NEFA) are associated with female subfertility. In this regard, our earlier studies showed that unsaturated fatty acids (USFs) inhibit and saturated fatty acids (SFAs) induce GCs‘ E2 production. In this study, we wondered how E2 production is regulated in the physio­logical context as USFs and SFAs mutually occur in the follicular fluid. We performed in-vitro primary GC cultures with palmitate, palmitoleate, stearate, oleate, linoleate, and alpha-linolenate, representing >80% of the NEFA fraction in the follicular fluid, and analyzed 62 different cell culture conditions. We found that the co-supplementation of SFAs with USFs antagonizes the actions of USFs and rescues the E2 production by restoring the expression of gonadotropin receptors and of aromatase to the control level. USFs induce ERK and Akt phosphorylation, which is inhibited by SFAs co-supplementation. Inhibitor studies identified that ERK but not AKT induced by USFs could act as a negative regulator of E2 synthesis. These results suggest that SFAs are vital components of follicular fluid, without which gonadotropin signaling and the ovarian cycle would probably be shattered by USFs.

Key words: granulosa cell, steroidogenesis, gonadotropins

02

MicroRNA profiling of oviductal ­extracellular vesicles and early embryos reveals molecular ‘snitches’ on embryo quality

MicroRNA-Profile von extrazellulären Vesikeln aus dem Eileiter und frühen Embryonen offenbaren molekulare „Spitzel“ für die Embryoqualität

M. Hamdi1, J. M. Sánchez2, B. Fernandez2, D. R. Câmara3, H. Bollwein3, D. Rizos2, S. Bauersachs1, C. Almiñana1

1Institute of Veterinary Anatomy, Vetsuisse-Faculty Zurich, University of Zurich, Lindau (ZH), Switzerland; 2Department of Animal Reproduction, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Madrid, Spain; 3Clinic of Reproductive Medicine, Vetsuisse-Faculty, University of Zurich, Lindau (ZH), Switzerland

Increasing evidence points at an active role of oviductal extracellular vesicles (oEVs) in the early embryo-maternal dialogue. We hypothesized that the molecular cargo of oEVs differs depending on the presence or absence of an embryo and on the embryo quality. An in vitro model was used to examine oEVs derived from the co-culture of primary bovine oviduct epithelial cells (BOEC) with good (G) and poor (P) quality in vitro produced embryos and without them. MicroRNA profiling of oEVs as well as G and P embryos was performed by RNA-sequencing. Presumptive zygotes were cultured until 53 h after fertilization and classified according to their morphology as G (? 8 cells) and P (< 8 cells) embryos. Both G and P embryos were co-cultured with BOEC monolayers or alone, as follows: BGE: BOEC with G embryos; BPE: BOEC with P embryos; GE: G embryos without cells; PE: P embryos without cells; B: BOEC alone and M: media alone (for all n = 6). After 24h of co-culture, conditioned media was collected from all groups, oEVs were isolated, characterized and oEV RNA cargo was analyzed. The presence of oEVs in all groups was confirmed by transmission electron microscopy, except for group M. In oEVs, 84 miRNAs were identified. Differentially abundant (DA) miRNAs were obtained for BGE vs. B (8) and BPE vs. B (4) (P-value < 0.01). In embryos, 187 miRNAs were identified, with 12 DA miRNAs for BGE vs. BPE, 3 for G vs. P, 8 for BGE vs. GE, and 11 for BPE vs. PE (P-value <0.01). These results suggest the existence of an oviductal recognition system of embryo quality via EV secretion. Besides, it showed the effect of BOEC on embryonic miRNA profiles, highlighting the impact of the lack of early embryo-maternal interactions in in vitro embryo production systems.

Key words: oviduct, extracellular vesicles, embryo quality

03

Nutritional imbalance and FSH-­induced ovarian hyperstimulation have an impact on the transcriptome of the ovine caruncular endometrium

Eine unausgewogene Ernährung und eine FSH-induzierte ovarielle Hyper­stimulation wirken sich auf das Transkriptom des karunkulären Endome­triums von Schafen aus

Ö. Bedir1, M. Tavares Pereira1, A. Grazul-Bilska2, M. P. Kowalewski1,3

1Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland; 2Department of Animal Sciences, North Dakota State University, Fargo, USA; 3Center for Clinical Studies (ZKS), Vetsuisse Faculty, University of ­Zurich (UZH), Zurich, Switzerland

Concepto-maternal contact can be impacted by a variety of factors including hormonal manipulations and maternal diet. We investigated the transcriptional effects of diet and FSH-mediated superovulation on the caruncular endometrium in sheep using deep RNA sequencing (NGS, RNA-Seq). Ewes (n = 3–5/group) were divided into control fed (CF), overfed (OF), and underfed (UF), with each group further divided between superovulated (SOV; FSH) or control (C; saline). Samples were collected on days (d) 5 and 10 of ­diestrus in the next cycle. Transcriptomic data were analyzed in differently expressed genes (P < 0.01, FDR < 0.05) between d5 and d10 (CF_C), and in response to FSH-mediated superovulation under different feeding conditions on d10. In CF, superovulation was predicted to activate pathways including FGF-, apoptosis- and sirtuin signaling, while estrogen-, integrin-, and HGF signaling were suppressed in response to superovulation. Superovulation decreased the expression of ESR2, MUC1, FOXO1 and FOXO4 while elevating the expression of ESR1, FGF10, FN1, CASP3 and BAD. In OF_C, terms related to cell adhesion, differentiation and communication were upregulated compared with CF_C. We previously confirmed that overfeeding increases the expression of integrins (e.g., ITGB3 and ITGA4,-5) and growth factors (e.g., IGF1,-2 and VEGFA) at d10 compared with control diet. In UF, genes related to cell adhesion (CADM1) and immune response were downregulated by superovulation. Overall, ­superovulation has multidirectional effects on local uterine function and the expression of factors involved in implantation, depending on diet.

Key words: sheep, nutrition, superovulation, transcriptome (RNA-Seq)

04

Endometrial gene expression in isolated hemoperfused equine uteri

Genexpression im Endometrium isoliert-hämoperfundierter equiner Uteri

E. Diel1, M. Köhne1, E. Packeiser2, H. Sieme1

1Unit for Reproductive Medicine – Clinic for Horses, University of Veterinary Medicine Hannover, Foundation, ­Hannover; 2Unit for Reproductive Medicine – Clinic for Small ­Animals, University of Veterinary Medicine Hannover, ­Foundation, Hannover, Germany

As importance and awareness of animal welfare are growing constantly, the need for alternatives to animal testing is bigger than ever. The aim of the study was to further validate an existing ex vivo model of the equine uterus on the molecular level. For this purpose, real-time quantitative PCR (RT-qPCR) was performed for oxytocin receptor (OXTR), progesterone receptor (PGR), estrogen receptor 1 (ESR1) and Caspase 3 (marker for apoptosis), to investigate changes in the temporal gene expression in the endometrium of isolated hemoperfused equine uteri (n = 12). Biopsy samples were obtained right after slaughter, after transportation to the laboratory, after 4, 5 and 6 hours of hemoperfusion and then stored in RNAlater at –80 °C until analysis. Primers for PGR, Caspase 3 and the housekeeping genes GAPDH and B2M were used from the literature and primers for OXTR and ESR1 were designed with Primer-BLAST. The average threshold (CT) of each triplet was normalized to GAPDH and B2M and the delta ratios were analyzed statistically. There were no significant changes in expression of Caspase 3 until 5 hours of perfusion after which the relative expression increased significantly (p < 0.05), indicating that there was no notable apoptosis during perfusion before but after 5 hours. Hormone receptors did not show significant changes in relative gene expression over the time of testing, except for PGR, which had significantly lower expression after 5 h (p < 0.05). In conclusion, no major changes in the mRNA level of the selected genes have been observed until 6 hours perfusion time, making the ex vivo model applicable for short-term studies on the molecular level.

Key words: ex vivo model, qPCR, equine endometrium

05

Characterization of bovine placental trophoblast giant cell migration by three-dimensional reconstruction

Charakterisierung der Migration der bovinen plazentären Trophoblast­riesenzellen mittels dreidimensionaler Rekonstruktion

A. Dörr1, L. Tiedje1, R. Koch1, I. Blume1, G. Wirth1, C. Wrede2, C. Pfarrer1

1Institute for Anatomy, University of Veterinary Medicine Hannover, Foundation, Hannover; 2Institute of Functional and Applied Anatomy, Research Core Unit Electron Micro­scopy, Hannover Medical School, Hannover, Germany

The bovine trophoblast giant cells (TGC) migrate towards the maternal epithelium to deliver hormonal products to the maternal compartment. To do so, TGC form pseudopodia towards the maternal epithelium. Since the literature contains little work about the actual migration dynamics, aim of the study was to analyse TGC migration three-dimensionally by serial block face scanning electron microscopy (SBF-SEM). Placenta samples from eight cows (gestation days 120, 150, 177, 205, 266, 270, 278 and 284) were collected, fixated in Karnovsky and embedded in Durcupan. Stacks, each of 3000 sections (60 nm thickness), were created by SBF-SEM. Analyses, illustration and model presentation were done with the softwares Microscopy Image Browser and IMOD. Volume calculations were performed with the software Amira. Transmission electronic microscopy images were generated for further details. Pseudopodia were observed in both, the TGC’s rear end closely associated with the fetal basement membrane and the migration front making contact with the uterine epithelium. They contained pale organelle poor fluid and were in contact with the cell through openings of varying sizes. Pseudopodia were observed in all stacks examined. One TGC could have up to four pseudopodia. The volume of pseudopodia varied between 50 µm³ and 2000 µm³. As part of the work, the term „pale bleb-like pseudopodium“ was elaborated. In theory, increased hydrostatic pressure leads to separation of the cell membrane from the actin cortex, which allows “pale” cytosol to flow in. Then, the actin cortex is degraded and organelle rich parts can enter.

Key words: trophoblast giant cells (TGC), bovine, pseudopodium, migration, 3D-Reconstruction

06

M1AP‘s role in male infertility: a comparative study in mice and men

Die Rolle von M1AP bei männlicher Infertilität: Ein Vergleich von Maus und Mensch

N. Rotte1, J. Dunleavy2, M.J. Wyrwoll1, S. Kliesch3, F. ­Tüttelmann 1, M. O‘Bryan 2, C. Friedrich1

1Institute of Reproductive Genetics, University of Münster, Münster, Germany; 2Faculty of Science, University of ­Melbourne, Melbourne, Australia; 3Centre of Reproductive Medicine and Andrology, University Hospital Münster, ­Münster, Germany

Male infertility is a complex disease affecting 7% of men. Although frequently suspected, only 20% of azoospermic men receive a genetic diagnosis. In 2020, we described rare bi-allelic variants in M1AP encoding meiosis 1-associated protein [Wyrwoll, 2020, AJHG, in press] as novel reason for crypto/azoospermia (very few/no sperm in the ejaculate) due to meiotic arrest (MeiA). This is in line with the described phenotype of knockout (KO) mice. Here, we aim to compare the clinical and histological phenotypes of both species to evaluate the KO mouse as appropriate model for investigating M1AP’s function in human MeiA. A homozygous M1ap KO mouse was generated. Analyses of postnatal mice included validation of litter size, daily sperm production, and histological examination. In-depth characterisation of testicular phenotypes included periodic-acid Schiff (PAS), CREM, yH2AX, cleaved caspase-3 (C3) or TUNEL staining. KO mice were overall healthy but exhibited severe subfertility, including decreased daily sperm production, litter sizes, and testicular weight. PAS staining confirmed spermatogenic failure, showing both arrested spermatocytes and spermatids. Similarly, men showed complete MeiA (n = 4) and occasionally round (CREM-positive, n = 5) or elongating (n = 4) spermatids. yH2AX expression exposed meiosis prophase I progression beyond pachynema and aberrant metaphase cells. Germ cells significantly underwent apoptosis, confirmed by C3 (mice) or TUNEL (men) staining. Our data suggest that M1AP directly or indirectly coordinates processes at pachytene-to-metaphase transition, probably activating the spindle assembly checkpoint when mutated/lost. Subsequent analyses have to target the exact cascade of disturbance.

Grants: This work was supported by the DFG Clinical Research Unit 326 ‚Male Germ Cells’

Key words: M1AP, meiotic arrest, male infertility

07

Morphological changes in the ­canine epididymis during recovery after 5-months treatment with a deslorelin implant

Morphologische Veränderungen am kaninen Nebenhoden während der Wiederherstellung nach 5-monatiger Behandlung mit einem Deslorelin-­Implantat

H. Greiner1, S. Aslan2, G. Saral3, F. Binli3, E. Akal4, M. Selçuk4, S. S. Ay3, M. Findik3, S. Goericke-Pesch1

1Unit for Reproductive Medicine – Clinic for Small Animals, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany; 2Department of Obstetrics Gynaeco­logy, Faculty of Veterinary Medicine, Near East University, Nicosia, Cyprus. 3Department of Obstrectics Gynaecology, Faculty of Veterinary Medicine, Ondokuz May?s University, Samsun, Turkey; 4 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Ondokuz May?s University, Samsun, Turkey

Administration of slow-release GnRH agonist implants (SRI) is a well-established alternative to surgical castration in small animal medicine. Whereas clinical-andrological, endocrine and testicular histological changes have been investigated in depth, research on epididymal changes during treatment and recovery are largely missing. To gain further insights, healthy dogs were treated with a deslorelin SRI over five months, the SRI was removed at 5 months and 4–5 dogs were castrated at implant removal (week, W 0) or 2, 4, 6 or 10 weeks after SRI removal. Testes of untreated healthy male dogs served as controls (CG). Besides testes, epididymides were collected and samples taken from the head, corpus and tail region. Slides from Bouin-fixed, paraffine-embedded tissues were hematoxyllin-eosin stained and histologically assessed. In each dog and region, epithelial height, presence of spermatozoa, cilial height, thickness of the muscle layer and presence of connective tissue was evaluated with a semiquantitative scoring by a blinded evaluator. GraphPad Prism was used for statistics. Epithelial (p < 0.01) and cilial height (p < 0.05), presence of spermatozoa (p < 0.05), and presence of connective tissue (p < 0.05) differed significantly between samples in all epididymal regions (Kruskal-Wallis) with lower epithelium and cilia, as well as less spermatozoa, but more connective tissue being present in early samples (W0 and/or W2) compared to later ones. The present results clearly show that downregulation by a SRI induces not only significant testicular changes, but also affects epididymal morphology indicating the need for further studies.

Key words: epididymis, canine, GnRH agonist implant

08

Establishment of placental 3-dimensional- (3D-) spheroid cultures to study the tropism of Coxiella burnetii for ovine trophoblasts

Etablierung plazentarer 3-dimensionaler- (3D-) Sphäroidkulturen zur Untersuchung des Tropismus von Coxiella burnetii zum ovinen Trophoblasten

J.-D. Haeger1, E. Liebler-Tenorio1, C. Pfarrer2, C. Berens1, A. Luehrmann3, K. Mertens-Scholz4, C. Menge1

1Friedrich-Loeffler-Institut, Institute of Molecular Patho­genesis, Jena; 2Institute for Anatomy, University of Veterinary Medicine Hannover, Foundation, Hannover; 3Institute for Clinical Microbiology, Immunology and Hygiene, ­Friedrich-Alexander-Universität, Erlangen; 4Friedrich-­Loeffler-Institut, Institute of Bacterial Infections and ­Zoonoses, Jena, Germany

The tropism of the zoonotic, obligate intracellular bacterium Coxiella burnetii (C. burnetii) [Q-fever agent] for ovine trophoblasts in placental tissues is difficult to study. Thus, we established 3D-spheroid models deploying ovine AH-1 trophoblasts to investigate the spread of C. burnetii in tissue-like culture models. Spheroids were formed via hanging drops (named: AH-1HD) or poly-HEMA (AH-1PH) and characterized by immunohistochemistry (IHC: cytoskeleton, cell turn over) and transmission electron microscopy [TEM]. AH-1HD (diameter: 200 µm) developed within 3 days and AH-1PH (diameter: 1000 µm) within 10 days. AH-1HD consisted of intact cells, whilst AH-1PH had an inner degenerative core surrounded by intact AH-1 cells. All cells of AH-1HD and the intact AH-1 cells of AH-1PH expressed cytokeratin 18, ezrin, SHMT2 and Ki-67. Active caspase-3 was mainly detected in the core of AH-1PH. AH1-HD spheroids were infected with C. burnetii (axenically [cell free] grown,100 genome equivalents as quantified by icd-­qPCR) for 5–17 days. C. burnetii-infection was detected by IHC. Following infection, C. burnetii spread rapidly through AH-1HD until 14 days post infection (dpi) at which all cells contained Coxiella-containing parasitophoric vacuoles (PV). Upon transfer of supernatants collected from infected AH-1HD at 14 dpi to AH-1PH spheroids, newly infected cells were detected in AH-1PH within 3 dpi. Both spheroidal models developed typical in vivo-like lesions like the formation of intracellular C. burnetii-containing parasitophoric vacuoles after infection. Therefore, the AH-1 trophoblast spheroids are promising ex vivo tools to study the tropism of C. burnetii for ovine trophoblasts under controlled conditions.

Key words: 3D-spheroids, Coxiella burnetii, Q-fever

09

The infection course of uropathogenic Escherichia coli induced epididymo-orchitis is affected by changes in iron homeostasis

Der Infektionsverlauf einer durch ­uropathogene Escherichia coli (UPEC) ­ausgelösten Epididymoorchitis wird durch Änderungen in der Eisenhomöostase beeinflusst

A. Harrer1, N. Ghatpande2, C. Pleuger1, M. Fijak1, D. Fietz2, N. Guttmann-Raviv3, S. Bhushan1, E. G. Meyron-Holtz3, A. Meinhardt1

1Institute of Anatomy and Cell Biology, Unit of Reproductive Biology, Justus-Liebig-University Giessen; 2Institute for ­Veterinary Anatomy, Histology and Embryology Justus-Liebig-University Giessen, Germany; 3Faculty of Biotechno­logy and Food Engineering, Technion-Israel Institute of Technology, Technion City, Haifa, Israel

Iron is an essential nutrient for both hosts and pathogens. Uropathogenic Escherichia coli (UPEC) is a gram-negative pathovar that expresses many iron acquisition systems to survive in a low-iron environment. It commonly infects the male genito-urinary tract which frequently results in long-term sperm-count reduction. As spermatogenesis starts with multiple cell divisions and high iron demands, and host response to bacterial infection often includes sequestration of iron as a step of nutritional immunity, we hypothesized that impaired iron homeostasis may affect the course of UPEC-mediated epididymo-orchitis. This is examined in transgenic mouse models with a deletion of the gene coding for the iron regulatory proteins (IRP) 1 or 2, which leads to opposing misregulation of iron metabolism in mice. In untreated Irp1-/- and Irp2-/- mice hypospermatogenesis was observed whilst no changes were seen in the epididymis. Western blot analysis demonstrated that levels of iron-related proteins did not change, except for increased ferritin levels in Irp2-/- testis. In UPEC elicited epididymo-orchitis lower infiltration of immune cells was detected in Irp1-/- testes compared to WT and Irp2-/- testes at 7 days after infection, which correlated with decreased testicular transcript levels of Tnf-?, Il1? and Il10 compared to WT. Furthermore, Irp1-/- mice showed a protective effect in UPEC epididymo-orchitis likely based on the reduced inflammatory response to the pathogen in testes. The mechanism behind the protective activity of Irp1 deletion is currently being investigated.

Key words: iron, inflammation, uropathogenic Escherichia coli (UPEC)

10

Correlation between fertility after artificial insemination and embryo­nic development characteristics post IVF in semen of Holstein Friesian sires

Zusammenhang zwischen der Fruchtbarkeit nach artifizieller Insemination und der embryonalen Entwicklungscharakteristik nach IVF mit Sperma von Holstein Friesen-Besamungsbullen

M. Hoelker2, L. Behren1, J. Kurzella1, D. Salilew-Wondim1,2, F. Rings1, E. Tholen1, C. Blaschka2

1Department of Animal Science, Biotechnology & Reproduction in farm animals, University of Goettingen, Goettingen; 2Institute of Animal Science, Animal Breeding, University of Bonn, Bonn, Germany

The present study aimed to analyze potential correlations between the field fertility (Non return rate, NRR) of Holstein Friesian sires after artificial insemination (AI) with conventional semen (NRR56-CONV) or sexed semen (NRR56-SEXED) and developmental characteristics of IVF embryos derived from semen of these sires in terms of developmental rates and developmental kinetics. Consequently, 10 sires with known records for field fertility were used for IVF of bovine embryos. In vitro maturation (TCM199, 38.8 °C, 5% CO?) and in vitro fertilization (Sperm-Talp, 38.8 °C, 5% CO?, 5% O?) was conducted via routine procedures. A total of 126 presumptive zygotes for each sire were placed individually in a time laps system (MiriTL, SOFaa+0,6 % BSA, 38.8 °C, 5% CO?, 5% O?) for in vitro culture. When comparing top-5 and sub-5 sires according to NRR56-CONV only minor differences were obtained with respect to cleavage rates (69.0 ± 15.6 % vs. 71.4 ± 12.8 %) and blastocyst rates (11.9 ± 8.2 % vs. 13.9 ± 7.6 %) following IVF. Likewise, there was now effect in terms of the duration required after IVF until first cleavage (34.8 ± 2.9 h vs. 34.6 ± 5.1 h) when comparing these two sire groups. In accordance, when bulls were allocated top-5 and sub-5 sires according to NRR56-SEXED, cleavage rates (69.9 ± 15.7 % vs. 70.3 ± 12.7 %), blastocyst rates (12.6 ± 8.0 % vs. 13.0 ± 8.0 %) as well as time until cleavage (34.8 ± 2.9 h vs. 34.5 ± 5.3 h) did not differ. Taken together, the present study did not observe clear correlations between sire field fertility following AI using conventional and/or sexed semen and developmental characteristics of subsequent embryos following IVF.

Key words: in vitro fertilization, reproductive technology, developmentral kinetics

11

T-cells in testicular germ cell tumors: New evidence of functional contributions by rare subsets

T-Zellen in testikulären Keimzelltumoren: Neue Befunde zur funktionellen Bedeutung seltener Subpopulationen

R. Islam1,2, M. Figura1,3, J. Heyer1,3, S. Indumathy1,2, B. Nathaniel2; K. Hartmann1, C. Pleuger4, M. Fijak4, S. Kliesch5, F. Dittmar3, A. Pilatz3, F. Wagenlehner3, M. Hedger2,7, B. Loveland6, K. Loveland2,7, J Guo8,9, H.-C, Schuppe3, D Fietz1

1Dept. of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University, Giessen, Germany; 2Centre for Reproductive Health, Hudson Institute for Medical Research, Clayton, Victoria, Australia; 3Dept. of Urology, Pediatric Urology and Andrology, Justus-Liebig-University, Giessen, Germany, 4Institute of Anatomy and Cell Biology, Hessian Centre of Reproductive Medicine, Justus-Liebig-University, Giessen, Germany; 5Centre of Reproductive Medicine and Andrology, University of Muenster, Muenster, Germany; 6Burnet Institute, Melbourne, Australia; 7Dept of Molecular and Translational Sciences, Monash University, Clayton, ­Victoria, Australia; 8State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; 9Division of Urology, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, USA

T cells represent the major component of tumor infiltrating lymphocytes (TIL) in testicular germ cell tumors (TGCT). Whereas the roles of rarer subtypes, such as regulatory (Treg) and follicular helper T (Tfh) cells have not been investigated in TGCT, these have each been associated with tumor progression and prognosis in other tumor entities. To analyze T cells and their subsets in TGCT, we employed three approaches: a) immunohistochemistry on a retrospective patient cohort including testis biopsies with preserved spermatogenesis lacking (n = 10) or with immune infiltrates (n = 12), germ cell neoplasia in situ lacking or with lympho­cytic infiltrates (n = 15, each), seminoma (SE, n = 28), and embryonal carcinoma (EC, n = 10); b) flow cytometry on a prospective patient cohort using samples from different locations in the tumor-bearing and contralateral testis (SE n = 12, EC n = 6); c) single cell RNAseq (normal testis n = 3, TGCT n = 4). All approaches, despite using different and heterogeneous patient samples, showed that the immune cell composition shifted from a macrophage-dominated environment in normal testis to a T cell-dominated milieu in TGCT, especially in SE. In regard to the rarer T cell subtypes, Treg and Tfh, these were most abundant in SE, i.e. in tumor-center sites. Additionally, deeper analysis of the scRNA-seq datasets showed Treg and Tfh signature molecules to be highly expressed in TGCT. Overall, this study describes the complexity of TIL in TGCT and provides first indications that rarer T cell subtypes and their signaling molecules are of functional importance in the TGCT immune environment. Funded by DFG IRTG 1871/P2.

Key words: testicular germ cell tumor, t cells, tumor microenvironment

12

Filamin A – a new player in the ovary

Filamin A – ein neuer Akteur im Ovar

Y. Jiang1, K. M. Caban2, T. Fröhlich2, D. Berg3, U. Berg3, D. Mayr4, A. Müller-Taubenberger1, A. Mayerhofer1,
H. Welter1

1Biomedical Center (BMC), Cell Biology, Anatomy III, Faculty of Medicine, Ludwig Maximilian University Munich; 2Laboratory for Functional Genome Analysis LAFUGA, Gene Center, Munich; 3A.R.T. Bogenhausen, Munich; 4Institute of Pathology, University Hospital, Munich, Germany

Filamin A (FLNA) is a well-characterized cytoskeletal protein, which cross-links actin filaments. In addition, it acts as an intracellular signaling scaffold and thus orchestrates pathways related to cell differentiation, development and morphogenesis. Specific functions of FLNA in various cell types, as well as in different tumors have been identified. Human ovary single cell RNAseq data also indicated its presence in the human ovary, specifically in granulosa cells (GCs). The adult ovary is a dynamic organ, hosting multiple morphogenic and physiological changes, occurring specifically in follicles and involving GCs. We confirmed FLNA-expression in cultured human IVF-derived GCs and in follicles of human and nonhuman primate ovary. Levels of FLNA in GCs were regulated by hormones and second messengers. Expression was also found in granulosa cell tumors (GCTs), which are derived from human GCs, as well as in KGN cells, a widely studied GCT cell line. To be able to examine functions of FLNA, we generated FLNA-knock out (FLNA-KO) KGN cells, using CRISPR/Cas9. Initial functional studies revealed altered morphology, increased cellular ATP-level and a different migration behavior of FLNA-KO cells. FLNA-KO KGN cells were further studied by a holistic mass spectrometry-based proteome analysis and prominent alterations in the cellular proteome were identified. In conclusion, our data show that FLNA is expressed by granulosa cells of the human ovary and by granulosa tumor cells and indicate multiple roles of ovarian FLNA with potential relevance for (in)fertility and tumorigenesis.

Grants: Supported by DFG project number 491030536

Key words: Filamin A, ovary, granulosa cells

13

Defining the transcriptome of in vivo derived bovine embryos through single-cell sequencing

Definition des Transkriptoms von in vivo gewonnenen Rinderembryonen durch Einzelzellsequenzierung

G. Neufeld, R. Herbicht, D. Herrmann, S. Altgilbers,
S. Heras-Garcia, C. Klein

Friedrich-Loeffler-Institut, Institute of Farm Animal Genetics (ING), Mariensee, Germany

The incredible potential of single-cell RNA-sequencing lies in the novel ability to study and exploit regulatory processes in complex tissues. The developing embryo completes a number of cell differentiation processes leading to the formation of various cell types such as epiblast, hypoblast, trophectoderm, endoderm, and mesoderm amongst others. With the advent of high throughput single cell sequencing approaches, it is now pos­sible to define the transcriptome of individual cell populations. In the present study, in vivo derived were recovered 17 days following artificial insemination. Following visual inspection of each embryo, the embryonic disc was separated from the remainder of the embryonic adnexa and both compartments were digested separately using TrypLE Express. Following generation of single cell suspensions, cells from both compartments were combined and 10x Genomics’ single cell RNA-seq technology was used to capture single cells and prepare barcoded, next-generation sequencing cDNA libraries. Sequencing results were aligned to the bovine genome and identified cells were used to generate the Cell Ranger filtered count matrix. Cells with more than 10% mitochondrial reads and/or less than 10,000 UMI were removed. Genes that were detected in less than 1% cells were removed (4000 cells on average passed QC). Based on lineage specific markers such as POU5F1, TMSB10, BMP4, and GATA4 clusters could be assigned to the corresponding lineages. Within trophectoderm cells, eight clusters were present, two of which separated more clearly; transcripts GCM1 and CCND2 were the top enriched transcripts for these two trophectoderm clusters.

Key words: embryonic disc, trophectoderm, genomics

14

Comparative assessment of the stress level of dairy cows from ­stimuli encountered in everyday life

Vergleichende Beurteilung der Stressbelastung von Milchkühen durch verschiedene Alltagsstimuli

K. Koenneker1, L. Pieper1, M. Jung1, K. Jäschke1, M. Schmicke2, M. Schulze1, F. Beyer1

1Institute for Reproduction of Farm Animals Schönow, ­Bernau bei Berlin, Germany; 2Department of Veterinary ­Endocrinology and Laboratory Diagnostics, University of Veterinary Medicine Hannover, Hannover, Germany

Failure to carry out common practices in stress research has led to a lack in comparability. The aim of this study was to analyse, whether, and to what extent everyday stimuli can be considered stressful. The stimuli tested in this study were artificial insemination (AI), embryo transfer (ET), natural breeding (NB), morning milking (MM), evening milking (EM), veterinary examination (VE), ultrasound examination (US) and hoof trimming (HT). The stimuli were compared by examining their impact on the hypothalamic-­pituitary-adrenal axis, through serum-cortisol analysis. The test-herd, comprising of 24 multiparous Holstein Friesian cows, was divided randomly every test day into Treatment- (Tr) and Control-groups (Co). Basal blood samples were taken 40 min (B1) and 20 min (B2) prior to stimulus application, immediately following the stimulus (S), as well as 20 min (R1) and 40 min (R2) post stimulus. Comparison of serum-cortisol in Tr-groups show a significant difference between NB to AI (p = 0.011); ET (p = 0.006); EM (p = 0.009); VE (p = 0.030) and US (p = 0.010). There is no significant difference between Co-groups (p > 0.05). Cortisol levels within Co-groups vary only slightly over time. The stimuli ET (p < 0.001), MM (p < 0.001), ME (p = 0.000), HT (p < 0.001) and NB (p < 0.001) show significant increases in cortisol after stimulus application, the levels fail to decrease significantly in the stimuli HT and NB. The presented data can be used to create recommendations for improving the welfare and productivity of dairy cows and influence the development of new animal husbandry techniques.

Grants: Funded by Förderverein Bioökonomie Forschung

Key words: stress, serum-cortisol, dairy cattle

15

Hypothermic, antibiotic-free preservation of boar semen is effective against multidrug resistant Serratia marcescens

Die hypotherme, antibiotikumfreie Konservierung von Ebersperma wirkt effektiv gegen multiresistente Serratia marcescens

I. K. Maaßen, A.-M. Luther, D. Waberski

Unit for Reproductive Medicine, Clinic for Pigs and Small ­Ruminants, University of Veterinary Medicine, Hannover, Germany

In boar semen, commonly preserved at 17 °C, contamination with Serratia (S.) marcescens is a major problem due to its multidrug resistance and spermicidal effects. Our study tested the hypothesis that hypothermic semen preservation at 5 °C inhibits the growth of S. marcescens. Ten semen samples from seven boars were diluted in antibiotic-free AndroStar® Premium extender (Minitüb, Germany) to 20 × 10? sperm/ml. Three subsamples were prepared: S1: contamination with S. marcescens (5 × 10² CFU/ml), storage at 17 °C (positive control), S2: contamination with S. marcescens, storage at 5 °C, and S3: storage at 5 °C (negative control). In S1, the number of bacteria increased exponentially, reaching more than 10¹² CFU/ml after 144 h. This coincided with an increase of agglutinated sperm, loss of motility evaluated by computer-assisted semen analysis and loss of membrane integrity evaluated by flow cyto­metry using PNA/Propidium iodide, starting at 72 h (p < 0.05) when bacterial counts were 10? CFU/ml. In contrast, bacterial growth in S2 was limited to 1.6 × 10³ CFU/ml until 144 h. In S3, bacterial counts were below 10 CFU/ml. In S2 and S3, sperm quality traits did not differ (p > 0.05) and were maintained throughout 144 h storage about threshold values for useable semen according to the guidelines of the German Livestock Association (BRS e.V.). In conclusion, low temperature storage of boar semen effectively inhibits the growth of bacteria including S. marcescens and maintains sperm quality. This is important if effective antibiotics are no longer available, or their use becomes unwanted.

Key words: semen preservation, boar semen, bacteria

16

Seasonal changes of microRNA expression in cryopreserved bovine spermatozoa

Untersuchung saisonaler Unterschiede der micro-RNA-Expression in kryokonservierten Bullenspermien

E. Malama1, S. Bauersachs2, S. Bozukova2, M. Siuda1, T. Kompara3, U. Witschi3, H. Bollwein1

1Klinik für Reproduktionsmedizin, Departement für Nutztiere, Vetsuisse-Fakultät, Universität Zürich, Switzerland; 2Institut für Veterinär-Anatomie, Vetsuisse-Fakultät, Universität Zürich, Switzerland; 3Swissgenetics, Zollikofen ­Switzerland

Our study aimed to investigate the seasonal changes of selected microRNAs (miRNA) in cryopreserved bovine sperm. Four Limousin, four Holstein, two Brown Swiss, one Simmental and one Swiss Fleckvieh bull of a single station, with fertility index within the mean ± SD of the bull population, were examined. Three weekly ejaculates per bull were cryopreserved at the onset of each of the calendar season (144 sperm samples in total). Total RNA was extracted with a modified TRIzol®-based protocol [Rauber, 2008, Dissertation, LMU Munich]. Following preparation of first-strand cDNA, relative quantification of the mature sequences of miR-30d-5p, miR-34b-5p, miR-92a-3p, miR-132-3p and miR-204-5p was performed by real-time PCR. The quantification cycles (Cq) of each miRNA were normalized to miR-30d-5p and miR-132-3p reference miRNAs using the ?Cq method [Livak, Methods 2001; 25: 402–8]. Based on mixed-effects modeling of ?Cq values (with season and bull as fixed and random effect, respectively), miR-34b appeared to be upregulated in spring (b = –1.78, P = 0.005) and summer (b = –2.01, P = 0.002) vs. winter months. The expression of the remaining miRNAs was constant throughout the year (P > 0.05 in all cases). Bull variability was responsible for 20.8%, 50.9%, 15.4%, 14.0% and 12.3% of the variance in the expression of miR-30d, miR-34b-5p, miR-92a-3p, miR-132-3p and miR-204-5p, respectively, with the Holstein bulls being the most common outliers. Our findings suggest a distinct seasonal pattern of miR-34b-5p expression, a miRNA preferentially expressed in the male germ line that has been described as potential marker of bull fertility; however, bull variability can be a considerable source of variance.

Key words: bull sperm, microRNA, seasonality

17

Functional analysis of the histidine N-methyltransferase SETD3 in endometriosis

Funktionelle Analyse der Histidin N-Methyltransferase SETD3 bei der ­Endometriose

M. Poloczek1, C. Ludwig2, T. Strauß1, M. Gabriel3, M. Poutanen3, L. Kiesel1, S. D. Schäfer1, J. M. Weitzel2, M. Götte1

1Department of Gynecology and Obstetrics, University Hospital Muenster, Muenster, Germany; 2 Institute of Reproductive Biology, Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany; 3Research Centre for Integrative Physiology and Pharmacology, Institute of Biomedicine, University of Turku, Finland

Endometriosis is associated with pain and reduced fertility. Here, we investigate the effects of alterations in the expression of SETD3, an actin-specific histidine N-methyltransferase, on cytoskeletal function and endometriotic cell motility. SETD3 expression in human tissue was analyzed using EndometDB, and evaluated in the uteri of the superfertile Dummerstorf mouse line FL1 by qPCR. SETD3 function in human endometriotic 12Z and primary cells were studied using siRNA. Cell motility, contractility, invasiveness, morpho­logy and gene expression were analyzed by rt-PCR, western blotting, immunofluorescence, scratch wound assay, collagen contraction assay and matrigel invasion assay, respectively. SETD3 gene expression was slightly increased in deep endometriotic lesions, and downregulated 1.6-fold in the uteri of superfertile mice. In vitro, immunofluorescence did not reveal major changes in cytoskeletal morphology. Only moderate changes in cytoskeletal element gene expression were observed, whereas SETD3 depletion resulted in a delay in cell motility in a scratch assay, in a reduction in invasiveness of 12Z cells, and in a reduced capability to contract collagen gels. SETD3 affects invasive growth, cell motility and contractility of endometriotic cells in-vitro and could be related to the pathogenesis of (deep) endometriosis. SETD3 downregulation in the uteri of superfertile mice may indicate a possible link to infertility.

Key words: SETD3; endometriosis; cytoskeleton;

18

Hypothermic preservation of boar semen at 5 °C: a field test

Hypotherme Konservierung von Ebersperma bei 5 °C: Ein Praxisversuch

F. Reckinger1, A. Riesenbeck2, D. Waberski3, R. Waßmuth1

1Faculty of Agricultural Sciences and Landscape Architecture, Osnabrück University of Applied Sciences, Osnabrück, Germany; 2GFS Genossenschaft zur Förderung der Schwei­nehaltung eG, Ascheberg, Germany; 3Unit for Reproductive Medicine, Clinic for Pigs and Small Ruminants, University of Veterinary Medicine, Hannover, Germany

Hypothermic preservation of boar semen at 5 °C instead of 17 °C, is a new approach to reduce antibiotics in semen doses. For practical use, information on the potentially boar-individual response to semen chilling and in vivo fertility is decisive. The aim was to assess sperm quality after long-term storage at 5 °C in four consecutive ejaculates of 20 boars and to test for fertility in a pretrial prior to a large-scale field study. In samples stored at 5 °C in Androstar Premium and analysed at 24 h, 72 h, 96 h and 144 h, overall motility assessed with computer-assisted semen analysis was 79.9% compared to 81.1% in controls stored at 17 °C in Beltsville Thawing Solution. Two boars had a lower sperm motility in 5 °C samples compared to controls (p < 0.001), reaching the lowest value of 65% (total motility) after 144 h. Acrosome defective sperm did not differ (p > 0.05) between the storage groups showing 6.5 % defects in 5 °C and 7.1 % in control samples at 144 h. Insemination with pooled semen of three boars stored split sample at 5 °C (n = 89 sows) and 17 °C (n = 91 sows) yielded high fertility. There were no differences (p > 0.05) in farrowing rate (5 °C: 92.1%; 17 °C: 92.3%) and average number of born piglets (5 °C: 18.7 ± 3.6; 17 °C: 18.5 ± 2.8). In conclusion, sperm quality in samples stored at 5 °C was maintained in all boars above thresholds for useable semen as defined by the German Livestock Association (BRS e.V.). First field data with 5 °C-boar semen in Europe indicate that hypothermic storage is applicable in routine insemination.

Key words: antibiotic-free; boar semen; hypothermic storage

19

Regulation of local produced novel adipokines during different reproductive stages in the bovine ovary

Regulation von lokal produzierten neuartigen Adipokinen während verschiedener reproduktiver Stadien im Ovar des Rindes

G. Thaqi1, B. Berisha1,2, M.W. Pfaffl1

1Chair of Animal Physiology and Immunology, School of Life Sciences, Technical University of Munich, Weihenstephan, Germany; 2Department of Animal Biotechnology, Faculty of Agriculture and Veterinary, University of Prishtina, Kosovo

The objective of this study was to determine the dynamic local regulation of novel adipokines as vaspin, adiponectin, visfatin, resistin and their known receptors heat shock protein 5, adiponectin receptor 1, adiponectin receptor 2 in bovine corpus luteum (CL) during the bovine estrous cycle and pregnancy. The CL of cycling cows was depicted in six groups (n = 10) representing different days of the cycle (D): group I (D1–2), group II (D3–4), group III (D5–7), group IV (D8–12), group V (D13–16), group VI (D >18) and four groups (n = 6–9) of pregnancy months (M): group VII (M1–2), group VIII (M3-4), group IX (M5–7) and group X (M > 7).Total RNA was isolated from 50 mg CL tissue using a guanidinium thiocyanate based method. Adipokine mRNA sequences were quantified by one-step RT-qPCR using hydrolysis probes. The relative mRNA expression levels were determined and normalized to the geometric mean of four constant expressed reference genes (cyclophilin A, ubiquitin, ubiquitin C and beta-actin). Our findings suggest that adipokines are present in CL during all cyclic and pregnancy stages and differently up/down-regulated. Vaspin and adiponectin are up-regulated (p < 0.05) in mid and late cycle stages (D8–12, D13–16, D > 18). Resistin showed high abundance (p < 0.05) during CL regression (D > 18) and the first months of pregnancy. In addition, the specific expression of adipokine receptors indicates their involvement in local mechanisms regulating CL function. Further investigations are required to elucidate the detailed regulation and mecha­nisms involved in different local actions of adipokines in CL tissue during estrous cycle and pregnancy.

Key words: adipokines, corpus luteum, gene expression

20

Compensability of morphological sperm defects: a basis for the re­vision of minimum requirements for the use of boar semen

Kompensierbarkeit von morphologischen Spermiendefekten: eine Grundlage für die Revision spermatologischer Mindestanforderungen beim Eber

D. Waberski1, A.-M. Luther1, B. Hensel2, M. Schulze2

1Unit for Reproductive Medicine, Clinic for Pigs and Small Ruminants, University of Veterinary Medicine Hannover, Hannover, Germany; 2Institute for Reproduction of Farm Animals Schönow, Bernau, Germany

It is well known that some sperm deficiencies can be compensated by an increase of sperm numbers in the insemination portion so that fertility is not affected. The situation, however, is less clear for retained cytoplasmic droplets (CD) which present the most common sperm abnormality in boar semen. Current minimum requirements defined by the German Livestock Association (BRS e.V.) set 15% of sperm with CD as the upper limit for useable boar semen. We hypothesized that a higher prevalence of CD can yield high fertility by compensation with high sperm numbers. Retrospective analysis of extended spermatology from 130 young boars and field insemination data (n = 1497 sows) using break point analysis and group comparisons with Dunn’s Multiple Comparison Test showed high farrowing rates and litter sizes in the three groups with low (< 10%), moderate (10–15%) and high (> 15%) prevalence of sperm with CD. Interestingly, a slightly higher farrowing rate (p < 0.001) was seen in the high CD group compared to the low and moderate CD groups, which coincided with a higher motility (p < 0.001) and a higher resistance to thermic stress (p < 0.001). Sperm numbers per dose were ? 2×10? sperm and had no effect on fertility. In conclusion, these results together with our previous single cell observation on high motility of CD-bearing sperm [Henning et al., under review] prompt to revise current minimum requirements for useable boar semen, ideally with consideration of motility, resistance to thermic stress and a minimum sperm number per insemination dose.

Key words: sperm morphology, cytoplasmic droplets, boar semen

21

Reproductive management of llama and alpaca flocks in Germany – results from a survey among animal owners

Reproduktionsmanagement in Alpaka- und Lama-haltenden Betrieben in Deutschland – Ergebnisse einer Um­frage unter Tierhaltern

H. Wagner, L. Ulrich, E.-M. Bartl, A. Wehrend

Clinic for Obstetrics, Gynecology and Andrology for large and small animals, Justus-Liebig-University Giessen, Germany

In recent years, breeding of New World camelids (NWC) became very popular in Germany. Good practice of reproductive management is required to receive healthy animals without behavioural disorders. Further, the diverse kinds of purposes place high demands on NWC for example such as private or touristic trekking tours and pet-faciliated therapy. However, it can be assumed that the rapidly increasing NWC population, together with a lack of knowledge, has resulted in over-demand on the owners. Therefore, our working group conducted a survey to assess the current reproductive management practice. The survey was addressed to NWC owners from all over Germany. 50.4% of the livestock owners indicated that they use a stallion from their own herd for mating, while 47.6% use a stallion from another herd. 20.2% of the participants did not choose the aspect of health as one of the three most important factors in the selection of mares for breeding. The first birth of mares occurs at the age of 3 years for 51.9% of the keepers, and on average mares are 34.3 months old. Mares are used for breeding by 51.9% of the livestock owners up to an age of 10–13 years, 40.7% use their mares up to an age of 16 years and 7.4% still up to an age of 20 years. 71.1% of the keepers do not have pregnancy tests performed on their animals, the spit-off test is considered a safe procedure. 87.4% of births take place from June to August, birth monitoring is done more than twice a day in 63.1% of farms. 39.1% of the farmers stated that obstetric intervention was necessary during foaling, 84.1% stated that the farmer himself had to intervene obstetricly, in 42.7% of the cases a veterinarian was consulted.

Key words: lifestock, camelids, breeding

Poster

Generation of an orthotopic mouse model to study Caspase-8 biology and cell contact in human ovarian cancer

Etablierung eines orthotopen Mausmodells zur Untersuchung des Einflusses von Caspase-8 und Zell-Zellkontakten im humanen Ovarialkarzinom

S. M. Aberoumandi1, I. Kostova2, E. Ullrich3, K. Strebhardt2, R. Mandal2, D. Fietz1, M. Kressin1

1Institute for Veterinary Anatomy, Histology and Embryo­logy, Justus-Liebig-University Giessen, Germany; 2Department of Gynecology and Obstetrics, University Hospital, Frankfurt/Main, Germany; 3Experimental Immunology, University Hospital for Children and Adolescents, Goethe University, Frankfurt/Main, Germany

Ovarian cancer (OC) is the deadliest gynaecological malignancy due to non-specific symptoms in early stages and late stage diagnosis. Therapy regimes rely on debulking surgery and chemotherapy (carboplatin/paclitaxel) with good first response but frequent relapses. Novel therapies are eagerly needed addressing cancer cell biology, such as programmed cell death (apoptosis). We aimed at analyzing the impact of Caspase-8 (a key protein in the apoptotic cascade) depletion in an orthotopic mouse model of human OC. Therefore, we used human OC cells modeling high grade serous OC (OVCAR8). Wildtype (WT) and Caspase-8 knockout (KO) OVCAR8 cells transfected with luciferase reporter were injected in both bursa of n = 4 NMRInu/nu mice (n = 2 WT, n = 2 KO). Starting one week after surgery and until mice were sacrificed, tumor growth was measured every week using an in vivo imaging system (IVIS). Ovaries and organs were harvested and fixed for IHC-P or stored in liquid nitrogen for protein/mRNA analyses. IVIS measurements showed an increase in tumor growth in Caspase-8 depleted cells compared to WT cells showing the biological significance of Caspase-8 in tumor progression. Future experiments will assess the expression and localization of cell contact proteins (e.g. Connexin 43) in this mouse models compared to healthy controls. Additionally, RNAseq datasets will be used for in-depth analysis of tumor cell transcriptome. These experiments will provide insight into OC cell biology linked to the Caspase-8-KO in an orthotopic mouse model which may be used for therapeutic studies.

Key words: ovarian cancer, orthotopic mouse model, caspase 8

Effect of zinc, selenium, and vitamin E administration on the seminal antioxidant status and lipid peroxidation in infertile male dromedary camels

Wirkung der Verabreichung von Zink, Selen und Vitamin E auf den antioxidativen Samenstatus und die Lipidperoxidation bei unfruchtbaren männlichen Dromedaren

A. Ali1,2,, D. R. Derar1,2, T. M. Alhassun1, M. M. Zeitoun3, E. M. Abdel-Elmoniem4, T. I. Almundarij1

1Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University, Saudi Arabia; 2Department of Theriogenology, Faculty of Veterinary Medicine, Assiut University, Egypt; 3Department of Animal Production, College of Agriculture and Veterinary Medicine, Qassim University, Saudi Arabia; 4Department of Soil, College of Agriculture and Veterinary Medicine, Qassim University, Saudi Arabia

The objective of this study was to investigate the effect of zinc (Zn), selenium (Se), and vitamin E (Vit E) administration on the antioxidant status and lipid peroxidation in the seminal plasma of infertile male dromedary (post-coital infertility, n = 33). Breeding history and andrological examination were accomplished. Infertile camels were treated with a combination of intramuscular injections of Vit E (?-tocopherol acetate, 1 mg/kg bw) and Se (sodium selenite, 0.088 mg /kg bw) once every week for three successive weeks and by daily oral administration of 360 mg of zinc gluconate for 5 successive weeks. Semen was collected and analyzed. Concentrations of total antioxidant capacity (TAC), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) were assessed in seminal plasma before and after treatment. Treated camels were allowed to mate females in estrus and conception rates were calculated. Treatment significantly increased the activity of TAC (0.23 ± 0.02 vs. 0.27 ± 0.12 Mm/L, P = 0.03), along with non-significantly improvement in the expression of SOD (219.61 ± 1.8 vs. 252.31 ± 15.2, P=0.08 U/mL) and CAT (192.64 ± 30 vs. 234.15 ± 34 U/L, P = 0.2). MDA activity non-significantly decreased after treatment (5.13 ± 1.3 vs. 4.36 ± 1 nmol/mL, P = 0.6). All parameters of seminogram and fertility improved after treatment (P < 0.01). In conclusion, Zn, Se and Vit E administration improved total antioxidant capacity, seminogram and fertility of male dromedary camels with post-coital infertility.

Key words: antioxidants, semen quality, dromedary camels

Clinicopathological features of an ovarian teratoma in an Arabian mare

Klinisch-pathologische Merkmale eines Ovarteratoms bei einer Araberstute

F. A. Al-Sobayil1, A. Ali1,3,*, D. R. Derar1,3, M. M. Khodeir2,4

1Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University, Saudi Arabia; 2Department of Pathology, College of Medicine, Qassim University, Saudi Arabia; 3Department of Theriogenology, ­Faculty of Veterinary Medicine, Assiut University, Egypt; 4Department of Pathology, Faculty of Medicine, Cairo University, Egypt

A multiparous Arabian mare (9-year-old) was admitted to the vet clinic due to emaciation and infertility. Blood samples were obtained for biochemical and hormonal analysis. The mare‘s estrous cycle was regular, as evidenced by estradiol 17? and progesterone levels and all biochemical parameters were within acceptable ranges. By transrectal palpation, the left ovary was large, round, and smooth, whereas the right ovary appeared normal in size and consistency. On US, the left ovary revealed a heterogeneous image with echogenic dispersed reflectors, calcifications, and echogenic spherical masses suggesting an ovarian tumor. The left ovary was surgically removed. The existed ovary was oval measuring 12 cm in length and 7 cm in breadth that is encircled by a capsule and contains hair, bone, and semi-oval sebaceous materials and a clotted ball floating in a red liquid. In sections, multiple benign tissue types emerging from all three germ layers were visible, confirming a case of mature cystic teratoma (MCT). The body‘s condition improved after the tumor was removed. Two months later, the mare became pregnant. Finally, MCT can impair the mare‘s overall health and fertility. The clinical and US characteristics pointed to ovarian neoplasm; however, gross- and histopathology are required for a final diagnosis.

Key words: teratoma, arabian mare, clinical pathology

Details matter – freezing rate in sperm cryopreservation

Auf Details kommt es an – Einfrierraten zur Kryokonservierung von Spermien

M. Bashawat, A. Weber, K. Müller

Leibniz Institute for Zoo and Wildlife Research, Department Reproduction Biology, Berlin, Germany

An optimal freezing rate is precondition to ensure the survival of cells during cryopreservation, in particular if cells are not able to proliferate such as spermatozoa. In wildlife species usually only small amounts of semen are available for preservation. Therefore, we tested different tubes (Greiner and LVL) for their suitability to preserve small sample volumes (300 and 100 µl) in a field-suited freezing device [1]. The tubes differ in their material and geometry. For instance, in Greiner round-bottomed tubes, samples form almost a sphere, while liquid in LVL tubes has a cylindrical shape. We tested tubes and volumes in a split-sample setting for epididymal semen of domestic cats (n = 8) and measured equilibration and freeze/thaw rates. Sperm motility was analysed (CASA) ten minutes post-thaw, and up to four hours after sub­sequent centrifugation/resuspension. A generalized linear model revealed slight differences in the percentage of total, progressive and (in part) fast sperm post-thaw, after centrifugation and one hour later in dependence of the vial-volume combination. Best values were achieved by the standard (Greiner, 300 µl) for which the method had been established [1]. Compared to the standard, a slight transient temperature increase was measured upon crystallization in LVL tubes with 300 µl samples. Freeze/thaw rates were expectedly faster in both tube types with 100 µl samples. Already small deviations of temperature curves in critical ranges might have caused the slight sperm quality differences. Because the differences disappear two hours post-thaw, we hypothesize that optimal freeze/thaw rates allowed more of the less fit sperm to survive.

Key words: semen cryopreservation, freezing rate, crystallization heat

Reference:

1. Klaus C. Reprod Dom Anim 2016; 51: 195–203.

Establishment of a bovine granu­losa- and theca co-culture

Etablierung einer bovinen Granulosa- und Theka-Co-Kultur

A. Baufeld, J. Vanselow

Research Institute for Farm Animal Biology (FBN), ­Dummerstorf, Germany

A reliable co-culture of granulosa (GCs) and theca cells (TCs) would contribute to a more comprehensive understanding of the interactions, signaling pathways and substrate exchange between both cell compartments in the bovine ovarian follicle. Using commercially available cell culture inserts, we established a reproducible co-culture model of bovine GCs and TCs. Primary theca and granulosa cells require considerably more time (up to 48 h) to attach onto the insert membrane compared to cell lines. Therefore, the TCs were initially seeded onto the inverted insert membrane, using a truncated tube as an inoculation container to prevent the medium from leaking. After 48 h, the insert was turned upside down and the GCs were cultured on the opposite side of the membrane. This co-culture was incubated for additional 6 days at 37 °C and 5% CO?, with a media exchange every other day. Subsequently, cross-sections of the membranes were fixed in bouin or paraformaldehyde to evaluate the success of the co-culture model. Bouin fixation, known as gentle procedure, seems to lead to detachment of the complete cell layer from the insert membrane, especially the GC compartment. This could result in a misinterpretation in evaluation of successful co-culture. In contrast, these effects did not occur with the paraformaldehyde-fixed membranes. Analyses of key gene expression and hormone production are ongoing. In future, this modified co-culture model will allow reliable and reproducible investigations of paracrine communication and substrate transport between the somatic cells of the follicle.

Key words: co-culture, granulosa, theca

Lipid interaction partners of recombinantly expressed porcine sperm­adhesin AQN-3

Lipid-Interaktionspartner von rekombinant exprimiertem Spermadhesin AQN-3

B.C. Braun1, P.D. Kroh1, P. Müller2, K. Müller1

1Leibniz Institute for Zoo and Wildlife Research, Department Reproduction Biology, Berlin; 2Humboldt University of ­Berlin, Department of Biology/Biophysics, Berlin, Germany

Spermadhesins are major proteins of ungulates seminal plasma with particularly high abundance in porcine species. As the name indicates, they bind to the spermatozoa and are involved in various cellular functions. Although aggregation between spermadhesin molecules as well as their binding to lipids was postulated [1], the detailed mechanism of interaction between protein and sperm cells is largely unknown. In the present study, we investigated the interaction of AQN-3, one of the five porcine spermadhesins, with lipids in vitro. For this purpose, AQN-3 was expressed recombinantly in E. coli (recAQN-3) having a His-tag for purification. Size exclusion chromatography revealed an aggregated/multimer status of recAQN-3. Applying two approaches, we observed an interaction of recAQN-3 with negatively charged lipids. First, presenting lipids on strips, the main interaction partner of recAQN-3 was phosphatidic acid (PA) followed by a less pronounced binding to phosphatidylinositol phosphates PI(4,5)P?, PI(4)P and PI(3,4,5)P?, cardiolipin (CL) and phosphatidylserine (PS). Second, presenting lipids as multilamellar vesicles (MLVs), binding of recAQN-3 to PI(4,5)P? and PI(4)P was stronger than to PA. In addition, an interaction with CL but not with PS was observed. Since the binding of recAQN-3 to MLVs remained stable under high salt condition, the molecular binding mechanism does not seem to be purely electrostatically driven. The functional relevance of AQN3-binding to negatively charged lipids will be investigated in future.

Key words: seminal plasma, spermadhesin, AQN-3

Reference:

1. Dostàlovà Z. Interaction of non-aggregated boar AWN-1 and AQN-3 with phospholipid matrices. A model for coating of spermadhesins to the sperm surface. Biol Chem Hoppe Seyler 1995; 376: 237–42.

Alpha 7 nicotinic receptors are involved in the regulation of ovarian functions (Part II)

Alpha-7-nikotinerge Rezeptoren sind an der Regulation der Ovarfunktion beteiligt (Teil II)

K.M. Caban1, P. Seßenhausen2, B. Popper3, A. Mayerhofer2, T. Fröhlich1

1Gene Center – Laboratory for Functional Genome Analysis, Ludwig-Maximilian-University (LMU), Munich; 2Biomedical Center (BMC), Cell Biology, Anatomy III, Faculty of Medicine, Ludwig-Maximilian-University (LMU) Munich;3 Biomedical Center (BMC), Core Facility Animal Models, Faculty of Medicine, Ludwig- Maximilian-University (LMU) Munich, ­Germany

The alpha7 nicotinic acetylcholine receptor (CHRNA7) is a member of the pentameric ligand-gated ion channels superfamily and one of the acetylcholine (Ach) receptors that are part of the cholinergic system of the brain. The expression of CHRNA7 is not restricted to the nervous system and was, among others, reported in the ovary, where its roles are not well known. To obtain insights into the roles of ovarian CHRNA7, we performed quantitative proteome analyses of ovaries from ­Chrna7 knock out (KO) and from age matched controls wildtype mice (WT). A holistic mass spectrometry-based proteome analysis was performed leading to the detection of 36,662 peptides which could be assigned to 3951 different proteins. Quantitative analysis showed 128 differentially abundant proteins (FDR < 0.05) of which 96 proteins were more abundant and 32 proteins less abundant in KO animals. An increased abundance of steroidogenesis-related proteins, e.g. CYP11A1 and HSD3B6 was observed, which implies that Chrna7 deficiency may lead to alterations in steroid production. Further bioinformatic analysis revealed that proteins increased in abundance are involved in mitochondrial and fatty acid metabolic processes, which might be related to steroidogenesis in theca cells. In the context of inflammatory response regulation, DDX3X, CTSS, NDUFC2, HNRNPA0 were more and PTGIS less abundant in KO samples. The proteomics results together with ones described in Part I, point to distinct ovarian roles of this receptor, which may include steroidogenesis, metabolism and inflammatory responses.

Grants: DFG, project number 432434245

Key words: CHRNA7, ovary, proteomics

Bull-, management- and environment-related factors affecting the DNA integrity of cryopreserved sperm

Einfluss von Bullen-, Management- und umweltbedingten Faktoren auf die DNA-Integrität kryokonservierter Spermien: Eine retrospektive Studie über 8 Jahre

B. Cinar1, E. Malama1, I. Ibanescu1, 2, M. Siuda1, C. Leiding3, H. Bollwein1

1Klinik für Reproduktionsmedizin, Departement für Nutztiere, Vetsuisse-Fakultät, Universität Zürich, Switzerland; 2Swissgenetics, Zollikofen, Switzerland; 3Besamungsverein Neustadt a.d. Aisch e.V., Germany

Our study aimed to explore the source of variation of DNA integrity in bovine sperm. Thus, the percentage of sperm with high DNA fragmentation index (%DFI) was determined in 3,482 frozen semen doses produced from 263 sires (13 ± 21 doses per sire) in years 2012–2019, using the Sperm Chromatin Structure Assay™. Hundred thirty-two doses were produced from sires at a young age (12–24 months), while 2,080 and 620 doses were collected from bulls at a mature (24–85 months) or older age (> 85 months). A total of 1,641 doses were subjected to a 24-hour equilibration prior to freezing. Doses (1841) collected on Fridays were equilibrated for 72 hours before freezing. The daily temperature-humidity index (THI) was computed throughout the 8-year experimental period out of the daily maximum ambient temperature and mean relative humidity. The effects of year, equilibration time, age class, season, THI of the sperm collection day and the interaction term age class×season×THI on %DFI were assessed using mixed-effects linear models; the effect of ejaculate (nested within bull) was included as random effect. The 72-hour equilibration had a beneficial effect on %DFI (b = –0.354, P < 0.001). Sperm collected from young and old bulls showed higher %DFI compared to mature sires; however, these differences were not significant (P > 0.05 in all cases). Elevated THI was linked with increased %DFI (b = 0.03, P = 0.029), with the highest %DFI values being observed in summer (b = 1.20, P = 0.017). Our observations suggest an adverse effect of warming temperatures on sperm chromatin stability. At the same time, the prolongation of the equilibration time prior to freezing appears to be beneficial for DNA integrity.

Key words: sperm DNA, temperature-humidity index, equilibration

Intracellular pathways associated with the function of canine decidual cells: involvement of selected protein kinases and their relation to progesterone signaling

Intrazelluläre Signalwege im Zusammenhang mit der Funktion von ­kaninen Deciduazellen: Beteiligung ausgewählter Proteinkinasen und ihre Beziehung zur Progesteron-Signalgebung

I. De Geyter, M. Tavares Pereira, M. P. Kowalewski

Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich (UZH), Zurich, Switzerland

The formation of the canine placenta involves the differentiation, i.e., decidualization, of endometrial stromal cells. Being the sole cell population expressing the nuclear progesterone receptor (PGR), decidual cells play a crucial role in the establishment and maintenance of canine pregnancy, offering good clinical opportunities for manipulating reproductive outcomes. In vitro, cAMP, acting through the downstream serine/threonine kinase (STK) PKA, can induce the decidualization of immortalized dog uterine stromal (DUS) cells. However, the role of other intracellular protein kinases in canine decidualization is still poorly understood. Here, using the PamChip assay (PamGene), 85 STKs were predicted to exhibit increased activity after the cAMP-induced decidualization of DUS cells, including PKA, PKC, ERK1/2, ATR and Akt1/2. Blocking PGR with antigestagens (aglepristone and mifepristone) for 6h decreased the activity of all modulated kinases in decidualized DUS cells. The functional significance of selected STKs in decidualized DUS cells was assessed using specific blockers for PKA (H89), ERK1/2 (U0126), and PKC (GF-109203X). Interfering with PKA and ERK1/2 resulted in downregulation of decidualization markers: IGF1, PTGES and PRLR (P < 0.05), whereas blocking of PKC decreased mRNA availability of IGF1 (P < 0.05), but not of PTGES or PRLR (P > 0.05). Overall, decidualization appears to be associated with the increased activity of several STKs, that could be ablated by the disruption of P4/PGR signaling. Furthermore, PKA and ERK1/2 appear to have a stronger involvement in decidualized DUS cell physiology than PKC.

Key words: decidualization, canine placenta, progesterone signaling

Extraction techniques of pork ­ovarian follicles for the construction of artificial ovaries

Extraktionstechniken von Schweine­follikeln zur Herstellung eines künstlichen Ovars

R. Dittrich, A. Fattahi, I. Hoffmann, M.W. Beckmann

Frauenklinik des Universitätsklinikums Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, ­Germany

Ovarian tissue freezing for fertility preservation before gonadotoxic therapy with subsequent transplanted back into the patient after therapy is an established method for fertility preservation. This method is not suitable in patients at intermediate/high risk of ovarian metastasis. An alternative method would be to isolate tumor cell free primordial follicles from the tissue and to transplant these embedded in an artificial ovarian scaffold (artificial ovary) back into the patient. A major factor in the development of the artificial ovary is a protocol that extracts as many intact and viable ovarian follicles as possible. A comparative analysis of purely mechanical and combined mechanicalenzymatic extraction methods of porky ovarian follicles was performed, varying the concentrations of the two enzymes used (collagenase, liberase) as well as the enzyme treatment protocol used. To evaluate survivability, intact follicles were cultured for 10 days with subsequent live/dead assay for viability analysis. The number of intact follicles was higher using Collagenase compared to Liberase, but follicle viability was higher for Liberase extraction (n = 138, 82.07%) compared to Collagenase extraction (n =127, 59.82 %, X²= 13.005, p < 0.001).

Although more follicles could be found after Collagenase digestion, more follicles survived after Liberase digestion. Therefore a combination of mechanical and enzymatic (0.5 mg/ml Liberase concentration) extraction proved to be the most productive extraction methodo­logy.

Key words: fertility preservation, primordial follicles, reproduction

Glutathione peroxidase activity and total antioxidant capacity in spermatozoa of dogs with benign prostatic hyperplasia

Glutathionperoxidase-Aktivität und ­totale antioxidative Kapazität in ­Spermien von Hunden mit benigner Prostatahyperplasie

A. Domoslawska1, S. Zdu?czyk1, M. Kankofer2, A. Rafalska1

1Department of Animal Reproduction with Clinic, University of Warmia and Mazury, Olsztyn, Poland; 2Department of Biochemistry, Faculty of Veterinary Medicine, University of Life Sciences, Lublin, Poland

Benign prostatic hyperplasia (BPH) is one of the most common problems in intact male dogs. Oxidative stress is considered to play a role in the development of BPH in men. However, there were only very few studies on oxidative status in dogs with BPH and the results were contradictory. The aim of this study was the evaluation of the glutathione peroxidase (GSH-Px) activity and the total antioxidant capacity (TAC) in spermatozoa of dogs with BPH. The study was conducted on 40 intact stud dogs of various breeds. The dogs were assigned to two groups: a BPH group (n = 20) and a non-affected group (n = 20). The diagnosis of BPH was based on history, clinical symptoms such as sanguineous discharge from the urethra, dysuria, tenesmus and enlargement of the prostate on rectal palpation and ultrasound examination (Mindray Bio-Medical 2 with a 7.5 MHz convex transducer). The control animals showed no clinical signs and the prostate was not enlarged. The age of the dogs ranged from 5 to 8 years. Animals in both groups were of similar weight. Semen was collected by manual manipulation. The activity of GSH-Px and TAC were determined spectrophotometrically. Protein concentration in the samples was determined by the biuret method. The mean activity of GSH-Px and the mean values of TAC in spermatozoa were significantly (p < 0.05) lower in dogs with BPH compared to non-affected dogs (0.50 ± 0.23 vs 0.73 ± 0.49 nkat/mg protein and 10.13 ± 5.59 vs 15.30 ± 8.40 µmol/g protein, respectively). In conclusion, the results suggest a potential involvement of oxidative stress in the pathogenesis of BPH in dogs. More studies are needed to clarify the role of oxidative stress in the pathogenesis of BPH in dogs.

Key words: dog, BPH, oxidative stress

The importance of retraining herd-owner inseminators in dairy cattle: status quo

Wichtigkeit von Schulungen der Eigenbestandsbesamer beim Rind: eine Bestandsaufnahme

A. Enzig-Strohm1, A. Peters1, C. Leiding2, M. Schulze1, M. Jung1

1Institute for Reproduction of Farm Animals Schönow, ­Bernau bei Berlin, Germany; 2Besamungsverein Neustadt a. d. Aisch e. V., Neustadt a. d. Aisch, Germany

Previous studies suggested that ongoing retraining of herd-owner inseminators is critically important for maintaining accurate performance of artificial insemination (AI). The aim of this study was to determine the training needs of cattle herd-owners in the field of AI as well as their interest in e-learning courses and the desired topics. Thirteen dairy farms in Bavaria were included in this study. An online questionnaire was used to assess the farmers knowledge regarding cattle reproduction and AI techniques, as well as to identify the farmers interests. During a farm visit, participants demonstrated their AI techniques, and the processes were evaluated to identify areas of improvement. All information was evaluated descriptively. Results indicate a need for training in the following areas in descending order: preliminary examination, fertility indicators/disorders, rules and regulations and anatomy. Farm visits revealed deficiencies in hygiene and proper AI technique. All participants estimated their experience with computers and the internet as average to very good. Twelve (92%) participants were interested in an e-learning course. Preferred topics, in descending order, were: pregnancy diagnosis, influence of feeding on fertility, fertility disorders, insemination ability, estrus cycle and AI procedure. According to this study, AI refresher courses for herd-owner inseminators would be useful and should focus on the aforementioned topics. Training could be offered as an e-learning course for herd-owner inseminators under the condition that participants have sufficient experience in using computers and the internet.

Grants: Funded by Dr. Dr. h.c. Karl Eibl Foundation.

Key words: Artificial insemination, training courses, dairy cattle

TRPV2 in the human ovary and ­human granulosa cells

TRPV2 im humanen Ovar und in humanen Granulosazellen

K. Eubler1, K. Caban2, N. Kreitmair1, A. Tiefenbacher1, U. Berg3, D. Berg3, D. Mayr4, T. Fröhlich2, A. Mayerhofer1

1Biomedical Center Munich (BMC), Cell Biology, Anatomy III, Ludwig-Maximilian-University (LMU), Planegg-Martins­ried; 2Gene Center – Laboratory for Functional Genome Analysis, LMU, Munich; 3Fertility Centre A.R.T. Bogenhausen, Munich; 4Institute of Pathology, LMU, Munich, Germany

Transient receptor potential channel vanilloid 2, TRPV2, is a non-selective cation channel involved in inflammatory processes and is considered the least-examined member of the TRP ion channel family. Recent studies linked this ion channel for the first time to the male gonad in both human and mouse [Eubler et al., 2018 & 2021]. This raises the question of potential role(s) for TRPV2 also in the female gonad. Based on transcriptomic data of human oocytes and granulosa cells from different developmental follicle stages [Zhang et al., 2018] and on subsequent own immunohistochemical studies, we identified TRPV2 in the ovary of several species. Its expression is localized to immune cells and, distinctly, to follicular granulosa cells. During follicular development TRPV2 expression appears to increase and in addition was also detected in the corpus luteum. Human granulosa cells (hGCs), derived from pre-ovulatory follicles of IVF-patients and transferred to in vitro cell culture conditions, retain TRPV2 and were used as a model. Exposure to cannabidiol (CBD), a known TRPV2 activator, led to transient calcium influxes and subsequently to significantly increased transcript and protein levels of several inflammatory factors, especially IL-6 and IL-8. Based on mass spectrometry analysis, 14 out of over 5000 identified proteins revealed a significantly reduced (8) or increased (6) abundance upon CBD exposure. Thus, TRPV2 is an ovarian ion channel and channel activation entails, among others, induction of inflammatory processes in ovarian granulosa cells. Further roles and involvement in diseases, e.g. granulosa cell tumors (GCTs), remain to be studied.

Grants: Supported by DFG, MA 108031/1, project #456828204

Key words: TRPV2 channel, human ovary, inflammation

Round-up and glyphosate sup­plementation during in vitro maturation impact bovine embryonic ­development

Round-up- und Glyphosat-Zugabe während der In-vitro-Maturation ­beeinflussen die bovine embryonale Entwicklung

A. S. Fries1, N. Blad-Stahl1,2, J. Beranek2, S. Mazurek2, C. Wrenzycki1

1Chair for Molecular Reproductive Medicine, Clinic for Veterinary Obstetrics, Gynecology and Andrology, Justus-Liebig-University Giessen, Giessen, Germany; 2Institute for Veterinary Physiology and Biochemistry, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Giessen, ­Germany

Glyphosate (Gly) and its best known formulation Roundup® (R-Gly) are widely used non-selective total herbicides. Several studies revealed that Gly and R-Gly can act as endocrine disruptors. It is suggested that the toxic effects of R-Gly are mainly caused by the additives of the formulation. The aim of this study was to compare the effects of different Gly and R-Gly concentrations used during in vitro maturation (IVM) on the developmental competence of the subsequent embryos. Cumulus-oocyte-complexes were collected from abattoir derived ovaries and matured in TCM-199 without oil overlay. This IVM medium was supplemented with Gly or R-Gly to end up with final concentrations of 0, 30 or 300 µg/ml of Gly respectively in the medium. IVM endured for 24 hours, followed by a standard IVP protocol. Cleavage and developmental rates were recorded at day 7 and 8 via morphological microscopic evaluation. Data was analysed with Oneway-Anova and Tukey-Test. Significantly less embryos cleaved in the group of oocytes matured with 300 µg/ml R-Gly (36.2% ± 16.6) compared to all other groups (67.1–78.5%). At day 7, developmental rate was clearly decreased for the group with maturation in the high R-Gly concentration (5.6% ± 2.4 vs. 22.5–30.0%). The developmental rate of day 8 embryos stemming from oocytes matured with 300 µg/ml R-Gly was also impaired in contrast to the other R-Gly and Gly-concentrations (5.6% ± 2.4 vs. 22.9–32.9%). These data show that R-Gly, especially the additives, alters the developmental competence of bovine oocytes and affects the subsequent embryo development.

Key words: glyphosate, IVM, bovine

Fertility in high-volume-insemination in cattle

Fruchtbarkeit bei High-volume-Besamung beim Rind

M. Fuchs1,2, M. Reichenbach1, Y. Zablotski2, C. Otzdorff2, H. Zerbe2, J. Scherzer1

1Bayern-Genetik GmbH, Altenbach; 2Clinic for Ruminants with Ambulatory and Herd Health Services, Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-University (LMU) Munich, Germany

Artificial insemination is one of the most important reproductive technologies in the world. To this day, deep cervical or intrauterine insemination, which was developed back in the 1940s, has become the standard insemination (SI) method. The aim of the present study was to test a high-volume-insemination (HVI) method, where semen is diluted with up to 30 ml of medium before insemination, in practice. Routine inseminations were carried out during natural oestrus (HVI n = 100, SI n = 100) and within superovulated donor animals during embryo transfer (HVI = 31, SI = 21) and the two insemination methods were compared based on conception rates and flushing results. Statistical analysis was performed with Software R using logistic regression and (robust) linear regression with interactions. Within routine inseminations during natural oestrus, the method of insemination had no significant influence on pregnancy rates (p = 0.670). Overall conception rate was 54.5% (HVI 53%, SI 56%). Using routine embryo transfer, in both insemination methods, more viable embryos were expected as the total number of embryos/oocytes obtained increased. This trend was significant for both methods of insemination (p < 0.001), however, there was no significant difference for the trends of HVI and SI method (p = 0.405). It could be shown that HVI method is equal to SI method due to the conception rates achieved in routine insemination during natural oestrus and the results of routine embryo recovery. Since the HVI is associated with significant additional expenses in terms of time, logistics and costs compared with SI, it is currently not recommended for widespread routine use.

Key words: high-volume-insemination, pregnancy rates, flushing results

Spatial and temporal distribution of the ruminant syncytin-like Berv-K1 in the bovine placenta and possible presence in placental extracellular vesicles

Räumliche und zeitliche Verteilung von dem Syncytin-ähnlichen Berv-K1 in der bovinen Plazenta und seine Präsenz in plazentären EVs

J. Galli, C. Almiñana, K. Klisch

Veterinär-Anatomisches Institut, Vetsuisse Fakultät, Universität Zürich, Switzerland

Syncytin-1, an endogenous retroviral envelope protein in the human placenta, is incorporated into extracellular vesicles (EVs) released from the syncytiotrophoblast and is involved in the modulation of the maternal immune system [1]. In bovine, it is unknown whether the analogous bovine endogenous retroviral envelope protein K1 (Berv-K1), mainly expressed in binucleate trophoblast giant cells (TGCs) of placentomes, might have a similar function via EVs. Therefore, we examined the spatial distribution of Berv-K1 in the bovine placenta and its presence in EVs deriving from the bovine placenta. EVs were isolated from bovine caruncle tissue and characterized using immunoblotting, transmission electron microscopy, nanoparticle tracking analysis and mass spectro­metry analysis. Immunohistochemistry and immunofluorescence were used to detect the expression pattern of Berv-K1 in placental tissue. A combination of laser capture microdissection and semiquantitative PCR confirmed a stronger expression of Berv-K1 in TGCs around the stem villi, compared to terminal villi, across gestation. Immunostaining revealed the expression of Berv-K1 in both, stem villi and terminal villi in prepartal and partal placentae. All methods used to charac­terize EVs support their presence in the bovine placental isolates. Besides, Berv-K1 was detected in EVs by mass spectrometry analysis. In conclusion, this study showed the spatial and temporal expression of Berv-K1 across gestation, with a differential profile between stem villi and terminal villi. Moreover, our results indicated that Berv-K1 is likely incorporated into placental EVs, suggesting a function at the feto-maternal interface.

Key words: Berv-K1, extracellular vesicles, bovine placenta

Reference:

1. Tolosa JM, et al. The endogenous retroviral envelope protein syncytin-1 inhibits LPS/PHA-stimulated cytokine responses in human blood and is sorted into placental ­exosomes. Placenta 2013; 33: 933–41.

Transcriptomic effects of moderate and severe hypoxia on MA-10 murine Leydig cells

Transkriptomische Auswirkungen ­moderater und schwerer Hypoxie auf murine MA-10-Leydig-Zellen

L. A. B. Gomes, M. P. Kowalewski

Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland

The Leydig cells are located in an environment that manages the demands of spermatogenesis under relatively low blood pressure, acting at the brink of hypoxia. They abundantly express HIF1?, a transcription factor that is a major player in the cellular response to hypoxia. Its stabilization was shown as an important regulator of cAMP- and STAR-dependent steroidogenesis. We investigated the transcriptomic effects (RNA-seq) of cAMP and reduced O? content (20%, 10%, 1%) in immortalized murine MA-10 Leydig cells; HIF1? was functionally blocked by echinomycin. Gene ontologies and functional terms were evaluated in differently expressed genes (P < 0.05, FDR < 0.01). Under 20% O?, the cAMP-treatment was positively associated with cholesterol metabolism, steroidogenesis, protein modification, cell migration, sprouting and angiogenesis, and cellular response to peptide hormone stimulus, and included top scored genes like Star, Akr1b7, Sik1, Nr4a1/Nur77, Cyp11b1 and Fos. Hypoxia modulated the response to cAMP; under 10% O? a positive association was found for cell proliferation and high DNA activity (replication, metabolism and repair) and protein deubiquitina­tion. Being associated with diminished steroidogenesis, 1% O? negatively affected terms related to mitochondrial function and cell cycle regulation. The withdrawal of HIF1? inhibited genes related to translation, response to DNA stimuli and metabolic process, and involved in regulation of cellular response to stress. The cellular and metabolic effects observed in cAMP stimulated Leydig cells seem to be strongly related to the O? content and HIF1? activity.

Key words: Leydig cells, transcriptomics, hypoxia

Vitrification of bovine in vitro produced embryos using a semi-automated instrument

Vitrifikation boviner in vitro produzierter Embryonen mit einem halb-automatischen Gerät

L. A. González-Grajales1, T. T. Peura2, E. Hasenpusch1

1Phönix Repro, GmbH, Bernau bei Berlin, Germany; 2Genea Biomedx, Sydney, NSW, Australia

The use of automation to equilibrate embryos prior to vitrification offers an opportunity to improve accuracy and efficiency. The objectives of this study were: 1) to determine survival and hatching rates after warming between bovine embryos cryopreserved by either slow-freezing methods or vitrification thorough a semi-automated closed system (GAVI®), and 2) to identify differences in survival rates between embryos classified as grade 1 and 2 prior to vitrification. A total of 196 embryos derived from oocytes collected from slaughterhouse ovaries were produced. Different developmental stages including early blastocyst to hatched embryos were selected for slow- freezing in 1.5 M ethylene glycol or vitrification with the GAVI® system according to manufacturer’s instructions. After warming, embryos were cultured individually at 38 °C, 5% CO? and 6% O?. We found no differences in the proportion of grade 1 and 2 embryos cryopreserved by slow-freezing or vitrification at 24 h post warming. However, a higher proportion of embryos (P < 0.01) from the vitrified group survived with good/fair quality until 36 h post warming when compared to slow-freezing. Differences in the proportion of hatched embryos at 24 or 36 h post warming between the two groups were not found. Embryos classified as grade 1 prior to vitrification showed a higher proportion (P < 0.01) of survival at 24 or 36 h post warming than grade 2 embryos.

In conclusion, vitrified embryos had similar developmental competence and extended survival rates compared to slow-frozen embryos. This is the first report on the use of a semi-automated system to vitrify bovine embryos.

Key words: vitrification, slow freezing, automation

Lactobacillus-conditioned medium supplemented during in vitro maturation of bovine oocytes helps to overcome the negative LPS effect

Laktobazillen-konditioniertes Medium während der In-vitro-Maturation boviner Oozyten hilft, den negativen LPS-Effekt zu überwinden

O. Granacher1, C. Gabler2, C. Wrenzycki1

1Chair for Molecular Reproductive Medicine, Clinic for Veterinary Obstetrics, Gynecology and Andrology, Faculty of ­Veterinary Medicine, Justus-Liebig-University Giessen, ­Germany; 2Institute of Veterinary Biochemistry, Department of Veterinary Medicine, Free University of Berlin, Germany

Lipopolysaccharide (LPS) has been detected in the follicular fluid of cows with infections. LPS administered during in vitro maturation (IVM) of bovine oocytes impairs their developmental competence; whereas, Lactobacillus (L.) spp. have been associated with increased oocyte quality when in follicular fluid. The aim of this study was to determine whether the negative effect of LPS could be overcome via L.-conditioned medium. Cumulus-­oocyte-complexes from slaughterhouse ovaries were isolated via slicing. Five % of the IVM medium (5 : 95) were replaced by L.-buchneri-conditioned medium (cond.; group 1), LPS was supplemented during IVM at a concentration of 10 µg/mL (group 2). In a third group, a combination of both supplements (combi) was employed. As control, standard IVM was performed. After 24 h of maturation, maturation rates were determined via Hoechst staining. IVF and IVC was the same for all IVM groups. Cleavage/developmental rates were recorded at day 7 (day 0 = IVF) via morphological microscopic evaluation. Maturation rate was significantly reduced in oocytes of the LPS group compared to all other groups (LPS: 51.2±3.6; cond.: 82.4±9.8; combi: 77.1 ± 4.2; control: 83.9 ± 13.0). No differences were detectable for cleavage rates. The developmental rates were significantly increased for embryos cultured in the combination of both supplements at day 7 and day 8 (day 7: LPS: 17.3 ± 5.9; cond.: 23.3 ± 4.6; combi: 30.3 ± 10.5; control: 22.9 ± 6.3; day 8: LPS: 21.2 ± 6.8; cond.: 26.9 ± 3.8; combi: 34.4 ± 9.5; control: 26.1 ± 6.8). These results suggest that L. buchneri conditioned medium helps to overcome the negative LPS effect.

Key words: in vitro maturation of bovine oocytes, LPS, Lactobacillus-conditioned medium

Artiodactyla, but not other mammals, have multiple copies of the aromatase gene

Artiodactyla, nicht aber andere Säugetiere, besitzen mehrere Kopien des Aromatase-Gens

J. Günther, R. Fürbass

Institut für Fortpflanzungsbiologie, Forschungsinstitut für Nutztierbiologie (FBN), Dummerstorf, Germany

Aromatase cytochrome P450 catalyzes the final step of estrogen biosynthesis, the conversion of testosterone and androstenedione into estradiol and estrone, respectively. After long assuming that a single CYP19A1 gene encodes aromatase in mammals, three functional CYP19 paralogs were first detected in the pig that are expressed in a tissue-specific manner. However, systematic analyses of paralogous CYP19 loci in other species were pending.

In this work, we screened genomic DNA sequences available in the NCBI database from species across the vertebrate clade, from non-teleost fish to mammals, and identified multiple copies of CYP19 only in artiodactyls, which include pigs, camels, whales, and ruminants. The number of CYP19 genes varies among taxa, with pigs having 3, camels 2, whales up to 3, and ruminants 2. The bovine and ovine CYP19 copies became pseudogenes. We also examined DNA sequences of putative core promoters for TF binding motifs. While promoters of the primordial CYP19 genes in all artiodactyl taxa contain highly conserved TF binding motifs (e.g. GATA, USF, SF1, and CRE), this is not the case for the promoters of the other CYP19 paralogs.

Conclusions:

1. Artiodactyla, which include some of the major livestock species, are unique in having multiple CYP19 paralogs.

2. This may have led to the evolution of novel aromatase isoforms.

3. The promoters of CYP19 paralogs have apparently adapted to different requirements of tissue-specific expression during ~70 million years of evolution of Artiodactyla.

Key words: evolution, CYP19 paralogs, transcription factor (TF) binding motifs

Radiological evaluation of extra genital endometriosis: four case ­series

Radiologische Bewertung der extra­genitalen Endometriose: Vier Fälle

L. Hacioglu1, E. Karaman2, S. Sendag3,4, A. Wehrend4

1SBÜ Training and Research Hospital Van, Turkey; 2Clinic for Obstetrics and Gynecology, Faculty of Medicine, Van YY-University, Van, Turkey; 3Clinic for Veterinary Obstetrics and ­Gynecology, Van YY- University, Van, Turkey; 4Clinic for Obstetrics, Gynecology, and Andrology of Small and Large Animals with an Ambulatory Service of Justus-Liebig-University, Giessen, Germany

Endometriosis is the development of endometrial tissue outside the uterine cavity. Ectopic endometrial tissue is usually located in the pelvis, but rarely it can be seen in all body organs such as the lung, ureter, brain, and abdominal wall. The majority of extrapelvic endometriosis cases are scar endometriosis after hysterectomy, cesarean section, and episiotomy. We aimed to report four cases who came to our clinic after developing a mass in the incision area after a cesarean section and were radiologically characterized as endometriosis. In our clinic, 4 patients aged between 22–38 years with a history of cesarean section were analyzed. The patient experienced a painful palpable mass on the anterior abdominal wall‘s old incision line. In the first radiological evaluations, such formations were diagnosed as endometriosis. Following preliminary diagnostics, superficial tissue ultrasonography (USG) and pelvic MRI (Magnetic Resonance Imaging) were performed. After pathological analysis of the samples post-surgical excision, the final diagnosis was reported as endometriosis. In conclusion, in patients who have a history of cesarean section, cyclic pain and swelling at the incision site should be fully examined, and endometriosis should not be overlooked in the differential diagnosis. Based on these cases, we may conclude that, while radiography makes it difficult to identify endometriosis from other masses, clinical history and other imaging findings (USG, MRI) can be helpful. The pathology results after surgical excision of the mass can be used to confirm a definitive diagnosis of endometriosis.

Key words: ultrasonography, pelvic MRI, caesaren section

Effect of vibration emissions on boar sperm quality during shipping: interaction of vibration intensity, transport and storage time

Auswirkung von Erschütterungsemissionen auf die Qualität von Eber­sperma während des Transports: Interaktion von Erschütterungsintensität, Transport- und Lagerzeit

T. Hafemeister1, P. Schulze2, C. Simmet3, M. Jung1, F. Fuchs-Kittowski2, M. Schulze1

1Institute for Reproduction of Farm Animals Schönow, ­Bernau, Germany; 2University of Applied Science, Berlin, Germany; 3Minitüb GmbH, Tiefenbach, Germany

Vibration emissions during the transport of boar semen for artificial insemination affect sperm quality. In the present study, the interactions of vibrations (displacement index (Di) = 0.5–6.0), duration of transport (0–12 h) and storage time (day 1–4) were investigated. Normospermic ejaculates were collected from 39 fertile Pietrain boars (aged 18.6 ± 4.5 months) and diluted in one-step with an isothermic (32 °C) BTS (Minitüb) extender (n = 546 samples). Sperm concentration was adjusted to 22×10? sperm/ml. Extended semen (85 ± 1 ml) was filled in 95 ml QuickTip Flexitubes (Minitüb). For transport simulation on day 0, a laboratory shaker IKA MTS 4 was used. Total sperm motility (TSM) on day 1–4 was evaluated using the CASA system AndroVision (Minitüb). Thermo-resistance test (TRT), mitochondrial activity (MITO) and plasma membrane integrity (PMI) were assessed on day 4. Sperm quality dropped with increasing vibration intensity and transport duration, enhanced by a longer storage time. A linear regression was performed using a mixed model accounting the boar as random effect. The interaction between Di and transport duration significantly (p < 0.001) explained data for TSM (–0.30 ± 0.02%), TRT300min (–0.39 ± 0.06%), MITO (–0.45 ± 0.06%) and PMI (–0.43 ± 0.05%). Additionally, TSM will decrease by 0.66 ± 0.08% with each day of storage (p < 0.001). It can be concluded that sperm doses extended in BTS should be transported carefully. If this is not possible or if the doses are transported a long way, the storage time should be reduced to a minimum.

Grants: Funded by BMWK (16KNO77341).

Key words: Artificial insemination; Boar semen transport; Vibrations

Impact of maternal age on the expression of the anti-oxidative and anti-glycative enzymes in human uterus

Einfluss des mütterlichen Alters auf die Expression der antioxidativen und antiglykativen Enzyme in humanem Uterusgewebe

E. Halbauer1, J. de Nivelle1, M. Buske1, G. Seliger2,
A. Navarrete Santos1, M. Schindler1

1Department of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg, Halle (Saale); 2Centre for Reproductive Medicine and Andrology, Halle University Hospital, Halle (Saale), Germany

Advanced maternal age is associated with adverse pregnancy outcomes and complications for the developing embryo and the mother. In part, this is related to an increase in metabolic and oxidative stress, which can be reduced by anti-oxidative and anti-glycative enzymes. This study characterises anti-oxidative and anti-glycative enzymes in the uterus at secretory and proliferative phase of women at young (YMA, 25–29 years), intermediate (IMA: 30–34) and advanced (AMA, 35–45 years) maternal ages. Immunohistochemistry was used to analyse the localisation of glyoxalase 1 (GLO1), receptor for advanced glycation end products (RAGE), heat shock protein 70 (HSP70), superoxide dismutase 2 (SOD2), and glutathione peroxidase 1/2 (GPX1/2) in women with AMA and IMA compared to YMA women.

All anti-oxidative and anti-glycatic enzymes studied were present in the human uterus depending on the female age. In the endometrium, GLO1, HSP70, SOD2 and GPX1/2 showed a prominent staining in the apical epithelium and glandular tubes. AMA and IMA showed increased GLO1, HSP70 and GPX1/2 staining predominantly in the apical epithelium of the endometrium. Furthermore, the smooth muscle cells of the myometrium expressed all stress-related enzymes, with the most intense staining for RAGE, GLO1 and HSP70 compared to the endometrium.

Our results proved that stress defence molecules were present in the human uterus. The upregulation of GLO1, HSP70 and GPX1/2 indicate an adaptive reaction to maternal ageing, especially the endometrium.

Key words: reproduction, ageing, stress defence

Glucose and calcium concentrations in cats with dystocia: a retrospective analysis

Glukose- und Kalziumkonzentrationen bei Katzen mit Dystokie: eine retro­spektive Analyse

D. Hardegen1, S. Sendag1,2, A. Wehrend1

1Clinic for obstetrics, gynecology, and andrology of small and large animals with an ambulatory service of Justus-Liebig-University, Giessen, Germany; 2Clinic for veterinary obstetrics and gynecology, Van YYÜ- University, Van, Turkey

There is currently no comparable examination of biochemical measured values in feline dystocia patients in the literature. The current study collected information on glucose and ionized calcium concentrations in the blood of cats with dystocia for the first time because hyoglycemia and hypocalcemia are associat­ed with uterine inertia in the literature. The blood gas monitor Radiometer Copenhagen ABL 800 Basic was used to measure glucose and calcium. Glucose was measured in 35 cats. 51% of the cats (n = 18, reference range: 3.1–6.9 mmol) showed normoglycemia. One of the cats was hypoglycaemic (3%). This cat also showed no uterine inertia. Hyperglycemia characterized 46% (n = 16) of the animals. Cats with hyperglycemia (46%, n = 16) were more common in the group with dead kittens. Ionized calcium was measured in 32 cats. 19% of the cats showed hypocalcemia (1.03–1.18 mmol/l). The rest showed normocalcemia (47%). Only two cats with evident hypocalcemia developed uterine inertia. Finally, hypoglycemia and hypocalcemia do not appear to have a significant role in the development of uterine inertia in cats.

Key words: uterus, hypoglycemia, hypocalcemia

Hypoxia regulates protein abundance and inflammatory factors in human granulosa cells

Hypoxie reguliert Proteinabundanz und inflammatorische Faktoren in humanen Granulosazellen

M. Höfner1, K. Eubler1, C. Herrmann1, U. Berg3, D. Berg3, A. Imhof2, I. Forné2, A. Mayerhofer1

1Biomedical Center Munich (BMC), Cell Biology, Anatomy III, Ludwig-Maximilian-University (LMU), Planegg-Martins­ried; 2BMC, Protein Analysis Unit, LMU, Planegg-Martins­ried; 3Fertility Centre A.R.T., Bogenhausen, Munich, ­Germany

Human granulosa cells (hGCs), obtained during In-vitro-Fertilization-Treatment, are often used as a cellular model for the study of human ovarian function. In most experiments, these cells are cultured under atmo­spheric oxygen conditions (21% O?). How­ever, the granulosa cell compartment of ovarian follicles is avascular and thus the oxygen concentration in the human ovarian follicle decreases with follicle growth. It is estimated that the oxygen concentration in the human preovulatory follicle ranges between 1–5%. To simulate this situation, we cultured hGCs under hypoxic (1% O?) and atmo­spheric conditions for four days. A proteomic analysis of three pools of hGCs was performed. While distinct differences between the pools of GCs were noted, the abundance of 49 proteins was significantly increased under hypoxia in all samples, whereas that of 204 proteins was decreased. According to GO term analysis, pathways associated with metabolic processes (for example aminoacid-catabolic-processes and glycolysis), mitochondrial protein biosynthesis and steroid biosynthesis were altered in hypoxia. Furthermore, hypoxia promoted increased levels of inflammatory cytokines IL-6 and IL-8, as demonstrated by ELISA assays performed in seven pools of hGCs. Our results reveal that hypoxic conditions are strong regulators of hGCs and thus oxygen concentrations must be taken into account when planning and interpreting cellular experiments.

Grants: Supported in part by DFG, project number 491030536 and 245169951

Key words: ovary, hypoxia, inflammation

Change of progesterone receptor expression during luteinisation of granulosa cells of domestic cat in vitro

Veränderung des Progesteronrezeptors während der In-vitro-Luteinisierung von Granulosazellen in der Hauskatze

M. M. Hryciuk, B. C. Braun

Leibniz Institute for Zoo and Wildlife Research, Department Reproduction Biology, Berlin, Germany

In the present study, we have investigated the potential receptivity of granulosa and luteinized cells from domestic cat towards progesterone (P4) during long term culture (n = 3). Granulosa cells (GCs) were cultured up to 18 days and the culture period was divided into two phases. The maintenance phase lasted 7 days, was done in 2D and was followed by a luteinisation phase for 11 days in ultralow attachment plates to induce spheroid formation. A strong increase of P4 production during luteinisation phase hinted to a successful luteinisation. Gene expression was measured for progesterone receptor (PGR) and progesterone receptor membrane components (PGRMC) 1 and 2. Furthermore, the protein expression for PGRMC1 was analysed by immunohistochemistry on a spheroid of luteinisation phase d7, and on ovaries of luteal and follicular phase for comparison. During the maintenance phase, gene expression was constant for PGR and PGMC1; PGRMC2 showed a decrease towards d7. The expression for all three genes was increasing during luteinisa­tion phase but with different patterns. PGRMC1 expression was strongly increased already at d2, whereas PGR and PGRMC2 showed that on d7. Immunohistochemical signals for PGRMC1 were strong for theca cells but weak for GCs. Luteal cells on ovaries were variously stained, some of them had a stronger signal than that of GCs. This was also observed for selected cells of the spheroid. We conclude from our results that the potential for reception towards progesterone is increasing during luteinisation in vitro.

Key words: granulosa cells, luteinisation, progesterone receptors

Host range determination and classification of bacteriophages specific to equine genital pathogens

Bestimmung des Wirtsspektrums und Klassifizierung von Bakteriophagen gegen spezifische equine Genitalpathogene

R. Hüsch1, M. Köhne1, S. Kittler2, M. Plötz2, H. Sieme1

1Unit for Reproductive Medicine – Clinic for Horses, University of Veterinary Medicine, Foundation, Hannover; 2Institute for Food Quality and Food Safety, University of Veterinary Medicine, Foundation, Hannover, Germany

The problem of increasing bacterial resistances towards antimicrobial drugs drives the search for alternatives to antibiotics. Bacterial infections of the uterus are among the major problems in equine reproduction. Due to their antibacterial capacities, bacteriophages are an alternative treatment option for bacterial infections. The aim of the study was to determine the respective host range of isolated bacteriophages specific to the most important equine genital pathogens (Streptococcus [Sc.]equi ssp. zooepidemicus, Klebsiella [Kl.] pneumoniae and Pseudomonas [Ps.] aeruginosa) and to characterize their morphology by electron microscopy. For determination of their host range the isolated bacteriophages (specific to Sc. equi spp. zooepidemicus [n = 13], Kl. pneumoniae [n = 6], and Ps. aeruginosa [n = 9]) were inoculated with bacterial clinical field isolates provided by two diagnostic laboratories (Sc. equi ssp. zooepidemicus [n = 37], Kl. pneumoniae [n = 26], and Ps. aeruginosa [n = 33]) and plaque formation was observed in 32/37 Streptococcus-, 16/26 Klebsiella-, and 14/33 Pseudomonas-isolates. Bacteriophages of Kl. pneumoniae and Ps. aeruginosa showed a smaller host range, whereas one single bacteriophage was able to form plaques on 30/37 of all tested Streptococcus-isolates. For charac­terizing their morphology, electron microscopy was performed. Preliminary results indicate that the newly isolated bacteriophages belong to the family Myoviridae.

In conclusion, the isolated bacteriophages specific for equine genital pathogens are promising candidates for further investigations in different in vitro and ex vivo models.

Key words: bacteriophages, host range, equine endometritis

The effect of oxytocin stimulation on canine myometrial contractions in the organ bath

Der Effekt von Oxytocin auf myometriale Kontraktionen des Hundes im ­Organbad

C. Jungmann1, G. Mazzuoli-Weber2, S.Goericke-Pesch1

1Unit for Reproductive Medicine – Clinic for Small Animals, 2Institute for Physiology and Cell Biology, University of ­Veterinary Medicine Hannover, Foundation, Hannover, ­Germany

Oxytocin is the most common medication given in case of dystocia. In the dog, response to oxytocin in case of uterine inertia is usually poor, indicating the need for caesarean section. Uterine interplacental tissue samples of 19 bitches were microscopically dissected, separating circular (CML) and longitudinal (LML) myometrial layers to analyse myometrial contractions in the organ bath. After equilibration, strips of each layer were stimulated with the same oxytocin concentration (1 nM, 10 nM, 100 nM) twice with a 20-minutes wash out in between. One untreated CML/LML strip each served as control. Contractions were recorded and evaluated for average amplitude (A), mean force (MF) and frequency (F). Results were statistically analysed for each stimulation separately using GraphPad Prism. Effects of different oxytocin concentrations were analysed and compared within layers, as well as between both layers (CML/LML). During the first stimulation, unlike LML, all oxytocin concentrations caused a significant increase in A, MF and F (all P <0.05) in CML compared to the control. Whereas no significant differences between oxytocin and controls were identified during the second stimulation, 10 nM and 100 nM caused significantly lower A, MF and F (all P < 0.05) in LML compared to 1 nM. Comparing the response of the layers, 100 nM and 10 nM caused a significantly reduced F (all P < 0.01) and significantly decreased A, MF, and F (all P < 0.001) in LML during the first and second stimulation, respectively. In summary, data indicate differences in functionality and oxytocin response of both myometrial layers.

Key words: oxytocin, canine uterine inertia, contractions

A multi-faceted approach to differentiation of male germ cells from primates

Ein vielschichtiger Ansatz zur Differenzierung männlicher Keimzellen von Primaten

P. W. Kibui, R. Sandhowe-Klaverkamp, J. Wistuba, S. Schlatt

Centrum für Reproduktionsmedizin und Andrologie, Medizinische Fakultät, Westfälische Wilhelms-Universität Münster, Germany

Recently, single-cell RNA sequencing un­ravelled a heterogenous spermatogonial stem cells (SSCs) compartment. In humans, the various spermatogonial states and the true SSCs remain to be elucidated due to limited tissue availability and ethical concerns. Using a multi-faceted ex-vivo approach, we evaluate spermatogenic progression and restoration in primates. We propose that either a specific subset of primitive SSCs modulates spermatogenic recovery following gonadotoxic injury or that the SSC niche is highly plastic. SSCs are very sensitive to gonadotoxic therapies except reserve SSCs that can survive. Thus, using radiation we induce damage likely to occur in (im)mature testis to study the poorly understood processes of germ cell loss. Organ culture (static and microfluidic) and xenografting of marmoset testicular tissues (MTT) in nude mice are powerful experimental models enabling us to evaluate the short and long term effects of radiation on SSC niche, respectively. Organ culture system successfully maintained MTT integrity upto 8 days while xenografting maintained MTT for about 6 months with 67% recovery. Our preliminary data indicate heterogenous spermatogonial states and SSCs populations change post-irradiation. Whole-mount imaging offers great insights into kinetics of SSC proliferation and clonal expansion with high potential for spermatogenic recovery. Novel findings from this study will improve our understanding of the fundamental SSC biology and regulation with a direct clinical relevance to fertility preservation/restoration in humans, livestock and conservation.

Key words: spermatgonial stem cells, marmoset, spermatogenic recovery

Evaluation of serum anti-Mullerian hormone concentrations following treatment with vitamin D in dairy heifers

Verlauf der Konzentration des Anti-Müller-Hormons im Serum nach einer Behandlung mit Vitamin D bei Milchfärsen

D. Koca1, Y. Nak2, S. Sendag1,4, D. Nak2, T. Arslan3, A. Wehrend4

1Clinic for Veterinary Obstetrics and Gynecology, Van YY- University, Van, Turkey; 2Clinic for Veterinary Obstetrics and Gynecology, Uludag-University, Bursa, Turkey; 3Department of Econometrics, Van YY-University, Van, Turkey; 4Clinic for Obstetrics, Gynecology, and Andrology of small and large animals with an Ambulatory Service of Justus-Liebig-University, Giessen, Germany

Anti-Müllerian hormone (AMH) levels in the blood are positively associated with number of antral follicles, ovarian function, and fertility. Numerous studies, particularly in human medicine, have shown that vitamin D (VD) has a positive effect on AMH levels, both directly and indirectly. To our knowledge, the effects of VD on serum AMH levels in heifers have not been investigated. Therefore, the current study aimed to investigate the following question: Is VD treatment associated with increased serum AMH concentrations in dairy heifers? The study included twenty healthy non-pregnant dairy Holstein ­heifers (11–13 months old) weighing an average of 319 kg (288–341 kg). The animals did not have direct contact with sunlight during the study because there were no open areas in the facility. In other standard rations (NRC) no vitamins or other additional nutrients were supplied to the animals. All heifers received a single intramuscular dosage of 5 ml (5 million IU) VD (Provet-D3®, Provet, Istanbul-Turkey). Blood samples were collected from the tail veins of all animals before (day 0) and after (days 7, 14, and 28) VD injection. The electrochemiluminescence immunoassay methodology was used to measure AMH. Comparison results showed that the AMH concentrations in the t0, t7, t14, and t28 days were not statistically different from each other’s (p = 0.10). Also, it was seen that there were no statistically significant correlations between the pairs (age-AMH) and (weight-AMH) in the t0 day, and (VD level-AMH) for all days. Grants: These findings demonstrated that exogenous VD did not affect on AMH in heifers.

Key words: AMH, vitamins, fertility

Expression of hormone receptors, PTGS2 and KI-67 in canine vaginal tumors

Expression von Hormonrezeptoren, PTGS2 und KI-67 in Vaginaltumoren der Hündin

H. Körber1, N. Chudigiewitsch1, E. M. Packeiser1, A. Beineke2, S. Goericke-Pesch1

1Unit for Reproductive Medicine – Clinic for Small Animals, 2Institute for Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany

Biomarkers are often used in diagnostics, deciding on possible treatment options and assessing the prognosis of tumors. However, little is known about the characterization of canine vaginal tumors (CVT). Therefore, we investigated the expression of estrogen receptor alpha (ESR1), progesterone receptor (PGR), prostaglandin-endoperoxide synthase 2 (PTGS2) and KI-67 in CVT by RT-qPCR and immunohistochemistry. Formalin-fixed, paraffine-embedded samples classified histologically (hematoxylin eosin and azan staining) and immunohistochemically (alpha-smooth-muscle-actin) as leiomyoma (LM), leiomyosarcoma (LMS), fibroma or polyp were included. RNA from tumor tissue only was extracted and RT-qPCR was performed using primers against ESR1, PR and PTGS2. Whereas mRNA expression of ESR1 and PTGS2 did not differ significantly between LM (n = 7), LMS (n = 7), fibroma (n = 6) and polyp (n = 10), PGR expression did (Kruskal-Wallis, p < 0.05) with expression being significantly higher in fibromas than in LMS (Dunn‘s multiple comparisons test, p < 0.05). (Semi-) quantitative evaluation of protein expression revealed significant differences between groups (LM: n = 12, LMS: n = 11, fibroma: n = 16, polyp: n = 14) for ESR1, PTGS2 and KI-67 (Kruskal-Wallis, all p < 0.0001), but not PR. LMS was characterized by highest KI-67 (p < 0.01) and lowest ESR1 expression (p < 0.01) compared to all other CVT. Besides, KI-67 expression was higher in LMs than in fibromas (p < 0.05). LMS and LM had more PTGS2 staining compared to fibroma (p < 0.001) and polyps (p < 0.0001). Our results provide a first step for a better characterization of CVT, but further research is required.

Key words: tumor characterization, dog, vagina

Utilizing mass spectrometry to identify proteomic changes in human sperm with abnormal morphology and motility

Nutzung der Massenspektrometrie zur Identifizierung proteomischer Veränderungen in menschlichen Spermien mit abnormaler Morphologie und Motilität

S. D. Kothalawala1,5, T. Timm2, G. Lochnit2, H.-C. Schuppe3, A. Pilatz3, F. Wagenlehner3, S. Kliesch4, L. O’Donnell5, K. Loveland5, D. Fietz1

1Institute for Veterinary Anatomy, Histology and Embryo­logy, Justus-Liebig-University Giessen; 2Protein Analytics, Biochemistry Institute, Justus-Liebig-University Giessen; 3Clinic of Urology, Pediatric Urology and Andrology, University Clinic Giessen-Marburg, Giessen; 4Centre of Reproductive Medicine and Andrology, University of Muenster, ­Muenster, Germany; 5Center for Reproductive Health, ­Hudson Institute of Medical Research, & Dept. of Molecular & Translational Sciences, Monash University, Clayton, Australia

Defects in sperm morphology and motility are commonly associated with male infertility yet the mechanisms underlying these disorders are not well understood. This study aimed to identify differentially expressed proteins in human spermatozoa with impaired morphology and motility compared to normal sperm. Human ejaculates classified as normozoospermia (NORM, n = 3) and defective morphology and motility (astheno-teratozoospermia [AT], n = 3) were subjected to mass spectrometry and the proteomes quantified and compared. 36 proteins were significantly altered in AT compared to NORM and were further characterized for their role in human sperm. The results showed that an epididymal-specific protein, ELSPBP1, was increased in AT sperm, suggesting its uptake in the epididymis is dependent on sperm phenotype. Testis-derived proteins ACTRT2, CCDC105, IFT57 and IPO4 were immunolocalized in biopsies from fertile and infertile men, and their role in spermiogenesis was confirmed. IPO4 is a nuclear-cytoplasmic transport protein and dual-label immunofluorescence revealed a stage-specific association of IPO4 and its putative cargo protein ASFT1B during human spermiogenesis. Changes in the protein levels of ACTRT2, IFT57, and ELSPBP1 in fertile vs infertile sperm were evaluated using Western Blots. In conclusion, we have identified proteins that could play important functional roles in human sperm and who’s altered expression is related to phenotypic abnormalities in men with astheno-teratozoospermia. Funded by DFG IRTG 1871/1.

Key words: mass spectrometry, astheno-teratozoospermia, spermiogenesis

Effect of the developmental environment on mitochondrial respiration characteristics of bovine cryopreserved blastocysts

Einfluss der Entwicklungsumgebung auf die mitochondriale Respiration von kryokonservierten Rinderblastozysten

J. Kurzella1, F. Rings1, D. Salilew-Wondim2, C. Blaschka2, E. Tholen1, M. Hoelker2

1Institute of Animal Science, Animal Breeding, University of Bonn, Bonn; 2Department of Animal Science, Biotechnology & Reproduction in farm animals, University of Goettingen, Goettingen, Germany

The aim of the present study was to compare cryopreserved embryos from different developmental environments (in vivo vs. in vitro) with respect to their mitochondrial metabolic activity. For this purpose, slow frozen in vitro derived expanded day 7 bovine blastocysts (VITRO) generated by routine procedures (SOFaa + 5% ÖCS, 5% CO? & 5% O?, 38.8 °C) were compared with slow frozen in vivo derived expanded day 7 blastocysts (VIVO). Metabolic analysis was first conducted on re-expanded (REE) embryos 4 hours after thawing (pools of 7, 6 replicates) with an extracellular FLUX analyzer taking advantage of a Cell-Mito Stress Test Kit (Seahorse XFp, Agilent). The results revealed a similar mitochondrial respiration between frozen-thawed VIVO-REE and VITRO-REE embryos (76% vs. 85.8%). However, there was a significant difference (p < 0.003, unpaired t-test) ­between VIVO-REE and VITRO-REE embryos in terms of relative spare capacity (340.2% vs. 241.5%) while ATP coupling efficiency remained similar between VIVO-REE and VITRO-REE embryos (78.31% vs. 73.80%). Secondly, metabolic profiling of embryos still collapsed (COL) 4 hours (pools of 15.3 replicates) after thawing did not reveal differences between VITRO-COL and ­VIVO-COL. In contrast, total oxygen consumption differed significantly (p < 0.05) ­between VIVO-REE and VIVO-COL (100% vs. 66.3%) and ­VITRO-REE and VITRO-COL (100% vs. 54.6%).

Collectively, our study showed that reexpanded embryos showed higher oxygen consumption rates compared to collapsed embryos with VITRO-REE embryos showing higher spare capacities than VIVO-REE embryos.

Key words: cryoconservation; vivo vs vitro embryo metabolism; extracellular FLUX analysis

Vaginal flora of the bitch: a retrospective data analysis

Vaginalflora der Hündin

A. Leps1, B. Klein2, C. Meyer3, C. Simon4, S. Goericke-Pesch1

1Unit for Reproductive Medicine – Clinic for Small Animals, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany; 2LABOKLIN GmbH & Co. KG, Bad Kissingen, Germany; 3SYNLAB.vet GmbH, Augsburg, Germany; 4Biocontrol, Biosciencia Healthcare GmbH, Ingelheim, ­Germany

Knowledge of the physiological vaginal flora of the bitch is crucial to reduce the prophylactic (over-)use of antibiotics before mating. The vaginal flora of the bitch was already described previously in several studies, usually from small groups of dogs. This study describes a large cohort for the first time. Vaginal swabs submitted to 3 commercial la­boratories for bacteriological analysis between 2015 and 2021 were analyzed retrospectively. Swabs were cultured for aerobic bacteria and identified by the growth on selective agar plates, biochemical parameters and the use of MALDI-TOF-MS. A total of 22074 samples were analyzed and 351 bacterial species were identified. In the majority of samples, a colonization with 2 or more isolates was found; monoculture was observed, but less common. The most frequently identified isolates were Escherichia coli, Staphylococcus pseudintermedius, ?-hemolytic (hem.) streptococci, Pasteurella spp. and ?-hem. streptococci. Besides those, also typical nosocomial pathogens, such as e. g. Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus spp., were identified in some vaginal samples. The results confirm earlier studies about the bacterial species identified in vaginal swabs, however, for the first time on a large population. Besides, typical nosocomial pathogens known to cause hospital associated infections were found indicating a possible role of dogs as hosts for multi-resistant pathogens. A major limitation is the lack of details, namely whether samples were collected from healthy or diseased animals. Further studies should include these aspects as well as corresponding fertility to gain further insights.

Key words: vaginal flora, dog, microbiology

The Dummerstorf high-fertility mouse line 1 – a worldwide unique model for increased female reproductive performance

Die Dummerstorfer Fruchtbarkeitsmauslinie 1 – ein weltweit einzigartiges Modell für erhöhte weibliche Fruchtbarkeit

C. L. M. Ludwig1, S. Bohleber2, R. Lapp1, A. Rebl1,
E. K. Wirth3,4, M. Langhammer1, U. Schweizer2, J. M. Weitzel1, M. Michaelis1

1Research Institute for Farm Animal Biology (FBN), Dummerstorf; 2Institut für Biochemie und Molekularbiologie (IBMB), Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn; 3Charité – Universitätsmedizin Berlin, Berlin; 4DZHK - German Centre for Cardiovascular Research, Berlin, ­Germany

During recent decades, several mouse models provided insights into the regulation of folliculogenesis. In contrast to the commonly used transgenic or knockout mouse models, the Dummerstorf high-fertility mouse line 1 (FL1) is a worldwide unique selection experiment for increased female reproductive performance and high fertility. FL1 mice almost doubled the number of ovulated ­oocytes compared to the unselected control mouse line. FL1 females are characterized by various alterations on endocrine and molecular levels, which have the potential to improve follicular development [1]. To gain insights into the cellular mechanisms leading to the high fertility phenotype, we used granulosa cells isolated from antral follicles for mRNA sequencing. Based on the results of the transcriptome analysis we measured hormones and growth factors associated with follicular development. While IGF1 levels are decreased in FL1 mice in estrus, we found no differences in Insulin, Prolactin and Oxytocin levels in FL1 mice compared to the control mouse line. Our results indicate that the crosstalk between different intracellular signaling pathways in granulosa cells is improved, follicular atresia in FL1 is decreased due to enhanced granulosa cell survival and by improving the efficiency of intracellular signaling, glucose metabolism and signal transduction, FL1 mice have advantages in reproductive performance. The understanding of these mechanisms and their interplay might be of fundamental interest for the understanding of proper fertility in human.

Key words: high-fertility, ovulation rate, folliculogenesis

Reference:

1. Ludwig CLM, et al. Endocrine and molecular factors of increased female reproductive performance in the Dum­mers­torf high-fertility mouse line FL1. J Mol Endocrinol 2022; 69: 285–98.

Case report: Congenital vulvar hypoplasia with and without secondary azotemia and urinary phlegmon in an adult and newborn female alpaca

Fallbericht: Kongenitale Vulvahypoplasie mit und ohne sekundärer Azotämie und Harnphlegmone in einer erwachsenen und neugeborenen Alpakastute

J. Lüttgenau, L. Bittner, H. Bollwein, U. Bleul

Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Switzerland

Malformation of the vulva is common among congenital abnormalities in alpacas. Interestingly, affected animals do not always become apparent during the neonatal period, since the vulva is often not completely imperforated and allows for low-grade urine outflow. In the first case, a 3-day-old female alpaca cria was presented with no rima vulvae but dripping urination from a <1 mm opening in a bulging of the vulva area. Ultrasonography of this area revealed anechogenic fluid of 1.86 cm vertical extent and a filled urinary bladder but undilated renal pelves. Blood urea was normal (7.5 mmol/L). In a second case, a 17-month-old alpaca came in with the same exterior appearance of the vulva area. Abdominal pressure allowed urination in an extremely narrow jet. Ultrasonography revealed extensive anechogenic fluid in the urinary bladder and uterus, but the renal pelves were not dilated. Blood urea was increased (9.2 mmol/L). Demarcation of the skin due to urinary phlegmon was observed around the bulging and at the inside of the upper thighs. In both cases, surgical vulvoplasty was performed under sedation and local anesthesia. Post-operative care included antibiotic treatment, pain management, daily wound cleaning and control of urination. Whereas the cria was released from the clinic 3 days after surgery, the adult was discharged not until 22 days after surgery due to the long-lasting wound healing of the phlegmonous skin. Although both, the neonate and adult alpaca, recovered from congenital vulvar hypoplasia after vulvoplasty, a standard examination of newborn alpacas for congenital diseases might prevent the secondary emergence of azotemia, urinary phlegmon or kidney damage.

Key words: urinary tract obstruction, vulvar deformity, New World camelids

Influence of different extenders for cryopreservation and equilibration times on sperm quality and ­fertility in Holstein Friesian bulls in Germany

Einfluss unterschiedlicher Verdünnermedien für die Kryokonservierung und Äquilibrierungszeiten auf die Spermaqualität und Fruchtbarkeit von Holstein-Friesen Bullen in Deutschland

T. Meschede1, L. Pieper2, M. Wiebke2, U. Janowitz1, M. Jung2, M. Schulze2

1Rinder-Union West eG, Muenster, Germany; 2Institute for Reproduction of Farm Animals Schoenow, Bernau, Germany

The aim of this study was to evaluate sperm quality and field fertility when comparing BioXcell extender using 4h equilibration time (Control, CON) with Triladyl (TRI) or OptiXcell (OPTX) using 24h equilibration time prior to cryopreservation of bull semen. A total of 60 ejaculates from 8 Holstein bulls (2.8 ± 0.78 years, 5–10 ejaculates per bull) were prepared in a split-sample procedure as CON and either TRI or OPTX (depending on the bull and the results of a pre-trial), resulting in 120 batches. Sperm quality after thawing and non-return rates (NRR?? and NRR??) per batch were recorded (n = 11,058 inseminations). CON batches were removed from the analysis of field fertility, where sperm concentration was more than 9% higher than in the treatment batches. Data were analyzed using generalized linear mixed models (bull and ejaculate = random effects, treatment group = fixed effect). Motility 30 (P = 0.027) and 120 (P = 0.003) min after thawing was highest for TRI and lowest for CON batches. The highest proportion of morphologically intact sperm were found in TRI and the lowest in OPTX (P < 0.001). Analogically, the lowest proportion of acrosome defects were observed in TRI batches (P < 0.001). Best results for plasma membrane integrity were detected in TRI and OPTX (P < 0.001). NRR?? (P = 0.088) and NRR?? (P = 0.269) for first inseminations as well as NRR?? (P = 0.336) and NRR?? (P = 0.582) for all inseminations were not different among groups. Sperm quality were superior for TRI compared to CON or OPTX. Field fertility did not differ significantly among groups. Using a bull-specific extender with longer equilibration time results in better sperm quality without compromising field fertility.

Key words: bovine, spermatozoa, equilibration time

Post-mortem assessment of the ­reproductive status of male wolves (Canis lupus) in Germany

Post-mortem-Analyse des Reproduk­tionsstatus männlicher Wölfe (Canis ­lupus) in Deutschland

K. Müller, A. Weber, G. Fritsch, C. A. Szentiks

Leibniz Institute for Zoo and Wildlife Research (IZW), Berlin, Germany

Since the late 1990s, the grey wolf (Canis lupus) has been returning and expanding in Germany as result of its strict protection. Nationwide population monitoring data are collected by the Federal Documentation and Consultation Centre on Wolves. Dead-found animals are an important source of information and in addition to the standardized post-mortem examination at IZW, 123 male wolves were examined since 2019 for their reproductive status. Testis and epididymidis weights were measured and spermatogenic activity evaluated by flow-cytometric ploidy analyses of dissociated testis tissue and occurrence of epididymal sperm. A subset of 38 juvenile (? 12 months), 25 subadult (? 24 months) and 42 adult males could be considered. A general linear model revealed the age group and the month of death as relevant factors for testis and epididymis weight as well as for spermatogenic activity and occurrence of sperm. Mainly juvenile males differ from adults. However, individual juveniles in their first season may perform spermatogenesis and produce sperm at a similar level as (sub)adults. Deciphering seasonality in subadult and adult males, haploid cells and epididymal sperm, results of meiosis, are not only prominent during the breeding period (Jan.–March) but already before (Oct.–Dec). In accordance, meiotic activity, reflected by the appearance of tetraploid cells, was also observed at the end of the year. During summer, nearly no spermatogenic activity was detected. Spearman correlation revealed that the testis/body weight ratio is strongly related to spermatogenetic activity and occurrence of sperm in general and within the juveniles during breeding season.

Key words: Grey wolf, spermatogenesis, seasonality

Sertoli cell number is altered in dogs with immune-mediated orchitis

Veränderung der Sertoli-Zell-Anzahl bei immun-mediierter Orchitits beim Rüden

P. Rehder, E. Packeiser, H. Körber, S. Goericke-Pesch

Unit for Reproductive Medicine – Clinic for Small Animals, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany

The most common diagnosis in infertile male dogs is the acquired non-obstructive azoospermia (NOA). NOA is not only associated with severe histological and inflammatory changes, but also with a significant impairment of spermatogenesis and immune cell infiltration, indicating an orchitis. Due to the lack of clinical signs and unknown, but chronic etiology, it is defined as chronic immune-mediated orchitis (IMO). Despite the presence of some spermatogonial stem cells, spermatogenesis is irreversibly affected indicating an important role for Sertoli cells (SC) in this process. SC number determines the fate of spermatogenesis. Therefore, Sertoli cell number was counted in 20 approximately round seminiferous tubules in testis samples obtained from IMO affected dogs (n = 12), healthy adult control dogs with histologically normal spermatogenesis and normospermia (n = 10) and healthy juvenile dogs (2–3 months; n = 3). Vimentin-stained slides were used for evaluation of Sertoli cell at 400-fold magnification. Numbers were statistically compared by one-way ANOVA followed by pairwise comparison by t-test. SC number differed significantly between groups (p < 0.0001) with significantly less SCs in AIO compared to CG and JG (each p < 0.001). SC numbers in CG and JG did not differ, how­ever. Our results indicate that SC survive in the damaged testis, but SC number is reduced. This might play an important role in treating AIO aiming for restoration of spermatogenesis. Further studies should focus on evaluating the differences in SC function to get gain further insights that might be suitable for (re-)initializing spermatogenesis.

Grants: This work is supported by the GkF (Society for cynological research).

Key words: canine orchitis, azoospermia, Sertoli cells

HIF-1? modulates the expression of cell survival and glucose uptake – related genes in bovine granulosa cells

HIF-1? moduliert die Expression von Genen, die mit dem Zellüberleben und der Glukoseaufnahme in Verbindung stehen, in Granulosazellen von Rindern

K. A. Samie1, M. P. Kowalewski1, D. Scarlet1,2

1Institute of Veterinary Anatomy, Vetsuisse Faculty Zurich; 2Clinic of Reproductive Medicine, Vetsuisse Faculty Zurich, Switzerland

The ovarian follicle maturation takes place under reduced oxygen (O?) tension (hypoxia), with hypoxia-inducible factor-1 alpha (HIF1?) playing important roles in maturation of bovine granulosa cells (GCs). HIF1? controls intercellular communication within cumulus oocyte complexes and oocyte development rates, exerting effects on blastocyst rates. The aim of this study was to deepen our understanding of factors regulating bovine GCs function. GCs were collected from slaughterhouse ovaries and were stimulated under normoxia (20% O?) or hypoxia (5% O?) with 100 ng/ml IGF1 for 24h. Additionally, the functional activity of HIF1? was blocked with 5 nM echinomycin (Ech). Expression of Star (P < 0.05) and progesterone (P4) production (P < 0.001) were increased by stimulation with IGF1, and this effect was inhibited by Ech. Reduced O? content further increased Star expression (P < 0.001) but lowered (P < 0.001) P4 output. Strikingly, being abundantly expressed, the Hif1? mRNA levels were not significantly affected by changing O? tension. Similarly, the autophagy-related gene Becn1, did not change. Ech enhanced (P < 0.05) Bnip3/Bcl2 ratio (the apoptotic index) and this was more evident under hypoxia. Furthermore, blocking of HIF1? decreased (P < 0.05) Glut1 expression. No effects were observed upon Ptgs2 and Fgf2 expression, regardless of the O? content. Taken together, our results demonstrate enhanced apoptosis and reduced glucose uptake in response to the functional suppression of HIF1?, shedding light on its function during bovine GC differentiation.

Key words: granulosa cells, HIF1?, apoptosis, GLUT1

Regulation of IL1B and IL1RA mRNA expression in bovine endometrial explants after treatment with selected bacterial components and cytokines

Regulation der mRNA-Expression von IL1B und IL1RA nach Inkubation mit ausgewählten bakteriellen Komponenten und Zytokinen in bovinen endometrialen Explants

A. M. Kneidl1, S. T. Schabmeyer1, S. Kirsch1, F. Weber1,
Y. Zablotski1, C. Gabler 2, F. Westerkamp2, W. Petzl1,
J. K. Schneider, H. Zerbe1, M. M. Meyerholz-Wohllebe1

1Clinic for Ruminants with Ambulatory and Herd Health ­Services, Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-University (LMU) Munich; 2Institute for Veterinary-Biochemistry, Freie Universität Berlin, Germany

An adequate regulation of inflammatory factors plays a central role in the prevention and healing of endometrial inflammation. This project investigated the role and regulation of the proinflammatory interleukin (IL)1B and its antagonist IL1RA mRNA expression in the bovine endometrium. Explants from 26 healthy uteri (42 explants per animal) were taken at the abattoir and treated in vitro with bacterial components (Escherichia coli (EC), Bacillus pumilus (BP), Lactobacillus buchneri (LB); heat inactivated and 10? KBE/ml) for 3, 14 and 24 hours (h) as well as with pro- and anti-inflammatory recombinant bovine cytokines (IL1B, IL17A, IL10; 200 ng/ml) for 24 h. The endometrial mRNA expression of IL1RA and IL1B was analysed via RT-qPCR and quantified by standard curve method. Statistical analysis was performed using ­linear or robust mixed effects models. All selected bacteria stimulated the mRNA expression of IL1B in an incubation time-independent manner (p < 0.001), whereas the mRNA expression of IL1RA was stimulated significantly by EC and BP after 3 h (p ? 0.001) and by LB after 14 h (p < 0.001) and 24 h (p = 0.0098). The IL1RA/IL1B ratio in explants co-incubated with any of the selected bacteria shifted in proinflammatory direction (p < 0.001). A reduced mRNA expression of both IL1B and IL1RA as well as a shift of the IL1RA/IL1B ratio in the anti-inflammatory direction was observed after co-incubation with IL10 (p < 0.001). These observations highlight the importance of future research on the role of facultative pathogenic bacteria like BP, potentially protective bacteria like LB, and anti-inflammatory factors like IL10 in the pathogenesis and prevention of endometritis.

Key words: bovine endometritis, inflammatory cytokine, inflammation model

Association between components of the IGF system in blood and in the oocyte microenvironment (follicular fluid) in dairy cows

Assoziation zwischen Bestandteilen des IGF-Systems im Blut und in der ­Eizellenmikroumgebung (Follikelflüssigkeit) bei Milchkühen

C. Schiffers1, I. Serbetci2, H. Grothmann3, A. Kassens3, K. Mense3, L. Sommer1, M. Sommer4, H. Bollwein2, M. Schmicke5

1Animal Health Management, Institute of Agricultural and Nutritional Sciences, Faculty of Natural Sciences III, Martin-Luther-University Halle-Wittenberg, Halle, Germany; 2Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland; 3Masterrind GmbH, Verden, Germany; 4Agrargenossenschaft Helmsdorf eG, Gerbstedt, ­Germany; 5Veterinary-endocrinology and laboratory diagnostics, Clinic for cattle, University of Veterinary Medicine Hannover, Germany

The somatotropic axis (endocrine) as well as the local IGF-system in the follicle (paracrine/autocrine) is known to be important for fertility in dairy cows. In the present study, IGF-1 and respective binding proteins (IGFBP) were analysed in plasma and parallel in the follicular fluid (FF) obtained from the ovulatory follicle to study the hypothesis that a transfer of endocrine IGF-1 to the oocyte microenvironment is possible. It is hypothesised that a correlation between IGF-1 and IGFBPs in plasma and FF can be interpreted as a transfer. Blood samples were taken from 78 dairy cows 30 ± 2 and 50 ± 3 days postpartum (p.p.) and 17 of these animals were chosen for OPU (aspiration of 27 follicles) after cycle synchronization (2xPGF2?, detailed ultrasound). In addition, plasma, and pooled FF from a cohort of 25 other animals obtained during a project by the Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Switzerland, were also analysed. BHB in serum and IGF-1, IGFBP-2, -3, -4, -5, and an IGFBP fragment (IGFBP-F) in plasma and FF were determined. In granulosa cells (n=6) the mRNA-expression for IGFBP-2, -4, IGFBP-protease PAPP-A, and IGF1R was measured. IGF-1 and IGFBP-2 showed a significant correlation between plasma and FF (IGF-1: r = 0.57; r² = 0.32; P < 0.001; IGFBP-2: r = –0.57; P < 0.05). In conclusion, the association between IGF-1 and IGFBP-2 between plasma and FF underlines the hypothesis of an existing transfer between the endocrine IGF-1 and the local IGF-system. This might rely to a causal association between metabolic disturbances in dairy cows and subsequent fertility.

Key words: IGF-1, IGFBP, Follicular fluid

Extraction forces at different traction modes during manually assisted calving – an in vivo study

In-vivo-Studie über die Zugkraft bei unterschiedlichen Zugmodi bei der Extraktion von Kälbern

S. Schmidt1, H. Bollwein2, M. Heppelmann3

1Clinic for Ruminants (Internal medicine and Surgery), University of Giessen, Giessen, Germany; 2Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland; 3Clinic for Cattle, University of Veterinary Medicine, Foundation, Hannover, Germany

There are controversial recommendations regarding the approach to manually assisted calvings. Some authors prefer simultaneous traction on the forelimbs of the calf while others recommend alternating traction as the method of choice. So far only in vitro studies have been performed on this issue showing divergent results. Therefore, the aim of this study was to compare the traction forces at different extraction modes during manual extraction of a calf in vivo.

Manual calving assistance was performed on 63 Holstein-Friesian cattle from two farms in Lower Saxony, Germany. Traction was applied simultaneously (SIM) to both legs, alternately (ALT) to one leg at a time or to one leg (ONE). Traction force was measured on a single limb digitally with a load cell.

There was no effect of traction mode on the maximum forces applied on single limb during the extraction of the calf (P > 0.05), but the passage of the thorax through the birth canal (P < 0.05). The maximum forces measured during ONE were higher than those measured during SIM (P < 0.05), while ALT did not differ from SIM and ONE (P > 0.05) while the thorax passes the birth canal. Therefore, traction should not be applied to only one leg to extract the thorax. Based on these results no recommendation neither for the alternate nor the simultaneous traction can be made.

Key words: calving assistance, traction forces, traction modes

Teat cistern and udder parenchyma differentially respond to Staphylococcus (S.) aureus and Escherichia (E.) coli challenge in a bovine explant model

Bovine Explants aus Zitzenzisterne und Euterparenchym reagieren unterschiedlich nach Stimulation mit Staphylococcus aureus und Escherichia coli

J. Schneider, S. Kirsch, Y. Zablotski, H. Zerbe, W. Petzl

Clinic for Ruminants with Ambulatory Clinic and Herd Health Management, Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-University Munich, Germany

S. aureus intramammary infection (IMI) still largely accounts for cases of subclinical mastitis in dairy cattle. The absence of an adequate inflammatory response is a major factor leading to bacterial persistence and chronic inflammation with comparably poor bacteriological cure rates in S. aureus IMI. The aim was to study the impact of S. aureus and E. coli challenge on inflammatory cytokine expression in explants of the teat cistern (TC) and udder parenchyma (UP). Individual tissue explants were obtained from TC (n = 12) and the UP (n = 8) and were cultured for 18 h in vitro with 10? colony forming units of heat killed S. ­aureus or E. coli or no stimulus respectively. The mRNA expression of IL1B, CXCL8 and IL6 was analysed by RT qPCR. Statistical analysis was performed using a mixed linear model (Software R Version 4.0.4). Genes coding for IL1B and CXCL8 were significantly (P < 0.05) induced in TC and UP by S. aureus and E. coli. IL6 was significantly (P < 0.01) induced in TC and UP by E. coli. Also S. ­aureus significantly (P < 0.05) induced IL6 in TC however to a lesser extent than E. coli (P < 0.05). In UP S. aureus failed to induce IL6. The data support a location and pathogen specific response in the udder. In conclusion, the reduced responsiveness of UP towards S. aureus in comparison to E. coli may partly explain bacterial persistence and chronic inflammation in UP during S. ­aureus IMI.

Key words: mastitis, innate immunity, 3Rs

Effect of mito-tempo on embryonic development and cryogenic variability of IVP derived bovine blastocysts

Auswirkung von Mito-Tempo auf die Embryonalentwicklung und die Kryotauglichkeit von Rinderblastozysten

M. Schreiber1, J. Kurzella2, D. Salilew-Wondim2, D. Teuteberg1, C. Blaschka1, M. Hoelker1

1Department of Animal Science, Biotechnology & Reproduction in farm animals, University of Goettingen, Goettingen; 2Institute of Animal Science, Animal Breeding, University of Bonn, Bonn, Germany

The addition of antioxidants to culture media has been reported to provide beneficial effects on developmental rates, ROS levels and cryogenic fitness of in vitro produced (IVP) bovine embryos. The addition of Mito-Tempo during in vitro culture, however, has not been investigated, yet. Consequently, the present study aimed to investigate the effect of Mito-Tempo supplementation to culture medium on early development and cryogenic viability of IVP derived bovine embryos. Maturation (TCM199, 39 °C, 5% CO?, 5% O?) and in vitro fertilization (Fert.-TALP, 39 °C, 5% CO?, 5% O?) of oocytes was conducted via routine procedures and media using frozen thawed semen (2×10?/ml). Presumptive zygotes were cultured for up to 8 days in SOFaa + 0.3 % BSA (39 °C, 5% CO?, 20% O?) without Mito-Tempo (control) or supplemented with 1 µM Mito-Tempo. Subsequent day 7-blastocysts of both groups were analyzed for intracellular ROS levels. In addition, day 7 blastocysts of both groups were vitrified using „­BO-VitriCool™“-media emerging the Cryotop®-vitrification system. Warming of these blastocysts (Bo-VitriWarm™-media) was followed by post-warming culture for 72 hours to determine viability rates, re-expansion rates and hatching rates. As a result, the present study did not obtain any significant effect of Mito-Tempo supplementation on embryo developmental rates. In contrast, supplementation of culture medium with Mito-­Tempo significantly (p < 0.05) enhanced re-expansion rates as well as hatching rates after warming compared to control embryos. These results confirm our hypothesis that the antioxidant Mito-Tempo exerts a positive effect on mitochondrial activity and reduces cryo-induced damage after vitrification.

Key words: antioxidants, vitrification, bovine blastocysts

Immunohistochemical and gene ­expression studies of oestrogen, progesterone, oxytocin and prostaglandin F2alpha receptors in the bovine uterus during the postpartum period

Immunhistochemische und molekulare Untersuchungen von Hormonrezeptoren im Uterus des Rindes während des Puerperiums

C. Schwär1, M. Wiebe1, C. Pfarrer2, S. Bauersachs3,
H. Bollwein4, M. Heppelmann1

1Clinic for Cattle; 2Institute for Anatomy, University of ­Veterinary Medicine, Foundation, Hannover, Germany; 3­Institute of Veterinary Anatomy; 4Clinic of Reproductive Medicine, Vetsuisse-Faculty, University of Zurich, ­Switzerland

The early postpartum (p.p.) period is one of the most critical for health and reproductive performance in dairy cows. Hormones and their corresponding receptors play a significant role in regulating postpartum uterine activity. Thirty-two tissue samples of the bovine intercaruncular uterine wall were collected during caesarean section (n = 16) and from euthanized cows in the first three weeks p.p. (n = 16). Ten of them were diagnosed with metritis. Immunohistochemistry (IHC) was performed for hormone receptors of oxytocin (OXTR), prostaglandin F2? (PTGFR), oestrogen (ESR1) and progesterone (PGR). Tissue sections were examined for positive staining and staining intensities at 9 tissue localizations. Additionally, quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) for the mRNAs of those receptors was performed for the p.p. samples for each endometrium and myometrium. Hormone receptor expression was detected via RT-qPCR and IHC in all time periods. There was no difference in the occurrence of OXTR apart from two localizations whereas PTGFR protein showed a decline over time p.p. for several localizations. RT-qPCR showed an increase over time p.p. for endometrial PTGFR expression. ESR1 protein showed an increase over time in IHC in all tissue layers as well as ESR1 mRNA showed an increase over time in endometrium. PGR appearance in IHC was strongest on day 0 and decreased at most sites p.p. Lower values for uteri of cows diagnosed with metritis compared to those without metritis could only be verified for ESR1 in one localization but not via RT-qPCR. The results indicate that the localization of the studied ­receptors changes during the p.p. period. Only very little effects of metritis were observed.

Key words: hormone receptors; bovine uterus; immunohistochemistry

Causes of dystocia in the cat

Ursachen von Schwergeburten bei der Katze

S. Sendag1,2, D. Hardegen2, A. Wehrend2

1Clinic for Veterinary Obstetrics and Gynecology, Van YY- University, Van, Turkey; 2Clinic for Obstetrics, Gynecology, and Andrology of small and large animals with an Ambulatory Service of Justus-Liebig-University, Giessen, Germany

To prevent dystocia, their causes must be known. In contrast to other animal species, there are only a few studies on the causes of difficult births in cats. To get more information about this field of reproduction medicine dystocias in cats referred to our clinic over the last 20 years (retrospectively from 2000 to 2010, prospectively from 2011 to March 2021) were investigated. The following was observed: In 74 (45%) of 166 cats, the cause of dystocia was of maternal origin and in 45 (27%) cats it was due to fetal causes. The cause of dystocia could not be identified in 47 cats (28%). It was found that uterine inertia was the most common problem (n = 55, 33%) in cats with dystocia due to maternal factors. The other maternal causes of dystocia were isolated cases. The most common fetal origin dystocias were single pregnancy (n = 20, 12%) and fetal oversize (both absolutely and relatively too large fetuses, n = 16, 10%). 83% of dystocia cases (n = 127) were surgically (Sectio caesarea conservative: 44; Sectio porro: 75; En bloc-resection: 8) treated, while 15% were initially done conservatively. In 26 (17 %) cases, conservative obstetric treatment was successful. Newborn mortality was 38.8 %. Maternal mortality was 2.4 %. 59 % of newborns with single pregnancies died. Single pregnancy, therefore, poses a particular risk to the fetus.

Key words: birth, uterus, pregnancy, fetal size

Alpha 7 nicotinic receptors are involved in the regulation of ovarian functions (Part I)

Alpha-7-nikotinerge Rezeptoren sind an der Regulation der Ovarfunktion beteiligt (Teil I)

P. Seßenhausen1, K. Caban2, N. Kreitmair1, B. Popper3, U. Berg4, D. Berg4, T. Fröhlich2, A. Mayerhofer1

1Biomedical Center (BMC), Cell Biology, Anatomy III, Faculty of Medicine, Ludwig-Maximilian-University Munich (LMU) Planegg; 2Gene Center, Laboratory for Functional Genome Analysis LAFUGA, Ludwig-Maximilian-University Munich (LMU); 3Biomedical Center (BMC), Core Facility Animal ­Models, Faculty of Medicine, Ludwig-Maximilian-University Munich (LMU), Planegg; 4Fertility Centre A.R.T., Bogen­hausen, Munich, Germany

Acetylcholine (ACh) is evolving as a regulator of the ovary, but the ACh receptors involved are not well known. Alpha 7 nicotinic receptors (CHRNA7) belong to the nicotinic receptor family of ion channels, which are associated with cellular functions, metabolic and anti-inflammatory responses. According to human ovary single cell RNAseq data, ­CHRNA7 is expressed in smooth muscle cells and follicular granulosa cells (GCs). Own PCR studies confirmed expression in IVF-derived human GCs and mouse ovary. To examine whether it may be involved in the regulation of ovarian functions, we studied Chrna7 knockout (KO), which lack exons 8-10, and wildtype mice as controls (both 3 months). We evaluated serial sections, performed qPCR and immunohistochemical studies. We found no changes in healthy or atretic primary, secondary, and tertiary follicles or in corpora lutea numbers. However, the numbers of primordial follicles were significantly reduced in KO animals. Dominant interstitial areas were apparent in KO, hinting to an alteration of the intra-ovarian environment. Next, qPCR studies showed a tendency to increased steroidogenic enzymes and immunostainings identified abundant CYP11A1-positive theca-­interstitial cells in KO ovaries. Expression levels (qPCR) of different macrophage markers and cytokines were not or only slightly changed. Thus, the lack of Chrna7 causes an ovarian phenotype, with changes of the intra-ovarian environment and primordial follicles. To examine the changes further, a proteomic study was performed, as described in Part II.

Grants: DFG project number 432434245

Key words: CHRNA7, ovary, interstitial cells

Ovarian stromal cells contribute to tumor progression in bovine ovary

Stromazellen der Eierstöcke tragen zur Tumorprogression im Rinderovar bei

A. Sharma, X. Tao, V. S. Baddela, F. Becker, C. Ludwig, J. Vanselow

Institut für Fortpflanzungsbiologie, Forschungsinstitut für Nutztierbiologie (FBN) Dummerstorf, Germany

Ovarian stromal fibroblast cells are known to be a major source of cancer progression in humans [1]. The current study presents the in vitro analysis of stromal cells obtained from an enlarged ovary of 4.5 cm in diameter excised from a Holstein cow during routine hygiene inspection. Due to circumferential increase in the size of ovary, the ovary was suspected of undergoing tumor progression. Initially, cells were isolated from the tumor ovarian stromal tissue and cultured in vitro. As evident, the cultured cells showed a ­spindle shaped fibroblast like morphology, which ceased to proliferate after the 8th passage. The cells were characterized as ­mesenchymal cells as they explicitly expressed the mesenchymal marker vimentin. Further, mRNA microarray analysis was performed comparing the transcriptomes of normal and tumor derived stromal cells. The microarray identified 1396 differentially expressed (fold change [FC] ? 2, p < 0.05, FDR [q] < 0.05) genes, of which 663 were up- and 733 were down-regulated. Earlier studies suggest that altered expression of asporin (ASPN) an extracellular secreted protein, plays a critical role in pathogenesis of various types of cancers [2]. Also in our microarray analysis, expression of the ASPN gene was higher (FC = –72.92, q-value = 0.0003) in stromal tumor cells. qPCR, western blot and immunohistochemistry analyses confirmed this result. Overall, the findings indicate that altered ASPN expression in ovarian stromal cells is associated with tumorigenesis and these cells might be aimed as potential targets for improving the therapeutic efficacy of ovarian tumor treatment in bovine.

Key words: stroma, tumor, asporin

References:

1. Fujisawa M, et al. Ovarian stromal cells as a source of cancer-associated fibroblasts in human epithelial ovarian cancer: a histopathological study. PLoS One 2018; 13: e0205494.

2. Zhan S, et al. Multifaceted roles of asporin in cancer: current understanding. Frontiers in Oncology 2019; 9: 948.

The potential of oxytocin-antagonists as a new therapeutic for the treatment of benign prostatic hyperplasia

Das Potential von Oxytocin-Antagonisten als neue Therapie zur Behandlung von benigner Prostatahyperplasie

B. Stadler1, K. Koslowa1, A. Mietens1, A. Pilatz2, F. Wagenlehner2, R. Middendorff1

1Institute of Anatomy and Cell Biology, Justus-Liebig-­University, Giessen; 2Department of Urology, Pediatric ­Urology and Andrology, Justus-Liebig-University, Giessen, Germany

The human prostate enlarges with age to a degree where 60% of 60-year old men report symptoms of benign prostatic hyperplasia (BPH). The muscle tension and number of the smooth muscle cells (SMCs) in the prostate increases with BPH, which can constrict the urethra often leading to micturition disorders. Therefore, the first line of medical treatment seeks to relax these SMCs by targeting the alpha-1 receptors using agents such as tamsulosin. Treatment with alpha-1 blockers especially tamsulosin is often accompanied by side effects such as ejaculatory disorders. We have investigated an alternative signaling pathway in the prostate targeting oxytocin receptor mediated SMC contractions to evaluate its potential as a novel treatment option for BPH. Contraction studies with samples of the human and rat prostate were performed using live imaging. Two experimental set-ups were compared: 1. no treatment ? oxytocin cligosiban; 2. no treatment ? cligosiban ? oxytocin. Oxytocin was able to increase spontaneously occurring contractions of both the human and the rat prostate. When given second cligosiban significantly reduced these oxytocin-induced contractions. When given first cligosiban alone did not alter spontaneous contractions of the prostate but was able to block the effect of a following oxytocin addition. Targeting the oxytocin pathway presents as a promising alternative to targeting alpha-1 receptors.

Key words: prostate, contractility, cligosiban

Effects of the glucocorticoids, dexamethasone and cortisol, on human testicular peritubular cells

Wirkungen der Glukokortikoide, Dexamethason und Cortisol auf menschliche testikuläre peritubuläre Zellen

Y. K. Stepanov1, T. Fröhlich1, A. Mayerhofer2, H. Welter2

1Laboratory for Functional Genome Analysis LAFUGA, Gene Center, Ludwig-Maximilian-University Munich (LMU); 2Biomedical Center, Cell Biology, Anatomy III, Faculty of Medicine, Ludwig Maximilian University Munich (LMU), Planegg-Martinsried, Germany

We recently described glucocorticoid receptor (GR) expression and activation in human testicular peritubular cells (HTPCs) [1] and provided evidence of profound short-term effects of the synthetic glucocorticoid Dexamethasone (Dex) on the immunological state and the proteome of HTPCs [2]. The widespread long-term use of Dex in multiple human medical contexts prompted us to examine consequences of long-term Dex-treatment (1 µM; 7 days) on HTPCs. We also aimed to study effects of the natural GR-ligand Cortisol (Cort). Holistic mass spectrometry-based proteome analyses were performed and complemented by qPCR analyses. We measured dose dependent effects of Cort on GR mRNA expression as well as on GR-regulated genes such as FKBP5 and IL6. Cort also lowered GR-protein levels. Proteomics analysis further revealed strong responses, which increased with prolonged Dex treatments. While not as pronounced, the effect of cortisol treatment was comparable to that of Dex. The differentially abundant proteins include extracellular matrix- and basement membrane components, cyto­skeletal organization related proteins. Our results demonstrate distinct GR-dependent actions of Dex and cortisol in HTPCs. They include inflammatory responses and regulation of the GR, massive changes in cellular phenotype. If transferable to the human testis, the results indicate that glucocorticoids are regulators of testicular functions.

Grants: Supported by DFG, project number 427588170

Key words: human male fertility; testis; glucocorticoids

References:

1. Welter H, et al. The glucocorticoid receptor NR3C1 in testicular peritubular cells is developmentally regulated and linked to the smooth muscle-like cellular phenotype. J Clin Med 2020; 9: 961.

2. Stepanov YK, et al. Profound effects of dexamethasone on the immunological state, synthesis and secretion capacity of human testicular peritubular cells. Cells 2022; 11: 3164.

Unsaturated fatty acids induce glycolytic consumption of glucose in granulosa cells

Ungesättigte Fettsäuren beeinflussen den Glukoseumsatz in Granulosazellen

X. Tao, J. Vanselow, V. S. Baddela

Institute of Reproductive Biology, Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany

Fatty acids are critical for maintaining membrane fluidity, cellular signaling, and lipid storage. Oleate (OA, C18:1), palmitate (PA, C16:0) and stearate (SA, C18:1) are the most abundant fatty acids in the blood circulation and also in the ovarian follicular fluid. Alpha-linolenic fatty acid (ALA, 18:3 n-3) is an omega-3 essential fatty acid shown to have health benefits. In the present study, we questioned about the role of these fatty acids in glucose utilization and lactate production in bovine granulosa cells (GCs). All fatty acids were conjugated to bovine serum albumin (BSA) to mimic in vivo conditions. OA and ALA treatments induced higher glucose utilization and lactate production compared to BSA (vehicle control), PA, and SA treatments. In contrast, PA and SA treatments did not significantly affect glucose utilization and lactate production compared to the BSA. Co-treatment of PA and SA, along with either OA or ALA, inhibited glucose utilization.Our recent transcriptome analysis revealed the enriched terms for insulin and EGF signaling in OA-treated granulosa cells. Accordingly, the western analysis showed that OA and ALA significantly induce p-AKT levels in GCs compared to PA and SA and the vehicle. Inhibition of AKT signaling using chemical inhibitor LY294002 ameliorated the OA-induced glucose uptake. Overall, these data show that unsaturated fatty acids cause glucose consumption via AKT signaling; however, saturated fatty acids like PA and SA prevent unsaturated fatty acid-induced glucose consumption.

Key words: oleic acid, glucose, granulosa cells

Influence of processing bovine ­colostrum on anti-trypsin activity

Beeinflussung der Anti-Trypsin-Aktivität im bovinen Kolostrum durch Prozessierung

L. Trzebiatowski1; P. Georgiev2; A. Wehrend1

1Clinic for Obstetrics, Gynaecology and Andrology of Large and Small Animals, Justus-Liebig-University, Giessen, ­Germany; 2Department of Obstetrics, Reproduction and Reproductive Disorders, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria

The crucial role of colostrum for the bovine neonate is well known for a long time. Immunoglobulins in particular must pass intact through the digestive tract with its proteolytic enzymes after ingestion before they can be absorbed into the neonatal circulation. For this reason, colostrum shows trypsin inhibitory activity. In calf rearing, various procedures are established to preserve and extend the durability of colostrum and to reduce microbial load. The aim of the study was to determine the influence of processing bovine colostrum on anti-trypsin activity. For this purpose, the first colostrum from 107 cows after collection was frozen, acidified and pasteurised following two different protocols. The determination of the anti-trypsin activity was performed photometrically via the inhibition of the turnover of an indicator substance (benzoyl DL-arginine p-nitroanilide hydrochloride) by trypsin. The study showed that freezing and thawing as well as acidification of bovine colostrum did not significantly change the anti-trypsin activity. Both pasteurisation protocols showed a decrease in anti-trypsin activity. This was significant in the protocol with the higher pasteurisation temperature. Anti-trypsin activity of colostrum seems to be an important factor for the protection of immunoglobulins from digestion. Different processing protocols of bovine colostrum have different impacts on the anti-trypsin activity. An additional goal of processing bovine colostrum should be to minimize the loss of this activity.

Key words: colostrum, processing, immunglobulins

The determination of SERPINA14 in ovarian tissue, immature and in vitro matured cumulus-oocyte complexes in cattle

Die Identifizierung von SERPINA14 in Ovargewebe, unreifen und in vitro gereiften Kumulus-Oozyten-Komplexen bei Rindern

A. Turhan1,2, J. Muggli2, J. Walter2, M. Kowalewski2, Ricardo Fernandez2, Kirstin Skaar2, U. Bleul1

1Clinic of Reproductive Medicine, Department of Farm ­Animals, Vetsuisse Faculty University of Zurich, Zurich, ­Switzerland; 2Institute of Veterinary Anatomy, Vetsuisse Faculty University of Zurich, Zurich, Switzerland

SERPINA14 is secreted by the uterine epithelium during pregnancy in cows and was suggested to have immunoregulatory effects. It has also been detected in corpora lutea and ovarian follicles as well as in cumulus oocyte complexes (COCs) [1]. Previous studies on the cumulus proteome suggested a link between SERPINA14 and oocyte maturation competence [2]. However, the role of ­SERPINA14 during maturation of COCs is unclear in cattle and other animal species. Here, the spatio-temporal distribution of SERPINA14 was investigated in ovaries of cows, and in COCs isolated from ovaries collected at a local abattoir, divided in the groups immature (control) and in vitro matured (IVM). All in vitro experiments were performed at least 3 times (control/IVM; n = 35 COCs/group from different animals). By immunohistochemistry, SERPINA14 protein was localized in follicular granulosa cells as well as in ovarian endothelial cells. In isolated COCs, the intensity of the signals appeared stronger in the cumulus cells of matured COCs than in their immature counterparts. This was confirmed by Western blot analysis, where stronger staining and thus stimulatory effect of IVM on the expression of SERPINA14 was observed. In conclusion, our results: 1) confirm the expression of SERPINA14 in bovine ovarian granulosa cells in vivo, 2) identified increased expression during IVM, suggesting a role of SERPINA14 during oocyte maturation and thus the resumption of meiosis.

Key words: Cattle, SERPINA14, in vitro maturation of oocytes

References:

1. Ulbrich SE, et al. Evidence for estrogen-dependent uterine serpin (Serpina14) expression during estrus in the bovine endometrial glandular epithelium and lumen1. Biology Reprod 2009; 81: 795–805.

2. Walter J, et al. The bovine cumulus proteome is influenced by maturation condition and maturational competence of the Oocyte. Scientific Reports 2020; 10: 9880.

Androgen signaling during downregulation and recovery subsequent to 5-months deslorelin treatment

Androgensignalisierung während der durch ein Deslorelin-Implantat induzierten Downregulation und nachfolgender Wiederherstellung

A. Vasetska1, E. M. Packeiser1, H. Körber1, S. Aslan2, G. Saral3, F. Binli3, E. Akal4, M. Selçuk4, S. S. Ay3, M. Findik3, S. Goericke-Pesch1

1Unit for Reproductive Medicine – Clinic for Small Animals, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany; 2Department of Obstetrics Gynaecology – Faculty of Veterinary Medicine, Near East University, ­Nicosia, Cyprus; 3Department of Obstrectics Gynaecology – Faculty of Veterinary Medicine, Ondokuz May?s University, Samsun, Turkey; 4 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Ondokuz May?s University, Samsun, Turkey

Slow-release GnRH agonist implants (SRI) are frequently used in small animal medicine to temporarily induce downregulation of testicular endocrine and germinative function resulting in basal testosterone concentrations and infertility due to arrest of spermatogenesis. Treatment-induced effects are fully reversible; testosterone is quickly restored. Recovery of spermatogenesis takes longer, but after 7-10 weeks first sperm are found in the ejaculates. Androgens act via androgen receptors (AR) and altered AR expression might be associated with downregulation due to SRI and subsequent recovery. Besides, proteins involved in AR signaling, e.g. heat shock protein hSP 70 and 90, might be altered due to treatment. To gain further insights, healthy dogs were treated with a deslorelin SRI over five months, the SRI was removed surgically at 5 months and 4-5 dogs were castrated at implant removal (week, W 0) or W1, 2, 3, 4, 5, 6, 7, 8 or 10 weeks after SRI insertion. Testes of untreated healthy male dogs served as controls (CG). Gene expressions of AR, hSP70, hSP90 and hSPA8 were investigated and group-wise comparison was performed using GraphPad Prism. Expression of AR (ANOVA, p < 0.0001), hSP70 (Kruskal-Wallis, p < 0.0001), hSP90 (ANOVA, p < 0.01) and hSP8A (Kruskal-Wallis, p < 0.01) differed significantly with higher AR, hSP70, but lower hSPA8 expression in the downregulated testis (W0) compared to later time points of recovery and CG. Further studies should clarify whether the differences are related to variable tissue composition or truly treatment-related, besides protein expression should be investigated.

Grants: Anastasiia Vasetska receives a scholarship of the Volkswagenstiftung.

Key words: androgens, GnRH agonist implant, dog

Correlations between colostrum composition and blood parameters of sows

Zusammenhänge zwischen der ­Kolostrumzusammensetzung und Blutparametern von Sauen

A. Vernunft1, L. Diesing2, C. C. Metges1,2, C. Gladbach1,3, M. Oster1, K. Wimmers 1,2

1Research Institute for Farm Animal Biology (FBN), ­Dummerstorf; 2Faculty of Agricultural and Environmental Sciences, University of Rostock, Rostock; 3Justus-Liebig-­University Giessen, Giessen, Germany

The aim of this study was to investigate the relationships between components of colostrum and blood parameters of sows. Colostrum was collected from 25 German Landrace and 18 German Saddleback sows (22 primipara, 21 secundipara) during parturition (D0) and on the first day post partum (D1). Corresponding blood samples were taken one day before farrowing (D-1), at D0 and D1. In colostrum, the levels of fat, protein, lactose, dry matter and total energy were determined and immunoglobulins (Ig) G, A and M were measured using an ELISA kit. In plasma, metabolic (glucose, lactate, ­creatinine, ?-hydroxybutyrate (bHBA), NEFA, triglycerides), biochemical (pH, Na, K, Ca, Cl, Mg, P) and endocrine parameters (cortisol, PGFM, oxytocin, estradiol, progesterone, adrenaline, noradrenaline) were analysed. Pearson correlation calculations showed that D0 colostrum fat content was positively correlated with plasma levels of bHBA and NEFA on D-1 (r = 0.62; r = 0.65), D0 (r = 0.51; r = 0.55) and D1 (r = 0.22; r = 0.41) and thus also with energy content. Plasma triglycerides on D-1 were positively correlated with IgA and IgM content of the D0 colostrum (r = 0.98; r = 0.90), while cortisol on D-1 showed a negative relationship with IgG (r = –0.91). High plasma levels of adrenaline or noradrenaline before birth (D-1) negatively affected IgG (r = –0.99; r = –0.96) and IgA (r = –0.91; r = –0.93) content in the D1 milk, while plasma glucose and IgG, A, and M in milk were positively correlated on D1 (r = 0.68; r = 0.71, r = 0.61). In conclusion, there are significant relationships of colostrum components to blood para­meters in sows, especially to blood indicators of metabolic stress and arousal.

Key words: colostrum, blood parameter, pig

Lactobacillus buchneri showed increased growth rate in the presence of bovine mucus collected at estrus in contrast to Trueperella pyogenes

Höhere Wachstumsrate bei Lactobacillus buchneri mit von bovinem, im Östrus gewonnenem Mucus im Gegensatz zu Trueperella pyogenes

T. Wahl1, K. Wagener2, M. Drillich2, M. Ehling-Schulz3,
C. Gabler1, M. Ibrahim1

1Institute of Veterinary Biochemistry, Freie Universität ­Berlin, Germany; 2Clinical Unit for Herd Health Management in Ruminants, Department for Farm Animals and Veterinary Public Health, Vetmeduni Vienna, Austria; 3Institute of Microbiology, Department for Pathobiology, Vetmeduni ­Vienna, Austria

Pathogenic bacteria like Trueperella pyogenes (TP) are associated with clinical endometritis in postpartum dairy cows. On the other hand, some bacterial species colonizing the bovine uterus are regarded as potential probiotic bacteria, such as Lactobacillus buchneri (LBB). The interaction between named bacterial populations and certain factors in the bovine uterus is not well known so far. The aim of this study was to analyze the growth rate of both TP and LBB in the presence of bovine mucus collected at estrus (BME) as well as of steroidal hormones estradiol (E2) and progesterone (P4). TP was isolated from a dairy cow developing clinical endometritis, whereas LBB was isolated from a healthy cow. LBB and TP were grown in MRS and BHI broth, respectively. Sterile BME at a rate of 20% was added to the medium. E2 contents of 10 pg/ml and P4 contents of 5 ng/ml were used to mimic the follicular and luteal phases, respectively. The OD??? of both cultures was measured at 0 h and 24 h. The experiment was repeated independently five times. LBB showed a significantly higher growth rate after 24 h in the presence of BME (P < 0.05) compared to controls. However, BME does not affect the growth rate of TP. Growth of neither LBB, nor TP was affected by E2 or P4. In conclusion, BME seems to contain factors that help protective bacteria to flourish, while pathogenic bacteria are not influenced. This may imply that healthy uterine mucus supports the uterine microbiome towards beneficial bacteria, however further research is needed. In addition, steroidal hormones E2 or P4 do not have a direct influence on the growth rates of LBB and TP.

Key words: bovine endometritis, Lactobacillus buchneri, Trueperella pyogenes

Spermatocele in a three-year-old Galloway bull – A case report

Spermatozele bei einem drei Jahre alten Galloway-Bullen – Ein Fallbericht

M. Wiebe1, R. Brehm2, M. Heppelmann1

1Klinik für Rinder, 2Anatomisches Institut, Stiftung Tierärztliche Hochschule Hannover, Germany

A three-year-old Galloway bull was admitted to the clinic for breeding soundness examination. According to the preliminary report the cows mated by him did not get pregnant, although ejaculate could be found in the vagina of the mated cows. General examination showed no divergent findings. During andrological examination the right testicle was found to have a higher thickness than the left one. There was also an asymmetry in epididymides, with the right one being enlarged and more rigid in consistency. In addition, there was a palpable fluctuating mass dorsolateral at the right testicle with a diameter of 3 cm, that was movable to the scrotum and to the testicles. In sonographical examination parenchyma of both testicles was slightly unsteady with multiple hyperechogenic specks and small anechogenic areas, in the right one more than in the left one. The mass dorsolateral at the right testicle was multi-chambered with anechogenic content in ultrasound. Anechogenic tubular structure was more prominent in right cauda epididymidis than in the left one. Examination of ejaculate, collected with an artificial vagina while the bull mounted a standing cow, revealed nearly no sperms in ejaculate. Histological examination of testicular biopsies showed intact tubuli seminiferi convoluti with predominantly normal spermatogenesis surrounded by leydig cells, but intense plasma stasis in testicular tissue of both sides. These results indicate the diagnosis of spermatocele with azoospermia, which is usually caused by obstruction of the efferent ducts and occurs commonly in rams and goats, but rarely in bulls.

Key words: cattle, testes, infertility

Stimulating the androgenic response through administration of an LHR agonist to Leydig cells obtained from the 41,XXY* Klinefelter syndrome mouse model

Stimulation der Androgenantwort von Leydig-Zellen durch Verabreichung ­eines LHR Agonisten im 41,XXY* Klinefelter Mausmodell

J. Wistuba, R. Sandhowe-Klaverkamp, J. Gromoll

Institute of Reproductive and Regenerative Biology, Centre of Reproductive Medicine and Andrology, University Clinics Münster, Münster, Germany

In Klinefelter syndrome (KS, 47,XXY) patients and in the respective XXY* mouse model, we previously excluded a general impairment of steroidogenic Leydig cell (LC) function and found the testes to produce normal intratesticular testosterone (ITT) levels. However, these values are only reached in vivo and by remarkably increased numbers of LCs. We could also show altered testicular vascularization in KS mice and men to contribute to the endocrine disbalance. Although ITT is maintained by LC hyperplasia and elevated LH levels this compensation is insufficient to restore serum T levels. With regard to the hampered vascularization, it appears possible that the transport/influx of the large hydratized proteohormone LH could be affected, i.e. that even elevated LH levels are insufficient to provoke adequate stimulatory action. We hypothesize that LH receptor stimulation by a small receptor agonist (Org 43553) could improve LH action and result in an enhanced testicular steroid response and normalized serum T. To explore the agonists´ stimulatory potential, LCs from XXY* mice and wild type controls were treated with effective agonist concentrations (0.1 or 1 mM for 3h) and T production was measured. We found that i) the LHR agonist is highly active and ii) XXY* LCs responded stronger. Subsequently, we used organ culture to test whether the effect holds also true in situ. We found a similar effect in these experiments showing an enhanced T response in 41,XXY* testicular tissues. We suggest that using LHR agonist might open up a potential new route to encounter hypogonadism in males with a supernumerary X chromosome.

Keywords: Androgens, Klinefelter Syndrome, Cell culture