– Journal of Reproductive Medicine and Endocrinology –

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

Reproduktionsmedizin

und Endokrinologie

– Journal of Reproductive Medicine and Endocrinology –

Andrologie

^•

Embryologie & Biologie

^•

Endokrinologie

^•

Ethik & Recht

^•

Genetik Gynäkologie

^•

Kontrazeption

^•

Psychosomatik

^•

Reproduktionsmedizin

^•

Urologie

Indexed in EMBASE/Excerpta Medica/Scopus

www.kup.at/repromedizin

Online-Datenbank mit Autoren- und Stichwortsuche

55th Annual Conference of Physiology and Pathology of

Reproduction and simultaneously 47th Joint Conference

of Veterinary and Human Reproductive Medicine 2nd–4th

March, 2022 – online Abstracts

J. Reproduktionsmed. Endokrinol 2022; 19 (Supplementum

1), 1-19

BACK TO THE FUTURE

10. DVR-KONGRESS

20.09.-22.09.2023

World Conference Center BONN

Prof. Dr. med. Jean-Pierre Allam PD Dr. rer. nat. Verena Nordhoff Prof. Dr. med. Nicole Sänger

SAVE THE DATE

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55

^th

Annual Conference of Physiology and Pathology of Reproduction and simultaneously

47

^th

Joint Conference of Veterinary and Human Reproductive Medicine

2

^nd

–4

^th

March, 2022 – online Abstracts

^*

01

Morphology, blood flow velocity and hormonal contents of the over- grown follicle in dromedary camels Morphologie, Blutflussgeschwindigkeit und Hormongehalt von sogenannten überwucherten Follikeln bei Dromeda- ren

A. Ali^1,2, D.R. Derar^1,2

1Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University, Buraidah, Saudi Arabia; ²Department of Theriogenology, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt Camel ovarian follicular growth has a dis- tinguishable turnover. Because ovulation is induced, the dominant follicle (DF) becomes atretic in non-mating females; however, in some females, the DF grows to be larger (OVGF, > 2 cm in diameter) than typical fol- licles. The cause of some females‘ continued follicular development is unknown, and there is some argument about the pathogenicity of this phenomenon. The objective of this study was to characterize the morphology, real- time changes in spectral blood flow velocity, and hormonal concentrations of OVGF so that we could better understand its occur- rence and fate. Twenty-three female camels were ultrasonographically classified as hav- ing an OVGF with clear content (OVGF_TH, n = 7), granular content (OVGF_GR, n = 4), fine trabeculae (OVGF_TKI, n = 3), mod- erate trabeculae (OVGF_TKII, n = 3), or dense trabeculae (OVGF_TKIII, n = 6). As controls, three camels with growing follicles (GF, 6 to  <  9 mm in diameter) and three with ovulatory follicles (OVF, 9–20 mm in diameter) were used. Spectral Doppler mode (PW) was used to assess peak systolic velocity (PSV) and time-averaged maximum velocity (TAmaxm). Follicular fluids (FF) were col- lected transvaginally and analyzed for estradi- ol 17β (E2) and progesterone (P4). The results showed that all types of OVGF had compara- tively low blood flow velocities (p = 0.0001).

The majority of OVGF were low in E2, and high in P4 (P4 > E2). PSV and E2 content cor- related positively (r = 0.621, p = 0.01). Finally, changes in OVGF blood flow may play a role in their development and atresia.

02

Retrospective study on the occur- rence of uterine torsion in sheep:

302 ovine dystocia cases submitted to a veterinary referral hospital Retrospektive Studie zum Auftreten von Uterustorsion beim Schaf: 302 Dystokiefälle aus einer Wiederkäuer- klinik

V. Balasopoulou, Y. Zablotski, H. Zerbe, K. Voigt Clinic for Ruminants with Ambulatory and Herd Health Ser- vices, LMU Munich, Oberschleissheim, Germany

Uterine torsion has been considered a rare cause of dystocia in sheep by many authors.

This study describes cases of uterine torsion in this species and analyzes potentially influen- tial factors on its occurrence. Clinical records of 302 sheep with dystocia admitted to a vet- erinary hospital were evaluated retrospective- ly. Sixty (19.9%) animals were diagnosed with uterine torsion. Statistical analyses were per- formed in R (version 3.6.3), using simple and multiple logistic regression models to identify potentially predisposing factors. Lamb birth weights did not differ significantly between ewes with uterine torsion and other causes of dystocia (p = 0.267). Following univari- ate analyses age, parity, season and nutrition were excluded as non-significant, while breed type, litter size and husbandry showed signifi- cance, with lower odds for meat breeds (OR 0.22; p < 0.001), twin- (OR 0.49; p = 0.020) or multiple-bearing ewes (OR 0.19; p = 0.013) and higher odds for fully housed animals (OR 17.87; p < 0.001). A subsequent multiple lo- gistic regression model identified year-round housing as the most influential factor (OR 10.71; p < 0.001). The breed type remained significant (p = 0.025), while the litter size was no longer significant in this multiple model (twins p = 0.191, multiples p = 0.089).

In contrast to previous publications, uterine torsion was identified as a relevant cause of dystocia in our caseload. Year-round housing was identified as the most influential predis- posing factor for the condition in the studied cohort.

03

Quantitative differences in sex hor- mone-binding globulin amounts between different ruminant breeds detected by mass spectrometry Quantitative Unterschiede im Sexual- hormon-bindenden Globulin zwischen verschiedenen Rinderrassen gemessen mittels Massenspektrometrie

B. Bartling, C. Siebenmorgen, J. Thielebein, M. Schmicke Animal Health Management, Martin-Luther-University Halle-Wittenberg, Halle, Germany

Sex hormone-binding globulin (SHBG) is a glycoprotein that binds particularly andro- gens and oestrogens and has both enhanc- ing and inhibiting endocrine functions. Its amount decreases with high levels of insulin and insulin-like growth factor 1, whereas high oestrogen and thyroxine levels cause its increase. Although a lot of studies focus on the regulation of SHBG in human medicine, few studies are performed in ruminants. We aimed at the detection of SHBG in blood of different cattle breeds by mass spectrometry.

After routine slaughtering, blood was collect- ed from three male/female Angus, Aurochs and Galloway having been kept under exten- sive management and in German Simmental and cross bred beef cattle kept in a stable.

After albumin depletion, blood samples were prepared using USP3-technology for LC-MS/

MS measurement. Received peptides were separated by using an UltiMate 3000 HPLC- System and analysed with the Orbitrap Ex- ploris™ 480 mass spectrometer. Among 135 proteins successfully identified by LC-MS/

MS, this technique also identified peptides related to the SHBG protein. The SHBG- related peptides were different in the number between the ruminant breeds with lowest mean number in Aurochs (2) and Angus (3), and highest mean number in German Sim- mental (8) and Galloway (7). Although the evaluation of sex- and age-related differences was not applicated because of the low number of animals, our data suggest the influence of breed on the SHBG amount in bovine blood.

Further studies are needed to confirm the dependency of SHBG on breed and other characteristics.

*Supporting Organisations: Deutsche Veterinärmedizinische Gesellschaft (DVG) and Deutsche Gesellschaft für Reproduktionsmedizin (DGRM). With permis- sion of Wiley, the abstracts of this conference will be jointly published in the Journal of Reproduction of Domestic Animals (RDA) and the Journal of Repro- ductive Medicine and Endocrinology (JRE). Peer-reviewed and compiled by the scientific committee. Index of authors (only primary authors) see page 19.

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04

The effects of L-lactate are putative- ly mediated via NMDA receptor associated signaling pathways in bovine granulosa cells

Die Effekte von L-Laktat werden ver- mutlich über NMDA-Rezeptor-asso- ziierte Signalwege in bovinen Granulo- sazellen vermittelt

A. Baufeld, J. Vanselow

Institute of Reproductive Biology, Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany Earlier, we showed that L-lactate acts as a signaling molecule in cultured bovine granu- losa cells (GCs). Since the signal transduction of L-lactate is dependent on lactate transport- ers, we further aim at elucidating the intra- cellular mechanisms of L-lactate. N-methyl- D-aspartate receptors (NMDARs) have been highlighted to be involved in L-lactate sign- aling in neurons. From our whole genome mRNA study, we obtained several hints that NMDAR signaling might be also present in GCs. In this study, we initially focused on NMDA receptor action in L-lactate treated GCs. Bovine GCs were cultured serum-free and supplemented with FSH, IGF-1 and an- drostenedione for 8 days (37 °C, 5% CO₂).

The cells were additionally cultured either with 30 mM sodium L-lactate or with 30 mM NaCl as vehicle control. Inhibitor studies were performed in a pre-treatment approach (48 h) with the potent NMDAR antagonists D-AP5 and MK801. Afterwards, GCs were characterized by analyzing the hormone con- centration in spent media by RIA as well as the transcript abundance of selected marker genes by qPCR. The L-lactate induced effects on the expression of CYP19A1, FSHR, LH- CGR, RGS2 and VNN2 were partially abol- ished by the pre-treatment with D-AP5 and MK801. So far, we can conclude from these results that the action of L-lactate might be additionally mediated via NMDARs. These new findings of NMDAR action in GCs are not only of interest for bovine reproduction, but may also be interesting for other species.

05

Growth dynamic of multidrug re- sistant bacteria and sperm quality in preserved boar semen

Wachstumsdynamik multiresistenter Bakterien und Spermienqualität in konserviertem Ebersamen

C. M. H. Beckermann¹, A.M. Luther¹, T.Q. Nguyen¹, J. Verspohl², D. Waberski¹

1Unit for Reproductive Medicine of Clinics/Clinic for Pigs and Small Ruminants, University of Veterinary Medicine, Han- nover, Germany; ²Institute for Microbiology, University of Veterinary Medicine Hannover, Hannover, Germany Multidrug resistant bacteria in preserved se- men may affect sperm quality and contrib- ute to the global antibiotic resistant threat.

With loss of antibiotic efficiency, alternative measures for control of bacterial growth are required, especially in boar semen which is usually stored at a temperature (17 °C) that

promotes bacterial growth. The aim was to study the growth dynamic of two resistant bacterial strains in preserved boar semen and to determine their impact on sperm quality.

In this, the role of storage temperature and extender were elucidated. Semen extender Beltsville Thawing Solution (BTS) was spiked with 10⁴ CFU/ml Klebsiella oxytoca (K. oxyto- ca) or Serratia marcescens (S. marcescens) iso- lated from semen samples. Bacterial counts increased exponentially during storage for 144 h at 17° C to 3 × 10⁶ CFU/ml (K. oxytoca) and 1.6  ×  10⁷ CFU/ml (S. marcescens). In extended semen (n =7 boars) spiked with the resistant bacteria, computer-assisted sperm motility traits decreased (p < 0.05) at bacterial counts > 10⁹ CFU/ml for K. oxytoca and > 10⁷ CFU/ml for S. marcescens. Flow cytometry values of plasma membrane and acrosome integrity (propidium iodide and FITC-PNA) were reduced at 10⁷ CFU/ml for S. marces- cens but not K. oxytoca compared to controls (p > 0.05). In semen extender Androstar Pre- mium hold for 144 h at 5 °C, K. oxytoca de- creased from the initial inoculation with 10⁴ CFU/ml by 1.5 log-levels and S. marcescens by 2 log levels (p < 0.05). In conclusion, storage of boar semen at 5 °C in Androstar Premium opens the perspective to control the growth of resistant bacteria below values that affect sperm quality.

06

Imbalanced nutrition, and FSH- induced ovarian hyperstimulation, exert transcriptomic effects on ovine caruncular endometrium

Unausgewogene Ernährung sowie FSH-induzierte ovarielle Hyperstimula- tion verursachen transkriptomische Effekte im karunkulären Endometrium beim Schaf

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

1Institute of Veterinary Anatomy, Vetsuisse Faculty, Univer- sity of Zurich, Switzerland; ²Department of Animal Sciences, North Dakota State University, Fargo, USA

Imbalanced nutrition and exogenous gonado trophins affect uterine function and morphology. In sheep, FSH-induced super- ovulation causes morphological alterations in the uterus, leading, i.e., to an increase in lipid droplets. Similarly, depending on diet, FSH treatment alters the expression of es- trogen and progesterone receptors. Based on these observations, we investigated the effects of imbalanced nutrition and FSH treatment on the caruncular endometrium, using deep RNA sequencing (NGS, RNA-Seq). Ewes (n  =  4–5/group) were divided as follows:

normal fed (NF), overfed (OF) and underfed (UF), and each group was further treated with FSH or saline (negative controls [C]). Samples were collected on days 5 and 10 of diestrus.

Gene ontologies, canonical pathways and upstream regulators were evaluated in differ- ently expressed genes (DEGs, p < 0.01, FDR

< 0.05) between days 5 and 10 (NF_C), as well as in response to FSH under different feeding conditions on day 10 (corresponding to the

time of embryonal recognition in pregnant animals). The 1484 DEGs identified between days 5 and 10 were associated with increased immune activity and cellular metabolic pro- cesses. A higher number of DEGs were iden- tified in response to FSH in NF (1374) than in OF (168) or UF (18) samples. In NF, enriched DEGs after FSH treatment were associated with increased cell growth, proliferation, and differentiation, and decreased immune activ- ity, cell-cell, and cell-ECM adhesion. These and other results suggest that FSH has multi- directional effects on local uterine function and the expression of factors involved in im- plantation, depending on diet.

07

Comparison of antioxidative and ox- idative profiles in plasma and saliva in dairy cows during pregnancy Vergleich der antioxidativen und oxi- dativen Profile in Plasma und Speichel bei Milchkühen während der Trächtig- keit

A. Bielecka, M. Jamioł, M. Kankofer

Department of Biochemistry, Faculty of Veterinary Medi- cine, University of Life Sciences in Lublin, Lublin, Poland Body fluids are a good source to get knowl- edge about metabolic status. Currently, non- invasive body fluid collection is not possible.

During pregnancy changes in the intensity of metabolic processes take place which can lead to the formation of oxidative stress. The study aimed to investigate the usefulness of saliva for monitoring the pregnancy status. The an- tioxidant and oxidant profiles of plasma and saliva during bovine pregnancy were com- pared. Seventy-six health pregnant and twelve non-pregnant control cows were included in the study. Blood and saliva samples were col- lected monthly during pregnancy. Fluids were used to evaluate the total antioxidant capacity (TAC) and the oxidative parameters related to protein and lipid peroxidative processes.

TAC, the content of hydroperoxides, and SH groups were determined spectrophoto- metrically. Dynamic changes depending on the period of pregnancy course were detected and revealed that the increase in oxidative in- tensity induced an appropriate answer of the body. The highest antioxidant activity in plas- ma was mostly noted in early and advanced pregnancy. All tested parameters except SH groups expressed higher values in saliva com- pared to plasma. However, with regards to antioxidant/oxidative parameters, saliva re- flects the content of plasma only in part, due to the local metabolism of the salivary gland.

Moreover, some examined parameters can indicate the intensity of oxidative stress and therefore could be used in a panel of markers.

Therefore, further studies are necessary to es- tablish physiological ranges of antioxidative/

oxidative profiles of body fluids in cows.

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08

Comparative treatment of kisspep- tin alone or in combination with cabergoline for estrus induction of bitches

Vergleichende Behandlung von Kiss- peptin allein oder in Kombination mit Cabergolin zur Brunstinduktion bei Hündinnen

G. Bozkurt¹, A.R. Ağaoğlu¹, A. Çortu¹, S. Aslan², A. Wehrend³, S. Schäfer Somi⁴

1Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Burdur Mehmet Akif Ersoy University, Burdur, Turkey; ²Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Near East University, Nico- sia, Turkey; ³Clinic for Obstetrics, Gynecology and Andrology, Justus-Liebig-University Giessen, Germany; ⁴Platform Insemination and Embryo Transfer, Vetmeduni Vienna, Austria

The aim of this study is to reveal the repro- ductive effectiveness of the use of cabergoline (CB, Galastop®) and kisspeptin (KP, KP-10) alone or in combination on reproductive hor- mones in bitches which were randomly di- vided into 5 groups as G1 (n = 4), G2 (n = 4), G3 (n = 4), G4 (n = 6), G5 (n = 7). Groups and treatments were as follows: G1: 0.09% NaCl (single dose, iv = SDI) + CB (5 µg/kg, once daily p.o., for 14 days = OD14), G2: KP (KP- 10, 0.5 µg/kg, SDI) + CB (5 µg/kg, OD14), G3: KP (KP-10, 1 µg/kg, SDI) + CB (5 µg/

kg, OD14), G4: KP (KP-10, 0.5 µg/kg, SDI) + 0.09% NaCl (OD14), G5: KP (KP-10, 1 µg/kg, SDI) + 0.09% NaCl (OD14). Blood samples (S) were taken 40 minutes before treatment (S1) and immediately before it (S2), as well as 10 (S3), 20 (S4), 60 (S5) and 120 (S6) minutes after treatment. Serum collection was con- tinued on the 3^rd (S7), 6^th (S8), 9^th (S9), 12^th (S10) and 15^th (S11) day after treatment. No significant difference could be determined between the S1 and S2 samples, and sam- ples taken in the later periods for E2 levels.

With regard to P4, a statistically significant difference was found between S1 (2.47  ng/

ml), S7 (1.41 ng/ml) and S10 (0.87 ng/ml) in group G1 (p < 0.05). A significant differ- ence was determined between the P4 values before and after the treatment in groups G3 (S1 0.41 to S7 0.14 ng/ml), G4 (S1 0.57 to S8 0.23 ng/ml) and G5 (S1 7.73 to S11 0.19 ng/

ml). Values continuously decreased up to the S10 (p < 0.05). Results reveal an impact of KP alone or in combination with CB P4 concen- trations, an eventual effect on FSH and LH secretion is currently investigated.

Grants: Funded by Polish National Agency for Academic Exchange under Grant No. PPI/

APM/2019/1/00044/U/00001‘

09

Sperm quality and bacterial growth in hypothermic, antibiotic-free pre- served semen of boars in Vietnam Spermienqualität und Bakterienwachs- tum in hypothermem, Antibiotikum- frei konserviertem Sperma von Ebern in Vietnam

H.D. Bui¹, T.Y. Dinh¹, H. Dang¹, K.D. Pham¹, D. Waberski²

1Faculty of Animal Science, Vietnam National University of Agriculture, Hanoi, Vietnam; ²Unit for Reproductive Medi- cine of Clinics, University of Veterinary Medicine Hannover, Hannover, Germany

Recently, preservation of boar semen at low temperature was introduced in Germany and South America to reduce the antibiotic use in pig reproduction. The efficiency of the new preservation method may depend on boar breeds and environmental bacteria. The aim was to evaluate the effect of hypothermic preservation on sperm quality and bacteria load in semen of boars located in Vietnam.

Semen of 30 boars was stored in Beltsville Thawing Solution with antibiotics at 17 °C (control) and antibiotic-free AndroStar Pre- mium (APrem) at 5 °C. Boar spermatozoa preserved in APrem at 5 °C maintained high motility above 75% after 5 days of storage;

values did not differ from control at 17  °C (p < 0.05). The proportion of abnormal sper- matozoa in semen samples stored at 5 °C in APrem extender was similar to control sam- ples stored at 17 °C (p > 0.05). The number of aerobic bacteria in semen samples stored at 5  °C in the antibiotic-free extender was lower than 10³ CFU/ml after 24, 72 and 120 h preservation, and did not differ from raw semen and control samples stored at 17  °C with antibiotics (p > 0.05). In conclusion, the low temperature semen preservation strategy proofed to be effective in Vietnamese boars to reduce the use of antibiotics in livestock.

Grants: Funded by ARES-CCD (Académie de Recherche et d‘Enseignement Supérieur – Com- mission de la Coopération au Développement in the frameword of the Institutional Support to Vietnam National University of Agriculture) and semen extender media were supported by Minitube (Tiefenbach, Germany).

10

Effects of an apolipoprotein E knockout on the expression of meta- bolic and developmental target genes in rabbit reproductive organs during preimplantation

Auswirkungen eines Apolipoprotein-E- Knockouts auf die Expression von Stoffwechsel- und Entwicklungsziel- genen in den Fortpflanzungsorganen des Kaninchens während der

Präimplantationsphase

M. Buske¹, J. Thoma¹, J. de Nivelle¹, J.-S. Jung¹, A. Ritsch², E. Halbauer¹, A. Toto Nienguesso¹, K. Zech¹, M. Schindler¹, A. Navarrete Santos¹

1Department of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany;

2Medical University Innsbruck, Austria

The laboratory rabbit (Oryctolagus cuniculus) is widely used as an animal model due to its close resemblance to human physio logy in cholesterol and fatty acid metabolism as well as in early embryo development. The aim of our study was to investigate the apolipopro- tein E knockout rabbit (ApoE^-/-) in terms of reproductive effects and expression of meta- bolic and developmental target genes. ApoE^{- /-} rabbits have an altered lipid metabolism,

resulting in elevated levels of plasma choles- terol and triglycerides [Niimi, Atherosclerosis 2016; 245: 187–93]. We obtained plasma, liver, ovary and endometrium samples, as well as preimplantation embryos from APOE(^-/-) and wild type rabbits at day 6 post coitum. Using RT-qPCR and Western Blot technologies, the expression of marker genes involved in lipid metabolism and embryo development were analysed. Plasma and liver analyses revealed metabolic changes with increased cholesterol, triglycerides, low-density lipoprotein, very low-density lipo-protein receptor and car- nitine acyltransferase 1 levels and decreased fatty acid synthase level. The systemic meta- bolic alterations were reflected in the female reproductive tract organs. In addition to meta- bolic changes, a strong increase of insulin-like growth factors, IGF1 and IGF2, indicated an effect on preimplantation processes in the en- dometrium. The ApoE^-/- rabbit produced sig- nificantly lower amounts of embryos (Mean ± SD: 7.1 ± 2.3 vs. 10.2 ± 3.7), demonstrating the loss in fertility at the preimplantation stage.

Data indicate that an ApoE dysregulation and systemic hypercholesterolemia are crucial fac- tors for female fertility.

Grants: Funded by DFG GRK2155 ProMoAge and JU3146

11

NET formation of polymorpho- nuclear neutrophils isolated from ovine colostrum

NET-Bildung polymorphkerniger neutrophiler Granulozyten aus ovinem Kolostrum

L. Demattio¹, D. Grob², I. Conejeros², A. Taubert², C. Hermosilla², A. Wehrend¹

1Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals of the Justus-Liebig-University Giessen, Germany; ²Institute of Veterinary Parasitology of the Justus- Liebig-University Giessen, Germany

Colostral leukocytes have been neglected in colostrum research for a long time, but are increasingly recognized as important fac- tors for the transfer of maternal immunity to the infants. Polymorphonuclear neutrophils (PMN) represent a subtype of colostral leu- kocytes, able to migrate into colostrum and through the neonate’s intestine. Our study focuses on PMN and their early innate effec- tor mechanisms. PMN have been shown to form neutrophil extracellular traps (NETs) against pathogens in other locations. In our study, PMN were isolated from ovine colos- trum by density gradient centrifugation us- ing Biocoll-Separation® solution. Afterwards, ovine PMN were co-cultured with Neospora caninum tachyzoites. The calcium ionophore A23187 (Merck, Munich) was used as positive control. For negative controls, unstimulated PMN were equally processed. NETs forma- tion was detected by immunofluorescence mi- croscopy. Here, DAPI was used to stain DNA and specific antibodies to detect neutrophil elastase (NE) and histones to demonstrate the characteristic co-localization of these specific molecules in NETs structures. These results show that ovine colostral PMN indeed

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respond to stimulation with A23187 or N.

caninum tachyzoites by forming NETs. This is the first evidence that ovine colostral PMN are capable of casting NETs. Further knowledge on the immunological relevance of colostral leukocytes is urgently needed to improve the current understanding the neonatal immune system. The current data might lead to a better understanding of lamb diseases.

12

Influence of desemination frequen- cy on aspects of semen analysis in stallions

Einfluss der Deseminationsfrequenz auf Aspekte der Samenuntersuchung beim Hengst

L. Demattio, C. Schepull, A. Wehrend

Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals, Justus-Liebig-University Giessen, Germany For stallions that are used for the production of frozen semen, it is recommended to collect semen several times before the actual semen production especially after a longer sexual abstinence, in order to improve the semen quality. But is this necessary? In the present study 29 stallions from different breeds were examined. The ejaculates were collected three times, after a variable time of sexual absti- nence on day 1, 2, and 4. Afterwards they were examined and frozen. The thawed semen por- tions were also examined. The examination was performed using a CASA system. Exami- nation of the native semen showed a non-sig- nificant decrease in sperm density (p = 0.23) and ejaculate volume (p  =  0.17) for three consecutive semen collections. Progressive motility of spermatozoa in native ejaculates was significantly increased by multiple semen collections (p < 0.0001), and the proportion of immobile spermatozoa was significantly reduced (p = 0.0002). In the thawed semen samples, the progressive motility of sperma- tozoa was significantly increased by multiple semen collection (p = 0.0016); so was the overall motility of spermatozoa (p = 0.0013).

Thus, it was shown that in deed multiple se- men collection in a certain time interval has a positive effect on some semen examination parameters in both native and thawed semen.

However, it was also shown that mostly, al- ready the first ejaculates in native state cor- responded to the actual expert opinions on the suitability for frozen semen production, this number was increased by 3.4% after 24h and 17.2% after 48h. After thawing none of the ejaculates was able to fulfill the experts´

opinions on semen quality.

13

Non-ischemic priapism in male dromedary: A case report Nicht-ischämischer Priapismus bei männlichen Dromedaren: Ein Fall- bericht

D. Derar^1,2, A. Ali^1,2, M. Al-Zaggafy^3,4, H. Al-Moghazy^3,5

1Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim, Saudi Arabia; ²Depart- ment of Theriogenology, Faculty of Veterinary Medicine,

Assiut University, Egypt; ³Veterinary teaching Hospital, Qassim University, Saudi Arabia; ⁴Department of Microbiol- ogy, Faculty of Veterinary Medicine, Khartoum University, Sudan; ⁵Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Benha University, Egypt

An 8-year-old dromedary camel presented to the veterinary teaching clinic, Qassim Uni- versity, Saudi Arabia with a history of persis- tent erection for almost 4 days after natural mating with a female in estrus pre-prepared by a mixture of dates, margarine, herbal weeds and olive oil (prescribed by an ethno veterinarian). Approximately, 20–30 episodes of erectile activities/hour were observed four days before the admission and each lasted for 1–3 minutes. Upon admission the animal showed the same symptoms in addition to it behaved as if it tries to masturbate. Clini- cal examination showed enlarged pendulous prepuce, persistent penile movement and restlessness. Abstaining from food, the ani- mal lost weight and general body condition looked unthrifty. The animal was venipunc- tured to obtain a complete hemogram. The complete blood count revealed decrease in the total leucocytic count, hemoglobinemia and total leukocytopenia. The animal pre- scribed fluid therapy, topical anesthetic oint- ment, analgesic, antipyretic and muscle relax- ant. The symptoms were temporarily relieved and resumed after 6 hours intensively. Finally, the animal was euthanized. To our knowl- edge, this is the first report of non-ischemic priapism in dromedary. Apart from the conventional reproductive disorders in male dromedary, folk medicine is a dilemma which should not be overlooked in camel breeding soundness examination.

14

The use of infrared thermography to determine the time of estrus in dromedary camels

Die Verwendung von Infrarot-Thermo- graphie zur Bestimmung der Brunst- zeit bei Dromedaren

D. Derar^1,2, A. Ali^1,2, M. Tharwat^1,3, Y. Al-Boty¹, A. Alsamri¹

1Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim, Saudi Arabia; ²Depart- ment of Theriogenology, Faculty of Veterinary Medicine, As- siut University, Egypt; ³Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Egypt The aim of the present study was to investi- gate the use of a thermal infrared imaging as a non-invasive diagnostic tool to relate the changes in the rectal, vulval and vaginal temperature to the ovarian findings in female dromedary during the breeding season. The study was carried out on 53 female drom- edaries admitted to the veterinary teaching clinic, Qassim University, Saudi Arabia for routine breeding soundness examination.

Immediately after admission, the tempera- ture of the vulva, vagina and rectum was measured and recorded using a FLIR C2 ther- mal imaging camera for each animal by the same personnel and at fixed distance at each examination. Then, data including breeding history, general health examination, external and internal gynecological examination using

ultrasonography were recorded and analyzed.

The stage of the follicular cycle of each animal was estimated according to the ovarian find- ings and history of last estrus. Data was tabu- lated and statistically analyzed. Results of the present study showed that the stages of the estrus cycle of the investigated animals could be categorized into 5 stages relative to the ovarian findings which are growing follicle, mature follicle, atretic follicle and non-follic- ular. Animals in the mature-follicle stage had a significantly (p < 0.05) higher temperature measured at any site compared with other phases of the cycle. It can be concluded that Infrared thermography could be a useful tool to detect estrus in female dromedary.

15

Performance of marmoset monkeys as embryo donors is reflected by stress-related parameters

Die Eignung von Weißbüschelaffen als Embryonenspender spiegelt sich in Stress-Indikatoren wider

C. Drummer^1,2, J. Münzker¹, M. Heistermann³, T. Becker⁴, S. Mißbach^2,4, R. Behr^1,2

1Platform Degenerative Diseases, German Primate Center – Leibniz Institute for Primate Research, Göttingen, Germany; ²DZHK, Partner Site Göttingen, Germany; ³Endo- crinology Laboratory, German Primate Center – Leibniz In- stitute for Primate Research, Göttingen, Germany; ⁴Animal Husbandry, German Primate Center – Leibniz Institute for Primate Research, Göttingen, Germany

Genetically modified non-human primates (NHPs) serve as models for human diseases.

Genetic modifications can be achieved by the manipulation of oocytes or embryos.

Therefore, reproductive health of the donors is crucial. Especially during long-term use of NHPs, their health and wellbeing are essen- tial and chronic distress needs to be avoided.

Here, we retrospectively assessed parameters indicating distress in common marmosets (n = 32) undergoing preimplantation embryo retrievals under anesthesia. Animals were either used for extended (long-term group, LTG; > 2.5 years in the study) or only for short periods (short-term group, STG; < 2.5 years in the study) as embryo donors based on their performance. Retrospectively, we calculated the embryo retrieval rates (ERR) and assessed Anti-Muellerian hormone (AMH) concentrations, cortisol levels and body weight fluctuations as a readout for ovarian reserve, distress and compromised well-being. As expected LTG-animals showed a higher ERR than STG-animals (p < 0.0001).

This was found already during the first year of experimental use (p = 0.0002). Importantly, ERR in the LTG remained stable through- out the experimental period up to 5 years indicating no accumulation of distress. High ERR was associated with high AMH and low cortisol levels and significantly reduced body weight fluctuations following anesthesia, sug- gesting a superior ability of the LTG-animals to cope with stress. We conclude that long- term experimental use of marmosets does not impair their fertility or health status per se, and might be in agreement with the 3R- princple of animal experimentation.

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16

Relationship between ultrasono- graphic findings and different blood parameters in pregnant dogs Zusammenhang zwischen Ultraschall- befunden und verschiedenen Blutpa- rametern bei trächtigen Hunden

E. Evci¹, S. Aslan¹, O. Ergene¹, I. Darbaz¹, K. Seyrek-Intaş¹, S. Sayıner², A. Wehrend³

1Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Near East University, Nicosia, Cyprus;

2Department of Biochemistry, Faculty of Veterinary Medi- cine, Near East University, Nicosia, Cyprus; ³Clinic of Obstet- rics, Gynecology and Andrology of Large and Small Animals with Ambulatory Service, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Germany

The aim of the study was to reveal the changes in C-reactive protein (CRP), Anti-Müllerian hormone (AMH), and hemogram (He) val- ues in female dogs at different periods of pregnancy. Thirteen (G1) dogs that became pregnant when mated and 7 dogs not preg- nant after mating (G2) were included in the study. Ultrasonography examinations (US) were performed on days 25, 35, 45, and 55 pc to determine the fetus number, and blood samples were taken from the 12^th day after mating (vaginal cytology, P4 > 5 ng/ml) up to the first postpartum day (GE1), including the US period. In the G2 group, blood was drawn at the same intervals until the 55^th day and until the 63rd day, respectively. No changes could be detected in terms of AMH in the periods in which blood was drawn in G1 and G2. However, there were significant differences in the number of litter and AMH values on the 12^th and 45^th days of pregnancy (p < 0.05; p < 0.01). In terms of CRP values, significantly higher values (38.26/15.66 and 32.54/15.97 mg/L; p < 0.05) were obtained in the G1 group on the 25^th and 35^th days com- pared to the G2 group. On the 45^th day, the difference between G1 and G2 was p < 0.01.

It was determined that CRP values were high- est on the 25^th and 35^th days in pregnant dogs and were significantly different (p < 0.01) compared to the 12^th day. Such a difference in G2 was not determined in terms of CRP. In terms of He parameters (WBC, Gran, HCT), a significant difference was obtained between G1 and G2 on the 55^th day of pregnancy. The results obtained reveal that CRP values may be a parameter in the monitoring of preg- nancy periods.

17

Influence of a sperm pre-incubation on development and sex-ratio of in vitro produced bovine embryos Einfluss einer Spermien-Präinkubation auf die Entwicklung und das Ge- schlechterverhältnis in vitro produzier- ter boviner Embryonen

A. Fries, B. Zimmer, B. Rabenau, F. Kotarski, C. Wrenzycki Chair for Molecular Reproductive Medicine, Clinic for Veteri- nary Obstetrics, Gynecology and Andrology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Germany

The aim of the present study was to investi- gate whether a bull-specific sperm pre-incu- bation prior to in vitro fertilization may influ- ence embryonic development and sex-ratio.

For each of the 3 bulls (Bull A, B, C) used for IVF, capacitation time was assessed by chlor- tetracycline staining. Therefore, 90 (Bull A) or 180 (Bull B, C) minutes before IVF cryo- preserved/thawed sperm was prepared using a standard protocol and was pre-incubated in FertTALP. This pre-incubation step was omit- ted for sperm of all 3 bulls serving as control.

After 19 hours of co-incubation of cumulus- oocyte-complexes and sperm, fertilization rate was analysed via Hoechst staining. In vitro culture was carried out up to 8 days, at days 7 (d7) and 8 (d8) cleavage and devel- opmental rates were recorded, and embryos were collected for sexing PCR. Cleavage rate was higher for embryos after IVF with pre- incubated sperm of bull A (82.6% vs. 77.8%;

p < 0.05) and at d7 more hatching blastocysts could be detected compared to Control (2.4%

vs. 0.0%; p < 0.05). There were more male embryos in total in the pre-incubated group of Bull B (91.7% vs. 66.7%; p < 0.05). Inter- estingly, the overall sex-ratio of bull C in the pre-incubated treatment was not different to Control however the sex-ratio of d8 embryos (blastocysts, expanded blastocysts) and blas- tocysts (d7, d8) was more balanced (58.3%, 50.0% males) compared to embryos resulting from non-incubated sperm (90.9%, 90.0%

males; p < 0.05). Taken together, pre-incu- bation of sperm might have a bull depend- ent influence on cleavage rate, proportion of hatching blastocysts at day 7 and embryonic sex-ratio.

18

Unhatched bovine blastocysts ex- press all transcripts of the estrogen biosynthetic pathway, whereas the corresponding enzymes are not yet detectable

Ungeschlüpfte Rinderblastozysten exprimieren alle Transkripte des Östro- gen-Biosynthesewegs, die entspre- chenden Enzyme sind jedoch noch nicht nachweisbar

R. Fürbass¹, M. Michaelis¹, G. Schuler²

1Institute of Reproductive Biology, Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany;

2Veterinary Clinic for Obstetrics, Gynecology and Andrology, Faculty of Veterinary Medicine, Justus-Liebig- University Giessen, Germany

Embryos of rodent species and rabbits, but also farm animals such as pigs, horses and cattle produce estrogens around implanta- tion. Embryonic estrogens are involved in regulating the implantation process. In cat- tle, the embryonic stage at which estrogen synthesis commences is yet unknown. How- ever, to understand early embryogenesis it is important to know whether, for example, locally produced estrogens are involved in the formation and further development of blas- tocysts. Therefore, in this study, we first used quantitative reverse transcription PCR to ex- amine the mRNA expression of the enzymes

necessary for the conversion of cholesterol into free and sulfonated estrogens (STAR, CYP11A1, CYP17A1, HSD3B, CYP19A1, and SULT1E1) as well as the estrogen recep- tors ESR1 and ESR2 in in vitro produced mor- ulae and blastocysts. Only in the blastocysts, the mRNAs of the entire estrogen biosynthe- sis chain and of both estrogen receptors were clearly present. We further assessed the ex- pression of the proteins CYP11A1, CYP17A1, CYP19A1, and SULT1E1 in blastocysts using capillary-based western analysis, but were unable to detect any of these enzymes. From our data, we conclude that early, unhatched bovine blastocysts are not capable of produc- ing estrogens or any steroid hormones from cholesterol on their own.

19

Increased insulin-like growth factor 1 and interferon tau signaling in pregnant cows with an extended voluntary waiting period of 120 days

Erhöhtes Insulin-ähnlicher Wachstums- faktor 1- und Interferon tau-Signaling bei trächtigen Kühen mit einer verlän- gerten freiwilligen Wartezeit von 120 Tagen

C. Gabler¹, S. Widera², J. Huber^1,2, C. Holder¹, L. Jessen², K. Mense², M. Jung², M. Schmicke³

1Institute of Veterinary Biochemistry, Freie Universität Berlin, Germany; ²Institute for the Reproduction of Farm Animals Schönow, Bernau, Germany; ³Animal Health Management, Martin-Luther University (MLU) Halle- Wittenberg, Germany

High rates of embryonic mortality after con- ception indicate that the embryo-maternal communication (EMC) is impaired leading to subfertility. It was hypothesized that ex- tension of the voluntary waiting period from 42 days (WTP42) to 120 days (WTP120) postpartum (pp) improves EMC and hence improve fertility. Insulin-like growth factor 1 (IGF1) is crucial for fertility. Therefore, we evaluated IGF1 signaling as well as interferon (INF) receptor subunit 2 (INFAR2) and pap- palysin 1 (PAPPA1) for INF tau signaling for embryo recognition. Endometrial samples were obtained by cytobrush from cows at day 35–41 pp, 115–120 pp and 1 day after artificial insemination (AI), respectively. In addition, IGF1 protein levels in the serum were assessed. Pregnancy diagnosis was per- formed 32 days after AI by sonography. Ten dairy cows each were classified as pregnancy positive (PREG-POS) or pregnancy negative (PREG-NEG) for each WTP. Isolated total RNA from endometrial samples was subjected to RT-qPCR. IGF1 protein levels were higher at day 1 after AI in WTP120 compared to WTP42 and lower at day 118 pp in WTP120 PREG-POS group compared to the PREG- NEG group. Increased mRNA expression of IGF1 receptor at day 1 after AI in WTP42 was observed in the PREG-POS group compared to the PREG-NEG group. An increase of the mRNA expression of INFAR2 and PAPPA1 was observed at day 1 after AI compared to day 115–120 pp in the PREG-POS group. The

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findings confirms that IGF1 and INF signal- ing are important for conceiving, possibly leading to reduced embryo mortality (0%) in the WTP120 group compared to WTP42 (10%).

Grants: Supported by LfULG.

20

Effects of the litter size of hemato- logical parameters in bitches

Einfluss der Wurfgröße auf die hämato- logischen Parameter bei der Hündin

P. Georgiev¹, S. Yotov¹, T.S. Ivanova¹, A. Wehrend²

1Department of Obstetrics, Reproduction and Reproductive Disorders, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria; ²Clinic for Veterinary Obstetrics, Gynecology and Andrology, Justus-Liebig-University Gies- sen, Germany

The aim of this work was to determine the effects of the litter size on hematological parameters in bitches according to course of pregnancy. In 75 animals (19 large and medium sized breeds) 90 pregnancies were followed. Starting at estrus to the end of preg- nancy for five monitored periods in parallel to clinical examinations blood samples were taken. We determined hematocrit (HCT), red blood cells (RBC), hemoglobin (HGB), mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, platelets (PLT) and mean platelet volume, leukocytes and differential blood count. Ani- mals were divided into two groups – first (I), normal and second (II) bitches with patho- logical pregnancy. Additionally each group was divided into subgroup A – small (up to 4 puppies) and subgroup B with normal lit- ter size (5–12 puppies). The changes of HCT, RBC and HGB were most pronounced. In subgroups IB and IIB in the last third and in subgoup IIA in the second half of pregnancy normocytic normochromic anemia was found. At the end of pregnancy in subgroups IB and IIB low mean HGB levels were detect- ed. Significant differences in HCT, RBC and HGB between distinct periods in subgroup IB and IIB were found. At the end of pregnancy the mean values of HCT, RBC and HGB in subgroup IA and in subgroup IB were 37.6 ± 4.2%, 5.7 ± 0.5T/L, 143.5 ± 13.2 g/l and 30.7

± 5.0 %, 4.6 ± 0.6T/L, 116.9 ± 17.9 g/l respec- tively (p < 0.01). Most other parameters were within norms. Although the PLT was highly, over the reference range individualized. We assume that in physiological pregnancy, the size of the litter affects the hematological pa- rameters.

21

Do probiotics supplemented during

in vitro maturation of bovine

oocytes improve the subsequent in

vitro development of embryos?

Kann ein Probiotika-Einsatz während der In-vitro-Maturation die resultieren- de In-vitro-Entwicklung der Embryo- nen verbessern?

O. Granacher¹, C. Gabler², C. Wrenzycki¹

1Chair for Molecular Reproductive Medicine, Clinic for Veteri- nary 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 Probiotics have positive effects on the organ- ism and reproduction. Bacteria of the genus lactobacillus are among others the best inves- tigated probiotics. As part of the uterine mi- crobiome of cows, Lactobacillus buchneri has been detected. The objective of this study is to determine the effect of Lactobacillus buch- neri on in vitro maturation (IVM) of bovine oocytes. Ovaries from a local slaughterhouse were used. By slicing, cumulus-oocyte-com- plexes were isolated from ovarian follicles for in vitro production. Different amounts (1–10%) of the IVM medium were replaced by Lactobacillus buchneri-conditioned tis- sue culture medium (TCM). The recorded cleavage and development rates differed, de- pending on how much conditioned medium was used. The experimental group with 5%

conditioned medium showed higher de- velopment rates than the groups with 10%

and 1%. Furthermore, the maturation rates showed the same concentration-dependent differences. In addition, the morphology of cumulus-oocyte-complexes varied after mat- uration in different amounts of Lactobacillus buchneri-conditioned medium. These results might suggest that Lactobacillus buchneri has an influence on maturation of bovine oocytes and resulting embryos.

22

V-set and immunoglobulin domain containing 10 like (VSIG10L) – an un- known player in the male reproduc- tive tract

V-set and immunoglobulin domain containing 10 like (VSIG10L) – ein unbekannter Spieler im männlichen Reproduktionstrakt

J. Günther, K. Zlatina, S.P. Galuska

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

VSIG10L is a transmembrane glycoprotein with an extracellular region consisting of several immunoglobulin-like domains and a large N-terminal intrinsically disordered region (IDR). Remarkably, a functional role of this protein has only been described in the context of esophagitis and esophageal ad- enocarcinoma. Here, VISG10L is assumed to contribute to the maintenance of the epithe- lial barrier and/or to modulate inflammatory processes. Interestingly, RNAseq data indicate that Sertoli cells are one of the main expres- sion sites of VSIG10L. Sertoli cells constitute the principal compartment of the blood-testis barrier (BTB) and express immunomodula- tory factors. The BTB is necessary for the formation of an immune privileged micro- environment, which is essential for spermato- genesis. Since VSIG10L seems to be relevant for both, the formation of an epithelial bar- rier and the modulation of inflammation, we want to investigate the role of this protein in the testis. This includes the identification of

interaction partners as well as a functional characterization of VSIG10L. We initially fo- cused on the N-terminal IDR, since multiple sequence alignments revealed that this region exists only in mammals. IDRs are known to be predominant sites of posttranslational modifications and to form bonds with vari- able interaction partners. The IDR of human VSIG10L has four potential N-glycosylation sites whose structure and function we aim to elucidate. For this purpose, we generated re- combinant VSIG10L proteins and performed mutations of relevant elements of the protein structure. This enables us now to characterize VSIG10L structurally and functionally.

23

Detection of vibration emissions during the transport of boar semen Erfassung von Erschütterungsemissio- nen während des Transportes von Ebersperma

T. Hafemeister¹, P. Schulze², R. Bortfeldt¹, R. Grossfeld³, C. Simmet³, M. Jung¹, F. Fuchs-Kittowski², M. Schulze¹

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

In pig breeding today, artificial insemination (AI) doses are delivered from AI centers to sow farms via logistics vehicles. Vibration emissions are suspected to affect the quality of boar semen. At present, however, it is not known which vibrations occur during the transport of AI doses. Therefore, a prototype of a measuring system was built to determine the vibrations by calculating a displacement index (Di). Vibrations were analyzed in stand- ardized road trials (n = 100) on several road types (A: dirt road, B: cobblestone, C: country road with interrupted asphalt, D: motorway) with different speeds (30, 60, 90, 120 and 150 km/h). A two-way ANOVA showed sig- nificant (p < 0.001) differences in Di, depend- ing on road surface and speed as well as an interaction of both factors. Long-term studies under field conditions indicated that 33% of the road surfaces are in good quality and gen- erate only few vibrations (Di ≤ 1), 40% are in a moderate quality with interrupted surface and varying acceleration-deceleration events (Di = 1–2), and 25% of the roads show a bad condition with the consequence of markedly increased vibrations (Di ≥ 2), corresponding to cobblestones or unpaved roads. Particu- larly strong vibrations on rough access roads to sow farms cannot be prevented, but lower speed is expected to reduce the damaging in- fluence on spermatozoa. Overall, more atten- tion should be paid to factors affecting sperm quality during transport. In the future, an Internet of Things (IoT) based solution could enable efficient and complete monitoring of the entire transport process in real time.

Grants: Funded by ZIM (16KNO77341).

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24

Influence of „vaginal seeding“ on immunological parameter of the new born calf

Einfluss von „vaginal seeding“ auf im- munologische Parameter des neuge- borenen Kalbes

T. Harborth¹, H. J. Schuberth², S. Dänicke³, F. Billenkamp³, M. Heppelmann¹

1Clinic for Cattle, ²Immunology Unit, University of Veterinary Medicine, Foundation, Hannover, Germany; ³Institute of An- imal Nutrition, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Braunschweig, Germany In human medicine caesarean section seemed to be a risk factor for the development of im- munologic and metabolic diseases in chil- dren, which can be partially counteracted with “vaginal seeding”, the exposure of the offspring to maternal vaginal fluids directly after caesarean section. The aim of this study was to investigate the effect of “vaginal seed- ing” on immunological parameter of calves born via caesarean section. “Vaginal seeding”

was performed on half of 14 Holstein-Friesian calves born via caesarean section, their muz- zle, nose and rest of the body were swabbed with a towel previously incubated in the ma- ternal vagina. The other calves formed the control group. In the first four weeks of life main leukocytic populations as well as mono- cytic and lymphocytic subpopulations were determined by flow cytometry. Numbers of circulating leukocytes, CD4^-/CD8^- T cells and monocytes, intermediate and non-classical monocytes were not affected by vaginal seed- ing (p > 0.05). PMN, lymphocytes, CD4⁺ T-cells and CD8⁺ T-cells showed differences between both groups (p < 0.05). The results of this study show an immunomodulatory effect of “vaginal seeding” during the first weeks of life. Results of the analysis of microbial sam- ples are still pending.

25

Relations between time of ovulation and fecal estrogen concentration, intravaginal partial pressure of oxy- gen and intravaginal temperature in sows

Zusammenhänge zwischen Ovula- tionszeitpunkt und fäkaler Östrogen- konzentration, intravaginalem Sauer- stoffpartialdruck und intravaginaler Temperatur bei Sauen

B. Hensel¹, R. Großfeld², C. Simmet², M. Jung¹, M. Schulze¹

1Institute for Reproduction of Farm Animals Schönow, Bernau, Germany; ²Minitüb GmbH, Tiefenbach, Germany Artificial insemination is the most important biotechnology in pig production. Appropriate timing is essential to achieve the best possible fertility results and novel methods support- ing manual estrus control by determining ovulation time more accurately would be of great benefit. Against this background, the evaluation of fecal estrogen concentra- tions (EC), intravaginal partial pressure of oxygen (IVPO₂ in mmHg) and intravaginal temperature (IVT in °C) and their respective

suitability for ovulation detection was the ob- jective of this study. Fourteen DanBred sows (parity: 5.2 ± 2.4) were checked for ovulation via ultrasound scan every 8h for 72h, start- ing 80h after weaning. Fecal samples were analyzed for their EC, furthermore, IVPO₂ and IVT were measured using a fluorescence- based technique (OxyLite™, Oxford Optro- nix, United Kingdom). On average, sows ovulated 121 ± 10h after weaning and 16 ± 9h after onset of heat. There was a significant drop in fecal EC 4h before ovulation (bo), when compared to all other time points (bo:

20h/12h: p = 0.05/0.003; after ovulation ao:

4h/12h: p < 0.001). We found no significant differences in the IVPO₂ or IVT at any of the measured time points (bo: 20h: p ≥ 0.31/0.37, 12h: p ≥ 0.31/0.36, 4h: p ≥ 0.30/0.40; ao: 4h:

p ≥ 0.70/0.40, 12h: p ≥ 0.30/0.36). Conclud- ing, neither the IVPO2 nor the IVT are suit- able parameters for ovulation detection. On the other hand, there is a relation between ovulation time and fecal EC, however, signifi- cant differences in the sow-individual estro- gen concentrations must be considered.

Grants: Funding by AiF (ZF4276703TS9).

26

Intrauterine ozone application as a possible treatment of chronic infec- tious endometritis in mares

Intrauterine Ozonapplikation als mög- liche Behandlung der chronisch-infek- tiösen Endometritis bei Stuten

L. Hofbauer^1,2, M. Köhne², A. Görgens¹, H. Sieme²

1Clinic for Horses Mühlen, Steinfeld, Germany; ²Unit for Re- productive Medicine – Clinic for Horses, University of Veteri- nary Medicine Hannover, Foundation, Hannover, Germany Chronic-infectious endometritis (CIE) is one of the major problems in equine reproduction and usually treated with antimicrobial drugs.

The aim of the study was to examine the effect of intrauterine (i.u.) ozone application on i.u.

bacterial growth and possible side effects on the endometrium in a clinical setting. Mares (n = 30) with signs of endometritis (i.u. fluid, positive uterine bacterial culture and cytolo- gy) were assigned randomly to different treat- ments (tx): i.u. insufflation of ozone-air-mix (120 ml, 80 µg ozone/ml) twice with a 48h- interval (group A; n = 10), systemic antibiotic tx with trimethoprim-sulfadiazine (30  mg/

kg, p.o., twice daily) for five days (group B; n = 10) or i.u. insufflation of air (120 ml, sterile-filtered) twice with a 48h-interval (group C; n = 10). Endometrial biopsy for histological examination was obtained before tx. A control examination (transrectal ultra- sound, uterine bacterial culture, cytology and biopsy) was performed 7 days after the last tx in all mares. Overall bacterial growth was reduced in every group after tx (p < 0.05), ir- respective of group (group A: 4/10 (positive culture after tx/number of mares); group B:

3/10 and group C: 6/10; p > 0.05). However, tx in groups A and B (p < 0.05) was more ef- fective in reducing growth of gram-negative bacteria compared to group C (p > 0.05). No tx effect on number of polymorphnuclear

granulocytes (cytology) was observed (p

> 0.05). Histological examination revealed no differences among groups, except for in- creased number of eosinophilic granulocytes in group C after tx. In conclusion, i.u. ozone application is a therapeutic option for CIE, especially for reducing growth of gram-neg- ative bacteria.

27

The ex vivo response of pregnant ca- nine myometrium to ecbolic drugs – preliminary results

Die Ex-vivo-Antwort des kaninen peripartalen Myometriums auf utero- tonisch wirksame Medikamente – vorläufige Ergebnisse

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

1Reproductive Unit – Clinic for Small Animals, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany; ²Institute for Physiology and Cell Biology, Univer- sity of Veterinary Medicine Hannover, Foundation, Hanno- ver, Germany

Uterine inertia (UI) is a common cause for dystocia in bitches, often requiring surgi- cal intervention. The etiology of UI and the role of the myometrium is not well un- derstood. Consequently, we aimed to study myo metrial contractions and uterokinetic effects of oxytocin (OXT) and PGF2α by us- ing the organ bath as an ex vivo approach.

Canine full thickness uterine biopsies from the interplacental sites were obtained during medically indicated c-sections. The circu- lar and longitudinal myometrial layers were dissected microscopically and separately mounted into tissue chambers. Connected to a force transducer, spontaneous and OXT/

PGF2α induced myometrial contractions were measured, data digitally recorded and stored. Spontaneous contractions occurred even 36 hours post-surgery, demonstrating a promising viability of the tissue. After one- hour equilibration, strips were stimulated with OXT n = 18 (10^{- 7}M, 10^-8M, 10^-9M) or PGF2α n = 5 (5×10^{- 5}M, 5×10^-7M, 5×10^-11M).

One strip of each layer served as untreated control. Stimulation was repeated three times with 20 minutes of wash out in between. All strips responded to OXT with contractions.

Whereas 10^-9M OXT induced rhythmical contractions in both layers, higher concen- trations triggered strong, tonic contractions of the longitudinal layer at first, but no more contractions afterwards. A reproducible con- tractile effect of natural PGF2α (dinoprost) was visible in the circular layer using 5×10^-5M only, a dose > LD50. No effect to PGF2α was seen in the longitudinal layer, although a final OXT stimulation (10^-7M) confirmed viability of the strips. In summary, the organ bath is a suitable tool to assess dose-response relation- ships of ecbolic agents.