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*

01

Morphology, blood flow velocity and hormonal contents of the overgrown follicle in dromedary camels

Morphologie, Blutflussgeschwindigkeit und Hormongehalt von sogenannten überwucherten Follikeln bei Dromedaren

A. Ali1,2, D.R. Derar1,2

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 distinguishable 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 follicles. 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 occurrence and fate. Twenty-three female camels were ultrasonographically classified as having an OVGF with clear content (OVGF_TH, n = 7), granular content (OVGF_GR, n = 4), fine trabeculae (OVGF_TKI, n = 3), moderate 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 collected transvaginally and analyzed for estradiol 17? (E2) and progesterone (P4). The results showed that all types of OVGF had comparatively 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 correlated 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 occurrence 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 Wiederkäuerklinik

V. Balasopoulou, Y. Zablotski, H. Zerbe, K. Voigt

Clinic for Ruminants with Ambulatory and Herd Health Services, 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 influential factors on its occurrence. Clinical records of 302 sheep with dystocia admitted to a veterinary hospital were evaluated retrospectively. Sixty (19.9%) animals were diagnosed with uterine torsion. Statistical analyses were performed 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 univariate analyses age, parity, season and nutrition were excluded as non-significant, while breed type, litter size and husbandry showed significance, 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 logistic 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 predisposing factor for the condition in the studied cohort.

03

Quantitative differences in sex hormone-binding globulin amounts ­between different ruminant breeds detected by mass spectrometry

Quantitative Unterschiede im Sexualhormon-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 androgens and oestrogens and has both enhancing 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 collected from three male/female Angus, Aurochs and Galloway having been kept under extensive 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 Exploris™ 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 Simmental (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.

04

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

Die Effekte von L-Laktat werden vermutlich über NMDA-Rezeptor-assoziierte Signalwege in bovinen Granulosazellen 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 granulosa cells (GCs). Since the signal transduction of L-lactate is dependent on lactate transporters, we further aim at elucidating the intracellular mechanisms of L-lactate. N-methyl-D-aspartate receptors (NMDARs) have been highlighted to be involved in L-lactate signaling 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 androstenedione for 8 days (37 °C, 5% CO?). 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 concentration 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, LHCGR, RGS2 and VNN2 were partially abolished 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 re­sistant 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, Hannover, Germany; 2Institute for Microbiology, University of Veterinary Medicine Hannover, Hannover, Germany

Multidrug resistant bacteria in preserved semen may affect sperm quality and contribute 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. oxytoca) or Serratia marcescens (S. marcescens) isolated 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. marcescens but not K. oxytoca compared to controls (p > 0.05). In semen extender Androstar Premium hold for 144 h at 5 °C, K. oxytoca decreased 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 Hyperstimulation 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, University of Zurich, Switzerland; 2Department of Animal Sciences, North Dakota State University, Fargo, USA

Imbalanced nutrition and exogenous gonado­trophins affect uterine function and morphology. In sheep, FSH-induced super­ovulation 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 estrogen 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 differently 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 processes. A higher number of DEGs were identified 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 activity, cell-cell, and cell-ECM adhesion. These and other results suggest that FSH has multidirectional effects on local uterine function and the expression of factors involved in implantation, depending on diet.

07

Comparison of antioxidative and oxidative profiles in plasma and saliva in dairy cows during pregnancy

Vergleich der antioxidativen und oxidativen Profile in Plasma und Speichel bei Milchkühen während der Trächtigkeit

A. Bielecka, M. Jamio?, M. Kankofer

Department of Biochemistry, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Lublin, Poland

Body fluids are a good source to get knowledge 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 antioxidant and oxidant profiles of plasma and saliva during bovine pregnancy were compared. Seventy-six health pregnant and twelve non-pregnant control cows were included in the study. Blood and saliva samples were collected 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 spectrophotometrically. Dynamic changes depending on the period of pregnancy course were detected and revealed that the increase in oxidative intensity induced an appropriate answer of the body. The highest antioxidant activity in plasma was mostly noted in early and advanced pregnancy. All tested parameters except SH groups expressed higher values in saliva compared to plasma. However, with regards to antioxidant/oxidative parameters, saliva reflects 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 establish physiological ranges of antioxidative/oxidative profiles of body fluids in cows.

08

Comparative treatment of kisspeptin alone or in combination with ­cabergoline for estrus induction of bitches

Vergleichende Behandlung von Kiss­peptin allein oder in Kombination mit Cabergolin zur Brunstinduktion bei Hündinnen

G. Bozkurt1, A.R. A?ao?lu1, 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, Nicosia, 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 reproductive effectiveness of the use of cabergoline (CB, Galastop®) and kisspeptin (KP, KP-10) alone or in combination on reproductive hormones in bitches which were randomly divided 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 continued on the 3rd (S7), 6th (S8), 9th (S9), 12th (S10) and 15th (S11) day after treatment. No significant difference could be determined between the S1 and S2 samples, and samples 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 difference 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 concentrations, 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 preserved semen of boars in Vietnam

Spermienqualität und Bakterienwachstum 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 Medicine 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 Premium (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 spermatozoa in semen samples stored at 5 °C in APrem extender was similar to control samples 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 – Commission 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 metabolic and developmental target genes in rabbit reproductive organs during preimplantation

Auswirkungen eines Apolipoprotein-E-Knockouts auf die Expression von Stoffwechsel- und Entwicklungsziel­genen in den Fortpflanzungsorganen des Kaninchens wa?hrend der Pra?implantationsphase

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 physio­logy in cholesterol and fatty acid metabolism as well as in early embryo development. The aim of our study was to investigate the apolipoprotein E knockout rabbit (ApoE-/-) in terms of reproductive effects and expression of metabolic and developmental target genes. ApoE- /- rabbits have an altered lipid metabolism, resulting in elevated levels of plasma cholesterol 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 carnitine acyltransferase 1 levels and decreased fatty acid synthase level. The systemic metabolic alterations were reflected in the female reproductive tract organs. In addition to metabolic changes, a strong increase of insulin-like growth factors, IGF1 and IGF2, indicated an effect on preimplantation processes in the endometrium. The ApoE-/- rabbit produced significantly 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 factors for female fertility.

Grants: Funded by DFG GRK2155 ProMoAge and JU3146

11

NET formation of polymorpho­nuclear 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 factors for the transfer of maternal immunity to the infants. Polymorphonuclear neutrophils (PMN) represent a subtype of colostral leukocytes, able to migrate into colostrum and through the neonate’s intestine. Our study focuses on PMN and their early innate effector mechanisms. PMN have been shown to form neutrophil extracellular traps (NETs) against pathogens in other locations. In our study, PMN were isolated from ovine colostrum by density gradient centrifugation using 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 formation was detected by immunofluorescence microscopy. 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 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 frequency 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 abstinence on day 1, 2, and 4. Afterwards they were examined and frozen. The thawed semen portions were also examined. The examination was performed using a CASA system. Examination of the native semen showed a non-significant 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 spermatozoa 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 semen 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, already the first ejaculates in native state corresponded 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 Fall­bericht

D. Derar1,2, A. Ali1,2, M. Al-Zaggafy3,4, H. Al-Moghazy3,5

1Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim, Saudi Arabia; 2Department of Theriogenology, Faculty of Veterinary Medicine, ­Assiut University, Egypt; 3Veterinary teaching Hospital, ­Qassim University, Saudi Arabia; 4Department of Microbiology, 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 University, Saudi Arabia with a history of persistent 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. Clinical examination showed enlarged pendulous prepuce, persistent penile movement and restlessness. Abstaining from food, the animal lost weight and general body condition looked unthrifty. The animal was venipunctured to obtain a complete hemogram. The complete blood count revealed decrease in the total leucocytic count, hemoglobinemia and total leukocytopenia. The animal prescribed fluid therapy, topical anesthetic ointment, analgesic, antipyretic and muscle relaxant. The symptoms were temporarily relieved and resumed after 6 hours intensively. Finally, the animal was euthanized. To our knowledge, 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-Thermographie zur Bestimmung der Brunstzeit bei Dromedaren

D. Derar1,2, A. Ali1,2, M. Tharwat1,3, Y. Al-Boty1, A. Alsamri1

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

The aim of the present study was to investigate 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 dromedaries admitted to the veterinary teaching clinic, Qassim University, Saudi Arabia for routine breeding soundness examination. Immediately after admission, the temperature of the vulva, vagina and rectum was measured and recorded using a FLIR C2 thermal 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 findings and history of last estrus. Data was tabulated 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-follicular. 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. Drummer1,2, J. Münzker1, M. Heistermann3, T. Becker4, S. Mißbach2,4, R. Behr1,2

1Platform Degenerative Diseases, German Primate Center – Leibniz Institute for Primate Research, Göttingen, ­Germany; 2DZHK, Partner Site Göttingen, Germany; 3Endocrinology Laboratory, German Primate Center – Leibniz Institute 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 essential 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 throughout 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, suggesting 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.

16

Relationship between ultrasonographic findings and different blood parameters in pregnant dogs

Zusammenhang zwischen Ultraschallbefunden und verschiedenen Blutparametern bei trächtigen Hunden

E. Evci1, S. Aslan1, O. Ergene1, I. Darbaz1, K. Seyrek-Inta?1, S. Say?ner2, A. Wehrend3

1Department of Obstetrics and Gynecology, Faculty of ­ Veterinary Medicine, Near East University, Nicosia, Cyprus; 2Department of Biochemistry, Faculty of Veterinary Medicine, Near East University, Nicosia, Cyprus; 3Clinic of Obstetrics, 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) values in female dogs at different periods of pregnancy. Thirteen (G1) dogs that became pregnant when mated and 7 dogs not pregnant 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 12th 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 55th 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 12th and 45th 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 25th and 35th days compared to the G2 group. On the 45th day, the difference between G1 and G2 was p < 0.01. It was determined that CRP values were highest on the 25th and 35th days in pregnant dogs and were significantly different (p < 0.01) compared to the 12th 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 55th day of pregnancy. The results obtained reveal that CRP values may be a parameter in the monitoring of pregnancy 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 Geschlechterverhältnis in vitro produzierter boviner Embryonen

A. Fries, B. Zimmer, B. Rabenau, F. Kotarski, C. Wrenzycki

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

The aim of the present study was to investigate whether a bull-specific sperm pre-incubation prior to in vitro fertilization may influence embryonic development and sex-ratio. For each of the 3 bulls (Bull A, B, C) used for IVF, capacitation time was assessed by chlor­tetracycline staining. Therefore, 90 (Bull A) or 180 (Bull B, C) minutes before IVF cryopreserved/thawed sperm was prepared using a standard protocol and was pre-incubated in FertTALP. This pre-incubation step was omitted 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 developmental 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). Interestingly, 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 blastocysts (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-incubation of sperm might have a bull dependent influence on cleavage rate, proportion of hatching blastocysts at day 7 and embryonic sex-ratio.

18

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

Ungeschlüpfte Rinderblastozysten ­exprimieren alle Transkripte des Östrogen-Biosynthesewegs, die entsprechenden 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 implantation. Embryonic estrogens are involved in regulating the implantation process. In cattle, the embryonic stage at which estrogen synthesis commences is yet unknown. However, to understand early embryogenesis it is important to know whether, for example, locally produced estrogens are involved in the formation and further development of blastocysts. Therefore, in this study, we first used quantitative reverse transcription PCR to examine 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 receptors ESR1 and ESR2 in in vitro produced morulae and blastocysts. Only in the blastocysts, the mRNAs of the entire estrogen biosynthesis chain and of both estrogen receptors were clearly present. We further assessed the expression 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 producing 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 Wachstumsfaktor 1- und Interferon tau-Signaling bei trächtigen Kühen mit einer verlängerten freiwilligen Wartezeit von 120 Tagen

C. Gabler1, S. Widera2, J. Huber1,2, 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 conception indicate that the embryo-maternal communication (EMC) is impaired leading to subfertility. It was hypothesized that extension 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 pappalysin 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 performed 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 findings confirms that IGF1 and INF signaling 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 hematological parameters in bitches

Einfluss der Wurfgröße auf die hämatologischen 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 Giessen, 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 pregnancy 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. Animals were divided into two groups – first (I), normal and second (II) bitches with pathological pregnancy. Additionally each group was divided into subgroup A – small (up to 4 puppies) and subgroup B with normal litter 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 detected. 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 respectively (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 parameters.

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 resultierende In-vitro-Entwicklung der Embryonen verbessern?

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

Probiotics have positive effects on the organism and reproduction. Bacteria of the genus lactobacillus are among others the best investigated probiotics. As part of the uterine microbiome of cows, Lactobacillus buchneri has been detected. The objective of this study is to determine the effect of Lactobacillus buchneri on in vitro maturation (IVM) of bovine oocytes. Ovaries from a local slaughterhouse were used. By slicing, cumulus-oocyte-complexes were isolated from ovarian follicles for in vitro production. Different amounts (1–10%) of the IVM medium were replaced by Lactobacillus buchneri-conditioned tissue culture medium (TCM). The recorded cleavage and development rates differed, depending on how much conditioned medium was used. The experimental group with 5% conditioned medium showed higher development 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 maturation 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 unknown player in the male reproductive 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 adenocarcinoma. Here, VISG10L is assumed to contribute to the maintenance of the epithelial barrier and/or to modulate inflammatory processes. Interestingly, RNAseq data indicate that Sertoli cells are one of the main expression sites of VSIG10L. Sertoli cells constitute the principal compartment of the blood-testis barrier (BTB) and express immunomodulatory factors. The BTB is necessary for the formation of an immune privileged micro­environment, which is essential for spermatogenesis. Since VSIG10L seems to be relevant for both, the formation of an epithelial barrier 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 focused 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 variable 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 recombinant 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 Erschütterungsemissionen 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 standardized 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 significant (p < 0.001) differences in Di, depending 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 generate 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. Particularly strong vibrations on rough access roads to sow farms cannot be prevented, but lower speed is expected to reduce the damaging influence on spermatozoa. Overall, more attention 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).

24

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

Einfluss von „vaginal seeding“ auf immunologische Parameter des neugeborenen 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 Animal 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 immunologic and metabolic diseases in children, 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 seeding” 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 muzzle, nose and rest of the body were swabbed with a towel previously incubated in the maternal vagina. The other calves formed the control group. In the first four weeks of life main leukocytic populations as well as monocytic 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 seeding (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 samples are still pending.

25

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

Zusammenhänge zwischen Ovula­tionszeitpunkt und fäkaler Östrogenkonzentration, intravaginalem Sauerstoffpartialdruck 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 supporting manual estrus control by determining ovulation time more accurately would be of great benefit. Against this background, the evaluation of fecal estrogen concentrations (EC), intravaginal partial pressure of oxygen (IVPO? in mmHg) and intravaginal temperature (IVT in °C) and their respective suitability for ovulation detection was the objective of this study. Fourteen DanBred sows (parity: 5.2 ± 2.4) were checked for ovulation via ultrasound scan every 8h for 72h, starting 80h after weaning. Fecal samples were analyzed for their EC, furthermore, IVPO? and IVT were measured using a fluorescence-based technique (OxyLite™, Oxford Optronix, 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 IVPO? 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). Concluding, neither the IVPO2 nor the IVT are suitable parameters for ovulation detection. On the other hand, there is a relation between ovulation time and fecal EC, however, significant differences in the sow-individual estrogen concentrations must be considered.

Grants: Funding by AiF (ZF4276703TS9).

26

Intrauterine ozone application as a possible treatment of chronic infectious endometritis in mares

Intrauterine Ozonapplikation als mögliche Behandlung der chronisch-infektiösen Endometritis bei Stuten

L. Hofbauer1,2, M. Köhne2, A. Görgens1, H. Sieme2

1Clinic for Horses Mühlen, Steinfeld, Germany; 2Unit for Reproductive Medicine – Clinic for Horses, University of Veterinary 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 cytolo­gy) were assigned randomly to different treatments (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 ultrasound, 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), irrespective 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 effective 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 increased 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-negative bacteria.

27

The ex vivo response of pregnant canine myometrium to ecbolic drugs – preliminary results

Die Ex-vivo-Antwort des kaninen ­peripartalen Myometriums auf utero­tonisch 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, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany

Uterine inertia (UI) is a common cause for dystocia in bitches, often requiring surgical intervention. The etiology of UI and the role of the myometrium is not well understood. Consequently, we aimed to study myo­metrial contractions and uterokinetic effects of oxytocin (OXT) and PGF2? by using 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 circular and longitudinal myometrial layers were dissected microscopically and separately mounted into tissue chambers. Connected to a force transducer, spontaneous and OXT/PGF2? 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- 7M, 10-8M, 10-9M) or PGF2? n = 5 (5×10- 5M, 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 concentrations triggered strong, tonic contractions of the longitudinal layer at first, but no more contractions afterwards. A reproducible contractile effect of natural PGF2? (dinoprost) was visible in the circular layer using 5×10-5M only, a dose > LD50. No effect to PGF2? 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 relationships of ecbolic agents.

28

Optimization of flow cytometric sexing unfit bull sperm by using microfluidic sperm sorting chips

Optimierung der durchflusszytometrischen Geschlechtsbestimmung ungeeigneter Bullenspermien mit Hilfe mikrofluidischer Spermasortierchips

O. Kalyoncu1, O. Calisici1, H. Oldenhof2, S. Alkabes3, M. Cordes-Blauert3, H. Sieme2, Á. Csaba Bajcsy1

1Clinic for Cattle and 2Unit for Reproductive Medicine, Clinic for Horses, University of Veterinary Medicine Hannover, Hanover, Germany; 3Masterrind GmbH, Verden, Germany

Several genetically valuable bull ejaculates don’t survive the negative effects of the higher requirements of the flow cytometric sexing process (FCSP) because they don’t have the necessary motility values or have high dead sperm percentages. Our aim was to determine the efficacy of microfluidic sperm sorting chips (MSSC) on FCSP by subjecting them to genetically valuable breeding bulls with a progressive motile rate (PMR) of approx. 70%. For this purpose, ejaculates of 6 bulls were collected and split into two groups. After collection, each ejaculate was treated with MSSC and the remaining volume of the initial ejaculates were used for the control group. In both groups, PMR and plasma membrane integrity (PMI) were measured by CASA and an automated sperm cell counter before they were subjected to FCSP. Data were analyzed using RM-ANOVA. PMR and PMI gave 93.4 ± 0.4% (mean ± SEM in %) and 95.7 ± 0.4% for MSSC, while the controls were 73.0 ± 1.8% and 68.0 ± 2.6%, respectively (p < 0.001). It was important to see that, the total oriented sperm population and X-sperm population increased by MSSC group compared to control (67.7 ± 1.5% and 25.4 ± 1.0% MSSC vs 45.1 ± 1.7% and 15.7 ± 0.8% in controls, p < 0.001). Furthermore, the sorted cell number/s increased nearly two times at the MSSC group compared to the control group (4395.8 ± 186.9 vs 2566.7 ± 159.4 events/s; p < 0.001).

In conclusion: MSSC increased the motility values and decreased the percentages of dead sperm in bulls with high genetic value, so that they could be used for successful sexing procedure.

Grants: Supported by Dr. Dr. h.c. Karl Eibl-Foundation and KOEK EU GmbH.

29

Nature’s curiosities – bilateral ­gynandromorphism in mice

Kuriositäten der Natur – Bilateraler ­Gynandromorphismus der Maus

B. Klein1, N. Terwort2, A. Kersebom2, J. Ehmcke1, S. Schlatt2, J. Wistuba2

1Zentrale Tierexperimentelle Einrichtung der Medizinischen Fakultät, WWU Münster; 2Centrum für Reproduktionsmedizin und Andrologie, Universitätsklinikum Münster

This case report summarises the topic of a male C57BL/6 mouse delivered from a commercial vendor reported with an enlarged scrotal area. At visual and palpatory inspection, ambigous sex characteristics were observed. Briefly, neither testes nor a vaginal opening were detected. During diagnostic section, a small penis, a left-sided testis, epididymis and vas deferens, as well as a right-sided ovary, fallopian tube, and enlarged uterine horn were detected. Organs were processed for histological evaluation of gonadal status. Briefly, male as well as female reproductive organs were developed physiologically. Spermatogenesis was found to be qualitatively normal but quantitatively substantially reduced. The ovary presented follicle-like structures. For determination of the chromosomal sex, X-inactivation was assessed by Xist expression. Xist expression was found to be negative, suggesting an XY karyotype for the gynandromorphic mouse. Gynandromorphism is an overall extremely rare finding mostly observed in insects, spiders, crustaceans, other arthropods and birds. Bilateral gynandromorphism further specifies a midline seperation of male vs female characteristics, as observed for the internal genitalia in the mouse presented here. We suggest that the loss of the Y-chromosom in one cell of an XY two-cell stage, consequently leading to XY i.e. male and X0 i.e. female cell progeny during cell division, might offer a possible explanation for the bilateral development of functional reproductive organs of both XY and X0 origin in the present gynandromorphic mouse.

30

How does hemicastration affect the testosterone, testicular volume and semen parameter in the dog?

Wie wirkt sich die Hemikastration beim Hund auf den Testosteronspiegel, das Hodenvolumen und die Spermaparameter aus?

H. Körber, S. Stempel, S. Goericke-Pesch

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

To study the consequences of hemicastration (HEC) and the effects on the residual testis in mature dogs, HEC of the right testes (D0) was performed in three 2.5-year-old, healthy normospermic male Beagle dogs. Over a period of 10 months (5 months before and 5 months following HEC), weekly general and andrological examination including calculation of the testicular volume (TV), semen collection and evaluation (volume, total sperm count (TSC), progressive motility (PM), morphology (MAS), sperm viability) as well as regular blood collections for testosterone analysis were performed. Following removal of the right testis on D0 and the left testis on D149, respectively, testicular tissues were bouin-fixed, paraffin-embedded, cut and hema­toxylin-eosin stained for histological evaluation. Results are presented as range [minimum – maximum]. Subjective evaluation of the testicular histology did not reveal obvious differences in spermatogenesis between testicular tissues before and after HEC. TV of the left testes was 8.9–11.2 cm3 on D0 and 13.5–17.2 cm3 on D 149 indicating a significant increase by 152–168% over time (p = 0.0134). Testosterone concentrations, semen volume, PM, viability, and MAS were not affected by HEC. Different to this and following HEC, TSC decreased initially, but returned to TSCs considered as normospermic (274.2–606.3 × 106 on D149). The current results clearly show that HEC affects TSC only initially, but reduction in TSC is compensated over time, likely associated with the significant testicular hypertrophy. Hypertrophy was not observed in earlier studies, likely associated with the shorter duration of observation compared to our study.

31

Mitochondrial metabolic characteristics of bovine expanded blastocysts are correlated with embryo quality as well as early developmental environment

Mitochondriale Stoffwechseleigenschaften von expandierten Rinder-Blastozysten korrelieren mit der ­Embryoqualität und der frühen Entwicklungsumgebung

J. Kurzella1, D. Miskel1, F. Rings1, D. Tesfaye1, D. Salilew-Wondim1, M. Hoelker2

1Institute of Animal Science, Animal Breeding, University of Bonn, Germany; 2Department of Animal Science, Biotechnology and Reproduction of Farm Animals, University of Goettingen, Germany

The present study aimed to examine the correlation of contrasting embryo qualities (D7-expanded blastocysts = VITRO-HIGH vs. D8-expanded blastocysts = VITRO-LOW) as well as different early developmental environments (VIVO vs VITRO) with embryo mitochondrial metabolic characteristics. Therefore, VITRO-HIGH and VITRO-LOW expanded blastocysts derived from in vitro culture (SOFaa + 5% ÖCS, 5% CO? + 5% O?) were compared with expanded VIVO blastocysts in terms of ATP production rate, ATP coupling efficiency as well as mitochondrial reserve capacity (8 embryos per group, 4–6 replicates). This was analysed by an extracellular FLUX Analyzer (Seahorse XFp) and application of a Mito-Stress Test Kit (Agilent).The results showed that ATP production rate was significantly lower (p < 0.05, ANOVA) in VITRO-LOW compared to VITRO-HIGH embryos and tended to be even higher in VIVO embryos (16.8% vs. 100.0% vs. 124.6%), respectively. In accordance, VITRO-LOW embryos reached a significant lower coupling efficiency compared to VITRO-HIGH and VIVO embryos (12.7% vs. 66.1% vs. 72.7 %), respectively. Finally, the relative reserve capacity was identified to be significantly higher for VIVO compared to VITRO-HIGH and VITRO-LOW embryo groups (326.9%; 279.0% and 184.0%), respectively. Taken together, the present study obtained that embryo quality as well as early developmental environment are correlated with contrasting mitochondrial metabolic characteristics in bovine expanded blastocysts. These findings in turn might explain to great extend contrasting prospective developmental potentials.

32

Membrane stability of preserved boar spermatozoa with cytoplasmic droplets

Membranstabilität von konservierten Eberspermien mit Plasmatropfen

A.M. Luther1, H. Henning2, P. Hasenleder1, D. Waberski1

1Unit for Reproductive Medicine of Clinics/Clinic for Pigs
and Small Ruminants, University of Veterinary Medicine, Hannover, Germany; 2Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Neustadt, Germany

Retained cytoplasmic droplets (CD) are the most frequent sperm abnormality in boars. Their impact on sperm fertility is still disputed. The aim was to assess the plasma membrane stability of preserved boar semen with a high (> 15%) or low incidence of sperm with a CD under various stress conditions, i.e. long-term storage at 17 °C, thermic stress after storage (3h, 38 °C) or incubation in presence of the capacitation inducer bicarbonate. In Experiment 1, semen samples (n = 78) from 13 AI centers were assessed for the sperm’s responsiveness to bicarbonate in a flow cytometric calcium influx assay. Samples with a high incidence of sperm with a CD contained fewer sperm which responded to bicarbonate and, after 96 h storage, more non-specifically destabi-lizing sperm (p < 0.05). The percentage responsive sperm negatively correlated with the percentage sperm with a CD (r = –0.61, p < 0.01). In Experiment 2, plasma membrane integrity (viability) was assessed with Eosin-Nigrosin stain in 26 semen samples on a single-cell level. The subset of sperm with a distal CD had a lower viability than sperm without a CD at all time points of storage until day 7 and after thermic stress (p < 0.05). Sperm without a CD did not differ in their viability regardless whether they originated from samples with a high or low incidence of sperm with a CD. In conclusion, the presence of a CD presumably has a negative impact on membrane stability of sperm in preserved semen samples. Since morphological normal sperm are not affected, a compensation of this sperm defect by higher sperm numbers in the semen dose appears possible.

Grants: Submitted for FBF workshop.

33

Sulfonation of estrogens in the equine testicular-epididymal compartment

Sulfonierung von Östrogenen in ­Hoden und Nebenhoden des Pferdes

P. Marburger, B. Zimmer, A.-S. Fries, C. Wrenzycki, G. Schuler

Chair for Molecular Reproductive Medicine, Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Germany

Estrogens are regarded as important regulators of reproductive functions also in males. In domestic mammals, there is yet little established information on their functions. Whereas in many mammalian species estradiol-17beta (E2) is the predominant estrogen in the systemic circulation of males, the concentrations of which roughly correspond to the basal level of females, high concentrations of estrogen sulfates (ES) occur as a special feature in stallions in addition to E2. To characterize the production of ES in the equine testis and epididymis, tissue samples of testes and seven defined epididymal segments were collected from five stallions of different breeds aged 3–12 years during routine castrations. In measurements of estrogen sulfotransferase (EST) activity in tissue homogenates, activities were higher on average in the testis than in the epididymal segments studied (% sulfonation of estrone: 57.2 ± 28.5 vs. 17.7 ± 13.7; p < 0.001). However, in isolated cases, the activities in epididymal samples exceeded those measured in the testes. Data from measurements of conjugated total estrogens in tissue samples clearly point to the testis as the major source of ES. Measurements of the Michaelis-Menten constant of EST in testicular and epididymal cytosolic preparations yielded values < 0.1 µmol/l consistent with the highly specific estrogen sulfotransferase SULT1E1. Results from RT-qPCR showed that in addition to SULT1E1, SULT1A1 (“phenol sulfotransferase”) is also expressed in all tissues examined, which may be relevant for estrogens especially in the presence of high substrate concentrations.

34

Effect of bull exposure to high temperature-humidity index on microRNA expression in sperm selected by density gradient centrifugation for in vitro fertilization

Effekt der Exposition von Besamungsbullen mit einem hohen Temperatur-Luftfeuchtigkeitsindex auf die Expression von microRNAs in Spermien, selektiert mit Dichtegradientenzentrifugation für In-vitro-Fertilisation

M. Melean1, C. Herrera1, M. Siuda1, S. Bozukova2,
S. Bauersachs2, H. Bollwein1, E. Malama1

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

The present study aimed to evaluate the effect of bull exposure to conditions of high tem­perature-humidity index (THI) on the expression of selected microRNAs (miRNAs) in frozen-thawed sperm selected by density gradient centrifugation (DGC) for in vitro fertilization (IVF). We examined 10 ejaculates from five mature Simmental bulls that were exposed to high (? 60 THI, n = 5) and low (? 55, n = 5) THI during the epididymal maturation phase (7 days prior to sperm collection). The motile sperm fraction was separated through DGC and total RNA was extracted from 100×106 sperm. Following preparation of first-strand cDNA, relative quantification of bta-miR-30d-5p, bta-miR-34b-5p, bta-miR-92a-3p, bta-miR-132-3p, and bta-miR-204-5p was performed by real-time PCR. The high-THI ejaculates showed lower relative expression (?Cq values) of miR-34b-5p, miR-92a-3p and miR-204-5p, whereas this was not the case for low-THI ejaculates. Interestingly, miR-204 has been shown to be directly involved in the chromatin condensation process in elongating spermatids, suggesting a role in chromatin stability of ejaculated sperm. Remarkably, the high-THI ejaculates of this study showed ­subtle DNA damage after induction of a 3-hour incubation stress (5.9% and 11.5% DNA fragmentation index at 0h and 3h, respectively; p < 0.05), distinct from sperm collected under low-THI conditions (5.2% and 9.2% at 0h and 3h, respectively; p > 0.05). Combined, these findings imply that miR-204 could be linked to sperm chromatin instability evident after incubation-induced stress, shedding light on the effects of high-THI exposure of the bull on mature sperm selected for IVF.

35

Homology directed repair using CRISPR Cas9 electroporation of zygotes reveals that COPA may be ­necessary for embryo development during genome activation

CRISPR Cas9-Elektroporation von ­Zygoten zeigt, dass COPA für die ­Em­bryonalentwicklung während der ­Genomaktivierung notwendig ist

D. Miskel1, J. Kurzella1, F. Rings1, D. Tesfaye1, D. Salilew-Wondim1, M. Hoelker2

1Institute of Animal Science, Animal Breeding, University of Bonn, Germany; 2Department of Animal Science, Biotechnology and Reproduction of Farm Animals, University of Goettingen, Germany

Early embryo development is characterized by morphological milestones representing major cellular achievements and reaching 8-cell stage by day 3 post fertilization is used to signify successful onset of genome activation in bovine embryos. Coatomer subunit alpha is involved in protein transport between the endoplasmic reticulum and Golgi and a single nucleotide polymorphism in exon 6 is responsible for the dominant red trait among Holstein cattle. This mutation was targeted in 2 trials: in the first trial, 524 IVF derived zygotes were electroporated with Cas9 ribonucleoproteins and developed to the 8-cell stage (39.5%). While half of them were removed for analysis the remaining embryos were cultured until day 8 to the blastocyst stage (24.8%) and removed. The second trial included a ssDNA repair template containing silent mutations while electroporating 880 zygotes. These embryos were also cultured to day 8 and the blastocysts (39.4%) removed. A subset of 8-cell embryos (43) and blastocysts (45 in trial 1; 91 in trial 2) were Sanger sequenced and analyzed for editing type. Although the first experiment showed an editing rate of 53.7%, all embryos exhibited the wild-type. The second experiment had an editing rate of 72.5% but notably showed a complete removal of wild-type in 7.7% of embryos through combinations of indels and template insertion, including a single homozygous insertion of the repair template. No instances of full knockout through indels were detected, suggesting that development to 8-cell stage requires at least one intact copy of COPA or a functionally identical variant.

36

Influence of calf rearing conditions on the suitability of artificial insemination bulls

Einfluss der Kälberaufzuchtbedingungen auf die Eignung von Besamungsbullen

V. Mittmann1, M. Schulze2, M. Jung2, M.K. Schaper3, M. Schmicke1

1Institute of Agriculture and Nutritional Sciences and Animal Health Management, Martin-Luther-University, Halle Wittenberg, Germany; 2Institute for Reproduction of Farm Animals Schönow, Bernau, Germany; 3Endocrinology Laboratory, Clinic for Cattle, University of Veterinary Medicine Hannover, Germany

A problem commonly encountered on artificial insemination (AI) centers is that some genetically interesting bulls do not provide sufficient high-quality semen. The present study aims to investigate to what extent the recording of negative stressors during rearing and the use of biomarkers allow conclusions about early semen production. In total, 103 bull calves from 6 AI centers were monitored for potential negative rearing stressors by use of a questionnaire (after 8 ± 1, 12 ± 2 months summated to a rearing score) and sampled (aged 4 ± 1, 8 ± 1, 12 ± 2 months). The following biomarkers were measured: cortisol, testosterone, DHEA, IGF-1, estradiol. With the beginning of semen collection, semen from each bull was analyzed (ejaculate volume, sperm concentration, total sperm output, total and progressive motility in raw and cryopreserved semen) directly at the AI center. The rearing score of bulls ranged from 36 to 117 points and was associated with sperm concentration at the first time of semen collection (rs = –0.24, p = 0.03). Bulls that mounted and delivered ejaculate at the first attempt, 11 ± 1 months, had higher testosterone and estradiol concentrations compared to bulls that did not mount (p < 0.05). A negative correlation was found between cortisol/DHEA ratio and total sperm motility in cryopreserved semen (rs = –0.44, p = 0.003). Bulls with good sperm quality had higher testosterone and IGF-1 at the first attempt than bulls with poorer quality. The farm-specific influence was highly significant in multiple parameters. In conclusion, testosterone, cortisol, estradiol and DHEA seem to be interesting candidates for prediction of sperm quality at an age of 8–9 months.

Grants: Submitted for FBF Workshop

37

Bovine plasma protein profile in two-dimensional electrophoresis

Plasmaproteinprofil von Rindern in der zweidimensionalen Elektrophorese

W. Mojsym, J. Wawrzykowski, M. Kankofer

Department of Biochemistry, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Poland

It is well known that pregnancy is regulated and controlled by hormones. Little is known, however, about the products of their biochemical action during pregnancy course – proteins. The study aims to describe protein maps in all months of pregnancy and compare them with each other. They are also compared to the non-pregnant group. Plasma samples were collected from healthy Holstein-Friesian cows in all 1–9 pregnancy months (n = 34). The control group involved samples from non-pregnant cows (C; n = 4). Plasma protein samples were separated into two fractions: supernatant (S) and pellet (P) to better display molecules at molecular mass similar to albumin [Tomascova A., 2019]. All fractions coming from one month were pooled in one sample and undergone two-dimensional electrophoresis, then analyzed in Delta 2D program. Both protein fractions were analyzed separately. In both protein fractions, significant differences were noticed – both quantitative and qualitative. PCA scatterplot showed the most significant differences in the S fraction between the 6th month and other samples. Significant differences were also noticed between the non-pregnant group and all pregnancy months, but the lowest between the 9th month. In the P fraction, there were distinguishable clusters: one related to the 1st, 5th, 8th, 9th month, and non-pregnant group. The second contained samples from the 2nd, 3rd, and 4th months. Further research about the identification of selected proteins on larger number of animals is needed.

38

Predictability of the outcome of pregnancies in mares with placentitis – a retrospective study

Vorhersagbarkeit des Ausgangs von Trächtigkeiten bei Stuten mit Plazentitis – eine retrospektive Studie

M. L. Niquille1, H. Bollwein1, M. Blanco2, J. Lüttgenau1

1Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Switzerland; 2Gestüt Lewitz, PS Pferdehaltung GmbH, Neustadt-Glewe, Germany

The aim of the study was to investigate the predictability of the outcome of pregnancies in mares with placentitis by evaluating their gestational history as well as clinical and sonographic findings. In 53 warmblood mares, which were examined during the breeding season 2018/19 in a stud in Northern Germany, placentitis was diagnosed by transrectal sonographic determination of the combined thickness of the uterus and placenta (CTUP). Examinations were performed due to clinical signs, such as premature udder development, lactation and vaginal discharge, or as part of routine controls. Of the 53 pregnancies in mares with placentitis, 15.1% ended with abortion (n = 5) or stillbirth (n = 3). Mares with abortion or placentitis during a previous pregnancy had a higher (p < 0.05) risk of abortion or stillbirth (23.8%; 5 out of 21 mares) compared to those without previous disturbances of pregnancy (9.4%; 3 out of 32). The CTUP of mares with placentitis was approx. 10 mm, regardless of the month (4 to 11) of pregnancy. Since the CTUP increases from 4 to 10 mm during gestation months 4 to 11 in healthy mares, there was no difference (p > 0.05) between the CTUP in mares with and without placentitis during the second half of gestation. All mares with a Caslick surgery due to vulvar malformation (21 out of 53) gave birth to live foals. In conclusion, previous disturbances of pregnancy increase the risk of abortion or stillbirth in mares with placentitis. The CTUP can be used as an indicator of pregnancy loss during the first but not the second half of pregnancy. Although Caslick surgery does not prevent placentitis, it improves the foals’ survival rate.

39

Comparative RNA-Seq of nine canine cell lines derived from prostate adenocarcinoma (PAC), PAC metastasis and transitional cell carcinoma (TCC)

Vergleichende Charakterisierung des Expressionsprofils kaniner Zell-Linien aus Prostata-Adenokarzinomen (PAC), PAC-Metastasen und Übergangszell-Karzinomen (TCC)

E. Packeiser1,2, L. Taher3, M. Ernst 4, J. Beck5, M. Hewicker-Trautwein6, B. Brenig7, E. Schütz5, I. Nolte1, H. Murua Escobar2,8

1Small Animal Clinic, University of Veterinary Medicine Hannover, Germany; 2Department of Hematology/Oncology/Palliative Care, Rostock University Medical Center, Germany; 3Institute of Biomedical Informatics, Graz University of Technology, Austria; 4Division of Bioinformatics, Department of Biology, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Germany; 5Chronix Biomedical GmbH, Göttingen, Germany; 6Institute of Pathology, University of Veterinary Medicine Hannover, Germany; 7Institute of Veterinary Medicine, University of Göttingen, Germany; 8Comprehensive Cancer Center Mecklenburg-Vorpommern (CCC-MV), Campus Rostock, University of Rostock, Germany

Tumor cell lines are important preclinical model systems. Nonetheless, clonal selection and artificial culturing conditions can cause significant differences compared to the original tissues. Our previous whole transcriptome characterization of 14 canine prostate cancer samples identified 49 altered pathways, grouped into the superpathways: „Regula­tion of the immune system and cell death“, „Inflammatory response and cytokines“, „Cell surface and PI3K signaling“, „Phagosome and autophagy“ and „Cell cycle“ [Thiemeyer et al. 2021]. Herein, we performed RNA-Seq of nine canine PAC, PAC metastasis and TCC cell lines and seven original tumor tissues to assess the cell lines’ model suitability. Concordances and differences between cell lines and tissues were analyzed with special focus on our previously identified molecular characteristics of canine prostate cancer. The majority of differentially expressed genes was found downregulated. Of the 49 characteristic pathways of canine prostate cancer, 28, 32 and 12 were deregulated in PAC, metastasis and TCC cell lines. “Phagosome and autophagy” and “Cell surface” were affected the most, while less pathways in “Cell cycle” were deregulated. Regarding therapeutic intervention, all cell lines featured unique target expression profiles. Pathways comprise several hundreds of genes. Consequently, the selection of cell lines for targeted therapy should be based on tumor entity, complemented by molecular profile.

40

Time-dependent expression and localization of membrane progesterone receptors in the canine corpus luteum in the presence and absence of pregnancy

Zeitabhängige Expression und Lokalisation von Membran-Progesteron-­Rezeptoren im Corpus luteum der Hündin in An- und Abwesenheit einer Trächtigkeit

P. Papa, M. Tavares Pereira , M.P. Kowalewski

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

The corpus luteum (CL) is the sole source of progesterone (P4) in the dog. Non-genomic actions of P4 in the CL were described in other species, and involve the regulation of cellular cycle, apoptosis and steroidogenic activity. These are mediated by 2 types of membrane P4 receptors (mPGR): P4 receptor membrane component (PGRMC) -1 and 2, and mPGR belonging to the class II progestin and adipoQ receptor (PAQR) family, including PAQR7 (PAQRa), -8 (PAQRb) and -5 (PAQRy). Since no information regarding these receptors in the canine CL is available, we investigated their presence by qPCR and immunohistochemistry during pre-implantation (days 10–12), post-implantation (days 18–25), mid-gestation (days 35–40) and prepartum luteolysis in pregnant animals and at days 10, 20, 30, 40, 50 and 60 post-ovulation in non-pregnant bitches (n = 4–6 animals/group). In pregnant animals, the expression of all members of the PAQR family was upregulated in post-implantation period, with PAQR5 being increased again during prepartum luteolysis. In non-pregnant animals, the expression of PGRMC2 increased after day 30 while PAQR5 expression increased only at day 60. No changes were observed in PGRMC1 expression. Regarding their localization, PGRMC1 and -2 were mainly localized in luteal cells, whereas stronger signals of PAQRa were observed in vascular cells. PAQRb positive signals could also be observed in luteal and interstitial cells. These results suggest a stage and pregnancy-dependent regulation of some of mPGR and a broader role of P4 in the non-genomic regulation of luteal function in the dog.

41

The utero-placental expression of HSD11B1 and HSD11B2 implies increased placental cortisol signaling in the dog prepartum

Die utero-plazentäre Expression von HSD11B1 und HSD11B2 impliziert eine erhöhte plazentäre Kortisol-Signalübertragung beim präpartalen Hund

M. Tavares Pereira1, A. Kazemian1, S. Aslan2, R. Payan-­Carreira3, I.M. Reichler4, M.P. Kowalewski1

1Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich (UZH), Switzerland; 2Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Near East University, Nicosia, Cyprus; 3School of Science and Technology, Department of Veterinary Medicine, University of ­Évora, Portugal; 4Clinic for Reproductive Medicine, Vetsuisse Faculty, University of Zurich (UZH), Switzerland

The initiation of the parturition cascade is associated with increased cortisol availability in several mammals. In the dog, upregulation of the glucocorticoid receptor at term in the trophoblast suggests that a local, i.e. placental, increase in cortisol signaling might additionally be involved in the onset of parturition. Therefore, the utero-placental expression of hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1; reduces cortisone into cortisol) and -2 (HSD11B2; oxidizes cortisol into the inactive cortisone) was evaluated in the canine utero-placental compartments during pre-implantation (d10–12 of pregnancy), and corresponding non-pregnant controls; implantation (d17); post-implantation (d18–25); mid-gestation (d35–40); prepartum luteolysis, and after administration of the antigestagen aglepristone at mid-gestation. Transcriptional availability of both HSD11B1 and HSD11B2 was increased during post-implantation and mid-gestation (p < 0.05). Pairwise comparisons revealed higher mRNA availability of HSD11B2 during post-implantation, whereas HSD11B1 was higher than -2 during mid-gestation and luteolysis (p < 0.05). Aglepristone downregulated HSD11B2 (p < 0.05), with HSD11B1 being significantly higher than -2 (p < 0.05). HSD11B1 was mainly expressed by cytotrophoblast cells, whereas HSD11B2 was predominantly localized in the syncytiotrophoblast. Our results support the presence of an active regulation of cortisol levels in the canine placenta, with cortisol-activation mechanisms predominating at term.

Grants: Funded by SNSF grant 31003A_182481

42

Hyperketonemia affects mRNA expression of selected pro-inflammatory factors and metabolic enzymes in the endometrium and oviduct of postpartum dairy cows

Hyperketonämie beeinflusst die mRNA-Expression ausgewählter pro-inflammatorischer Faktoren und metabolischer Enzyme im Endometrium und Ovidukt im Puerperium von Milchkühen

H. Pothmann1, M. Ibrahim2, U. Besenfelder3, V. Havlicek3, M. Drillich1, C. Gabler2

1Clinical Unit for Herd Health Management in Ruminants, Vetmeduni Vienna, Austria; 2Institute of Veterinary Biochemistry, Freie Universität Berlin, Germany; 3Reproduction Centre Wieselburg, Institute for Animal Breeding and Genetics, Vetmeduni Vienna, Austria

Inflammatory processes in the reproductive tract of postpartum (pp) dairy cows is a risk factor for subfertility. Negative energy balance (NEB), indicated by hyperketonemia also negatively affects reproductive performance. Little is known about synergistic effects of inflammation and NEB in the uterus and oviduct. The objective of this study was to assess the mRNA expression of selected pro-inflammatory factors and metabolic enzymes in the endometrium and oviduct of dairy cows. Endometrial samples were taken on day 7, 14, 28, 42 pp from 8 dairy cows with the cytobrush method. From the same cows, oviductal samples were collected on day 28 pp by using a small cytobrush that was introduced into the oviduct under transvaginal endoscopic control. Four cows with > 1.2 mmol/L beta-hydroxybutyrate were classified as hyperketonemic (HKET) and compared with four healthy control cows (CON). Total RNA was isolated from all cytobrush samples and quantitative RT-PCR was carried out. 3-oxoacyl-transferase mRNA expression was not detected in most endometrial samples but was higher expressed in oviductal ­samples in CON compared with HKET. Beta-hydroxybutyrate-dehydrogenase mRNA was higher expressed in endometrial than in oviductal samples. Interleukin (IL) 1A, IL8, and chemokine CXL ligand 1/2 mRNA was more highly expressed in endometrial than in oviductal samples. On day 7 pp, the mRNA expression of these factors was higher in HKET than in CON. The findings of this study indicate that an inflammatory response is mainly restricted to the endometrium and that the oviduct is able to utilize ketone bodies as energy supply.

43

Dead boar spermatozoa do not affect live cells during a thermo-resistance test

Tote Eberspermien beeinflussen lebende während eines Thermoresistenztests nicht

M. Quirino1,2, U. Jakop¹, A. Retzlaff¹, A. Hudler¹, A. Mellagi², F. Bortolozzo², M. Jung¹, M. Schulze¹

¹Institute for Reproduction of Farm Animals Schönow, ­Bernau, Germany; ²Setor de Suínos, Faculdade de ­Veterinária, Universidade Federal do Rio Grande do Sul, ­Porto Alegre, Brazil

Boar semen storage can increase the number of dead spermatozoa by storage conditions or cells’ aging. An increase in the proportion of dead spermatozoa in artificial insemination (AI) doses also occurs in practice when low sperm quality of ejaculates is compensated by increasing sperm concentration. However, it is not known if the proportion of dead spermatozoa could affect the live cells. Therefore, this study evaluated the effect of the proportion of dead spermatozoa on sperm quality during a thermo-resistance test (TRT). After 3 days of storage at 17 °C, 54 conventional AI doses, with 20–30 × 106 sperm/mL in 90 mL of BTS, were split into three 15 mL treatments (A: 75%, B: 50% and C: 25% live cells) by mixing two subsamples: I and II. Subsample I had 75% of live cells, while subsample II, submitted to a flash-frozen process, had 0% live cells. Spermatozoa were evaluated after TRT at 30 (on-test) and 300 min (off-test) incubation at 38 °C. At the on-test, treatments A, B and C showed total sperm motility of 77.7 ± 0.5, 49.7 ± 0.6 and 25.6 ± 0.4%, respectively. Considering the relative difference (resilience) of sperm motility during TRT, all treatments lost similar proportion (p = 0.31) of motile cells: (A) 15.6 ± 2.2%, (B) 19.0 ± 2.9% and (C) 19.2 ± 2.7%. Furthermore, membrane integrity and mitochondrial activity of live cells did not change between the on- and off-test (p ? 0.40). Concluding, dead spermatozoa had no influence on the quality of live cells during the period and conditions of a TRT.

Grants: Supported by DAAD (57552395).

44

Validation of a 5-color flow cyto­metry panel for boar sperm quality analysis

Entwicklung eines 5-Farben-Durchflusszytometrie-Panels zur Messung der Spermaqualität beim Eber

M. Quirino1,2, U. Jakop¹, A. Mellagi², F. Bortolozzo², M. Jung¹, M. Schulze¹

¹Institute for Reproduction of Farm Animals Schönow, ­Bernau, Germany; ²Setor de Suínos, Faculdade de ­Veterinária, Universidade Federal do Rio Grande do Sul, ­Porto Alegre, Brazil

For more rapid and practical analysis of different physiological sperm characteristics, this study aimed to validate a 5-color flow cytometry (FC) panel for concurrently analyze 4 different boar sperm parameters (mitochondrial status [a], degree of membrane lipid disorder [b], plasma membrane [c] and acrosome integrity [d]), using also a DNA marker (e) to select spermatozoa. From each of thirty boar semen doses six aliquots (250 µL) were taken. Five aliquots were stained individually with Rhodamine 123 (a), Merocyanine 540 (b), Propidium Iodide (c), PNA-Alexa Fluor 647 (d) and Hoechst 33342 (e), respectively; while the sixth one was stained with all the 5 fluorochromes simultaneously (f). After 10 min at 37 °C, analysis was carried out with a CytoFLEX S, obtaining 10,000 events into the sperm gate. Spectral overlap of multicolor samples (f) was corrected by applying a compensation matrix, defined manually through equalization of fluorescence median intensity of positive controls. Strong and significant correlations (rs) were found between the parameters obtained by the single and the 5-color staining assay: active mitochondria = 0.98, low degree of lipid disorder = 0.94, intact plasma membrane = 0.90, intact acrosomal membrane = 0.98, and DNA marker = 0.99 (p < 0.01). Concordance Correlation Coefficients (CCC) considered as strong or almost perfect were presented for all para­meters (0.98, 0.91, 0.89, 0.97 and 0.99, respectively), confirming that this 5-color panel can be successfully implemented for analyzing boar sperm quality in a single, practical and quick assay.

Grants: Supported by DAAD (57552395).

45

Gynaecological examination of the female dog – a tutorial for vaginal cytology and revised definitions of cells

Die gynäkologische Untersuchung der Hündin – ein Tutorial für die vaginale Zytologie und überarbeitete Definitionen der Zellen

F. Reckers1, R. Klopfleisch2, V. Belik3, S. Arlt1

1Clinic for Animal Reproduction, Faculty of Veterinary Medicine, Freie Universität Berlin; 2Institute of Veterinary Patho­logy, Faculty of Veterinary Medicine, Freie Universität Berlin; 3Institute for Veterinary Epidemiology and Biostatistics, ­Faculty of Veterinary Medicine, Freie Universität Berlin, ­Germany

In the context of gynecological examination of the female dog, vaginal cytology plays an important role. While collection and preparation of samples are easy, the evaluation of smears appears to be challenging. Incon­sistent definitions of cell characteristics such as size, cornification and the appearance of the nucleus have been published. The aim of the project was to develop revised definitions of vaginal cells and a tutorial for cell determination. To get a deeper insight into the use of cytology in veterinary practice, an online survey was distributed to colleagues interested in small animal reproduction. Participants were asked to define eight cells and answer questions. The agreement of the sixteen participants determining the cells was poor (k = 0.398). Eleven respondents were of the opinion that vaginal cytology has a low reliability. Nevertheless, thirteen participants stated to use this tool regularly. Based on the feedback of the survey participants, information from scientific literature and own measurements, revised definitions of vaginal cells have been developed. In addition, a tutorial has been designed as a flowchart. It guides the user systematically through the evaluation of specific cell characteristics. An evaluation of the results of five raters with different experience levels led to a high agreement (k = 0.858). Vaginal cytology is a useful diagnostic tool in terms of cycle staging and gynecological disorders, but it seems helpful to standardize cell types.

46

First insights into the spermatogonial stem cell compartment in the canine testis

Erste Einblicke in die Regulation des Stammzellkompartiments im Hunde- Hoden

L. Reifarth, H. Körber, S. Goericke-Pesch

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

Our previous research proved that spermatogonial stem cells (SSC) with the potential of self-renewal and differentiation survive in testicular tissues altered by “immune mediated orchitis” (AIO) in the male dog. The aim of this study was to shed light on the mechanisms underlying the disturbance of the SSC niche examining regulators of spermatogenesis. We investigated established markers of the retinoic acid (RA) system (ALDH1A1, ALDH1A2, PLZF) and indicators for a disruption of the mitosis-meiosis-switch (PCNA, DMRT1, CREM) using immunhistochemistry (IHC). Testicular biopsies were taken from 15 dogs with AIO and 10 adult healthy dogs with normospermic ejaculates (CG). IHC was evaluated descriptively and revealed specific immunopositive staining for each marker with a reduced expression in AIO. Regarding the RA system, immunopositive signals were detected in the spermatogonia (PLZF), the pachytene spermatocytes and round spermatids (ALDH1A2) and Sertoli cells and Leydig cells (ALDH1A1), respectively. The nuclei of spermatogonia stained immunopositive against PCNA and DMRT1 protein. CREM revealed early arrest in n = 5 and late arrest in n = 10 AIO samples. The number of PCNA-immunopositive signals (NIP) was counted in 20 fields of vision (200x magnification) and statistically compared revealing significantly less NIP in AIO compared to CG (unpaired t-test p < 0.0001). Our data proof for the first time the presence of most studied parameters in the canine testis and indicate a contribution of retinoic acid in the underlying pathophysiology of AIO.

Grants: Supported by AfT.

47

Influence of myopathies on the ­reproductive performance of warmblood mares

Einfluss von Myopathien auf die Reproduktionsleistung von Warmblutstuten

P. Richterich

Pferdepraxis auf Boyenstein, Beckum, Germany

Myopathies caused by metabolic disorders are characterized by substantial disturbances in movement and well-being. Studies in connection with polysaccharide storage myopathy type 2 (PSSM2) showed that certain genotypes occur more frequently in affected mares than others. In the current study, only n/P4–n/Px mares were included and were observed during the past 3 years. In these mares (n = 12), nervousness, moderate muscling with increased tone (?HBDH 315.4 ± 47.8 U/l [<170]) and partially impaired standing ability with trembling was observed. Gynecologically, the mares had cycle irregularities; the cycle was prolonged in 4 mares with clear estrus symptoms (23.7 ± 1.3 days) or characterized by indistinct, irregular estrus in others (n = 7). In some of the irregular mares, external but not internal estrus symptoms could be observed, or the synchronicity between ovarian and uterine symptoms was not given. The follicles grew rapidly, often accompanied by atresia or impaired ovulation. The use of hCG showed only unsatisfactory results. Appropriate management of the mares led to a notable improvement in muscle tone (?HBDH 194.22 ± 22.6 U/l). The mares further presented with an improvement of general satisfaction and relaxation. The cycle normalized within one month for 9/12 of the mares and after about nine weeks for another two. An insemination index of 1.3 was achieved in five mares inseminated with n/n stallions. In conclusion, a specific diet consisting of roughage, proteins, energy and selected additives obviously has positive effects on muscle metabolism and thus indirectly on fertility.

48

Case report: Schistosoma reflexum and rectovaginal tear in a 5-year old female Alpaca

Fallbericht: Schistosoma reflexum und Scheidendachriss bei einer fünfjährigen Alpakastute

S. Schabmeyer, M.M. Meyerholz, M. Beer, W. Petzl, H. Zerbe, K. Voigt, V. Balasopoulou

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

Dystocia caused by foetal abnormalities is rare in New World Camelids. In this case, a 5-year old female alpaca was presented following prolonged labour for 6 hours. At the time of presentation, the head and two limbs of the foetus were protruding from the vagina and a deep rectovaginal tear was present. Manual vaginal examination revealed malformation of the cria, as an open thoracic cavity and stiff joints were palpable. The narrow birth canal did not allow manual delivery or foetotomy. Caesarean section was performed to develop the dead cria. The malformation was diagnosed as schistosoma reflexum, as it included exposure of the abdominal and thoracic viscera, spinal inversion and arthrogryposis. The placenta was removed during surgery. Continuous suture of the rectovaginal tear was performed under local anesthesia in two layers with polyfilamentous absorbable suture material. Post-operative treatment included intravenous fluid therapy, antibiotics, analgesics and daily attention to the rectovaginal suture. Initially, the alpaca showed diminished appetite and signs of discomfort during defaecation and urination. Oedema of the perianal and vulvar area declined over a 4-day period. The animal was discharged after five days of hospitalization. In its familiar environment, it immediately returned to its normal appetite. Three months after surgery no alterations in the anogenital region were visible. In conclusion, even in difficult obstetrical situations involving foetal monsters and injury of the dam, caesarean section can be life-saving. More frequent causes of dystocia, such as foetal maldisposition, should be considered as differential diagnosis.

49

Blockade of the cholesterol transporter ABCA1 in canine spermatozoa membranes by use of Probucol is dose dependant

Die Blockade des Cholesterin-Transportmoleküls ABCA1 in der Spermatozoen-Membran von Hunden mittels Probukol ist dosisabhängig

S. Schäfer-Somi

Platform for Artificial Insemination and Embryo Transfer, Vetmeduni Vienna, Austria

To investigate the effect of different concentrations of PRO on cholesterol efflux, 13 ejaculates from dogs of different breeds were used. Kinetic parameters were examined by CASA, the viability by fluorescent stain CYBR14/PI [Schäfer-Somi, 2020]. After examination, the concentration was adapted to 100 × 106/mL with PBS, then a boron dipyrromethene difluoride (BODIPY) fluorophore (P9672, Sigma Aldrich, Vienna, A [Bernecic, 2019]) was added at a final concentration of 0.4 µM. After 10 min of incubation at 37 °C, samples were washed, diluted to 5 × 106/mL, and divided into 5 aliquots with 0, 100, 250, 500 and 1000 µM PRO in DMSO. Samples were incubated at 37 °C for 2h, then PI was added (5 µg/mL) 10 min before flow cyto­metry. Sampes with DMSO only and viability were measured in parallel. A mixed linear model (Sirdak) was used to assess differences between PRO groups by considering the dog effect. A p value of < 0.05 was considered significant. Results are given as % live cells with cholesterol efflux. Viability did not differ between controls (0 µM), and 100, 250 and 500 µM PRO, but decreased sign with 1000 µM PRO. In all PRO groups, cholesterol efflux was reduced against the controls (100 µM, p < 0.05, all others, p < 0.01); cells with efflux decreased with increasing PRO (0 µM: 77.8 ± 10.6%, 100 µM: 63.7 ± 11.7%, 250 µM: 52.1 ± 12.9%, 500 µM: 37.7 ± 11.6%, 1000 µM: 33.1 ± 14.4%). Significance between 100 µM, and 500 and 1000 µM PRO: p < 0.01, between 250 µM and 1000 µM: p < 0.05.

Conclusion ABCA1 was maximum blocked with 500 µM PRO; with 1000 µM PRO viability was significantly decreased.

50

Cycle-spanning longterm effects of early corpus luteum development in dairy bovines

Zyklus-übergreifende Langzeiteffekte der frühen Corpus luteum-Entwicklung beim Milchvieh

J. Schneebeli

Summaprada, Switzerland

As for a bovine oestrus follicle’s luteal capacity, the progesterone (P4) blood level before the vesicle’s 1st manual detection (rectal palpation), was shown to be a relevant factor. The present study aimed at gaining additional insight into this phenomenon. Daily collected data were allotted according to the later ovulating follicle’s rise (R) and compared with the P4 output after oestrus (100 data-sets; Swiss Brown dairy cattle). The time around 7 days before R proved to be most informative as for cycle-spanning events. Depending on how data were classified in different groups, (high vs. low or high vs. medium vs. low P4; before R), successive cycles’ P4-levels correlated differently. The study’s results seem to reflect the bovine-specific physiological (most typical) 2- or 3-wave properties. In view of a marked positive correlation between forming luteal tissue and the next cycle’s corpus luteum development, currently practiced standard reproduction procedures (MOET) should be rethougth skeptically. The only way to increase physiological understanding is to focus on spontaneous ovulations. Effects which are known to influence the ovulatory follicle’s fate additionally (left/right relationships etc.), were not considered in this report. In unmated animals, the lowest P4 levels at the time of a follicle’s emergence, correlate most markedly with the next corpus luteum’s P4 output. How far the mating-success depends on that fact must be investigated further.

51

Osteopontin expression in bovine endometrial gland cells is not altered by progesterone in vitro

Die Osteopontinexpression in bovinen endometrialen Drüsenzellen wird in ­vitro nicht von Progesteron beeinflusst

N. Schroth, C. Hildebrand, J. Hollenbach, C. Pfarrer

Institute of Anatomy, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany

Osteopontin (OPN) is an extracellular matrix protein that plays a major role in implantation and placentation. It is secreted by endometrial glands as a major component of the histotrophe and required for adhesion and signal transduction at the uterine-placental interface. In sheep, its expression in endometrial glands is regulated by progesterone (P4) downregulating its own receptor (PR) in endometrial epithelial cells which is a requirement for OPN secretion. As ovine and bovine implantation and placentation differ from each other, the present study aims to investigate the regulation of the OPN expression in bovine endometrial gland cells in vitro. For the experiments, a recently established bovine endometrial gland cell line (BEGC) was incubated with 20 ng/ml P4 for 24, 48 and 96h. Cells incubated in full medium and serum reduced medium served as controls. The mRNA expression of the PR and OPN was determined by quantitative real-time PCR. Additionally, OPN protein expression was analyzed by immunofluorescence und semiquantitative Western Blot. The results show that BEGC express OPN on mRNA and protein level. However, mRNA and protein expression levels were not altered by P4 stimulation. In conclusion, BEGC express OPN but its expression seems not to be influenced by P4. This might be due to the fact that either, in cattle, OPN expression is not regulated by P4 or the applied P4 concentration and incubation time was not suitable to cause any effects. Further studies involving more variables (preconditioning, concentrations, stimulation periods) are planned.

52

Bedding material contaminated with Fenpropimorph causes reduction of fertilizing potential in AI boars

Mit Fenpropimorph kontaminiertes Einstreumaterial reduziert die Fertilitätsleistung von KB-Ebern

M. Schulze1, B. Hensel1, D. Schröter2, C. Leiding3, M. Jung1, M. Lautner3

1Institute for Reproduction of Farm Animals Schönow, ­Bernau, Germany; 2Institut für Getreideverarbeitung GmbH, Nuthetal, Germany; 3Besamungsverein Neustadt a. d. Aisch e. V., Neustadt a. d. Aisch, Germany

In summer 2021, a significant decline in the production output of boars located in an artificial insemination (AI) center in southern Germany was detected. In July, over 54% of the AI boars were no longer in production and more than 45% of ejaculates did not meet the quality requirements (sperm motility, sperm output and sperm concentration) for use in AI. These numbers represent a significant drop in comparison to the other production months and were accompanied by oligozoospermia (azoospermia), asthenozoospermia and teratozoospermia. The aim of this study was to describe the resulting causal research and investigations, which concluded that the origin of the reduction of fertilizing potential was most likely related to a contamination of the bedding material with Fenpropimorph, an ergosterol biosynthesis-inhibiting fungicide with reprotoxic potential. The bedding of the affected AI center was exchanged at the beginning of April 2021. The temporal connection between the new bedding and the beginning of the fertility problems is consistent with the connection to Fenpropimorph as the spermatogenesis cycle in boars takes 41 days and first problems became noticeable in June, about 6 to 8 weeks after sawdust replacement. Fenpropimorph was found in the bedding material, as well as in liver samples of affected animals. The respective concentrations, ranged between 0.20 ± 0.36 mg/kg and 0.019 ± 0.001 mg/kg (mean ± SD). Furthermore, autopsy findings revealed hyperemia of the testis, histologically focal degeneration of the germinal epithelium and signs of reduced spermatogenesis and spermiogenesis.

Grants: Supported by FBF.

53

Neonatal survival rates associated with caesarean section in the dog

Neonatale Überlebensraten im Zusammenhang mit der Sectio caesarea beim Hund

S. Sendag1,2, V. Fux1, T. Conze1, A. Wehrend1

1Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals with Veterinary Ambulance, Justus- Liebig-University Giessen, Germany; 2Clinic for Veterinary Obstetrics and Gynecology, University of Van YYÜ, Van, ­Turkey

Current information on the survival rate of puppies after caesarean section (CS) is lacking in the literature. Therefore, the aim of this study was to present the neonatal mortality rate in puppies after CS. To this end, 277 births that were delivered by CS were evaluated. The obstetric examination and the anaesthetic operation protocol were carried out uniformly and are described by Conze et al. (2019) in detail. The total number of puppies is 1260, of which 25.7% were born before surgery and 74.3% were developed by CS. The average litter size was 4.5 ± 2.9. Most often, two puppies were born per birth (15.9%). 162 bitches did not give puppies before CS. A total of 324 puppies were born before the operation per vias naturales. Of these, 237 were vital and 87 were dead. On average, 1.2 ± 1.9 puppies were born before CS. That only one puppy was born before the operation was the most common case. The death rate was highest at 20.7% if two pups were born before surgery. 936 puppies were developed via a CS. Of these, 688 were developed vital and 248 dead or died within 24 hours post natum. On average 3.4 ± 2.5 puppies were developed by CS. Only one puppy was developed by CS in 73 cases, followed by 55 times in which two puppies were developed and 48 times in which three puppies were developed. The survival rate for puppies born before the procedure is 73.1%. The survival rate of the puppies, which were developed by the CS, is only slightly above this at 73.5%. Based on the total number of puppies, the survival rate is 73.4%. The data presented here help to identify the risk of dying for puppies that are developed in the context of a CS.

54

Effect of uterine torsion intra partum on placental steroidogenesis in cattle

Beeinträchtigung der plazentaren Steroidsynthese in Fällen einer Torsio uteri intra partum beim Rind

S. Sendag1,3, M. Sickinger2, G. Schuler3, A. Wehrend3

1Clinic for Obstetrics and Gynecology, Veterinary Medicine, Van YYÜ, Van, Turkey; 2Clinic for Ruminants (Internal Medicine and Surgery), Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Germany; 3Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals with Ambulatory Service, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Germany

In late pregnant or intrapartal cattle, depending on the case uterine torsion can endanger the life of the offspring and may lead to severe degenerative and inflammatory lesions in the uterus. In addition to the uterus and fetus, the placenta may also be affected by the condition. The aim of this work was to examine the extent to which impaired placental function is reflected in maternal levels of placental estrogens. The study included a total number of 37 dairy cows of various breeds and age. The uterine torsion (UT) group consisted of 20 animals diagnosed with UT after spontaneous birth onset. The comparison group (CG) included 17 animals in which birth was undisturbed or could be terminated with moderate obstetrical assistance. Immediately after the initial obstetrical examination a blood sample was collected for the radioimmunological determination of estradiol-17? (E2), free total estrogen (fTE) and conjugated total estrogen (cTE). In addition, progesterone (P4) was measured, which in bovine gestation is predominantly derived from the ovary. Group comparisons were performed using Student’s t-test. Estrogens were significantly lower in UT vs. CG cows (E2: 1.16 ± 0.82 nmol/l vs. 2.16 ± 0.85 nmol/l, p = 0.0009; fTE: 5.50 ± 4.23 nmol/l vs. 11.9 ± 5.0 nmol/l, p = 0,0002; cTE: 34.9 ± 22.3 nmol/l vs. 55.7 ± 31.5 nmol/l, p = 0,0251). P4 concentrations were not statistically different (1.4 ± 1.4 nmol/l [UT] vs. 1.5 ± 0.6 nmol/l [CG]; p > 0.05). The data clearly demonstrate that impaired uterine blood flow during UT significantly compromises placental steroidogenesis. Possibly this observation can be used for diagnostic or prognostic purposes.

55

Steroid hormone concentrations in parturient cows affected by insufficient dilatation of the cervix

Steroidhormonkonzentrationen bei Kühen mit mangelhafter Zervixöffnung unter der Geburt

S. Sendag1,2, M. Yildiz1, G. Schuler2, A. Wehrend2

1Clinic for Obstetrics and Gynecology, Faculty of Veterinary Medicine, Van YYÜ, Van, Turkey; 2Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals with ­Ambulatory Service, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Germany

Adequate myometrial activity and sufficient dilatation of the cervix are the most critical processes in physiological termination of birth. Insufficient dilatation of the cervix (IDC) is a frequent cause of dystocia in cattle. The mechanisms underlying cervical opening and the pathogenesis of IDC are still widely unclear. Studies on the relationship between IDC and steroid hormones have been limited and have yielded inconsistent findings. Thus, the aim of this study was to investigate the maternal steroid hormone concentrations in cows affected by IDC. The study included a total number of 26 dairy cows of various breeds and age. The IDC group consisted of 12 animals in which the condition was dia­gnosed during obstetric examination. The comparison group (CG) included 14 animals in which birth was completed with moderate obstetric assistance, if at all. In each of the animals, immediately after the initial obstetrical examination a blood sample was collected for the radioimmunological determination of estradiol-17?, free total estrogens, conjugated total estrogens and progesterone. Group comparisons were performed using Mann Whitney U test. Estradiol-17? was significantly higher in IDC vs. CG cows (2034.9 ± 1330.5 pmol/l vs. 1081.8 ± 723.3 pmol/l; p = 0.046). There were no statistically significant differences in the other steroids assessed. The results suggest that in cattle the widely held view that estrogens are important stimulators of cervical softening is inaccurate or that steroid effects on the cervix are modulated to a significant degree at the local level.

56

Influence of negative energy balance and periparturient diseases on follicular dynamics, oocyte quality and subsequent in vitro embryo development in dairy cows

Einfluss von Energiebilanz und Produktionskrankheiten im peripartalen Zeitraum auf die Follikeldynamik, die Qualität der Eizellen sowie die Entwicklung und Kompetenz der Embryonen bei Milchkühen

I. Serbetci, A. Gonzalez-Grajeles, C. Herrera, I. Ibanescu, D. Scarlet, H. Bollwein

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

During transition period, dairy cows suffer from profound negative energy balance (NEB) which leads to suboptimal fertility by affecting follicular environment. The aim of this study was to identify the relationship between postpartum diseases and embryonic development. Multiparous Holstein cows (n = 30) were weekly examined after calving for 8 weeks to detect metabolic or inflammatory disease. Cumulus oocyte-complexes (COCs) were recovered by Ovum Pick-up (OPU) twice weekly from the 5th to the 8th week postpartum. All COCs subsequently underwent IVM, IVF and IVC until blastocyst stage. Cows were retrospectively assigned to one of the following groups: healthy (C, n = 8), metabolic disorders (MD, beta-hydroxybutyrate acid [BHBA] > 1.2 mmol/L, n = 14), or inflammatory disease (ID, somatic cell count [SCC] > 200,000 cells/mL and/or vaginal discharge, n = 8). There was no significant difference in the cleavage rate among groups: C: 84.0 ± 20.5%, MD: 81.8 ± 18.1%, and ID: 82.6 ± 14.4%. Blastocyst rates were also not statistically different between C, MD, and ID cows: 58.6 ± 26.5% vs. 56.9 ± 26.5% vs. 64.1 ± 22.6%. Hatching rate of blastocysts was neither affected by metabolic disorders, nor by inflammatory diseases (C vs. MD vs. ID: 63.7 ± 28.1% vs. 76.1 ± 22.0% vs. 72.9 ± 31.4%). Our results show no effect of postpartum diseases on developmental competence of bovine oocytes after OPU, as assessed by cleavage, blastocyst, and hatching rates. This suggests that in vitro embryo production can successfully be performed starting 5 weeks after calving, regardless of periparturient disorders.

57

Effect of plasma transfusion on foals with hypogammaglobulinemia

Auswirkung der Plasmatransfusion bei Fohlen mit Hypogammaglobulinämie

M. Sievert, G. Schuler, A. Wehrend

Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals, Justus-Liebig-University Giessen, Germany

Newborn horses are born near agammaglobulinemic due to the noninvasive placentation type in horses. Hypogammaglobulinemia is a life-threatening condition in newborn foals caused by inadequate postpartum colostrum intake. Because of the time limitation of intestinal absorption, the treatment of choice for hypogammaglobulinemia is plasma transfusion. In this study, we determined how many grams of immunoglobulin G (IgG) are needed to cause a measurable increase in IgG concentration in the blood of foals. For this purpose, the IgG concentration of 21 foals was determined before and 24 hours after transfusion. In addition, the IgG content of the transfused plasma was measured. The measurements were performed with a self-developed sandwich ELISA. In 16 of the 21 foals (76.2%) an increase in IgG concentration was noted at 24 hours after transfusion, whereas in the remaining 5 foals no increase was detected, this is probably caused by a rapid consumption of the transfused IgG. A mean of x? = 9.8 g (0,2–59,9 g) IgG was required to cause a measurable increase of the IgG level. In addition, it was shown that the IgG level in the foal’s blood 24 hours after transfusion increased by a mean of x? = 0.1 mg/dl (0.002–0.537 mg/dl) when 1 g of plasma was transfused. These values provide guidance on how much plasma should be transfused; however, factors such as other diseases of the foal have an influence on IgG concentration. As shown in this study, there is not always an increase in IgG concentration after plasma transfusion. Therefore, a follow-up measurement should always be performed after transfusion.

58

Reversibility of effects by 5-months deslorelin implant treatment in male beagle dogs

Reversibilität der durch eine 5-monatige Behandlung mit einem Deslorelin-Implantat induzierten Effekte beim Beagle-Rüden

S. Stempel1, G. Schuler2, S. Goericke-Pesch1

1Reproductive Unit – Small Animals, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany; 2Clinic for Obstetrics, Gynecology and Andrology of Small and Large Animals, Justus-Liebig-University Giessen, ­Germany

Although deslorelin slow release implants (DSRI) are considered a suitable alternative to surgical castration in male dogs, there is still some missing detailed information. Here, we investigated the reversibility of effects induced by a 5-month treatment with a 4.7 mg deslorelin implant (DSRI) and subsequent hemicastration in male Beagle dogs. Healthy normospermic Beagle dogs were treated with a DSRI (TG, n = 5) or saline (CG, n = 3) over 5 months. DSRIs were subsequently removed and all dogs hemicastrated for histological assessment of spermatogenesis (D ex). Animals were physically and andrologically examined (testis volume, TV/prostate size), semen collected and blood sampled for testosterone (T) analysis before and after treatment in regular intervals. Implant removal was easily possible in all dogs of TG. On D ex, 2 dogs had basal T (? 0.1 ng/ml), 2 had T < 0.2 ng/ml and the last had T = 1.76 ng/ml, T, TV and prostate size increased rapidly after implant removal, not being different from CG on d14 (T), d70 (TV) and d49 (prostate size), respectively. TV and prostate size were increased by factor 4.48 and 6.22 compared to D ex. Dogs were a-/azoospermic on D ex and returned to normospermia between D84 and D133. Our data confirm reversibility of effects induced by a 4.7 mg DSRI and – despite hemicastration – return to normospermia, but they also clearly show high individual variability in the restart of endocrine and germinative testicular function that is clinically relevant for the veterinary practitioner.

59

Effect of heat stress on fertility and the occurrence of periparturient disorders in dairy cows

Einfluss von Hitzestress auf die Fertilität und das Entstehen peripartaler Erkrankungen bei Milchkühen

M. Tekin, C. Guse, M. Iwersen, M. Drillich, K. Wagener

University Clinic for Ruminants, Clinical Unit for Herd Health Management in Ruminants, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria

Heat stress (HS) decreases fertility of dairy cows and can lead to substantial economic losses. The objectives of the study were to assess the effect of different levels of heat stress I) on the occurrence of periparturient diseases and II) on reproductive performance. A total of 1556 dairy cows were examined for signs of puerperal metritis (PM) and subclinical ketosis (SK) on day 5 postpartum (pp). Animals were examined for clinical and subclinical endometritis (CE and SE) on day 28 pp. The extent of HS was estimated on basis of the temperature-humidity index (THI) obtained from half-hourly measurements with data loggers (Plus 2, Tinytag-Gemini Datalogers Ltd., UK) installed in the barns. The time and amplitude a cow was exposed to THI between 72 and highest were determined from calving to diagnoses (objective I), during 6 weeks before artificial insemination (AI), and 40 days after AI (objective II). For each time period, animals were grouped into ‘no-HS’, ‘low-HS’ and ‘moderate-HS’. There was no effect of HS on the occurrence of the uterine diseases PM, CE and SE (p > 0.05). The severity of HS, however, was associated with the prevalence of SK (no-HS: 4.3%, low-HS: 7.3%, moderate-HS: 8.3%, p < 0.05). Logistic regression analysis revealed that cows exposed to low and moderate HS 40 days after AI had a reduced first service conception chance (odds ratio: 0.78, CI: 0.68–0.88, p < 0.001). The Kaplan Meier analysis showed that the time to first AI and time to pregnancy were associated with HS before and after AI (p < 0.05). This study showed associations between HS and reproduction. More research is needed to clarify reduced fertility in heat-stressed cows.

60

Endometrial thickness in dairy cows with clinical endometritis and its association with reproductive performance

Prognostizierbarkeit der Fruchtbarkeit von Kühen mit klinischer Endometritis anhand der Endometriumsdicke

A. Tschuor, L. Pieper, H. Bollwein, D. Scarlet

Clinic of Reproductive Medicine, Department for Farm ­Animals, Vetsuisse Faculty Zürich, Switzerland

The aim of this study was to analyze reproductive performance of cows with clinical endometritis based on endometrial diameter (ED) determined at three timepoints after calving: T1: 21–34 days, T2: 35–48 days and T3: 49–62 days. In 100 cows with endometritis (E) and 100 healthy (H) cows, PGF2alpha was given after each evaluation to prevent/treat endometritis. Cows subsequently underwent AI (n = 1–7) followed by pregnancy diagnosis 30 and 60 days thereafter, respectively. Cows were considered not pregnant if conception did not occur within 200 days after calving. At T1 and T2, E cows had higher ED than H cows (T1: 9.6 ± 0.1 vs 8.8 ± 0.1 mm, p < 0.001; T2: 9.0 ± 0.1 vs 8.5 ± 0.1 mm, p < 0.05). Parity of the cow was positively correlated with ED in H cows (p < 0.05), but not in E cows. A significant effect of endometritis on ED (p < 0.05) was observed at T1 and T2 in cows with 1–3 lactations, but there was no effect in cows with ? 4 lactations. At T1, body condition score (? 3 vs > 3), difficulty of parturition (spontaneous vs dystocia), and occurrence of metritis all affected ED (p < 0.05) in the presence of endometritis. At the same time, endometritis lead to a significant (p < 0.001) increase in ED in cows without retained placenta or hypocalcemia. All these effects were less visible at T2. There was a negative association (p < 0.05) at T1 and T2 between progesterone concentration (<1 or >1 ng/ml) and ED in E cows, but not in H cows. Calving to first service interval and first service conception rate were not influenced by the presence of endometritis and therefore cannot be reliably predicted based on ED determination postpartum.

61

A sertoli cell specific knockout of connexin 43 leads to an altered ­distribution pattern of ?-tubulin in ­sertoli cells

Der Sertoli-zellspezifische Knockout von Connexin 43 führt zu einem ­veränderten Verteilungsmuster von
?-Tubulin in Sertoli-Zellen

M. Ueffing, M. Langeheine, R. Brehm

Institute of Anatomy, University of Veterinary Medicine Hanover, Hanover, Germany

A Sertoli cell (SC) specific knockout (KO) of the gene coding for the gap-junction protein Connexin 43 (Cx43) in mice using the Cre/loxP recombinase system leads to spermatogenic impairment. Moreover, these Cx43-deficient SC show signs of deviant cell shapes, but the underlying cause is not fully decoded yet. Involvement of the cytoskeleton might be one important factor for the observed changes. Especially microtubules are essential for structural support of SC and play a key role in the translocation of developing germ cells (GC) to the luminal edge of the seminiferous tubule in preparation of spermiation. In order to detect potential changes of microtubule distribution in SCC x 43KO mice, immunohistochemistry for ?-tubulin was performed on 5 ?m sections of Bouin-fixed testes. Testis sections of SCCx43KO mice and their wild type (WT) littermates from postnatal day 2 up to adulthood were compared. Seminiferous tubules of WT mice showed a stage-specific spoke-like staining pattern manifesting characteristic disparities between the different stages of spermatogenesis similar to rat. The radial pattern consisted of band- or cable-like structures from the tubular base to the lumen where it branched out and surrounded spermatids. In contrast, Cx43-deficient SC showed a diffuse staining pattern of ?-tubulin or cable-like-structures that were laterally distracted and not in line with the lumen. Differences between both genotypes became more evident during puberty concomitant with the absence of developing GC in mutant mice. These findings suggest that Cx43 in SC has an impact on ?-tubulin distribution and microtubule organisation.

62

Effects of cycle stage on uterine contractility in an equine ex vivo-model

Effekte des Zyklusstandes auf uterine Kontraktionen in einem Ex-vivo-Modell beim Pferd

C. Unruh, M. Köhne, H. Sieme

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

An influence of cycle stage on uterine contractility has been documented in mares. The aim of this study was to investigate the suitability of an isolated-haemoperfused ex vivo-model to study uterine contractility by comparing contractility patterns and response to oxytocin application among estrous (n = 4), diestrous (n = 4) and anestrous (n = 4) uteri, collected at a commercial abattoir. Analyses of perfusate (arterial/venous) were performed every hour for 6 hours to determine cell viability (glucose consumption, lactate production) and cell death (potassium (K+), lactate dehydrogenase concentration [LDH]). Sonomicrometry crystals were implanted into one uterine horn to record spontaneous contractions and the response to 0.5 IU oxytocin after 6h. Frequency, amplitude and duration of uterine contractions were analysed for 10 min at one-hour-intervals. While cellular ­viability and LDH remained physiological, K+ increased over time. Overall, the amplitude of contractions of uteri in oestrus (9.5 ± 7.5%; mean ± SD) differed from diestrous (4.8 ± 4.5%) and anestrous (4.8 ± 4.1%) uteri significantly (p < 0.05). Frequency of contractions in oestrus (2.0 ± 1.3 [contractions/600 sec]) was higher compared to diestrus (1.2 ± 1.3; p < 0.05) but not to anestrus (1.4 ± 0.8). No significant effects of cycle stage on contraction amplitude and duration after administration of oxytocin as well as on duration of contractions were detected. An influence of cycle stage on frequency, amplitude and duration was not observed for different time points. In conclusion, the ex vivo-model is suitable to investigate uterine contractility in the mare.

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Comparison of colostrum qualities of German Landrace and German Saddleback sows

Vergleich der Kolostrumqualitäten von Sauen der Deutschen Landrasse und des Deutschen Sattelschweins

A. Vernunft1, L. Diesing2, C.C. Metges1,2, C. Gladbach1,3

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

The quality of colostrum and milk is crucial for the health and development of piglets, but receives little attention in pig breeding [Theil et al., 2014]. The aim of this study was to compare the quality of the first milk (colostrum) of modern German Landrace (GL) and indigenous German Saddleback (GS) sows. In a pilot study, colostrum samples were collected from 25 GL and 18 GS (22 primipara, 21 secundipara) after the birth of the third piglet (D0) and on the first day post partum (D1). The sows were housed collectively in a conventional barn. In the samples, milk fat (XL), crude protein (XP), lactose (Lac), dry matter (DM) and total energy (TE) contents were determined. Immunoglobulins (Ig) G, A and M were measured by an ELISA kit (Bethyl Lab.). Overall (D0+D1), colostrum had 7.6 ± 2.4% XL, 2.5 ± 0.8% Lac, 13.7 ± 5% XP, 27.1 ± 5.4% DM and 6.8 ± 1.5 MJ/kg TE. No differences were found between breeds or parity in macronutrients but XP, DM and TE decreased and Lac increased from D0 to D1 (p < 0.05). In GL sows, medians of 21.3 and 12.6 ng/ml IgG, 5.7 and 3.1 ng/ml IgA, 4.9 and 3.1 ng/ml IgM were found in D0 and D1 milk, respectively. In GS sows, 24.8 and 4.4 ng/ml IgG, 7.2 and 1.7 ng/ml IgA, 3.4 and 1.7 ng/ml IgM were found in D0 and D1 milk, respectively. Overall, Ig levels were lower as reported (Hurley, 2015). Ig levels decreased 2-fold in GL (p = 0.02) but 3.6-fold in Saddleback colostrum from D0 to D1 (p < 0.001). In conclusion, the macronutrient quality of colostrum did not differ between modern and indigenous pig breed. However, immunoglobulin levels on D1 might be better in modern than in indigenous breeds under conventional conditions.

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Dynamics and diversity of intra­uterine anaerobic microbiota in dairy cows with clinical and subclinical endometritis

Dynamiken und Diversität intrauteriner anaerober Mikrobiota bei Milch­kühen mit klinischen und subklinischen Endometritiden

K. Wagener1, P. Ballas1,2, I. Pothmann1, H. Pothmann1, M. Ehling-Schulz2, M. Drillich1

1Clinical Unit for Herd Health Management in Ruminants, University Clinic for Ruminants, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Austria; 2Functional Microbiology, Institute for Microbiology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria

Despite the dynamic character of uterine bacterial infections in dairy cows, studies on the compositional changes in the postpartum uterine microbiota are limited. Especially the role of anaerobes for the pathogenesis of uterine disease, such as clinical (CE) and subclinical endometritis (SE), is not entirely clear. Therefore, the aim of this study was to evaluate fluctuations within the anaerobically cultivated microbiota in cows with CE and SE. In total, 122 dairy cows were sampled with the cytobrush technique on the day of parturition (day 0), and on days 3, 9, 15, 21, and 28 postpartum (pp). Health status (CE, SE and healthy) was determined on day 21 pp by vaginoscopy and cytological examination. Samples were cultivated anaerobically and analyzed with MALDI-TOF MS. In total, we found 1858 isolates, including 78.8% facultative and 18.6% obligate anaerobes. Trueperella (27.8%), Streptococcus (25.4%), and Escherichia (13.1%) were the most prevalent facultative anaerobic genera. The obligate anaerobes were mainly represented by Peptoniphilus (9.3%) and Bacteroides (3.3%). Trueperella. pyogenes was related to CE, E. coli to SE and Streptococcus pluranimalium was associated with uterine health (p < 0.05). Primiparous cows showed higher prevalence of T. pyogenes, Fusobacterium necrophorum, Porphyromonas levii and Peptinophilus than multiparous cows (p < 0.05) but were not more susceptible to CE and SE. The study showed the diversity of the anaerobe uterine microbiota in cattle and demonstrated that anaerobic bacteria are associated with the development of endometritis whereas others may contribute to uterine health.

65

Phoenixin-14 and its receptor in the bovine endometrium during the ­estrous cycle: mRNA expression and protein localization

Phoenixin-14 und sein Rezeptor im Rinderendometrium während des Brunstzyklus: mRNA-Expression und Proteinlokalisation

N. Wal?dziak, J. Mlynarczuk, R. Rekawiecki, M.K. Kowalik

Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland

Phoenixin-14 (PXN-14) is a newly identified neuropeptide, which is involved in the regulation of reproductive function in mammals. It is mainly produced in the hypothalamus from the precursor protein, a small integral membrane protein 20 (SMIM20). PNX-14 acts through the orphan G-protein-coupled receptor 173 (GPR173). Despite the confirmation of the effect of PNX-14 on different reproductive tissues, there are no evidence about the presence and the potential role of PNX-14 in the endometrium. In this study, mRNA expression and cellular localization of PNX-14/SMIM20 and GPR173 in the bovine endometrium on days 2–5, 6–10, 11–16 and 17–20 of the estrous cycle (n = 4/group) were determined by real time PCR or immunohistochemical analysis, respectively. We observed that PNX-14/SMIM20 and GPR173 mRNA level changes during the estrous cycle, with the highest expression on days 6–10 (p < 0.05). Immunostaining for PNX-14 and GPR173 proteins was detectable in the endometrium from all stages of the estrous cycle, presenting strong reaction in the luminal and glandular epithelial cells and endothelial cells of blood vessels. In conclusion, obtained data show variable expression of PNX-14 and its receptor in bovine endometrium during the estrous cycle and indicate the possible participation of PNX-14 in the regulation of endometrial function.

Grants: Supported by the National Science Centre, Poland grant PRELUDIUM BIS-2 no 2020/39/O/NZ4/03388

66

The proteome of the in vivo and in vitro environment of equine ­cumulus-oocyte complexes

Das Proteom der Umgebung equiner Kumulus-Oozyten-Komplexe in vivo und in vitro

J. Walter1, S. Hellmueller1, L. Kunz2, C. Fortes2, P. Nanni2, J. Grossmann2,3, C. Herrera1, U. Bleul1

1Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Switzerland; 2Functional Genomics Center ­Zurich, University and ETH Zurich, Switzerland; 3Swiss Institute of Bioinformatics, (SIB), Zurich, Switzerland

In vitro Maturation (IVM) is a crucial and rate limiting factor in in the in vitro production of equine embryos. A previous study elucidated important metabolic aberrations in cumulus cells due to IVM [Walter et al. 2019]. This study investigated the basal protein content in maturation medium and the proteins secreted by equine cumulus-oocyte-complexes (COCs) during IVM and compared it with the follicular fluid proteome. COCs were incubated in groups of ten for 30 hours in maturation medium (M199 + FSH + FBS). For each maturation group (n = 6) a control sample of maturation medium without COCs was incubated. Follicular fluid (n = 4) was collected from preovulatory follicles. Label-free proteomics analysis was performed on all samples. Data were acquired on an Orbitrap Lumos mass spectrometer, and further processed for qualitative analysis using MaxQuant and Scaffold software. This approach identified a total of 252 proteins with at least 2 peptides, of which 127 proteins were detected in the follicular fluid, 152 in the maturation medium and 126 in the medium control. Eighty proteins were solely present in the follicular fluid. Downstream analysis using STRING-DB revealed an overrepresentation of proteins of the KEGG pathways complement and coagulation cascade as well as of cholesterol metabolism in these follicular fluid proteins. The maturation medium contained 52 proteins that were not detected in their controls. For these proteins, participants from KEGG pathways glycolysis, oocyte meiosis and the Inhibin-A complex were overrepresented. In summary, this study identified major differences in the in vivo and in vitro surrounding of equine COCs during maturation.

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Comparative proteomics in the ­endometrium of young, old and apolipoprotein E-knockout rabbits at preimplantation

Vergleichende Proteomanalyse des ­Endometriums von jungen, alten und Apolipoprotein-E-Knockout-Kaninchen in der Präimplantationsphase

K. Zech1, M. Buske1, A. Toto Nienguesso1, E. Halbauer1, J.-S. Jung1, M. Fuszard2, D. Dobritzsch2, 3, P. Winterhalter2, A. Ritsch4, M. Schindler1, A. Navarrete Santos1

1Department of Anatomy und Cell Biology, Martin Luther University Halle-Wittenberg, Halle, Germany; 2ZMG and Core Facility – Proteomic Mass Spectrometry, Medical ­Faculty, Martin Luther University Halle-Wittenberg, Halle, Germany; 3Department of Plant Biochemistry, Faculty of Natural Sciences – Biosciences, Martin Luther University Halle-Wittenberg, Halle, Germany; 4Medical University of Innsbruck, Austria

The endometrium is substantially involved in the embryogenesis and the nourishment of the fetus. The risk of abortion is increasing with age and is frequently caused by an endometrial defect. Apolipoprotein (Apo) E-knockout rabbits display hyperlipidemia with increased levels of cholesterol and trigly­cerides leading to arteriosclerosis and premature ageing [Ji et al., Transgenic Res 2015; 24: 227–35; Beierfuß et al., PLoS ONE 2017; 12: e0187564]. The aim of this study was to investigate the endometrium-specific proteome in ApoE-deficient, young (5 months) and old rabbits (25–27 months) at day 6 post coitum. The differential proteome profiles of eighteen females from three groups of young, old and ApoE-knockout rabbits were analysed by mass spectrometry. We established a detailed protein database of the rabbit endometrium during the time of embryo implantation. By comparing the proteomic profiles of ApoE-/-, young and old rabbits, we revealed metabolic and functional differences. While endometrium from old females showed indicators of stress and DNA-damage repair, the ApoE- /- proteome showed increased levels of proteins involved in metabolism, cell division, proliferation and apoptosis inhibition with decreased stress markers, indicating that the endometrium functional benefits from the ApoE deficiency. We suppose that ApoE ­either acts directly on the endometrial cells or indirectly by an altered metabolism. The endometrial dataset provides new information for studying the ApoE-based mechanisms of metabolic diseases and female subfertility.

Grants: Supported by the Deutsche Forschungsgemeinschaft GRK2155 ProMoAge and JU3146.