53rd Annual Conference of Physiology and Pathology of Reproduction and 45th Mutual Conference of Veterinary and Human Reproductive Medicine
26th–28th February, 2020, Rostock
Journal für Reproduktionsmedizin und Endokrinologie - Journal of Reproductive Medicine and Endocrinology 2020; 17 (1): 14-38
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53rd Annual Conference of Physiology and Pathology of Reproduction and 45th Mutual Conference of Veterinary and Human Reproductive Medicine
26th–28th February, 2020, Rostock
*Supporting Organisations: Deutsche Veterinärmedizinische Gesellschaft (DVG) and Deutsche Gesellschaft für Reproduktionsmedizin (DGRM). With permis-sion of Wiley, the abstracts of this conference will be jointly published in the Journal of Reproduction of Domestic Animals (RDA) and the Journal of Reproduc-tive Medicine and Endocrinology (JRE). Peer-reviewed and compiled by the scientific committee.
Is the presence of overgrown follicles in dromedary camel a pathological phenomenon?
Ist das Vorhandensein von übergroßen Follikeln im Dromedarkamel ein pathologisches Phänomen?
1Department of Veterinary Medicine; 2Department of Animal Production, College of Agriculture and Veterinary Medicine, Qassim University, Qassim, Saudi Arabia; 3Department of Theriogenology, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt
The objective of this study was to test whether overgrown follicles (OVGF) are a pathological phenomenon in dromedary camels. Female dromedaries with OVGF (n = 125) were examined by manual palpation and ultrasonography. The OVGF were subdivided into those with thin walls and clear hypoechogenic content (OVGF-TH, n = 18) and those with thick walls and fibrous trabeculae (OVGF-TK, n = 107). Transvaginal follicle aspiration was performed in females with OVGF and from a control group with growing follicles (GF group, n = 5). Serum was collected and analysed for FSH, LH, P4 and E2. The follicular fluid (FF) was analysed for E2 and P4. The results showed that mean E2 concentration in FF and serum were lower in OVGF-TH and OVGH-TK groups than in the GF group (P < 0.05). Mean FSH concentration in serum was higher in OVGF-TH and OVGH-TK groups than in the GF group (P = 0.03). Mean LH concentration was not significantly (P = 0.1) greater in OVGF-TH and OVGH-TK groups than in the GF group. It seems that the high FSH and/or LH concentrations stimulated the continuing growth of the developing follicles to reach these large sizes with the inability to ovulate, suggesting that the phenomenon of OVGF in camels is a pathological finding.
Accidental progesterone ingestion by a neutered male dog
Versehentliche Progesteronaufnahme durch einen kastrierten Rüden
Clinic for Animal Reproduction, Department of Veterinary Medicine, Freie Universität Berlin, Germany
A 10 years old neutered Petit Basset Griffon was introduced to our clinic three days after ingestion of 12 capsules Famenita 200 mg progesterone capsules (Exeltis Germany GmbH, Ismaning, Germany). The usual dosage of the owner, who took the medication against menopausal problems, was 200 mg per day. The dog consumed 2400 mg in total in one dose. Clinical examination revealed that the dog was in good general health condition. Blood samples were taken to measure the progesterone concentration (with Immulite Progesterone Assay (LKPW), Siemens, Berlin, Germany) and other blood parameters to assess a potential toxic effect. Progesterone concentrations were 4.7 ng/ml three days after ingestion, 1.16 ng/ml after seven days and were below 0.2 mg/ml after 11 days. All other blood parameters were almost within reference ranges. Based on these findings it can be assumed that the progesterone concentrations may have been high directly after ingestion. However, the owner had not observed any clinical side effects. Information in the literature about potential progesterone intoxication is scarce. According to one case report, a cat showed signs of sedation after a potential progesterone intoxication [Dhumeaux, et al. JFMS 2010; 12: 811–3]. In addition, high progesterone doses may modify sleep patterns and be sedative and anxiolytic in humans [Bitran, et al. J Neuroendocrinol 1995; 7: 171–7]. In the present case, the dog seemed to be able to metabolize and excrete progesterone within a few days without any negative implications on his health.
HIF1A driven gene expression is essential for granulosa cell function in bovine
Die HIF1A-getriebene Genexpression ist essentiell für die Funktion der Granulosazellen bei Rindern
Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany
Hypoxia inducible transcription factor 1 (HIF1) is a pleiotropic transcription factor consisting of a constitutively expressed ?-subunit (HIF1B) and a regulatory ?-subunit (HIF1A). In the present study, we analysed the expression and function of HIF1A in bovine granulosa cells (GC) for further understanding of follicular biology. Treatment of GC with FSH and IGF1 hormones resulted in the dose-dependent up-regulation of HIF1A expression. Affirming this gene expression, immunohistochemistry of bovine ovarian sections showed distinct staining of HIF1A protein in the GC layer of growing ovarian follicles. These data suggest an essential role of HIF1A in FSH and IGF1 induced follicular functions. Suppression of HIF1 function using echinomycin and gene knockdown procedures resulted in the down- and up-regulation of VEGFA and VNN2 expression, respectively, indicating the angiogenic and anti-inflammatory roles of HIF1 in GC. Importantly, HIF1A could be able to regulate the expression of critical steroidogenic genes such as STAR, HSD3B1, and CYP19A1. Our data indicate that CYP19A1 is one of the plausible direct downstream targets of HIF1 in GC as shown by chromatin immunoprecipitation analysis. We could also show that HIF1 plays a distinctive role in cell proliferation under normoxic and hypoxic conditions. Knockdown of HIF1A resulted in decreased and increased GC proliferation under normoxic and hypoxic conditions, respectively, by regulating PCNA and CCND2 expression. Based on these results, we propose that HIF1A driven transcriptional activity plays a key role in GC of bovine ovarian follicles.
Case report: Perineal urethrostomy in a 1-day old alpaca cria
Fallbericht: Anlegen einer Harnröhrenfistel bei einem 1-Tag alten Alpaca cria
1Clinic for Ruminants with Ambulatory and Herd Health Services, Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-University Munich, Germany, 2Experimental Farm Oberschleissheim, Ludwig-Maximilians-University Munich, Germany
Congenital abnormalities and malformations are relatively common in alpacas. In this case, a 1-day old cria (10.1 kg) was presented with strangury and bladder distention. Although exterior male genitals were present, urination through the prepuce was not possible. Slight pressure on an obvious bulging of a pouch in the perineal area revealed drip-like urination from a 1 mm opening. Abdominal ultrasonographical examination of the urinary tract showed a dilated bladder (diameter 6 cm). Blood urea nitrogen and creatinine levels were increased [16.4 mmol/L (4.5–11.5 mmol/L), 155.8 µmol/L (91–203 µmol/L)]. Under general and local anaesthesia, the opening in the perineal area was extended. A Rüsch ureter catheter (No 6 Teleflex Medical, Ireland) was placed through this opening into the bladder and the mucosa was adapted to the skin (Monosyn metric 3/0, B. Braun surgical, Spain). Post-operative care included antibiotic treatment, pain management, daily cleaning of the wound and control of the urination through the catheter for seven days. One day after catheter removal (day 8) ultrasonographical imaging revealed an empty bladder and blood urea nitrogen and creatinine levels were normalized (5.50 mmol/L, 89.6 µmol/L). The cria was discharged after a total of 13 days of hospital treatment and had developed satisfactorily (weight at discharge 13.0 kg). Two months later, the cria shows normal behaviour and normal urination via the urethrostomy. In conclusion, the congenital partial vulvar atresia of the intersex cria was surgically resolved via perineal urethrostomy.
L-lactate – a signalling molecule altering the gene expression profile in a genome wide manner during granulosa cell differentiation
L-Lactat – ein Signalmolekül verändert das Genexpressionsprofil genomweit während der Granulosazell-Differenzierung
1Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany; 2Institute for Immunology, University of Rostock, Rostock, Germany
L-lactate was shown to influence the differentiation of cultured estrogen-active bovine granulosa cells (GC) leading to an early pre-ovulatory phenotype. Subsequently, we examined, whether L-lactate treated GC showed a significant alteration in their genome wide expression profile. Therefore, GC were cultured serum-free with FSH and IGF-1 for 8 days. Additionally, the cells were treated with 30 mM L-lactate or 30 mM NaCl (vehicle control). The ensuing microarray analysis was performed with the Bovine Gene 1.0 ST Array. 487 clusters (= 461 annotated genes) were identified as differentially expressed in L-lactate treated GC compared to the vehicle control. Two thirds (= 333 clusters) were up- and one third (= 154 clusters) down-regulated. The top up-regulated genes were TXNIP (22.0), H19 (12.4) and AHSG (8.5), whereas VNN1 (-2.8), SLC27A2 (-2.7) and CYP19A1 (-2.3) were among the top down-regulated genes. Subsequent pathway analysis by IPA revealed the involvement of ‘cAMP-mediated signalling’ as well as ‘Axon guidance signalling’. Further, estradiol and progesterone were identified as potential upstream regulators of gene expression. An effector network analysis provided first hints that processes of “angiogenesis” and “vascularization” appeared to be activated, whereas “organismal death” was predicted to be inhibited. Our data reveal that L-lactate can act as a signalling molecule by altering the gene expression profile in a broad but specific manner. Moreover, angiogenic processes but also migratory events like cell movement and axonal guidance signalling are initiated.
Effects of follicle stimulating hormone- (FSH-) induced controlled ovarian hyperstimulation and nutrition on gene expression in ovine endometrium
Effekte der mit Follikel-stimulierendem Hormon- (FSH-) induzierten kontrollierten ovariellen Überstimulation und Ernährung auf die Genexpression im Endometrium bei Schafen
1Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, Switzerland; 2Department of Histology and Embryology, Faculty of Veterinary Medicine, Erciyes University, Kayseri, Turkey; 3Department of Animal Sciences, North Dakota State University, Fargo, USA
Overweight and underweight are commonly known risk factors for fertility disturbances. Moreover, controlled ovarian stimulation with exogenous gonadotropins during assisted reproductive technologies may alter endometrial receptivity in cycles of in vitro fertilization (IVF). Here, using an ovine model, we investigated the effects of ovarian hyperstimulation under different feeding regimes, on expression of selected genes involved in uterine receptivity. Several (n = 14) genes were screened for their mRNA levels in caruncular endometrial areas of adult sheep in 12 experimental groups (n = 3–5 each): hyperstimulated with FSH or non-treated (naturally cycling), in normally fed (NF), overfed (OF) or underfed (UF) animals. In each feeding group, samples were collected at the early- and mid-luteal phases (days [d]5 and 10 of the luteal life-span). Within groups, more effects were observed at d10. When compared with d5, FSH-treatment at d10 revealed, i.a., decreased expression of MUC1 in NF animals, with concomitantly increased FGF10 and FN1 (P < 0.05). At d10, FSH increased FGF10 in NF while decreased it in UF (P < 0.05). Also at d10, levels of ITGB3 and ITGA4 were increased in OF (P < 0.05) and UF (P < 0.001) compared with NF control animals. Their expression was also increased in OF animals (P < 0.05) treated with FSH. Overall, imbalanced nutrition affects uterine expression of genes responsible for intercellular communication, cell adhesion, growth factors, and impacts the uterine responsiveness to exogenously applied hormonal stimulation and, likely, implantation.
Bioassay for early bovine pregnancy detection by using epithelial cells
Bioassay zum frühen bovinen Trächtigkeitsnachweis unter Verwendung epithelialer Zellen
1Clinic for Cattle, University of Veterinary Medicine Hannover, Germany; 2Laboratory for Functional Genome Analysis, Gene Center, Ludwig-Maximilians-University Munich, Germany; 3Institute of Anatomy, University of Veterinary Medicine Hannover, Germany; 4Institute of Pathology, Ludwig-Maximilians-University Munich, Germany; 5Martin-Luther-University Halle-Wittenberg, Faculty of Natural Sciences III, Animal Health Management, Halle, Germany
An early pregnancy detection (before day 21 after artificial insemination; AI), for diagnostic purpose or in research projects, is still not available for cows. However, to examine the underlying reasons for early embryonic mortality (day 7–16 after AI), an earlier pregnancy detection method is necessary. We previously described a bioassay using serum/plasma of a pregnant cow incubated with leucocytes of a donor cow, following measurements of pregnancy induced gene expression. However, due to differences in leucocyte batches, standardization of the test was poor. Therefore, the aim of the present study was to test three different cell types (bovine trophoblast cells [F3], bovine caruncular epithelial cells [BCEC], Madin-Darby bovine kidney cells [MDBK]) in a pregnancy bioassay. After incubation (24h) with serum of pregnant (n = 38) and not pregnant (n = 65) cows (day 1/16 after AI) and a control (recombinant interferon-?; IFN?), qRT-PCR was performed to determine mRNA expression of two IFN? induced genes (MX2, ISG15). In all three cell types IFN? lead to induction of MX2 and ISG15 mRNA expression. However, a threshold for pregnant and not pregnant cows cannot be found. A transcriptome analysis of gene expression of with pregnant serum stimulated BCEC and MDBK was performed. Remarkably, the transcriptome data revealed that SPINK5 and HSD17? were solely upregulated in cells stimulated with serum of pregnant cows. Therefore, these genes are interesting candidates for further testing of an early pregnancy bioassay.
Teaching artificial insemination in cattle: assessment of stress level
Beurteilung des Stresslevels von Rindern im Rahmen praktischer Übungen zur künstlichen Besamung
1Institute for Reproduction of Farm Animals Schönow, Bernau, Germany; 2Clinic for Cattle, Endocrinology Laboratory, University of Veterinary Medicine Hannover, Germany; 3Martin-Luther-University Halle-Wittenberg, Faculty of Natural Science, Animal Health Management, Germany
Artificial insemination (AI) is the most important biotechnology in modern animal breeding. As the public interest in animal welfare rises in the last years, the questions were addressed, if and how the stress level of cows used for teaching artificial insemination can be assessed. For that purpose, a herd of 32 cows were divided into three groups: Experienced (E, completed more than 8 sessions), Not Experienced (NE, completed less than 8 sessions) and Control (C, not used for AI training). All animals were monitored during 15 training sessions. In each session, blood and saliva samples for cortisol analysis were taken 60 and 30 min before and 30 as well as 60 min during handling and 30 min after the end of the training. Cortisol was measured using a validated immunoassay. Furthermore, heart rate variability (HRV) was analysed to determine the activity of the parasympathetic (PNS) and sympathetic nervous system (SNS). Intervals of five min length were analysed 60 and 30 min before starting, in the beginning, the middle and at the end, as well as 30 min after finishing a training session. Cortisol increased during training (P < 0.005) and decreased within 30 min after finishing a session (P < 0.001). During training, only NE had higher Cortisol levels than C (P < 0.05). HRV showed no differences between the several analysed five-minute intervals. The results show an immediate return of cortisol to basal levels after finishing a session and a reduced cortisol rise in experienced animals. In addition, the training sessions did not result in a significant shift of the autonomic nervous system to an activation of the SNS or a downregulation of the PNS.
Grants: Supported by FBF.
Does testicular greyscale analysis during puberty predict future reproductive performance of AI boars?
Ermöglicht eine testikuläre Graustufenanalyse während der Pubertät eine Vorhersage der zukünftigen Reproduktionsleistung von Besamungsebern?
1Institute for Reproduction of Farm Animals Schönow, Bernau, Germany; 2Pig Improvement Company, Hendersonville, TN, USA
New ways of predicting fertilizing performance in young artificial insemination (AI) boars are very important for breeding companies to ensure genetic dissemination in the field. The aim of the current study was to characterize the testicular development of 218 Piétrain boars (Line 408, Pig Improvement Company, PIC) for commercial use in AI at defined ages throughout pubertal development. Scrotum, testes, and epididymis were examined at day (d) 100 and 170 using B-mode ultrasound with LOGIQ®e R7 (GE healthcare) and a 5–13 MHz 12L-RS linear transducer. Greyscale analysis (GSA) was performed with Image-Pro® premier (Mediacybernetics). Statistical analysis showed significant (P < 0.05) differences between 100 and 170 d for Maximum grey value, Minimum Grey value (MIN GV), Mean Grey value, Standard deviation of Mean grey value, Heterogeneity, Normalized grey scale histogram width, Area under the curve (AUC), and Mean Gradient value (GRAD). Furthermore, AUC (P = 0.037) and GRAD (P = 0.030) measured at 100 d revealed significant differences between boars with high and low sperm producing capability (SPC). MIN GV was higher for boars with low SPC at 170 d (P = 0.046). Segmental nonlinear regression analysis was used for determining breakpoints for GSA. Boars meeting defined breakpoints, i.e. noticeable shifts in ultrasonic image composition, had a higher risk for a low SPC, calculated as odds ratio (OR) at 170 d (OR = 2.4 [1.18, 5.00]). These results seem promising for predicting future SPC. Now it will be important to validate these preliminary results on a larger boar population.
Dystocia and stillbirth on four East German dairy cattle farms
Schwergeburten und Totgeburten auf vier ostdeutschen Milchviehbetrieben
Clinic for Ruminants and Swine, Faculty of Veterinary Medicine, University of Leipzig
Dystocia and stillbirth can be of considerable economic importance for the farmer. Not just because of direct losses, but also because of negative effects on the following production period. Furthermore, it has relevance for animal welfare. Still around 6–8% of dairy calves are born dead or die shortly after delivery. Sex, parity of the dam, season of birth and monitoring intensity are a few factors influencing stillbirth and dystocia rates on a farm. In a retrospective observational study on four farms in eastern Germany we analysed calving data submitted by the farmers to the herd management software “Herde”. 12,922 calvings over two years were included, and data to parity of the dam, stillbirth rate and ease of calving were collected. Dystocia occurred in 1–6% (mean 3%) of the calvings and stillbirth rate was calculated to be 4–7% (mean 6%). In male calves, dystocia rates were 2–7% (mean 4%) and 47–70% (mean 59%) of all stillbirths occurred in male offspring. Female calves were born in 1–3% (mean 2%) under dystocic conditions. In heifers dystocia occurred with 1–17% (mean 8%) of the calvings. In comparison in cows with 1–4% (mean 2%) (p = 0.16). Interestingly stillbirths in heifers occurred less often with 31–48% (mean 38%) of the stillbirths than in cows with 54–69% (mean 63%) (p = 0.002). Similarly surprising, stillbirth occurred more often in eutocic calvings (mean 44%, range 35–57% of the stillbirths) than in dystocic calvings (mean 17%, range 13–23% of the stillbirths) (p = 0.025). In calvings with assistance, not rated to be dystocic, stillbirth occurred in 20–52 % (mean 39%). In our preliminary study, in a limited number of farms, classical factors favouring stillbirth as parity of the dam and dystocia might not be the main risk factors, and herd specific factors need to be studied in more detail. Further studies with a larger number of farms are necessary.
Glyphosate affects in vitro maturation of bovine cumulus-oocyte-complexes
Glyphosat beeinflusst die In-vitro-Maturation boviner Kumulus-Oozyten-Komplexe
1Chair for Molecular Reproductive Medicine, Clinic for Veterinary Obstetrics, Gynecology and Andrology; 2Institute for Veterinary Physiology and Biochemistry, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Germany
Glyphosate (Roundup®) is a non-selective systemic herbicide widely used. There is some evidence that Roundup® (R-Gly) can act as an endocrine disruptor. Therefore, the aim of this study was to evaluate the effect of different Roundup® concentrations supplemented during in vitro maturation (IVM) on maturation rates and subsequent embryo development as well as on the concentrations of steroids (progesterone-P4, estradiol-E2) and metabolites (glucose, lactate, pyruvate, glutamate, glutamine, citrate) in maturation medium (MM). Embryos were generated using a standard IVP protocol (at least 3 replicates). MM (supplemented with 0, 30, 300 µg/mL R-Gly) was collected after 24 h of maturation and used for steroid analyses via RIA and photometric metabolite measurement. Maturation rates (MR) were determined after Hoechst staining and cleavage/developmental rates (CR, DR) were recorded. MR did not differ significantly between oocytes. CR/DR were similar between embryos generated with 0 and 30 µg/mL R-Gly supplementation during IVM (73.9% ± 11.1, 80.3% ± 7.1; 31.7% ± 11.2; 29.5% ± 11.5). However, cleavage and developmental rates were significantly decreased for embryos generated with 300 µg/mL during IVM (36.2% ± 16.6; 4.9% ± 4.5) compared to both other groups. A significant P4 and E2 increase was detected in MM from the 300 µg/mL group (P4: 26.0 ± 6.0 ng/mL; E2: 147.4 ± 45.1 pg/mL). No differences could be determined for the MM metabolites. These data indicate that a supraphysiological R-Gly concentration during IVM affects steroid synthesis of cumulus cells and subsequent embryo development.
Influence of breed on endocrine, metabolic and ethological parameters of sows during farrowing in consideration of husbandry conditions
Einfluss der Rasse auf endokrine, metabolische und ethologische Parameter im Geburtsverlauf von Sauen unter Berücksichtigung der Haltungsbedingungen
1Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany; 2Justus-Liebig-University Giessen, Germany; 3Christian-Albrecht-University Kiel, Germany; 4Tierpark Arche Warder e.V., Warder, Germany
The shift of the birthing conditions of sows from farrowing crates to farrowing pens is intended to improve animal welfare. Concerns have been raised whether modern breeding lines will be capable to fully adapt to the new environment. This study evaluates the spontaneous farrowing process in both farrowing crates and farrowing pens in German Landrace (GL; n = 28) and Angeln Saddleback (AS; n = 13), i.e. pig breeds which differ fundamentally in performance and breeding history. Beginning from the birth of the first piglet until one hour after the last delivery, half hourly blood samples were collected via an ear vein catheter. The duration of farrowing (AS: 199 ± 85 min; GL: 227 ± 57 min) as well as the rate of prolongations during farrowing (birth interval > 60 min, AS: 7.7%; GL: 46.4%, p = 0.03) have been considerably dependent on breed, regardless of the type of housing. Birth intervals were comparable between breeds (GL: 17.1 min; AS: 15.1 min). Differences in piglet losses during lactation (GL: 11.6%; AS: 16.2%) occurred mainly due to crushing (GL: 24.3%; AS: 41.7%). Preliminary results show an impact of breed on metabolic and endocrine parameters of the birthing process. Cortisol for example shows rather higher concentrations for AS vs GL under both housing conditions, whereas GL show rather higher Estradiol levels throughout the farrowing process. Results retrieved from this study form a further basis for the public discussion on improving animal welfare in pig production.
Grants: The project is supported by the Hessian Ministry for the Environment, Climate Protection, Consumer Protection and Agriculture.
Lipid accumulation and mitochondrial activity during in vitro maturation of bovine oocytes
Lipidakkumulation und mitochondriale Aktivität während der In-vitro-Maturation von Rindereizellen
1Universidade Federal de Mato Grosso do Sul, Faculdade de Medicina Veterinária e Zootecnia, Campo Grande, MS, Brazil; 2Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany; 3Universidade Estadual de Mato Grosso do Sul, Aquidauana, MS, Brazil
The aim of this study was to measure lipid content and mitochondrial activity during in vitro maturation (IVM) of bovine oocytes. Therefore, ovaries were collected in a local slaughterhouse and transported to the laboratory in 0.9% saline plus antibiotics at 30°C. The follicles were aspirated and the cumulus-oocyte-complexes (COCs) screened and classified. IVM was performed in BO-IVM medium (Bioscience) for 24 hrs at 38.8°C, 6% CO2 and maximum humidity. COCs (n = 102) were collected at 0, 4, 8 and 24 hrs after in vitro maturation, and time 0 was considered the moment immediately after follicle aspiration. Cumulus cells were removed and oocytes stained with MitoTracker® (300 nM) for 40 min (mitochondrial activity) and Bodipy® (3 µg/ml) for 10 min (lipid detection). After washing and fixation, the stained oocytes were analysed in a confocal microscope coupled to an inverted Axiovert 200M microscope. The fluorescence intensity was quantified using ImageJ software and statistical analysis was performed using SAS software (version 9.4). No difference was found of mitochondrial activity (p > 0.05) between immature and in vitro matured oocytes for 4, 8 and 24 hrs. However, after 4 hrs of maturation, the lipid content was higher (p < 0.001) than that observed in immature oocytes and was similar at all maturation time points studied. It was concluded that oocyte lipid accumulation occurs largely in the first 4 hours of in vitro maturation without variation of mitochondrial activity, suggesting that mitochondrial beta oxidation is not the main pathway involved in this process.
Examination about bacterial colonization on prolapsed vaginal tissue in ewes with Prolapse vaginae ante partum
Untersuchung zur bakteriellen Besiedlung von prolabiertem Vaginalgewebe bei Schafen mit Prolaps vaginae ante partum
1Clinic for Obstetrics, Gynecology, and Andrology of small and large animals with an ambulatory service of Justus-Liebig-University Giessen, Germany;
The prolapse of vaginal tissue ante partum is a common disorder in sheep. The treatment usually consists of reposition of prolapsed tissue and a temporary vulval lock. Whether an antibiotic drug needs to be administrated or if antimicrobial therapy is unnecessary remains doubtful. The aim of this study was to get information about bacterial colonization of the prolapsed tissue by examining smears from the prolapsed tissue (P) using an aerobe bacterial culture (n = 15). As a control, vaginal smears from healthy high-pregnant sheep were examined in the same way (K) (n = 5). In group P average 5.9 different bacterial species were detected, in group K 2.4 different species. The most frequently isolated bacteria in P were E. coli and alpha-haemolytic Streptococcus, and in K group were E. coli and aerobic Bacillus. In P, 37 cases of high-grade bacterial growth were found, in K only one such case. In conclusion the antibiotic treatment seems to be a suitable metaphylactic procedure to prevent ascending transcervical infections of placenta and fetuses.
Changes in seminal Semenogelin I and II of asthenozoospermic camels
Veränderungen des seminalen Semenogelin I and II von asthenozoospermen Kamelen
1Department of veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University, Saudi Arabia; 2Department of Theriogenology, Faculty of Veterinary Medicine, Assiut University, Egypt
Semenogelin I (SEM I) and II (SEM II) are two proteins of the seminal plasma in human and animals. They are mainly secreted from the seminal glands and required for preventing premature capacitation of the sperm. The objective of the present study was to evaluate the changes in SEM I or II concentrations of the infertile dromedary males relative to the percentage of motile sperms in their semen. Total of 47 male dromedaries were assigned for the present investigation (39 infertile and 8 control). The main complaint of these camels was the inability to impregnate fertile females for at least one season. History and signalment, clinical examination and semen analysis were carried out for each individual animal. Based on the semenogram, asthenozoospermia (ASTH) was diagnosed and categorized into light (motility 31–50%, n = 9), moderate (11–30%, n = 11) and severe (? 10%, n = 13). Normal motility was set at ? 50% (6 infertile and 8 fertile males). Seminal plasma was harvested and analysed for Semenogelin I and II. Both biomarkers were significantly lower in ASTH than control males (P < 0.05). It can be concluded that seminal SEM I and II concentrations could be used as indicators for deteriorated sperm motility in male dromedary probably due to premature capacitation. Future work on the source of both SEM I and II in camel and its actual mechanism in the process of capacitation are to be investigated.
Does glyphosate affect the expression of developmental genes in bovine oocytes?
Beeinflusst Glyphosat die Expression entwicklungsrelevanter Gene in bovinen Oozyten?
1Department of Animal Sciences, Georg-August-University Göttingen, Germany; 2Department of Economics, Georg-August-University Göttingen, Germany
The aim of this study is to determine, whether the active ingredient glyphosate alone or in formulation with Roundup affects the expression of developmentally important genes in bovine oocytes. Ovaries of healthy cows were collected at a slaughterhouse. Glyphosate and Roundup were added to the maturation medium in active agent concentration of 10 µg/ml each. After 24 hours of incubation (39 °C, 5% CO2), oocytes were denuded individually for separate treatment of cumulus cells and oocytes (n = 12 for each group). Total RNA was isolated with TriZol and cDNA was synthesized. The relative expression of the gene transcripts was determined by qPCR with EvaGreen detection. The samples were quantified according to the method of Pfaffl [Pfaffl, Nucleic Acids Res 2001; 29: e45]. The expression levels were analysed via ANOVA and Tukey‘s HSD test. Bone morphogenetic protein 15 was significantly higher expressed in the oocytes of the Roundup group compared to the control (p < 0.05). Zygote arrest 1 was significantly higher expressed in oocytes of the glyphosate group (p < 0.01) and of the Roundup group (p < 0.001) compared to the control. Estrogen receptor 1 was significantly higher expressed in cumulus cells of the glyphosate group than in the control (p < 0.05). No significant differences in expression could be observed for growth differentiation factor 9, nuclear progesterone receptor and hydroxy-delta-5-steroid dehydrogenase. In this study, negative effects of glyphosate and Roundup on bovine oocyte development could not be detected.
The expression of genes involved in chronic inflammation and extracellular matrix composition in mare endometrium
Die Expression von Genen, die an chronischen Entzündungen beteiligt sind, und die Zusammensetzung der extrazellulären Matrix im Stutenendometrium
1Department of Large Animal Diseases with Clinic, Veterinary Research Centre and Center for Biomedical Research, Faculty of Veterinary Medicine, Warsaw University of Life Sciences WULS – SGGW, Warsaw, Poland; 2Clinic for Veterinary Obstetrics, Gynecology and Andrology, Justus-Liebig-University, Giessen, Germany
Chronic endometritis (CE) is a serious medical condition implicated in 12–46% cases of infertile mares. In these cases the pro-inflammatory molecules, including monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) act on cells resided in the extracellular matrix (ECM), and probably affect the hyaluronic acid (HA) content in ECM via changes in the activity of three different hyaluronan synthases (HAS1-3). The aim was to compare the expression of genes of MCP-1, IL-6, HAS1-3 in the endometrium between health mares (HE) and mares with CE. Total RNA was extracted from all endometrial biopsy samples (HE: n = 10 and CE: n = 10). RT-PCR amplification was done using specific oligonucleotide primers (TaqMan). The semi-quantitation of target gene expression was performed using two independent endogenous reference genes (GAPDH, HPRT1) in the comparative CT method (??CT method). The high (p < 0.05) expression of mRNA (mean ± SD) of MCP-1 and IL-6 was observed in CE (MCP-1: 3.85 ± 1.31 AU; IL-6: 3.01 ± 2.42 AU) in comparison to HE (MCP-1: 2.25 ± 2.04 AU; IL-6: 1.46 ± 1.24 AU). The expression of mRNA of HAS1 and HAS3 was higher (p < 0.05) in CE (HAS1: 2.89 ± 1.49 AU; HAS3: 5.74 ± 3.13 AU) than in HE (HAS1: 1.30 ± 0.90 AU; HAS3: 1.58 ± 1.43 AU), whereas of HAS2 was lower (p = 0.032) in CE (HAS2: 0.48 ± 0.34 AU) than HE (HAS2: 1.81 ± 1.31 AU). The activity of hyaluronan synthases, responsible for the production of HA which is considered as a major macromolecular component of the ECM, undergoes dynamic regulation during chronic inflammation in mare’s endometrium.
Clinical and spermatological findings in male dogs with acquired infertility: a retrospective analysis
Klinische und spermatologische Befunde bei Rüden mit erworbener Infertilität: eine retrospektive Analyse
Department of Animal Reproduction with Clinic, University of Warmia and Mazury in Olsztyn, Poland
A variety of causative factors of infertility in male dogs have been reported. In the study, the results of clinical examination and semen evaluation of 61 infertile stud dogs were described. The dogs were presented at our clinic from 2012 to 2019 because of infertility, defined as conception failure at least 3 matings with different bitches. The dogs belonged to various breeds, were 4-8 years old and had a history of prior normal fertility. The dogs were subjected to clinical examination including ultrasonography of prostate and testes. Semen was collected by manual manipulation. The sperm concentration and motility parameters were evaluated using computer assisted sperm analyser. The morphology of spermatozoa and the percentage of live and dead spermatozoa were assessed microscopically. In all dogs semen parameters were outside of reference range, mostly oligoastheno-teratozoospermia was found. Thirty dogs showed no clinical abnormalities of genital organ and no signs of systemic diseases and the testicular degeneration was assumed as the possible cause of infertility. In 20 dogs benign prostatic hyperplasia was diagnosed, in 3 dogs infertility was associated with hypothyroidism. Three dogs had a history of babesiosis, 1 dog was previously treated with ketoconazole. Moreover, one case each chronic prostatitis, prostatic adenocarcinoma, epididymitis and retrograde ejaculation was diagnosed. The results showed that the cause of acquired infertility could not be identified in almost half of the dogs. In other dogs infertility was often associated with prostate diseases.
IVF with oocytes from ovum-pick-up or complete dissociation of the ovary results in similar embryo development rates in the common marmoset monkey (Callithrix jacchus)
Vergleichbare Entwicklungsraten bei IVF-Embryonen von Ovum-pick-up Eizellen und von Eizellen aus kompletter Präparation der Ovarien beim Weißbüschelaffen (Callithrix jacchus)
Platform Degenerative Diseases, German Primate Center – Leibniz Institute for Primate Research, Göttingen, Germany
Common marmosets are a valuable animal model in biomedical research, particularly since their genetic modification was invented. Essential for the generation of genetically modified animals is access to early, i.e. single cell embryos, which can be obtained by assisted reproductive technologies. This includes oocyte retrieval after hormonal stimulation of the ovaries in vivo with FSH and hCG. We monitored the stimulation of the ovaries by ultrasound in 9 wake female common marmosets. Follicular development was assessed and numbers of visible follicles were documented. Comparison of the ultrasound-based data with the in vivo findings during subsequent laparotomic oocyte retrieval revealed accurate representation of the in vivo situation by ultrasound. In addition, we used two oocyte retrieval methods: (1) ovum-pick-up (OPU) by puncturing the surface of the ovaries and (2) ovariohysterectomy (OvEx) with complete dissection of the ovaries. OPU provided an average of 12.4 oocytes per round. In contrast, in the OvEx group the outcome was on average 30.1 oocytes. After fertilization with freshly prepared marmoset sperm, 11.3% of the oocytes in the OPU group developed to multicellular embryos, respective 11.6% in the OvEx group. In summary, (1) ultrasonography of marmoset ovaries is a reliable tool for the exact determination of follicular development under hormonal stimulation and (2) full dissection of the ovary after OvEx provides significantly more oocytes than OPU by needle aspiration and (3) the relative embryo outcome after IVF is almost equal irrespective to the method applied for oocyte retrieval.
Mimicking maternal stress in vitro: Using a compartmentalized oviduct epithelium model to investigate transepithelial cortisol distribution
Maternaler Stress in vitro: Verwendung eines kompartimentierten Eileiter-Epithelzellmodells zur Untersuchung der transepithelialen Cortisolverteilung
Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany
High levels of the stress hormone cortisol in the early embryonic environment may impair maternal fertility. Aim of the study was to use a compartmentalized in vitro model of porcine oviduct epithelial cells (POEC) to explore the distribution of cortisol across the oviduct epithelium. In experiment 1, different levels of cortisol (0, 50, 100 and 250 nM) were applied to the basolateral compartment of inserts with or without POEC (n = 5) for 3d. Levels of cortisol in the apical and basolateral compartments were quantified by ELISA. Results showed that in the absence of cells, all cortisol concentrations were stable for 3d. However, in the presence of POEC, cortisol in all groups was decreased to < 28 nM in both basal and apical compartment on d3. RT-qPCR analysis revealed generally high abundance of HSD11B1 and -2 (converting cortisone to cortisol and vice versa) and a down-regulation of HSD11B1 in treated cells. In experiment 2, POEC (n = 2) were treated with 250 nM cortisol for 12, 24, 48 and 72h, respectively. Cortisol levels in cells, apical and basal compartments were assessed. The cortisol concentration of cells was low (< 0.4 nM in all groups). In the apical compartment cortisol increased to 84 ± 33 nM within 12h and then gradually decreased to 21 ± 6 nM after 72h. The basolateral cortisol concentration steadily decreased over time from 256 ± 10 nM (0h) to 25 ± 9 nM (72h). We hypothesize that basolateral cortisol is rapidly metabolized by the epithelium, thereby stabilizing the luminal cortisol level. The transepithelial distribution profile of cortisol provides the basis for investigating the impact of maternal stress on early embryo development.
Grants: This study was supported by the Deutsche Forschungsgemeinschaft (DFG CH2321/1-1 and TR 1656/1-1).
Excessive proliferation of spermatogonia in an azoospermic patient treated with clomiphene – an immunohistochemical approach
Massive Proliferation von Spermatogonien in einem azoospermen Patienten nach Behandlung mit Clomiphen – ein immunhistochemischer Ansatz
1Institute for Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University Giessen, Germany; 2Clinic for Urology, Pediatric Urology and Andrology, University Clinic Giessen/Marburg, Giessen, Germany
Clomiphene citrate is used as pre-treatment for infertile men with non-obstructive azoospermia (NOA) prior to testicular sperm extraction (TESE). We report on a 33-year old azoospermic patient with a previous history of TESE and clomiphene citrate therapy before undergoing microscopically assisted, bilateral testicular biopsy (M-TESE). For histological analysis, four biopsies of each testis were fixed with Bouin’s solution, processed and evaluated according to standard protocols. Protein detection for germ cells, germ cell neoplasia in situ, proliferation and apoptosis, and Sertoli cell markers has been performed using specific antibodies. Histology revealed mixed atrophy of spermatogenesis, predominantly with spermatogonia or primary spermatocyte arrest, respectively. Foci with qualitatively preserved, quantitatively severely reduced spermatogenesis were found in the right testis. In the left testis, seminiferous tubules were massively packed with cells proved to be normally differentiated spermatogonia with slightly increased proliferative activity and a higher degree of apoptosis. Sertoli cells proved to be correctly matured. Formation of blood-testis barrier (BTB), however, seemed to be disturbed, as the expression of BTB specific proteins was absent or dislocated in the tubules with proliferation of spermatogonia. To our knowledge this is the first observation of excessive, non-malignant proliferation of spermatogonia in an NOA patient, maybe related to impairment of Sertoli cell function and a spermatogonial dysfunction caused by high-dose clomiphene citrate treatment preceding testicular biopsy.
Challenges of a tail mounted calving sensor
Herausforderung von Abkalbesensoren, die am Kuhschwanz befestigt werden
Clinic for Animal Reproduction, Faculty of Veterinary Medicine, Freie Universität Berlin, Germany
Objectives Calving sensors can help to recognize onset of parturition and thus in-time calving assistance if needed. A good calving sensor must not only detect onset of calving reliably but must ensure easy handling and be harmless for the dam. Tail mounted calving sensors measure increasing tail raising of a dam as a sign of imminent parturition. This study was to test functionality and compatibility of a tail mounted calving sensor.
Methods 180 dairy cows and heifers were enrolled in this study. The study took place in a Transition Maternity Facility on a commercial dairy farm milking 2500 cows. Five days before the estimated calving date (275 post insemination) a tail mounted calving sensor was attached to the cow’s tail. Compatibility and position of the sensor was controlled in every hour until calving.
Results In only 25 cows the sensor stayed on tail until calving, in 114 animals sensor fell off or slipped down > 3 cm, in 31 animals sensor was removed, because the tail was swollen or painful, in 10 cows the sensor messaged technical default.
Conclusion In this study the calving sensor could not detect parturition in 86% of the cows because it did not stay in position or caused irritation at the cow’s tail. The device weighs more than 300 grams. It is a great challenge to fixate such an object safely at the cow’s tail, without producing irritations, swellings or even wounds on the tail.
Serum progesterone level but not luteal blood flow is increased 6 days after ovulation in mares with intrauterine fluid accumulation after AI with frozen semen
Bei Stuten, die eine intrauterine Flüssigkeitsansammlung nach künstlicher Besamung mit Gefriersperma aufweisen, ist der Serum-Progesteronspiegel im Gegensatz zum lutealen Blutfluss 6 Tage nach der Ovulation erhöht
1DIMEVET Department of Veterinary Medical Sciences, University of Bologna, Italy; 2AUB INFA National Institute of Artificial Insemination, University of Bologna, Italy; 3Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Switzerland
The objective of the study was to investigate the effect of an intrauterine inflammation on luteal blood flow (LBF) and serum progesterone (P4) concentrations in mares. Therefore, 40 Standardbred mares referred for artificial insemination (AI) were bred with frozen semen of proven fertility for a total of 53 estrous cycles. Ovulation (Day 0) was induced by application (iv) of 2500 IU hCG at the first time when a follicular diameter of at least 30–35 mm was observed in the presence of endometrial oedema and a positive response to a teaser stallion. At 30–36 h after application of hCG, all mares were inseminated and additionally treated (iv) with 50 mg dexamethasone. Ovulation and the presence of intrauterine fluid accumulation (IUFA) as an indicator for endometritis were detected by ultrasonography at 12 h after AI. Mares with IUFA received a uterine lavage followed by 20 IU oxytocin (im), whereas mares without IUFA received only 20 IU oxytocin. Blood sampling was performed on Day 6 to analyse serum P4 concentrations. Ultrasonography was used on Days 3 and 6 to assess LBF, and on Day 14 for pregnancy diagnosis. In 53% of the estrous cycles, IUFA was detected and negatively affected the per-cycle pregnancy rate (35% vs 68%; P < 0.05). Serum P4 levels on Day 6 were higher (P < 0.05) in mares with IUFA compared to mares without IUFA (9.3 ± 5.7 vs 6.6 ± 2.4 ng/mL; mean ± SD), whereas LBF increased (P < 0.001) between Days 3 and 6 but did not differ (P > 0.05) between mares with and without IUFA. Results indicate that an intrauterine inflammation does not affect luteal perfusion but increases the secretory function of the equine corpus luteum at 6 d after ovulation.
Gene editing in cultured bovine trophoblast cells and IVP embryos using CRISPR/Cas9
Gen-Editierung in kultivierten Rindertrophoblastzellen und IVP-Embryonen mittels CRISPR/Cas9
Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany
The CRISPR/Cas9 technology is well suited for the targeted introduction of mutations into genomes and is used in a wide variety of organisms, including mammals. We have adapted the method to the requirements of bovine cell cultures and embryos to generate gene knockout mutants that enable the investigation of gene functions. A) For genome editing in cell cultures, cells were transfected with plasmids that co-expressed target-specific guide RNAs and Cas9. In mutant cells (crispants), we usually found deletions or insertions (indels) of a few base pairs (bp). Unexpectedly, however, some crispants had incorporated larger insertions of up to 100 bp derived from endogenous repetitive DNA or the expression plasmid used. B) In order to allow analysis of knockout phenotypes during ontogenesis, it was necessary to perform gene editing experiments in zygotes. The aim was to inactivate both alleles of the target gene in all body cells of the developing individual. To this end, pre-assembled CRISPR/Cas9 complexes were microinjected into the cytoplasm of in vitro produced (IVP) zygotes. In vitro production of embryos was carried out according to a standard procedure using oocytes from slaughtered cows and the BO media system. The mean blastocyst rate of our IVP experiments was 45%. The mean blastocyst rates of microinjected embryos were inversely proportional to the amounts of injected CRISPR/Cas9 complexes and ranged from 44% to 19%. About 14% of blastocysts derived from CRISPR/Cas9-microinjected zygotes had mutated target sequences. However, since crispant blastocysts were mosaics, the procedure must be further optimized.
The new four-channel EMG telemetry system used to determine uterine activity in different phases of the estrous cycle
Das neue 4-Kanal-EMG-Telemetriesystem dient zur Bestimmung der Uterusaktivität in verschiedenen Phasen des Östruszyklus
1Department of Large Animal Diseases with Clinic, Veterinary Research Centre and Center for Biomedical Research, Faculty of Veterinary Medicine, Warsaw University of Life Sciences WULS – SGGW, Warsaw, Poland; 2Clinic for Veterinary Obstetrics, Gynecology and Andrology, Justus-Liebig-University, Giessen, Germany
The new four-channel telemetry transmitter was applied to measure the EMG activity of reproductive tract in sows. Four silver bipolar electrodes were surgically positioned in the muscle layer of the caudal and cranial part of the cervix (CdC, CrC); right and left uterine horn (RUH, LUH). The new digital software platform (Ponemah) was used to record the EMG signals during a continuous in vivo experiment. The data series were obtained from a single, representative day in the follicular phase (FLP) and mid-luteal phase (MLP). The semiautomatic analytical software (NeuroScore 3.3.1) was used to extract the EMG signal features: amplitude (A), root mean square (RMS) and duration (D), and the data from all animals of each day were pooled, compared and presented further as mean ± SD. In CdC no differences (p > 0.05) in A (FLP: 0.26 ± 0.10 mV; MLP: 0.15 ± 0.06 mV), RMS (FLP: 0.04 ± 0.01 mV; MLP: 0.02 ± 0.01 mV) and D (FLP: 4.11 ± 2.88s; MLP: 5.32 ± 2.51s) were noted. In CrC, RUH and LUH the voltage-dependent features (A, RMS) were higher (p < 0.01) in FLP (A: CrC: 1.55 ± 1.01 mV; RUH: 2.01 ± 0.85 mV; LUH: 1.81 ± 0.54 mV; RMA: CrC: 0.99 ± 0.76 mV; RUH: 1.95 ± 1.25 mV; LUH: 1.79 ± 0.94 mV) than in MLP (A: CrC: 0.18 ± 0.09 mV; RUH: 0.22 ± 0.14 mV; LUH: 0.21 ± 0.03 mV; RMS: CrC: 0.02 ± 0.01 mV; RUH: 0.02 ± 0.01 mV; LUH: 0.02 ± 0.01 mV), whereas the time-dependent feature (D) was comparable (p > 0.05) on FLP (CrC: 3.85 ± 3.21s; RUH: 4.85 ± 2.88s; LUH: 4.07 ± 2.91s) in comparison to MLP (CrC: 6.25 ± 3.39s; RUH: 4.42 ± 4.05s; LUH: 4.35 ± 3.44s). The new telemetry system is accurate to determine the EMG signal features including estrous cycle phases.
Influence of thermal treatment of colostrum on triglyceride and cholesterol serum concentration of bovine neonates
Einfluss einer thermischen Behandlung von Kolostrum auf die Serumkonzentration von Triglyceriden und Cholesterin boviner Neonaten
1Clinic of Obstetrics, Gynaecology and Andrology of small and large animals, Justus-Liebig-University of Giessen, Germany; 2Institute of Veterinary Food Science, Justus-Liebig-University Giessen, Germany; 3Unit for biomathematics, Justus-Liebig-University Giessen, Germany.
Colostrum intake influences not only the immunoglobulin but also fatty acid and cholesterol concentration in the blood of newborn calves. Pasteurization of colostrum is gaining importance in killing certain microorganisms. The aim of this study was to investigate the influence of pasteurization of first colostrum on the concentration of triglycerides and cholesterol in the serum of newborn calves. For this purpose, 20 newborn bull calves were randomly divided into two groups (n = 10). The experimental group received 6 liters at 63.5 °C for 30 minutes pasteurized colostrum within the first 12 hours post natum (Pasteurizer HT 250, Förster), the control group 6 liters of untreated colostrum. Blood samples were taken from the V. jugularis at three time points (before and 24 or 48 hours after colostrum intake) and examined for the concentration of triglycerides and cholesterol. No statistically significant difference in triglycerides could be observed between the groups (p > 0.05). The calves of the control group had significantly higher cholesterol concentration (p = 0,041). The results indicate that the absorption of ingredients other than immunoglobulins is also influenced by pasteurization.
Results of transvaginal ultrasound-guided reduction of twin pregnancies in mares
Ergebnisse zur transvaginalen ultrasonographisch geleiteten Reduktion von Zwillingsträchtigkeiten bei der Stute
Tierarztpraxis Hohen Schönberg, Germany
Twin pregnancies cause economic losses in mares. They terminate in abortion, stillbirth or in the delivery of dead, weak or deformed foals. Transvaginal ultrasound-guided twin reduction techniques are described for twin pregnancies that advance to the phase of fixation and nidation. In total 64 twin pregnancies were ultrasound-guided managed by aspiration of embryonic fluid. Mares with twin pregnancies from day 23 to day 37 after breeding were directed to the ultrasound-guided procedure. All mares got a therapeutic dose of Romifedin (Sedivet Fa. Böhringer Ingelheim/Vetmedica) for sedation and furthermore Flunixine Meglumine (Finadyne Fa. MSD Tiergesundheit) before and for 3 days after aspiration. The aspiration was done with an ultrasound equipment by Pie Medical Dorsten/Germany (Parus Vet 240) using a probe of 5 MHz and aspiration with a needle of 0.9 mm diameter and a length of 80 mm. The location of the twins in the uterus was characterized by 29% in unilateral and 71% in bilateral position. There was a significant influence (p < 0.001) of the age of the embryos and of the duration of the aspiration procedure on the survival rate of the remaining embryo. A further influencing factor was the amount of aspirated fluid. In total the success rate was 89%.
The role of endometrial claudin-3 for implantation, decidualization and embryo development
Die Rolle des endometrialen Claudin-3 für die Implantation, Dezidualisierung und Embryoentwicklung
1Institute of Anatomy, University Hospital Essen, University Duisburg-Essen, Germany; 2Departement of Pediatrics, Division of Nephrology, Charité – Universitätsmedizin Berlin, Germany
The composition of cell contacts in the endometrium plays an important role in the process of embryo implantation and the establishment of pregnancy. In previous studies, we showed an induction of the tight junction protein claudin-3 in the decidua during murine trophoblast invasion from 6.5 dpc onwards. Here we evaluated the function of claudin-3 in implantation and embryo development with the help of a claudin-3 knockout (KO) mouse. Claudin-3 KO mice were fertile and their litter sizes did not differ significantly from those of control mice. Despite the presence of claudin-3 in the luminal and glandular epithelium of murine endometrium during the estrous cycle, KO mice revealed normal cycle phases. However, the weight of the implantation sites on 6.5 and 8.5 dpc as well as the weight of the embryos on 17.5 dpc, but not of the placentas, was significantly reduced in claudin-3 KO mice. This was shown to be a maternal effect. During postnatal development the differences in weight adjust between KO and control mice. In the claudin-3 KO mice, an upregulation of claudin-4 during the early stages of pregnancy was shown which could partly compensate for the lack of claudin-3. Since the main mass of the implantation sites at these stages of early pregnancy consists of decidual tissue, the reduced weight of implantation sites in KO mice hints to an impairment of decidualization possibly subsequently causing placental malnutrition of the embryo.
Progestogen profiling in the European roe deer (Capreolus capreolus) during diapause and the reactivation period
Progestogen-Profiling im europäischen Reh (Capreolus capreolus) während der Diapause und der embryonalen Reaktivierungsphase
1ETH Zurich, Institute of Agricultural Sciences, Animal Physiology, Switzerland; 2University of Zurich, Department of Chemistry, Switzerland
The European roe deer (Capreolus capreolus) is the sole ungulate species exhibiting a period of temporary embryonic arrest during pregnancy referred to as embryonic diapause. Fertilization takes place in July and August. At the 20-30 cell stage, the embryo enters diapause for approximately five months from August to December. During diapause, the developmental pace of the embryo is reduced. After diapause in late December or beginning of January, the embryo resumes growth and its development resembles that of other ruminant species. The molecular regulation of diapause and the subsequent reactivation of embryonic growth in the roe deer is not fully understood. Unlike in other mammalian species exhibiting diapause like the mink or the mouse, progesterone and estradiol levels do not change in the roe deer during diapause and the reactivation of embryonic growth [Drews et al. RB 2019; 19: 149–57]. Nevertheless, it is conceivable that bioactive metabolites of the classical ovarian steroids such as 5?-dihydroprogesterone contribute to the regulation of diapause. A recently developed LC-MS based approach allows the profiling of 12 progestogens in plasma. A first subset of five roe deer plasma samples spanning the period of embryonic diapause was analysed for progestogens and revealed that at least eight bioactive metabolites are present in roe deer plasma. More than one hundred additional samples across diapause and reactivation are currently under analysis. Thereby, we aim at unravelling a possible contribution of progestogens to the regulation of uterine gland secretion to understand the regulation of embryonic reactivation in the roe deer.
R123 and JC-1 staining of mitochondria in domestic cat sperm before and after freezing
R123- und JC-1-basierte Mitochondrienfärbung von Spermien der Hauskatze vor und nach Gefrierkonservierung
Dept. Reproduction Biology, Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany
To assess the mitochondrial activity of epididymal sperm from domestic cat, rhodamine 123 (R123) and a carbocyanine dye (JC-1) were comparatively applied before and after cryopreservation. Propidium iodide (PI) was used to label the dead sperm. Both dyes generate similar results for the total number of sperm with active mitochondria: 62 ± 11% and 66 ± 10% for R123 and JC-1, respectively, before freezing and 33 ± 8% and 38 ± 10% after thawing (n = 10). As additional information, JC-1 reveals two viable sperm populations with either high or low mitochondrial activity. The ratio between these populations is strongly dependent on incubation time and requires exact timing of flowcytometric analysis. The percentage of sperm with low mitochondrial activity was 16 ± 14% before freezing and 8 ± 7% after thawing. These numbers correspond to about ¼ of all active sperm in both cases. The percentage of all active fresh sperm stained by JC-1 is correlated to the percentages of motile (CASA, mean: 28 ± 13%), progressive (25 ± 13%) and fast progressive (20 ± 11%) as well as R123 positive sperm after thawing. The percentage of R123 positive fresh sperm correlates to the percentage of fast progressive sperm after thawing (Spearman, p < 0.05). The percentages of R123 as well as JC-1 positive sperm determined after thawing correlate to the percentages of motile, progressive, and in case of R123, fast progressive sperm (Spearman, p < 0.05). We conclude that the predictive potential of JC-1 staining in fresh samples to post-thaw motility is superior to that of R123 staining but the relation to fertility remains to be investigated.
Hepatic transcriptome analysis after intramammary pathogen challenge of cows carrying the cholesterol deficiency haplotype
Analyse des Lebertranskriptoms nach intramammärer Pathogen-Inokulation von Kühen mit dem Cholesterindefekt-Haplotyp
1Leibniz Institute for Farm Animal Biology (FBN), Institute of Genome Biology, Dummerstorf, Germany; 2Clinic for Cattle, University of Veterinary Medicine Hannover, Germany; 3Immunology Unit, University
The identification and elimination of hereditary diseases, especially lethal ones, is essential for high reproductive performance in cattle population. The cholesterol deficiency (CD) defect on Bos taurus autosome (BTA) 11 results in obligatory sick homozygous animals (CDS) and heterozygous carrier animals (CDC) [Kipp, J Dairy Sci 2016; 99: 8915–31 and Menzi, Anim Genet 2016; 47: 253–57]. In our study, we analysed the hepatic transcriptome of cows after intramammary challenge with common udder pathogens (Staphylococcus aureus, S. aureus or Escherichia coli, E. coli) early in their first lactation. In the S. aureus group, 4 CDC were compared to 20 wildtype homozygous animals. In the E. coli group, we evaluated 3 CDC to 8 animals not carrying the defect. RNA sequencing generated 3.8 billion reads (Ø 109 million reads per sample) with 98% of reads mapping at least once to the reference genome UMD3.1. Comparing CDC to the control revealed 577 (S. aureus group) respectively 75 (E. coli group) tentatively (p < 0.1) differentially expressed loci in the hepatic transcriptome. In both challenge groups, the APOB (Apolipoprotein B) gene was significantly (p < 0.05) differentially expressed between CDC and the control. From our data, we can conclude that APOB gene expression in liver is altered not only in CDS animals, but also in heterozygous carrier animals (CDC) – even under conditions of an intramammary pathogen challenge.
Low temperature preservation in boar semen: Are lipopeptides a possible alternative to conventional antibiotics?
Niedrigtemperaturlagerung von Ebersperma: Sind Lipopeptide eine mögliche Alternative zu konventionellen Antibiotika?
1Institute for Reproduction of Farm Animals Schönow e. V., Bernau, Germany; 2Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany; 3Physical Chemistry Department, Faculty of Pharmacy, Medical University of Gdansk, Poland
Antimicrobial resistance is a steadily increasing problem and poses a serious threat to the public health. Therefore, it is highly necessary to question the standardized supplementation of boar semen extenders with conventional antibiotics and to advance the development of novel antimicrobial compounds. The aim of this study was to evaluate a low-temperature, antibiotic-free storing procedure using Androstar® Premium extender (Minitube) combined with antimicrobial peptides (AMPs) as alternative supplements. The effect of 5 °C storage without antibiotics on sperm quality was compared to control samples using a BTS extender (Beltsville Thawing Solution, Minitube) with antibiotics at 17 °C. Secondary, seven lipopeptides were tested on sperm-compatibility at 17 °C. Two AMPs, Pal-KKK-NH2 and Pal-KKKK-NH2, did not negatively affect sperm quality and were evaluated for their efficiency of bacterial growth inhibition at 5 °C. As expected, 5 °C-storage affected some sperm quality parameters, however, the total motility after a fertility-relevant thermo-resistance test was not compromised (P = 0.429). Addition of Pal-KKK-NH2 and Pal-KKKK-NH2 did not impede sperm quality compared to the control at 5 °C (P > 0.130) and both AMPs managed to significantly (P ? 0.05) reduce the amount of bacterial contamination at all tested time points (0, 24, 48 and 72 hours) during storage. Therefore, both tested AMPs are promising prospects in view of the current situation regarding antibiotic resistance. Planned in vivo trials will show if there is any effect on fertility.
Grants: Supported by funds of German government’s Special Purpose Fund held at Landwirtschaftliche Rentenbank (28-RZ-3.053).
Loss of connexin-43 in Sertoli cells leads to altered prepubertal Sertoli cell maturation and impairment of the mitosis-meiosis switch
Abwesenheit von Connexin-43 in Sertoli-Zellen führt zu einer Veränderung der juvenilen Sertoli-Zell-Reifung und des Übergangs zwischen Mitose und Meiose
1Institute for Anatomy, University of Veterinary Medicine Hannover, Germany; 2Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Germany; 3Clinic for Cattle, University of Veterinary Medicine Hannover, Germany
In recent years, a significant decrease of male reproductive function has been reported but many underlying causes are still unknown. Generation of a conditional Sertoli cell (SC)-specific connexin43 (Cx43) knockout mouse line (SCCx43KO) has provided a translational model. As the predominant testicular gap junction protein, expression of Cx43 between SC and between SC and germ cells (GC) is known to be essential for initiation and maintenance of spermatogenesis in different species. Adult SCCx43KO males show altered spermatogenesis and are infertile. Thus, the present study aims to identify molecular mechanisms leading to testicular alterations in SCCx43KO mice. Transcriptome analysis of 8-, 10- and 12-day-old mice was performed by next-generation sequencing (NGS). Additionally, candidate genes were examined by qRT-PCR and immunohistochemistry. NGS revealed many significantly differentially expressed genes in the SCCx43KO mice. For example, GC-specific genes were mostly downregulated and found to be involved in meiosis and spermatogonial differentiation (e.g. Dmrtb1, Sohlh1). In contrast, SC-specific genes, implicated in SC maturation and proliferation, were mostly upregulated (e.g. Amh, Fshr). Moreover, time-specific gene ontology (GO) analysis yielded ten significant GO terms at all three time points and eight of these can be related to meiosis. In conclusion, Cx43 in SC appears to be required for normal progression of the first wave of spermatogenesis, especially for the mitosis-meiosis switch, and also for the regulation of prepubertal SC maturation.
Fractional semen collecting: A chance to reduce the damaging effect of seminal plasma on boar spermatozoa during long-term storage
Fraktionelle Samengewinnung: Eine Chance zur Reduktion der schädigenden Wirkung von Seminalplasma auf Eberspermien während der Langzeitlagerung
Unit for Reproductive Medicine of Clinics, University of Veterinary Medicine Hannover, Germany
Long-term exposure of boar sperm to autologous seminal plasma can cause a dramatic loss in motility. The aim of this study was to examine whether the selection of the first 150 (± 20) ml of the ejaculate (E1) compared to the total ejaculate (TE) increases the seminal plasma tolerance of sperm from sensitive boars during long-term storage. E1 was collected separately from the remaining ejaculate (E2) of seven boars. Semen samples from E1 only and from the remaining E1 + E2 (representing the total ejaculate (TE)) were diluted to 18×106 sperm/ml ad 100 ml in modified pH-stabilized Beltsville Thawing Solution. After 144 h storage at 17 °C, the motility between E1-samples and TE-samples did not differ (p > 0.05) but they variated more in the TE-group (82.2 ± 8.4%) compared to the E1-group (87.6 ± 2.4%). Two boars with the lowest motility in TE-samples (Boar 1: 78.6%, Boar 2: 64.8%) showed a higher motility in E1-samples (Boar 1: 86.3%, Boar 2: 86.2%). Spermatozoa of all boars showed a high level of sperm with intact membranes assessed with flow cytometry with propidium iodide and FITC-PNA (E1: 85.2 ± 1.3%, TE: 83.1 ± 1.8%, p > 0.05) and a high proportion of viable sperm with high mitochondria membrane potential assessed with propidium iodide and JC-1 (E1: 91.6 ± 3.7%, TE: 92.4 ± 2%, p > 0.05). In conclusion, the selection of the first 150 (± 20) ml of the ejaculate may present a possibility to encounter the damaging effect of seminal plasma on liquid preserved sperm of sensitive boars during long-term storage.
Grants: Supported by FBF.
Effects of beta-Hydroxybutyrate on the metabolism and motility of bovine endometrial gland cells in vitro
Einfluss von beta-Hydroxybutyrat auf den Metabolismus und die Motilität boviner endometrialer Drüsenzellen in vitro
Institute of Anatomy, University of Veterinary Medicine Hannover, Germany
Ketosis is a common metabolic disease in dairy cows leading to decreased reproductive performance in the transition period. Besides other factors, beta-Hydroxybutyrate (BHBA) blood concentration is significantly higher in these animals. The present study aims to investigate the effects of BHBA on bovine endometrial gland cells in vitro. Functional uterine glands are essential for uterine receptivity, implantation and placental development. An established permanent bovine endometrial gland cell (BEGC) line was used for the experiments. BEGC were stimulated with different concentrations of BHBA: 0.6 mM, 1.2 mM, 1.8 mM and 2.4 mM for 24h. Cell motility was examined by live cell imaging and cell metabolism was determined using the MTT assay. Serum free medium (SF) and modified Ham’s F12 medium (FM) served as controls. There were no significant differences in cell metabolism after incubation with BHBA. Additionally, cell motility was not affected when BEGC were incubated with BHBA concentrations of 0.6 mM, 1.2 mM and 1.8 mM. However, when incubating BEGC with 2.4 mM, cells began to get apoptotic after approximately 13h. These data show that BHBA concentrations of 0.6 mM, 1.2 mM and 1.8 mM seems to have no impact on metabolism and motility of BEGC cells, though live cell imaging showed that incubation with 2.4 mM BHBA results in apoptosis after a certain time. The apoptosis of endometrial gland cells might be one of the reasons for lower fertility in ketotic cows. Further investigations are planned regarding the influence of BHBA on endometrial gland cells, for instance steroid receptor expression and structure of the cytoskeleton.
Influence of corpus luteum stage on diameter of isolated steroidogenic cells in domestic cat
Der Einfluss des Gelbkörperstadiums auf den Durchmesser von isolierten steroidogenen Zellen bei der Hauskatze
Leibniz Institute for Zoo and Wildlife Research, Department of Reproduction Biology, Berlin, Germany
Corpus luteum (CL) is a transient gland on the mammalian ovary which produces progesterone. It is composed of small (SLC) and large (LLC) steroidogenic cells, as well as fibroblasts, endothelial cells and immune cells. To study the physiology of the gland, in vitro culture of luteal cells is a useful tool. However, the isolation step may hold limitations, because of high fragility of steroidogenic cells towards mechanical forces and enzymatic digestion. To investigate whether the stage of CL has an impact on diameter of isolated cells, we have isolated cells from CLs at formation (n = 2), maintenance/development (n = 11) and early regression (n = 5) stage. During isolation, luteal tissue pieces were incubated with collagenase 0.1% (type I and II, SERVA Electrophoresis GmbH) and 0.005% DNase (Merck) for 55 min at 39 °C. Afterwards, the pieces were gently smashed through 40 µm cell strainer and then, isolated cells were purified on 40% Percoll layer. We found that the enzymatic method isolates SLC only, with an exception for formation stage where differentiating SLC or still growing LLC were present too. The diameter for isolated steroidogenic cells was 12.9 ± 3.7, 11.9 ± 2.3, 9.3 ± 2.1 µm for formation, maintenance/development and early regression stage accordingly. By comparing the diameter of cells isolated in maintenance/development and early regression stage we detected significant difference (p < 0.05). We found that the average diameter of isolated SLC is decreasing with ongoing age of CL.
Grants: The study was funded by DFG (BR 4021/5-1).
DNA integrity in viable and non-viable bull sperm – a direct analysis after sorting of sperm by flow cytometry
DNA-Integrität in vitalen versus non-vitalen Spermien von Bullen – eine direkte Analyse nach dem Sortieren der Spermien mittels Durchflusszytometrie
1Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Switzerland; 2Besamungsverein Neustadt an der Aisch e.V., Germany
Previous studies revealed only poor or no correlations between sperm viability and DNA integrity. As in those experiments DNA integrity was analysed on the whole sperm population, we aimed to analyse the percentage of sperm with a high DNA fragmentation index (%DFI) separately in viable and non-viable sperm. Cryopreserved semen samples from 24 bulls were thawed and sperm viability was determined based on their mitochondrial membrane potential (MMP) using MitoProbe™ DiIC1(5). The viable and non-viable sperm were separated by a flow cytometric cell-sorter (S3e™ Cell Sorter). Non-sorted stained counterparts were used as controls and their overall MMP was determined using a flow cytometer-analyser (CytoFLEX). Afterwards, %DFI was measured using the Sperm Chromatin Structure Assay in the sorted subpopulations and their controls. The viable sperm showed a lower %DFI (0.74 ± 0.49%) compared to the non-viable sperm (5.51 ± 2.09%) and their non-sorted controls (2.48 ± 0.59%). No correlation (P > 0.05) was detected between MMP and %DFI in non-sorted samples. These preliminary results prove that DNA integrity is related to sperm viability, but the analysis of the entire sperm population containing a mix of viable and non-viable sperm conceals it. In further studies we will investigate if there is a relationship between DNA integrity of viable sperm and fertility.
Tumour infiltrating T lymphocytes in human testis cancer – identification and functional analysis
Tumor-assoziierte T-Lymphozyten im humanen Hodenkrebs – Identifizierung und funktionelle Untersuchung
R. Islam1, J. Borderies1, D. Püschl1,2, S. Indumathy1,2, K. Hartmann1, S. Kliesch3, B. Loveland4, F. Wagenlehner5, A. Pilatz5, M. P. Hedger2, K. L. Loveland2,6,
1Dept. of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University, Giessen, Germany; 2Centre for Reproductive Health, Hudson Institute for Medical Research, Clayton, Victoria, Australia;
In human testicular germ cell tumours (TGCT), i.e. seminoma and pre-invasive germ cell neoplasia in situ (GCNIS), infiltrating immune cells are frequently found with T cells representing a major component of the tumour-infiltrating lymphocyte (TIL) population. Recent studies indicate that functional polarization/subtypes of TIL including regulatory T cells (Treg) and T follicular helper cells (Tfh) play an important role in cancer development and immune surveillance. Therefore, we aim to characterize subsets of TIL in seminoma and GCNIS. Human testis samples (seminoma, GCNIS with lymphocytic infiltrates vs. normal spermatogenesis) were processed for immunohistochemistry (IHC) and RT-PCR, including quantitative analyses (RT-qPCR). Single cell suspensions were obtained from fresh TGCT specimens and used for flow cytometry. IHC confirmed the presence of CD8+ and CD4+ cells in immune cell infiltrates in GCNIS and seminoma with a scattered distribution in the latter, and revealed that CD4+/FOXP3+ Treg are present in seminoma in high number. These results were supported by flow cytometry, which also showed presence of Tfh. Moreover, increased transcript levels of corresponding markers and cytokines (RT-qPCR) were detected in seminoma. Further functional characterization of TIL in testicular neoplasia will help to elucidate “immune editing” during TGCT development.
Grants: Supported by DFG IRTG “Molecular Pathogenesis of Male Reproductive Disorders” GRK 1871/2.
Eligibility of boars for hypothermic, antibiotic-free semen storage under field conditions
Eignung von Ebern für hypotherme, antibiotikafreie Spermalagerung unter Feldbedingungen
1Unit of Reproductive Medicine of the Clinics, University of Veterinary Medicine Hannover, Germany; 2Setor de Suínos, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brasil
Hypothermic storage of boar semen at 5 °C is an innovative method for reducing the usage of antibiotics (AB) in semen extenders and facilitates temperature stability during semen transport. The aim of this study was to examine the quality of boar semen stored at 5 °C compared to 17 °C (control) in a boar population of an AI center in preparation of a field insemination trial. Semen (n = 34 normospermic boars) was diluted to 1.5×109 sperm/dose in Androstar Premium®, adapted at 22 °C for 3 ± 1 h and stored at 17 °C with AB or at 5 °C without AB, respectively. At 72 h, total motility for 17 °C-samples was 87.7 ± 7.3% and 77.9 ± 10.4% for 5 °C-samples (p < 0.05) and at 120 h it was 87.8 ± 7.4% in 17 °C-samples and 76.5 ± 10.6% in 5 °C-samples, respectively (p < 0.05). At 72 h in 5 °C storage, 4 samples (11.8%) failed the requirements for usable semen (total motility ? 65%) and 5 samples (14.7%) at 120 h. In 17 °C storage, 1 sample (2.9%) did not pass the threshold at 72 h and 120 h. Acrosome integrity remained high (? 90%) for all samples and time points. Then, 6 semen pools from selected boars (n = 15) were used for postcervical insemination (2.5×109 sperm/dose) up to 72 h with AB-free at 5 °C stored doses (n = 99 sows) and with AB-containing at 17 °C stored doses (n = 95 sows). Total motility for both pool groups remained high (> 88.9%) for 120 h. Preliminary data revealed high pregnancy rates of ? 98.9% in both groups. In conclusion, based on the threshold for semen quality, for the majority of boars (85.3%) the antibiotic-free semen storage at 5 °C seems to be applicable. Confirmation in field insemination trials under different conditions must be obtained.
Bacteriocins – a possible alternative for antibiotics in boar semen extenders?
Können Bakteriozine im Verdünnermedium für die Konservierung von Ebersperma eingesetzt werden?
1Institute for Reproduction of Farm Animals Schönow, Bernau, Germany; 2Institute of Food Safety and Food Hygiene, Department of Veterinary Medicine, Freie Universität Berlin, Germany; 3Minitube GmbH, Germany
As boar spermatozoa are usually stored at 16-18 °C for several days and a sterile production is impossible, a reasonable inhibition of bacterial growth is recommended. Due to rising numbers of antimicrobial resistance, the conventional use of antibiotics in semen extenders is under current debate. As a possible alternative to conventional antibiotics, four different bacteriocins (B1-4) with known bacteriolytic activity against Escherichia (E.) coli were investigated for their tolerance by boar spermatozoa and their impact against bacterial contamination in BTS-extended semen w/o gentamicin. The results showed a 50% reduction of E. coli by B4 compared to the control. No impact on spermatozoa was found in lower concentrations of 0.01 and 0.25% bacteriocin in the extender for storage up to 72 hours, the higher concentrations of 0.5 and 1% bacteriocin led to a significant (P < 0.05) reduction in semen quality for B1-3. B4 with no impact on semen quality even at higher concentrations was also tested in different short- and long-term extenders as well as at 17 and 6 °C storage temperature. For all tested extenders and temperatures, no significant (P > 0.05) differences between samples with and w/o B4 could be shown. Therefore, the targeted use of bacteriocins in semen extenders against specific bacteria seems a reasonable alternative to conventional antibiotics. Planned in vivo trials with semen diluted in extenders with bacteriocins will give the information, if there is any impact on fertility.
Grants: Supported by AIF (ZF4276702SK6).
Progestagen-profiling in pregnant Asian elephants (Elephas maximus) using a novel method combining ultra-high-performance liquid chromatography (UHPLC) with high resolution mass spectroscopy (HRMS)
Progestagen-Profiling tragender asiatischer Elefanten (Elephas maximus) durch Anwendung einer neuen Methode, die Ultra-Hochleistungs-Flüssigchromatographie (UHPLC) und hochauflösende Massenspektrometrie (HRMS) kombiniert
1University of Veterinary Medicine Hannover, Clinic for Cattle, Endocrinology Laboratory, Germany;
The supervision of pregnant elephants in zoos includes routine endocrine measurements. Various matrices and different methods were used. In some assays, a specific progesterone (P4) metabolite such as 5?-pregnane-3,20-dione (5?-DHP), 3?-hydroxy-5?-pregnan-20-one (3?,5?-THP) or 5?-pregnan-3?-ol-20-on is determined whereas other approaches made use of antibody cross reactivity to different progesterone metabolites. It was shown that the group of 5?-reduced metabolites are the most adequate metabolites to monitor CL function or predict the calving date. The aim was to determine patterns of progesterone metabolites for the first time in more detail using a novel UHPLC-HRMS based approach. Blood samples were available weekly for 5 weeks and daily in the week before the expected date of parturition from six zoo housed elephants in seven pregnancies. Ten reduced progestagens (20?-DHP, 20?-DHP, 3?,5?-THP, 3?,5?-THP, 3?,5?-THP, 3?,5?-THP, 3?-DHP, 3?-DHP, 5?-DHP and 5?-DHP), P4 and pregnenolone (P5) were determined with defined standards, but only P4, P5, 5?-DHP, 3?-DHP, 5?-THP were detected. The most abundant metabolite was 5?-DHP as expected from literature, but in contrast to previous studies, much higher levels of 5?-DHP were observed in relation to P4. Interestingly, the 5?-DHP/P4 ratio was highly individual for each elephant. In conclusion, the novel method using parallel metabolite assessment promises to be advantageous with respect to diagnose pathological pregnancies in mammals.
Pro-angiogenic alterations in the uterus of porcine von Willebrand disease (VWD)
Proangiogene Veränderungen im Uterus bei porcinem von-Willebrand-Syndrom (VWS)
1Institute of Anatomy, University of Veterinary Medicine Hannover, Germany; 2Werlhof Institute, Hannover, Germany
Clinical studies show that women affected by von Willebrand disease (VWD) are more likely to miscarry, possibly because of an impaired angiogenic effect of von Willebrand factor (VWF), due to reduced (type 1, T1) or non-measurable (type 3, T3) levels of VWF. As Endothelin (EDN1) and associated molecules could contribute to the impaired vascularization, the aim of this study was to analyse the expression of EDN1, Endothelin Converting Enzyme 1 (ECE1) and connective tissue growth factor (CTGF) in a porcine model of VWD. The phenotype and genotype of the model is identical with the human VWD. Uterine samples were taken from 14 sows in estrus, representing T1 (n = 2) and T3 (n = 2) and compared to controls (n = 10). Genetic expressions were measured via RT-qPCR and analysed with the ??CT method using PECAM and PROCR as reference genes. The mean expression level of ECE1 was four-fold (T3) to six-fold (T1) the mean expression of WT. EDN1 was upregulated 30% (T1) to 40% (T4) in the affected individuals. CTGF showed the strongest upregulation with ten-fold (p = 0.05) expression in T1 and five-fold in T3 pigs compared to WT. We demonstrate an upregulation of three pro-angiogenic factors in individuals with VWD, which might play a role in the impaired blood vessel formation and resulting angiodysplasia seen in VWD patients. This might be a possible link to the higher risk of miscarriage suggesting defective angiogenesis as causative for poor pregnancy outcome. A larger sample size and further angiogenic factors are warranted.
Conservation genetics: Elucidating gene expression dynamics and regulatory pathways during primary to secondary ovarian follicle transition in Felis catus by RNA-seq
Dynamik der Genexpression während der Follikulogenese in Felis catus
1Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany; 2Berlin Center for Genomics in Biodiversity Research, BeGenDiv, Berlin, Germany
The Felis catus (domestic cat) animal model is utilised to study artificial reproductive technologies (ART) for translation to threatened and endangered felids. Currently, in vitro growth of female felid germ cells remains sub-optimal. Additionally, an inadequate understanding of the early stages of folliculogenesis in felids has abated conservation efforts. Here, we collected primary and early secondary follicles from domestic cat ovaries for RNA sequencing. For this study, we aimed to identify and characterise differentially expressed genes (DEGs) involved in the early stages of folliculogenesis (primary to secondary follicle transition). We identified a total of 154 significantly DEGs comparing primary versus secondary follicle groups which included 122 up-regulated and 32 down-regulated genes. Gene ontology conditional enrichment analysis functionally annotated a number of DEGs associated with biological processes involved in folliculogenesis. For example, gene expression of furin paired basic amino acid cleaving enzyme (FURIN), WEE1 homolog 2 (WEE2), phospholipase C zeta 1 (PLCZ1), and G protein-coupled receptor 149 (GPR149) was shown to increase with ongoing follicle development (5% FDR, 2-fold difference in expression). In conclusion, we have identified genes which promote folliculogenesis through early ovarian follicle growth and through the positive regulation of transforming growth factor ?1 activation and positive regulation of cytosolic calcium ion concentration involved in egg activation.
MicroRNAs of bovine sperm and their relation to sperm fertility after sex-sorting
Spermien microRNAs beim Bullen und ihr Zusammenhang mit dem Befruchtungspotential des gesexten Spermas
1Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Switzerland; 2Swissgenetics, Zollikofen, Switzerland
Our study aimed to investigate the relation between the microRNA (miRNA) profile of unsorted sperm and bull fertility after artificial insemination (AI) with sex-sorted semen. For this purpose, 18 proven sperm donors were selected. The annual 56-day non-return rates of the 18 sires after AI with unsorted (NRRconv) and sex-sorted sperm (NRRss) were 69.1% ± 2.83% and 58.4% ± 5.79%, respectively, thus showing a relative reduction of 5.25% to 31.44% after sex-sorting. Post-thaw motility and functional status (viability, mitochondrial function, intracellular Ca2+ levels) of unsorted sperm were assessed through computer-assisted sperm analysis and multicolour flow cytometry. Total RNA was extracted with a modified TRIzol® protocol from a pool of four cryopreserved ejaculates per bull. Small RNA libraries were prepared (NEXTflex™ Small RNA-Seq Kit v3, Bioo Scientific) and sequenced with an Illumina® HiSeq 2500 system. Unique sequences were mapped to a collection of non-coding RNA sequence databases using BLAST to identify miRNAs. The relation between miRNA expression levels and NRR was assessed using Pearson’s correlation coefficient (r). In total, 85 miRNAs were identified. The expression levels of nine miRNAs (miR-9-5p, miR-423-5p, miR-34c, miR-449a, miR-1246, miR-92a, miR-2483-5p, miR-21-5p, miR-5193-5p) were related to the NRRss (0.515 ? |r |? 0.693, P < 0.05) but not to NRRconv (P > 0.05). None of these miRNAs was related to sperm motility and functional traits, except for miR-1246 that was related to the percentage of viable sperm with functional mitochondria and low intracellular Ca2+ levels (r = 0.541, P < 0.05). Concluding, our results show that the investigation of miRNAs in conventionally cryopreserved bovine sperm provides valuable information on their fertility after sex-sorting.
Ruminant binucleate trophoblast cells release exosomes into the maternal caruncular stroma
Diplokaryozyten der Wiederkäuer setzen Exosomen in das mütterliche Karunkelstroma frei
1Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, Switzerland; 2Institute of Virology, Vetsuisse Faculty, University of Zurich, Switzerland
It is well known that Binucleate Trophoblast Cells (BNC) in the ruminant placenta fuse with cells of the maternal caruncular epithelium. One function of this fusion is the exocytotic release of BNC-derived secretory proteins at the basement membrane of the caruncular epithelium. The BNC-granules contain a variety of proteins, which are released into the maternal organism by this complex process. We use transmission electron microscopy to reveal that the BNC granules in several ruminant species from two different clades (bovidae, cervidae) contain intraluminal microvesicles of about 65 nm diameter. These microvesicles, together with the secretory proteins, are released by exocytosis into the maternal stroma basal to the caruncular epithelium. The released exosomes can be seen at the basement membrane of the caruncular epithelium and in the underlying stroma. This finding suggests the presence of an additional avenue of feto-maternal communication in ruminants. The released exosomes might function as local mediators of intercellular communication in the placenta and might have also systemic functions in pregnant ruminants. Typically, exosomes are generated by the exocytosis of multivesicular bodies. The origin of exosomes from intraluminal microvesicles is a new, so far unknown, process.
Impact of a maternal supplementation of 10% inulin/FOS to high-fat or high-protein diet in mice on pregnancy outcome and offspring development
Einfluss einer maternalen Supplementierung mit 10 % Inulin/FOS zu einer Hochfett- oder Hochproteindiät in Mäusen auf den Trächtigkeitsverlauf und die Entwicklung der Nachkommen
1Institute of Nutritional Physiology “Oskar Kellner”, Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany; 2Institute of Genetics and Biometry, Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany
Nutritional imbalances or malnutrition, e.g. maternal high-protein or high-fat diets, during pregnancy are important factors for infant development disorders. Maternal supplementation of prebiotics (e.g. inulin, oligofructose) promotes gut health and reduces body weight. Prebiotics may be beneficial during pregnancy and lactation and may thus influence pregnancy outcomes. The aim of our study was to elucidate the impact of 10% inulin/oligofructose (FOS) supplementation to maternal high-fat (HF) or high-protein (HP) diet on pregnancy rate, litter size and pup’s development in mice. Five-week-old female C57BL/6NCrl mice were fed control diet (CD) for 2 weeks (n = 128). Thereafter, full siblings of litter mice were randomly assigned to 6 feeding groups: CD, CD with 10% inulin/FOS (1:1, CD+I; Orafti®HP inulin, Orafti®L95), HF (40% calories from fat), HF with 10% inulin/FOS (HF+I), HP (40% calories from protein) or HP with 10% inulin/FOS (HP+I). At 8 weeks of age, female mice were mated. Pregnancy rate, pregnancy time, stillbirth, pup survival, litter size and pup weight until day 20 were evaluated by a proc mixed model in SAS. Inulin/FOS supplementation alone did not significantly affect the measured parameters. Pups of HF and HF+I-fed dams had higher body weight than pups of all other treatments at 20 days (P < 0.05), whereas liver weight was lower in HF+I than HF pups (P < 0.05). Pregnancy outcomes were not directly affected by the supplementation of inulin/FOS. Lighter liver weight of HF+I pups indicated beneficial effects on pup health, potentially reducing the risk of obesity-related diseases.
Comparison of different thawing methods for bovine semen
Vergleich verschiedener Auftaumethoden von Rindertiefgefriersperma
1Alta Germany GmbH; 2Clinic for Cattle, University
In dairy reproduction management different methods for thawing semen are practically relevant. To identify the most successful procedure the aim of the present study was to verify how different thawing regimens influence conception rate in dairy cattle. The study was conducted at a large dairy farm in Brandenburg with about 1460 milking cows from October 2017 to January 2019. Animals in heat were automatically detected by an accelerometer system with neck straps. Three thawing methods were randomly tested: (A) thawing in water bath (38 °C) for 11s, transportation of the portion to the cow with a gun warmer at 35 °C, (B) thawing in water bath (38 °C) for 35s, transportation of the portion to the cow with a gun warmer at 35 °C and (C) transportation of the portion to the cow at ambient temperature and thawing the semen in the reproduction tract of the cow for 30s. In total 3337 inseminations were evaluated. For statistical analysis logistic regression was performed. No statistically significant effect of thawing method on conception rate and no interaction between method and insemination or lactation number, insemination code (natural heat vs. Ovsynch) or season could be determined. However, independently from method, lactation number, insemination code and season significantly affected conception rate. The reason for thawing semen in water bath is to reach a higher thawing rate to inhibit osmotic damage of the sperm cells and to prevent the formation of ice crystals, which could also harm sperm cells. Under field conditions at large dairy farms other factors than thawing method are more important to reach high conception rates after artificial insemination.
Endometritis in mares – diagnostic and treatment practices