Isolation and cultivation of adult primary bovine hepatocytes from abattoir derived liver

The aim was to establish a cell culture of adult primary bovine hepatocytes obtained from liver following slaughter and to cultivate the cells in a sandwich culture. Cells and medium samples were taken after separation of cells (day 0), during monolayer (days 1, 2 and 3) and during sandwich culture (days 1, 2, 3, 4, 7, 10 and 14). The mRNA expression of BAX, BCL2L, FAS, IGF-1 and GHR1A was measured as well as urea and LDH. Hepatocytes were obtained by using a two-step collagenase perfusion and were purified thereafter by density gradient centrifugation. The viability was 68.2 ± 9.5 %. In sandwich culture, cells have a typical polygonal hepatocytelike shape, build cell-cell contacts, and show irregularity of cell borders suggesting bile canaliculi generation. The BAX mRNA expression increased on day 1 as well but decreased steadily until day 3 and remained constant for 14 days. Urea- and LDH-concentrations increased from day 4 to day 7. In conclusion, we found that it is possible to gather viable primary hepatocytes from adult bovine liver after slaughter, and that cells gathered this way show typical morphologies, urea-production and low LDH-leakage especially at day 4 in a sandwich system

Stress Response of Stone Martens and Red Foxes in Two Different Live Traps

Trapping of terrestrial animals is an important tool for harvest, pest control and research worldwide. To catch animals alive, animal welfare has to be ensured, which is reflected in different agreements on trading and trapping of animals between sovereign nations (Council Regulation [EEC] No 3254/91). The red fox (Vulpes vulpes) and stone marten (Martes foina) represent important predatory animals. Their influence on protected species as well as their increasing appearance in urban areas demand responsible handling. In our study, we evaluated 2 trap systems used for trapping red foxes and stone martens in accordance with criteria stipulated in the Agreement on International Humane Trapping Standards (AIHTS) and International Organisation for Standardisation 10990 Part 5 – Methods for Testing Restraining Traps (ISO 10990). In total, we captured 20 red foxes in a concrete pipe vault trap and 13 stone martens in a Strack’s wooden box trap in Schleswig-Holstein, Germany, and observed their behavior inside each trap. After anesthesia, a clinical examination of each animal was conducted, and blood and hair samples were taken. After euthanasia, radiological examinations of the full body were taken, and necropsies and histopathological investigations were performed. No trap-associated lesions were found. Hormone analysis showed no remarkable signs of stress for the animals, according to state-of-the-art methods. Apart from serum cortisol, the quotient of dehydroepiandrosterone in serum and hair seems to be the most predictive value on stress response of the 2 different species. Video observation of the trapped animals emerged as a valuable tool to estimate animal welfare by behavior. This study complements AIHTS and ISO 10990 criteria with results on behavior and hormone analysis, being an additional benefit when evaluating animal welfare of each trapping system

Peripheral thyroid hormone levels and hepatic thyroid hormone deiodinase gene expression in dairy heifers on the day of ovulation and during the early peri-implantation period

Background: Before the onset of fetal thyroid hormone production, the transplacental delivery of maternal thyroid hormones is necessary for embryonic and fetal development. Therefore, the adaptation of maternal thyroid hormone metabolism may be important for pregnancy success and embryo survival. The aims of this study were to determine the thyroid hormone levels during the early peri-implantation period until day 18 and on the day of ovulation, to determine whether pregnancy success is dependent on a "normothyroid status" and to determine whether physiological adaptations in maternal thyroid hormone metabolism occur, which may be necessary to provide sufficient amounts of biologically active T3 to support early pregnancy. Therefore, blood samples obtained on the day of ovulation (day 0) and days 14 and 18 of the Holstein-Friesian heifers (n = 10) during the respective pregnant, non-pregnant and negative control cycles were analyzed for thyroid-stimulating-hormone (TSH), thyroxine (T4) and triiodothyronine (T3). Liver biopsies (day 18) from pregnant and respective non-pregnant heifers were analyzed for mRNA expression of the most abundant hepatic thyroid hormone deiodinase (DIO1) by real time qPCR. Results: Although liver DIO1 mRNA expression did not differ between the pregnant and non-pregnant heifers on day 18, the serum concentrations of TSH and T3 on day 18 were higher in non-pregnant heifers compared to pregnant heifers (P < 0.05). Moreover, T3 decreased between day 0 and 18 in pregnant heifers (P < 0.001). Conclusions: In conclusion, no associations between thyroid hormone patterns on day 18 and pregnancy success were detected. During the early peri-implantation period, TSH and T3 may be affected by the pregnancy status because both TSH and T3 were lower on day 18 in pregnant heifers compared to non-pregnant dairy heifers. In further studies, the thyroid hormone axis should be evaluated throughout the entire gestation to confirm these data and identify other possible effects of pregnancy on the thyroid hormone axis in cattle

Sedation With Xylazine Hydrochloride Decreases the Stress Response in Merino Meat Sheep During Routine Hoof Trimming in a Tilt Table

We hypothesized that the hoof trimming in sheep in dorsal recumbency implicates a short but intensive stress situation and that the sedation with xylazine causes a decrease in the stress response in this situation. Ten healthy female merino meat sheep were randomly divided into two groups receiving either xylazine hydrochloride (0.1 mg/kg body mass (BM) applied intramuscularly) or a placebo treatment with 0.9% NaCl. Routine hoof trimming was performed in a tilt table and vital signs (rectal temperature (RT), heart rate (HR), and respiratory rate (RR)), 33 different behavioral traits and blood cortisol concentrations were recorded throughout the experiment at six different time points (total of 55min). The procedure itself elicited a clear stress response (increase in the RR, RT, defensive movements, lip twitching, swallowing, and flight behavior). Parallelly, the blood cortisol concentrations were increased, reaching their maximum with 81.5 ng/ml in the control group when the sheep were tilted back into a standing position. In the sedated sheep, no increase in the RR and RT and a decrease in the HR were observed. In addition, the behavioral signs showed a decrease in flight, defensive, and general stress behavior (decrease in licking,movement of head and legs, and sitting on knees), complemented by the serum cortisol concentrations showing 2.28 times reduced concentration at the end of the procedure, compared to the control sheep. The results confirm our hypothesis and support the conduction of future trials investigating the feasibility and benefit of a sedation of sheep prior to routine hoof trimming under practical circumstances

Schmerzausschaltung beim thermischen Enthornen: Untersuchungen zu verschiedenen Verfahren der Schmerzausschaltung bei der thermischen Enthornung von Kälbern

In einer umfangreichen Untersuchung wurden verschiedene Methoden des Schmerzmanagements bei der thermischen Enthornung von Kälbern überprüft. Eine wirksame Schmerzausschaltung wurde mit der Anwendung von Lokalanästhetika erreicht. Das Sedativum stellte die Kälber ruhig und ermöglichte ein weitestgehend präzises Arbeiten. Die Anwendung von Lokalanästhetika darf ausschließlich durch einen Tierarzt erfolgen. Im Interesse des Tierschutzes und der Tiergesundheit sollten die Kälber aber nur mit einem vollständigen Schmerzmanagement enthornt werden. Alternativ wird empfohlen, die Milchkuhherden zukünftig stärker mit genetisch hornlosen Kälbern zu reproduzieren. Redaktionsschluss: 07.05.201

Transrectal three-dimensional fetal volumetry in early pregnant mares: Relationships between maternal factors and equine fetal volume measurements

The aim of this study was to investigate the effects of maternal, hormonal, and fetal factors on early fetal volume (FV) measurements in mares obtained by three-dimensional (3D) ultrasound. Furthermore, postpartum parameters were explored in regard to their association with early FV. For this purpose, 149 German warmblood mares that were artificially inseminated and confirmed to be pregnant between days 14-16 of gestation, were examined transrectally at day 45 ± 1 of gestation with the portable 3D ultrasound device Voluson® i (GE, Zipf, Austria). FV was calculated by using the extension software Virtual Organ Computer-aided AnaLysis (VOCAL™). Two different mixed linear models were used to analyze associations between the investigated maternal and fetal factors and the FV. Explanatory variables investigated in the first model were: maternal age, parity, maternal weight, and body condition score, type of pregnancy (recipient or biological mother), barren status (lactating or non-lactating), fetal sex, progesterone (P4) and equine chorionic gonadotropin (eCG) concentrations; and in the second model outcome variables such as gestational length, birth weight, placental weight, fetal sex, and abortion were included in the analysis. The final models revealed a significant relation between FV and eCG (b = 0.011, P = 0.030), as well as with P4 (b = -0.053, P = 0.016), but interestingly P4 was negatively related to FV. Fetal sex showed the most prominent effect on FV (b = -0.256, P = 0.039), with female fetuses being smaller than male fetuses. In the second model none of the investigated parameters were related to early FV except for fetal sex (b = -0.328, P = 0.047), again with female fetuses being smaller. In summary, it was found that FV is related with eCG, P4 and fetal sex, but was no suitable predicting factor for the investigated outcome parameters. Furthermore, the findings suggest that sex specific growth differences exist already in early gestation. The detailed biological mechanism by which P4 and eCG affect fetal size has to be investigated in prospective studies

Oral Supplementation with a Special Additive of Retinyl Palmitate and Alpha Tocopherol Reduces Growth Retardation in Young Pancreatic Duct Ligated Pigs Used as a Model for Children Suffering from Exocrine Pancreatic Insufficiency

Pancreatic exocrine insufficiency (PEI) is a disease of diverse aetiology—e.g., majority of patients suffering from cystic fibrosis (CF) show PEI congenitally. Malnutrition and malabsorption of nutrients impair growth and nutritional status. As reduced fat digestion leads to a deficiency of fat-soluble vitamins the supplementation is standard, but absorption is a critical point in PEI-patients. The pancreatic duct ligated (PL) pig is an established model for PEI in humans and has been proven to be a suitable model to compare different vitamin additives for supplementation. In a former study, PEI caused distinct growth retardation in young piglets, but did not affect growth in older ones. Our study hypothesised that this age-dependent effect is caused by exhausted body reserves of fat-soluble vitamins and, therefore, extra supply reduces growth retardation. PEI was induced by PL at the age of seven (PL-7) or 16 weeks (PL-16). Controls (C) underwent a sham surgery. Some PL-7 pigs (PL-7 + Vit) were fed a special vitamin additive. PEI reduced the mean final body weight (kg) at 26 weeks of age significantly with lower effect in PL-16-pigs (C:117; PL-7:49.5; PL-7 + Vit:77.1; PL-16:96.4). Extra vitamin supply resulted in an increased growth and normalised serum concentration of alpha-tocopherol, underlining the importance of special supplementation in PEI-patients

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction

Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are determined in many sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiency in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to twenty-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane intact spermatozoa supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa

Sea Buckthorn Pomace Supplementation in the Diet of Growing Pigs—Effects on Fatty Acid Metabolism, HPA Activity and Immune Status

There is evidence that sea buckthorn, as a source of n-3 polyunsaturated fatty acids (n-3 PUFA), possesses health-enhancing properties and may modulate neuroendocrine and immune functions. In the present study, we investigated the effect of sea buckthorn pomace (SBP) supplementation in the diet of growing German Landrace pigs on fatty acids in the blood and hypothalamus, peripheral immune parameters and mRNA expression of corticotropin-releasing hormone (CRH), mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the hypothalamus and spleen. Pigs were fed diets supplemented with 12% of dried SBP or 0% SBP (control group) over an intervention period of eight weeks. The fatty acid profiles in blood plasma were significantly affected by SBP supplementation only for C18:2n-6 and n-6/n-3 PUFA ratio compared with the control group. SBP supplementation did not significantly affect the fatty acid concentrations in the hypothalamus. Furthermore, there were no significant differences in mRNA expression of CRH, MR and GR in the hypothalamus or of GR mRNA expression in the spleen. Concerning the immune status, the plasma IgG levels tended to be higher in SBP pigs, whereas the leukocyte distribution, mitogen-stimulated lymphocyte proliferation, and serum IgM levels remained unchanged. In conclusion, the SBP supplementation of the diet only caused moderate effects on fatty acid metabolism, but no significant effects on hypothalamic–pituitary–adrenal (HPA) activity and immunity in growing pigs. It seems that a beneficial effect of dietary n-3 PUFA on health and welfare is more likely to be expected during stressful situations

Progestogen Profiling Over the Course of Diapause and Resumption of Embryo Development in the European Roe Deer

Progesterone (P4) plays a pivotal role in maintenance of pregnancy in many mammalian species. Species-specific P4 metabolites have been shown to function as primary acting progestogen and the receptor binding capacity varies between species. The European roe deer (Capreolus capreolus) displays a 4-5 month period of embryonic diapause, which decouples fertilization from implantation. The majority of roe deer have two corpora lutea that secrete P4. No changes in P4 concentrations have been observed during pre-implantation embryo development. As 5α-DHP is known to play a major role during pregnancy in elephants and horses, we hypothesized that 5α-DHP functions as additional progestogen facilitating embryo reactivation. The profile of 11 progestogens was quantified in roe deer plasma over the course of diapause and resumption of embryo development including P4, 3α- and 3β-DHP, 20α- and 20β-DHP, 5α- and 5β-DHP, 3α,5α- and 3α,5β-THP, as well as 3β,5α- and 3β,5β-THP. While P4 was most abundant during diapause and resumption of development, 20α-DHP was the most abundant P4 metabolite. This is different than in pregnant elephants, where 5α-DHP was most abundant, and the luteal phase in cattle, where 3α,5α-THP was most abundant. With the exception of a weak correlation of 3β,5α-THP, none of the progestogens significantly correlated with embryonic development in the roe deer. Thus, plasma 5α-DHP does not seem to play a role in embryo reactivation. We propose that progestogens might contribute to priming the endometrium for supporting embryo development and preparation for implantation