Investigations of extracellular matrix proteases, apoptotic and anti-apoptotic factors in the bovine corpus luteum

The study is subdivided into two different parts: the first part deals with the development of a method to gain uterus milk in vivo during the preimplantation periode in cattle for the investigation of regulatory factors. The second part investigates different proteases in bovine follicles 20 hours after GnRH (Gonadotropin releasing hormone) injection (shortly bevor ovulation) for comparable as well as in the corpus luteum (CL) during oestrous cycle and induced luteolysis. In addition apoptotic as well as anti-apoptotic factors were evaluated in the CL during oestrous cycle and induced luteolysis. For the development of a method for gaining uterus milk in vivo during the first 24 days of gravidity in cattle, nine heifers were cycle synchronised using the Ovsynch method and artificially inseminated. Before flushing an epiduralanaesthesia was given and both uterus horns were flushed with 13ml 0.9% NaCl using a balloon embryo transfer catheter at day 5, 7, 12, 17 and 24 of gravidity. The catheter was placed 1cm cranial to the bifurcatio uteri in both horns. It was possible to retrive between 3ml and 13ml of the used flushing fluid. The uterus milk from the ipsilateral horn was inspected for an embryo and an EDTA-stabilisator was given to the uterus milk of both horns. An infection of the uterus occured in three heifers after the second and in five heifers after the third flushing. In one heifer no infection was found. Between day 17 and day 24 all heifers showed clear signs of oestrus. It was possible to detect progesterone, oestradiol-17-beta, PGF2alpha and VEGF via enzyme immunoassay (EIA) and radio immunoassay (RIA), respectively. Because of the occurred infection no statistic analysis was made. But it could be seen that the level of progesterone ranged between <0.1 and 0.4ng/ml, whereas no correlation to the inflammation was found. The values of the native uterus milk should be about 10-fold higher, because of the diluting effect of the flushing fluid. The pattern of oestradiol-17-beta was erratic, because the oestradiol levels of the flushing fluid from heifers with no infection till day 12 of gravidity could not be related to each other. It could be shown in further tests that high oestradiol-17-beta levels were measured via EIA in 0.9% NaCl with EDTA-stabilisator compared to 0.9% NaCl without the stabilisator. VEGF revealed levels between 0.08 and 2.58ng/ml, whereas the higher levels were seen during inflammation. The level of PGF2alpha showed a clear correlation to the infection. But there were also differences seen between the different heifers or even between the two uterus horns of one animal during the first flushing, which might be due to the massage of the uterus horns while flushing, which can induce prostaglandin secretion. The method of gaining uterus milk in vivo should be changed as follows: to prevent an endometritis just one flushing should be done per gravidity with gaining of the embryo, so that the heifer can be inseminated again after two oestrous cycle. Again both horns should be flushed, whereas one sterile disposable catheter should be used for each horn and the flushing should be devided into two parts: first flushing with 13ml of sterile 0.9% NaCL for biomolecular methodes, then flushing of the embryo using 5 x 50ml sterile PBS buffer. The embryo should be frozen in RNA-Later at -80° for further investigations. The received flushing fluid should be separated in two aliquotes, whereas one is used for the oestradiol-17-beta measurement and must not contain the EDTA-stabilisator. These two aliquotes should be centrifugated and the fluid should be stored seperatly at –20° from the received cell pellet. Part of the cell pellet can be used for the identification of the cells found in the uterus lumen and the rest stored in RNA-Later at –80° for further biomolecular investigations. For the evaluation of prostaglandins in the uterus milk it should be considered to treat the heifers with a prostaglandin synthetase inhibitor shortly before flushing. For the investigations of different extracellular matrix proteases and apoptotic as well as anti-apoptotic factors in bovine follicles and CL, RNA samples of a previous experiment were taken. In these experiment 26 cows received luteolytic dosis of a PGF2alpha-analogon and the CLs were removed by transvaginal ovaryectomy 0, 2, 4, 12, 24, 48 and 64 hours after induction of luteolysis. Additionally to these experiment five more cows were ovarectomised and the CLs were taken 0.5 hours after PGF2alpha treatment. For the investigation during oestrous cycle CL samples were collected at the slaughter house. For comparable reasons also follicles 20 hours after GnRH injection were taken using a superovulation model. The mRNA quality was inspected using the Bioanalyzer and good to very good results were seen for all samples. The mRNA expression levels were detected using quantitativ real time PCR (Rotor-Gene with SYBR Green I). The expression datas were normalised with the Bestkeeper index of four housekeeping genes using the delta delta CP method. The matrix metalloproteases (MMP-1, MMP-2, MMP-9, MMP-14, MMP-19), their tissue inhibitors (TIMP-1, TIMP-2), the plasminogen activator (PA) system (tissue –PA (tPA), urokinase-PA (uPA), urokinase-PA-receptor (uPAR), PA-inhibitor–1(PAI-1), PA-inhibitor-2 (PAI-2)), which are able degradate the extracellular matrix were investigated in follicles 20h after GnRH injection and in the CL during oestrous cycle and induced luteolysis. Furthermore the mRNA expression of the monocyte chemoattractant protein-1 (MCP-1), factors of the extrinsic apoptotic pathway (Tumor necrosis factor alpha (TNFalpha), TNF-Receptor-1 (TNFR1), TNF-Receptor-2 (TNFR2), Fas, Fas-Ligand (FasL)) and intrinsic apoptotic pathway (p53, Bax, Bcl-XL, Smac, Survivin) as well as the caspses 3, -6 and 7 were evaluated in the CL during oestrous cycle and induced luteolysis. During the first seven days of the oestrous cycle angiogenesis plays a critical role in the development of the CL. Basal membranes of blood vessels have to be removed to enable endothelial cell migration, which is necessary for the formation of new capillaries. MMP-1, MMP-2 and MMP-14 were not regulated, but TIMP-2, MMP-9, MMP-19 and TIMP-1 showed an increasing expression from 20h after GnRH till day 8-12 of the oestrous cycle in the CL. VEGF and bFGF seem to be able to stimulate the expression of MMP-1 and MMP-2 in luteal endothelial cells, which enable these cells to degrade the vascular basement membranes for sprouting new capillaries. MMP-19 is also expressed by these cells and has the same action like MMP-2 on basement membranes. The action of MMP-2 is not only regulated by VEGF and bFGF, but also by TIMP-2, which is lower expressed during angiogenesis in the CL and therefore might not block the action of MMP-2. TIMP-1 and TIMP-2 seem to be not only necessary for the inhibition of MMPs, but they are also able to enhance the proliferation of endothelial cell during angiogenesis. VEGF and bFGF also stimulate the expression of uPA in endothelial cells during angiogenesis. By forming a complex with uPAR and PAI-1 on the outer cell membrane these cells might be now able to detach from the vitronectin rich perivascular region and migrate towards the fibronection rich stroma of the CL in order to form new capillaries. Not only angiogenesis plays a critical role during CL formation, but also the inhibition of apoptotic action during vessel sprouting. Progesterone, bFGF and VEGF seem to inhibit an increased expression of apoptotic factors like p53 and Bax, while progesterone also inhibits Fas and caspase3. Addtitionally to the suppression of apoptotic factors an up-regulation of anti-apoptotic factors like Bcl-XL. and Survivin occurs. TNFalpha, produced by invading macrophages during CL formation, acts not only as an apoptotic factor, but is also able to enhance the production of PGE2 in endothelial and luteal cells, which stimulates together with TNFalpha the proliferation of endothelial cells during luteal angiogenesis. During induced luteolysis all investigated MMPs showed an increased expression as well as TIMP-1. Only TIMP-2 was down-regulated till 64h after PGF2alpha. PGF2alpha as luteolytic agents leads to the expression of MCP-1 on luteal endothelial cells. Thereupon monocytes and cytotoxic T cells migrate into the regressing CL and also start to express MCP-1 to recruite further mononuclear cells. For these migration into the stroma of the CL monocytes express uPA, uPAR and PAI-2. Macrophages are necessary for the phagocytosis of apoptotic cells and the degradation of ECM components in the CL during luteolysis. They are able to express different MMPs like MMP-1, MMP-9 and MMP-2. These MMPs degrade collagen I, the main structur collagen in the stroma of the CL, which might enhance the migration abilities of these macrophages. MMP-2 is also up-regulated in microvascular endothelial cells by TNFalpha, which is produced by macrophages. At the same time a down-regulation of TIMP-2 occurs, so that no inhibition of MMP-2 happens and a detachment of the endothelial cells from the vessel basement membrane follows. This leads to apoptosis of the endothelial cells, which seem to be the first cells to undergo cell death during luteolysis of the CL. Apoptosis seems to play an important role during luteolysis of the CL. The extrinsic pathway mediated by TNFalpha, its two receptors, Fas and FasL seem to be more important than the intrinsic pathway, due to the massiv up-regulation of these factors seen during induced luteolysis. TNFalpha and FasL show a high increased expression from the beginning of induced luteolysis onwards, which might by due to the infiltration of T cells and macrophages expressing these factors. Endothelial cells express TNFR1 and TNFR2 on their surface during luteolysis, which leads to the activation of the extrinsic apoptotic pathwas by binding of TNFalpha. INFgamma enhances these apoptotic action on endothelial cells. It seems possible that apoptosis of luteal cells occurs after the degradation of capillaries, which leads to an undersupply with oxygen and an increase of ROS. These reactive oxygen spieces up-regulate p53 and Bax as apoptotic factors of the intrinsic pathway, which leads to an activation of the caspases and apoptosis of luteal cells during induced luteolysis. These apoptotic action is further enhanced by the activation of the extrinsic pathway. Luteal cells stimulated by TNFalpha and INFgamma express Fas on their cell surface when the progesterone level declines. Cytotoxic T cells, express FasL and promote luteal cell death by activation the extrinsic pathway. This happens only when the progesterone level is decreased, which is known to happen at the end of the functional luteolysis. The results of these study show that ECM degrading proteases have two different roles in the life span of the CL: first they enable the sprouting of capillaries during angiogenesis in the developing CL, but during induced luteolysis their role seems to be to degrade ECM components of the CL stroma, which detaches endothelial cells from vessel basement membranes. These leads together with the infiltration of macrophages and cytotoxic T cells to apoptosis of endothelial and luteal cells

Comparison of the Meat Quality of Turopolje, German Landrace x Turopolje and German Landrace x Pietrain Pigs

Aim of the study was to evaluate, if the mixed breed German Landrace x Turopolje (L x T) was suitable for conventional fattening and the production of high quality palatable meat. Hence, we chose to study the carcass characteristics of three different breeds: true bred Turopolje (T x T) (n=15), an autochthonous Croatian breed, German Landrace x Pietrain (L x P) (n=19), a typical German pig hybrid and German Landrace x Turopolje (L x T) (n=23) as mixed breed. All three breeds were kept in a conventional fattening indoor system. The data consisted of the chemical and physical values of the carcass and the difference between breeds during breeding and fattening. All pigs were fattened with a conventional ad libidum feeding system. The feed consisted of an optimal mixture for the fattening of L x P. The daily feed intake and the weight from birth until the end of the fattening was recorded every 14 days. The quality of the carcass was evaluated at the age of 20 and 25 weeks. The measurement of the carcass was based on the “Richtlinie für die Stationsprüfung auf Mastleistung, Schlachtkörperwert und Fleischbeschaffenheit beim Schwein” published by the national German control office. L x T showed the lowest feed intake per kg carcass compared to the other breeds. The quality of meat was characterized by pH, conductance, intramuscular fat and water holding capacity. L x T showed a trend for a lower conductance in week 25. The value of pH and water holding capacity was not significant between the breeds. Surprisingly, the intramuscular fat of L x T was by trend higher compared to L x P and significantly lower than T x T (p < 0.05). L x T had by trend a higher carcass weight and a larger carcass length compared to L x P, which was significantly higher than T x T (p < 0.05). In conclusion, the new breed L x T seems to be suitable for an indoor fattening system and produces a high quality palatable meat. The energy and protein intake should be slightly reduced, which would reduce the cost of meat production

Effect of the Ketone Body Beta-Hydroxybutyrate on the Innate Defense Capability of Primary Bovine Mammary Epithelial Cells.

Negative energy balance and ketosis are thought to cause impaired immune function and to increase the risk of clinical mastitis in dairy cows. The present in vitro study aimed to investigate the effect of elevated levels of the predominant ketone body β-hydroxybutyrate on the innate defense capability of primary bovine mammary epithelial cells (pbMEC) challenged with the mastitis pathogen Escherichia coli (E. coli). Therefore, pbMEC of healthy dairy cows in mid- lactation were isolated from milk and challenged in culture with 3 mM BHBA and E. coli. pbMEC stimulated with E. coli for 6 h or 30 h showed an up-regulation of several innate immune genes, whereas co-stimulation of pbMEC with 3 mM BHBA and E. coli resulted in the down-regulation of CCL2, SAA3, LF and C3 gene expression compared to the challenge with solely the bacterial stimulus. These results indicated that increased BHBA concentrations may be partially responsible for the higher mastitis susceptibility of dairy cows in early lactation. Elevated levels of BHBA in blood and milk during negative energy balance and ketosis are likely to impair innate immune function in the bovine mammary gland by attenuating the expression of a broad range of innate immune genes

Physiological and behavioral responses of dairy cattle to the introduction of robot scrapers

Autonomous mobile robot scrapers are increasingly used in order to clean the floors on dairy farms. Given the complexity of robot scraper operation, stress may occur in cows due to unpredictability and lack of control of the situation. Experiencing stress can impair animal welfare and in the long-term the health and milk production of the cows. Therefore, this study addressed potential stress responses of dairy cattle to the robot scraper after introducing the autonomous mobile machine. 36 cows in total were studied on three different farms to explore possible modifications in cardiac function, behavior, and adrenocortical activity. The research protocol on each farm consisted of four experimental periods including one baseline measurement without robot scraper operation followed by three test measurements, in which cows interacted with the robotic cleaning system. Interbeat intervals were recorded in order to calculate the heart rate variability parameter RMSSD, behavior was observed to determine time budgets, and fecal samples were collected for analysis of the cortisol metabolites concentration. A statistical analysis was carried out using linear mixed-effects models. Heart rate variability decline immediately after the introduction of the robot scraper and modified behavior in the subsequent experimental periods indicated a stress response. The cortisol metabolites concentration remained constant. It is hypothesized that after the initial phase of decrease, heart rate variability stabilized through the behavioral adjustments of the cows in the second part of the study. Persistent alterations in behavior gave rise to the assumption that the animals’ habituation process to the robot scraper was not yet completed. In summary, the present study illustrated that the cows showed minor signs of disturbance towards the robotic cleaning system. Thus, our findings suggest that dairy cattle can largely adjust their behavior to avoid aversive effects on animal welfare. Additional research can provide further insight into the development of the animal-machine interaction beyond the initial phase of robot scraper operation considered in this study

Immune cell counts and signaling in body fluids of cows vaccinated against Clostridium difficile

Abstract Background New treatment options are needed to prevent relapses following failed antibiotic therapies of Clostridium difficile infections (CDI) in humans. The concomitant therapy with an anti-C. difficile IgA containing whey protein concentrate can support the sustainable recovery of CDI patients. For 31 weeks, nine dairy cows were continuously vaccinated with several anti-C. difficile vaccines by certain routes of administration to produce anti-C. difficile IgA enriched milk. The study aimed at finding decisive differences between low responder (LR) and high responder (HR) cows (> 8.0 µg ml−1 total milk C. difficile specific IgA) concerning their immune response to vaccination on cellular and molecular biological levels. Results The results of total and differential cell counting (DCC) in blood and milk and the outcomes of the gene expression analysis of selected immune factors were assessed relating to the usage of two vaccine batches for injection (MucoCD-I batch A and B), marking two immunization (IM) periods, and compared to a control group (Ctr). The MucoCD-I batch A caused short-term leukopenia followed by leukocytosis in the blood of LR and HR. The total somatic cell counts in milk were not altered by the treatment. The DCC revealed that the leukocytes of the treated groups were partly impaired by the treatment. The gene expression analysis exposed cumulative and sustainable differences (p < 0.05) between LR and HR for the genes encoding for lactoferrin, CXCL8, IL1β, IL2, IL6, IL12β, IFNγ, CD4 and CD163. The regulation of the epithelial IgA cell receptor PIGR was not impaired by the IM. In contrast to the vaccination with MucoCD-I batch A, the second IM period with MucoCD-I batch B resulted in mitigation and synchronization of the treated groups’ immune responses. Conclusions The inversely regulated cytokines in the blood and milk cells of the treated groups led to a variously directed, local T cell response resulting in their different production intensities of C. difficile specific IgA in milk

Primer for RT-qPCR measurements.

<p>Primer for RT-qPCR measurements.</p

IC of pbMEC.

<p>The typical cobblestone-like morphology and the epithelial character were confirmed, by the positive cytokeratin staining, the insert shows the negative control. [Leica light microscope, magnification x200].</p

Comparison of the RT-qPCR data of LF and CCL2 with the LF and CCL2 content in pbMEC cell culture supernatants.

<p>The fold changes of the LF gene expression (A) indicated a significant down-regulation of LF gene expression when pbMEC where co-stimulated with <i>E</i>. <i>coli</i> and 3 mM BHBA. The same effect could be detected within the competitive LF ELISA (B) of pbMEC cell culture supernatants. The amount of secreted LF decreased significantly in case of the co-stimulation. The distinct and significant down-regulation in CCL2 gene expression (C) could also be confirmed by the results of the bovine CCL2 VetSet^™ ELISA Kit (D). The CCL2 gene expression and the amount of secreted protein decreased distinctly as well as significantly within the co-stimulatory group. Significant changes: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, distinct changes: + 0.1 ≤ p ≤ 0.05.</p

Fold changes in gene expression upon immune stimulation.

<p>Fold changes in gene expression upon immune stimulation.</p

Milk yield and proximates related to different groups and lactation stages.

<p>Milk yield and proximates related to different groups and lactation stages.</p